CRTAM induces an effector memory T cell phenotype of splenic CD8 T cells

CRTAM induces an effector memory T cell phenotype of splenic CD8 T cells | 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 Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed.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 CRTAM induces an effector memory T cell phenotype of splenic CD8 T cells Authors : Gabriela Hernández-Galicia 0009-0005-8140-4721 , Jocelyn C. Pérez-Lara , Irlanda Olvera-Gomez 0000-0003-1698-6559 , and Vianney Ortíz-Navarrete 0000-0001-6546-9713 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.174446286.60104297/v1 1264 views 597 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract CRTAM, an adhesion molecule with immunoregulatory properties, plays a crucial role in late-stage polarization processes, including the localization of proteins like CD44, talin, CD3, and PKC-ζ, as well as the secretion of cytokines such as IFN-γ, IL-17, and IL-22. These processes are essential for the development of memory CD8 T lymphocytes. This study aimed to investigate the impact of CRTAM expression on the generation of memory CD8 T lymphocytes. Our findings demonstrate that CRTAM is indispensable for establishing the CD8 effector memory subpopulation and maintaining CD8 central memory cells long-term after live bacteria immunization. Moreover, during the acute phase of the immune response against an attenuated strain of Salmonella , CRTAM expression promotes a transient KLRG1 + CD127 + phenotype. Furthermore, CRTAM influences protection against virulent Salmonella , impacting the frequency of CD8+ IFN-γ+ and Grz-B+ cells and CD8 T cell retention. Specifically, CRTAM deficiency diminishes the protective capacity and alters the balance of effector and memory CD8 T cell populations. These results collectively establish CRTAM as a crucial regulator of memory CD8 T cell subset dynamic, influencing both the establishment and maintenance of them while also playing a role in the acute and recall response. Introduction Memory T CD8 lymphocytes are essential for protective immunity against intracellular pathogens (1). Upon antigen exposure, activated T CD8 cells differentiate into effector cells. A subset of these cells survives the contraction phase to form a heterogeneous pool of memory cells that differs phenotypically in migration patterns and localization. Central memory and effector memory T cells are characterized by distinct surface markers, such as CD45RO (humans), CCR7, CD44, CD62L (L-selectin), CX3CR1, or CD27 (1–6). The generation of memory T CD8 cells is influenced by various factors, including asymmetric division of proteins such as T-bet, BCL-xL, or mtorc1 complex (Mammalian Target of Rapamycin Complex 1), as well as inflammatory cytokines in the microenvironment, such as IFN-γ, IL-7 or IL-15 (4,7,8). During the acute phase of the immune response, IL-7Rα expression (CD127) marks memory precursor effector cells (MPECs) destined to become memory cells. In contrast, KLRG1 + T CD8 cells are more prone to die by apoptosis during the contraction phase (1,9). Cytotoxic and Regulatory T cell Molecule (CRTAM; CD355) is a Nectin-like family member, characterized by two extracellular immunoglobulin-like domains: a variable (V) and a constant (C) domain, both essential for binding its ligand, Nectin-like 2 (NecL-2) (10–13). Although initially identified in invariant Natural Killer T (iNKT) cells, Natural Killer (NK) cells, and CD8 T cells, the expression of CRTAM has been observed in cytotoxic CD4 T cells, neutrophils, and eosinophils (14–16). T-cell activation results in CRTAM upregulation, which contributes to the sustained polarization of CD44, CD3, and Talin. This upregulation also promotes the secretion of IFN-γ, IL-22, and IL-17, facilitating the retention of CD8 T cells within lymph nodes through interaction with NecL-2 (17). Previous studies have indicated immunoregulatory function for CRTAM, including potential modulation of central memory (CD44+ CD62L+) and effector memory (CD44+ CD62L-) T CD4 cell differentiation. Specifically, experiments using CRTAM knock-in mice stimulated with agonist antibodies against CD3 and CD28 (αCD3/αCD28) suggest the involvement of CRTAM in regulating the acquisition of T CM and T EM memory phenotypes (12). This effect is likely mediated by signal transduction through the cytoplasmic tail of CRTAM, CRTAM-CRTAM, or CRTAM-NecL-2 interactions (15). Given the established roles of CRTAM in protein polarization, cytokine secretion, and T cell retention within lymph nodes, we hypothesized that this adhesion molecule plays a critical role in the generation of memory T cells. Therefore, we investigated the impact of CRTAM expression on the development of T CD8 memory cells. Here, we report that CRTAM deficiency significantly reduced effector memory cells and impaired secondary responses to Salmonella infection. These findings highlight the importance of CRTAM in shaping the CD8 T cell memory responses. Methods Study design To elucidate the role of CRTAM in T CD8 + memory generation, transgenic deficient Crtam or wild-type mice were immunized with an attenuated strain of Salmonella enterica ser. Typhimurium aroA - or infected with a virulent strain of Salmonella (14028). The phenotype of memory subsets was determined by flow cytometry. Mice In this study, six to eight-week-old males or females wild-type (C57BL6/J) or Crtam.Ko.Lex.B6 ( Crtam -/ ) mice were used. Mice of the same age and sex were used for all experiments. The mice were housed under pathogen-free conditions in the Laboratory Animal Production and Experimentation Unit (UPEAL), with food and water ad libitum , on a 12-h light/12-h dark cycle. The protocol (00-7814) was approved by the Intern Committee for Care and Use of Experimental Animals of Cinvestav. Bacterial growth Salmonella enterica ser. Typhimurium strains SL3261 ( aroA - ; attenuated) or 14028 (virulent) were grown overnight in Luria Bertani broth. The next day, a subculture was carried out by 1:30, diluted and growing until the logarithmic growth phase. At this point, serial dilutions were performed in a sterile Phosphate-buffered saline (PBS) solution to obtain suitable Colony-Forming Units (CFU). Mice immunization/ infection C57BL6/J or Crtam.Ko.Lex.B6 mice were immunized intraperitoneally with 100 CFU of Salmonella strain SL3261. After immunization, the mice’s weight was monitored every two days. For the protection assay, thirty days after immunization, mice were challenged with 500 or 150 CFU of Salmonella strain 14028 and the survival curve was estimated. Cell isolation and flow cytometry Cell suspensions from the spleen or mesenteric lymph nodes (draining lymph nodes; DLN) were collected from C57BL6/J or Crtam.Ko.Lex.B6 mice and organs were disaggregated to obtain a cell suspension. Then, an ammonium chloride buffer solution was used for red blood cell lysis. One million cells were stained for flow cytometry analysis. Briefly, to determine viability, Zombie NIR™ (Biolegend) was used according to the manufacturer’s instructions. T cells were stained with the following antibodies: CD3-FITC, APC-Cy7 (Biolegend), CD8-PE, APC or FITC (Biolegend), CD44-APC or PB (Biolegend), CD62L-PE or APC (Biolegend), KLRG1-PerCPCy5.5 (Biolegend), CD127 (IL-Ra) FITC or PE (Biolegend), and CD69 PE (Biolegend). For Intracellular IFN-γ and Granzyme B detection, cells were permeabilized with PermWash (Biolegend) according to the manufacturer’s instructions. Cells were fixed with a 2% paraformaldehyde solution. Flow cytometry data were acquired in Sysmex XF-1600 flow cytometry. The gating analysis is shown in Supplementary 1. Cytokine quantification Serum cytokine levels were quantified using two methods. A LEGENDplex™ Mouse Inflammation Panel was used to simultaneously measure IL-23, IL-1α, IFN-γ, TNF-α, CCL2 (MCP-1), IL-12p70, IL-1β, IL-10, IL-6, IL-27, IL-17A, IFN-β, and GM-CSF, following the manufacturer’s instructions. Additionally, serum IFN-γ levels from mice in the protection experiments were measured using an ELISA MAX™ Standard set (Mouse) according to the manufacturer’s instructions. Data analysis FlowJo v.10.6 (BD) was used for flow cytometry data analysis. GraphPad Prism was used for statistical analysis and graph generation. The specific statistical tests used are shown in the figure legends. ImageJ was used for spleen size determination. Images were created using BioRender (YK27HU2NNF). CRTAM is indispensable for acquiring the antigen-driven effector memory CD8 T cell phenotype. CRTAM plays a role in several T-cell processes, including aspects of T-memory cell generation, such as protein polarization, cytokine secretion, and lymph node retention (17–19). To investigate the role of CRTAM in developing CD8 memory T cells, we first assessed the frequency of memory T CD8 cell subsets in the spleen under homeostatic conditions ( Supplementary 2A ). Our results show no alterations in the frequency of memory subsets between CRTAM-deficient and wild-type mice. T-cell activation induces CRTAM expression (14,17). Therefore, to activate the T cell response, we used an attenuated strain of the intracellular bacteria, S almonella Typhimurium, to examine the impact of CRTAM on the acquisition of CD8 memory cell phenotype 37 days after immunization (dpi) (SL3261) ( Figure 1A ) (14,17). CD8 memory T cells were characterized by flow cytometry as central memory (CD44 + CD62L + ), effector memory (CD44 + CD62L - ), and naïve lymphocytes (CD44 - CD62L + ). Our results show that CRTAM deficiency leads to a significant reduction in effector memory (T EM ) cells, accompanied by a corresponding increase in the naïve T cell population ( Figure 1B, C ), suggesting a critical role of CRTAM in the development of the effector memory T CD8 cell compartment. Previous in-vitro studies have shown a critical role for CRTAM in maintaining late polarization of CD44 (17). Consequently, we analyzed the surface expression of CD44 and L-selectin (CD62L) on CD8 T cells 37 days post-immunization with the attenuated Salmonella (SL3261) strain. Our results revealed a significant reduction in CD44 surface density on CD8 T cells from CRTAM-deficient mice, while CD62L expression remained unchanged ( Figure 1D-G ). This observation indicates that the reduced CD44 phenotype persists in CRTAM-deficient mice even when immunization was performed many days earlier. Subsequently, we evaluate whether these differences in the frequencies of the memory subsets persisted over time ( Figure 1H-J ). Sixty days after immunization, we also observed preferential retention in the naïve phenotype ( Figure 1H ), a sustained reduction in CD8 central memory (T CM ) cells ( Figure 1I ), and a restoration of T EM frequency. These findings suggest that CRTAM may play a role in maintaining the memory phenotype or retention of these cells within the spleen, thus indicating a potential function for CRTAM in the establishment of CD8 T memory cells. The potential role of CRTAM in restraining the equilibrium between CD8 T cell effector and memory precursor lineages. Previous studies have shown that the establishment of CD8 T cell memory can be followed during the acute phase of the immune response by differential expression of CD127 (IL-7Rα) and KLRG1 (1). Therefore, seven days after immunization with an attenuated strain of Salmonella Typhimurium (SL3261), we analyzed the frequency of CD8 effectors T cells, central memory, and effector memory cells. In Figure 2A-D , the results show a reduction in effector CD8 T cells (CD44 - CD62L - ) in the spleen and draining lymph node (DLN; mesenteric lymph node). CD8 T cells from CRTAM-deficient mice in DLN retained a naïve phenotype, as previously observed at 37 days ( Figure 2D ). Therefore, we evaluated any change in CD8 stem cell memory cells (T SCM ). In fact, in DLN and the spleen, we found a higher frequency of CD8 SCM T cells in CRTAM-deficient mice ( Figure 2E ). These results suggest that alterations in the CD8 memory pool start during the primary immune response, and therefore, CRTAM expression is involved in the generation of the memory CD8 T cell subset. We also analyze the frequency of CD8 effector memory precursor T cells (1). We found a higher frequency of these double-positive CD8 T cells in the spleen and DLN in CRTAM-deficient mice ( Figure 2F ). These results suggest that CRTAM might affect the balance between effector and memory cells during the acute phase of the immune response. CRTAM modifies the microenvironment of cytokines during the acute phase of the immune response During CD8+ T cell activation, the cytokine milieu is crucial for memory cell generation, and T cells from CRTAM-deficient mice do not produce IFNγ, IL-17, and IL-22. Thus, we investigated whether CRTAM deficiency alters cytokine production during the immunization with the attenuated Salmonella Typhimurium (SL3261) ( Figure 3 ) (1,18–22). First, we evaluate the frequency of CD8+ IFN-γ+ T cells at 7 dpi ( Figure 3A ). While a higher frequency of these cells was observed in the absence of CRTAM, the amount of secreted IFN-γ was paradoxically lower ( Figure 3B ), suggesting that CRTAM may be involved in the regulation of IFN-γ secretion but not in the production of this cytokine. Additionally, levels of IL-1α, IFN-β, IL-17A, IL-1β, and TNF-α were significantly reduced ( Figure 3C - D ), supporting a role for CRTAM in the establishment of a pro-inflammatory microenvironment. CRTAM expression improves secondary response against Salmonella To assess the impact of CRTAM expression on recall responses, mice were immunized with attenuated Salmonella SL3261 and, 30 days later, challenged with a lethal dose (500 CFU) of virulent Salmonella 14028 ( Figure 4A ). Seven days post-challenge, bacterial burden in the draining lymph nodes (DLN) was evaluated. CRTAM deficiency resulted in a significantly higher bacterial burden in the DLN and a lower survival rate ( Figure 4B - C ), demonstrating a role for CRTAM in Salmonella clearance during recall responses. Given the reduced survival rate observed in CRTAM-deficient mice challenged with a lethal dose of Salmonella 14028, subsequent experiments employed a sub-lethal dose of 150 CFU of virulent Salmonella 14028. Weight percentage was assessed as an indicator of bacterial control ( Figure 4D ). Following the sub-lethal challenge, CRTAM-deficient mice exhibited significantly lower weight but improved survival ( Figure 4D and Supplementary 3A ). To evaluate the capacity function of T CD8 cells in recall responses, we measured the CD8+ IFN-γ+ frequency ( Figure 4E ). We observed a significant increase in the frequency and amount of intracellular IFN-γ in CD8 T cells from the spleen and DLN ( Figure 4F; Supplementary 3B-C ). In contrast, we detected a significant decrease in CD8 + Granzyme-B + (Grz-B + ) frequency in DLN and a trend towards reduced intracellular Grz-B levels ( Supplementary 3D-G ). All the above suggests that CRTAM expression impacts the effector capacity of T CD8+ lymphocytes in recall responses. We next investigated whether the observed functional impairment in CD8+ T cells was due to altered in vivo activation. CD8+ CD69+ cell frequency analysis in the spleen and DLN revealed no significant difference between CRTAM-deficient and control mice ( Supplementary 3H-I ). These results indicate that CRTAM expression does not affect CD8+ T cell activation, although effector functions are impacted. Also, the frequency of the memory subsets was evaluated in DLN. We observed a significant decrease in CD8 T EM in the spleen and draining lymph nodes ( Supplementary 3J ). These findings suggest that CRTAM regulates the dynamics of CD8 memory subsets during recall responses. CRTAM is involved in the retention of CD8 in the spleen during the response against Salmonella In the recall response, we found that wild-type mice exhibited splenomegaly, which was prevented in the absence of CRTAM, as seen in Figure 4A. This observation was further supported by measurements of the spleen’s area and an increase in cellularity ( Figure 4B-C ). These effects were not observed in DLN ( Supplementary 4A ). Initially, we hypothesized that this splenomegaly was a consequence of increased proliferation. However, the frequency of CD8 + Ki-67 + was independent of CRTAM expression in the spleen ( Figure 4D ). Subsequently, we evaluated the frequency of CD8 + cells and found a significant reduction in the spleen ( Figure 4E ). These parameters were not affected in DLN ( Supplementary Figure 4B-C ). Our results suggest that CRTAM plays a role in the retention of CD8 T cells during recall responses, as previously reported in primary infections (23). Discussion This study explored the role of CRTAM expression in shaping the dynamics of T CD8 memory cells. Consistent with previous findings that CRTAM deficiency does not affect steady-state T cell compartment frequencies or the expression of CD44 and CD62L, we found no significant differences in the frequency of CD8 T memory subsets under homeostatic conditions(17,24). This could be attributed to the absence of CRTAM expression in resting T cells, as its significant upregulation requires cellular activation. Therefore, its impact on establishing and maintaining memory subsets under steady-state conditions appears to be limited. In contrast, during the memory phase after antigenic stimulation, we observed a significant decrease in effector memory cells in the spleen and maintenance of central memory cells over time, skewed by CRTAM deficiency ( Figure 5 ). These findings align with previous in vitro studies in CRTAM knock-in mice, which showed a higher frequency of CD44 + subsets in activated CD4 T cells. These studies suggest that CRTAM may contribute to the acquisition of a memory phenotype and the retention of T lymphocytes in lymphoid organs (17,23,25). To our knowledge, this is the first study to evaluate the in vivo effects of CRTAM expression on regulating the memory subset. Furthermore, we observed reduced CD44 expression on CD8 T cells in CRTAM-deficient mice. This could be attributed to aberrant CD44 segregation, as CRTAM may contribute to the late polarization maintenance of CD44 but not CD62L (17). Although we cannot exclude the possibility that CRTAM-skewed segregation of CD3, Talin, or PKC-θ might affect memory cell generation in this in vivo model, further investigation is needed to confirm this hypothesis (17). Our data suggests that CRTAM could be involved in the splenic and DLN retention of effectors and memory CD8 T cells, thereby increasing their likelihood of receiving crucial survival signals derived from cell-cell contact or being exposed to interleukins such as IL-7 in the microenvironment(26–28). Furthermore, the absence of CRTAM could lead to a reduced interaction time between dendritic cells and T lymphocytes, potentially resulting in a less robust and long-lasting memory CD8 T cell pool, consistent with previous findings by Henrickson SE et al. (29). During the effector phase of the immune response, we analyze the frequency of memory and memory precursor cells. We found decreased effector cells in CRTAM-deficient mice, partially explaining their impaired control of infections with Salmonella and other pathogens (15,17,30,31). We also observed a skew toward a double-positive phenotype (KLRG1 + IL-7Rα + ) during the acute response, an intermediate state prone to memory cells with a reduced lifespan and proliferative capacity (1,32). We hypothesize that the higher frequency of these “short-lived memory cells” without CRTAM could explain the spleen’s decreased central memory cell numbers and the impaired recall responses observed in these mice. Furthermore, our data suggests that CRTAM plays a role in the effector function of CD8 T cells during immune response recovery. We observed differences in intracellular granzyme-B and IFN-γ + levels, along with weight loss in CRTAM-deficient mice, suggesting that CRTAM may be necessary for IFNγ secretion and could regulate the induction or degranulation of granzyme B in CD8 T cells, as previously reported (17,24). Finally, we found that CRTAM is involved in recruiting or retaining CD8 T cells in the spleen during recall responses. Previous studies have shown that the CRTAM-NecL-2 interaction is essential to maintaining CD8 T cells in lymph nodes and affects clonal expansion (23,30). However, in our model, we did not observe differences in CD8 T cell proliferation, activation, or retention in the DLN. In conclusion, our findings demonstrate that the adhesion molecule CRTAM plays a crucial role in regulating the dynamics of CD8 T memory subsets. While some of its effects may be specific to lymph nodes, others appear to be shared with the spleen, suggesting a complex in-vivo regulation of CRTAM in CD8 T cell biology. Author contributions GHG performed experiments and statistical analysis. GHG, JCPL, IOG, and VON participated in the study design and wrote the manuscript. All authors have read and approved the final manuscript. Acknowledgments To Felipe Cruz-Martinez (Animal Production and Experimentation Unit from Cinvestav) for his assistance in animal care. We also want to thank biologist Benjamin Ortiz-Lopez for his technical support with cytokine quantification. Funding information GHG receives support from CONAHCYT (Consejo Nacional de Humanidades, Ciencia y Tecnología), CVU 93040. CONAHCYT-México granted J.C.P.-L (CVU 781816) a postdoctoral scholarship. Conflict of Interest Statement The authors have no conflicts of interest to declare. Ethics statement The Committee for Laboratory Animal Care (CICUAL) approved the study protocol from the Center for Research and Advanced Studies of the National Polytechnic Institute (Cinvestav), approval number 00-7814. References 1. Joshi NS, Cui W, Chandele A, Lee HK, Urso DR, Hagman J, et al. Inflammation Directs Memory Precursor and Short-Lived Effector CD8+ T Cell Fates via the Graded Expression of T-bet Transcription Factor. Immunity. 2007 Aug 24;27(2):281–95. 2. Sallusto F, Lenig D, Förster R, Lipp M, Lanzavecchia L. 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C57BL6/J (+/+) or CRTAM knock-out (-/-) mice were immunized with an attenuated strain of Salmonella (SL3261). B. Representative plots of T CD8 memory subsets identified by CD44 or CD62L expression in the spleen. C. Frequency of CD8 T naïve: T N , central memory: T CM and effector emory: T EM . C57BL6/J (+/+; ●) and CRTAM knock-out (-/-; ■ ). D-E. Representative histograms (left) and Median Intense Fluorescence (MFI; Right) of CD44 expression on T CD8 from +/+ or -/- mice. F-G. Left. Representative histograms and MFI (right) of CD62L expression on T CD8 from CRTAM +/+ or -/- mice. H-J. Kinetics of naïve CD8+ T cells ( H ), Central Memory T cells (T CM , I ), and effector memory T cells (T EM , J ) in the spleen over 60 days following immunization in CRTAM +/+ or -/- mice. Each point represents a mouse. H-J. Data are presented as mean +/- SD. Statistical significance was tested by a two-tailed t -test. *, p <0.05. Figure 2. CRTAM regulates the balance of the T CD8 effector and memory cells. A and C. Representative plots of memory subsets in the spleen and draining lymph node (DLN) after immunization with an attenuated Salmonella (SL3261). B and D. Frequency of T CD8 cells naïve, T CM , T EM, and effectors at seven days post-immunization. E. Frequency of T CD8 memory stem cells (CD44 - CD62L + Sca-1 + ) from DLN and the spleen. F. Representative dot plots of KLRG1 and CD127 (IL-7Rα) of CD8 + cells from the spleen. Symbols: C57BL6/J ( +/+; ●) and CRTAM knock-out mice (-/-; ■ ). Each point represents a mouse. H-J. A two-tailed t-test tested statistical significance. *, p <0.05. Figure 3. CRTAM modifies the microenvironment of cytokines. A. Left. Representative plots of intracellular IFN-γ detection in CD8 + T cells from the spleen of C57BL6/J (+/+) and CRTAM knock-out (-/-) mice seven days after immunization with an attenuated Salmonella Typhimurium (SL3261). Right. Frequency of CD8 + IFN-γ + cells from the spleen. B. Serum quantification of IFN-γ seven days after immunization with an attenuated Salmonella Typhimurium (SL3261). C. Heatmap of serum normalized quantification of IFN-γ, IL-1α, IL-27, IL-1β, GM-CSF, IFN-β, TNF-α, MCP-1, IL-17A, IL-6, IL-10, IL-23 and IL-12p70 from C57BL6/J or CRTAM knock-out mice seven days after immunization with Salmonella Typhimurium (SL3261). D-F. Serum quantification of IL-17A (D), IL-1β (E), and IFN-β (F) seven days after immunization with Salmonella Typhimurium (SL3261). Each point represents a mouse. A two-tailed t-test tested statistical significance t -test. *, p <0.05 ; ** p<0.01. Figure 4. CRTAM impacts the efficacy of recall response. A. Experimental design. B. Colony Forming Units (CFU) of Salmonella Typhimurium (14028) recovered from draining lymph nodes of challenged C57BL6/J (+/+; ●) or CRTAM knock-out (-/-; ■ ) mice with 500 CFUs. C. Survival curve of mice challenged with 500 CFUs of Salmonella Typhimurium strain 14028 (virulent). D. Percentage of weight change from C57BL6/J or CRTAM knock-out mice challenge (+30 days) with 150 CFU (sub-lethal challenge). E. Frequency of CD8+ IFN-γ + cells from the spleen. F. Representative IFN-γ histograms (Left) and MFI (Right) in T CD8 cells from the spleen . B and D. Data are presented as mean +/- SD. C57BL6/J (+/+; ●) and CRTAM knock-out mice (-/-; ■ ). Each point represents a mouse. A two-tailed t-test tested statistical significance. *, p <0.05; p**<0.01; ****, p<0.0001. Figure 5. Schematic summary . In activated T CD8 cells, CRTAM-NecL-2 interaction contributes to effector memory phenotype acquisition, possibly due to aberrant segregation of CD44. CRTAM is necessary for phenotype maintenance or retention in the spleen of central memory cells. Also, IFN-γ secretion is CRTAM-dependent and recruitment or retention in the spleen in recall responses. Supplementary 1. Flow cytometry gating strategy. A. T lymphocytes were initially identified by CD3 expression and CD8 + T cells from this population. The expression of CD62L and CD44 was used to characterize these cells further to distinguish memory T cell subsets. Finally, a functional analysis of CD8+ T cells was performed by assessing the expression of IFN-γ and Granzyme B. Supplementary 2. The frequency of the T CD8 memory subsets under homeostatic conditions is CRTAM independent. A. Left. Diagram of the identification of memory subsets by expression of CD62L and CD44. Right. Representative plot of T CD8 memory subsets from C57BL6/J or CRTAM knock-out mice. B. Frequency of CD8 T naïve, T CM and T EM . Wild-type mice (C57BL6/J; +/+; ●) and CRTAM knock-out (-/-; ■ ). Each point represents a mouse. A two-tailed t-test tested statistical significance. ns, not significant . Supplementary 3. T CD8 + IFN-γ + frequency in DLN is affected by CRTAM expression. A. Survival curve of mice challenged with 150 CFU Salmonella strain 14028 (virulent). Wild-type mice (C57BL6/J; +/+; ●) and CRTAM knock-out (-/-; ■ ). B. Frequency of CD8 + IFN-γ + from DLN, seven days after a sublethal challenge with Salmonella (14028). C. IFN-γ MFI in CD8 T cells from the spleen was analyzed seven days after the sub-lethal challenge with Salmonella (14028). D-E. Frequency of CD8 + Granzyme-B + cells from the spleen (D) and DLN (E) after a sub-lethal challenge, respectively. F. Left. Representative histograms of Granzyme-B in CD8 T cells. Right. MFI of Granzyme-B in CD8 T cells from the spleen. G. Granzyme-B MFI in T CD8 cells from DLN. H-I. The frequency of CD8+ CD69+ was analyzed seven days after the sub-lethal challenge with Salmonella strain 14028 from DLN and the spleen, respectively. J. The frequency of T CM , T EM , T N , and effector T CD8 cells was analyzed seven days after the sublethal challenge with Salmonella strain 14028 (virulent) from the DLN. Data are presented as mean +/- SD. Each point represents a mouse. A two-tailed t-test tested statistical significance. Supplementary 4. CRTAM affects the retention of T CD8 cells in recall responses A. Representative images of the spleen of six mice seven days post-challenge. The bar represents 10 mm. B. Data from the area analysis of panel A. C. Total number of splenic cells. D. Frequency of CD8 + Ki-67 + from the spleen. E. Frequency of CD3 + CD8 + from the spleen. Wild-type mice (B6; +/+; ●) and CRTAM knock-out (-/-; ■ ). Each point represents a mouse. A two-tailed t-test tested statistical significance. *, p <0.05; **, p<0.01. Information & Authors Information Version history V1 Version 1 12 April 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cd8 cell cell surface molecules memory spleen and lymph nodes Authors Affiliations Gabriela Hernández-Galicia 0009-0005-8140-4721 Cinvestav View all articles by this author Jocelyn C. Pérez-Lara Cinvestav View all articles by this author Irlanda Olvera-Gomez 0000-0003-1698-6559 Hospital Juarez de Mexico View all articles by this author Vianney Ortíz-Navarrete 0000-0001-6546-9713 [email protected] Cinvestav View all articles by this author Metrics & Citations Metrics Article Usage 1264 views 597 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Gabriela Hernández-Galicia, Jocelyn C. Pérez-Lara, Irlanda Olvera-Gomez, et al. CRTAM induces an effector memory T cell phenotype of splenic CD8 T cells. Authorea . 12 April 2025. DOI: https://doi.org/10.22541/au.174446286.60104297/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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