CD40 agonism enhances immune checkpoint blockade and generates immunologic memory via CD4+ T cells in ERα+ mammary tumors

CD40 agonism enhances immune checkpoint blockade and generates immunologic memory via CD4+ T cells in ERα+ mammary tumors | 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 CD40 agonism enhances immune checkpoint blockade and generates immunologic memory via CD4 + T cells in ERα+ mammary tumors Casey Lam, Olivia Lanchoney, Vishnu Maddipatla, Nune Markosyan, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6823527/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2026 Read the published version in npj Breast Cancer → Version 1 posted 9 You are reading this latest preprint version Abstract There has been marked improvement in the clinical outcome of triple-negative breast cancer (TNBC) with the use of immune checkpoint blockade (ICB) although serious immune-related adverse effects are not uncommon. Unlike TNBC, ERα + breast tumors are largely unresponsive to ICB. Here we demonstrate defective priming by cross-presenting conventional dendritic cells (cDCs) and a blunted response to ICB in ERα + mouse mammary tumors compared to TNBC. Systemic administration of an agonistic CD40 antibody (aCD40) induced T cell proliferation and activation in tumor-draining lymph nodes and attracted effector T cells to the tumor bed from the periphery. This effect was largely due to activation, maturation and migration of type 1 conventional dendritic cells (cDC1s). aCD40 alone slowed tumor growth in ERα + tumors but its combination with ICB cured tumor-bearing mice, accomplishing a “vaccine effect” and the immune-mediated rejection of tumor rechallenge. The anti-tumor effect of aCD40 effect was cDC1 and CD8 + T cell-dependent, whereas the rejection of secondary tumor rechallenge in cured mice required CD4 + T cells. Importantly, intra-tumoral administration of aCD40 combined with systemic or intra-tumoral ICB – to mimic neoadjuvant therapeutic approaches—induced complete regressions of both treated and distant tumors. These findings indicate that aCD40 achieves DC activation required for the response to immunotherapy in ERα + tumors and further supports intra-tumoral administration of both aCD40 and ICB as an effective treatment that might limit systemic exposure and lower risk of immune-related toxicity. Biological sciences/Cancer/Breast cancer Health sciences/Oncology/Cancer/Breast cancer Health sciences/Oncology/Cancer/Cancer models Health sciences/Oncology/Cancer/Cancer therapy Health sciences/Oncology/Cancer/Tumour immunology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Breast cancer remains a leading cause of cancer-related mortality in women worldwide, with estrogen receptor positive (ER+) subtypes accounting for approximately 70% of all cases 1 , 2 . Although many patients have a good prognosis, a significant proportion of patients will have locally advanced disease with a high risk of distant disease and death 3 or develop endocrine therapy resistance 4 , 5 . Unfortunately, traditional chemotherapy has poor efficacy in ER + subtypes with low rates of complete pathologic response 6 , highlighting the urgent need for alternative therapeutic approaches. Immune checkpoint blockade (ICB), which has revolutionized treatment paradigms across multiple cancer types, has demonstrated only modest efficacy in breast cancer, with anti-PD-1/PD-L1 and anti-CTLA4 monoclonal antibodies benefiting a small subset of patients with predominantly PD-L1-positive tumors 7 – 10 . ICB in ER + breast cancer has shown largely limited efficacy 11 , 12 , primarily attributed to an immunologically "cold" tumor microenvironment (TME) including low density of tumor-infiltrating lymphocytes (TILs) and low tumor mutational burden 13 – 15 . Conversely, checkpoint blockade has demonstrated clinical efficacy in triple-negative breast cancer (TNBC) 16 – 18 , likely due to having a more activated tumor immune microenvironment compared to ER + disease. We thus pursued an alternative strategy to convert immunologically cold ER + breast cancers into more highly inflammatory tumors capable of improved responsiveness to checkpoint blockade. CD40, a member of the tumor necrosis factor receptor superfamily expressed predominantly on antigen-presenting cells (APCs) 19 , represents a promising immunotherapeutic target 20 . Activation of CD40 through agonistic antibodies initiates a cascade of immunological events critical for effective anti-tumor immunity 21 . Specifically, CD40 agonism promotes dendritic cell (DC) maturation, enhances antigen presentation, upregulates costimulatory molecules, and facilitates cross-presentation of tumor-associated antigens 22 – 27 . CD40 agonists have also demonstrated synergistic potential when administered with immune checkpoint inhibitors 28 , 29 and have been found to have acceptable safety profiles with evidence of clinical activity in human clinical trials 30 – 33 . Agonistic CD40 therapy has the potential to bridge the gap between innate and adaptive immunity by enabling robust CD8 + T cell priming and activation, potentially overcoming the immunosuppressive barriers characteristic of ER + breast cancers. Our present study evaluates the ability of aCD40 to improve anti-tumor immunity via DC activation and subsequent T cell priming and activation and to synergize with checkpoint blockade in a syngeneic mouse model of ER + breast cancer. Our findings suggest agonistic CD40 therapy may be a viable strategy for immune activation and long-lasting immunity in ER + breast cancer. Results aCD40 enhances ERα + mammary tumor sensitivity to immune checkpoint blockade TNBC and ERα + breast cancer differ clinically in their response to chemotherapy and immunotherapy, with hormone receptor-positive cancers being more resistant to ICB 11 , 12 . It is well established that tumor mutational burden and T-cell infiltration can predict the response to ICB treatment, with preliminary evidence suggesting this is also relevant in breast cancer 13 – 15 , 37 . To understand the potential underlying resistance mechanisms of ERα + tumors to ICB, we aimed to compare the mutational burden and tumor immune environment of ERα + Brpkp110 and TNBC E0771 mammary tumor cell lines. In vivo , Brpkp110 tumors express ERα by flow cytometry (Suppl. Figure 1A) and IHC (Suppl. Figure 1B left) and are responsive to estradiol supplementation (Suppl. Figure 1C). As previously described 35 , E0771 tumors lack ERα expression in vivo (Suppl. Figure 1B right). Next, using an in-house developed algorithm called antigen.garnish 36 , we determined the number of single nucleotide and complex variants and predicted MHCI and MHCII neoantigens for both tumor cell lines against wild-type C57BL/6J. The two tumor cell lines displayed comparable levels of variants and predicted neoantigens (Suppl. Figure 1D), but exhibited differences in the tumor immune environment when implanted orthotopically into the fat pads of congenic mice. Consistent with human breast cancer, ERα + Brpkp110 tumors are more immunologically “cold” compared to triple-negative E0771 tumors, with decreased intratumoral T cell infiltration and increased frequency of tumor-associated macrophages (TAM) (Fig. 1A-B). Both the abundance of CD8 + T cells and the proportion of Granzyme B + CD8 + cytotoxic T cells (CTL) were lower in the ERα + Brpkp110 tumors compared to TNBC. ERα + Brpkp110 tumors also had significantly decreased numbers of type 1 and type 2 conventional dendritic cells (cDC1 and cDC2) suggesting a weakened capacity for T cell priming considered critical for anti-tumor immunity (Fig. 1C). Although TNBC E0771 tumors had a more highly activated immune environment, the tumors continued to demonstrate more aggressive tumor characteristics, including higher proportions of Ki67 + proliferating EpCam + tumor cells (Fig. 1D). As expected, when implanted orthotopically, untreated E0771 grew faster acquiring almost 3-fold larger volumes within the same period as ERα + Brpkp110 tumors (Fig. 1E-F). Upon treatment with ICB (αCTLA-4 and αPD-1), 70% of E0771 tumors regressed, and 53% of all treated mice were cured by day 29 post-implantation (Fig. 1E). In contrast, only 22% of ICB-treated Brpkp110 tumors regressed, and none of the treated hosts were cured (Fig. 1F), recapitulating the ICB-resistant phenotype of ERα + human tumors. The very low cDC content in ERα + tumors may result in poor T cell priming, activation, and recruitment, rendering ICB treatment an insufficient mechanism for reversal of tumor-associated immunosuppression. When treated with aCD40, the growth of Brpkp110 tumors was suppressed across the group with 37% tumor regressions. In contrast, the response of E0771 tumors to aCD40 was very modest, with no regressions. However, combining aCD40 and ICB cured 100% of E0771 and 63% of Brpkp110 tumor-bearing mice. Collectively, these data demonstrate that Brpkp110 can be utilized as a model to study the immunobiology of ERα + breast cancer and that aCD40 can overcome the obstacles underlying the ERα + tumor resistance to ICB. aCD40 increases tumor-infiltrating cytotoxic T-cells To understand how aCD40 changes the TME and makes ERα + tumors more responsive to ICB, the Brpkp110 tumor-bearing mice were treated with aCD40 only. Interestingly, aCD40 alone cured 25% of treated mice (Fig. 2A). The TME analysis 7 days after a single dose of aCD40 revealed fewer EpCam + tumor cells and a lower proportion of Ki67 + proliferating tumor cells in treated tumors (Fig. 2B). Tumor cell apoptosis was also increased in treated tumors as evidenced by increased staining for cleaved caspase 3 (Fig. 2C). These changes in tumor cell population are unlikely to be a direct effect of aCD40 on tumor cells as only 1% of them expressed CD40 in vitro (Suppl. Figure 2A). In contrast about one-third of total cDCs, cDC1s, and cDC2s in untreated Brpkp110 tumors were CD40+ (Suppl. Figure 2B). Interestingly, there were no changes in total cDC and cDC2 populations between control and treated groups but there was a significant decrease in cDC1 population on treatment day 7 (Fig. 2D). The time course of the changes in CD103 + migratory subpopulation of cDC1s showed that already on day 2 and 5 of aCD40 treatment, there was a significant decrease in the proportion of these cells in the tumor with a concomitant increase of the same cell population in tumor draining lymph node (TDLN) on days 5 and 7 (Fig. 2E). On day 13 post-treatment, the treated tumors had a higher percentage of CD3 + and CD8 + T cells and a trend towards a higher percentage of CD4 + T cells compared to the controls (Fig. 2F). In addition to an increase in activated CD8 + T cells, aCD40 treated tumors demonstrated decreased proportions of immunosuppressive CD4 + Foxp3 + regulatory T cells (Fig. 2G). CD4 + and CD8 + T cells appeared more activated in aCD40 treated tumors as the proportion of Granzyme B + cells was higher in these populations (Fig. 2H). We confirmed the increase in CD8 + T cells in treated tumors by IF staining (Fig. 2I). In addition to increasing the number of tumor-infiltrating CD8 + T cells, aCD40 prompted their translocation from the tumor periphery to the center of the tumor (Fig. 2J). The increase in T cells in the TME after aCD40 treatment was possibly due to the increase in the production of chemokines such as CCL5, CCL21, CXCL9, and CXCL10 within the tumor (Fig. 2K). Thus, aCD40 treatment reshaped the TME of ERα + Brpkp110 tumors by prompting DC migration to TDLN and subsequent T cell priming, activation, and recruitment into the tumor. cDC1s and T cells are critical for the anti-tumor activity of aCD40 aCD40 treatment induced maturation and activation of cDC1s and cDC2s in the lymph nodes, with elevated expression of CD40, CD80, and CD86 in TDLN infiltrating cDCs two days after aCD40 administration (Fig. 3A). At the same time point, CD4 + and CD8 + cells in TDLN appeared to be more activated after aCD40 treatment and exhibited higher Granzyme B expression and higher proportions of PD-1 and CD44 expressing cells (Fig. 3B). In addition, cell proliferation as measured by Ki67 was increased in CD4 + and CD8 + T cells after aCD40 treatment (Fig. 3B). To assess the dependency on T cells for the therapeutic effect of aCD40 in ERα + tumors, tumor-bearing control and treated mice were depleted of CD8 + or CD4 + or both CD4 + and CD8 + T cells (Suppl. Figure 3A). The tumor suppressive effect of aCD40 was abolished in the absence of CD8 + or CD8 + and CD4 + T cells, but was unaffected in hosts that were depleted of only CD4 + T cells (Fig. 3C). Similarly, there was a significant impact on tumor growth suppression after aCD40 when tumors were implanted into Batf3 knock out (KO) mice that lack cross-presenting cDC1s (Suppl. Figure 3B and Fig. 3D). These data indicate that aCD40 tumor-suppressive effect heavily relies on the presence and function of cDC1 and CD8 + cytotoxic T cells (CTL) and may be able to substitute for CD4 + help. Combination aCD40 and ICB treatment results in a vaccine effect and allows for subsequent rejection of rechallenge tumors The influx of T cells into the TME and their activation after aCD40 treatment inevitably leads to T cell exhaustion, potentially increasing sensitivity to ICB. aCD40 alone suppressed the growth of ERα + tumors; however, only one-fourth of tumors had complete regressions (Fig. 2A), while the combination of aCD40 and ICB increased the complete response rate up to 83% (Fig. 1F, Fig. 4A-D). Interestingly, the effect of the combination therapy was only partially reversed by depletion of only CD8 + or CD4 + T cells (Fig. 4A and B, respectively), whereas combined CD8 + and CD4 + T cell depletion completely abolished the effect of aCD40 + ICB treatment (Fig. 4C). Tumors implanted in Batf3 KO hosts were resistant to combination therapy (Fig. 4D). The effect of combination immunotherapy on T cells persisted beyond the onset of tumor clearance. Two months after complete tumor regressions and cessation of treatment, the proportion of CD44 + CD62L- effector memory and CD44 + CD62L + central memory CD4 + and CD8 + T cells were higher in the blood of cured mice compared to treatment-naïve mice implanted with tumors (Fig. 4E). At the same time, cured mice had fewer circulating CD44-CD62L- naïve T cells compared to treatment-naïve mice (Fig. 4E). The presence of effector and central memory T cells in cured mice is consistent with a vaccine effect and continued anti-tumor immunosurveillance and immunologic memory. To test the robustness of this immunologic memory, cured mice were rechallenged with the same burden of ERα + Brpkp110 tumor cells. The cells grew in control, tumor naïve mice but were universally rejected in cured mice (Fig. 4F). The rejection of tumors upon secondary rechallenge was only CD4 + T cell-dependent and not CD8 + T cell dependent, as the cured hosts depleted of only CD8 + T cells were able to reject a secondary tumor rechallenge similar to the cured, T cell sufficient hosts (Fig. 4G). Intratumoral administration of aCD40 + ICB suppresses the growth of treated and distant tumors Intratumoral (IT) administration of ICB and aCD40 have individually been shown to be efficacious in human clinical trials without significant systemic absorption 38 – 40 . Here we tested the efficacy of IT aCD40 alone or in combination with IT ICB in double-flanked Brpkp110 tumor-bearing mice. IT aCD40 suppressed the growth of both the treated ipsilateral tumor as well as the distant contralateral tumor (Fig. 4H). Next, various combinations of IT and intraperitoneal (IP) administrations of aCD40 and ICB were tested in double-flanked hosts. IT aCD40 and ICB were able to suppress both the treated and distant tumors, similar to the effect observed in IP treatments (Fig. 4I), indicating that local administration of aCD40 + ICB results in an abscopal effect on distant tumors. Discussion In the current study, we sought to elucidate the mechanisms underlying ER + tumor resistance to ICB by comparing immunological profiles of ER + Brpkp110 and TNBC E0771 murine mammary tumors. Differences were observed in their immune infiltration profiles despite similar mutational burdens and neoantigen loads between these tumor models. TNBC E0771 tumors exhibited a T cell-inflamed, "hot" TME characterized by robust CD8 + T cell infiltration and higher proportions of Granzyme B + cytotoxic T lymphocytes (CTLs), alongside greater densities of dendritic cells. In contrast, ER + Brpkp110 tumors displayed an immunologically "cold" TME with minimal lymphocytic infiltration. These distinctions in immune contexture, rather than mutational burden, correlated with differential ICB responsiveness. These findings suggest that immune resistance in ER + tumors stems at least in part from deficient T cell priming, activation, and recruitment, rather than acquired dysfunction of activated T cells within the TME. This hypothesis provided the rationale for targeting the CD40 pathway in order to enhance dendritic cell maturation and antigen presentation 19 , 23 . In various immunologically "cold" tumor models, including pancreatic adenocarcinoma and glioblastoma, agonistic CD40 antibodies (aCD40) have successfully activated cDC1s, improved T cell priming, and enhanced anti-tumor immunity 21 , 41 . Consistent with our mechanistic hypothesis, we found that aCD40 monotherapy exhibited robust tumor-suppressive effects in ER + tumors compared to TNBC. The combination of aCD40 and ICB (αPD1 and αCTLA-4), addressing both pre- and post-T cell activation defects, resulted in complete tumor clearance in the majority of TNBC and ER + tumor-bearing mice, representing unprecedented efficacy in an ER + murine breast cancer model. Our temporal analysis of aCD40 effects revealed a sequential cascade of immunological events. Following a single dose of aCD40, we observed a significant reduction in intratumoral CD103 + migratory DCs by days 5–7 post-treatment, concurrent with increased accumulation of mature, activated cDC1 and cDC2 in tumor-draining lymph nodes (TDLNs), accompanied by robust proliferation and activation of CD4 + and CD8 + T cells. These findings parallel prior observations in pancreatic cancer models 22 and highlight aCD40’s ability to increase DC mobilization into the TDLN. The immunomodulatory effects of aCD40 also result in the induction of a pro-inflammatory chemokine milieu. aCD40 treatment significantly increased tumoral expression of CCL5, potentially contributing to the recruitment of Granzyme B + CD4 + T cells from TDLNs to the tumor bed, as previously reported in pancreatic cancer models 42 . Concomitantly, we observed a marked reduction in Foxp3 + regulatory T cells within treated tumors, suggesting a shift in the CD4 + T cell population from an immunosuppressive to an immunostimulatory phenotype. Additionally, aCD40 enhanced tumoral production of CXCL9 and CXCL10, potent T cell chemoattractants that likely facilitated T cell trafficking from the periphery to the tumor microenvironment. An intriguing observation was the aCD40-induced upregulation of CCL21 within treated tumors. CCL21, traditionally produced by lymphatic endothelial cells and lymph node stromal cells, acts as a chemotactic signal for CCR7-expressing DCs to traffic to lymph nodes 43 . While CD103 + CCR7 + cDC1s represent the canonical antigen-trafficking migratory DCs responsible for CD8 + T cell priming 44 , recent evidence indicates that a subset of CCR7 + immature DCs that downregulate antigen presentation machinery and upregulate PD-L1 can undermine T cell priming in lymph nodes 45 . The increased CCL21 production within aCD40-treated tumors may serve to retain these regulatory DCs within the tumor microenvironment, thereby facilitating more effective T cell priming in TDLNs. Simultaneously, the presence of PD-L1-expressing DCs within tumors could impair T cell effector function, potentially explaining why ICB complements aCD40 in achieving maximal therapeutic efficacy. However, further investigation is warranted to fully elucidate the functional significance of enhanced CCL21 expression in this context. Through selective depletion studies, we established that initial therapeutic tumor regression following aCD40 monotherapy critically depends on CD8 + T cells but not CD4 + T cells, similar to prior data demonstrating that aCD40 can substitute for CD4 + T cell help 46 . The absence of Batf3-dependent cross-presenting cDC1s also abolished a significant proportion of the aCD40 anti-tumor effect, underscoring the essential role of cDC1s in initiating CD8 + T cell-mediated tumor immunity. In contrast, the efficacy of combined aCD40 and ICB treatment required both CD4 + and CD8 + T cells along with cDC1s for primary tumor rejection, highlighting the cooperative nature of these immune populations in mediating maximal therapeutic responses in the context of combination immunotherapy. The durable nature of the anti-tumor immunity was evidenced by elevated proportions of circulating effector and central memory T cells two months after tumor clearance, as well as the ability of cured mice to reject secondary tumor rechallenge, confirming the establishment of immunological memory via a vaccine effect. In contrast, while primary tumor rejection required both CD4 + and CD8 + T cells, rechallenge tumor clearance demonstrated dependency on CD4 + T cells alone. This unexpected finding suggests that in the context of established immune memory, CD4 + T cells may either acquire direct cytotoxic functionality or orchestrate anti-tumor responses through cell populations other than CD8 + T cells. This observation warrants further investigation to delineate the precise mechanisms by which CD4 + T cells mediate tumor rejection in the memory phase. Finally, our demonstration that intratumoral administration of aCD40, either alone or in combination with ICB, recapitulates the effects of systemic treatment has significant clinical implications. This finding indicates that localized immunomodulation within the primary tumor and associated TDLNs is sufficient to induce regression of distant lesions, potentially through the systemic dissemination of tumor-specific T cells. This approach could mitigate immune-related adverse events associated with systemic administration of immunotherapeutics, while maintaining therapeutic efficacy. Moreover, our data suggest that TDLNs play a crucial role in controlling distant metastatic lesions and preventing recurrence, arguing for the potential conservation of TDLNs during surgical resection to maximize the endogenous vaccine effect of the primary tumor. In summary, we demonstrate for the first time complete reversal of ICB resistance in ER + breast cancer through CD40 agonism. By converting immunologically "cold" ER + tumors into responsive entities through the activation of dendritic cells and enhancement of T cell priming, our findings provide a strong rationale for clinical investigation of combined aCD40 and ICB in ER + breast cancers. This approach may dramatically expand the proportion of breast cancer patients who can benefit from immunotherapy, potentially addressing a critical unmet need in the management of the most common breast cancer subtype. Furthermore, the mechanistic insights gleaned from this study may inform combination immunotherapy strategies across other immunologically "cold" tumor types characterized by deficient T cell priming and recruitment. Methods Cell lines Brpkp110 is an estrogen receptor positive (ER+), progesterone receptor positive (PR+) and human epidermal growth receptor 2 negative (HER2-) mammary cell line derived from a tumor induced in the mammary gland of a KRas G12D−LSL/wt ; p53 flx/flx ; myr-p110⍺ wt/fl mouse (gift from Jose Conejo Garcia, Duke University) which leads to constitutively activated PI3K signaling 34 . Brpkp110 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 0.05% sodium pyruvate, and 3.3uL 2-mercaptoethanol. E0771 is a widely-used ERα- cell line 35 and was cultured in DMEM, 10% FBS, 1% glutamine, and 0.2% gentamicin. Mice and treatments Age- and sex-matched 7- to 18-week-old C57BL/6 mice (Jackson Laboratory, cat# 00664) and B6.129S(C)- Batf3 tm1Kmm /J (Jackson Laboratory, cat# 013755) were housed in specific pathogen-free conditions and treated as per an approved Institutional Animal Care and Use Committee protocol at the University of Pennsylvania (Protocol #804666). Tumors in C57BL/6 and B6.129S(C)- Batf3 tm1Kmm /J mice were established orthotopically in paired bilateral abdominal mammary glands 9 and 4 via injection of 5e5 cells of Brpkp110 or E0771 cell lines. The treatment antibodies listed below were obtained from Bio X Cell unless otherwise indicated. Tumor-bearing C57BL/6 or B6.129S(C)- Batf3 tm1Kmm /J mice received one dose of an agonistic CD40 antibody (clone FGK4.5, 100 µg intraperitoneal (i.p.) or intratumoral (i.t.)) or rat IgG2a (clone 2A3, 100 µg i.p. or i.t.) on day 0 as previously described 28 . Anti-CTLA-4 (clone 9H10, 200 µg i.p. or i.t.) or rat IgG2b (clone LTF-2, 200 µg i.p. or i.t.) were given on day 0, 4, 7 28 . Anti-PD-1 (clone RMPI-14, 200 µg i.p. or i.t.) or rat IgG2b (clone LTF-2, 200 µg i.p. or i.t.) were given on day 0 and every 3 to 4 days thereafter 28 . Anti-CD4 (clone GK1.5, 200 µg i.p.) or rat IgG2b (clone LTF-2, 200 µg i.p.) was given on day − 4, day 0, and every 3 to 4 days thereafter for CD4 T-cell depletion. Anti-CD8 (clone 2.43, 200 µg i.p.) or rat IgG2b (clone LTF-2, 200 µg i.p.) was given on day − 4, day 0, and every 3 to 4 days thereafter for CD8 T-cell depletion. Tissue processing and flow cytometry Mice were sacrificed according to IACUC approved protocols and tumors and tumor-draining lymph nodes (TDLN) were collected. Tumors were digested in 1 mg/mL collagenase with protease inhibitor (MilliporeSigma) and filtered through a 70 µm cell filter. TDLNs were mechanically dissociated and filtered using 100 µm filters. Cells were resuspended in PBS and incubated in Fc block (Biolegend 3527448) for 5 minutes on ice prior to staining for flow cytometry. The live/dead stain Zombie UV was used. Cells were stained with Zombie UV live/dead stain and for cell surface markers at 4°C in the dark for 30 minutes. Cell suspensions were then fixed and permeabilized for 30 minutes (eBioscience Intracellular Fixation & Permeabilization Buffer Set, Invitrogen) and overnight staining was performed for intracellular targets. Conjugated antibodies used for flow cytometry were obtained from BD Biosciences [PD-L1 (clone MIH5), CD4 (clones GK1.5, RM4-5), CD103 (clone M290), CD80 (clone 16-10A1), FoxP3 (clone R16-715)], Biolegend [EpCam (clone G8.8), CD45 (clone 30-F11), Ki67 (clone 16A8), Granzyme B (clone AQA16A02), CD8 (clone 53 − 6.7), CD3 (clone 145-2C11), CD40 (clone 3/23), CD86 (clone G2-1), PD-1 (clone 29F.1A12), CD19 (clone 6D5), B220 (clone RA3-6B2), CD11c (clone N418), NK1.1 (clone PK136), Gr-1 (clone RB6-8C5), XCR1 (clone ZET), CD11b (clone M1/70), CD64 (clone X54-5/7.1), SIRPα (clone P84), CD44 (clone IM7), CD62L (clone MEL-14)], and Abcam [ERα (clone E115). Flow cytometry was performed using an LSRFortessa or FACSymphony A3 Cell Analyzer and data were analyzed using FlowJo v10.8 software (BD Biosciences). Immunohistochemistry and immunofluorescence Tumor tissues were fixed in Zinc formalin for 24 hours and embedded in paraffin. Immunohistochemistry (IHC) for ERα+ (clone E115, Abcam 32063, 1:9000) and cleaved caspase-3 (Cell Signaling Technology 9661, 1:500) was performed by the Comparative Pathology Core of the University of Pennsylvania School of Veterinary Medicine. Immunofluoresence (IF) for CD8 and DAPI (Thermo Scientific D21490,1:1000) was performed according to the Immunofluorescent Staining of Paraffin-embedded Tissue Protocol from Novus Biologicals, with certain modifications as follows. Antigen retrieval was performed using the IHC-Tek Epitope Retrieval Solution (IHC World 1W-1100), with slides incubated for 45 minutes in an IHC World steamer at 95-98°C. Tissue sections were blocked with 5% donkey serum diluted in PBS with 0.3% Triton X-100 and stained with rabbit anti–mouse primary CD8 (clone: D4W2Z, Cell Signaling Technology 98941, 1:100) followed by donkey anti-rabbit Alexa Fluor 594 (Thermo Scientific A-21207, 1:250) secondary antibody. Slide scanning was performed on Aperio Versa 8 slide scanner at the Molecular Pathology and Imaging Core facility at the University of Pennsylvania. Data were analyzed using QuPath. In short, QuPath’s wand function was utilized to draw a border around the DAPI-stained tissue area. Then, the “Thresholder” function was utilized to gate for positive antibody-staining, in both IF and IHC, which could be visualized in real-time. Once an appropriate staining threshold value was determined for positive-staining gating it was applied to whole tissue sections. QuPath quantified percent positive area for each antibody was used for analysis. Cytokine analysis Brpkp110 tumor-bearing mice underwent treatment as described in results. Mice were euthanized and tumors were collected at day 7 post treatment. Tumors were minced and incubated in media for 48 hours. Supernatant was collected and pooled per treatment group and cytokine array was performed with technical duplicates via the proteome profiler (R&D, catalog # ARY028). Whole exome sequencing Genomic DNA was extracted from Brpkp110 and E0771 cell pellets and livers of wild-type C57BL/6 mice. Whole exome sequencing was performed on the Illumina platform (2x150bp) by Azenta Life Sciences. Neoantigen prediction was performed via antigen.garnish 36 . Briefly, we applied antigen garnish to identify single nucleotide variants (SNVs) and SNV-induced protein alterations and ran mutated peptides (8mers to 14mers) through a series of MHC-binding affinity platforms (netMHCI/netMHCII/netMHCIpan/netMHCIIpan). Identified peptides with predicted binding affinities less than 500 nM were prioritized as strong binders. Statistical analysis Tumor growth curves were analyzed using two-way ANOVA with Tukey multiple comparisons of means to compare differences between two individual groups. A two-tailed Student t test was used to analyze differences between two groups. One-way ANOVA with the Bonferonni multiple comparison test was used to assess differences between any two individual groups. Statistical analyses were performed using GraphPad Prism 10. P ≤ 0.05 was considered statistically significant. Declarations Competing Interests Dr. Vonderheide has received consulting fees from BMS, EMD Serono, Grey Wolf Therapeutics and Crossbow Therapeutics, research funding from Revolution Medicines, is an inventor on patents relating to cancer cellular immunotherapy, cancer vaccines, and KRAS immune epitopes, and receives royalties from Children’s Hospital Boston for a licensed research-only monoclonal antibody, but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests. Funding Early-stage surgeon scientist program to JQZ (P30CA016520-46), Breast cancer research foundation to RHV (BCRF-24-167), University of Pennsylvania Department of Surgery Pilot Grant to JQZ, Abramson Cancer Center Pilot Grant to JQZ, Thomas B. and Jeannette E. Laws McCabe Fund to JQZ (Funders played no role in study design, data collection, analysis, and interpretation of data, or writing of this manuscript.) Author Contribution JQZ, CL, OL, VM, NJ, CRF performed experiments, designed methodology, and performed data analysis. NJ performed computational analysis. JQZ, NM, SZ, RPD, RHV were involved in conceptualization and manuscript preparation. All authors were involved in data interpretation, manuscript review and editing. Acknowledgement Acknowledgments to the University of Pennsylvania core services: Penn Vet comparative pathology Core, Molecular pathology and imaging core (Center for Molecular Studies in Digestive and Liver Diseases, P30DK050306), and Penn Cytomics Core Data Availability The datasets generated and/or analyzed during the current study are available in the Sequence Read Archive (SRA) repository accession code SUB15367120. Code availability The underlying code used for this study is available at the link below: https://github.com/andrewrech/antigen.garnish References Siegel, R. L., Giaquinto, A. N. & Jemal, A. Cancer statistics, 2024. CA A Cancer J Clinicians 74, 12–49 (2024). Howlader, N. et al. US Incidence of Breast Cancer Subtypes Defined by Joint Hormone Receptor and HER2 Status. JNCI: Journal of the National Cancer Institute 106, (2014). Pan, H. et al. 20-Year Risks of Breast-Cancer Recurrence after Stopping Endocrine Therapy at 5 Years. N Engl J Med 377, 1836–1846 (2017). 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Avelumab, an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: a phase 1b JAVELIN Solid Tumor study. Breast Cancer Res Treat 167, 671–686 (2018). Rugo, H. S. et al. Safety and Antitumor Activity of Pembrolizumab in Patients with Estrogen Receptor–Positive/Human Epidermal Growth Factor Receptor 2–Negative Advanced Breast Cancer. Clinical Cancer Research 24, 2804–2811 (2018). Loi, S. et al. Prognostic and Predictive Value of Tumor-Infiltrating Lymphocytes in a Phase III Randomized Adjuvant Breast Cancer Trial in Node-Positive Breast Cancer Comparing the Addition of Docetaxel to Doxorubicin With Doxorubicin-Based Chemotherapy: BIG 02–98. JCO 31, 860–867 (2013). Denkert, C. et al. Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy. The Lancet Oncology 19, 40–50 (2018). Samstein, R. M. et al. Tumor mutational load predicts survival after immunotherapy across multiple cancer types. Nat Genet 51, 202–206 (2019). Schmid, P. et al. Atezolizumab plus nab-paclitaxel as first-line treatment for unresectable, locally advanced or metastatic triple-negative breast cancer (IMpassion130): updated efficacy results from a randomised, double-blind, placebo-controlled, phase 3 trial. The Lancet Oncology 21, 44–59 (2020). Cortes, J. et al. Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer (KEYNOTE-355): a randomised, placebo-controlled, double-blind, phase 3 clinical trial. The Lancet 396, 1817–1828 (2020). Schmid, P. et al. Pembrolizumab for Early Triple-Negative Breast Cancer. N Engl J Med 382, 810–821 (2020). Elgueta, R. et al. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunological Reviews 229, 152–172 (2009). Vonderheide, R. H. CD40 Agonist Antibodies in Cancer Immunotherapy. Annu. Rev. Med. 71, 47–58 (2020). Vonderheide, R. H. CD40 Agonist Antibodies in Cancer Immunotherapy. Annu. Rev. Med. 71, 47–58 (2020). Lin, J. H. et al. Type 1 conventional dendritic cells are systemically dysregulated early in pancreatic carcinogenesis. Journal of Experimental Medicine 217, e20190673 (2020). Ma, D. Y. & Clark, E. A. The role of CD40 and CD154/CD40L in dendritic cells. Seminars in Immunology 21, 265–272 (2009). Van Kooten, C. & Banchereau, J. CD40-CD40 ligand. Journal of Leukocyte Biology 67, 2–17 (2000). Zhang, J. Q. et al. Macrophages and CD8 + T Cells Mediate the Antitumor Efficacy of Combined CD40 Ligation and Imatinib Therapy in Gastrointestinal Stromal Tumors. Cancer Immunology Research 6, 434–447 (2018). Schoenberger, S. P., Toes, R. E. M., Van Der Voort, E. I. H., Offringa, R. & Melief, C. J. M. T-cell help for cytotoxic T lymphocytes is mediated by CD40–CD40L interactions. Nature 393, 480–483 (1998). Bennett, S. R. M. et al. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393, 478–480 (1998). Morrison, A. H., Diamond, M. S., Hay, C. A., Byrne, K. T. & Vonderheide, R. H. Sufficiency of CD40 activation and immune checkpoint blockade for T cell priming and tumor immunity. Proc. Natl. Acad. Sci. U.S.A. 117, 8022–8031 (2020). Winograd, R. et al. Induction of T-cell Immunity Overcomes Complete Resistance to PD-1 and CTLA-4 Blockade and Improves Survival in Pancreatic Carcinoma. Cancer Immunology Research 3, 399–411 (2015). Bajor, D. L. et al. Long-term outcomes of a phase I study of agonist CD40 antibody and CTLA-4 blockade in patients with metastatic melanoma. OncoImmunology 7, e1468956 (2018). Padrón, L. J. et al. Sotigalimab and/or nivolumab with chemotherapy in first-line metastatic pancreatic cancer: clinical and immunologic analyses from the randomized phase 2 PRINCE trial. Nat Med 28, 1167–1177 (2022). O’Hara, M. H. et al. CD40 agonistic monoclonal antibody APX005M (sotigalimab) and chemotherapy, with or without nivolumab, for the treatment of metastatic pancreatic adenocarcinoma: an open-label, multicentre, phase 1b study. The Lancet Oncology 22, 118–131 (2021). Van Laethem, J.-L. et al. Combining CD40 agonist mitazalimab with mFOLFIRINOX in previously untreated metastatic pancreatic ductal adenocarcinoma (OPTIMIZE-1): a single-arm, multicentre phase 1b/2 study. The Lancet Oncology 25, 853–864 (2024). Sheen, M. R. et al. Constitutively activated PI3K accelerates tumor initiation and modifies histopathology of breast cancer. Oncogenesis 5, e267–e267 (2016). Le Naour, A., Rossary, A. & Vasson, M. EO771, is it a well-characterized cell line for mouse mammary cancer model? Limit and uncertainty. Cancer Medicine 9, 8074–8085 (2020). Richman, L. P., Vonderheide, R. H. & Rech, A. J. Neoantigen Dissimilarity to the Self-Proteome Predicts Immunogenicity and Response to Immune Checkpoint Blockade. Cell Systems 9, 375–382.e4 (2019). Barroso-Sousa, R. et al. Tumor Mutational Burden and PTEN Alterations as Molecular Correlates of Response to PD-1/L1 Blockade in Metastatic Triple-Negative Breast Cancer. Clinical Cancer Research 26, 2565–2572 (2020). Omland, S. H. et al. Feasibility of Intratumoral Anti-PD1 as Treatment of Human Basal Cell Carcinoma: An Explorative Study with Adjuvant Ablative Fractional Laser. Cancers 14, 5815 (2022). Khalil, D. N. et al. In situ vaccination with defined factors overcomes T cell exhaustion in distant tumors. Journal of Clinical Investigation 129, 3435–3447 (2019). Irenaeus, S. M. M. et al. First-in‐human study with intratumoral administration of a CD40 agonistic antibody, ADC‐1013, in advanced solid malignancies. Intl Journal of Cancer 145, 1189–1199 (2019). Beatty, G. L., Li, Y. & Long, K. B. Cancer immunotherapy: activating innate and adaptive immunity through CD40 agonists. Expert Review of Anticancer Therapy 17, 175–186 (2017). Huffman, A. P., Lin, J. H., Kim, S. I., Byrne, K. T. & Vonderheide, R. H. CCL5 mediates CD40-driven CD4 + T cell tumor infiltration and immunity. JCI Insight 5, e137263 (2020). Comerford, I. et al. A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system. Cytokine & Growth Factor Reviews 24, 269–283 (2013). Roberts, E. W. et al. Critical Role for CD103+/CD141 + Dendritic Cells Bearing CCR7 for Tumor Antigen Trafficking and Priming of T Cell Immunity in Melanoma. Cancer Cell 30, 324–336 (2016). Lee, C. Y. C. et al. Tumour-retained activated CCR7 + dendritic cells are heterogeneous and regulate local anti-tumour cytolytic activity. Nat Commun 15, 682 (2024). French, R. R., Chan, H. T. C., Tutt, A. L. & Glennie, M. J. CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med 5, 548–553 (1999). Additional Declarations Competing interest reported. Dr. Vonderheide has received consulting fees from BMS, EMD Serono, Grey Wolf Therapeutics and Crossbow Therapeutics, research funding from Revolution Medicines, is an inventor on patents relating to cancer cellular immunotherapy, cancer vaccines, and KRAS immune epitopes, and receives royalties from Children’s Hospital Boston for a licensed research-only monoclonal antibody, but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests. Supplementary Files 060425FiguresforNPJBCSupp.pdf Cite Share Download PDF Status: Published Journal Publication published 13 Jan, 2026 Read the published version in npj Breast Cancer → Version 1 posted Editorial decision: Revision requested 13 Jul, 2025 Reviews received at journal 06 Jul, 2025 Reviews received at journal 27 Jun, 2025 Reviewers agreed at journal 12 Jun, 2025 Reviewers agreed at journal 11 Jun, 2025 Reviewers invited by journal 09 Jun, 2025 Editor assigned by journal 08 Jun, 2025 Submission checks completed at journal 06 Jun, 2025 First submitted to journal 04 Jun, 2025 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. 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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-6823527","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":470343480,"identity":"a48607cf-06f5-4fb2-b333-012634a4e702","order_by":0,"name":"Casey Lam","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Casey","middleName":"","lastName":"Lam","suffix":""},{"id":470343481,"identity":"a144e53f-6e02-47fb-acd2-f16ee19f8743","order_by":1,"name":"Olivia Lanchoney","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Olivia","middleName":"","lastName":"Lanchoney","suffix":""},{"id":470343482,"identity":"16b04fce-b2d7-4e7c-815a-649c9f130f06","order_by":2,"name":"Vishnu Maddipatla","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Vishnu","middleName":"","lastName":"Maddipatla","suffix":""},{"id":470343483,"identity":"975ad83e-be67-4848-8f94-c9bec53fad8d","order_by":3,"name":"Nune Markosyan","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Nune","middleName":"","lastName":"Markosyan","suffix":""},{"id":470343484,"identity":"fb200b9f-e5bf-462d-9c44-08b27392091b","order_by":4,"name":"Nikhil Joshi","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Nikhil","middleName":"","lastName":"Joshi","suffix":""},{"id":470343485,"identity":"d710e64f-7602-4dfe-ae2b-da7c1e88ec74","order_by":5,"name":"Courtney Ray Fofana","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Courtney","middleName":"Ray","lastName":"Fofana","suffix":""},{"id":470343486,"identity":"02cb640d-e262-4135-ac7d-ea673899d0bd","order_by":6,"name":"Shan Zeng","email":"","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Zeng","suffix":""},{"id":470343487,"identity":"88870f98-2525-4d08-a685-cb8a4580c8ff","order_by":7,"name":"Ronald P. 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Zhang","email":"data:image/png;base64,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","orcid":"","institution":"University of Pennsylvania","correspondingAuthor":true,"prefix":"","firstName":"Jennifer","middleName":"Q.","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-06-04 21:08:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6823527/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6823527/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41523-025-00889-7","type":"published","date":"2026-01-13T16:28:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84682251,"identity":"b95037f8-3ef6-47cd-b544-3cfadd469db6","added_by":"auto","created_at":"2025-06-16 08:35:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55264,"visible":true,"origin":"","legend":"\u003cp\u003eaCD40 increases ERα+ mammary tumor sensitivity to ICB. A-C. Proportions of indicated \u0026nbsp;cell types in the TME of Brpkp110 and E0771 tumors by flow cytometry on day 13 post implantation \u0026nbsp;(n=7-8). D. Proportions of Ki67+ proliferating EpCAM+ tumor cells in vivo measured by flow cytometry \u0026nbsp;on day 13 post implantation (n=7-8). E. Tumor volume changes compared to pretreatment on day 29 \u0026nbsp;post implantation (left) and growth curves (right) of E0771 tumor cells. Indicated treatments initiated on \u0026nbsp;day 8 post implantation (n=12-15, data representative of 2 experiments with similar results). F. Tumor \u0026nbsp;volume changes compared to pretreatment on day 26 post implantation (left) and growth curves (right) \u0026nbsp;of Brpkp110 tumor cells. Indicated treatments initiated on day 7 post implantation (n=18-20, data \u0026nbsp;representative of 3 experiments with similar results). Data: (A-D) median, (E-F, left) each column \u0026nbsp;represents individual tumor, (E-F, right) mean ± SEM. For all panels, p\u0026lt;0.05 was considered \u0026nbsp;statistically significant, and * p\u0026lt;0.05, ** p\u0026lt;0.01, ***p\u0026lt;0.001 and ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6823527/v1/8bf1da586b833fe876868620.png"},{"id":84682253,"identity":"6a05cc60-821d-4dfa-a861-5e88eb40d26e","added_by":"auto","created_at":"2025-06-16 08:35:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":347064,"visible":true,"origin":"","legend":"\u003cp\u003eaCD40 increases tumor-infiltrating cytotoxic T-cells in orthotopic ERα+ Brpkp110 tumors. A. \u0026nbsp;Tumor volume changes compared to pretreatment on day 25 post implantation (left) and growth curves (right) of \u0026nbsp;Brpkp110 tumor cells. aCD40 or vehicle treatments initiated on day 6 post implantation (n=16-18, data \u0026nbsp;representative of 3 experiments with similar results). B. Flow cytometry analysis of implanted control and aCD40 \u0026nbsp;treated Brpkp110 tumors on day 7 post treatment (n=10, data representative of 2 experiments with similar \u0026nbsp;results). C. Cleaved caspase 3 measured by IHC in control and aCD40 treated Brpkp110 tumors on day 5 post\u0002treatment (n=6-7, data representative of 2 experiments with similar results). D. DC subtype proportions \u0026nbsp;measured by flow cytometry in implanted control and aCD40 treated Brpkp110 tumors on day 7 post-treatment \u0026nbsp;(n=10, data representative of 2 experiments with similar results). E. Proportions of CD103+ cDC1s measured in \u0026nbsp;Brpkp110 tumors (left) and TDLN (right) on days 2, 5, and 7 post aCD40 administration, compared to untreated \u0026nbsp;(control) tumors (n=6-7). F. Proportions of CD3+, CD8+, and CD4+ T cells by flow cytometry in control and \u0026nbsp;aCD40 treated Brpkp110 tumors on day 13 post treatment (n=10, data representative of 2 experiments with \u0026nbsp;similar results). G. FoxP3+ CD4+ regulatory T cells on day 7 post-treatment (n=7, data representative of 2 \u0026nbsp;experiments with similar results). H. Proportions of Granzyme B+ T cells in subpopulations by flow cytometry in \u0026nbsp;control and aCD40 treated Brpkp110 tumors on day 7 post implantation (n=10, data representative of 2 \u0026nbsp;experiments with similar results). I. Images of immunofluorescent staining for CD8 (red) and nuclei (blue) and \u0026nbsp;CD8 staining quantification in untreated (control) and aCD40 treated Brpkp110 tumors on day 7 post treatment \u0026nbsp;(n=4-6). J. Quantification of CD8 immunofluorescent staining in outer, middle, and inner thirds of control and \u0026nbsp;aCD40 treated tumors on day 7 post treatment (n=5-6). K. After 7 days of treatment with aCD40, Brpkp110 \u0026nbsp;tumors were minced and cultured ex vivo. Supernatant was collected and pooled for each treatment group after \u0026nbsp;48 hours and cytokines were measured. (n=2, with 3 tumors pooled per group). Data: (A, left) each column \u0026nbsp;represents individual tumor and (A, right) mean±SEM, (B-G, H right, K) median, (I, J) mean±SEM. * p\u0026lt;0.05, ** \u0026nbsp;p\u0026lt;0.01, *** p\u0026lt; 0.001, and ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6823527/v1/9a886a5b83fb764cb735cdc4.png"},{"id":84684105,"identity":"e6ced951-2d8c-4012-82cb-494273968514","added_by":"auto","created_at":"2025-06-16 08:43:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":68107,"visible":true,"origin":"","legend":"\u003cp\u003ecDC1s and T cells are critical for the anti-tumor activity of aCD40. A. DC maturation and activation \u0026nbsp;markers measured by flow cytometry in cDC1 and cDC2 cell populations of control and aCD40 treated \u0026nbsp;Brpkp110 TDLN on day 2 post-treatment (n=8-10, data representative of 2 experiments with similar results). B. \u0026nbsp;T cell activation and proliferation markers measured by flow cytometry in CD4+ and CD8+ T cell populations of \u0026nbsp;control and aCD40 treated Brpkp110 TDLN on day 7 post-treatment (n=10, data representative of 2 experiments \u0026nbsp;with similar results). C. Growth curves of control and aCD40 treated Brpkp110 tumors implanted into WT hosts \u0026nbsp;with or without T cell depletions (n=17-20). D. Growth curves of control and aCD40 treated Brpkp110 tumors \u0026nbsp;implanted into WT and BATF3 KO hosts (n=16-20). Data: (A-B) median, (C-D) mean±SEM. * p\u0026lt;0.05, ** p\u0026lt;0.01, \u0026nbsp;*** p\u0026lt; 0.001, and ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6823527/v1/9164d821238cffd31123392f.png"},{"id":84682258,"identity":"4d6c6488-6558-4b8b-97f7-894ff113be8b","added_by":"auto","created_at":"2025-06-16 08:35:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":93519,"visible":true,"origin":"","legend":"\u003cp\u003eaCD40 and ICB results in a vaccine effect and rejection of a secondary tumor rechallenge. A. \u0026nbsp;Brpkp110 tumor growth curves (left) and volume changes compared to pretreatment (right) on day 27 post \u0026nbsp;implantation in control and aCD40+ICB treated hosts with and without CD8+ T cell depletions. Indicated \u0026nbsp;treatments initiated on day 7 post implantation (n=12-15). B. Brpkp110 tumor growth curves (left) and volume \u0026nbsp;changes compared to pretreatment (right) on day 25 post implantation in control and aCD40+ICB treated hosts \u0026nbsp;with and without CD4+ T cell depletions. Indicated treatments initiated on day 8 post implantation (n=14-18). C. \u0026nbsp;Brpkp110 tumor growth curves (left) and volume changes compared to pretreatment (right) on day 27 post \u0026nbsp;implantation in control and aCD40+ICB treated hosts with and without CD4+ and CD8+ T cell depletions. \u0026nbsp;Indicated treatments initiated on day 7 post implantation (n=12-18). D. Tumor growth curves (left) and volume \u0026nbsp;changes compared to pretreatment (right) on day 26 post Brpkp110 tumor implantation into WT and BATF3 KO \u0026nbsp;hosts. Indicated treatments initiated on day 7 post implantation (n=14-18). E. Proportions of circulating effector \u0026nbsp;memory (CD44+CD62L-), central memory (CD44+CD62L+), and naïve (CD44-CD62L-) CD4+ (left) and CD8+ \u0026nbsp;(right) in blood, 3 months post treatment induced tumor clearance (n=5-6, data representative of 2 experiments \u0026nbsp;with similar results). F. Secondary Brpkp110 tumor rechallenge of naïve and previously Brpkp110 tumor bearing \u0026nbsp;mice cured after aCD40 + ICB, at least 2 months post primary tumor clearance (n=12-14, data representative of \u0026nbsp;3 experiments with similar results). G. Control and rechallenge tumor growth in T cell sufficient (n=6-12) and T \u0026nbsp;cell depleted hosts (n=12-14, data representative of 2 experiments with similar results). H. Brpkp110 tumor \u0026nbsp;growth curves in intra-tumoral (IT) vehicle (control) and IT aCD40 treated hosts. aCD40 administered tumors \u0026nbsp;denoted as aCD40 IT and contralateral untreated tumors denoted as CD40 IT Distant (n=9-12, data \u0026nbsp;representative of 2 experiments with similar results). I. Brpkp110 tumor growth curves in intra-tumoral (IT) \u0026nbsp;vehicle (control) and IT or intraperitoneal (IP) aCD40 or ICB received hosts (n=4-9, data representative of 2 \u0026nbsp;experiments with similar results). * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt; 0.001, and ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6823527/v1/099db26553aab63cf4532db0.png"},{"id":100614307,"identity":"530ff8d6-60b2-4c5a-94d1-fb804ade4dab","added_by":"auto","created_at":"2026-01-19 17:18:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1287027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6823527/v1/e35818eb-4b89-42c0-9304-ba092047e8f1.pdf"},{"id":84682252,"identity":"f34cace1-c7b6-411b-8984-e9d747df0a74","added_by":"auto","created_at":"2025-06-16 08:35:27","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":372377,"visible":true,"origin":"","legend":"","description":"","filename":"060425FiguresforNPJBCSupp.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6823527/v1/e9eaf5aebe73a9bf361fad80.pdf"}],"financialInterests":"Competing interest reported. Dr. Vonderheide has received consulting fees from BMS, EMD Serono, Grey Wolf Therapeutics and Crossbow Therapeutics, research funding from Revolution Medicines, is an inventor on patents relating to cancer cellular immunotherapy, cancer vaccines, and KRAS immune epitopes, and receives royalties from Children’s Hospital Boston for a licensed research-only monoclonal antibody, but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests.","formattedTitle":"\u003cp\u003eCD40 agonism enhances immune checkpoint blockade and generates immunologic memory via CD4\u003csup\u003e+\u003c/sup\u003e T cells in ERα+ mammary tumors\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer remains a leading cause of cancer-related mortality in women worldwide, with estrogen receptor positive (ER+) subtypes accounting for approximately 70% of all cases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Although many patients have a good prognosis, a significant proportion of patients will have locally advanced disease with a high risk of distant disease and death\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e or develop endocrine therapy resistance\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Unfortunately, traditional chemotherapy has poor efficacy in ER\u0026thinsp;+\u0026thinsp;subtypes with low rates of complete pathologic response\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, highlighting the urgent need for alternative therapeutic approaches. Immune checkpoint blockade (ICB), which has revolutionized treatment paradigms across multiple cancer types, has demonstrated only modest efficacy in breast cancer, with anti-PD-1/PD-L1 and anti-CTLA4 monoclonal antibodies benefiting a small subset of patients with predominantly PD-L1-positive tumors\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. ICB in ER\u0026thinsp;+\u0026thinsp;breast cancer has shown largely limited efficacy\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, primarily attributed to an immunologically \"cold\" tumor microenvironment (TME) including low density of tumor-infiltrating lymphocytes (TILs) and low tumor mutational burden\u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Conversely, checkpoint blockade has demonstrated clinical efficacy in triple-negative breast cancer (TNBC)\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, likely due to having a more activated tumor immune microenvironment compared to ER\u0026thinsp;+\u0026thinsp;disease. We thus pursued an alternative strategy to convert immunologically cold ER\u0026thinsp;+\u0026thinsp;breast cancers into more highly inflammatory tumors capable of improved responsiveness to checkpoint blockade.\u003c/p\u003e \u003cp\u003eCD40, a member of the tumor necrosis factor receptor superfamily expressed predominantly on antigen-presenting cells (APCs)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, represents a promising immunotherapeutic target\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Activation of CD40 through agonistic antibodies initiates a cascade of immunological events critical for effective anti-tumor immunity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Specifically, CD40 agonism promotes dendritic cell (DC) maturation, enhances antigen presentation, upregulates costimulatory molecules, and facilitates cross-presentation of tumor-associated antigens\u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. CD40 agonists have also demonstrated synergistic potential when administered with immune checkpoint inhibitors\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and have been found to have acceptable safety profiles with evidence of clinical activity in human clinical trials\u003csup\u003e\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Agonistic CD40 therapy has the potential to bridge the gap between innate and adaptive immunity by enabling robust CD8\u0026thinsp;+\u0026thinsp;T cell priming and activation, potentially overcoming the immunosuppressive barriers characteristic of ER\u0026thinsp;+\u0026thinsp;breast cancers. Our present study evaluates the ability of aCD40 to improve anti-tumor immunity via DC activation and subsequent T cell priming and activation and to synergize with checkpoint blockade in a syngeneic mouse model of ER\u0026thinsp;+\u0026thinsp;breast cancer. Our findings suggest agonistic CD40 therapy may be a viable strategy for immune activation and long-lasting immunity in ER\u0026thinsp;+\u0026thinsp;breast cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eaCD40 enhances ERα\u0026thinsp;+\u0026thinsp;mammary tumor sensitivity to immune checkpoint blockade\u003c/h2\u003e \u003cp\u003eTNBC and ERα\u0026thinsp;+\u0026thinsp;breast cancer differ clinically in their response to chemotherapy and immunotherapy, with hormone receptor-positive cancers being more resistant to ICB\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. It is well established that tumor mutational burden and T-cell infiltration can predict the response to ICB treatment, with preliminary evidence suggesting this is also relevant in breast cancer \u003csup\u003e\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. To understand the potential underlying resistance mechanisms of ERα\u0026thinsp;+\u0026thinsp;tumors to ICB, we aimed to compare the mutational burden and tumor immune environment of ERα\u0026thinsp;+\u0026thinsp;Brpkp110 and TNBC E0771 mammary tumor cell lines. \u003cem\u003eIn vivo\u003c/em\u003e, Brpkp110 tumors express ERα by flow cytometry (Suppl. Figure\u0026nbsp;1A) and IHC (Suppl. Figure\u0026nbsp;1B left) and are responsive to estradiol supplementation (Suppl. Figure\u0026nbsp;1C). As previously described\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, E0771 tumors lack ERα expression \u003cem\u003ein vivo\u003c/em\u003e (Suppl. Figure\u0026nbsp;1B right). Next, using an in-house developed algorithm called antigen.garnish\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, we determined the number of single nucleotide and complex variants and predicted MHCI and MHCII neoantigens for both tumor cell lines against wild-type C57BL/6J. The two tumor cell lines displayed comparable levels of variants and predicted neoantigens (Suppl. Figure\u0026nbsp;1D), but exhibited differences in the tumor immune environment when implanted orthotopically into the fat pads of congenic mice. Consistent with human breast cancer, ERα\u0026thinsp;+\u0026thinsp;Brpkp110 tumors are more immunologically \u0026ldquo;cold\u0026rdquo; compared to triple-negative E0771 tumors, with decreased intratumoral T cell infiltration and increased frequency of tumor-associated macrophages (TAM) (Fig.\u0026nbsp;1A-B). Both the abundance of CD8\u0026thinsp;+\u0026thinsp;T cells and the proportion of Granzyme B\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;cytotoxic T cells (CTL) were lower in the ERα\u0026thinsp;+\u0026thinsp;Brpkp110 tumors compared to TNBC. ERα\u0026thinsp;+\u0026thinsp;Brpkp110 tumors also had significantly decreased numbers of type 1 and type 2 conventional dendritic cells (cDC1 and cDC2) suggesting a weakened capacity for T cell priming considered critical for anti-tumor immunity (Fig.\u0026nbsp;1C). Although TNBC E0771 tumors had a more highly activated immune environment, the tumors continued to demonstrate more aggressive tumor characteristics, including higher proportions of Ki67\u0026thinsp;+\u0026thinsp;proliferating EpCam\u0026thinsp;+\u0026thinsp;tumor cells (Fig.\u0026nbsp;1D). As expected, when implanted orthotopically, untreated E0771 grew faster acquiring almost 3-fold larger volumes within the same period as ERα\u0026thinsp;+\u0026thinsp;Brpkp110 tumors (Fig.\u0026nbsp;1E-F). Upon treatment with ICB (αCTLA-4 and αPD-1), 70% of E0771 tumors regressed, and 53% of all treated mice were cured by day 29 post-implantation (Fig.\u0026nbsp;1E). In contrast, only 22% of ICB-treated Brpkp110 tumors regressed, and none of the treated hosts were cured (Fig.\u0026nbsp;1F), recapitulating the ICB-resistant phenotype of ERα\u0026thinsp;+\u0026thinsp;human tumors. The very low cDC content in ERα\u0026thinsp;+\u0026thinsp;tumors may result in poor T cell priming, activation, and recruitment, rendering ICB treatment an insufficient mechanism for reversal of tumor-associated immunosuppression. When treated with aCD40, the growth of Brpkp110 tumors was suppressed across the group with 37% tumor regressions. In contrast, the response of E0771 tumors to aCD40 was very modest, with no regressions. However, combining aCD40 and ICB cured 100% of E0771 and 63% of Brpkp110 tumor-bearing mice. Collectively, these data demonstrate that Brpkp110 can be utilized as a model to study the immunobiology of ERα\u0026thinsp;+\u0026thinsp;breast cancer and that aCD40 can overcome the obstacles underlying the ERα\u0026thinsp;+\u0026thinsp;tumor resistance to ICB.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eaCD40 increases tumor-infiltrating cytotoxic T-cells\u003c/h3\u003e\n\u003cp\u003eTo understand how aCD40 changes the TME and makes ERα\u0026thinsp;+\u0026thinsp;tumors more responsive to ICB, the Brpkp110 tumor-bearing mice were treated with aCD40 only. Interestingly, aCD40 alone cured 25% of treated mice (Fig.\u0026nbsp;2A). The TME analysis 7 days after a single dose of aCD40 revealed fewer EpCam\u0026thinsp;+\u0026thinsp;tumor cells and a lower proportion of Ki67\u0026thinsp;+\u0026thinsp;proliferating tumor cells in treated tumors (Fig.\u0026nbsp;2B). Tumor cell apoptosis was also increased in treated tumors as evidenced by increased staining for cleaved caspase 3 (Fig.\u0026nbsp;2C). These changes in tumor cell population are unlikely to be a direct effect of aCD40 on tumor cells as only 1% of them expressed CD40 \u003cem\u003ein vitro\u003c/em\u003e (Suppl. Figure\u0026nbsp;2A). In contrast about one-third of total cDCs, cDC1s, and cDC2s in untreated Brpkp110 tumors were CD40+ (Suppl. Figure\u0026nbsp;2B). Interestingly, there were no changes in total cDC and cDC2 populations between control and treated groups but there was a significant decrease in cDC1 population on treatment day 7 (Fig.\u0026nbsp;2D). The time course of the changes in CD103\u0026thinsp;+\u0026thinsp;migratory subpopulation of cDC1s showed that already on day 2 and 5 of aCD40 treatment, there was a significant decrease in the proportion of these cells in the tumor with a concomitant increase of the same cell population in tumor draining lymph node (TDLN) on days 5 and 7 (Fig.\u0026nbsp;2E). On day 13 post-treatment, the treated tumors had a higher percentage of CD3\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells and a trend towards a higher percentage of CD4\u0026thinsp;+\u0026thinsp;T cells compared to the controls (Fig.\u0026nbsp;2F). In addition to an increase in activated CD8\u0026thinsp;+\u0026thinsp;T cells, aCD40 treated tumors demonstrated decreased proportions of immunosuppressive CD4\u0026thinsp;+\u0026thinsp;Foxp3\u0026thinsp;+\u0026thinsp;regulatory T cells (Fig.\u0026nbsp;2G). CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells appeared more activated in aCD40 treated tumors as the proportion of Granzyme B\u0026thinsp;+\u0026thinsp;cells was higher in these populations (Fig.\u0026nbsp;2H). We confirmed the increase in CD8\u0026thinsp;+\u0026thinsp;T cells in treated tumors by IF staining (Fig.\u0026nbsp;2I). In addition to increasing the number of tumor-infiltrating CD8\u0026thinsp;+\u0026thinsp;T cells, aCD40 prompted their translocation from the tumor periphery to the center of the tumor (Fig.\u0026nbsp;2J). The increase in T cells in the TME after aCD40 treatment was possibly due to the increase in the production of chemokines such as CCL5, CCL21, CXCL9, and CXCL10 within the tumor (Fig.\u0026nbsp;2K). Thus, aCD40 treatment reshaped the TME of ERα\u0026thinsp;+\u0026thinsp;Brpkp110 tumors by prompting DC migration to TDLN and subsequent T cell priming, activation, and recruitment into the tumor.\u003c/p\u003e\n\u003ch3\u003ecDC1s and T cells are critical for the anti-tumor activity of aCD40\u003c/h3\u003e\n\u003cp\u003eaCD40 treatment induced maturation and activation of cDC1s and cDC2s in the lymph nodes, with elevated expression of CD40, CD80, and CD86 in TDLN infiltrating cDCs two days after aCD40 administration (Fig.\u0026nbsp;3A). At the same time point, CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;cells in TDLN appeared to be more activated after aCD40 treatment and exhibited higher Granzyme B expression and higher proportions of PD-1 and CD44 expressing cells (Fig.\u0026nbsp;3B). In addition, cell proliferation as measured by Ki67 was increased in CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells after aCD40 treatment (Fig.\u0026nbsp;3B). To assess the dependency on T cells for the therapeutic effect of aCD40 in ERα\u0026thinsp;+\u0026thinsp;tumors, tumor-bearing control and treated mice were depleted of CD8\u0026thinsp;+\u0026thinsp;or CD4\u0026thinsp;+\u0026thinsp;or both CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells (Suppl. Figure\u0026nbsp;3A). The tumor suppressive effect of aCD40 was abolished in the absence of CD8\u0026thinsp;+\u0026thinsp;or CD8\u0026thinsp;+\u0026thinsp;and CD4\u0026thinsp;+\u0026thinsp;T cells, but was unaffected in hosts that were depleted of only CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;3C). Similarly, there was a significant impact on tumor growth suppression after aCD40 when tumors were implanted into Batf3 knock out (KO) mice that lack cross-presenting cDC1s (Suppl. Figure\u0026nbsp;3B and Fig.\u0026nbsp;3D). These data indicate that aCD40 tumor-suppressive effect heavily relies on the presence and function of cDC1 and CD8\u0026thinsp;+\u0026thinsp;cytotoxic T cells (CTL) and may be able to substitute for CD4\u0026thinsp;+\u0026thinsp;help.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCombination aCD40 and ICB treatment results in a vaccine effect and allows for subsequent rejection of rechallenge tumors\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe influx of T cells into the TME and their activation after aCD40 treatment inevitably leads to T cell exhaustion, potentially increasing sensitivity to ICB. aCD40 alone suppressed the growth of ERα\u0026thinsp;+\u0026thinsp;tumors; however, only one-fourth of tumors had complete regressions (Fig.\u0026nbsp;2A), while the combination of aCD40 and ICB increased the complete response rate up to 83% (Fig.\u0026nbsp;1F, Fig.\u0026nbsp;4A-D). Interestingly, the effect of the combination therapy was only partially reversed by depletion of only CD8\u0026thinsp;+\u0026thinsp;or CD4\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;4A and B, respectively), whereas combined CD8\u0026thinsp;+\u0026thinsp;and CD4\u0026thinsp;+\u0026thinsp;T cell depletion completely abolished the effect of aCD40\u0026thinsp;+\u0026thinsp;ICB treatment (Fig.\u0026nbsp;4C). Tumors implanted in Batf3 KO hosts were resistant to combination therapy (Fig.\u0026nbsp;4D). The effect of combination immunotherapy on T cells persisted beyond the onset of tumor clearance. Two months after complete tumor regressions and cessation of treatment, the proportion of CD44\u0026thinsp;+\u0026thinsp;CD62L- effector memory and CD44\u0026thinsp;+\u0026thinsp;CD62L\u0026thinsp;+\u0026thinsp;central memory CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells were higher in the blood of cured mice compared to treatment-na\u0026iuml;ve mice implanted with tumors (Fig.\u0026nbsp;4E). At the same time, cured mice had fewer circulating CD44-CD62L- na\u0026iuml;ve T cells compared to treatment-na\u0026iuml;ve mice (Fig.\u0026nbsp;4E). The presence of effector and central memory T cells in cured mice is consistent with a vaccine effect and continued anti-tumor immunosurveillance and immunologic memory. To test the robustness of this immunologic memory, cured mice were rechallenged with the same burden of ERα\u0026thinsp;+\u0026thinsp;Brpkp110 tumor cells. The cells grew in control, tumor na\u0026iuml;ve mice but were universally rejected in cured mice (Fig.\u0026nbsp;4F). The rejection of tumors upon secondary rechallenge was only CD4\u0026thinsp;+\u0026thinsp;T cell-dependent and not CD8\u0026thinsp;+\u0026thinsp;T cell dependent, as the cured hosts depleted of only CD8\u0026thinsp;+\u0026thinsp;T cells were able to reject a secondary tumor rechallenge similar to the cured, T cell sufficient hosts (Fig.\u0026nbsp;4G).\u003c/p\u003e\n\u003ch3\u003eIntratumoral administration of aCD40 + ICB suppresses the growth of treated and distant tumors\u003c/h3\u003e\n\u003cp\u003eIntratumoral (IT) administration of ICB and aCD40 have individually been shown to be efficacious in human clinical trials without significant systemic absorption\u003csup\u003e\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Here we tested the efficacy of IT aCD40 alone or in combination with IT ICB in double-flanked Brpkp110 tumor-bearing mice. IT aCD40 suppressed the growth of both the treated ipsilateral tumor as well as the distant contralateral tumor (Fig.\u0026nbsp;4H). Next, various combinations of IT and intraperitoneal (IP) administrations of aCD40 and ICB were tested in double-flanked hosts. IT aCD40 and ICB were able to suppress both the treated and distant tumors, similar to the effect observed in IP treatments (Fig.\u0026nbsp;4I), indicating that local administration of aCD40\u0026thinsp;+\u0026thinsp;ICB results in an abscopal effect on distant tumors.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the current study, we sought to elucidate the mechanisms underlying ER + tumor resistance to ICB by comparing immunological profiles of ER + Brpkp110 and TNBC E0771 murine mammary tumors. Differences were observed in their immune infiltration profiles despite similar mutational burdens and neoantigen loads between these tumor models. TNBC E0771 tumors exhibited a T cell-inflamed, \"hot\" TME characterized by robust CD8 + T cell infiltration and higher proportions of Granzyme B + cytotoxic T lymphocytes (CTLs), alongside greater densities of dendritic cells. In contrast, ER + Brpkp110 tumors displayed an immunologically \"cold\" TME with minimal lymphocytic infiltration. These distinctions in immune contexture, rather than mutational burden, correlated with differential ICB responsiveness.\u003c/p\u003e \u003cp\u003eThese findings suggest that immune resistance in ER + tumors stems at least in part from deficient T cell priming, activation, and recruitment, rather than acquired dysfunction of activated T cells within the TME. This hypothesis provided the rationale for targeting the CD40 pathway in order to enhance dendritic cell maturation and antigen presentation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In various immunologically \"cold\" tumor models, including pancreatic adenocarcinoma and glioblastoma, agonistic CD40 antibodies (aCD40) have successfully activated cDC1s, improved T cell priming, and enhanced anti-tumor immunity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Consistent with our mechanistic hypothesis, we found that aCD40 monotherapy exhibited robust tumor-suppressive effects in ER + tumors compared to TNBC. The combination of aCD40 and ICB (αPD1 and αCTLA-4), addressing both pre- and post-T cell activation defects, resulted in complete tumor clearance in the majority of TNBC and ER + tumor-bearing mice, representing unprecedented efficacy in an ER + murine breast cancer model.\u003c/p\u003e \u003cp\u003eOur temporal analysis of aCD40 effects revealed a sequential cascade of immunological events. Following a single dose of aCD40, we observed a significant reduction in intratumoral CD103 + migratory DCs by days 5–7 post-treatment, concurrent with increased accumulation of mature, activated cDC1 and cDC2 in tumor-draining lymph nodes (TDLNs), accompanied by robust proliferation and activation of CD4 + and CD8 + T cells. These findings parallel prior observations in pancreatic cancer models\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and highlight aCD40’s ability to increase DC mobilization into the TDLN.\u003c/p\u003e \u003cp\u003eThe immunomodulatory effects of aCD40 also result in the induction of a pro-inflammatory chemokine milieu. aCD40 treatment significantly increased tumoral expression of CCL5, potentially contributing to the recruitment of Granzyme B + CD4 + T cells from TDLNs to the tumor bed, as previously reported in pancreatic cancer models\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Concomitantly, we observed a marked reduction in Foxp3 + regulatory T cells within treated tumors, suggesting a shift in the CD4 + T cell population from an immunosuppressive to an immunostimulatory phenotype. Additionally, aCD40 enhanced tumoral production of CXCL9 and CXCL10, potent T cell chemoattractants that likely facilitated T cell trafficking from the periphery to the tumor microenvironment.\u003c/p\u003e \u003cp\u003eAn intriguing observation was the aCD40-induced upregulation of CCL21 within treated tumors. CCL21, traditionally produced by lymphatic endothelial cells and lymph node stromal cells, acts as a chemotactic signal for CCR7-expressing DCs to traffic to lymph nodes\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. While CD103 + CCR7 + cDC1s represent the canonical antigen-trafficking migratory DCs responsible for CD8 + T cell priming\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, recent evidence indicates that a subset of CCR7 + immature DCs that downregulate antigen presentation machinery and upregulate PD-L1 can undermine T cell priming in lymph nodes\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The increased CCL21 production within aCD40-treated tumors may serve to retain these regulatory DCs within the tumor microenvironment, thereby facilitating more effective T cell priming in TDLNs. Simultaneously, the presence of PD-L1-expressing DCs within tumors could impair T cell effector function, potentially explaining why ICB complements aCD40 in achieving maximal therapeutic efficacy. However, further investigation is warranted to fully elucidate the functional significance of enhanced CCL21 expression in this context.\u003c/p\u003e \u003cp\u003eThrough selective depletion studies, we established that initial therapeutic tumor regression following aCD40 monotherapy critically depends on CD8 + T cells but not CD4 + T cells, similar to prior data demonstrating that aCD40 can substitute for CD4 + T cell help\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The absence of Batf3-dependent cross-presenting cDC1s also abolished a significant proportion of the aCD40 anti-tumor effect, underscoring the essential role of cDC1s in initiating CD8 + T cell-mediated tumor immunity. In contrast, the efficacy of combined aCD40 and ICB treatment required both CD4 + and CD8 + T cells along with cDC1s for primary tumor rejection, highlighting the cooperative nature of these immune populations in mediating maximal therapeutic responses in the context of combination immunotherapy. The durable nature of the anti-tumor immunity was evidenced by elevated proportions of circulating effector and central memory T cells two months after tumor clearance, as well as the ability of cured mice to reject secondary tumor rechallenge, confirming the establishment of immunological memory via a vaccine effect.\u003c/p\u003e \u003cp\u003eIn contrast, while primary tumor rejection required both CD4 + and CD8 + T cells, rechallenge tumor clearance demonstrated dependency on CD4 + T cells alone. This unexpected finding suggests that in the context of established immune memory, CD4 + T cells may either acquire direct cytotoxic functionality or orchestrate anti-tumor responses through cell populations other than CD8 + T cells. This observation warrants further investigation to delineate the precise mechanisms by which CD4 + T cells mediate tumor rejection in the memory phase.\u003c/p\u003e \u003cp\u003eFinally, our demonstration that intratumoral administration of aCD40, either alone or in combination with ICB, recapitulates the effects of systemic treatment has significant clinical implications. This finding indicates that localized immunomodulation within the primary tumor and associated TDLNs is sufficient to induce regression of distant lesions, potentially through the systemic dissemination of tumor-specific T cells. This approach could mitigate immune-related adverse events associated with systemic administration of immunotherapeutics, while maintaining therapeutic efficacy. Moreover, our data suggest that TDLNs play a crucial role in controlling distant metastatic lesions and preventing recurrence, arguing for the potential conservation of TDLNs during surgical resection to maximize the endogenous vaccine effect of the primary tumor.\u003c/p\u003e \u003cp\u003eIn summary, we demonstrate for the first time complete reversal of ICB resistance in ER + breast cancer through CD40 agonism. By converting immunologically \"cold\" ER + tumors into responsive entities through the activation of dendritic cells and enhancement of T cell priming, our findings provide a strong rationale for clinical investigation of combined aCD40 and ICB in ER + breast cancers. This approach may dramatically expand the proportion of breast cancer patients who can benefit from immunotherapy, potentially addressing a critical unmet need in the management of the most common breast cancer subtype. Furthermore, the mechanistic insights gleaned from this study may inform combination immunotherapy strategies across other immunologically \"cold\" tumor types characterized by deficient T cell priming and recruitment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Methods","content":"\u003ch2\u003eCell lines\u003c/h2\u003e\u003cp\u003eBrpkp110 is an estrogen receptor positive (ER+), progesterone receptor positive (PR+) and human epidermal growth receptor 2 negative (HER2-) mammary cell line derived from a tumor induced in the mammary gland of a \u003cem\u003eKRas\u003c/em\u003e\u003csup\u003e\u003cem\u003eG12D−LSL/wt\u003c/em\u003e\u003c/sup\u003e ;\u003cem\u003ep53\u003c/em\u003e\u003csup\u003e\u003cem\u003eflx/flx\u003c/em\u003e\u003c/sup\u003e ;\u003cem\u003emyr-p110⍺\u003c/em\u003e\u003csup\u003e\u003cem\u003ewt/fl\u003c/em\u003e\u003c/sup\u003e mouse (gift from Jose Conejo Garcia, Duke University) which leads to constitutively activated PI3K signaling\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Brpkp110 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 0.05% sodium pyruvate, and 3.3uL 2-mercaptoethanol. E0771 is a widely-used ERα- cell line\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and was cultured in DMEM, 10% FBS, 1% glutamine, and 0.2% gentamicin.\u003c/p\u003e\u003ch3\u003eMice and treatments\u003c/h3\u003e\u003cp\u003eAge- and sex-matched 7- to 18-week-old C57BL/6 mice (Jackson Laboratory, cat# 00664) and B6.129S(C)-\u003cem\u003eBatf3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Kmm\u003c/em\u003e\u003c/sup\u003e/J (Jackson Laboratory, cat# 013755) were housed in specific pathogen-free conditions and treated as per an approved Institutional Animal Care and Use Committee protocol at the University of Pennsylvania (Protocol #804666). Tumors in C57BL/6 and B6.129S(C)-\u003cem\u003eBatf3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Kmm\u003c/em\u003e\u003c/sup\u003e/J mice were established orthotopically in paired bilateral abdominal mammary glands 9 and 4 via injection of 5e5 cells of Brpkp110 or E0771 cell lines. The treatment antibodies listed below were obtained from Bio X Cell unless otherwise indicated. Tumor-bearing C57BL/6 or B6.129S(C)-\u003cem\u003eBatf3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Kmm\u003c/em\u003e\u003c/sup\u003e/J mice received one dose of an agonistic CD40 antibody (clone FGK4.5, 100 µg intraperitoneal (i.p.) or intratumoral (i.t.)) or rat IgG2a (clone 2A3, 100 µg i.p. or i.t.) on day 0 as previously described\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Anti-CTLA-4 (clone 9H10, 200 µg i.p. or i.t.) or rat IgG2b (clone LTF-2, 200 µg i.p. or i.t.) were given on day 0, 4, 7\u003csup\u003e28\u003c/sup\u003e. Anti-PD-1 (clone RMPI-14, 200 µg i.p. or i.t.) or rat IgG2b (clone LTF-2, 200 µg i.p. or i.t.) were given on day 0 and every 3 to 4 days thereafter\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Anti-CD4 (clone GK1.5, 200 µg i.p.) or rat IgG2b (clone LTF-2, 200 µg i.p.) was given on day − 4, day 0, and every 3 to 4 days thereafter for CD4 T-cell depletion. Anti-CD8 (clone 2.43, 200 µg i.p.) or rat IgG2b (clone LTF-2, 200 µg i.p.) was given on day − 4, day 0, and every 3 to 4 days thereafter for CD8 T-cell depletion.\u003c/p\u003e\u003ch2\u003eTissue processing and flow cytometry\u003c/h2\u003e\u003cp\u003e Mice were sacrificed according to IACUC approved protocols and tumors and tumor-draining lymph nodes (TDLN) were collected. Tumors were digested in 1 mg/mL collagenase with protease inhibitor (MilliporeSigma) and filtered through a 70 µm cell filter. TDLNs were mechanically dissociated and filtered using 100 µm filters. Cells were resuspended in PBS and incubated in Fc block (Biolegend 3527448) for 5 minutes on ice prior to staining for flow cytometry. The live/dead stain Zombie UV was used. Cells were stained with Zombie UV live/dead stain and for cell surface markers at 4°C in the dark for 30 minutes. Cell suspensions were then fixed and permeabilized for 30 minutes (eBioscience Intracellular Fixation \u0026amp; Permeabilization Buffer Set, Invitrogen) and overnight staining was performed for intracellular targets. Conjugated antibodies used for flow cytometry were obtained from BD Biosciences [PD-L1 (clone MIH5), CD4 (clones GK1.5, RM4-5), CD103 (clone M290), CD80 (clone 16-10A1), FoxP3 (clone R16-715)], Biolegend [EpCam (clone G8.8), CD45 (clone 30-F11), Ki67 (clone 16A8), Granzyme B (clone AQA16A02), CD8 (clone 53 − 6.7), CD3 (clone 145-2C11), CD40 (clone 3/23), CD86 (clone G2-1), PD-1 (clone 29F.1A12), CD19 (clone 6D5), B220 (clone RA3-6B2), CD11c (clone N418), NK1.1 (clone PK136), Gr-1 (clone RB6-8C5), XCR1 (clone ZET), CD11b (clone M1/70), CD64 (clone X54-5/7.1), SIRPα (clone P84), CD44 (clone IM7), CD62L (clone MEL-14)], and Abcam [ERα (clone E115). Flow cytometry was performed using an LSRFortessa or FACSymphony A3 Cell Analyzer and data were analyzed using FlowJo v10.8 software (BD Biosciences).\u003c/p\u003e\u003ch2\u003eImmunohistochemistry and immunofluorescence\u003c/h2\u003e\u003cp\u003eTumor tissues were fixed in Zinc formalin for 24 hours and embedded in paraffin. Immunohistochemistry (IHC) for ERα+ (clone E115, Abcam 32063, 1:9000) and cleaved caspase-3 (Cell Signaling Technology 9661, 1:500) was performed by the Comparative Pathology Core of the University of Pennsylvania School of Veterinary Medicine. Immunofluoresence (IF) for CD8 and DAPI (Thermo Scientific D21490,1:1000) was performed according to the Immunofluorescent Staining of Paraffin-embedded Tissue Protocol from Novus Biologicals, with certain modifications as follows. Antigen retrieval was performed using the IHC-Tek Epitope Retrieval Solution (IHC World 1W-1100), with slides incubated for 45 minutes in an IHC World steamer at 95-98°C. Tissue sections were blocked with 5% donkey serum diluted in PBS with 0.3% Triton X-100 and stained with rabbit anti–mouse primary CD8 (clone: D4W2Z, Cell Signaling Technology 98941, 1:100) followed by donkey anti-rabbit Alexa Fluor 594 (Thermo Scientific A-21207, 1:250) secondary antibody. Slide scanning was performed on Aperio Versa 8 slide scanner at the Molecular Pathology and Imaging Core facility at the University of Pennsylvania. Data were analyzed using QuPath. In short, QuPath’s wand function was utilized to draw a border around the DAPI-stained tissue area. Then, the “Thresholder” function was utilized to gate for positive antibody-staining, in both IF and IHC, which could be visualized in real-time. Once an appropriate staining threshold value was determined for positive-staining gating it was applied to whole tissue sections. QuPath quantified percent positive area for each antibody was used for analysis.\u003c/p\u003e\u003ch2\u003eCytokine analysis\u003c/h2\u003e\u003cp\u003eBrpkp110 tumor-bearing mice underwent treatment as described in results. Mice were euthanized and tumors were collected at day 7 post treatment. Tumors were minced and incubated in media for 48 hours. Supernatant was collected and pooled per treatment group and cytokine array was performed with technical duplicates via the proteome profiler (R\u0026amp;D, catalog # ARY028).\u003c/p\u003e\u003ch2\u003eWhole exome sequencing\u003c/h2\u003e\u003cp\u003eGenomic DNA was extracted from Brpkp110 and E0771 cell pellets and livers of wild-type C57BL/6 mice. Whole exome sequencing was performed on the Illumina platform (2x150bp) by Azenta Life Sciences. Neoantigen prediction was performed via antigen.garnish\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, we applied antigen garnish to identify single nucleotide variants (SNVs) and SNV-induced protein alterations and ran mutated peptides (8mers to 14mers) through a series of MHC-binding affinity platforms (netMHCI/netMHCII/netMHCIpan/netMHCIIpan). Identified peptides with predicted binding affinities less than 500 nM were prioritized as strong binders.\u003c/p\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eTumor growth curves were analyzed using two-way ANOVA with Tukey multiple comparisons of means to compare differences between two individual groups. A two-tailed Student \u003cem\u003et\u003c/em\u003e test was used to analyze differences between two groups. One-way ANOVA with the Bonferonni multiple comparison test was used to assess differences between any two individual groups. Statistical analyses were performed using GraphPad Prism 10. \u003cem\u003eP\u003c/em\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e≤\u003c/span\u003e 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eDr. Vonderheide has received consulting fees from BMS, EMD Serono, Grey Wolf Therapeutics and Crossbow Therapeutics, research funding from Revolution Medicines, is an inventor on patents relating to cancer cellular immunotherapy, cancer vaccines, and KRAS immune epitopes, and receives royalties from Children\u0026rsquo;s Hospital Boston for a licensed research-only monoclonal antibody, but declares no non-financial competing interests. All other authors declare no financial or non-financial competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003e Early-stage surgeon scientist program to JQZ (P30CA016520-46), Breast cancer research foundation to RHV (BCRF-24-167), University of Pennsylvania Department of Surgery Pilot Grant to JQZ, Abramson Cancer Center Pilot Grant to JQZ, Thomas B. and Jeannette E. Laws McCabe Fund to JQZ (Funders played no role in study design, data collection, analysis, and interpretation of data, or writing of this manuscript.)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJQZ, CL, OL, VM, NJ, CRF performed experiments, designed methodology, and performed data analysis. NJ performed computational analysis. JQZ, NM, SZ, RPD, RHV were involved in conceptualization and manuscript preparation. All authors were involved in data interpretation, manuscript review and editing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgments to the University of Pennsylvania core services: Penn Vet comparative pathology Core, Molecular pathology and imaging core (Center for Molecular Studies in Digestive and Liver Diseases, P30DK050306), and Penn Cytomics Core\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analyzed during the current study are available in the Sequence Read Archive (SRA) repository accession code SUB15367120.\u003c/p\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eCode availability\u003c/h2\u003e\n\u003cp\u003eThe underlying code used for this study is available at the link below: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/andrewrech/antigen.garnish\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel, R. 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CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. \u003cem\u003eNat Med\u003c/em\u003e 5, 548\u0026ndash;553 (1999).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-breast-cancer","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjbcancer","sideBox":"Learn more about [npj Breast Cancer](http://www.nature.com/npjbcancer/)","snPcode":"41523","submissionUrl":"https://mts-npjbcancer.nature.com/","title":"npj Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6823527/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6823527/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThere has been marked improvement in the clinical outcome of triple-negative breast cancer (TNBC) with the use of immune checkpoint blockade (ICB) although serious immune-related adverse effects are not uncommon. Unlike TNBC, ERα\u0026thinsp;+\u0026thinsp;breast tumors are largely unresponsive to ICB. Here we demonstrate defective priming by cross-presenting conventional dendritic cells (cDCs) and a blunted response to ICB in ERα\u0026thinsp;+\u0026thinsp;mouse mammary tumors compared to TNBC. Systemic administration of an agonistic CD40 antibody (aCD40) induced T cell proliferation and activation in tumor-draining lymph nodes and attracted effector T cells to the tumor bed from the periphery. This effect was largely due to activation, maturation and migration of type 1 conventional dendritic cells (cDC1s). aCD40 alone slowed tumor growth in ERα\u0026thinsp;+\u0026thinsp;tumors but its combination with ICB cured tumor-bearing mice, accomplishing a \u0026ldquo;vaccine effect\u0026rdquo; and the immune-mediated rejection of tumor rechallenge. The anti-tumor effect of aCD40 effect was cDC1 and CD8\u0026thinsp;+\u0026thinsp;T cell-dependent, whereas the rejection of secondary tumor rechallenge in cured mice required CD4\u0026thinsp;+\u0026thinsp;T cells. Importantly, intra-tumoral administration of aCD40 combined with systemic or intra-tumoral ICB \u0026ndash; to mimic neoadjuvant therapeutic approaches\u0026mdash;induced complete regressions of both treated and distant tumors. These findings indicate that aCD40 achieves DC activation required for the response to immunotherapy in ERα\u0026thinsp;+\u0026thinsp;tumors and further supports intra-tumoral administration of both aCD40 and ICB as an effective treatment that might limit systemic exposure and lower risk of immune-related toxicity.\u003c/p\u003e","manuscriptTitle":"CD40 agonism enhances immune checkpoint blockade and generates immunologic memory via CD4+ T cells in ERα+ mammary tumors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-16 08:35:23","doi":"10.21203/rs.3.rs-6823527/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-13T21:02:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-07T03:07:27+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T20:06:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113329587860452323291019010886382429956","date":"2025-06-12T11:30:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305598053864734741494301748672208998028","date":"2025-06-12T02:11:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-09T23:14:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-08T18:00:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-06T14:22:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Breast Cancer","date":"2025-06-04T21:06:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-breast-cancer","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjbcancer","sideBox":"Learn more about [npj Breast Cancer](http://www.nature.com/npjbcancer/)","snPcode":"41523","submissionUrl":"https://mts-npjbcancer.nature.com/","title":"npj Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c8f6521-4afc-4ab3-9ebe-b3b81516f777","owner":[],"postedDate":"June 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50100368,"name":"Biological sciences/Cancer/Breast cancer"},{"id":50100369,"name":"Health sciences/Oncology/Cancer/Breast cancer"},{"id":50100370,"name":"Health sciences/Oncology/Cancer/Cancer models"},{"id":50100371,"name":"Health sciences/Oncology/Cancer/Cancer therapy"},{"id":50100372,"name":"Health sciences/Oncology/Cancer/Tumour immunology"}],"tags":[],"updatedAt":"2026-01-19T16:44:02+00:00","versionOfRecord":{"articleIdentity":"rs-6823527","link":"https://doi.org/10.1038/s41523-025-00889-7","journal":{"identity":"npj-breast-cancer","isVorOnly":false,"title":"npj Breast Cancer"},"publishedOn":"2026-01-13 16:28:27","publishedOnDateReadable":"January 13th, 2026"},"versionCreatedAt":"2025-06-16 08:35:23","video":"","vorDoi":"10.1038/s41523-025-00889-7","vorDoiUrl":"https://doi.org/10.1038/s41523-025-00889-7","workflowStages":[]},"version":"v1","identity":"rs-6823527","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6823527","identity":"rs-6823527","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}