Depletion of CD8+ CAR T-cells leads to superior anti-tumor efficacy of pure CD4+ CAR T-cells against Acute Leukemias

Depletion of CD8+ CAR T-cells leads to superior anti-tumor efficacy of pure CD4+ CAR T-cells against Acute Leukemias | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Depletion of CD8+ CAR T-cells leads to superior anti-tumor efficacy of pure CD4+ CAR T-cells against Acute Leukemias Tim Sauer, Qian Chen, Hao Yao, Lianghao Mao, Dominic Depke, Lei Wang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6283250/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Chimeric antigen receptor T-cell (CART) therapy has shown impressive therapeutic efficacy in several hematologic malignancies, however primary or secondary treatment failure remains a significant challenge driving translational research to improve the functionality of CARTs. Here, we show the optimal composition of CARTs targeting acute leukemias with respect to the content of CD4 + and CD8 + T-cells. Our analysis demonstrated that pure CD4 + CARTs exhibited superior antitumor activity and proliferative capacity in vitro and in vivo compared to CD8 + -containing CART products. Furthermore, the secretome of pure CD4 + CARTs, enriched for Th1 and Th2 cytokines, was more potent in stimulating the anti-leukemic activity of CARTs. Mechanistically, we found that the interaction with CD8 + CARTs induces apoptosis in CD4 + CARTs leading to their impaired functionality. Our findings demonstrate the superior efficacy and persistence of pure CD4 + CARTs against acute leukemias warranting further exploration of their therapeutic potential within early phase clinical trials. Health sciences/Oncology/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia Health sciences/Oncology/Cancer/Haematological cancer/Leukaemia/Acute lymphocytic leukaemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The adoptive transfer of chimeric antigen receptor (CAR) modified T lymphocytes (CARTs) has emerged as an innovative cancer immunotherapy, offering an effective treatment strategy for various malignancies. CARTs targeting CD19 and BCMA have proven to be effective for the treatment of CD19-expressing malignancies such as acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin’s lymphoma and Multiple Myeloma. This success has led to the approval of multiple CD19- and BCMA-specific CART cell products by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA). 1 While CARTs can induce long-lasting remissions in many patients, primary resistance and disease relapse remains major challenges. Insufficient expansion and persistence of CARTs are frequently observed in patients with primary and secondary treatment failure to CART cell treatment, highlighting an urgent need for strategies to overcome these major limitations. 2 – 6 Optimizing the composition of CART products with respect to CD4 and CD8 expressing CARTs has been one approach to achieve prolonged antitumor activity of CARTs. So far, most clinically used CART products contain T cells with a donor-specific CD4/CD8 ratio following leukapheresis, resulting in a heterogeneous mixture of these two distinct subsets. 4 – 10 These subsets differ significantly in their activation and exhaustion states, differentiation potential, proliferative capacity, and cytokine release profiles. 11 – 14 To date, only Lisocabtagene maraleucel (Liso-cel), a CD19-targeting CART product approved for the treatment of relapsed/refractory large B cell non-Hodgkin lymphoma, contains CD4 + and CD8 + central memory T (T CM ) cells at a predefined ratio of 1:1, but efficacy data is very similar to non-adjusted commercial products. 15 Traditionally, CD4 + T cells have been considered to be "helper" cells, orchestrating the immune response by releasing cytokines and supporting other immune cells, while CD8 + T cells are mainly responsible for direct cytotoxic effects, targeting and destroying infected or malignant cells. 16 , 17 However, emerging studies have demonstrated that CD4 + CART cells can directly kill tumor cells in an MHC-independent manner through death receptor-mediated cytolysis, granule exocytosis, and proinflammatory cytokines such as IFN-γ and TNF-α. 11 – 14 , 18 – 22 Interestingly, a recent study reported that CD4 + CARTs were the most abundant subpopulation in the blood of two patients about 10 years after CD19-CART infusion to treat chronic lymphocytic leukemia (CLL). 23 These results suggest that CD4 + CARTs may play an indispensable role as both “helpers” and “fighters”. In this study, we sought to elucidate the significance of CD4 + CARTs and investigate the crosstalk between CD4 + and CD8 + subsets within CART cell products. Our goal was to determine the optimal CD4 + /CD8 + CART cell ratio that enhances the CART functionality against acute leukemias, potentially improving therapeutic outcomes. Results Healthy-donor derived CART products with superior functionality contain a higher percentage of CD4 + CARTs To investigate donor-associated factors that potentially influence the functionality of CART products, we generated CART products, targeting CD70, CD19, EphA2 and GD2 ( i.e .: CD70.CART, CD19.CART, EphA2.CART, GD2.CART), from activated T-cells of 23 healthy donors (HD1-HD23). We assessed the in vitro cytotoxic and proliferative capacity of all 92 CART products by repetitive stimulation with tumor cells expressing their respective target antigen in a serial co-culture assay (Supplementary Figure 1A-C). Based on these results, a functionality score (FS) for each donor and CAR construct was calculated and donors were subsequently ranked according to the result of their respective FS (Figure 1A). A subsequent co-culture assay at a lower and thus more challenging effector to target (E:T) cell ratio confirmed the FS-based functional ranking of donors (Figure 1B). Next, we compared the secretion of TNF-α + and IFN-γ + upon antigen stimulation between CART products generated from donors with high and low FS and found no statistical significant difference (Figure 1C). Furthermore, we determined the expression of proteins associated with T-cell exhaustion on the surface of CARTs. While the percentage of PD-1, TIM-3 and LAG-3 (PD-1+/TIM-3+/LAG-3+) expressing cells was significantly higher only in CD19.CARTs with low FS, there was no difference for CD70.CARTs, EphA2.CARTs and GD2.CARTs (Figure 1D). Subsequently, characterization of CART products revealed no significant differences in T-cell differentiation (Figure 1E). However, CART products with high FS did contain a significantly increased percentage of CD4 + CARTs compared to those with impaired functionality (Figure 1F), suggesting that CD4 + CART prevalence is associated with a high FS in healthy donor-derived CAR T-cells. Increasing CD4 + /CD8 + CART ratio is associated with enhanced anti-leukemic activity in vitro Having established that healthy donor-derived CART products with high FS contained a significantly increased percentage of CD4 + CARTs, we next sought to determine the impact of the CD4/CD8 ratio on the phenotype and functionality of CART targeting AML-specific CD33 and CD70, as well as B cell malignancies-specific target CD19 (Supplementary Figure 1A-B and Figure 2). CD4 + and CD8 + CART subsets did not show significant differences in the frequency of CAR expressing T-cells or the intensity as determined by the integrated mean fluorescence intensity (iMFI) (Supplementary Figure 3A-B). On day 16 of CART manufacturing, CD4 + CART cells predominantly displayed a memory phenotype (central memory (T CM ) and effector memory (T EM ) T-cells) as determined by the expression of CD45RA and CCR7 using flow cytometry whereas the CD8 + CART subsets consisted predominantly of terminally differentiated T-cells (T EMRA ) (Supplementary Figure 3C-D). Upon stimulation with target antigen expressing tumor cells, the CD107a expression was significantly increased in CD8 + CARTs indicating a stronger degree of degranulation (Supplementary Figure 3E). In contrast, the secretion of TNF-α was significantly higher in CD4 + CARTs while IFN-γ secretion was elevated for CD19 and CD33 expressing CD8 + CARTs but decreased for CD8 + CD70.CARTs (Supplementary Figure 3F). To evaluate the short-term cytotoxic potential of CD4 + and CD8 + CARTs, we stimulated the cells with target antigen positive tumor cells and found a trend towards enhanced tumor cell killing for CD8 + CD70.CARTs and CD19.CARTs and even a significant advantage for the CD8 + CD33.CARTs (Supplementary Figure 4A). Next, we sought to evaluate the impact of the CD4/CD8 composition of CARTs products on the cytolytic and proliferative capacity upon repeated antigen stimulation. We used CD4 + and CD8 + CARTs that were separated by FACS to generate CART products consisting of CD4/CD8 ratios ranging between 9:1 and 1:9 as well as pure CD4 + and CD8 + CART products and repeatedly stimulated these products with target antigen expressing tumor cells (Supplementary Figure 4B-C). Cytotoxicity and proliferation of all CART products improved continuously with increasing CD4/CD8 ratios and pure CD4 + CARTs exhibited superior functionality while pure CD8 + CARTs showed the lowest proliferation and anti-tumor activity (Figure 2A-B). To exclude a potential association between the functional superiority of CD4 + CARTs and NF kappa B activation mediated by the 4-1BB or CD27 co-stimulatory domains, we additionally chose a second-generation CLL-1-directed CAR construct containing only the CD28 co-stimulatory domain (Supplementary Figure 5A-B) and consistently found an association between the CD4/CD8 ratio and the functionality of the CART product (Supplementary Figure 5C-D). Having demonstrated the enhanced antitumor activity of CD4 + CARTs derived from healthy donors, we asked the questions whether depletion of CD8 + CARTs may also enhance the functionality of patient derived CARTs. To this end, cryopreserved 3 rd -generation CD19.CAR Ts derived from 6 patients with B cell malignancies who were treated within the HD-CAR1 clinical trial (NCT02208362) were thawed, CD4 + and CD8 + CARTs were separated by FACS and repeatedly stimulated with Nalm-6 tumor cells at different CD4/CD8 ratios. Documented clinical responses in these patients ranged from complete remissions (CR) to progressive disease (Supp. Table 1). In 5 out of 6 patients, in vitro cytotoxicity and proliferation of CD19.CARTs could be enhanced by increasing the percentage of CD4 + CARTs within the product and similar to our results with healthy donors, we observed superior proliferation and antitumor efficacy for pure CD4 + CART products compared to the original non-modified CART products suggesting that depleting CD8 + CARTs to obtain a pure CD4 + CART product may also significantly enhance the functionality of patient-derived CART products (Figure 2C-D). CD4 + CART cells exhibit superior antitumor activity and proliferative capacity in vivo Next, we investigated the functionality of CD70- and CD19-directed CART products at various CD4/CD8 ratios as pure CD4 + and CD8 + CARTs in vivo using a murine NSG xenograft model. To this end, NSG mice were intravenously (IV) injected either with 5x10 5 CD70-expressing Molm-13 cells genetically modified to express the click beetle green (CBG) luciferase or 1x10 5 CD19-postive Nalm-6 cells expressing firefly luciferase on day 0. On day 5, after tumor cell engraftment, mice were infused IV with a single dose of 1x10 6 CD70.CART or CD19.CART cells composed of either pure CD4 + or CD8 + CART subsets or a mixture of CD4 + and CD8 + at ratios of 3:1, 1:1 and 1:3. Animals treated with non CAR-transduced (NT) T cells served as controls. Tumor growth was observed by weekly BLI (Figure 3A and Supplementary Figure 7A). Intriguingly, for both the CD70.CART and the CD19.CART model, the pure CD4 + CART cell products exhibited the strongest anti-leukemic activity in all treated animals compared to the CD4 + and CD8 + containing CART cell products or the pure CD8 + CART cells leading to a statistically significant survival benefit. Importantly, the efficacy of CARTs gradually decreased by reducing CD4 + /CD8 + T-cell ratios with the pure CD8 + CART product showing only a marginally better tumor cell control than non-transduced T (NT) cells. (Figure 3B-C and Supplementary Figure 7B-E). The administered CART infusions were well tolerated, with no considerable weight loss observed in any of the mice (Supplementary Figure 7F-G). To investigate a potential correlation between the enhanced antitumor activity of CD4 + CARTs and their in vivo proliferation and persistence, we used our Molm-13 xenograft model with unmodified tumor cells and CD70.CARTs that were genetically modified to express a CBG luciferase fusion protein (Figure 3D). We observed a significant proliferative advantage of pure CD4 + CARTs over CART products that contained CD8 + CARTs and analogously to the tumor cell elimination, T cell proliferation and persistence gradually decreased with increasing content of CD8 + CARTs. The least CART proliferation was observed in mice treated with pure CD8 + CARTs. (Figure 3E-F). The CD4 + CART secretome significantly enhances the functionality of CARTs To further elucidate the mechanism behind the superior efficacy of CD4 + CART cells, we sought to determine whether soluble factors secreted by CD4 + CART cells may be pivotal for their enhanced functionality. CD4 + and CD8 + CARTs were stimulated separately with their corresponding target tumor cells and the supernatant from these co-cultures were harvested 24 hours (24h) after stimulation. Subsequently, a second co-culture was set up in which CD4 + or CD8 + CARTs were stimulated with target tumor cells either in the presence of supernatant harvested from CD4 + CART co-culture (4S), CD8 + CART co-culture (8S) or in the presence of fresh medium without any cytokine substitution (M). 4S, 8S and M was freshly added with every antigen stimulation (Figure 4A). As expected, we observed improved cytotoxicity and proliferation for CD4 + and CD8 + CARTs in the presence of the 8S supernatant compared to media control. Notably, the presence of 4S lead to even further enhanced cytotoxic and proliferative capacity of CD4 + and CD8 + CARTs (Figure 4B-C). To further characterize the soluble factors secreted by CD4 + and CD8 + CARTs, we measured the supernatant concentration of several cytokines secreted by CD4 + and CD8 + CARTs when they mixed in various ratios in the co-culture 24h after stimulation using a flow cytometry-based multiplex immunoassay. With increasing CD4/CD8 ratios， CARTs secreted higher levels of Th1, Th2, and Th17 related cytokines compared to products containing a higher percentage of CD8 + CARTs, namely IL-2, TNF-α, IL-4, IL-6, and IL-17. Additionally, CD19.CART and CD33.CART products with increased CD4/CD8 ratio also led to higher IL-10 secretion (Figure 4D and Supplementary Figure 8A-B). Direct cell-to-cell interaction with CD8 + CART cells impairs the functionality of CD4 + CART cells While a mixture of CD4 + and CD8 + CART cells exhibited improved cytotoxic activity than pure CD8 + CART product, they were less efficacious in tumor cell elimination compared to pure CD4 + CART product. This finding prompted us to further investigate the impact of CD8 + CARTs on CD4 + CARTs. Utilizing our co-culture assay, we compared the anti-tumor efficacy and proliferation of CARTs at a CD4/CD8 ratio of 1:1 with pure CD4 + CART products that contained only half the amount of total CARTs. Despite this hypothetical disadvantage, the pure CD4 + CART product demonstrated superior cytotoxicity and proliferation compared to the product containing both CD8 + and CD4 + CART cells suggesting that CD8 + cells diminished the functionality of CD4 + CARTs (Figure 5A-B and Supplementary Figure 9). However, when examining the exhaustion phenotype of CD4 + and CD8 + CART at different ratio compositions during repetitive antigen stimulation, the expression of inhibitory receptors (PD-1+/TIM-3+/LAG-3+) between these two subsets did not differ significantly (Supplementary Figure 10). To elucidate the lack of synergistic effect observed after mixing CD4 + and CD8 + CART cells, we postulated that the CD4/CD8 ratio of the CARTs within the mixture might have undergone a shift during repeated stimulation. Indeed, the composition of the cells was analyzed during the co-culture assays, which revealed an increase in the percentage of CD8 + T cells along with a decrease in CD4 + T cells (Supplementary Figure 11). Specifically, this alteration in the CD4/CD8 ratio was attributable, at least in part, to the greater proliferation of CD8 + CART cells, which were tracked using the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE). The proliferation of CD8 + CART cells was augmented in the presence of CD4 + CARTs, while the expansion of CD4 + CARTs was diminished when they were co-applied with CD8 + T cells (Supplementary Figure 12). Next, to further investigate whether the inhibitory effect of CD8 + CARTs on CD4 + CARTs may be attributed to competitive consumption of cytokines, or rather direct cell-to-cell interaction, we utilized a cell culture device with two chambers separated by a semipermeable membrane that inhibits a physical cell-to-cell contact but allows cytokines and other soluble factors to traffic. CD19- and CD70- CD4 + CARTs were stimulated with target cells in the bottom chamber (TCD4) while CD8 + CARTs were co-cultured with tumor cells in the upper chamber (Figure 5C) or vice versa (Supplementary Figure 13A). Pure CD4 + CARTs (PCD4) or a mixture of CD8 + and CD4 + CARTs (MCD4) co-cultured with target cells served as controls (Figure 5C and Supplementary Figure 13A). In line with our previous results, PCD4 demonstrated superior anti-tumor efficacy and proliferation while the presence of CD8 + CARTs without direct cell-to-cell contact impaired the proliferative capacity but not the cytotoxicity of CD4 + CARTs at later stages of the co-culture assays (D20 and D54 for the CD70.CARTs and the CD19.CARTs, respectively). Strikingly, direct physical contact with CD8 + CARTs significantly reduced the anti-leukemic efficacy and proliferation of CD4 + CARTs with repetitive antigen stimulation, suggesting that the inhibition of CD4 + CART functionality by CD8 + CARTs is a result of direct physical interaction between the two T-cell subsets (Figure 5D-G and Supplementary Figure 13B-E). Interaction with CD8 + CART cells induces apoptosis of CD4 + CART cells Next, we sought to investigate the potential mechanism of CD4 + CART impairment through direct physical contact with CD8 + CARTs. Using in-depth quantitative analysis of the proteome in CD4 + CD70.CART cells, which were cocultured with tumor cells either exclusively (PCD4) or in a mixture with CD8 + CART cells (MCD4), we quantified nearly 7000 proteins at a peptide and protein false discovery rate of 1%. Among these, we found 1335 proteins to be differentially regulated (permutation-based false discovery rate [FDR] < 0.05) between the groups (Figure 6A-C). Further analysis of biological processes and pathways revealed a marked enrichment of cell cycle-associated pathways in PCD4, with key proteins involved in cell cycle progression being notably higher in expression, suggesting that PCD4 cells have a enahnced capacity for proliferation compared to MCD4 cells (Figure 6D, F, G). This aligns with the functional data previously presented in Figure 2B. Conversely, pathways related to apoptosis were particularly prominent in MCD4, with most apoptosis-related proteins being upregulated, indicating that the MCD4 cells experienced increased apoptotic stress when co-cultured with CD8 + CART cells targeting the tumor cells (Figure 6E, H,I). Based on the proteome analysis, we determined the expression of Annexin V which is commonly used to detect apoptotic cells by flowcytometry on the surface of stimulated CD4 + CD19.CART and CD70.CART in the presence or absence of CD8 + CARTs. The percentage of Annexin V positive CD4 + CARTs significantly increased with decreasing CD4/CD8 ratio (Supp. Figure 14A-C). Furthermore, while we did not observe an increase of apoptosis in CD4 + CARTs that were separated from CD8 + CARTs through the semi-permeable membrane (PCD4 vs. TCD4), inhibition of cell-to-cell interaction with CD8 + CART significantly mitigated the apoptosis in CD4 + CARTs (MCD4 vs. TCD4 and PCD4) (Supp. Figure 14D-E). These results confirmed the induction of apoptosis in CD4 + CARTs mediated by physical contact with CD8+ CARTs on the functional level. Discussion Aiming to identify donor-specific factors that are associated with superior functionality of CARTs independent of structural differences of the CAR constructs, we found that CART products targeting hematologic malignancies as well as solid tumors exhibited enhanced in vitro anti-tumor efficacy and proliferative capacity if they contained a higher percentage of CD4 + CARTs. Focusing on CAR constructs that are directed against various antigens expressed by myeloid and lymphatic leukemias, in vitro co-culture assays and murine xenograft models confirmed the functional superiority of pure CD4 + CARTs compared to all CAR products that contained CD8 + CARTs. The elimination of CD8 + CARTs even augmented the in-vitro activity of CART products in most patients treated with 3rd generation CD19.CARTs within a clinical trial. Subsequent exploration of the underlying mechanisms revealed that the physical interaction with CD8 + CARTs induced apoptosis of CD4 + CARTs, hereby impairing their antileukemic activity. The optimal composition of CART products with respect to the content of CD4 + and CD8 + CARTs remains a matter of ongoing investigation and in the majority of clinical trials, the CD4/CD8 ratio of the CART products was determined by the lymphocyte composition of the donor. 24 For lisocabtagen maraleucel, a CD19-directed second generation CART product containing a 4-1BB co-stimulatory, a 1:1 ratio of CD4 + naïve (T N ) and CD8 + central-memory (T CM ) T cells was demonstrated to have optimal anti-tumor activity in B cell lymphoma animal models. 13 The superior antileukemic activity of pure CD4 + CARTs that we observed in this study was not necessarily expected as CD4 + T cells have been traditionally considered to function as helper cells, while CD8 + T cells are supposed to be the mediator of cytotoxicity. 16 , 25 However, evolving evidence has demonstrated that CD4 + T cells can also serve as the “cytotoxic” cells and exhibit a direct tumor cell-targeting cytotoxicity. 20 , 26 , 27 Indeed, Both CD4 + and CD8 + CART cells are capable of forming immune synapses after engagement with the target cells, hence mediating tumor cell elimination by degranulation and ligand-based lytic pathways. 19 , 28 , 29 Furthermore, CD4 + CART cells have shown superior efficacy over CD8 + CART cells in enhancing host immunity, which could potentially reduce the risk of antigen-negative relapse. 28 Several other studies align with our findings demonstrating that CD4 + CARTs alone exhibit a superior capability for eliminating target cells. 11 , 12 , 14 , 18 Agarwal et al. demonstrated that administering CD4-targeted (CD4-LV) and CD8-targeted lentiviral vector (CD8-LV) into NSG mice successfully generated murine CD4 + CART cells and CD8 + CART cells in vivo. Their results indicated that mice treated with CD4-LV achieved faster and more effective tumor cell elimination compared to those receiving CD8-LV alone or a combination of both vectors. 11 In a murine orthotopic glioblastoma (GBM) model, Wang et al. showed that CD4 + IL13Rα2 CAR T cells outperformed CD8 + CART cells, exhibiting sustained cytotoxicity and recursive killing potential even under repetitive tumor challenges. Furthermore, they showed the maintenance of the CD4 + subset positively correlated with the recursive killing ability of CART products derived from GBM patients. 14 Both of these studies attribute the superior performance of CD4 + CART cells to the greater susceptibility of CD8 + CART cells to exhaustion. Notwithstanding, the coinhibitory-related markers (PD-1, TIM-3, LAG-3) are comparable between CD4 + and CD8 + CART cells in our coculture experiment. In contrast to our findings, other groups have demonstrated the superiority of CD8 + CARTs over CD4 + CARTs or at least the need for the presence of both subsets in the most potent CART products. However, the comparability of these studies with our data is limited. Sommermeyer et al. compared CD19.CART products that differed not only with respect to their CD4 and CD8 expression but also contained T-cell subsets of distinct differentiation status (naïve, central and effector memory and terminally differentiated) which further alters the functional characteristics of these CART products while we characterized bulk CD4 + and CD8 + CART products. 13 Boulch et al. demonstrated that murine CD19-target CD8 + CART cells exhibited a higher capacity to eliminate tumor as compared with CD4 + CART cells in a immune competent MYC-driven B cell lymphoma animal model. 28 , 30 However, they also reported that IFN-γ production was the dominant mechanism for tumor elimination by CD4 + CD19.CART cells in their model but the tumor cells were not sensitive to pro-apoptotic effect of IFN-γ. 30 Such discrepancies could be attributed to the utilization of disparate tumor models and treatment regimens. While we did not observe any clinical signs of hyperinflammation such as weight loss, hunched posture, ruffled fur or changes in the body temperature in our murine xenograft models, the enhanced anti-leukemic activity of pure CD4 + CARTs in vitro and in vivo was associated with increased CART proliferation and higher levels of Th1- and Th2-related cytokines which raises the concern of more severe treatment-related toxicity. In fact, evidence suggests that higher in vivo expansion of CART cells correlates with increased overall response rates but also a heightened incidence of CRS in patients with B-cell malignancies and multiple myeloma. 4 , 31 Owing to their robust proliferative capacity and cytokine release profile, CD4 + CART cells may effectively stimulate myeloid cells to produce inflammatory mediators. Clinical studies have indicated associations between CRS and other factors such as disease burden, co-stimulatory domains, manufacturing procedures, and T cell differentiation status. 12 , 15 , 32 , 33 Findings from two independent studies suggested that CD4 + CART cells may be more prone to inducing cytokine-release syndrome (CRS), 12 , 34 a major adverse event associated with CAR T cell therapy in B-cell malignancies. One study utilized a murine-derived CD19.CART cell therapy in an immunocompetent mouse model, reporting that CD4 + CART cell therapy favors the occurrence of CRS in cases of high tumor burden, while at low tumor burden, no discernible toxicities were observed and a therapeutic benefit was still noted. 34 Another study using immunodeficient mice reconstituted with human CD34 + stem cells and engrafted with Nalm-6 tumor cells demonstrated superior long-term responses accompanied by CRS in pure CD4 + CART cells compared to pure CD8 + CD19-directed CART cells. 12 However, in another research using the same humanized mouse model, both the CD4 + CART group and mixed CD4 + /CD8 + CART group exhibited similar CRS-like symptoms in the context of an effective antitumor response. 11 To date, no clinical trials have been conducted using pure CD4 + CART cells for treatment in real-world settings. Therefore, the induction mechanisms and pathophysiology of cytokine release syndrome (CRS) associated with CD4 + CART cells warrant further investigation. Considering the critical role of CD4 + CART cells in sustaining antitumor responses and long-term persistence, adjusting cell dosages may be advisable before clinical translation of single CD4 + CART cell treatment. Mechanistically, the secretome of CD4 + CARTs was more potent in stimulating the anti-leukemic activity of CARTs with a more pronounced effect on CD8 + CARTs leading to a shift of the CD4/CD8 ratio within the CART products in favor of CD8 + CARTs over time. CART products with increasing content of CD4 + CARTs secreted higher levels of type 1 and type 2 cytokines suggesting that both types of immune response play a relevant role for their functionality. In fact, it has been shown that type 2 immune responses are associated with long-lasting remission and that IL4 and IL10 may lead to enhanced effector function and improved metabolic fitness of CART cells. 35 – 38 On the other hand, proteomic analysis revealed that interaction with CD8 + CART cells induced apoptosis of CD4 + CART cells, which was further confirmed by flow cytometric detection of apoptotic proteins. These two simultaneously occurring mechanisms may explain that the intercellular crosstalk between CD4 + and CD8 + CART cells significantly hinders the anti-leukemic efficacy of CD4 + CART cells. In the context of CARs targeting myeloid and lymphoblastic leukemias, the results of our extensive in vitro and in vivo analyses provide evidence for the superior anti-leukemic efficacy of pure CD4 + CARTs compared to products containing a mixture of CD4 + and CD8 + CARTs. We are demonstrating the enhanced functionality of CD4 + CARTs is primarily attributed to the secretome of CD4 + CARTs, which induces a more potent anti-leukemic response. However, the presence of CD4 + CARTs also induces excessive proliferation of CD8 + CARTs, leading to a reduction in the CD4/CD8. Furthermore, the interaction between CD4 + and CD8 + CART cells leads to apoptosis of CD4 + CARTs and subsequently to the impaired functionality of the CART product.. Thus, our findings warrant testing of pure CD4 + CART products targeting hematologic malignancies in early phase clinical trials that are carefully designed to investigate the safety of this approach, considering the potential proliferative capacity of pure CD4 + CART products and their efficacy. Material and methods Generation of CART cells Retroviral vector production and T-cell transduction have been described previously. 39 , 40 In brief, 293T cells were transfected with packaging plasmids (PegPam, RD114) and the SFG vector containing the CAR construct (SFG.CD27.CD3zeta.IRES.tCD19, SFG.CD19.CH2-CH3.CD28.4-1BB.CD3zeta, SFG.CD33.CH2-CH3.CD28.4-1BB.CD3zeta, SFG.CLL-1.CH3.CD28.CD3zeta, SFG.4H5.IgG1h.CD28.CD3zeta.T2A.tNGFR, SFG.14g2a.IgG1.CD8a.4-1BB.CD3zeta.IRES.tCD19, SFG.CD19.CH3.CD28.4-1BB.CD3zeta). The retroviral supernatant was collected after 48 and 72 hours. Peripheral blood mononuclear cells were isolated from the peripheral blood using density gradient centrifugation. T cells were then activated using CD3 and CD28 antibodies. After 48 hours of expansion in complete medium, which consisted of 45% RPMI-1640 (Thermo Fisher Scientific, Waltham, MA), 45% Click’s medium (FujiFilm; Irvine Scientific, Santa Ana, CA), 2 mM GlutaMAX-I CTS (Thermo Fisher Scientific, Waltham, MA), and 10% fetal bovine serum (Hyclone, Logan, UT), the T cells were transduced in 24-well plates coated with RetroNectin (Takara). Additionally, the medium was supplemented with 10 ng/mL interleukin-7 (IL-7) and 5 ng/mL IL-15 (R&D Systems, Minneapolis, Minnesota). Cells and culture conditions De-identified apheresis products from voluntary healthy donors were sourced from the Heidelberg blood bank. CD19.CART cells, derived from patients in the Heidelberg CART cell trial 1 (HD-CAR-1; NCT03676504), were manufactured in accordance with GMP (Good Manufacturing Practice) guidelines and stored in a nitrogen tank ready for use. All participants provided signed informed consent and were treated in compliance with the Declaration of Helsinki. 293T cells were acquired from the Deutsche Sammlung von Mikrooganismen und Zellkulturen (DSMZ) and cultured using Iscove's Modified Dulbecco's Medium (IMDM). Nalm-6, Raji, K562, Molm-13, and HL-60 cell lines were purchased from DSMZ and cultured in RPMI 1640 medium. LAN-1 cells, also obtained from DSMZ, were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine. A-549 cells, sourced from DSMZ, were cultured in Dulbecco's Modified Eagle Medium (DMEM). All cell lines were cultivated in their respective media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, within a humidified atmosphere containing 5% CO2 at 37°C. All the above-mentioned cell lines were authenticated at DKFZ (German Cancer Research Center) and were free from mycoplasma contamination checked by polymerase chain reaction (PCR). Flow Cytometry The fluorochrome-conjugated isotype control and antihuman antibodies against CD3, CD4, CD8, CD45RA, CCR7, CD70, CD19, NGFR, PD1, TIM3, LAG3, Ki67, CD107a, TNF-α, IFN-γ and AnnexinV were purchased from BD Bioscience or Biolegend. Biotin-labeled protein L and fluorochrome-labeled streptavidin from Thermo Fisher Scientific and Biolegend were used to identify CAR expression of CD33.CAR, CLL-1.CAR and long-spacer CD19.CAR (SFG.CD19.CH2-CH3.CD28.4-1BB.CD3zeta). Additionally, to precisely detect the short-spacer CD19.CAR SFG.CD19.CH3.CD28.4-1BB.CD3zeta) expression, the CD19 CAR Detection Reagent (biotinylated) together with anti-biotin-PE from Miltenyi Biotec were applied. The expression of CD70.CAR and GD2.CAR was assessed by identifying truncated CD19 via an APC-conjugated or PE-conjugated antibody specific to CD19. Similarly, EphA2.CAR expression was ascertained by detecting truncated NGFR using an antibody specific to NGFR, conjugated with either APC or PE. Dead cells were removed from the analysis using either the LIVE/DEAD™ fixable near-infrared (IR) dead cell stain kit (Thermo Fisher Scientific) or 7AAD (BD Biosciences). Fluorescence compensation was conducted for every specific experiment with different FACS panels before cell acquisition in a flow cytometer. Fluorescence minus one (FMO) control, isotype control or non-transduced control were included. Flow cytometry data were acquired using BD FACSymphony™ A3 and BD LSRII cell analyzers and visualized with FlowJo V.10.10.0 software. Surface marker staining Cells were harvested in the FACS tube and washed once with FACS buffer. The supernatant was removed from the cells after centrifuge. If necessary, 5 ul of Fc Receptor Blocking Solution (Biolegend) per million cells was added in 100 µl staining volume prior to staining with antibody of interest and incubated at room temperature for 5–10 minutes. A cocktail of fluorochrome-labeled antibodies against the surface markers of interest was prepared and added to cells and mixed well. After incubation for 30 min at 4°C in the dark, cells were washed with FACS buffer. Finally, cells were resuspended in 400 µl of FACS buffer supplemented with 7-AAD Viability Staining Solution for measurement. Intracellular cytokine staining CD70.CART, CD19.CART, and CD33.CART cells were seeded into a 96-well U-bottom plate. The cells were then stimulated with either the respective target tumor cells or non-target K562 cells in the presence of monensin, brefeldin A, and CD107a antibody for 5 hours at 37°C. Afterward, the cells were washed with PBS and stained with NEAR-IR for 30 minutes at 4°C in the dark. Finally, surface markers were stained for 20 minutes at 4°C in the dark. The cells were fixed and permeabilized at room temperature following the instructions of the Miltenyi Fixation/Permeabilization Solution Kit. Antibodies were used to stain cytokines IFN-γ and TNF-α for 30 minutes in the dark at room temperature. Lastly, the cells were washed and resuspended in FACS buffer for acquisition. CFSE staining CAR-T cells were resuspended in a CFSE (Carboxyfluorescein succinimidyl ester) staining solution (Biolegend) comprising 1000 µl of PBS and 1 µl of a 5 mM CFSE stock. This cell preparation was then incubated for 20 minutes at 37°C in the dark. The staining reaction was halted by the introduction of complete medium, followed by two additional washes with the medium. For the assay, 1 x 10 6 CFSE-stained cells were allocated per well into a 24-well plate and were then stimulated by X-ray irradiated target tumor cells at a 1:1 ratio. The proliferative response of the CAR-T cells was evaluated after a five-day stimulation period. Coculture assay An illustrative scheme of the experimental approach is presented in Supplemental Fig. 1C. CART cells were co-cultured with tumor cells at the appropriate Effector-to-Target (E: T) ratio in plates without the addition of external cytokines. A single well of plates for each condition was sampled every certain days, and the total count of T cells and tumor cells was determined through flow cytometry using CountBright counting beads. The population of dead cells was excluded through 7AAD staining. ZsGreen expression was used to identify tumor cells, while CD3 antibodies were employed for T-cell detection. CART cells were subjected to a repetitive challenge with new tumor cells at the same E: T ratio. If CART cells lost their ability to eliminate tumor cells or ceased to proliferate and subsequently disappeared, the respective condition was terminated. Cytokine release assay Supernatants were collected 24h following the co-culture. To quantify cytokines in the supernatant, bead-based multiplex LEGENDplex™ analysis (BioLegend) was performed following the manufacturer's instructions. A range of pro-inflammatory cytokines, including IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF-α, soluble Fas, soluble FasL, granzyme A, granzyme B, perforin, and granulysin were measured. The analysis was conducted on the BD FACSCantoII flow cytometer. Data were interpreted by the LEGENDPlex™ V8.0 software (Biolegend). Xenograft model In vivo experiments were conducted in accordance with a protocol endorsed by the federal and Institutional Animal Care and Use Committee (IACUC). The female and male NOD.Cg-Prkdc scid lL2rg tm1Wjl /SzJ (NSG) mice, aged 6 to 10 weeks, were acquired from the Heidelberg University Interfaculty Biomedical Research Facility (IBF) breeding colony and maintained in IBF during the experiments. Animals were administered injections of tumor cells and T cells intravenously through the tail vein, as detailed in the "Results" and “Supplementary Information” sections. Subsequent measurements of tumor mass or T-cell expansion were taken regularly by bioluminescent imaging, using the in vivo imaging system IVIS Lumina II (Caliper LifeScience, Hopkinton, MA). The mice were euthanized when they reached predefined endpoint criteria complied with IACUC. Rather than employing the CD19.CAR construct with a hinge-CH2-CH3 spacer (long spacer), which was utilized in both the in-vitro experiments and the Heidelberg CAR T cell trial 1 (HD-CAR-1; NCT03676504), 41 a short-spacer CD19.CAR construct featuring a hinge-CH3 was used to modify the T cells for the murine leukemia xenograft treatment (Supplementary Fig. 6). Previous observations, such as those reported by Almasbak et al. in 2015, have identified that the CH2 domain in commonly used IgG1-Fc spacers can bind to soluble mouse Fcγ-receptor I, leading to off-target T-cell activation directed at murine macrophages and consequently diminishing anti-leukemia activity. 42 To address this issue, our study adopted a short-spacer CAR construct that omits the CH2 region in the NSG mouse model. Label-free proteome measurement FACS-sorted CD4 + CART cells from the coculture condition were thoroughly washed in plain PBS, lysed in 1%SDC buffer (1%SDC, 100mM Tris pH8.5, 40mM CAA and 10mM TCEP), incubated on ice for 20 minutes, boiled at 95°C, sonicated for 5 mins on a Biorupter plus as described previously. 43 Samples were digested with trypsin and LysC for 16 hours at 37°C. Digestion was stopped by adding 5X volumes of isopropanol/1% TFA and vortexing vigorously. The peptides were de-salted on equilibrated styrene divinylbenzene-reversed phase sulfonated (SDB-RPS) StageTips, washed with isopropanol/1% TFA and 0.2% TFA, and eluted with 60µl of elution buffer (1.25% Ammonia, 80% ACN). The dried elutes were resuspended in MS loading buffer (3% ACN, 0.3% TFA) and stored at -20°C until MS measurement. 400 ng peptides were loaded onto a nanoElute system (Bruker Daltonics Inc, Bremen, Germany) coupled with a TIMS TOF HT mass spectrometer (Bruker Daltonics, Bremen, Germany) using a CaptiveSpray nano-electrospray ion source. Peptides were separated on an IonOpticks Aurora 25 cm column using a binary buffer system: buffer A (0.1% formic acid, 2% ACN) and buffer B (0.1% FA in 99.9% ACN), using a 120-minute gradient starting from 2% buffer B, increasing to 12% in 60 min, 20% in 30 min, 30% in 10 min, and finally reaching 85% in 10 min, which was held for an additional 10 min at a flow rate of 0.3 µl/min. DDA-PASEF mode was employed, and MS1 spectra were recorded from 150 m/z to 1700 m/z, TIMS functioning at Scan range 100–1700 m/z, Ramp Time 100 ms, Duty cycle 100%, Cycle time 100.00 ms and Spectra Rate 9.43 Hz. MS data analysis and bioinformatics MS raw files were processed using Maxquant (version 1.5.5.2) supported by Andromeda search engine against the UniProt human reference fasta (version 2016). MaxQuant default settings were employed for identification and quantification, allowing a maximum of 2 missed cleavages. The minimal peptide length was set to 7. Label-free quantification (LFQ) was performed using the MaxLFQ algorithm implemented in MaxQuant, with the following settings: a minimal ratio count of 2 and fast LFQ enabled. The peptide and protein FDR was set to 1% to ensure high confidence in the identified peptides and proteins. The protein group file generated from MaxQuant software was further analyzed in Perseus (version 2.0.7.0). Values were log2 transformed, and data were pre-cleaned by filtering out contaminants, reverse, and only identified by site modifications before statistical analysis. Enrichment analysis and keywords were performed by comparing the protein expressions between groups. Paired sample t-tests and multi-sample t-tests were used to compare between two groups with a permutation-based FDR cutoff of 5%. The proteome data has been submitted to the PRIDE database with the project accession number: PXD060508. Statistical analysis Statistical analysis was performed using Prism 10 software (GraphPad Software, San Diego, CA). For analyzing continuous variables across three or more groups, one-way analysis of variance (ANOVA) was employed. When comparing two groups, the analysis was carried out using either a t-test or a Wilcoxon rank-sum test. Survival times resulting from the injection of tumor cells in mouse experiments were analyzed through the use of Kaplan-Meier curves and the Mantel-Cox log-rank test. P-values < 0.05 were defined as statistically significant. Declarations The HD-CAR-1 clinical trial, from which primary patient samples were derived, was approved by the ethics committee of the medical faculty of the University of Heidelberg. The experiments involving the murine xenograft models were approved by the animal care committee of the Regierungspräsidium Karlsruhe. Data and code availability Data generated from proteome analysis are deposited in the PRIDE database with the project accession number: PXD060508. Acknowledgement We thank Dr. Volker Eckstein and Stefanie Hofmann for assistance with the fluorescence-activated cell sorting. We thank all patients for providing their specimens. Authorship Contribution QC, TS, MS and AKJ designed the reserch; QC and TS analyzed and interpreted the data and wrote the paper; QC, HY, LHM, DD, DS, MS, BLH and JMU performed the experiments; LW, AS, CMT and AKJ contributed to the interpretation of the results. Disclosure of conflicts of interest The authors declare no competing financial interests. References Cappell, K.M., and Kochenderfer, J.N. (2023). Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol 20 , 359-371. 10.1038/s41571-023-00754-1. Ali, S.A., Shi, V., Maric, I., Wang, M., Stroncek, D.F., Rose, J.J., Brudno, J.N., Stetler-Stevenson, M., Feldman, S.A., Hansen, B.G., Fellowes, V.S., et al. (2016). T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma. Blood 128 , 1688-1700. 10.1182/blood-2016-04-711903. Cappell, K.M., Sherry, R.M., Yang, J.C., Goff, S.L., Vanasse, D.A., McIntyre, L., Rosenberg, S.A., and Kochenderfer, J.N. (2020). Long-Term Follow-Up of Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy. J Clin Oncol 38 , 3805-3815. 10.1200/JCO.20.01467. Munshi, N.C., Anderson, L.D., Jr., Shah, N., Madduri, D., Berdeja, J., Lonial, S., Raje, N., Lin, Y., Siegel, D., Oriol, A., Moreau, P., et al. (2021). Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med 384 , 705-716. 10.1056/NEJMoa2024850. Neelapu, S.S., Locke, F.L., Bartlett, N.L., Lekakis, L.J., Miklos, D.B., Jacobson, C.A., Braunschweig, I., Oluwole, O.O., Siddiqi, T., Lin, Y., Timmerman, J.M., et al. (2017). Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med 377 , 2531-2544. 10.1056/NEJMoa1707447. Shah, B.D., Ghobadi, A., Oluwole, O.O., Logan, A.C., Boissel, N., Cassaday, R.D., Leguay, T., Bishop, M.R., Topp, M.S., Tzachanis, D., O'Dwyer, K.M., et al. (2021). KTE-X19 for relapsed or refractory adult B-cell acute lymphoblastic leukaemia: phase 2 results of the single-arm, open-label, multicentre ZUMA-3 study. Lancet 398 , 491-502. 10.1016/S0140-6736(21)01222-8. Fowler, N.H., Dickinson, M., Dreyling, M., Martinez-Lopez, J., Kolstad, A., Butler, J., Ghosh, M., Popplewell, L., Chavez, J.C., Bachy, E., Kato, K., et al. (2022). Tisagenlecleucel in adult relapsed or refractory follicular lymphoma: the phase 2 ELARA trial. Nat Med 28 , 325-332. 10.1038/s41591-021-01622-0. Martin, T., Usmani, S.Z., Berdeja, J.G., Agha, M., Cohen, A.D., Hari, P., Avigan, D., Deol, A., Htut, M., Lesokhin, A., Munshi, N.C., et al. (2023). Ciltacabtagene Autoleucel, an Anti-B-cell Maturation Antigen Chimeric Antigen Receptor T-Cell Therapy, for Relapsed/Refractory Multiple Myeloma: CARTITUDE-1 2-Year Follow-Up. J Clin Oncol 41 , 1265-1274. 10.1200/JCO.22.00842. Maude, S.L., Laetsch, T.W., Buechner, J., Rives, S., Boyer, M., Bittencourt, H., Bader, P., Verneris, M.R., Stefanski, H.E., Myers, G.D., Qayed, M., et al. (2018). Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med 378 , 439-448. 10.1056/NEJMoa1709866. Wang, M., Munoz, J., Goy, A., Locke, F.L., Jacobson, C.A., Hill, B.T., Timmerman, J.M., Holmes, H., Jaglowski, S., Flinn, I.W., McSweeney, P.A., et al. (2020). KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma. N Engl J Med 382 , 1331-1342. 10.1056/NEJMoa1914347. Agarwal, S., Hanauer, J.D.S., Frank, A.M., Riechert, V., Thalheimer, F.B., and Buchholz, C.J. (2020). In Vivo Generation of CAR T Cells Selectively in Human CD4(+) Lymphocytes. Mol Ther 28 , 1783-1794. 10.1016/j.ymthe.2020.05.005. Bove, C., Arcangeli, S., Falcone, L., Camisa, B., El Khoury, R., Greco, B., De Lucia, A., Bergamini, A., Bondanza, A., Ciceri, F., Bonini, C., et al. (2023). CD4 CAR-T cells targeting CD19 play a key role in exacerbating cytokine release syndrome, while maintaining long-term responses. J Immunother Cancer 11 . 10.1136/jitc-2022-005878. Sommermeyer, D., Hudecek, M., Kosasih, P.L., Gogishvili, T., Maloney, D.G., Turtle, C.J., and Riddell, S.R. (2016). Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30 , 492-500. 10.1038/leu.2015.247. Wang, D., Aguilar, B., Starr, R., Alizadeh, D., Brito, A., Sarkissian, A., Ostberg, J.R., Forman, S.J., and Brown, C.E. (2018). Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JCI Insight 3 . 10.1172/jci.insight.99048. Abramson, J.S., Palomba, M.L., Gordon, L.I., Lunning, M.A., Wang, M., Arnason, J., Mehta, A., Purev, E., Maloney, D.G., Andreadis, C., Sehgal, A., et al. (2020). Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet 396 , 839-852. 10.1016/S0140-6736(20)31366-0. Borst, J., Ahrends, T., Babala, N., Melief, C.J.M., and Kastenmuller, W. (2018). CD4(+) T cell help in cancer immunology and immunotherapy. Nat Rev Immunol 18 , 635-647. 10.1038/s41577-018-0044-0. Csaplar, M., Szollosi, J., Gottschalk, S., Vereb, G., and Szoor, A. (2021). Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models. Cancers (Basel) 13 . 10.3390/cancers13174301. Adusumilli, P.S., Cherkassky, L., Villena-Vargas, J., Colovos, C., Servais, E., Plotkin, J., Jones, D.R., and Sadelain, M. (2014). Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity. Sci Transl Med 6 , 261ra151. 10.1126/scitranslmed.3010162. Benmebarek, M.R., Karches, C.H., Cadilha, B.L., Lesch, S., Endres, S., and Kobold, S. (2019). Killing Mechanisms of Chimeric Antigen Receptor (CAR) T Cells. Int J Mol Sci 20 . 10.3390/ijms20061283. Li, T., Wu, B., Yang, T., Zhang, L., and Jin, K. (2020). The outstanding antitumor capacity of CD4(+) T helper lymphocytes. Biochim Biophys Acta Rev Cancer 1874 , 188439. 10.1016/j.bbcan.2020.188439. Xhangolli, I., Dura, B., Lee, G., Kim, D., Xiao, Y., and Fan, R. (2019). Single-cell Analysis of CAR-T Cell Activation Reveals A Mixed T(H)1/T(H)2 Response Independent of Differentiation. Genomics Proteomics Bioinformatics 17 , 129-139. 10.1016/j.gpb.2019.03.002. Yang, Y., Kohler, M.E., Chien, C.D., Sauter, C.T., Jacoby, E., Yan, C., Hu, Y., Wanhainen, K., Qin, H., and Fry, T.J. (2017). TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance. Sci Transl Med 9 . 10.1126/scitranslmed.aag1209. Melenhorst, J.J., Chen, G.M., Wang, M., Porter, D.L., Chen, C., Collins, M.A., Gao, P., Bandyopadhyay, S., Sun, H., Zhao, Z., Lundh, S., et al. (2022). Decade-long leukaemia remissions with persistence of CD4(+) CAR T cells. Nature 602 , 503-509. 10.1038/s41586-021-04390-6. Liu, Y., Sperling, A.S., Smith, E.L., and Mooney, D.J. (2023). Optimizing the manufacturing and antitumour response of CAR T therapy. Nature Reviews Bioengineering 1 , 271-285. 10.1038/s44222-023-00031-x. Moeller, M., Kershaw, M.H., Cameron, R., Westwood, J.A., Trapani, J.A., Smyth, M.J., and Darcy, P.K. (2007). Sustained antigen-specific antitumor recall response mediated by gene-modified CD4+ T helper-1 and CD8+ T cells. Cancer Res 67 , 11428-11437. 10.1158/0008-5472.CAN-07-1141. Brown, D.M. (2010). Cytolytic CD4 cells: Direct mediators in infectious disease and malignancy. Cell Immunol 262 , 89-95. 10.1016/j.cellimm.2010.02.008. Moeller, M., Haynes, N.M., Kershaw, M.H., Jackson, J.T., Teng, M.W., Street, S.E., Cerutti, L., Jane, S.M., Trapani, J.A., Smyth, M.J., and Darcy, P.K. (2005). Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection. Blood 106 , 2995-3003. 10.1182/blood-2004-12-4906. Boulch, M., Cazaux, M., Loe-Mie, Y., Thibaut, R., Corre, B., Lemaître, F., Grandjean, C.L., Garcia, Z., and Bousso, P. (2021). A cross-talk between CAR T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity. Sci Immunol 6 . 10.1126/sciimmunol.abd4344. Liadi, I., Singh, H., Romain, G., Rey-Villamizar, N., Merouane, A., Adolacion, J.R., Kebriaei, P., Huls, H., Qiu, P., Roysam, B., Cooper, L.J., et al. (2015). Individual Motile CD4(+) T Cells Can Participate in Efficient Multikilling through Conjugation to Multiple Tumor Cells. Cancer Immunol Res 3 , 473-482. 10.1158/2326-6066.CIR-14-0195. Boulch, M., Cazaux, M., Cuffel, A., Guerin, M.V., Garcia, Z., Alonso, R., Lemaitre, F., Beer, A., Corre, B., Menger, L., Grandjean, C.L., et al. (2023). Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-gamma is a major determinant of CD4(+) CAR T-cell antitumor activity. Nat Cancer 4 , 968-983. 10.1038/s43018-023-00570-7. Ogasawara, K., Lymp, J., Mack, T., Dell'Aringa, J., Huang, C.P., Smith, J., Peiser, L., and Kostic, A. (2022). In Vivo Cellular Expansion of Lisocabtagene Maraleucel and Association With Efficacy and Safety in Relapsed/Refractory Large B-Cell Lymphoma. Clin Pharmacol Ther 112 , 81-89. 10.1002/cpt.2561. Arcangeli, S., Bove, C., Mezzanotte, C., Camisa, B., Falcone, L., Manfredi, F., Bezzecchi, E., El Khoury, R., Norata, R., Sanvito, F., Ponzoni, M., et al. (2022). CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. J Clin Invest 132 . 10.1172/JCI150807. Hernani, R., Benzaquen, A., and Solano, C. (2022). Toxicities following CAR-T therapy for hematological malignancies. Cancer Treat Rev 111 , 102479. 10.1016/j.ctrv.2022.102479. Boulch, M., Cazaux, M., Cuffel, A., Ruggiu, M., Allain, V., Corre, B., Loe-Mie, Y., Hosten, B., Cisternino, S., Auvity, S., Thieblemont, C., et al. (2023). A major role for CD4(+) T cells in driving cytokine release syndrome during CAR T cell therapy. Cell Rep Med 4 , 101161. 10.1016/j.xcrm.2023.101161. Bai, Z., Feng, B., McClory, S.E., de Oliveira, B.C., Diorio, C., Gregoire, C., Tao, B., Yang, L., Zhao, Z., Peng, L., Sferruzza, G., et al. (2024). Single-cell CAR T atlas reveals type 2 function in 8-year leukaemia remission. Nature 634 , 702-711. 10.1038/s41586-024-07762-w. Bai, Z., Woodhouse, S., Zhao, Z., Arya, R., Govek, K., Kim, D., Lundh, S., Baysoy, A., Sun, H., Deng, Y., Xiao, Y., et al. (2022). Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL. Sci Adv 8 , eabj2820. 10.1126/sciadv.abj2820. Feng, B., Bai, Z., Zhou, X., Zhao, Y., Xie, Y.Q., Huang, X., Liu, Y., Enbar, T., Li, R., Wang, Y., Gao, M., et al. (2024). The type 2 cytokine Fc-IL-4 revitalizes exhausted CD8(+) T cells against cancer. Nature 634 , 712-720. 10.1038/s41586-024-07962-4. Zhao, Y., Chen, J., Andreatta, M., Feng, B., Xie, Y.Q., Wenes, M., Wang, Y., Gao, M., Hu, X., Romero, P., Carmona, S., et al. (2024). IL-10-expressing CAR T cells resist dysfunction and mediate durable clearance of solid tumors and metastases. Nat Biotechnol 42 , 1693-1704. 10.1038/s41587-023-02060-8. Han, H., Wang, L., Ding, Y., Neuber, B., Huckelhoven-Krauss, A., Lin, M., Yao, H., Chen, Q., Sauer, T., Schubert, M.L., Guo, Z., et al. (2024). Extracorporeal photopheresis as a promising strategy for the treatment of graft-versus-host disease after CAR T-cell therapy. Blood Adv 8 , 2675-2690. 10.1182/bloodadvances.2023012463. Sauer, T., Parikh, K., Sharma, S., Omer, B., Sedloev, D., Chen, Q., Angenendt, L., Schliemann, C., Schmitt, M., Muller-Tidow, C., Gottschalk, S., et al. (2021). CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity. Blood 138 , 318-330. 10.1182/blood.2020008221. Schubert, M.L., Schmitt, A., Huckelhoven-Krauss, A., Neuber, B., Kunz, A., Waldhoff, P., Vonficht, D., Yousefian, S., Jopp-Saile, L., Wang, L., Korell, F., et al. (2023). Treatment of adult ALL patients with third-generation CD19-directed CAR T cells: results of a pivotal trial. J Hematol Oncol 16 , 79. 10.1186/s13045-023-01470-0. Almasbak, H., Walseng, E., Kristian, A., Myhre, M.R., Suso, E.M., Munthe, L.A., Andersen, J.T., Wang, M.Y., Kvalheim, G., Gaudernack, G., and Kyte, J.A. (2015). Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model. Gene Ther 22 , 391-403. 10.1038/gt.2015.4. Jayavelu, A.K., Schnoder, T.M., Perner, F., Herzog, C., Meiler, A., Krishnamoorthy, G., Huber, N., Mohr, J., Edelmann-Stephan, B., Austin, R., Brandt, S., et al. (2020). Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms. Nature 588 , 157-163. 10.1038/s41586-020-2968-3. Additional Declarations There is NO Competing Interest. Supplementary Files NatureCommunicationsSupplementaryMaterialsCD4CARTcellpaper.docx Supplementary Material Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6283250","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":444330727,"identity":"3b7db3ee-3fc8-45a8-9c55-23345574bdd6","order_by":0,"name":"Tim Sauer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABQElEQVRIie3RPUvDQBjA8ScELoNPOqe0tF/hQiFRWupXyVFIp7gIwUGw002+rBUKfoWWQl0jB3VqZ8HBFEEEM8RFAoI1KW2xJh26ieQ/JE8SftwlAcjL+4vJq0HpAGB8JsnRIlCmi9skJXBN0NskuJWsJ81aXS3hNnKo7FlSdCoq5vUb8wMQRwVQ73zfbaCpXDy/gttIb0ztyzgWtfKjM9R7II4JFFrUmtp4cH5v7MPUziRABOuWnFEJQTBeDQyNcYH0wSZU4iKLSNGXOOsWJ7efCwJoRozPkT69JGSeRUDlwtI0dSQviQGMe/EqRPYl7qWIUPtCvWzrXXSGxR5tJ6SmWdNW/C42gXj4TZSryWAWfdSrmjIZhMFJnd10UH+P3GbFVMZyGLrN1FeO+7E03XxCkp+1W3K4q8jLy8v7j30DR4xkm415gwMAAAAASUVORK5CYII=","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":true,"prefix":"","firstName":"Tim","middleName":"","lastName":"Sauer","suffix":""},{"id":444330728,"identity":"ee58710c-805a-4ec5-a142-709d9f63891d","order_by":1,"name":"Qian Chen","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Chen","suffix":""},{"id":444330729,"identity":"fec314d0-e357-46fd-b19a-a7739e3803a9","order_by":2,"name":"Hao Yao","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Yao","suffix":""},{"id":444330730,"identity":"4a3d0a29-8443-49af-8b89-e5a66cb57f04","order_by":3,"name":"Lianghao Mao","email":"","orcid":"","institution":"Hopp Children’s Cancer Center Heidelberg (KiTZ), German Cancer Research Center (DKFZ)","correspondingAuthor":false,"prefix":"","firstName":"Lianghao","middleName":"","lastName":"Mao","suffix":""},{"id":444330731,"identity":"d31685ae-a393-42a8-ab54-ae2ac20b66ec","order_by":4,"name":"Dominic Depke","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Dominic","middleName":"","lastName":"Depke","suffix":""},{"id":444330732,"identity":"af935b22-98af-4b9b-b8dc-f0e00a883e5a","order_by":5,"name":"Lei Wang","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Wang","suffix":""},{"id":444330733,"identity":"5be8213b-5fe6-4fb1-aff4-1c135b55be23","order_by":6,"name":"David Sedloev","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Sedloev","suffix":""},{"id":444330734,"identity":"8ce4cdd3-19e7-4ede-8755-ee5bdb7b998c","order_by":7,"name":"Marina Marina Scheller","email":"","orcid":"https://orcid.org/0000-0001-6108-0831","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"Marina","lastName":"Scheller","suffix":""},{"id":444330735,"identity":"5c15ba65-5fff-435a-99ec-335d7b5594b6","order_by":8,"name":"Bailin He","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Bailin","middleName":"","lastName":"He","suffix":""},{"id":444330736,"identity":"6fdf5973-15c8-42aa-a6b9-265f1d4f8b19","order_by":9,"name":"Yi Liu","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Liu","suffix":""},{"id":444330737,"identity":"10c51e34-fb39-48cc-8901-d4e9674172f0","order_by":10,"name":"Julia Unglaub","email":"","orcid":"","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Julia","middleName":"","lastName":"Unglaub","suffix":""},{"id":444330738,"identity":"ebc9702f-91e2-42ed-b7d2-49b4a7ba6043","order_by":11,"name":"Anita Schmitt","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anita","middleName":"","lastName":"Schmitt","suffix":""},{"id":444330739,"identity":"0f75a9b9-7ba5-4ed4-8dcf-1cf4c19e7091","order_by":12,"name":"Ashok Jayavelu","email":"","orcid":"","institution":"Max Planck Institute","correspondingAuthor":false,"prefix":"","firstName":"Ashok","middleName":"","lastName":"Jayavelu","suffix":""},{"id":444330740,"identity":"45f312a7-9113-41c3-9d81-fb087216e7a2","order_by":13,"name":"Carsten Müller-Tidow","email":"","orcid":"https://orcid.org/0000-0002-7166-5232","institution":"Heidelberg University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Carsten","middleName":"","lastName":"Müller-Tidow","suffix":""},{"id":444330741,"identity":"28c56e6e-8392-40cf-b1c0-8111854e9f6b","order_by":14,"name":"Michael Schmitt","email":"","orcid":"https://orcid.org/0000-0002-1579-1509","institution":"University Hospital Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Schmitt","suffix":""}],"badges":[],"createdAt":"2025-03-22 11:20:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6283250/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6283250/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81090663,"identity":"18830fa1-644d-40c6-8501-9bdc36f72dde","added_by":"auto","created_at":"2025-04-22 07:08:24","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1113359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative analysis of CART products derived from most potent effectors and least potent effectors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Serial co-culture assay with healthy-donor derived CD70.CART, CD19.CART, EphA2.CART or GD2.CART products and their respective target cells. The absolute tumor and T cell numbers at the end of each coculture were determined by flow cytometry using CountBright\u003csup\u003e®\u003c/sup\u003e counting beads. Double-gradient heatmaps are depicting the functionality score (FS) for each co-culture that integrates T-cell and tumor cell count and is calculated by substracting the fold change in tumor cells from the fold change in CAR T cells, both relative to their initial counts (red, high FS; white, FS=0; blue, low FS). A high, positive FS indicates effective CAR T cell proliferation and tumor cell reduction, while a low or negative FS indicates inadequate CAR T cell proliferation, uncontrolled tumor growth, or both. (A) CART products were generated from 23 healthy donors. CD70.CARTs and Molm-13 cells were cocultured at a 1:4 E:T ratio, CD19.CARTs and Raji cells at a 1:1 E:T ratio, EphA2.CARTs and A-549 cells at a 1:1 E:T ratio and GD2.CARTs and LAN-1 cells at a 2:1 E:T ratio. Fresh target cells were added every 5 days for the CD70.CART, CD19.CART and EphA2.CART and every 3 days for the GD2.CART experiments. (B) CART products from the 5 most and 4 least potent donors were cocultured at a more challenging ET ratios of 1:6 (CD70.CART), 1:2 (CD19.CART), 1:4 (EphA2.CART) and 1:1 (GD2.CART) and fresh target cells were added every 5 (CD70.CART), 3 (CD19.CART and GD2.CART) and 4 days (EphA2.CART). (C) Percentage of TNF-α\u003csup\u003e+\u003c/sup\u003e and IFN-γ\u003csup\u003e+\u003c/sup\u003e producing CART cells for the most and least potent effectors after 4 hours of antigen stimulationat an E:T ratio of 1:1 as determined by intracellular cytokine staining followed by flow-cytometry. (D) Expression of PD-1, TIM-3 and LAG-3, markers associated with T-cell exhaustion, on CARTs of most and least potent effectors as determined by flow cytometry on day 10 (CD70.CARTs), day 12 (CD19.CARTs and GD2.CARTs) and day 20 (EphA2.CARTs) after co-culture assay. (E-F) Immunophenotypic characterization of CART cells between most and least potent effectors by flow cytometry on day 16 post transduction (Naive: CD45RA+CCR7+; central memory [CM]: CD45RA–CCR7+; effector memory [EM]: CD45RA−CCR7−; EM T cells re-express CD45RA [EMRA]: CD45RA+CCR7−). A two-tailed t-test was used for statistical analysis. *P \u0026lt; 0.05; **P \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/efb6e04f11dd33839f08078f.jpeg"},{"id":81090080,"identity":"0342e0c6-12b7-44b5-86b0-dd439e9c27d2","added_by":"auto","created_at":"2025-04-22 07:00:24","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":653482,"visible":true,"origin":"","legend":"\u003cp\u003eHigher CD4\u003csup\u003e+ \u003c/sup\u003eCART composition leads to improved antitumor efficacy and enhanced T cell proliferation.\u003c/p\u003e\n\u003cp\u003e(A-B) Serial co-culture assay with CD70.CARTs and Molm-13, CD19.CARTs and Nalm-6 and CD33.CARTs and HL-60 at an E:T ratio of 1:3 cells from 6 healthy donors. Fresh tumor cells were added every 3 (CD19.CARTs and CD33.CARTs) or every 4 days (CD70.CARTs). The absolute tumor and T cell numbers at the end of each coculture were determined by flow cytometry using CountBright\u003csup\u003e®\u003c/sup\u003e counting beads. (A) Double-gradient heat map of tumor cell killing (blue, 100% target cell lysis; white, 0% target cell lysis); (B) Double-gradient heat map of T cell proliferation (red, highest proliferation value; orange, baseline proliferation value; white, no proliferation). (C-D) CD19.CARTs derived from 6 patients (Pat) treated within the HD-CAR1 clinical trial and Nalm-6 cells were cocultured at a 1:3 E:T ratio, followed by fresh Nalm6 cells being added every 3 days. The absolute tumor and T cell numbers at the end of each coculture were determined by flow cytometry using CountBright counting beads. Bulk CART product indicate the CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e ratio of the transfused CART product. (C) Double-gradient heat map of tumor cell killing (blue, 100% target cell lysis; white, 0% target cell lysis); (D) Double-gradient heat map of T cell proliferation (red, highest proliferation value; orange, baseline proliferation value; white, no proliferation).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/84651d0f652f986ca67b8b12.jpeg"},{"id":81090664,"identity":"0359cfcc-a822-4da6-9137-55da942368d8","added_by":"auto","created_at":"2025-04-22 07:08:24","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":903021,"visible":true,"origin":"","legend":"\u003cp\u003eCD4\u003csup\u003e+ \u003c/sup\u003eCD70.CARTs have higher anti-tumor and proliferative activity in vivo.\u003c/p\u003e\n\u003cp\u003e(A) Schematic representation of the experimental setup of the Molm-13 xenograft model. 1 × 10\u003csup\u003e6 \u003c/sup\u003enon-transduced (NT) T cells or CD70.CART cells at various CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART ratios were administered IV 5 days after IV injection of 5 × 10\u003csup\u003e5 \u003c/sup\u003eMolm-13 cells expressing click beetle green (CBG) luciferase. Bioluminescence imaging (BLI) was perfromed prior to T cell injection on day 0 to confirm tumor cell engraftment and continued weekly thereafter (N = 6 for each treatment group). (B) Kaplan-Meier survival graph of mice treated with either NT T cells or CD70.CARTs at various CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART ratios. The statistical analysis of survival between the treatment groups was performed using the log-rank (Mantel-Cox) test (N=6, **P \u0026lt; 0.01; ***P \u0026lt; 0.001). (C) Images depicting the BLI signal derived from Molm-13 cells on the specified days in mice treated with either NT T cells or CD70.CARTs at various CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART ratios. (D) Schematic representation of in vivo T-cell persistence experiment. NSG mice were injected with 5 × 10\u003csup\u003e5\u003c/sup\u003e Molm-13 cells 5 days prior to a single dose of 1 × 10\u003csup\u003e6\u003c/sup\u003e NT T cells or CD70.CARTs at various CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART ratios also expressing CBG luciferase. T cell trafficking and proliferation was determined by BLI on days 0, 4, 9, and 13 of T-cell injection (N = 6 for each group). (E) Fold change of total flux for each group treated with luciferase-labeled NTs or CD70.CARTs. The data represent the mean ± standard deviation (SD) obtained from 6 mice per treatment group. (F) Images depicting the BLI signal derived from T cells on the specified days in mice treated with either NTs or CD70.CARTs at various CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART ratios.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/ec04dbaef0f1eb24a498d1bc.jpeg"},{"id":81090082,"identity":"86de018d-783a-4f0c-aff7-fe4e7b95d1ec","added_by":"auto","created_at":"2025-04-22 07:00:24","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1258466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFunctional impact of secretome derived from CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCARTs and CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCARTs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schematic outline of the experimental setup. Supernatant harvested from co-cultures of CD4\u003csup\u003e+ \u003c/sup\u003eCARTs or CD8\u003csup\u003e+ \u003c/sup\u003eCARTs with appropriate target cells after 24 hours are indicated as 4S or 8S. (B-C) Serial co-culture assays with CD4\u003csup\u003e+\u003c/sup\u003e or CD8\u003csup\u003e+\u003c/sup\u003e CART cells from 6 healthy donors and target tumor cells in the presence of 4S, 8S or fresh medium as control. CD70.CARTs and Molm-13, CD19.CARTs and Nalm-6 and CD33.CARTs and HL-60 were co-cultured at an E:T ratio of 1:3. Fresh tumor cells were added every 3 (CD19.CARTs and CD33.CARTs) or every 4 days (CD70.CARTs). The absolute tumor and T cell numbers at the end of each coculture were determined by flow cytometry using CountBright\u003csup\u003e®\u003c/sup\u003e counting beads. (B) Double-gradient heat map of tumor cell killing (blue, 100% target cell lysis; white, 0% target cell lysis); (C) Double-gradient heat map of T cell proliferation (red, highest proliferation value; orange, baseline proliferation value; white, no proliferation). (D)\u003cstrong\u003e \u003c/strong\u003eMultiplex analysis of cytokine production by CD19.CART products from 6 healthy donors at indicated CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e ratios after 24 hour co-culture with Nalm-6 cells at a E:T ratio of 1:3. The mean value of concentrations are shown.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/a798146e4fc56a116ee8f07e.jpeg"},{"id":81090091,"identity":"2b4c37f0-ba33-493e-a8c7-323c484b8f48","added_by":"auto","created_at":"2025-04-22 07:00:24","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":487965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction with CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCARTs impairs the functionality of CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eCARTs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Serial co-culture assay of CD4\u003csup\u003e+ \u003c/sup\u003eCD70.CARTs, CD19.CARTs and CD33.CARTs from 6 healthy donors with appropriate target cells in the absence or presence of an equal amount of CD8\u003csup\u003e+ \u003c/sup\u003eCARTs resulting in a 2-fold increase of the total CART number compared to the pure CD4\u003csup\u003e+\u003c/sup\u003e CART conditions. Thus, E:T ratio was 1:3 for CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e and 1:6 for the pure CD4\u003csup\u003e+\u003c/sup\u003e CART conditions. Fresh tumor cells were added every 3 (CD19.CARTs and CD33.CARTs) or every 4 days (CD70.CARTs). The absolute tumor and T cell numbers at the end of each coculture were determined by flow cytometry using CountBright\u003csup\u003e®\u003c/sup\u003e counting beads.. (A) Double-gradient heat map of tumor cell killing (blue, 100% target cell lysis; white, 0% target cell lysis); (B) Double-gradient heat map of T cell proliferation (red, highest proliferation value; orange, baseline proliferation value; white, no proliferation). (C) Experimental setup of the serial co-culture assay in the Transwell cell-culture device. CD70.CARTs and CD19.CARTs were co-cultured with Molm-13 or Nalm-6 cells at an E:T ratio of 1:1 and fresh tumor cells were added every 3 (CD19.CARTs) or every 4 days (CD70.CARTs). CD4\u003csup\u003e+ \u003c/sup\u003eCARTs (in the lower chamber) separated from CD8\u003csup\u003e+ \u003c/sup\u003eCARTs by the semipermeable membrane are indicated as TCD4, CD4\u003csup\u003e+ \u003c/sup\u003eCARTs cultured in the lower chamber without any CD8\u003csup\u003e+\u003c/sup\u003e CARTs are indicated as PCD4 and CD4\u003csup\u003e+ \u003c/sup\u003eCARTs that were co-culture in the presence of CD8\u003csup\u003e+ \u003c/sup\u003eCARTsare indicated as MCD4. The absolute tumor and T cell numbers at the end of each co-culture were determined by flow cytometry using CountBright counting beads. (D) Tumor cell killing and (E) T-cell proliferation of CD70.CARTs on day 12 and day 20 of co-culture. (F) Tumor cell killing and (G) T-cell proliferation of CD19.CARTs on day 33 and day 54 of co-culture. (N = 5, paired t test, *P \u0026lt; 0.05; **P \u0026lt; 0.01; ****P \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/7f070ab7f581581aacb0c21f.jpeg"},{"id":81090086,"identity":"593f6aa4-9ef5-4aaa-a258-6eee9bc4393e","added_by":"auto","created_at":"2025-04-22 07:00:24","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1128644,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProteomic analysis reveals induction of apoptosis pathways in CD4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e CARTs upon interaction with CD8\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e CARTs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomic analysis of CD4\u003csup\u003e+\u003c/sup\u003e CARTs after co-culture with appropriate target cells in the presence (MCD4) or absence of CD8\u003csup\u003e+\u003c/sup\u003e CARTs (PCD4). (A) Schematic representation of the experimental setup. PCD4 and MCD4 CD70.CART cells were co-cultured with Molm-13 cells at an E:T ratio of 1:3 and fresh tumor cells were added every 4 days. On day 12 of co-culture, PCD4 and MCD4 CARTs were isolated for proteomics analysis using fluorescence-activated cell sorting (FACS). (B) Principal component analysis (PCA) showing the first 2 principal components (component 1, component 2) for PCD4 and MCD4. (C) Volcano plot displaying the pairwise comparison of regulated proteins in PCD4 and MCD4 CARTs (permutation-based FDR\u0026lt;0.05). (D-E) Horizontal bar graphs illustrating the enrichment analysis of PCD4 (D) and MCD4 CARTs (E) based on terms of Gene Ontology Biological Processes (GOBP). The y-axis displays the GOBP term, while the x-axis indicates the number of enriched genes related to the corresponding term. (F) Network diagram of all cell cycle-related proteins in the dataset. Each node represents a specific protein, and the lines between the nodes indicate protein-protein interactions. (G) Comparison of expression intensity for proteins related to cell cycle in PCD4 and MCD4 CARTs. (H) Network diagram of all proteins involved in apoptosis. (I) Comparison of expression intensity for proteins related to apoptosis in PCD4 and MCD4 CARTs.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/64d06680401f88740b998dc3.jpeg"},{"id":81091843,"identity":"624bfdb4-de3e-4990-9268-d64d2edef4df","added_by":"auto","created_at":"2025-04-22 07:16:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6722685,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/9de5aa8f-a86a-44d6-b8c3-9ec74a384e56.pdf"},{"id":81090667,"identity":"19ced245-6cee-4ca2-b02a-31e4bf52a2a5","added_by":"auto","created_at":"2025-04-22 07:08:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7280654,"visible":true,"origin":"","legend":"Supplementary Material","description":"","filename":"NatureCommunicationsSupplementaryMaterialsCD4CARTcellpaper.docx","url":"https://assets-eu.researchsquare.com/files/rs-6283250/v1/1a9c58e973d70ffa4d657550.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Depletion of CD8+ CAR T-cells leads to superior anti-tumor efficacy of pure CD4+ CAR T-cells against Acute Leukemias","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe adoptive transfer of chimeric antigen receptor (CAR) modified T lymphocytes (CARTs) has emerged as an innovative cancer immunotherapy, offering an effective treatment strategy for various malignancies. CARTs targeting CD19 and BCMA have proven to be effective for the treatment of CD19-expressing malignancies such as acute lymphoblastic leukemia (ALL), B-cell non-Hodgkin\u0026rsquo;s lymphoma and Multiple Myeloma. This success has led to the approval of multiple CD19- and BCMA-specific CART cell products by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e While CARTs can induce long-lasting remissions in many patients, primary resistance and disease relapse remains major challenges. Insufficient expansion and persistence of CARTs are frequently observed in patients with primary and secondary treatment failure to CART cell treatment, highlighting an urgent need for strategies to overcome these major limitations.\u003csup\u003e\u003cspan additionalcitationids=\"CR3 CR4 CR5\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOptimizing the composition of CART products with respect to CD4 and CD8 expressing CARTs has been one approach to achieve prolonged antitumor activity of CARTs. So far, most clinically used CART products contain T cells with a donor-specific CD4/CD8 ratio following leukapheresis, resulting in a heterogeneous mixture of these two distinct subsets.\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e These subsets differ significantly in their activation and exhaustion states, differentiation potential, proliferative capacity, and cytokine release profiles.\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e To date, only Lisocabtagene maraleucel (Liso-cel), a CD19-targeting CART product approved for the treatment of relapsed/refractory large B cell non-Hodgkin lymphoma, contains CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e central memory T (T\u003csub\u003eCM\u003c/sub\u003e) cells at a predefined ratio of 1:1, but efficacy data is very similar to non-adjusted commercial products.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Traditionally, CD4\u003csup\u003e+\u003c/sup\u003e T cells have been considered to be \"helper\" cells, orchestrating the immune response by releasing cytokines and supporting other immune cells, while CD8\u003csup\u003e+\u003c/sup\u003e T cells are mainly responsible for direct cytotoxic effects, targeting and destroying infected or malignant cells.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e However, emerging studies have demonstrated that CD4\u003csup\u003e+\u003c/sup\u003e CART cells can directly kill tumor cells in an MHC-independent manner through death receptor-mediated cytolysis, granule exocytosis, and proinflammatory cytokines such as IFN-γ and TNF-α.\u003csup\u003e\u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Interestingly, a recent study reported that CD4\u003csup\u003e+\u003c/sup\u003e CARTs were the most abundant subpopulation in the blood of two patients about 10 years after CD19-CART infusion to treat chronic lymphocytic leukemia (CLL).\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e These results suggest that CD4\u003csup\u003e+\u003c/sup\u003e CARTs may play an indispensable role as both \u0026ldquo;helpers\u0026rdquo; and \u0026ldquo;fighters\u0026rdquo;.\u003c/p\u003e \u003cp\u003eIn this study, we sought to elucidate the significance of CD4\u003csup\u003e+\u003c/sup\u003e CARTs and investigate the crosstalk between CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e subsets within CART cell products. Our goal was to determine the optimal CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART cell ratio that enhances the CART functionality against acute leukemias, potentially improving therapeutic outcomes.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eHealthy-donor derived CART products with superior functionality contain a higher percentage of CD4\u003csup\u003e+\u003c/sup\u003e CARTs\u003c/h2\u003e\n\u003cp\u003eTo investigate donor-associated factors that potentially influence the functionality of CART products, we generated CART products, targeting CD70, CD19, EphA2 and GD2 (\u003cem\u003ei.e\u003c/em\u003e.: CD70.CART, CD19.CART, EphA2.CART, GD2.CART), from activated T-cells of 23 healthy donors (HD1-HD23). We assessed the in vitro cytotoxic and proliferative capacity of all 92 CART products by repetitive stimulation with tumor cells expressing their respective target antigen in a serial co-culture assay (Supplementary Figure 1A-C). Based on these results, a functionality score (FS) for each donor and CAR construct was calculated and donors were subsequently ranked according to the result of their respective FS (Figure 1A). A subsequent co-culture assay at a lower and thus more challenging effector to target (E:T) cell ratio confirmed the FS-based functional ranking of donors (Figure 1B).\u003c/p\u003e\n\u003cp\u003eNext, we compared the secretion of\u0026nbsp;TNF-\u0026alpha;\u003csup\u003e+\u003c/sup\u003e and IFN-\u0026gamma;\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eupon antigen stimulation between CART products generated from donors with high and low FS and found no statistical significant difference\u0026nbsp;(Figure 1C).\u0026nbsp;Furthermore, we determined the expression of proteins associated with T-cell exhaustion on the surface of CARTs. While the percentage of\u0026nbsp;PD-1, TIM-3 and LAG-3 (PD-1+/TIM-3+/LAG-3+)\u0026nbsp;expressing cells was significantly higher only in CD19.CARTs with low FS, there was no difference for CD70.CARTs,\u0026nbsp;EphA2.CARTs and GD2.CARTs\u0026nbsp;(Figure 1D). Subsequently, characterization of CART products revealed no significant differences in T-cell differentiation (Figure 1E). However, CART products with high FS did contain a significantly increased percentage of CD4\u003csup\u003e+\u003c/sup\u003e CARTs compared to those with impaired functionality (Figure 1F), suggesting that CD4\u003csup\u003e+\u003c/sup\u003e CART prevalence is associated with a high FS in healthy donor-derived CAR T-cells.\u003c/p\u003e\n\u003ch2\u003eIncreasing CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART ratio is associated with enhanced anti-leukemic activity in vitro\u003c/h2\u003e\n\u003cp\u003eHaving established that healthy donor-derived CART products with high FS contained a significantly increased percentage of CD4\u003csup\u003e+\u003c/sup\u003e CARTs, we next sought to determine the impact of the CD4/CD8 ratio on the phenotype and functionality of CART targeting AML-specific CD33 and CD70, as well as B cell malignancies-specific target CD19 (Supplementary Figure 1A-B and Figure 2).\u003c/p\u003e\n\u003cp\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART subsets did not show significant differences in the frequency of CAR expressing T-cells or the intensity as determined by the integrated mean fluorescence intensity (iMFI) (Supplementary Figure 3A-B). On day 16 of CART manufacturing,\u0026nbsp;CD4\u003csup\u003e+\u003c/sup\u003e CART cells predominantly displayed a memory phenotype (central memory (T\u003csub\u003eCM\u003c/sub\u003e) and effector memory (T\u003csub\u003eEM\u003c/sub\u003e) T-cells) as determined by the expression of CD45RA and CCR7 using flow cytometry whereas the CD8\u003csup\u003e+\u003c/sup\u003e CART subsets consisted predominantly of terminally differentiated T-cells (T\u003csub\u003eEMRA\u003c/sub\u003e) (Supplementary Figure 3C-D). Upon stimulation with target antigen expressing tumor cells, the CD107a expression was significantly increased in CD8\u003csup\u003e+\u003c/sup\u003e CARTs indicating a stronger degree of degranulation (Supplementary Figure 3E). In contrast, the secretion of TNF-\u0026alpha; was significantly higher in CD4\u003csup\u003e+\u003c/sup\u003e CARTs while IFN-\u0026gamma; secretion was elevated for CD19 and CD33 expressing CD8\u003csup\u003e+\u003c/sup\u003e CARTs but decreased for CD8\u003csup\u003e+\u003c/sup\u003e CD70.CARTs (Supplementary Figure 3F).\u003c/p\u003e\n\u003cp id=\"_Toc142327602\"\u003eTo evaluate the short-term cytotoxic potential of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs, we stimulated the cells with target antigen positive tumor cells and found a trend towards enhanced tumor cell killing for CD8\u003csup\u003e+\u003c/sup\u003e CD70.CARTs and CD19.CARTs and even a significant advantage for the CD8\u003csup\u003e+\u003c/sup\u003e CD33.CARTs\u0026nbsp;(Supplementary Figure 4A). Next, we sought to evaluate the impact of the CD4/CD8 composition of CARTs products on the cytolytic and proliferative capacity upon repeated antigen stimulation. We used CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs that were separated by FACS to generate CART products consisting of CD4/CD8 ratios ranging between 9:1 and 1:9 as well as pure CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART products and repeatedly stimulated these products with target antigen expressing tumor cells (Supplementary Figure 4B-C). Cytotoxicity and proliferation of all CART products improved continuously with increasing CD4/CD8 ratios and pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs exhibited superior functionality while pure CD8\u003csup\u003e+\u003c/sup\u003e CARTs showed the lowest proliferation and anti-tumor activity (Figure 2A-B). To exclude a potential association between the functional superiority of CD4\u003csup\u003e+\u003c/sup\u003e CARTs and NF kappa B activation mediated by the 4-1BB or CD27 co-stimulatory domains, we additionally chose a second-generation CLL-1-directed CAR construct containing only the CD28 co-stimulatory domain (Supplementary Figure 5A-B) and consistently found an association between the CD4/CD8 ratio and the functionality of the CART product (Supplementary Figure 5C-D).\u003c/p\u003e\n\u003cp\u003eHaving demonstrated the enhanced antitumor activity of CD4\u003csup\u003e+\u003c/sup\u003e CARTs derived from healthy donors, we asked the questions whether depletion of CD8\u003csup\u003e+\u003c/sup\u003e CARTs may also enhance the functionality of patient derived CARTs. To this end, cryopreserved 3\u003csup\u003erd\u003c/sup\u003e-generation CD19.CAR Ts derived from 6 patients with B cell malignancies who were treated within the HD-CAR1 clinical trial (NCT02208362) were thawed, CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs were separated by FACS and repeatedly stimulated with Nalm-6 tumor cells at different CD4/CD8 ratios. Documented clinical responses in these patients ranged from complete remissions (CR) to progressive disease (Supp. Table 1).\u0026nbsp;In 5 out of 6 patients, in vitro cytotoxicity and proliferation of CD19.CARTs could be enhanced by increasing the percentage of CD4\u003csup\u003e+\u003c/sup\u003e CARTs within the product and similar to our results with healthy donors, we observed superior proliferation and antitumor efficacy for pure CD4\u003csup\u003e+\u003c/sup\u003e CART products compared to the original non-modified CART products suggesting that depleting CD8\u003csup\u003e+\u003c/sup\u003e CARTs to obtain a pure CD4\u003csup\u003e+\u003c/sup\u003e CART product may also significantly enhance the functionality of patient-derived CART products (Figure 2C-D).\u003c/p\u003e\n\u003ch2\u003eCD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCART cells exhibit superior antitumor activity and proliferative capacity in vivo\u003c/h2\u003e\n\u003cp\u003eNext, we investigated the functionality of CD70- and CD19-directed CART products at various CD4/CD8 ratios as pure CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs in vivo using a murine NSG xenograft model. To this end, NSG mice were intravenously (IV) injected either with 5x10\u003csup\u003e5\u003c/sup\u003e CD70-expressing Molm-13 cells genetically modified to express the click beetle green (CBG) luciferase or 1x10\u003csup\u003e5\u003c/sup\u003e CD19-postive Nalm-6 cells expressing firefly luciferase on day 0. On day 5, after tumor cell engraftment, mice were infused IV with a single dose of 1x10\u003csup\u003e6\u003c/sup\u003e CD70.CART or CD19.CART cells composed of either pure CD4\u003csup\u003e+\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eor CD8\u003csup\u003e+\u003c/sup\u003e CART subsets or a mixture of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e at ratios of 3:1, 1:1 and 1:3.\u0026nbsp;Animals treated with non CAR-transduced (NT) T cells served as controls. Tumor growth was observed by weekly BLI (Figure 3A and Supplementary Figure 7A). Intriguingly, for both the CD70.CART and the CD19.CART model, the pure CD4\u003csup\u003e+\u003c/sup\u003e CART cell products exhibited the strongest anti-leukemic activity in all treated animals compared to the CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e containing CART cell products or the pure CD8\u003csup\u003e+\u003c/sup\u003e CART cells leading to a statistically significant survival benefit. Importantly, the efficacy of CARTs gradually decreased by reducing CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e T-cell ratios with the pure CD8\u003csup\u003e+\u003c/sup\u003e CART product showing only a marginally better tumor cell control than non-transduced T (NT) cells. (Figure 3B-C and Supplementary Figure 7B-E). The administered CART infusions were well tolerated, with no considerable weight loss observed in any of the mice (Supplementary Figure 7F-G).\u003c/p\u003e\n\u003cp\u003eTo investigate a potential correlation between the enhanced antitumor activity of CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCARTs and their in vivo proliferation and persistence, we used our Molm-13 xenograft model with unmodified tumor cells and CD70.CARTs that were genetically modified to express a CBG luciferase fusion protein (Figure 3D). We observed a significant proliferative advantage of pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs over CART products that contained CD8\u003csup\u003e+\u003c/sup\u003e CARTs and analogously to the tumor cell elimination, T cell proliferation and persistence gradually decreased with increasing content of CD8\u003csup\u003e+\u003c/sup\u003e CARTs. The least CART proliferation was observed in mice treated with pure CD8\u003csup\u003e+\u003c/sup\u003e CARTs. (Figure 3E-F).\u003c/p\u003e\n\u003ch2\u003eThe CD4\u003csup\u003e+\u003c/sup\u003e CART secretome significantly enhances the functionality of CARTs\u003c/h2\u003e\n\u003cp\u003eTo further elucidate the mechanism behind the superior efficacy of CD4\u003csup\u003e+\u003c/sup\u003e CART cells, we sought to determine whether soluble factors secreted by CD4\u003csup\u003e+\u003c/sup\u003e CART cells may be pivotal for their enhanced functionality. CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eand CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCARTs were stimulated separately with their corresponding target tumor cells and the supernatant from these co-cultures were harvested 24 hours (24h) after stimulation. Subsequently, a second co-culture was set up in which CD4\u003csup\u003e+\u003c/sup\u003e or CD8\u003csup\u003e+\u003c/sup\u003e CARTs were stimulated with target tumor cells either in the presence of supernatant harvested from CD4\u003csup\u003e+\u003c/sup\u003e CART co-culture (4S), CD8\u003csup\u003e+\u003c/sup\u003e CART co-culture (8S) or in the presence of fresh medium without any cytokine substitution (M). 4S, 8S and M was freshly added with every antigen stimulation (Figure 4A). As expected, we observed improved cytotoxicity and proliferation for CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs in the presence of the 8S supernatant compared to media control. Notably, the presence of 4S lead to even further enhanced cytotoxic and proliferative capacity of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs (Figure 4B-C).\u003c/p\u003e\n\u003cp\u003eTo further characterize the soluble factors secreted by CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs, we measured the supernatant concentration of several cytokines secreted by CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs when they mixed in various ratios in the co-culture 24h after stimulation using a flow cytometry-based multiplex immunoassay. With increasing CD4/CD8 ratios， CARTs secreted higher levels of Th1, Th2, and Th17 related cytokines compared to products containing a higher percentage of CD8\u003csup\u003e+\u003c/sup\u003e CARTs, namely IL-2, TNF-\u0026alpha;, IL-4, IL-6, and IL-17. Additionally, CD19.CART and CD33.CART products with increased CD4/CD8 ratio also led to higher IL-10 secretion (Figure 4D and Supplementary Figure 8A-B).\u003c/p\u003e\n\u003ch2\u003eDirect cell-to-cell interaction with CD8\u003csup\u003e+\u003c/sup\u003e CART cells impairs the functionality of CD4\u003csup\u003e+\u003c/sup\u003e CART cells\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eWhile a mixture of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART cells exhibited improved cytotoxic activity than pure CD8\u003csup\u003e+\u003c/sup\u003e CART product, they were less efficacious in tumor cell elimination compared to pure CD4\u003csup\u003e+\u003c/sup\u003e CART product. This finding prompted us to further investigate the impact of CD8\u003csup\u003e+\u003c/sup\u003e CARTs on CD4\u003csup\u003e+\u003c/sup\u003e CARTs. Utilizing our co-culture assay, we compared the anti-tumor efficacy and proliferation of CARTs at a CD4/CD8 ratio of 1:1 with pure CD4\u003csup\u003e+\u003c/sup\u003e CART products that contained only half the amount of total CARTs. Despite this hypothetical disadvantage, the pure CD4\u003csup\u003e+\u003c/sup\u003e CART product demonstrated superior cytotoxicity and proliferation compared to the product containing both CD8\u003csup\u003e+\u003c/sup\u003e and CD4\u003csup\u003e+\u003c/sup\u003e CART cells suggesting that CD8\u003csup\u003e+\u003c/sup\u003e cells diminished the functionality of CD4\u003csup\u003e+\u003c/sup\u003e CARTs (Figure 5A-B and Supplementary Figure 9). However, when examining the exhaustion phenotype of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART at different ratio compositions during repetitive antigen stimulation, the expression of inhibitory receptors (PD-1+/TIM-3+/LAG-3+) between these two subsets did not differ significantly (Supplementary Figure 10).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo elucidate the lack of synergistic effect observed after mixing CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART cells, we postulated that the CD4/CD8 ratio of the CARTs within the mixture might have undergone a shift during repeated stimulation. Indeed, the composition of the cells was analyzed during the co-culture assays, which revealed an increase in the percentage of CD8\u003csup\u003e+\u003c/sup\u003e T cells along with a decrease in CD4\u003csup\u003e+\u003c/sup\u003e T cells (Supplementary Figure 11). Specifically, this alteration in the CD4/CD8 ratio was attributable, at least in part, to the greater proliferation of CD8\u003csup\u003e+\u003c/sup\u003e CART cells, which were tracked using the intracellular fluorescent label carboxyfluorescein diacetate succinimidyl ester (CFSE). The proliferation of CD8\u003csup\u003e+\u003c/sup\u003e CART cells was augmented in the presence of CD4\u003csup\u003e+\u003c/sup\u003e CARTs, while the expansion of CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCARTs was diminished when they were co-applied with CD8\u003csup\u003e+\u003c/sup\u003e T cells (Supplementary Figure 12).\u003c/p\u003e\n\u003cp\u003eNext, to further investigate whether the inhibitory effect of CD8\u003csup\u003e+\u003c/sup\u003e CARTs on CD4\u003csup\u003e+\u003c/sup\u003e CARTs may be attributed to competitive consumption of cytokines, or rather direct cell-to-cell interaction, we utilized a cell culture device with two chambers separated by a semipermeable membrane that inhibits a physical cell-to-cell contact but allows cytokines and other soluble factors to traffic. CD19- and CD70- CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCARTs were stimulated with target cells in the bottom chamber (TCD4) while CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCARTs were co-cultured with tumor cells in the upper chamber (Figure 5C) or vice versa (Supplementary Figure 13A). Pure CD4\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCARTs (PCD4) or a mixture of CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eand CD4\u003csup\u003e+\u003c/sup\u003e CARTs (MCD4) co-cultured with target cells served as controls (Figure 5C and Supplementary Figure 13A). In line with our previous results, PCD4 demonstrated superior anti-tumor efficacy and proliferation while the presence of CD8\u003csup\u003e+\u003c/sup\u003e CARTs without direct cell-to-cell contact impaired the proliferative capacity but not the cytotoxicity of CD4\u003csup\u003e+\u003c/sup\u003e CARTs at later stages of the co-culture assays (D20 and D54 for the CD70.CARTs and the CD19.CARTs, respectively). Strikingly, direct physical contact with CD8\u003csup\u003e+\u003c/sup\u003e CARTs significantly reduced the anti-leukemic efficacy and proliferation of CD4\u003csup\u003e+\u003c/sup\u003e CARTs with repetitive antigen stimulation, suggesting that the inhibition of CD4\u003csup\u003e+\u003c/sup\u003e CART functionality by CD8\u003csup\u003e+\u003c/sup\u003e CARTs is a result of direct physical interaction between the two T-cell subsets (Figure 5D-G and Supplementary Figure 13B-E).\u003c/p\u003e\n\u003ch2\u003eInteraction with CD8\u003csup\u003e+\u003c/sup\u003e CART cells induces apoptosis of CD4\u003csup\u003e+\u003c/sup\u003e CART cells\u003c/h2\u003e\n\u003cp\u003eNext, we sought to investigate the potential mechanism of CD4\u003csup\u003e+\u003c/sup\u003e CART impairment through direct physical contact with CD8\u003csup\u003e+\u003c/sup\u003e CARTs. Using in-depth quantitative analysis of the proteome in CD4\u003csup\u003e+\u003c/sup\u003e CD70.CART cells, which were cocultured with tumor cells either exclusively (PCD4) or in a mixture with CD8\u003csup\u003e+\u003c/sup\u003e CART cells (MCD4), we quantified nearly 7000 proteins at a peptide and protein false discovery rate of 1%. Among these, we found 1335 proteins to be differentially regulated (permutation-based false discovery rate [FDR] \u0026lt; 0.05) between the groups (Figure 6A-C). Further analysis of biological processes and pathways revealed a marked enrichment of cell cycle-associated pathways in PCD4, with key proteins involved in cell cycle progression being notably higher in expression, suggesting that PCD4 cells have a enahnced capacity for proliferation compared to MCD4 cells (Figure 6D, F, G). This aligns with the functional data previously presented in Figure 2B. Conversely, pathways related to apoptosis were particularly prominent in MCD4, with most apoptosis-related proteins being upregulated, indicating that the MCD4 cells experienced increased apoptotic stress when co-cultured with CD8\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eCART cells targeting the tumor cells (Figure 6E, H,I).\u003c/p\u003e\n\u003cp\u003eBased on the proteome analysis, we determined the expression of Annexin V which is commonly used to detect apoptotic cells by flowcytometry on the surface of stimulated CD4\u003csup\u003e+\u003c/sup\u003e CD19.CART and CD70.CART in the presence or absence of CD8\u003csup\u003e+\u003c/sup\u003e CARTs. The percentage of Annexin V positive CD4\u003csup\u003e+\u003c/sup\u003e CARTs significantly increased with decreasing CD4/CD8 ratio (Supp. Figure 14A-C). Furthermore, while we did not observe an increase of apoptosis in CD4\u003csup\u003e+\u003c/sup\u003e CARTs that were separated from CD8\u003csup\u003e+\u003c/sup\u003e CARTs through the semi-permeable membrane (PCD4 vs. TCD4), inhibition of cell-to-cell interaction with CD8\u003csup\u003e+\u003c/sup\u003e CART significantly mitigated the apoptosis in CD4\u003csup\u003e+\u003c/sup\u003e CARTs (MCD4 vs. TCD4 and PCD4) (Supp. Figure 14D-E). These results confirmed the induction of apoptosis in CD4\u003csup\u003e+\u003c/sup\u003e CARTs mediated by physical contact with CD8+ CARTs on the functional level.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAiming to identify donor-specific factors that are associated with superior functionality of CARTs independent of structural differences of the CAR constructs, we found that CART products targeting hematologic malignancies as well as solid tumors exhibited enhanced in vitro anti-tumor efficacy and proliferative capacity if they contained a higher percentage of CD4\u003csup\u003e+\u003c/sup\u003e CARTs. Focusing on CAR constructs that are directed against various antigens expressed by myeloid and lymphatic leukemias, in vitro co-culture assays and murine xenograft models confirmed the functional superiority of pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs compared to all CAR products that contained CD8\u003csup\u003e+\u003c/sup\u003e CARTs. The elimination of CD8\u003csup\u003e+\u003c/sup\u003e CARTs even augmented the in-vitro activity of CART products in most patients treated with 3rd generation CD19.CARTs within a clinical trial. Subsequent exploration of the underlying mechanisms revealed that the physical interaction with CD8\u003csup\u003e+\u003c/sup\u003e CARTs induced apoptosis of CD4\u003csup\u003e+\u003c/sup\u003e CARTs, hereby impairing their antileukemic activity.\u003c/p\u003e \u003cp\u003eThe optimal composition of CART products with respect to the content of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs remains a matter of ongoing investigation and in the majority of clinical trials, the CD4/CD8 ratio of the CART products was determined by the lymphocyte composition of the donor.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e For lisocabtagen maraleucel, a CD19-directed second generation CART product containing a 4-1BB co-stimulatory, a 1:1 ratio of CD4\u003csup\u003e+\u003c/sup\u003e na\u0026iuml;ve (T\u003csub\u003eN\u003c/sub\u003e) and CD8\u003csup\u003e+\u003c/sup\u003e central-memory (T\u003csub\u003eCM\u003c/sub\u003e) T cells was demonstrated to have optimal anti-tumor activity in B cell lymphoma animal models.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe superior antileukemic activity of pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs that we observed in this study was not necessarily expected as CD4\u003csup\u003e+\u003c/sup\u003e T cells have been traditionally considered to function as helper cells, while CD8\u003csup\u003e+\u003c/sup\u003e T cells are supposed to be the mediator of cytotoxicity.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e However, evolving evidence has demonstrated that CD4\u003csup\u003e+\u003c/sup\u003e T cells can also serve as the \u0026ldquo;cytotoxic\u0026rdquo; cells and exhibit a direct tumor cell-targeting cytotoxicity.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e Indeed, Both CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART cells are capable of forming immune synapses after engagement with the target cells, hence mediating tumor cell elimination by degranulation and ligand-based lytic pathways.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Furthermore, CD4\u003csup\u003e+\u003c/sup\u003e CART cells have shown superior efficacy over CD8\u003csup\u003e+\u003c/sup\u003e CART cells in enhancing host immunity, which could potentially reduce the risk of antigen-negative relapse.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Several other studies align with our findings demonstrating that CD4\u003csup\u003e+\u003c/sup\u003e CARTs alone exhibit a superior capability for eliminating target cells.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e Agarwal et al. demonstrated that administering CD4-targeted (CD4-LV) and CD8-targeted lentiviral vector (CD8-LV) into NSG mice successfully generated murine CD4\u003csup\u003e+\u003c/sup\u003e CART cells and CD8\u003csup\u003e+\u003c/sup\u003e CART cells in vivo. Their results indicated that mice treated with CD4-LV achieved faster and more effective tumor cell elimination compared to those receiving CD8-LV alone or a combination of both vectors.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e In a murine orthotopic glioblastoma (GBM) model, Wang et al. showed that CD4\u003csup\u003e+\u003c/sup\u003e IL13Rα2 CAR T cells outperformed CD8\u003csup\u003e+\u003c/sup\u003e CART cells, exhibiting sustained cytotoxicity and recursive killing potential even under repetitive tumor challenges. Furthermore, they showed the maintenance of the CD4\u003csup\u003e+\u003c/sup\u003e subset positively correlated with the recursive killing ability of CART products derived from GBM patients.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Both of these studies attribute the superior performance of CD4\u003csup\u003e+\u003c/sup\u003e CART cells to the greater susceptibility of CD8\u003csup\u003e+\u003c/sup\u003e CART cells to exhaustion. Notwithstanding, the coinhibitory-related markers (PD-1, TIM-3, LAG-3) are comparable between CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART cells in our coculture experiment. In contrast to our findings, other groups have demonstrated the superiority of CD8\u003csup\u003e+\u003c/sup\u003e CARTs over CD4\u003csup\u003e+\u003c/sup\u003e CARTs or at least the need for the presence of both subsets in the most potent CART products. However, the comparability of these studies with our data is limited. Sommermeyer et al. compared CD19.CART products that differed not only with respect to their CD4 and CD8 expression but also contained T-cell subsets of distinct differentiation status (na\u0026iuml;ve, central and effector memory and terminally differentiated) which further alters the functional characteristics of these CART products while we characterized bulk CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART products.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Boulch et al. demonstrated that murine CD19-target CD8\u003csup\u003e+\u003c/sup\u003e CART cells exhibited a higher capacity to eliminate tumor as compared with CD4\u003csup\u003e+\u003c/sup\u003e CART cells in a immune competent MYC-driven B cell lymphoma animal model.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e However, they also reported that IFN-γ production was the dominant mechanism for tumor elimination by CD4\u003csup\u003e+\u003c/sup\u003e CD19.CART cells in their model but the tumor cells were not sensitive to pro-apoptotic effect of IFN-γ.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Such discrepancies could be attributed to the utilization of disparate tumor models and treatment regimens.\u003c/p\u003e \u003cp\u003eWhile we did not observe any clinical signs of hyperinflammation such as weight loss, hunched posture, ruffled fur or changes in the body temperature in our murine xenograft models, the enhanced anti-leukemic activity of pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs in vitro and in vivo was associated with increased CART proliferation and higher levels of Th1- and Th2-related cytokines which raises the concern of more severe treatment-related toxicity. In fact, evidence suggests that higher in vivo expansion of CART cells correlates with increased overall response rates but also a heightened incidence of CRS in patients with B-cell malignancies and multiple myeloma.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Owing to their robust proliferative capacity and cytokine release profile, CD4\u003csup\u003e+\u003c/sup\u003e CART cells may effectively stimulate myeloid cells to produce inflammatory mediators. Clinical studies have indicated associations between CRS and other factors such as disease burden, co-stimulatory domains, manufacturing procedures, and T cell differentiation status.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Findings from two independent studies suggested that CD4\u003csup\u003e+\u003c/sup\u003e CART cells may be more prone to inducing cytokine-release syndrome (CRS),\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e a major adverse event associated with CAR T cell therapy in B-cell malignancies. One study utilized a murine-derived CD19.CART cell therapy in an immunocompetent mouse model, reporting that CD4\u003csup\u003e+\u003c/sup\u003e CART cell therapy favors the occurrence of CRS in cases of high tumor burden, while at low tumor burden, no discernible toxicities were observed and a therapeutic benefit was still noted.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Another study using immunodeficient mice reconstituted with human CD34\u003csup\u003e+\u003c/sup\u003e stem cells and engrafted with Nalm-6 tumor cells demonstrated superior long-term responses accompanied by CRS in pure CD4\u003csup\u003e+\u003c/sup\u003e CART cells compared to pure CD8\u003csup\u003e+\u003c/sup\u003e CD19-directed CART cells.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e However, in another research using the same humanized mouse model, both the CD4\u003csup\u003e+\u003c/sup\u003e CART group and mixed CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e CART group exhibited similar CRS-like symptoms in the context of an effective antitumor response.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e To date, no clinical trials have been conducted using pure CD4\u003csup\u003e+\u003c/sup\u003e CART cells for treatment in real-world settings. Therefore, the induction mechanisms and pathophysiology of cytokine release syndrome (CRS) associated with CD4\u003csup\u003e+\u003c/sup\u003e CART cells warrant further investigation. Considering the critical role of CD4\u003csup\u003e+\u003c/sup\u003e CART cells in sustaining antitumor responses and long-term persistence, adjusting cell dosages may be advisable before clinical translation of single CD4\u003csup\u003e+\u003c/sup\u003e CART cell treatment.\u003c/p\u003e \u003cp\u003eMechanistically, the secretome of CD4\u003csup\u003e+\u003c/sup\u003e CARTs was more potent in stimulating the anti-leukemic activity of CARTs with a more pronounced effect on CD8\u003csup\u003e+\u003c/sup\u003e CARTs leading to a shift of the CD4/CD8 ratio within the CART products in favor of CD8\u003csup\u003e+\u003c/sup\u003e CARTs over time. CART products with increasing content of CD4\u003csup\u003e+\u003c/sup\u003e CARTs secreted higher levels of type 1 and type 2 cytokines suggesting that both types of immune response play a relevant role for their functionality. In fact, it has been shown that type 2 immune responses are associated with long-lasting remission and that IL4 and IL10 may lead to enhanced effector function and improved metabolic fitness of CART cells.\u003csup\u003e\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e On the other hand, proteomic analysis revealed that interaction with CD8\u003csup\u003e+\u003c/sup\u003e CART cells induced apoptosis of CD4\u003csup\u003e+\u003c/sup\u003e CART cells, which was further confirmed by flow cytometric detection of apoptotic proteins. These two simultaneously occurring mechanisms may explain that the intercellular crosstalk between CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART cells significantly hinders the anti-leukemic efficacy of CD4\u003csup\u003e+\u003c/sup\u003e CART cells.\u003c/p\u003e \u003cp\u003eIn the context of CARs targeting myeloid and lymphoblastic leukemias, the results of our extensive in vitro and in vivo analyses provide evidence for the superior anti-leukemic efficacy of pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs compared to products containing a mixture of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CARTs. We are demonstrating the enhanced functionality of CD4\u003csup\u003e+\u003c/sup\u003e CARTs is primarily attributed to the secretome of CD4\u003csup\u003e+\u003c/sup\u003e CARTs, which induces a more potent anti-leukemic response. However, the presence of CD4\u003csup\u003e+\u003c/sup\u003e CARTs also induces excessive proliferation of CD8\u003csup\u003e+\u003c/sup\u003e CARTs, leading to a reduction in the CD4/CD8. Furthermore, the interaction between CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e CART cells leads to apoptosis of CD4\u003csup\u003e+\u003c/sup\u003e CARTs and subsequently to the impaired functionality of the CART product.. Thus, our findings warrant testing of pure CD4\u003csup\u003e+\u003c/sup\u003e CART products targeting hematologic malignancies in early phase clinical trials that are carefully designed to investigate the safety of this approach, considering the potential proliferative capacity of pure CD4\u003csup\u003e+\u003c/sup\u003e CART products and their efficacy.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of CART cells\u003c/h2\u003e \u003cp\u003eRetroviral vector production and T-cell transduction have been described previously.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e In brief, 293T cells were transfected with packaging plasmids (PegPam, RD114) and the SFG vector containing the CAR construct (SFG.CD27.CD3zeta.IRES.tCD19, SFG.CD19.CH2-CH3.CD28.4-1BB.CD3zeta, SFG.CD33.CH2-CH3.CD28.4-1BB.CD3zeta, SFG.CLL-1.CH3.CD28.CD3zeta, SFG.4H5.IgG1h.CD28.CD3zeta.T2A.tNGFR, SFG.14g2a.IgG1.CD8a.4-1BB.CD3zeta.IRES.tCD19, SFG.CD19.CH3.CD28.4-1BB.CD3zeta). The retroviral supernatant was collected after 48 and 72 hours. Peripheral blood mononuclear cells were isolated from the peripheral blood using density gradient centrifugation. T cells were then activated using CD3 and CD28 antibodies. After 48 hours of expansion in complete medium, which consisted of 45% RPMI-1640 (Thermo Fisher Scientific, Waltham, MA), 45% Click\u0026rsquo;s medium (FujiFilm; Irvine Scientific, Santa Ana, CA), 2 mM GlutaMAX-I CTS (Thermo Fisher Scientific, Waltham, MA), and 10% fetal bovine serum (Hyclone, Logan, UT), the T cells were transduced in 24-well plates coated with RetroNectin (Takara). Additionally, the medium was supplemented with 10 ng/mL interleukin-7 (IL-7) and 5 ng/mL IL-15 (R\u0026amp;D Systems, Minneapolis, Minnesota).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCells and culture conditions\u003c/h2\u003e \u003cp\u003eDe-identified apheresis products from voluntary healthy donors were sourced from the Heidelberg blood bank. CD19.CART cells, derived from patients in the Heidelberg CART cell trial 1 (HD-CAR-1; NCT03676504), were manufactured in accordance with GMP (Good Manufacturing Practice) guidelines and stored in a nitrogen tank ready for use. All participants provided signed informed consent and were treated in compliance with the Declaration of Helsinki. 293T cells were acquired from the Deutsche Sammlung von Mikrooganismen und Zellkulturen (DSMZ) and cultured using Iscove's Modified Dulbecco's Medium (IMDM). Nalm-6, Raji, K562, Molm-13, and HL-60 cell lines were purchased from DSMZ and cultured in RPMI 1640 medium. LAN-1 cells, also obtained from DSMZ, were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine. A-549 cells, sourced from DSMZ, were cultured in Dulbecco's Modified Eagle Medium (DMEM). All cell lines were cultivated in their respective media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, within a humidified atmosphere containing 5% CO2 at 37\u0026deg;C. All the above-mentioned cell lines were authenticated at DKFZ (German Cancer Research Center) and were free from mycoplasma contamination checked by polymerase chain reaction (PCR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry\u003c/h2\u003e \u003cp\u003eThe fluorochrome-conjugated isotype control and antihuman antibodies against CD3, CD4, CD8, CD45RA, CCR7, CD70, CD19, NGFR, PD1, TIM3, LAG3, Ki67, CD107a, TNF-α, IFN-γ and AnnexinV were purchased from BD Bioscience or Biolegend. Biotin-labeled protein L and fluorochrome-labeled streptavidin from Thermo Fisher Scientific and Biolegend were used to identify CAR expression of CD33.CAR, CLL-1.CAR and long-spacer CD19.CAR (SFG.CD19.CH2-CH3.CD28.4-1BB.CD3zeta). Additionally, to precisely detect the short-spacer CD19.CAR SFG.CD19.CH3.CD28.4-1BB.CD3zeta) expression, the CD19 CAR Detection Reagent (biotinylated) together with anti-biotin-PE from Miltenyi Biotec were applied. The expression of CD70.CAR and GD2.CAR was assessed by identifying truncated CD19 via an APC-conjugated or PE-conjugated antibody specific to CD19. Similarly, EphA2.CAR expression was ascertained by detecting truncated NGFR using an antibody specific to NGFR, conjugated with either APC or PE. Dead cells were removed from the analysis using either the LIVE/DEAD\u0026trade; fixable near-infrared (IR) dead cell stain kit (Thermo Fisher Scientific) or 7AAD (BD Biosciences). Fluorescence compensation was conducted for every specific experiment with different FACS panels before cell acquisition in a flow cytometer. Fluorescence minus one (FMO) control, isotype control or non-transduced control were included. Flow cytometry data were acquired using BD FACSymphony\u0026trade; A3 and BD LSRII cell analyzers and visualized with FlowJo V.10.10.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSurface marker staining\u003c/h2\u003e \u003cp\u003eCells were harvested in the FACS tube and washed once with FACS buffer. The supernatant was removed from the cells after centrifuge. If necessary, 5 ul of Fc Receptor Blocking Solution (Biolegend) per million cells was added in 100 \u0026micro;l staining volume prior to staining with antibody of interest and incubated at room temperature for 5\u0026ndash;10 minutes. A cocktail of fluorochrome-labeled antibodies against the surface markers of interest was prepared and added to cells and mixed well. After incubation for 30 min at 4\u0026deg;C in the dark, cells were washed with FACS buffer. Finally, cells were resuspended in 400 \u0026micro;l of FACS buffer supplemented with 7-AAD Viability Staining Solution for measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular cytokine staining\u003c/h2\u003e \u003cp\u003eCD70.CART, CD19.CART, and CD33.CART cells were seeded into a 96-well U-bottom plate. The cells were then stimulated with either the respective target tumor cells or non-target K562 cells in the presence of monensin, brefeldin A, and CD107a antibody for 5 hours at 37\u0026deg;C. Afterward, the cells were washed with PBS and stained with NEAR-IR for 30 minutes at 4\u0026deg;C in the dark. Finally, surface markers were stained for 20 minutes at 4\u0026deg;C in the dark. The cells were fixed and permeabilized at room temperature following the instructions of the Miltenyi Fixation/Permeabilization Solution Kit. Antibodies were used to stain cytokines IFN-γ and TNF-α for 30 minutes in the dark at room temperature. Lastly, the cells were washed and resuspended in FACS buffer for acquisition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCFSE staining\u003c/h2\u003e \u003cp\u003eCAR-T cells were resuspended in a CFSE (Carboxyfluorescein succinimidyl ester) staining solution (Biolegend) comprising 1000 \u0026micro;l of PBS and 1 \u0026micro;l of a 5 mM CFSE stock. This cell preparation was then incubated for 20 minutes at 37\u0026deg;C in the dark. The staining reaction was halted by the introduction of complete medium, followed by two additional washes with the medium. For the assay, 1 x 10\u003csup\u003e6\u003c/sup\u003e CFSE-stained cells were allocated per well into a 24-well plate and were then stimulated by X-ray irradiated target tumor cells at a 1:1 ratio. The proliferative response of the CAR-T cells was evaluated after a five-day stimulation period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCoculture assay\u003c/h2\u003e \u003cp\u003eAn illustrative scheme of the experimental approach is presented in Supplemental Fig.\u0026nbsp;1C. CART cells were co-cultured with tumor cells at the appropriate Effector-to-Target (E: T) ratio in plates without the addition of external cytokines. A single well of plates for each condition was sampled every certain days, and the total count of T cells and tumor cells was determined through flow cytometry using CountBright counting beads. The population of dead cells was excluded through 7AAD staining. ZsGreen expression was used to identify tumor cells, while CD3 antibodies were employed for T-cell detection. CART cells were subjected to a repetitive challenge with new tumor cells at the same E: T ratio. If CART cells lost their ability to eliminate tumor cells or ceased to proliferate and subsequently disappeared, the respective condition was terminated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCytokine release assay\u003c/h2\u003e \u003cp\u003eSupernatants were collected 24h following the co-culture. To quantify cytokines in the supernatant, bead-based multiplex LEGENDplex\u0026trade; analysis (BioLegend) was performed following the manufacturer's instructions. A range of pro-inflammatory cytokines, including IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF-α, soluble Fas, soluble FasL, granzyme A, granzyme B, perforin, and granulysin were measured. The analysis was conducted on the BD FACSCantoII flow cytometer. Data were interpreted by the LEGENDPlex\u0026trade; V8.0 software (Biolegend).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eXenograft model\u003c/h2\u003e \u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e experiments were conducted in accordance with a protocol endorsed by the federal and Institutional Animal Care and Use Committee (IACUC). The female and male NOD.Cg-Prkdc\u003csup\u003escid\u003c/sup\u003e lL2rg\u003csup\u003etm1Wjl\u003c/sup\u003e/SzJ (NSG) mice, aged 6 to 10 weeks, were acquired from the Heidelberg University Interfaculty Biomedical Research Facility (IBF) breeding colony and maintained in IBF during the experiments. Animals were administered injections of tumor cells and T cells intravenously through the tail vein, as detailed in the \"Results\" and \u0026ldquo;Supplementary Information\u0026rdquo; sections. Subsequent measurements of tumor mass or T-cell expansion were taken regularly by bioluminescent imaging, using the in vivo imaging system IVIS Lumina II (Caliper\u003c/p\u003e \u003cp\u003eLifeScience, Hopkinton, MA). The mice were euthanized when they reached predefined endpoint criteria complied with IACUC.\u003c/p\u003e \u003cp\u003eRather than employing the CD19.CAR construct with a hinge-CH2-CH3 spacer (long spacer), which was utilized in both the in-vitro experiments and the Heidelberg CAR T cell trial 1 (HD-CAR-1; NCT03676504),\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e a short-spacer CD19.CAR construct featuring a hinge-CH3 was used to modify the T cells for the murine leukemia xenograft treatment (Supplementary Fig.\u0026nbsp;6). Previous observations, such as those reported by Almasbak et al. in 2015, have identified that the CH2 domain in commonly used IgG1-Fc spacers can bind to soluble mouse Fcγ-receptor I, leading to off-target T-cell activation directed at murine macrophages and consequently diminishing anti-leukemia activity.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e To address this issue, our study adopted a short-spacer CAR construct that omits the CH2 region in the NSG mouse model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLabel-free proteome measurement\u003c/h2\u003e \u003cp\u003eFACS-sorted CD4\u003csup\u003e+\u003c/sup\u003e CART cells from the coculture condition were thoroughly washed in plain PBS, lysed in 1%SDC buffer (1%SDC, 100mM Tris pH8.5, 40mM CAA and 10mM TCEP), incubated on ice for 20 minutes, boiled at 95\u0026deg;C, sonicated for 5 mins on a Biorupter plus as described previously.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Samples were digested with trypsin and LysC for 16 hours at 37\u0026deg;C. Digestion was stopped by adding 5X volumes of isopropanol/1% TFA and vortexing vigorously. The peptides were de-salted on equilibrated styrene divinylbenzene-reversed phase sulfonated (SDB-RPS) StageTips, washed with isopropanol/1% TFA and 0.2% TFA, and eluted with 60\u0026micro;l of elution buffer (1.25% Ammonia, 80% ACN). The dried elutes were resuspended in MS loading buffer (3% ACN, 0.3% TFA) and stored at -20\u0026deg;C until MS measurement.\u003c/p\u003e \u003cp\u003e400 ng peptides were loaded onto a nanoElute system (Bruker Daltonics Inc, Bremen, Germany) coupled with a TIMS TOF HT mass spectrometer (Bruker Daltonics, Bremen, Germany) using a CaptiveSpray nano-electrospray ion source. Peptides were separated on an IonOpticks Aurora 25 cm column using a binary buffer system: buffer A (0.1% formic acid, 2% ACN) and buffer B (0.1% FA in 99.9% ACN), using a 120-minute gradient starting from 2% buffer B, increasing to 12% in 60 min, 20% in 30 min, 30% in 10 min, and finally reaching 85% in 10 min, which was held for an additional 10 min at a flow rate of 0.3 \u0026micro;l/min. DDA-PASEF mode was employed, and MS1 spectra were recorded from 150 m/z to 1700 m/z, TIMS functioning at Scan range 100\u0026ndash;1700 m/z, Ramp Time 100 ms, Duty cycle 100%, Cycle time 100.00 ms and Spectra Rate 9.43 Hz.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMS data analysis and bioinformatics\u003c/h2\u003e \u003cp\u003eMS raw files were processed using Maxquant (version 1.5.5.2) supported by Andromeda search engine against the UniProt human reference fasta (version 2016). MaxQuant default settings were employed for identification and quantification, allowing a maximum of 2 missed cleavages. The minimal peptide length was set to 7. Label-free quantification (LFQ) was performed using the MaxLFQ algorithm implemented in MaxQuant, with the following settings: a minimal ratio count of 2 and fast LFQ enabled. The peptide and protein FDR was set to 1% to ensure high confidence in the identified peptides and proteins.\u003c/p\u003e \u003cp\u003eThe protein group file generated from MaxQuant software was further analyzed in Perseus (version 2.0.7.0). Values were log2 transformed, and data were pre-cleaned by filtering out contaminants, reverse, and only identified by site modifications before statistical analysis. Enrichment analysis and keywords were performed by comparing the protein expressions between groups. Paired sample t-tests and multi-sample t-tests were used to compare between two groups with a permutation-based FDR cutoff of 5%. The proteome data has been submitted to the PRIDE database with the project accession number: PXD060508.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using Prism 10 software (GraphPad Software, San Diego, CA). For analyzing continuous variables across three or more groups, one-way analysis of variance (ANOVA) was employed. When comparing two groups, the analysis was carried out using either a t-test or a Wilcoxon rank-sum test. Survival times resulting from the injection of tumor cells in mouse experiments were analyzed through the use of Kaplan-Meier curves and the Mantel-Cox log-rank test. P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were defined as statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe HD-CAR-1 clinical trial, from which primary patient samples were derived, was approved by the ethics committee of the medical faculty of the University of Heidelberg.\u003c/p\u003e\n\u003cp\u003eThe experiments involving the murine xenograft models were approved by the animal care committee of the Regierungspr\u0026auml;sidium Karlsruhe.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData and code availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData generated from proteome analysis are deposited in the PRIDE database with the project accession number: PXD060508.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Volker Eckstein and Stefanie Hofmann for assistance with the fluorescence-activated cell sorting. We thank all patients for providing their specimens.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQC, TS, MS and AKJ designed the reserch; QC and TS analyzed and interpreted the data and wrote the paper; QC, HY, LHM, DD, DS, MS, BLH and JMU performed the experiments; LW, AS, CMT and AKJ contributed to the interpretation of the results.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of conflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCappell, K.M., and Kochenderfer, J.N. (2023). Long-term outcomes following CAR T cell therapy: what we know so far. Nat Rev Clin Oncol \u003cem\u003e20\u003c/em\u003e, 359-371. 10.1038/s41571-023-00754-1.\u003c/li\u003e\n\u003cli\u003eAli, S.A., Shi, V., Maric, I., Wang, M., Stroncek, D.F., Rose, J.J., Brudno, J.N., Stetler-Stevenson, M., Feldman, S.A., Hansen, B.G., Fellowes, V.S., et al. (2016). T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma. Blood \u003cem\u003e128\u003c/em\u003e, 1688-1700. 10.1182/blood-2016-04-711903.\u003c/li\u003e\n\u003cli\u003eCappell, K.M., Sherry, R.M., Yang, J.C., Goff, S.L., Vanasse, D.A., McIntyre, L., Rosenberg, S.A., and Kochenderfer, J.N. (2020). Long-Term Follow-Up of Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy. J Clin Oncol \u003cem\u003e38\u003c/em\u003e, 3805-3815. 10.1200/JCO.20.01467.\u003c/li\u003e\n\u003cli\u003eMunshi, N.C., Anderson, L.D., Jr., Shah, N., Madduri, D., Berdeja, J., Lonial, S., Raje, N., Lin, Y., Siegel, D., Oriol, A., Moreau, P., et al. (2021). Idecabtagene Vicleucel in Relapsed and Refractory Multiple Myeloma. N Engl J Med \u003cem\u003e384\u003c/em\u003e, 705-716. 10.1056/NEJMoa2024850.\u003c/li\u003e\n\u003cli\u003eNeelapu, S.S., Locke, F.L., Bartlett, N.L., Lekakis, L.J., Miklos, D.B., Jacobson, C.A., Braunschweig, I., Oluwole, O.O., Siddiqi, T., Lin, Y., Timmerman, J.M., et al. (2017). Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma. N Engl J Med \u003cem\u003e377\u003c/em\u003e, 2531-2544. 10.1056/NEJMoa1707447.\u003c/li\u003e\n\u003cli\u003eShah, B.D., Ghobadi, A., Oluwole, O.O., Logan, A.C., Boissel, N., Cassaday, R.D., Leguay, T., Bishop, M.R., Topp, M.S., Tzachanis, D., O\u0026apos;Dwyer, K.M., et al. (2021). KTE-X19 for relapsed or refractory adult B-cell acute lymphoblastic leukaemia: phase 2 results of the single-arm, open-label, multicentre ZUMA-3 study. Lancet \u003cem\u003e398\u003c/em\u003e, 491-502. 10.1016/S0140-6736(21)01222-8.\u003c/li\u003e\n\u003cli\u003eFowler, N.H., Dickinson, M., Dreyling, M., Martinez-Lopez, J., Kolstad, A., Butler, J., Ghosh, M., Popplewell, L., Chavez, J.C., Bachy, E., Kato, K., et al. (2022). Tisagenlecleucel in adult relapsed or refractory follicular lymphoma: the phase 2 ELARA trial. Nat Med \u003cem\u003e28\u003c/em\u003e, 325-332. 10.1038/s41591-021-01622-0.\u003c/li\u003e\n\u003cli\u003eMartin, T., Usmani, S.Z., Berdeja, J.G., Agha, M., Cohen, A.D., Hari, P., Avigan, D., Deol, A., Htut, M., Lesokhin, A., Munshi, N.C., et al. (2023). Ciltacabtagene Autoleucel, an Anti-B-cell Maturation Antigen Chimeric Antigen Receptor T-Cell Therapy, for Relapsed/Refractory Multiple Myeloma: CARTITUDE-1 2-Year Follow-Up. J Clin Oncol \u003cem\u003e41\u003c/em\u003e, 1265-1274. 10.1200/JCO.22.00842.\u003c/li\u003e\n\u003cli\u003eMaude, S.L., Laetsch, T.W., Buechner, J., Rives, S., Boyer, M., Bittencourt, H., Bader, P., Verneris, M.R., Stefanski, H.E., Myers, G.D., Qayed, M., et al. (2018). Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia. N Engl J Med \u003cem\u003e378\u003c/em\u003e, 439-448. 10.1056/NEJMoa1709866.\u003c/li\u003e\n\u003cli\u003eWang, M., Munoz, J., Goy, A., Locke, F.L., Jacobson, C.A., Hill, B.T., Timmerman, J.M., Holmes, H., Jaglowski, S., Flinn, I.W., McSweeney, P.A., et al. (2020). KTE-X19 CAR T-Cell Therapy in Relapsed or Refractory Mantle-Cell Lymphoma. N Engl J Med \u003cem\u003e382\u003c/em\u003e, 1331-1342. 10.1056/NEJMoa1914347.\u003c/li\u003e\n\u003cli\u003eAgarwal, S., Hanauer, J.D.S., Frank, A.M., Riechert, V., Thalheimer, F.B., and Buchholz, C.J. (2020). In Vivo Generation of CAR T Cells Selectively in Human CD4(+) Lymphocytes. Mol Ther \u003cem\u003e28\u003c/em\u003e, 1783-1794. 10.1016/j.ymthe.2020.05.005.\u003c/li\u003e\n\u003cli\u003eBove, C., Arcangeli, S., Falcone, L., Camisa, B., El Khoury, R., Greco, B., De Lucia, A., Bergamini, A., Bondanza, A., Ciceri, F., Bonini, C., et al. (2023). CD4 CAR-T cells targeting CD19 play a key role in exacerbating cytokine release syndrome, while maintaining long-term responses. J Immunother Cancer \u003cem\u003e11\u003c/em\u003e. 10.1136/jitc-2022-005878.\u003c/li\u003e\n\u003cli\u003eSommermeyer, D., Hudecek, M., Kosasih, P.L., Gogishvili, T., Maloney, D.G., Turtle, C.J., and Riddell, S.R. (2016). Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia \u003cem\u003e30\u003c/em\u003e, 492-500. 10.1038/leu.2015.247.\u003c/li\u003e\n\u003cli\u003eWang, D., Aguilar, B., Starr, R., Alizadeh, D., Brito, A., Sarkissian, A., Ostberg, J.R., Forman, S.J., and Brown, C.E. (2018). Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JCI Insight \u003cem\u003e3\u003c/em\u003e. 10.1172/jci.insight.99048.\u003c/li\u003e\n\u003cli\u003eAbramson, J.S., Palomba, M.L., Gordon, L.I., Lunning, M.A., Wang, M., Arnason, J., Mehta, A., Purev, E., Maloney, D.G., Andreadis, C., Sehgal, A., et al. (2020). Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet \u003cem\u003e396\u003c/em\u003e, 839-852. 10.1016/S0140-6736(20)31366-0.\u003c/li\u003e\n\u003cli\u003eBorst, J., Ahrends, T., Babala, N., Melief, C.J.M., and Kastenmuller, W. (2018). CD4(+) T cell help in cancer immunology and immunotherapy. Nat Rev Immunol \u003cem\u003e18\u003c/em\u003e, 635-647. 10.1038/s41577-018-0044-0.\u003c/li\u003e\n\u003cli\u003eCsaplar, M., Szollosi, J., Gottschalk, S., Vereb, G., and Szoor, A. (2021). Cytolytic Activity of CAR T Cells and Maintenance of Their CD4+ Subset Is Critical for Optimal Antitumor Activity in Preclinical Solid Tumor Models. Cancers (Basel) \u003cem\u003e13\u003c/em\u003e. 10.3390/cancers13174301.\u003c/li\u003e\n\u003cli\u003eAdusumilli, P.S., Cherkassky, L., Villena-Vargas, J., Colovos, C., Servais, E., Plotkin, J., Jones, D.R., and Sadelain, M. (2014). Regional delivery of mesothelin-targeted CAR T cell therapy generates potent and long-lasting CD4-dependent tumor immunity. Sci Transl Med \u003cem\u003e6\u003c/em\u003e, 261ra151. 10.1126/scitranslmed.3010162.\u003c/li\u003e\n\u003cli\u003eBenmebarek, M.R., Karches, C.H., Cadilha, B.L., Lesch, S., Endres, S., and Kobold, S. (2019). Killing Mechanisms of Chimeric Antigen Receptor (CAR) T Cells. Int J Mol Sci \u003cem\u003e20\u003c/em\u003e. 10.3390/ijms20061283.\u003c/li\u003e\n\u003cli\u003eLi, T., Wu, B., Yang, T., Zhang, L., and Jin, K. (2020). The outstanding antitumor capacity of CD4(+) T helper lymphocytes. Biochim Biophys Acta Rev Cancer \u003cem\u003e1874\u003c/em\u003e, 188439. 10.1016/j.bbcan.2020.188439.\u003c/li\u003e\n\u003cli\u003eXhangolli, I., Dura, B., Lee, G., Kim, D., Xiao, Y., and Fan, R. (2019). Single-cell Analysis of CAR-T Cell Activation Reveals A Mixed T(H)1/T(H)2 Response Independent of Differentiation. Genomics Proteomics Bioinformatics \u003cem\u003e17\u003c/em\u003e, 129-139. 10.1016/j.gpb.2019.03.002.\u003c/li\u003e\n\u003cli\u003eYang, Y., Kohler, M.E., Chien, C.D., Sauter, C.T., Jacoby, E., Yan, C., Hu, Y., Wanhainen, K., Qin, H., and Fry, T.J. (2017). TCR engagement negatively affects CD8 but not CD4 CAR T cell expansion and leukemic clearance. Sci Transl Med \u003cem\u003e9\u003c/em\u003e. 10.1126/scitranslmed.aag1209.\u003c/li\u003e\n\u003cli\u003eMelenhorst, J.J., Chen, G.M., Wang, M., Porter, D.L., Chen, C., Collins, M.A., Gao, P., Bandyopadhyay, S., Sun, H., Zhao, Z., Lundh, S., et al. (2022). Decade-long leukaemia remissions with persistence of CD4(+) CAR T cells. Nature \u003cem\u003e602\u003c/em\u003e, 503-509. 10.1038/s41586-021-04390-6.\u003c/li\u003e\n\u003cli\u003eLiu, Y., Sperling, A.S., Smith, E.L., and Mooney, D.J. (2023). Optimizing the manufacturing and antitumour response of CAR T therapy. Nature Reviews Bioengineering \u003cem\u003e1\u003c/em\u003e, 271-285. 10.1038/s44222-023-00031-x.\u003c/li\u003e\n\u003cli\u003eMoeller, M., Kershaw, M.H., Cameron, R., Westwood, J.A., Trapani, J.A., Smyth, M.J., and Darcy, P.K. (2007). Sustained antigen-specific antitumor recall response mediated by gene-modified CD4+ T helper-1 and CD8+ T cells. Cancer Res \u003cem\u003e67\u003c/em\u003e, 11428-11437. 10.1158/0008-5472.CAN-07-1141.\u003c/li\u003e\n\u003cli\u003eBrown, D.M. (2010). Cytolytic CD4 cells: Direct mediators in infectious disease and malignancy. Cell Immunol \u003cem\u003e262\u003c/em\u003e, 89-95. 10.1016/j.cellimm.2010.02.008.\u003c/li\u003e\n\u003cli\u003eMoeller, M., Haynes, N.M., Kershaw, M.H., Jackson, J.T., Teng, M.W., Street, S.E., Cerutti, L., Jane, S.M., Trapani, J.A., Smyth, M.J., and Darcy, P.K. (2005). Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection. Blood \u003cem\u003e106\u003c/em\u003e, 2995-3003. 10.1182/blood-2004-12-4906.\u003c/li\u003e\n\u003cli\u003eBoulch, M., Cazaux, M., Loe-Mie, Y., Thibaut, R., Corre, B., Lema\u0026icirc;tre, F., Grandjean, C.L., Garcia, Z., and Bousso, P. (2021). A cross-talk between CAR T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity. Sci Immunol \u003cem\u003e6\u003c/em\u003e. 10.1126/sciimmunol.abd4344.\u003c/li\u003e\n\u003cli\u003eLiadi, I., Singh, H., Romain, G., Rey-Villamizar, N., Merouane, A., Adolacion, J.R., Kebriaei, P., Huls, H., Qiu, P., Roysam, B., Cooper, L.J., et al. (2015). Individual Motile CD4(+) T Cells Can Participate in Efficient Multikilling through Conjugation to Multiple Tumor Cells. Cancer Immunol Res \u003cem\u003e3\u003c/em\u003e, 473-482. 10.1158/2326-6066.CIR-14-0195.\u003c/li\u003e\n\u003cli\u003eBoulch, M., Cazaux, M., Cuffel, A., Guerin, M.V., Garcia, Z., Alonso, R., Lemaitre, F., Beer, A., Corre, B., Menger, L., Grandjean, C.L., et al. (2023). Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-gamma is a major determinant of CD4(+) CAR T-cell antitumor activity. Nat Cancer \u003cem\u003e4\u003c/em\u003e, 968-983. 10.1038/s43018-023-00570-7.\u003c/li\u003e\n\u003cli\u003eOgasawara, K., Lymp, J., Mack, T., Dell\u0026apos;Aringa, J., Huang, C.P., Smith, J., Peiser, L., and Kostic, A. (2022). In Vivo Cellular Expansion of Lisocabtagene Maraleucel and Association With Efficacy and Safety in Relapsed/Refractory Large B-Cell Lymphoma. Clin Pharmacol Ther \u003cem\u003e112\u003c/em\u003e, 81-89. 10.1002/cpt.2561.\u003c/li\u003e\n\u003cli\u003eArcangeli, S., Bove, C., Mezzanotte, C., Camisa, B., Falcone, L., Manfredi, F., Bezzecchi, E., El Khoury, R., Norata, R., Sanvito, F., Ponzoni, M., et al. (2022). CAR T cell manufacturing from naive/stem memory T lymphocytes enhances antitumor responses while curtailing cytokine release syndrome. J Clin Invest \u003cem\u003e132\u003c/em\u003e. 10.1172/JCI150807.\u003c/li\u003e\n\u003cli\u003eHernani, R., Benzaquen, A., and Solano, C. (2022). Toxicities following CAR-T therapy for hematological malignancies. Cancer Treat Rev \u003cem\u003e111\u003c/em\u003e, 102479. 10.1016/j.ctrv.2022.102479.\u003c/li\u003e\n\u003cli\u003eBoulch, M., Cazaux, M., Cuffel, A., Ruggiu, M., Allain, V., Corre, B., Loe-Mie, Y., Hosten, B., Cisternino, S., Auvity, S., Thieblemont, C., et al. (2023). A major role for CD4(+) T cells in driving cytokine release syndrome during CAR T cell therapy. Cell Rep Med \u003cem\u003e4\u003c/em\u003e, 101161. 10.1016/j.xcrm.2023.101161.\u003c/li\u003e\n\u003cli\u003eBai, Z., Feng, B., McClory, S.E., de Oliveira, B.C., Diorio, C., Gregoire, C., Tao, B., Yang, L., Zhao, Z., Peng, L., Sferruzza, G., et al. (2024). Single-cell CAR T atlas reveals type 2 function in 8-year leukaemia remission. Nature \u003cem\u003e634\u003c/em\u003e, 702-711. 10.1038/s41586-024-07762-w.\u003c/li\u003e\n\u003cli\u003eBai, Z., Woodhouse, S., Zhao, Z., Arya, R., Govek, K., Kim, D., Lundh, S., Baysoy, A., Sun, H., Deng, Y., Xiao, Y., et al. (2022). Single-cell antigen-specific landscape of CAR T infusion product identifies determinants of CD19-positive relapse in patients with ALL. Sci Adv \u003cem\u003e8\u003c/em\u003e, eabj2820. 10.1126/sciadv.abj2820.\u003c/li\u003e\n\u003cli\u003eFeng, B., Bai, Z., Zhou, X., Zhao, Y., Xie, Y.Q., Huang, X., Liu, Y., Enbar, T., Li, R., Wang, Y., Gao, M., et al. (2024). The type 2 cytokine Fc-IL-4 revitalizes exhausted CD8(+) T cells against cancer. Nature \u003cem\u003e634\u003c/em\u003e, 712-720. 10.1038/s41586-024-07962-4.\u003c/li\u003e\n\u003cli\u003eZhao, Y., Chen, J., Andreatta, M., Feng, B., Xie, Y.Q., Wenes, M., Wang, Y., Gao, M., Hu, X., Romero, P., Carmona, S., et al. (2024). IL-10-expressing CAR T cells resist dysfunction and mediate durable clearance of solid tumors and metastases. Nat Biotechnol \u003cem\u003e42\u003c/em\u003e, 1693-1704. 10.1038/s41587-023-02060-8.\u003c/li\u003e\n\u003cli\u003eHan, H., Wang, L., Ding, Y., Neuber, B., Huckelhoven-Krauss, A., Lin, M., Yao, H., Chen, Q., Sauer, T., Schubert, M.L., Guo, Z., et al. (2024). Extracorporeal photopheresis as a promising strategy for the treatment of graft-versus-host disease after CAR T-cell therapy. Blood Adv \u003cem\u003e8\u003c/em\u003e, 2675-2690. 10.1182/bloodadvances.2023012463.\u003c/li\u003e\n\u003cli\u003eSauer, T., Parikh, K., Sharma, S., Omer, B., Sedloev, D., Chen, Q., Angenendt, L., Schliemann, C., Schmitt, M., Muller-Tidow, C., Gottschalk, S., et al. (2021). CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity. Blood \u003cem\u003e138\u003c/em\u003e, 318-330. 10.1182/blood.2020008221.\u003c/li\u003e\n\u003cli\u003eSchubert, M.L., Schmitt, A., Huckelhoven-Krauss, A., Neuber, B., Kunz, A., Waldhoff, P., Vonficht, D., Yousefian, S., Jopp-Saile, L., Wang, L., Korell, F., et al. (2023). Treatment of adult ALL patients with third-generation CD19-directed CAR T cells: results of a pivotal trial. J Hematol Oncol \u003cem\u003e16\u003c/em\u003e, 79. 10.1186/s13045-023-01470-0.\u003c/li\u003e\n\u003cli\u003eAlmasbak, H., Walseng, E., Kristian, A., Myhre, M.R., Suso, E.M., Munthe, L.A., Andersen, J.T., Wang, M.Y., Kvalheim, G., Gaudernack, G., and Kyte, J.A. (2015). Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model. Gene Ther \u003cem\u003e22\u003c/em\u003e, 391-403. 10.1038/gt.2015.4.\u003c/li\u003e\n\u003cli\u003eJayavelu, A.K., Schnoder, T.M., Perner, F., Herzog, C., Meiler, A., Krishnamoorthy, G., Huber, N., Mohr, J., Edelmann-Stephan, B., Austin, R., Brandt, S., et al. (2020). Splicing factor YBX1 mediates persistence of JAK2-mutated neoplasms. Nature \u003cem\u003e588\u003c/em\u003e, 157-163. 10.1038/s41586-020-2968-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6283250/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6283250/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChimeric antigen receptor T-cell (CART) therapy has shown impressive therapeutic efficacy in several hematologic malignancies, however primary or secondary treatment failure remains a significant challenge driving translational research to improve the functionality of CARTs. Here, we show the optimal composition of CARTs targeting acute leukemias with respect to the content of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T-cells. Our analysis demonstrated that pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs exhibited superior antitumor activity and proliferative capacity in vitro and in vivo compared to CD8\u003csup\u003e+\u003c/sup\u003e-containing CART products. Furthermore, the secretome of pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs, enriched for Th1 and Th2 cytokines, was more potent in stimulating the anti-leukemic activity of CARTs. Mechanistically, we found that the interaction with CD8\u003csup\u003e+\u003c/sup\u003e CARTs induces apoptosis in CD4\u003csup\u003e+\u003c/sup\u003e CARTs leading to their impaired functionality. Our findings demonstrate the superior efficacy and persistence of pure CD4\u003csup\u003e+\u003c/sup\u003e CARTs against acute leukemias warranting further exploration of their therapeutic potential within early phase clinical trials.\u003c/p\u003e","manuscriptTitle":"Depletion of CD8+ CAR T-cells leads to superior anti-tumor efficacy of pure CD4+ CAR T-cells against Acute Leukemias","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-22 07:00:19","doi":"10.21203/rs.3.rs-6283250/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"89c3f155-44b8-4da8-be93-2f1e8b13f08a","owner":[],"postedDate":"April 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47305370,"name":"Health sciences/Oncology/Cancer/Haematological cancer/Leukaemia/Acute myeloid leukaemia"},{"id":47305371,"name":"Health sciences/Oncology/Cancer/Haematological cancer/Leukaemia/Acute lymphocytic leukaemia"}],"tags":[],"updatedAt":"2025-06-05T02:15:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-22 07:00:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6283250","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6283250","identity":"rs-6283250","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}