Memory CD8+ T cells exhibit increased antigen threshold requirements for recall proliferation

A hallmark of immunological memory is the ability of previously primed T cells to undergo rapid recall responses upon antigen reencounter. Classic work has suggested that memory T cells proliferate in response to lower doses of antigen than naive T cells and with reduced requirements for co-stimulation. In contrast to this premise, we observed that naive but not memory T cells proliferate in vivo in response to limited antigen presentation. To reconcile these observations, we tested the antigen threshold requirement for cell cycle entry in naive and central memory CD8⁺ T cells. Although both naive and memory T cells detect low dose antigen, only naive T cells activate cell cycle effectors. Direct comparison of TCR signaling on a single cell basis indicated that central memory T cells do not activate Zap70, induce cMyc expression, or degrade p27 in response to antigen levels that activate these functions in naive T cells. The reduced sensitivity of memory T cells may result from both decreased surface TCR expression and increased expression of protein tyrosine phosphatases as compared with naive T cells. Our data describe a novel aspect of memory T cell antigen threshold sensitivity that may critically regulate recall expansion.The ability of the adaptive immune system to respond more rapidly and effectively to pathogens that have been previously encountered is the basis of immunological memory. This attribute of CD8⁺ T cell memory is primarily due to an estimated 5–100-fold increase in the frequency of antigen-specific cells after memory formation over that found in naive individuals (Ahmed and Gray, 1996). Additionally, evidence suggests that clonal competition during the expansion phase of T cell priming may increase the affinity of the resulting antigen-specific effector and memory CD8⁺ T cell pool compared with the naive pool (Busch and Pamer, 1999; Zehn et al., 2009). Indeed, based on functionality, memory CD8⁺ T cells appear to be more sensitive to TCR-mediated stimulation than naive cells. Multiple studies have observed that resting memory but not naive CD8⁺ T cells can secrete cytokines and produce cytolytic effectors more rapidly than naive cells upon antigen encounter (Zimmermann et al., 1999; Veiga-Fernandes et al., 2000; Slifka and Whitton, 2001). Consistent with this ability, memory CD8⁺ T cells show epigenetic changes at cytokine gene loci that are consistent with more rapid gene expression (Kersh et al., 2006; Northrop et al., 2006). In addition, memory T cells redistribute their TCR into higher order oligomers that may increase antigen sensitivity (Kumar et al., 2011). Multiple phenotypic differences between naive and memory CD8⁺ T cells have also been described that may influence TCR reactivity including up-regulation of adhesion molecules and increased surface expression of the IL-2Rβ chain CD122 (Berard and Tough, 2002).However, the characteristics ascribed to naive and memory T cells may have been influenced by the experimental systems used to test them. For example, although memory CD8⁺ T cells reportedly proliferate in response to lower doses of antigen than naive T cells (Pihlgren et al., 1996; Curtsinger et al., 1998; London et al., 2000), little difference in peptide sensitivity was observed in the absence of exogenous IL-2 (Curtsinger et al., 1998; Zimmermann et al., 1999). Thus, the increased sensitivity of memory T cells to cytokine may be responsible for their superior response. Additionally, although some in vitro studies have found that memory CD8⁺ T cells do not require CD28-mediated co-stimulation to initiate recall expansion (Flynn and Müllbacher, 1996; Bachmann et al., 1999), B-7 expression appears to be necessary for recall expansion in vivo (Borowski et al., 2007; Boesteanu and Katsikis, 2009). These inconsistent data may be attributable to comparison of in vitro and in vivo results or inadequate analysis of the contribution of distinct memory CD8⁺ T cell subsets. Extensive phenotyping of antigen-specific T cell responses has suggested that multiple markers may co-segregate with proliferative capacity. CD8⁺ central memory T cells expressing CD44^hi, CD62L^hi, CD27^hi, CXCR3^hi, CD43^lo, KLRG1^lo, and CD127^hi exhibit the most robust recall proliferation, whereas CD44^hi, CD62L^lo, CD27^hi, CXCR3^hi, CD43^hi, KLRG1^lo, and CD127^hi effector memory T cells exhibit sustained cytotoxicity but poorer recall expansion (Wherry et al., 2003; Sallusto et al., 2004; Hikono et al., 2007; Olson et al., 2013).Intriguingly, it has been reported that after clearance of acute influenza infection, residual viral antigen presentation can drive proliferation and expansion of naive but not memory CD8⁺ T cells of the same specificity (Belz et al., 2007; Khanna et al., 2008). This observation is in contrast to the expectation that memory T cells exhibit greater responsiveness than naive cells. It has been suggested that naive and memory T cells may respond to antigen presentation by distinct DC subsets or migrate to different areas of the lymph node (Belz et al., 2007; Kastenmüller et al., 2013). Currently, it remains unclear why residual, long-lived antigen does not stimulate memory T recall proliferation. To better understand the requirements for efficient proliferative recall expansion, we have compared the activation and proliferation of TCR transgenic CD62L^hiCD44^lo naive and CD62L^hiCD44^hi central memory CD8⁺ T cells in multiple models of noninfectious antigen presentation at limiting levels in vitro and in vivo. Although we found that both naive and central memory cells received a TCR stimulus and were activated by limiting levels of antigen presentation, only naive CD8⁺ T cells entered cell cycle and expanded. This effect appeared T cell intrinsic, as naive T cells preferentially proliferated in response to limiting levels of peptide presented by multiple DC subsets. Direct comparison of naive and memory T cells indicated that resting memory CD8⁺ T cells express more cyclin-dependent kinase inhibitor p27 and do not activate effectors of cell cycle progression or Zap70 upon low dose peptide stimulation, although no defect was observed at saturating concentrations. Additionally, we found that memory T cells expressed lower levels of surface TCR and higher levels of non-receptor tyrosine phosphatases involved in negative regulation of TCR signaling. Our data clearly indicates that, surprisingly, memory CD8⁺ T cells actually exhibit a higher antigen threshold than naive CD8⁺ T cells to stimulate cell cycle entry in vitro and in vivo.     

Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell–dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10– and IL-21–mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21–induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells.Long-lived immunological memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T-dependent antigens (Ags) and underlies the success of most currently available vaccines (Ahmed and Gray, 1996; Rajewsky, 1996; Tangye and Tarlinton, 2009; Goodnow et al., 2010). PCs reside in survival niches in bone marrow and secondary lymphoid tissues and constantly produce high titers of neutralizing antibodies (Abs; Tangye and Tarlinton, 2009; Tangye, 2011). In contrast, memory B cells recirculate throughout peripheral blood, secondary lymphoid tissues, and bone marrow. Upon reexposure to Ag, they can proliferate and differentiate into Ab-secreting plasmablasts more rapidly than naive cells, thereby replenishing the PC pool and simultaneously expanding the memory cell population (Ahmed and Gray, 1996; Rajewsky, 1996; Tangye and Tarlinton, 2009).Analysis of gene-targeted mice and humans with monogenic primary immunodeficiencies has identified some of the molecular requirements for memory B cell generation. Thus, mutations in B cell–intrinsic genes (CD19/CD81, CD40, IKBKG, DOCK8, and IL2RG) or genes expressed by CD4⁺ T helper cells (CD40LG, ICOS, and SH2D1A [SAP]) all result in reductions in the frequencies of memory B cells and associated deficiencies in total serum Ig levels or Ag-specific Ab (Tangye and Tarlinton, 2009; Recher et al., 2011; Jabara et al., 2012; Tangye et al., 2012). We also have some understanding of the mechanisms that enable memory B cells to respond more rapidly and vigorously than naive cells to cognate Ag. First, memory B cells are recruited into division significantly earlier and undergo more rounds of division than naive cells (Bernasconi et al., 2002; Tangye et al., 2003a,b; Macallan et al., 2005). Second, memory B cells have higher expression of cell surface receptors, TLRs (TLR7/9/10), CD21, CD27, and TACI, that could enable them to respond more efficiently to co-stimulatory signals (Tangye et al., 1998; Bernasconi et al., 2002, 2003; Darce et al., 2007; Good et al., 2009). Third, memory B cells express heightened levels of CD80 and CD86 (Liu et al., 1995; Tangye et al., 1998; Ellyard et al., 2004; Good et al., 2009), which facilitate soliciting help from T helper cells. Fourth, memory B cells express lower levels of genes that restrict the entry of naive B cells into division, limiting their activation (Good and Tangye, 2007; Horikawa et al., 2007). Lastly, distinct signaling pathways downstream of the B cell receptor expressed by naive (i.e., IgM) or memory (IgG) cells have been identified that preferentially promote responsiveness of memory cells (Martin and Goodnow, 2002; Engels et al., 2009; Davey and Pierce, 2012). However, the requirements for cytokine-mediated regulation of naive and memory B cells remain to be determined.Human B cell differentiation is regulated by the actions of numerous cytokines, with IL-10 and IL-21, produced by T follicular helper cells (Tfh cells), being key factors in promoting proliferation, isotype switching, PC differentiation, and secretion of most Ig isotypes by not only naive B cells, but also memory B cells, including both IgM⁺ and isotype-switched subsets (Banchereau et al., 1994; Arpin et al., 1997; Pène et al., 2004; Ettinger et al., 2005; Bryant et al., 2007; Avery et al., 2008a,b). Although the functions of IL-10 and IL-21 on human B cells are similar, the effects of IL-21 exceed those of IL-10 by 10–100-fold (Bryant et al., 2007). The importance of IL-21 to immune regulation has been validated by the recent identification of IL-21R–deficient humans, who exhibit infectious susceptibility to several pathogens (Kotlarz et al., 2013). The predominance of IL-21 in regulating human B cell function over IL-10 is also indicated by the fact that IL21R mutations result in poor Ab responses after vaccination (Kotlarz et al., 2013), whereas specific Abs are produced at normal levels in individuals with mutations in IL10/IL10R (Kotlarz et al., 2012). IL-10 and IL-21 activate STAT1, STAT3, STAT5, as well as MAPK/ERK and PI3K/Akt pathways (Asao et al., 2001; Zeng et al., 2007; Avery et al., 2008b, 2010; Diehl et al., 2008). Autosomal-dominant hyper-IgE syndrome (AD-HIES) is caused by heterozygous mutations in STAT3 (Holland et al., 2007; Minegishi et al., 2007; Casanova et al., 2012). These mutations operate in a dominant-negative manner, effectively reducing the level of functional STAT3 by 75%. Loss-of-function mutations in STAT1 also underlie several immunodeficiency states, such as those characterized by selective susceptibility to infection with environmental mycobacteria and, depending on the nature of the mutation (i.e., dominant/recessive), some viruses (Boisson-Dupuis et al., 2012; Casanova et al., 2012). By examining these patients, we previously found that functional STAT3 deficiency not only severely compromised the generation of memory (i.e., CD27⁺) B cells in vivo, but prevented IL-10– and IL-21–mediated induction of PRDM1 (Blimp-1 [B lymphocyte induced maturation protein-1]) and XBP1 (X-box binding protein 1) in naive B cells and their subsequent differentiation to the PC lineage in vitro. However, STAT3 mutant (STAT3_MUT) naive B cells could still acquire expression of AICDA (activation-induced cytidine deaminase) and undergo IL-21–induced isotype switching in vitro. In contrast, STAT1 was dispensable for human B cell differentiation in vivo and in vitro (Avery et al., 2010).These findings led us to investigate further the role of STATs in governing human B cell differentiation. We have now discovered that the small number of memory B cells generated in STAT3-deficient patients are unaffected by these mutations; thus, they are capable of differentiating into Ab-secreting cells in response to STAT3-actvating cytokines as efficiently as normal memory cells. These findings demonstrate that the threshold of STAT3 activation required for B cell differentiation is significantly lower in memory compared with naive cells. Consequently, limiting amounts of functional STAT3 are sufficient to mediate memory, but not naive, B cell differentiation, thereby revealing an intrinsic difference in the requirements for activating naive versus memory B cells. The memory B cell deficiency in AD-HIES patients likely contributes to impaired Ag-specific Ab responses characteristic of these individuals. Thus, by targeting the residual population of STAT3-deficient memory B cells to respond to IL-21, it may be possible to improve humoral immunity in AD-HIES.     

Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells

The enormous diversity of the naive T cell repertoire is instrumental in generating an immune response to virtually any foreign antigen that can be processed into peptides that bind to MHC molecules. The low frequency of antigen-specific naive T cells, their high activation threshold, and the constrains of antigen-processing and presentation have hampered analysis of naive repertoires to complex protein antigens. In this study, libraries of polyclonally expanded naive T cells were used to determine frequency and antigen dose–response of human naive CD4⁺ T cells specific for a variety of antigens and to isolate antigen-specific T cell clones. In the naive repertoire, T cells specific for primary antigens, such as KLH and Bacillus anthracis protective antigen, and for recall antigens, such as tetanus toxoid, cytomegalovirus, and Mycobacterium tuberculosis purified protein derivative, were detected at frequencies ranging from 5 to 170 cells per 10⁶ naive T cells. Antigen concentrations required for half-maximal response (EC50) varied over several orders of magnitude for different naive T cells. In contrast, in the memory repertoire, T cells specific for primary antigens were not detected, whereas T cells specific for recall antigens were detected at high frequencies and displayed EC50 values in the low range of antigen concentrations. The method described may find applications for evaluation of vaccine candidates, for testing antigenicity of therapeutic proteins, drugs, and chemicals, and for generation of antigen-specific T cell clones for adoptive cellular immunotherapy.The naive T cell repertoire is extraordinarily diverse because it contains an enormous number of distinct T cell clones, each represented by only a few cells (Goldrath and Bevan, 1999). Upon antigenic stimulation in secondary lymphoid organs, rare antigen-specific naive T cells undergo clonal expansion and differentiate to effector and memory cells (Jenkins et al., 2001; Kaech and Ahmed, 2001; Sallusto et al., 2004). Thus, the memory T cell repertoire contains a collection of expanded T cell clones that reflect the antigenic experience of the individual. Molecular studies indeed established that the TCR diversity is at least 100-fold lower in the memory compared with the naive repertoire (Arstila et al., 1999).The identification and characterization of antigen-specific T cells in the naive repertoire is of fundamental relevance to understanding the process of clonal selection and of practical relevance to predicting the immunogenicity of vaccines and therapeutic proteins. Human peptide-specific CD8⁺ T cells can be directly identified using soluble peptide/MHC class I tetramers (Altman et al., 1996). However, because of the low frequency of naive T cell precursors, this method can be successfully used only in special cases when the frequency is exceptionally high, such as for a Melan-A/MART-1 peptide bound to HLA-A2 (Pittet et al., 1999; Dunbar et al., 2000). Several laboratories have also developed human MHC class II multimers to detect peptide-specific memory CD4⁺ T cells (Novak et al., 1999; Meyer et al., 2000; Cameron et al., 2002; Lemaitre et al., 2004; Moro et al., 2005), but no data are yet available as to whether these multimers can be used to identify naive CD4⁺ T cells. More recently, a method based on MHC tetramers followed by magnetic bead enrichment was used to identify antigen-specific CD4⁺ and CD8⁺ T cells in naive mice (Hataye et al., 2006; Moon et al., 2007; Obar et al., 2008). Using this method, it has been estimated that the frequency of CD4⁺ T cells specific for epitopes within ovalbumin, I-E α chain, or Salmonella typhimurium varies from 20 to 200 cells per mouse, whereas the frequency of CD8⁺ T cells specific for ovalbumin or viral peptides varies from 80 to 1,200 cells per mouse. These figures are consistent with those previously estimated using an indirect method based on adoptive transfer of TCR transgenic T cells (McHeyzer-Williams and Davis, 1995; Butz and Bevan, 1998; Blattman et al., 2002; Stetson et al., 2002; Whitmire et al., 2006).In spite of several advantages, the MHC multimer technology is not generally applicable in view of the large numbers of MHC alleles and of the requirement of an a priori knowledge of the peptide sequence. In addition, MHC tetramers can assess only one epitope at a time and may identify cells that bind, but do not recognize, the naturally processed antigen. An alternative approach is to prime naive T cells in vitro using antigen-pulsed DCs. Using this method, it has been shown that antigen-specific CD8⁺ T cells can be isolated after consecutive rounds of in vitro antigenic stimulation and enrichments (Ho et al., 2006; Wolfl et al., 2007). However, because of the extensive in vitro selection, this method is not suitable to study the human naive repertoire.With the aim of developing a method to study the human naive T cell repertoire, we set up a novel in vitro T cell assay that is based on the screening of libraries of polyclonally expanded naive CD4⁺ T cells. The assay was used to determine frequency, antigen dose–response, and epitope specificity of human naive CD4⁺ T cells specific for a variety of antigens, to isolate representative T cell clones from the naive CD4⁺ repertoire, and to compare antigen-specific T cells in the naive and memory T cell compartments.     

Naive tumor-specific CD4+ T cells differentiated in vivo eradicate established melanoma

In vitro differentiated CD8⁺ T cells have been the primary focus of immunotherapy of cancer with little focus on CD4⁺ T cells. Immunotherapy involving in vitro differentiated T cells given after lymphodepleting regimens significantly augments antitumor immunity in animals and human patients with cancer. However, the mechanisms by which lymphopenia augments adoptive cell therapy and the means of properly differentiating T cells in vitro are still emerging. We demonstrate that naive tumor/self-specific CD4⁺ T cells naturally differentiated into T helper type 1 cytotoxic T cells in vivo and caused the regression of established tumors and depigmentation in lymphopenic hosts. Therapy was independent of vaccination, exogenous cytokine support, CD8⁺, B, natural killer (NK), and NKT cells. Proper activation of CD4⁺ T cells in vivo was important for tumor clearance, as naive tumor-specific CD4⁺ T cells could not completely treat tumor in lymphopenic common gamma chain (γ_c)–deficient hosts. γ_c signaling in the tumor-bearing host was important for survival and proper differentiation of adoptively transferred tumor-specific CD4⁺ T cells. Thus, these data provide a platform for designing immunotherapies that incorporate tumor/self-reactive CD4⁺ T cells.Adoptive cellular therapy (ACT) of cancer using in vitro differentiated CD8⁺ T cells is a powerful treatment against established cancer in humans and mice. In recent years, great progress has been attained in the understanding of the mechanisms involved in enhancing treatment of large established tumors (Gattinoni et al., 2006). Lymphodepletion before adoptive therapy greatly enhances ACT in humans and mice through the creation of cytokine sinks, removal of regulatory T cells (T reg cells), and the release of toll-like receptor agonists (Gattinoni et al., 2005a; Paulos et al., 2007; Dudley et al., 2008). Recent evidence suggests that irradiation also enhances the expression of ICAM and VCAM in the tumor vasculature allowing tumor-reactive T cells to enter more readily (Quezada et al., 2008). Although CD8⁺ T cells are potent mediators of antitumor immunity, there has been little focus on tumor-specific CD4⁺ T cells. CD4⁺ Th cells are important in immunity because in the absence of help, CD8⁺ T cells can be deleted or lose the capacity to develop into memory CD8⁺ T cells upon rechallenge (Janssen et al., 2003; Antony et al., 2005; Williams et al., 2006). Therefore, the use of tumor/self-reactive CD8⁺ T cells in the adoptive immunotherapy of cancer may face similar fates because T cells must remove tumor antigen in the context of persisting self-antigen, which in some cases leads to autoimmunity (Gattinoni et al., 2006; Rosenberg et al., 2008). Adoptive cell therapies that incorporate CD4⁺ T cells are far superior to therapies that only use CD8⁺ T cell clones (Dudley et al., 2002). Therefore, one theoretical means of improving immunotherapy to self may involve the provision of tumor-reactive or self-reactive CD4⁺ T cells (Nishimura et al., 1999; Marzo et al., 2000; Antony et al., 2005), but a more direct role for CD4⁺ T cells in tumor immunity remains unclear (Ho et al., 2002; Muranski and Restifo, 2009).Recently, adoptive transfer of in vitro differentiated tumor-specific CD4⁺ T cells in humans and mice has shown promise against cancer as a therapy (Nishimura et al., 1999; Perez-Diez et al., 2007; Hunder et al., 2008; Muranski et al., 2008). This has rekindled the idea of using antigen-specific CD4⁺ Th during immunotherapy because CD4⁺ Th cells can mediate the proper signals required in vivo to activate CD8⁺ T cells and other cells of the innate immune system (Kahn et al., 1991; Hung et al., 1998; Nishimura et al., 1999; Antony et al., 2006; Williams et al., 2006). In fact, several preclinical and clinical trials have shown the importance of CD4 help during immunotherapy of cancer (Nishimura et al., 1999; Antony et al., 2006; Dudley et al., 2008). However, isolation of tumor-specific CD4⁺ T cells has been difficult (Wang, 2001) and only a few MHC class II vaccines have been produced as a result of the lack of knowledge of how to generate vaccines that specifically activate Th cells instead of tumor-specific Foxp3⁺ T reg cells (Rosenberg, 2001; Vence et al., 2007). In addition, lack of appropriate mouse models to study tumor-specific CD4⁺ T cell responses to self-antigens has hindered progress in our understanding of the role of CD4⁺ T cells in maintaining immunity to cancer.Now, with a better understanding of CD4⁺ T cell biology, the use of cytokines to differentiate and expand T cells in vitro has led to a panoply of CD4 lineages with specific in vivo functions (Weaver and Rudensky, 2009). For example, in vitro differentiated CD4⁺ Th17 tumor-specific T cells have shown superiority over CD4⁺ Th1 differentiated T cells in the adoptive immunotherapy of cancer in a mouse model of melanoma (Muranski et al., 2008). IL-2 and IL-7 in vitro expanded NY-ESO-1–specific CD4⁺ T cells in humans have also shown clinical promise in one patient who had not received prior lymphodepleting conditioning or a vaccine (Hunder et al., 2008). Although these are promising studies, the mechanisms involved in the direct therapy of cancer by CD4⁺ T cells remain elusive. Likewise, methods for enhancing adoptive immunotherapy without prior in vitro manipulation that may lead to the terminal differentiation of T cells also remain unclear (Gattinoni et al., 2005b, 2009; Klebanoff et al., 2005). Although such manipulations can lead to vaccine independence (Klebanoff et al., 2009), long-term benefits from in vivo differentiation may outweigh in vitro stimulation because the in vivo environment may provide the correct signals that cannot be attained in a culture dish.To test a direct role for CD4⁺ T cells in the immunotherapy of cancer, we used a gp75/tyrosinase-related protein (TRP) 1–specific CD4⁺ TCR transgenic (Tg) mouse that produces class II–restricted T cells that recognize mouse TRP-1 in the context of I-A^b (Muranski et al., 2008). TRP-1 is expressed in malignant melanoma and in the skin and eyes of mice and humans; therefore, this model mimics the human condition as closely as possible. Surprisingly, we found that adoptive transfer of naive TRP-1–specific CD4⁺ T cells into lymphopenic animals bearing large established melanoma caused tumor regression and depigmentation independent of vaccination, cytokine administration, and CD8⁺, B, NK, and NKT cells. This therapy was dependent on common gamma chain (γ_c) signaling in the host for survival and differentiation of CD4⁺ T cells in vivo. These data provide a better understanding for the design of immunotherapies that incorporate tumor/self-reactive CD4⁺ T cells.     

Tumor masses support naive T cell infiltration,activation, and differentiation into effectors

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.CD8 T cells are a crucial component of the adaptive immune response to malignancies. Antigen-experienced CD8 T cells specific for tumor antigens can be recovered from the blood, lymphoid organs, and tumors of both cancer patients and tumor-bearing mice. In multiple mouse tumor models, CD8 T cells are required for the immune control or rejection of the tumor (Dunn et al., 2004). In addition, recent studies have shown that the presence of tumor-infiltrating lymphocytes (TIL) is a positive prognostic factor for patients with colorectal, ovarian, esophageal, and pancreatic cancer, as well as for melanoma and glioma patients (Galon et al., 2006; Pagès et al., 2010). Multiple groups, including our own, have shown robust activation of CD8 T cells in the tumor-draining LNs over the course of tumor outgrowth (Marzo et al., 1999; Bai et al., 2001; Wolkers et al., 2001; Spiotto et al., 2002; Hargadon et al., 2006). Although two previous studies attempted to address the tumor as a potential site of naive T cell activation, they relied on temporal separation of the appearance of activated T cells in the draining LN and tumor (Bai et al., 2001) or used tumors at an early stage of growth (Shrikant and Mescher, 1999) that may not accurately reflect the structure, environment, and activating potential of an established tumor (Schreiber et al., 2006). Thus, the potential for the tumor to serve as a site of naive T cell activation remains largely unexplored.The tumor mass is an attractive site of T cell priming, as it provides a large depot of antigen and contains multiple cell types that could present that antigen (Balkwill and Mantovani, 2001). Cross-presentation of tumor antigen by DCs in the tumor-draining LN (Nelson et al., 2001; Wolkers et al., 2001; Nguyen et al., 2002; Spiotto et al., 2002; Nowak et al., 2003; Hargadon et al., 2006), as well as direct presentation by tumor cells that have migrated to the tumor-draining LN (Wolkers et al., 2001; Hargadon et al., 2006), have been demonstrated. In addition, BM-derived stromal cells within the tumor have been shown to present antigen to differentiated effectors (Zhang et al., 2007) and activated CD8 T cells have been shown to proliferate further within the microenvironment of brain tumors (Masson et al., 2007). However, it is not known whether intratumoral DCs/myeloid cells or tumor cells can activate naive CD8 T cells within the tumor mass.Naive T cells follow a restricted migratory pattern through blood, LN, and efferent lymph because of their high expression of CD62L and CCR7 and their low expression of tissue-specific adhesion molecules and chemokine receptors (von Andrian and Mackay, 2000). Therefore, a central question is whether naive T cells could access tumor masses in nonlymphoid tissues. However, multiple studies have also demonstrated that naive T cell infiltrate peripheral tissues as part of their normal migratory activity (Westermann et al., 1996; Zippelius et al., 2004; Cose et al., 2006; Galkina et al., 2006; Staton et al., 2006). Most recently, T cells with a naive phenotype have been detected in skin, lung, liver, lamina propria, and brain (Cose et al., 2006). Although tumors manipulated to express LIGHT (Yu et al., 2004) or lymphotoxin α (Schrama et al., 2001; Kim et al., 2004) can attract naive T cells, the ability of naive T cells to infiltrate unmanipulated tumors has not been addressed.A final consideration is whether the tumor microenvironment will support or suppress the activation of naive T cells. Tumors contain a diverse array of immune cells and proinflammatory cytokines (Balkwill and Mantovani, 2001; Coussens and Werb, 2002). Tumor invasion into normal tissue, hypoxia, and necrosis can augment this inflammatory response. However, tumors are also considered to be immunosuppressive. Myeloid-derived suppressor cells, T reg cells, IDO, and TGF-β have all been associated with the presence of dysfunctional CD4 and CD8 TILs (Zou, 2005; Rabinovich et al., 2007). If naive T cells access the antigen presentation within tumors, the presence of immunosuppression might limit their potential for activation and differentiation.In this study, we directly address whether naive T cells can infiltrate normal tumors and become activated there. We separated T cell activation in the tumor-draining LN from that potentially occurring in the tumor mass using FTY720, a drug that prevents the egress of T cells from LNs, and using mice lacking LNs. These methods also allowed us to track the outcomes of T cell activation in each location and examine the effector differentiation and functional activity of T cells activated within tumors. Our work demonstrates that naive CD8 T cells can infiltrate tumors, become activated there, and differentiate into functional effectors.     

Interleukin (IL)-23 mediates Toxoplasma gondii–induced immunopathology in the gut via matrixmetalloproteinase-2 and IL-22 but independent of IL-17

Peroral infection with Toxoplasma gondii leads to the development of small intestinal inflammation dependent on Th1 cytokines. The role of Th17 cells in ileitis is unknown. We report interleukin (IL)-23–mediated gelatinase A (matrixmetalloproteinase [MMP]-2) up-regulation in the ileum of infected mice. MMP-2 deficiency as well as therapeutic or prophylactic selective gelatinase blockage protected mice from the development of T. gondii–induced immunopathology. Moreover, IL-23–dependent up-regulation of IL-22 was essential for the development of ileitis, whereas IL-17 was down-regulated and dispensable. CD4⁺ T cells were the main source of IL-22 in the small intestinal lamina propria. Thus, IL-23 regulates small intestinal inflammation via IL-22 but independent of IL-17. Gelatinases may be useful targets for treatment of intestinal inflammation.Within 8 d after peroral infection with Toxoplasma gondii, susceptible C57BL/6 mice develop massive necrosis in the ileum, leading to death (Liesenfeld et al., 1996). T. gondii–induced ileitis is characterized by a CD4 T cell–dependent overproduction of proinflammatory mediators, including IFN-γ, TNF, and NO (Khan et al., 1997; Mennechet et al., 2002). Activation of CD4⁺ T cells by IL-12 and IL-18 is critical for the development of small intestinal pathology (Vossenkämper et al., 2004). Recently, we showed that LPS derived from gut flora via Toll-like receptor (TLR)–4 mediates T. gondii–induced immunopathology (Heimesaat et al., 2006). Thus, the immunopathogenesis of T. gondii–induced small intestinal pathology resembles key features of the inflammatory responses in inflammatory bowel disease (IBD) in humans and in models of experimental colitis in rodents (Liesenfeld, 2002). However, most animal models of IBD assessed inflammatory responses in the large intestine, and models of small intestinal pathology are scarce (Kosiewicz et al., 2001; Strober et al., 2002; Olson et al., 2004; Heimesaat et al., 2006).IL-12 shares the p40 subunit, IL-12Rβ1, and components of the signaling transduction pathways with IL-23 (Parham et al., 2002). There is strong evidence that IL-23, rather than IL-12, is important in the development of colitis (Yen et al., 2006). The association of IL-23R encoding variant Arg381Gln with IBD (Duerr et al., 2006) and the up-regulation of IL-23p19 in colon biopsies from patients with Crohn''s disease (Schmidt et al., 2005) underline the importance of IL-23 in intestinal inflammation. Effector mechanisms of IL-23 include the up-regulation of matrixmetalloproteinases (MMPs; Langowski et al., 2006), a large family of endopeptidases that mediate homeostasis of the extracellular matrix. MMPs were significantly up-regulated in experimental models of colitis (Tarlton et al., 2000; Medina et al., 2003) and in colonic tissues of IBD patients (von Lampe et al., 2000).Studies in mouse models of autoimmune diseases have associated the pathogenic role of IL-23 with the accumulation of CD4⁺ T cells secreting IL-17, termed Th17 cells (Aggarwal et al., 2003; Cua et al., 2003). Moreover, increased IL-17 expression was reported in the intestinal mucosa of patients with IBD (Fujino et al., 2003; Nielsen et al., 2003; Kleinschek et al., 2009).In addition to IL-17, Th17 cells also produce IL-22, a member of the IL-10 family (Dumoutier et al., 2000). IL-22, although secreted by certain immune cell populations, does not have any effects on immune cells in vitro or in vivo but regulates functions of some tissue cells (Wolk et al., 2009). Interestingly, IL-22 has been proposed to possess both protective as well as pathogenic roles. In fact, IL-22 mediated psoriasis-like skin alterations (Zheng et al., 2007; Ma et al., 2008; Wolk et al., 2009). In contrast, IL-22 played a protective role in experimental models of colitis (Satoh-Takayama et al., 2008; Sugimoto et al., 2008; Zenewicz et al., 2008; Zheng et al., 2008), in a model of Klebsiella pneumoniae infection in the lung (Aujla et al., 2007), and against liver damage caused by concanavalin A administration (Radaeva et al., 2004; Zenewicz et al., 2007). IL-22 has been reported to be produced by CD4⁺ T cells (Wolk et al., 2002; Zheng et al., 2007), γδ cells (Zheng et al., 2007), CD11c⁺ cells (Zheng et al., 2008), and natural killer cells (Cella et al., 2008; Luci et al., 2008; Sanos et al., 2009; Satoh-Takayama et al., 2008; Zheng et al., 2008). The role of IL-22 in small intestinal inflammation remains to be determined.In the present study, we investigated the role of the IL-23–IL-17 axis in T. gondii–induced small intestinal immunopathology. We show that IL-23 is essential in the development of small intestinal immunopathology by inducing local MMP-2 up-regulation that could be inhibited by prophylactic or therapeutic chemical blockage. Interestingly, IL-23–dependent IL-22 production was markedly up-regulated and essential for the development of ileal inflammation, whereas IL-17 production was down-regulated after T. gondii infection. IL-22 was mostly produced by CD4⁺ T cells in the small intestinal lamina propria.     

Skin-resident memory CD4+ T cells enhance protection against Leishmania major infection

Leishmaniasis causes a significant disease burden worldwide. Although Leishmania-infected patients become refractory to reinfection after disease resolution, effective immune protection has not yet been achieved by human vaccines. Although circulating Leishmania-specific T cells are known to play a critical role in immunity, the role of memory T cells present in peripheral tissues has not been explored. Here, we identify a population of skin-resident Leishmania-specific memory CD4⁺ T cells. These cells produce IFN-γ and remain resident in the skin when transplanted by skin graft onto naive mice. They function to recruit circulating T cells to the skin in a CXCR3-dependent manner, resulting in better control of the parasites. Our findings are the first to demonstrate that CD4⁺ T_RM cells form in response to a parasitic infection, and indicate that optimal protective immunity to Leishmania, and thus the success of a vaccine, may depend on generating both circulating and skin-resident memory T cells.The development of effective vaccines for several intracellular microbial pathogens, such as Mycobacteria, Toxoplasma, Plasmodium, and Leishmania, remains an elusive goal. Despite substantial efforts to define the mechanisms required for resistance, to develop new adjuvants, and to identify protective antigens, the long-lived cellular immunity generated in response to infection has not been recapitulated by vaccination. To address this problem in leishmaniasis, we have focused on defining the memory T cells that mediate infection-induced immunity.C57BL/6 mice show robust immunity to reinfection after resolution of a primary Leishmania major infection (referred to here as immune mice), providing a useful model to interrogate the factors that might contribute to a successful vaccine. Previous studies have shown that immune mice contain circulating CD4⁺ T cells with effector, effector memory, and central memory phenotypes (Scott et al., 2004; Colpitts et al., 2009; Colpitts and Scott, 2010). Each of these T cell subsets likely plays a role in resistance to reinfection, with the effector subsets rapidly migrating into tissues to provide protection and central memory T cells proliferating in the draining lymph node to provide a pool of new effector cells. However, whereas adoptive transfer of either effector or central memory T cells to naive mice enhances immunity to reinfection (Zaph et al., 2004), neither subset alone or in combination provides the same level of protection as that seen in intact immune animals.In addition to circulating memory T cells, an additional memory T cell subset resides in the tissues as resident memory T cells (T_RM; Kim et al., 1999; Hogan et al., 2001a; Masopust et al., 2001; Clark et al., 2006; Gebhardt et al., 2009; Wakim et al., 2010; Hofmann and Pircher, 2011; Jiang et al., 2012; Mackay et al., 2012; Schenkel et al., 2013). Several studies have described T_RM cells that mediate immunity to acute viral infections, such as vaccinia, herpes simplex, influenza, and lymphocytic choriomeningitis virus (Gebhardt et al., 2009; Teijaro et al., 2011; Jiang et al., 2012; Schenkel et al., 2013). These T_RM cells can be found in the gut, brain, lung, and skin (Kim et al., 1999; Clark et al., 2006; Wakim et al., 2010; Teijaro et al., 2011), and their location allows them to respond immediately to control a challenge infection without the delay associated with the mobilization of circulating T cells. Additionally, T_RM cells can promote rapid recruitment of effector cells from the circulation (Schenkel et al., 2013) and induce antigen-independent innate immunity (Ariotti et al., 2014; Schenkel et al., 2014), thereby accelerating and amplifying resistance to infections.CD8⁺ T_RM cells are fairly well characterized, but less is known about CD4⁺ T_RM cells. Nevertheless, recent studies using Kaede-Tg mice to facilitate tracking of T cells in the skin indicate that a population of CD4⁺ T cells appear to be skin-resident under homeostatic conditions (Bromley et al., 2013). In addition, CD4⁺ T_RM cells in the lung and vaginal mucosa have been reported to enhance resistance to influenza and herpes simplex virus, respectively (Hogan et al., 2001b; Teijaro et al., 2011; Iijima and Iwasaki, 2014; Laidlaw et al., 2014), and a population of human tissue-resident CD4⁺ T cells remain in the skin after circulating T cells have been depleted (Clark et al., 2012; Watanabe et al., 2015). However, the potential role of CD4⁺ T_RM cells in establishing resistance to chronic parasitic infections, such as L. major, is virtually unknown.Here, we identify for the first time a tissue-resident population of CD4⁺ T cells that seed the skin after L. major infection. These cells are observed in skin sites far from the primary infection site, and persist long term in immune mice. They produce IFN-γ in response to stimulation with L. major and, during a secondary challenge, act as sentinels to rapidly recruit circulating memory cells, resulting in enhanced protection against reinfection. Thus, our results suggest that these previously unidentified memory CD4⁺ T cells are instrumental in protection against L. major parasites and should now be considered during vaccine development.     

IL-27 inhibits HIV-1 infection in human macrophages by down-regulating host factor SPTBN1 during monocyte to macrophage differentiation

The susceptibility of macrophages to HIV-1 infection is modulated during monocyte differentiation. IL-27 is an anti-HIV cytokine that also modulates monocyte activation. In this study, we present new evidence that IL-27 promotes monocyte differentiation into macrophages that are nonpermissive for HIV-1 infection. Although IL-27 treatment does not affect expression of macrophage differentiation markers or macrophage biological functions, it confers HIV resistance by down-regulating spectrin β nonerythrocyte 1 (SPTBN1), a required host factor for HIV-1 infection. IL-27 down-regulates SPTBN1 through a TAK-1–mediated MAPK signaling pathway. Knockdown of SPTBN1 strongly inhibits HIV-1 infection of macrophages; conversely, overexpression of SPTBN1 markedly increases HIV susceptibility of IL-27–treated macrophages. Moreover, we demonstrate that SPTBN1 associates with HIV-1 gag proteins. Collectively, our results underscore the ability of IL-27 to protect macrophages from HIV-1 infection by down-regulating SPTBN1, thus indicating that SPTBN1 is an important host target to reduce HIV-1 replication in one major element of the viral reservoir.Macrophages, as a major target of HIV-1, play an important role in HIV-1 infection. Macrophage infection is found extensively in body tissues and contributes to HIV-1 pathogenesis (Koenig et al., 1986; Salahuddin et al., 1986; Wang et al., 2001; Smith et al., 2003). Macrophage lineage cells are among the first cells to be infected because most viruses involved in the first round of infection use CCR5 as the co-receptor to initiate HIV-1 replication in vivo (Philpott, 2003). Once infected, macrophages have been shown to promote rapid virus dissemination by transmitting virus particles to CD4⁺ T cells via a transit “virological synapse” (Groot et al., 2008). Although most CD4⁺ T cells are eventually killed by HIV-1, infected macrophages survive longer and can harbor virus particles in intracellular compartments (Raposo et al., 2002; Pelchen-Matthews et al., 2003), thus maintaining a hidden HIV-1 reservoir for ongoing infection (Wahl et al., 1997; Lambotte et al., 2000; Zhu et al., 2002; Smith et al., 2003; Sharova et al., 2005). Collectively, macrophage infection is involved throughout the progression of disease. Therefore, restriction of macrophage infection may provide a key to eradication of HIV-1 infection.HIV-1 infection is modulated by a variety of host cellular factors. HIV-1 has evolved to have specific viral proteins to counteract certain host restriction factors. Human HIV-1 restriction factors, including APOBEC3G and BST-2, have been reported (Neil et al., 2008; Sheehy et al., 2002) and models of how HIV-1 overcomes these restrictions have been described in reviews (Evans et al., 2010; Goila-Gaur and Strebel, 2008). More recently, SAMHD1, a restriction factor of myeloid cells, was found to limit HIV replication by depleting intracellular dNTPs, and it is largely opposed by Vpx (Hrecka et al., 2011; Laguette et al., 2011; Lahouassa et al., 2012). Release of these host restrictions, however, does not guarantee productive infection. HIV-1, with a limited genome of nine open reading frames, has to fully exploit an array of cellular proteins to facilitate its life cycle at almost every step (Goff, 2007). Genome-wide siRNA screens, using 293T or HeLa cells as HIV-1 targets, have revealed hundreds of potential supportive host factors (Brass et al., 2008; Zhou et al., 2008), only some of which have been validated in primary target cells. Regulation of host factors, both inhibitory and supportive, may offer great opportunities to prevent HIV-1 infection of macrophages.Cytokine-mediated immunoregulation is an effective way to inhibit HIV-1 infection in cells of myeloid lineage (Kedzierska and Crowe, 2001). Our previous studies have demonstrated that IL-27 strongly inhibits HIV-1 replication in terminally differentiated monocyte-derived macrophages (MDMs) (Fakruddin et al., 2007). IL-27 is an IL-12 family cytokine mainly produced by dendritic cells and macrophages (Kastelein et al., 2007). It was originally characterized as a proinflammatory cytokines to induce Th1 responses in T cells (Pflanz et al., 2004; Villarino et al., 2004). However, the IL-27 receptor complex, consisting of WSX-1 and glycoprotein 130 (gp130), is also expressed on monocytes (Pflanz et al., 2004) and recent evidence has supported a role for IL-27 in monocyte activation (Kalliolias and Ivashkiv, 2008; Guzzo et al., 2010a). In the current study, we aim to investigate the role of IL-27 stimulation during monocyte differentiation in modulating macrophage susceptibility to HIV-1 infection, and our study will help to evaluate whether IL-27 can be used to prevent HIV-1 infection of macrophages.     

One naive T cell,multiple fates in CD8+ T cell differentiation

The mechanism by which the immune system produces effector and memory T cells is largely unclear. To allow a large-scale assessment of the development of single naive T cells into different subsets, we have developed a technology that introduces unique genetic tags (barcodes) into naive T cells. By comparing the barcodes present in antigen-specific effector and memory T cell populations in systemic and local infection models, at different anatomical sites, and for TCR–pMHC interactions of different avidities, we demonstrate that under all conditions tested, individual naive T cells yield both effector and memory CD8⁺ T cell progeny. This indicates that effector and memory fate decisions are not determined by the nature of the priming antigen-presenting cell or the time of T cell priming. Instead, for both low and high avidity T cells, individual naive T cells have multiple fates and can differentiate into effector and memory T cell subsets.Activation of naive antigen-specific T cells is characterized by a vigorous proliferative burst, resulting in the formation of a large pool of effector T cells. After pathogen clearance, ∼95% of activated T cells die, leaving behind a stable pool of long-lived memory cells (Williams and Bevan, 2007). Two fundamentally different mechanisms could give rise to the production of effector and memory T cells during an immune response. First, single naive T cells may be destined to produce either effector T cells or memory T cells, but not both (“one naive cell, one fate”). As an alternative, effector and memory T cells could derive from the same clonal precursors within the naive T cell pool (“one naive cell, multiple fates”). As the fate decisions that control T cell differentiation could either be taken during initial T cell priming (i.e., before the first cell division) or at later stages, at least four conceptually different models describing effector and memory T cell differentiation can be formulated (Fig. S1).A first model predicts a separate origin of effector and memory T cells as a result of differential T cell priming by APCs. In this scenario, fate decisions would be taken before the first cell division, and even though cells destined to become memory cells may transiently display traits associated with effector T cells (e.g., expression of granzyme B or IFN-γ; see the following paragraphs), their ability for long-term survival would be predetermined. In line with this model, several studies have provided evidence that the fate of CD8⁺ T cells may, to some extent, be programmed during initial activation (Kaech and Ahmed, 2001; van Stipdonk et al., 2003; Masopust et al., 2004; Williams and Bevan, 2007; Bannard et al., 2009).A second model, which relies on recent data from Chang et al. (2007), likewise suggests that the priming APC plays the crucial role in determining effector or memory T cell fate, but by a strikingly different mechanism and with an opposite prediction concerning the lineage relationship of effector and memory T cells. Specifically, analysis of T cell–APC conjugates has shown that the first division of activated T cells can be asymmetric, with the daughter T cell that is formed proximal to the APC being more likely to contribute to the effector T cell subset and the distal daughter T cell being more likely to generate memory T cells (Chang et al., 2007). Assuming that all primary daughter cells survive and yield further progeny, these data would predict that single naive T cells contribute to both the effector and the memory subset.In contrast to these two models that are based on a determining role of the priming APC, two other models predict that T cell fate is determined by the cumulative effect of signals that not only naive T cells but also their descendants receive. The first of these models, termed the “decreasing potential model,” argues that T cell progeny that receive additional stimulation after priming undergo terminal differentiation toward the effector subset, whereas descendants that do not encounter these signals may transiently display certain effector functions but will ultimately become memory T cells (Ahmed and Gray, 1996). In support of this model, it has been demonstrated that continued inflammatory signals (Badovinac et al., 2004; Joshi et al., 2007) and prolonged antigenic stimulation (Sarkar et al., 2008) can lead descendant CD8⁺ T cells to preferentially develop into effector cells.If the descendants of all individual naive T cells have an equal chance of receiving signals for terminal differentiation, the standard decreasing potential model predicts that memory and effector T cells will be derived from the same population of naive T cells. However, there is evidence that the environmental factors that promote either terminal differentiation or memory T cell development may alter over the course of infection (Sarkar et al., 2008). A fourth model therefore argues that the progeny of T cells that are activated early or late during infection will receive distinct signals and, hence, assume (partially) different fates (van Faassen et al., 2005; D’Souza and Hedrick, 2006; Quigley et al., 2007; Stemberger et al., 2007a).A large number of studies in which cell differentiation was analyzed at the population level have been informative in revealing which effector properties can be displayed by T cells that subsequently differentiate into memory T cells (for review see Jameson and Masopust, 2009). In particular, two recent studies using IFN-γ or granzyme B reporter mice have shown that memory T cells can arise from cells that have previously transcribed IFN-γ or granzyme B genes (Harrington et al., 2008; Bannard et al., 2009). However, it is important to realize that these studies reveal little with regard to the developmental potential of individual naive T cells. Specifically, the fact that T cells that have a particular effector capacity can become memory T cells does not indicate that all naive T cells yield such effector cells, nor does it indicate that all memory T cells have gone through an effector phase.To determine the developmental potential of naive T cells, it is essential to develop technologies in which T cell responses can be analyzed at the single naive T cell level. In early work that aimed to follow T cell responses at the clonal level, TCR repertoire analysis has been used to assess the kinship of T cell populations (Maryanski et al., 1996; Kedzierska et al., 2004). However, as several naive T cell clones can share the same TCR, it has been argued that such analyses do not necessarily monitor T cell fate at the single T cell level (Stemberger et al., 2007b; Obar et al., 2008). Recently, Stemberger et al. (2007a) have reported on a more elegant approach to address naive T cell potency. Using the transfer of single naive CD8⁺ T cells into mice, this study provides direct evidence that a single naive CD8⁺ T cell can form both effector and memory cell subsets. However, the statistical power of single-cell transfer studies obviously has limitations. In addition, if homeostatic proliferation would occur before antigen-driven proliferation in this system, this would limit the conclusions that can be drawn with regard to the pluripotency of a single naive T cell.In this study, we have developed a technology that allows the generation of naive T cells that carry unique genetic tags (barcodes), and we describe how this technology can be used for the large-scale assessment of the developmental potential of single naive T cells. Using physiological frequencies of barcode-labeled naive CD8⁺ T cells of different functional avidities, we demonstrate that in both systemic and local infection models, effector and memory CD8⁺ T cell subsets share the same precursors in the naive T cell pool. These data demonstrate that under all conditions analyzed, single naive T cells do not selectively yield effector or memory T cells. Rather, T cell differentiation into effector and memory T cell subsets occurs by a one naive cell, multiple fates principle.