CD1d-Restricted NKT Cells Promote Central Memory CD8+ T Cell Formation via an IL-15-pSTAT5-Eomes Axis in a Pathogen-Exposed Environment

CD1d-Restricted NKT Cells Promote Central Memory CD8+ T Cell Formation via an IL-15-pSTAT5-Eomes Axis in a Pathogen-Exposed Environment | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 12 April 2025 V1 Latest version Share on CD1d-Restricted NKT Cells Promote Central Memory CD8+ T Cell Formation via an IL-15-pSTAT5-Eomes Axis in a Pathogen-Exposed Environment Authors : Yingyu Qin 0009-0001-9989-7473 [email protected] , Yilin Qian , Jingli Zhang , and Shengqiu Liu Authors Info & Affiliations https://doi.org/10.22541/au.174446289.99341938/v1 Published International Journal of Molecular Sciences Version of record Peer review timeline 208 views 125 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Generation of memory CD8 + T cells is necessary for the establishment of protective T cell immunity against pathogens. The understanding of the cellular and molecular processes involved in the formation of memory CD8 + T cells remains elusive. Current knowledge gaps may stem from reliance on specific pathogen-free (SPF) models, where restricted microbial exposure creates an immunological milieu that diverges from natural environments. Here, we demonstrate that under non-SPF housing, CD1d +/− and Ja18 +/− mice show enhanced central memory (TCM) formation versus CD1d-restricted NKT-deficient (CD1d −/− /Ja18 −/− ) controls, implicating CD1d-restricted NKT cells in TCM formation. Mechanistically, CD1d-restricted NKT cells elevated CD4 + T cell-derived IL-15 in CD1d +/- mice, activating the IL-15/pSTAT5/Eomes axis essential for TCM maintenance. Functional validation through CFSE-labeled OT-1 memory cell transfers revealed an NKT-dependent survival advantage in CD1d +/− hosts, providing direct evidence for microbial-experienced niches in shaping immune memory. These findings establish CD1d-restriced NKT cells as physiological regulators of TCM generation, suggesting their potential utility as vaccine adjuvants to enhance protective immunity. Introduction CD8 + T cells are a key type of lymphocytes in adaptive immune system, which plays critical role in combating and controlling malignancies and intracellular infections. Once naïve CD8 + T cells are activated, a largely autonomous program of proliferation and differentiation is induced, which results of effector and memory CD8 + T cell generation [1]. Memory CD8 + T cells are a more potent subset defensing against infections and tumors, which possess long-term survivability and can undergo proliferation and acquire effector function rapidly and robustly upon antigen re-exposure [2]. Thus, understanding the factors driving efficient memory CD8 + T cell generation is a major goal with a view to improvement of vaccine and immunotherapeutic design. It is important to realize that most studies were conducted in specific pathogen–free (SPF) housing mice to reveal the fundamental principles about the generation and maintenance of T-cell memory, and to deduce these findings are also processed in humans. However, the immune system of humans is naturally live in a pathogenic rich environment. Some studies have been suggested that the immune system of dirty mice which are exposure to multiple mouse pathogens is more similar to that observed in adult humans [3-5]. Therefore, “dirty”mice can be used to better define the role of the adaptive immune system in the generation and maintenance of memory T cells within innate immune cells. It is believed that help cells are important to promote effective cellular immunity by enhancing CD8 + T cell clonal expansion, differentiation and survival. Besides CD4 + T help cells, a large body of studies have determined that type I Natural killer T (NKT) cells, also known as invariant NKT cells can also achieve this task [6]. NKT cells are a lymphocyte lineage belong to innate immune system specific to recognize lipid antigens presented by CD1d molecule expressed by antigen presenting cells [7, 8]. As an innate type lymphocyte, NKT cell response is immediate and robust. Upon stimulation, NKT cells rapidly produce large amounts of T-helper (Th)-1 and Th2-type cytokines induce subsequent activation of other type of immune cells including NK cells, DCs as well as T and B cells [9, 10]. α-galactosylceramide (αGalCer) is a synthetic antigen that can potently and selectively activate iNKT cells [11]. Several studies have shown that iNKT cells stimulated by α-galactosylceramide (αGalCer), may affect memory CD8 + T cell differentiation [12-14]. Early work suggested that activated iNKT cells promote the differentiation of Murine cytomegalovirus (MCMV) specific central memory CD8 + T cells(TCM) [13]. The homeostatic proliferation of memory CD8 + T cells is facilitated by IL-4-producing iNKT cells [14]. iNKT cells govern the fate determination of CD8 + T cells, initiating from effector CD8 + T cells with memory potential or limited lifespan potential [12]. However, the synthetic lipid antigen not can fully encompass the authentic physiological function of NKT cells. Given that activation can occur through the constitutive expression of inflammatory cytokine receptors, such as IL-12R and IL-18R, even in the absence of TCR signals, this enables direct or indirect microbial-induced activation of NKT cells [15]. Additionally, our previous study has reported that iNKT cells can enhance the effector function of CD8 + T cells through direct interaction without exogenous antigens, thereby promoting CD8 + T cell-mediated antitumor activity [16]. However, the impact of CD1d-restricted NKT cells on memory CD8 + T cell differentiation in the absence of synthetic lipid antigen stimulation remains to be elucidated. In this study, we observed higher frequencies of TCM in CD1d +/− or Ja18 +/− compared to CD1d −/− mice or Ja18 −/− (deficient in iNKT cells) mice within a “dirty” environment, suggesting the essential role of CD1d-restricted NKT in TCM formation. Eomesodermin (Eomes) is a transcription factor crucial for the development and function of CD8 + memory T cells [17]. Memory CD8 + T cells lacking Eomes exhibit impaired long-term persistence and fail to undergo effective secondary expansion upon re-challenge [18]. We observed that Eomes expression is strictly correlated with TCM formation. IL-15 contributes to the homeostatic proliferation and survival of memory CD8 + T cells under steady state conditions [19]. Steady-state analysis revealed elevated IL-15 levels in CD1d +/− mice compared to CD1d -/- controls, primarily sourced from CD4 + T helper cells. Subsequent investigations established that CD1d-restricted NKT cells promote TCM formation through an IL-15-STAT5 phosphorylation-Eomes upregulation axis. This functional relationship was validated through adoptive transfer experiments using CFSE-labeled memory OT-1 cells. Recipient CD1d +/− mice demonstrated superior survival and homeostatic proliferation of transferred memory CD8 + T cells relative to CD1d −/− hosts, directly implicating iNKT cells in sustaining the memory T cell pool. Materials and Method Mice B6(Cg)-Traj18 tm1.1Kro /J (Jα18 -/- ) mice[20] were kindly provided by Pro. Xinzhi Wang (China Pharmaceutical University, Nanjing, China) and B6.129S6-Del(3Cd1d2-Cd1d1)1Sbp/J (CD1d -/- ) mice were purchased from Jackson laboratories and kindly provided by Pro. Zhigang Lei (Nanjing Medical University, Nanjing, China). The littermates of CD1d +/- and CD1d -/- mice were generated through the crossing of CD1d +/- and CD1d -/- mice. The same applies to Jα18 +/- and Jα18 -/- mice. These mice were housed with open cages in conventional animal facilities of Southeast University. Ovalbumin (OVA)–specific TCR transgenic mice (OT-I) were purchased from Aniphe BioLab (Jiangsu, China) and these mice were kept in specific pathogen free animal facilities of Southeast University. All animal experiments were approved by institutional guidelines established by the Committee of Ethics on Animal Experiments of Southeast University. Flow cytometric analysis Lymphocytes were analyzed with below antibodies: CD3-PE/ Percp5.5 (17A2), CD8α-Percp5.5/FITC (53-6.7), CD44-APC (IM7), CD62L-APC.Cy7/BV605 (MEL-14), TCRVβ5.1,5.2-APC (MR9-4), CD45.1-PE/BV786 (A20), TCRVα2-PE (B20.1), NK1.1-FITC (S17016D), IL-15Rα-PE (6B4C88), CD122-FITC (TM-β1), pSTAT5-PE (A17016B.Rec), TCF1-BV421 (S33-966), Eomes-PE (W17001A), T-bet-PE.Cy7 (4B10). LIVE/DEAD Fixable Blue viability-BV510. For transcription factor staining, the lymphocytes underwent intracellular staining using the Fixation/Permeabilization Solution Kit (BD Pharmingen) after surface staining. For IL-15Rα staining, the lymphocytes were incubated in culture medium and treated with PMA/ionomycin for 4 hours followed by surface staining. For phosphorylated STAT5 detection, the splenocytes were stimulated with 50 ng/ml IL-15 for 37℃ 30min followed by staining of pSTAT5 and surface staining using a standard protocol [21]. Data acquisition was performed on a five-laser BD LSRFortessa™ Cell Analyzer Flow Cytometer. Real-time quantitative PCR The spleens were isolated from indicated mice, followed by TRIzol treatment and subsequent RNA extraction. Quantitative real-time PCR (qPCR) was performed with the use of SYBR Green (Vazyme), on a QuantStudio™ 3 Real-Time PCR System (Thermo fisher). All reactions were run in triplicates. Primers for real-time qPCR are as follows: IL-7: F: 5’-CAGGAACTGATAGTAATTGCCCG-3’ and R: 5’-CTTCAACTTGCGAGCAGCACGA-3’; IL-15: F: 5’-GTAGGTCTCCCTAAAACAGAGGC-3’ and R: 5’- TCCAGGAGAAAGCAGTTCATTGC-3’; IL-21: F: 5’- CGCCTCCTGATTAGACTTCG-3’ and R:5’-TGGGTGTCCTTTTCTCATACG-3’; β-actin: F: 5’-GCCAACCGTGAAAAGATGACCCAG-3’ and R: 5’-ACGACCAGAGGCATACAGGGACAG-3’. IL-15 serum ELISA About 200 μL of peripheral blood was collected from the littermates of CD1d +/- and CD1d -/- mice. The samples were incubated at room temperature for 30min, followed by centrifugation at 2000 x g for 20min to remove the clot. The concentration of IL-15 in the serum was subsequently measured using an IL-15 ELISA kit (ELK Biotechnology, China). Adoptive transfer memory OT-1 cell strategy For memory OT-1 cell transfer, OT-1 mice were immunized with 100 μg of OVA 257-264 peptides emulsified in Freund’s complete adjuvant (CFA, Sigma-Aldrich). Following one month of immunization, the memory differentiation of OT-1 cells was examined through the expression of CD44 and CD62L using FACS analysis. The total population of memory OT-1 T cells was isolated from the spleen and inguinal lymph nodes (ILNs) via a negative CD8a + T Cell Isolation Kit (Miltenyi Biotec, 130-104-075) according to the manufacturer’s instructions, subsequentlymemory CD8 + T cells were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE). Subsequently, 6×10 5 CFSE-labeled memory OT-1 cells were intravenously injected into CD1d +/− and CD1d −/− mice respectively. Statistical analysis Statistical analyses were performed using GraphPad Prism (version 8). Unpaired Student’s t-test was employed to determine the differences between two data sets. One-way analysis of variance (ANOVA) was used for more than 2 comparison groups. The significance levels considered were p<0.05 (*), p<0.01 (**), p<0.001 (***) and p<0.0001 (****). The mean±standard error of the mean (SEM) was used to express the data. CD1d-restricted NKT cells promote TCM formation in steady state To investigate the physiological role of NKT cells in memory CD8 + T cell generation , we compared littermate CD1d +/− and CD1d −/− mice (lacking NKT cells) housed under conventional conditions. Without experimental intervention , both strains exhibited an age-dependent increase in central memory T cell (TCM) frequencies , but CD1d +/− mice consistently showed higher TCM frequencies than CD1d −/− mice from early to late ages (Fig. 1A, B). Since CD1d deficiency depletes all CD1d-restricted NKT cells , we further assessed Jα18 −/− mice , which lack only iNKT cells . Consistent with our earlier findings, Jα18 −/− mice had reduced TCM populations compared to Jα18 +/− controls across all ages (Fig. 1C, D). However, the complete ablation of all CD1d-restricted NKT cells ( CD1d −/− ) led to more severe impairments in the formation of central memory T cells (TCMs) compared to the ablation of iNKT cells alone ( Jα18 −/− ) . These results indicate that CD1d-restricted NKT cells promote TCM formation under conventional conditions. CD1d-restricted NKT cells promote homeostatic proliferation of memory CD8 + T cells To determine whether CD1d-restricted NKT cells regulate memory T cell survival and homeostatic proliferation, we transferred CFSE-labeled memory OT-1 cells into CD1d −/− and CD1d +/− littermates (Fig. 2A). Peripheral blood (PBL) analysis at days 7, 21, and 35 post-transfer revealed a progressive decline in OT-1 cell numbers, with CD1d +/− mice consistently retaining more OT-1 cells than CD1d −/− mice (Fig. 2B). At day 35, we also observed higher numbers of memory OT-1 cells in the spleen and inguinal lymph nodes (ILN) of CD1d +/− mice (Fig. 2C). To assess homeostatic proliferation, we analyzed CFSE dilution in transferred OT-1 cells. CD1d +/− mice exhibited significantly more OT-1 cells in division cycles D2 and D3 compared to CD1d −/− mice (Fig. 2D). Together, these results indicate that CD1d-restricted NKT cells enhance memory T cell survival and homeostatic proliferation. Eomes expression is strictly correlated with TCM formation Underlying CD8 T cell differentiation and function are transcription factors. T cell factor-1 (TCF-1) (encoded by the TCF7 gene) is a transcription factor that plays important role during the formation and maintenance of CD8 + T cell memory in acute infections [22]. The T-box transcription factors T-box expressed in T cells (T-bet) and eomesodermin (Eomes) have been implicated as master regulators of CD8 T cell differentiation and function [17]. T-bet is associated with effector function and terminal effector T cell subsets, and is also expressed in memory T cells [23]. Eomes primarily is associated with memory T cell formation [18]. We then examined whether these transcription factors are involved in generation of TCM mediated by CD1d-restricted NKT cells. We found CD1d-restricted NKT cells slightly enhanced the frequencies of TCF1 + CD62L + (Fig. 3A) and T-bet + CD62L + population (Fig. 3B). However, CD1d-restricted NKT cells significantly enhanced the frequencies of Eomes + CD62L + populations (Fig. 3C). Furthermore, we found that the populations of TCF1 + CD62L + , T-bet + CD62L + , and Eomes + CD62L + were positively correlated with the proportion of TCM. Notably, among these correlations, the association between Eomes and TCM was the strongest (Pearson r=0.9745, P<0.0001) (Fig. 3D), which implies that Eomes may play a role in TCM generation mediated by CD1d-restricted NKT cells. iNKT cells promote the homeostatic proliferation of memory OT-1 cells IL-15 and IL-7 are critical cytokines for maintaining homeostatic proliferation and survival of memory CD8 + T cells under steady-state conditions [18], while IL-21, a hallmark cytokine of follicular helper T (TFH) cells, drives differentiation of central and effector memory CD8+ T cells [24, 25]. To investigate potential regulatory differences between CD1d +/- and CD1d −/− mice, we compared splenic cytokine gene expression profiles. Although IL-7 and IL-21 mRNA levels showed no significant differences between genotypes, CD1d +/− mice exhibited markedly elevated IL-15 mRNA expression compared to CD1d −/− counterparts (Fig. 4A). This transcription difference was corroborated at the protein level, with higher serum IL-15 concentrations observed in CD1d +/− mice (Fig. 4B) Then we identified the source cells responsible for IL-15 production. Given that secreted IL-15 binds to transmembrane IL-15Rα with high affinity and is presented on the cell surface, where it acts in trans on adjacent IL-2Rβ:γ-expressing cells[26], we therefore examined IL-15Rα surface expression to evaluate IL-15 trans-presentation capacity. Given the absence of NKT cells in CD1d -/- mice and our previous observation that NKT cells do not promote TCM formation under SPF conditions (data not shown), we therefore examined whether the environmental factors influence NKT cells production of IL-15 to regulation generation of TCM. We therefore compared IL-15Rα surface expression on wild-type NKT cells under conventional (”dirty”) versus SPF housing conditions. While NKT cells demonstrated baseline IL-15 production capacity, no significant environmental differences were detected in this population (Fig. 4C). Intriguingly, T cells from ”dirty” environment mice displayed elevated IL-15Rα levels (Fig. 4C), suggesting potential NKT cell-mediated regulation of T cell IL-15 production. We subsequently performed a comparison of IL-15Rα expression levels on CD4 + T and CD8 + T cells between CD1d +/− and CD1d −/− mice. Notably, CD1d +/− mice exhibited significantly higher IL-15Rα levels on CD4 + T help cells compared to CD1d −/− mice (Fig. 4D), whereas CD8+ T cells showed minimal receptor expression in both groups (data not shown). Macrophages and DCs are also IL-15 producing cells [27]. However, the level of IL-15Rα was comparable between CD1d +/− and CD1d −/− mice (Fig. 4E). Collectively, these findings suggest that CD1d-restricted NKT cells may regulate IL-15 production through a CD4 + T cell-dependent mechanism, potentially contributing to enhanced survival and homeostatic proliferation of CD8 + TCM cells in D1d −/− mice. CD1d-restricted NKT cells mediate homeostatic formation of TCM through the IL-15-pSTAT5-Eomes axis Our data demonstrate that CD1d-restricted NKT cells significantly increase the frequency of Eomes⁺CD62L⁺CD8⁺ T cells (Fig. 3C), with Eomes expression levels strongly correlating with the proportion of central memory T (TCM) cells (Fig. 3D). Notably, TCM compartments in the presence of CD1d-restricted NKT cells exhibited elevated Eomes expression (Fig. 5A). Several reports have addressed that stimulation-mediated by IL-15 through CD122 leads to the induction of Eomes expression [27-31]. Therefore, we postulated a functional link between NKT cell-mediated IL-15 signaling and Eomes regulation. Consistent with this hypothesis, we observed significantly increased phosphorylation of STAT5 (pSTAT5)—a downstream effector of IL-15 signaling triggered by IL-15Rβ:γ engagement—in TCM cells from mice with CD1d-restricted NKT cells (Fig. 5B). Furthermore, CD1d-restricted NKT cells markedly enhanced Eomes expression specifically within the CD62L⁺CD122⁺ T cell subset (Fig. 5C). Together, these findings establish that CD1d-restricted NKT cells drive TCM generation potentially through an IL-15-pSTAT5-Eomes signaling axis. Discussion The differentiation and maintenance of memory CD8 + T cells represent critical determinants of long-term immunological protection against pathogens and malignancies. While prior studies have established the importance of helper cells in shaping CD8+ T cell memory, the present work provides novel insights into the physiological role of CD1d-restricted NKT cells in TCM formation under conventional (”dirty”) housing conditions. Our findings demonstrate that CD1d-restricted NKT cells, particularly iNKT cells, are essential for sustaining TCM populations through an IL-15-dependent mechanism involving STAT5 phosphorylation and Eomes upregulation. These observations extend current paradigms by highlighting how environmental pathogen exposure modulates innate-adaptive crosstalk to influence memory T cell biology. A striking feature of this work is the environmental specificity of NKT cell function. Unlike SPF-housed mice, where NKT cells showed negatively influence on TCM frequencies, conventionally housed (”dirty”) mice exhibited a pronounced reliance on CD1d-restricted NKT cells for TCM maintenance. This dichotomy underscores how microbial exposure primes innate lymphocytes to acquire regulatory roles in adaptive immunity—a concept increasingly recognized in mucosal tissues but less explored systemically. For instance, commensal microbiota educate group 3 innate lymphoid cells (ILC3s) to support intestinal T cell memory [32]. Here, we extend this paradigm by demonstrating that systemic pathogen exposure licenses NKT cells to orchestrate CD8 + TCM homeostasis, likely via tonic activation through cytokine receptors (e.g., IL-12R, IL-18R) or indirect microbial ligand engagement. Such a mechanism positions NKT cells as environmental rheostats, dynamically calibrating memory T cell reserves to match historical pathogen encounters. This aligns with evolutionary demands for immune systems to optimize resource allocation: in pathogen-rich settings, sustained NKT-mediated TCM support ensures rapid recall responses, whereas sterile environments may prioritize effector readiness over memory longevity. We found that TCM damage was more pronounced in CD1d −/− mice than in Jα18 −/− mice, suggesting that both invariant NKT (iNKT) cells and various CD1d-restricted NKT cell subsets, as well as certain γδ T cell subsets that have been reported to engage CD1d-restricted lipid antigen presentation, contribute functionally, potentially through distinct cytokine microenvironments. These findings align with emerging evidence supporting NKT cell heterogeneity in tissue-specific immunity [33]. Eomes emerged as the dominant transcription correlate of NKT-mediated TCM maintenance, outperforming TCF1 and T-bet in predictive strength. This aligns with Eomes’ dual role in promoting memory precursor survival and metabolic fitness[34, 35]. IL-15 signaling via STAT5 phosphorylates and stabilizes Eomes, enabling TCM cells to compete for survival signals in IL-15-limited niches. Notably, NKT cells selectively boosted Eomes expression in CD62L + CD122 + TCM subsets, suggesting a focused effect on self-renewing memory populations. This specificity may explain the superior homeostatic proliferation of transferred OT-1 cells in CD1d +/− hosts, as Eomes promote T cell survival through enhancement of mitochondrial fitness and adaptability to cytokine fluctuations [34-36]. Mechanistically, the IL-15-STAT5-Eomes axis likely synergizes with TCF1 to enforce memory stemness while counteracting T-bet-driven effector differentiation, thereby stabilizing the TCM phenotype. The IL-15-Eomes axis identified in this study complements established pathways for memory T cell maintenance [28-30, 35] and positions NKT cells as novel regulators within this cytokine network. Notably, the environmental dependency of this phenomenon—absent in specific pathogen-free (SPF) reared mice—underscores the critical role of microbial exposure in enabling NKT cells to regulate memory T cell function, possibly via sustained activation of pattern recognition receptors[37]. While this study advances our understanding of NKT cell biology, several limitations warrant consideration. For instance, the precise crosstalk between NKT and CD4⁺ T cells, as well as the spatial dynamics of IL-15, remain unvalidated (e.g., through IL-15 knockout models). Despite these gaps, they do not diminish the central role of NKT cells in maintaining TCM via the IL-15/Eomes axis. Further mechanistic validation would reinforce, but not negate, the proposed model. In summary, this study demonstrates that CD1d-restricted NKT cells serve as pivotal regulators of the homeostasis of CD8 + central memory T cells (TCM) in hosts exposed to pathogens. By translating microbial exposure into IL-15 signaling-driven, Eomes-dependent memory maintenance, these cells link innate environmental perception with adaptive immune memory. This homeostatic function complements their acute helper role, underscoring the multifaceted nature of NKT cells. Future investigations into the temporal and spatial regulation of microbial licensing signals and IL-15 delivery could refine strategies for vaccine development and T cell-based therapies, while emphasizing the profound impact of environmental context on the shaping of immune memory. Data Availability Statement All data used during the study appear in the submitted article. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (32100712) and Fundamental Research Funds for the Central Universities (2242022R40060). Authorship Contributions YQ designed research studies, YQ, JZ, SL and YLQ conducted the experiments. YQ acquired and analyzed data. YQ wrote the manuscript. All authors contributed to the article and approved the submitted version. Disclosure of Conflicts of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References 1. Marianne J B van Stipdonk, G.H., Martijn S Bijker, Edward E Lemmens, Nathalie M Droin, Douglas R Green, Stephen P Schoenberger, Dynamic programming of CD8+ T lymphocyte responses. Nature Immunology, 2003. 4 : p. 361-365.2. 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Immunity, 2012 36 : p. 68-78.35. Joulia E, M.M., Agesta A, et al, Eomes-dependent mitochondrial regulation promotes survival of pathogenic CD4+ T cells during inflammation. J Exp Med, 2024. 221 : p. e20230449.36. Buck MD, O.S.D., Klein Geltink RI, Curtis JD, Chang CH, et al, Mitochondrial Dynamics Controls T Cell Fate through Metabolic Programming. Cell Death Dis, 2016. 166 : p. 63-76.37. Godfrey DI, R.J., New ways to turn on NKT cells. J Exp Med, 2011. 208 : p. 1121-1125. Figure legends Figure 1. Comparison of the TCM population in littermates of CD1d +/- (Jα18 +/- ) and CD1d -/- (Jα18 -/- ) mice under steady-state conditions. 1. Representative flow cytometry plots showing the analysis of central memory (CD44 + CD62L + ) CD8 + T cells (TCM) in in the peripheral blood (PBL) (left panel). Statistical data representng the percentages of TCM in littermates of CD1d +/- and CD1d -/- mice at various ages (right panel). 2. Representative flow cytometry plots showing the analysis of TCM in the spleens of 72-week old CD1d +/- and CD1d -/- mice, along with their gating strategy (left panel). Statistical data depicting the percentages of TCM in littermates of CD1d +/- and CD1d -/- mice (right panel). 3. Representative flow cytometry plots showing the analysis of TCM in the PBL (left panel). Statistical data representng the percentages of TCM in littermates of Jα18 +/- and Jα18 -/- mice at various ages (right panel). 4. Representative flow cytometry plots showing the analysis of TCM in the spleens of 46-week old Jα18 +/- and Jα18 -/- mice (left panel). Statistical data depicting the percentages of TCM in littermates of Jα18 +/- and Jα18 -/- mice (right panel). The number of CD1d +/- and CD1d -/- mice: 5-7 weeks n=11/13, 9-11 weeks n=10/5, 15-17 weeks n=18/10, 20-30 weeks n=11/9, 40-50 weeks n=15/9, 72 weeks n=13/7; The number of Jα18 +/- and Jα18 -/- mice: 6 weeks n=19/15, 31 weeks n=22/15, 46 weeks n=23/15. Mean with SEM are shown; Statistics: Statistical significance was determined using one-way ANOVA with Tukey tests for multiple-group comparisons (A, C); non-paired two-tailed Student’s test (B, D). *P < 0.05, **P<0.01, ***P<0.001, ****P < 0.0001; Figure 2. CD1d-restricted NKT cells promote survival and homeostatic proliferation of memory OT-1 cells A-D. 6×10 5 CFSE-labeled memory CD45.1 + OT-1 cells were intravenously injected into CD45.2 + CD1d +/- and CD45.2 + CD1d -/- mice respectively. 4 mice per group. 1. The schematic diagram of the experimental workflow and flow cytometry plots illustrating the memory phenotypes of OT-1 cells following CFSE labeling prior to transfer. 2. The transferred OT-1 cells were detected in PBL at the indicated time points. 3. The transferred OT-1 cells were detected in the spleen and inguinal lymph nodes (ILN) on day 35. 4. On day 35, the frequencies of OT-1 cells in CFSE division zones (D2, D2+D3) were examined in PBL, spleen and ILN. Each dot representing a mouse. CD1d +/- (n=4), CD1d -/- (n=4). Representative results are from two independent experiments with consistent results. Mean with SEM are shown; Statistics: non-paired two-tailed Student’s test: *P < 0.05, **P 0.05. Figure 3. Examination of transcription factors related to TCM A-D. The proportions of TCF1 + CD62L + (A), T-bet + CD62L + (B) and Eomes + CD62L + (C) within CD8 + T cells were examined via FACS analysis in the spleens of 10-12-week old CD1d +/- and CD1d -/- mice. The frequencies of TCF1 + CD62L + , T-bet + CD62L + and Eomes + CD62L + CD8+T cells were correlated with CD44 + CD62L + CD8 + T cells (TCM) (D). Each dot representing a mouse. CD1d +/- (n=5), CD1d -/- (n=4). Representative results are from three independent experiments with consistent results. Data are presented as mean ± SEM; Statistical significance was measured by unpaired two-tailed Student’s t test (A-C) and Pearson’s correlation coefficient. **p < 0.01, ****P < 0.0001. Figure 4. Examination of cytokines related to TCM formation 1. qPCR analysis of the mRNA level of IL-7, IL-15 and IL-21 in fresh isolated splenocytes from 10-12-week old CD1d -/- and CD1d +/- mice. The data from mice CD1d +/- (n=9) and CD1d -/- (n=8) were collected. 2. Protein levels of IL-15 were detected in serum of CD1d +/- and CD1d -/- mice, with 10 mice in each group. 3. The expression levels of IL-15Rα on the surface of NKT or T cells in the spleen of wild-type mice bred in SPF or “dirty” conditions were examined. Four mice per group. The data are representative of two independent experiments. 4. The expression levels of IL-15Rα on the surface of NKT cells in the spleen of CD1d +/- and CD1d -/- mice were compared. Four mice per group. The data are representative of two independent experiments. Mean with SEM are shown; Statistics: non-paired two-tailed Student’s test: *P < 0.05, **P < 0.01, ***P<0.001; Figure 5. CD1d-restricted NKT cells facilitate homeostatic formation of TCM through the IL-15-pSTAT5-Eomes axis 1. Expression levels of Eomes in TCM of CD8 + T cells were expressed via mean fluorescence intensity (MFI). 2. The phospho-flow cytometry of STAT5 in TCM of CD8 + T cells from CD1d +/- and CD1d -/- splenocytes response to IL-15 stimulation. The histogram illustrates the MFI of STAT5 phosphorylation in TCM. The dashed lines represent cells that are not stimulated by IL-15. 3. The expression of Eomes in CD62L + CD122 + gated on TCM of CD8 + T cells from CD1d +/- and CD1d -/- mice was displayed and expression levels of Eomes were compared through MFI. Each dot representing a mouse. Four mice per group. The data are representative of three independent experiments. Mean with SEM are shown; Statistics: non-paired two-tailed Student’s test: *P < 0.05, **P < 0.01, ***P<0.001; Information & Authors Information Version history V1 Version 1 12 April 2025 Peer review timeline Published International Journal of Molecular Sciences Version of Record 28 Jul 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cd8 cell immune homeostasis memory nkt cell Authors Affiliations Yingyu Qin 0009-0001-9989-7473 [email protected] Southeast University School of Medicine View all articles by this author Yilin Qian Southeast University School of Medicine View all articles by this author Jingli Zhang Southeast University School of Medicine View all articles by this author Shengqiu Liu Southeast University School of Medicine View all articles by this author Metrics & Citations Metrics Article Usage 208 views 125 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yingyu Qin, Yilin Qian, Jingli Zhang, et al. CD1d-Restricted NKT Cells Promote Central Memory CD8+ T Cell Formation via an IL-15-pSTAT5-Eomes Axis in a Pathogen-Exposed Environment. Authorea . 12 April 2025. DOI: https://doi.org/10.22541/au.174446289.99341938/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. 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