Bcl6 acts as an amplifier for the generation and proliferative capacity of central memory CD8+ T cells

Central memory CD8(+) T cells (T(CM)) are considered to be more efficient than effector ones (T(EM)) for mediating protective immunity. The molecular mechanism involved in the generation of these cells remains elusive. Because Bcl6 plays a role in the generation and maintenance of memory CD8(+) T cells, we further examined this role in the process in relation to T(CM) and T(EM) subsets. In this study, we show that T(CM) and T(EM) were functionally identified in CD62L(+) and CD62L(-) memory (CD44(+)Ly6C(+)) CD8(+) T cell subsets, respectively. Although T(CM) produced similar amounts of IFN-gamma and IL-2 to T(EM) after anti-CD3 stimulation, the cell proliferation capacity after stimulation and tissue distribution profiles of T(CM) differed from those of T(EM). Numbers of T(CM) were greatly reduced and elevated in spleens of Bcl6-deficient and lck-Bcl6 transgenic mice, respectively, and those of T(EM) were constant in nonlymphoid organs of these same mice. The majority of Ag-specific memory CD8(+) T cells in spleens of these mice 10 wk after immunization were T(CM), and the number correlated with Bcl6 expression in T cells. The proliferation of Ag-specific memory CD8(+) T cells upon secondary stimulation was dramatically up-regulated in lck-Bcl6 transgenic mice, and the adoptive transfer experiments with Ag-specific naive CD8(+) T cells demonstrated that some of the up-regulation was due to the intrinsic effect of Bcl6 in the T cells. Thus, Bcl6 is apparently a crucial factor for the generation and secondary expansion of T(CM).     

OX40 costimulatory signals potentiate the memory commitment of effector CD8+ T cells

A T cell costimulatory molecule, OX40, contributes to T cell expansion, survival, and cytokine production. Although several roles for OX40 in CD8(+) T cell responses to tumors and viral infection have been shown, the precise function of these signals in the generation of memory CD8(+) T cells remains to be elucidated. To address this, we examined the generation and maintenance of memory CD8(+) T cells during infection with Listeria monocytogenes in the presence and absence of OX40 signaling. We used the expression of killer cell lectin-like receptor G1 (KLRG1), a recently reported marker, to distinguish between short-lived effector and memory precursor effector T cells (MPECs). Although OX40 was dispensable for the generation of effector T cells in general, the lack of OX40 signals significantly reduced the number and proportion of KLRG1(low) MPECs, and, subsequently, markedly impaired the generation of memory CD8(+) T cells. Moreover, memory T cells that were generated in the absence of OX40 signals in a host animal did not show self-renewal in a second host, suggesting that OX40 is important for the maintenance of memory T cells. Additional experiments making use of an inhibitory mAb against the OX40 ligand demonstrated that OX40 signals are essential during priming, not only for the survival of KLRG1(low) MPECs, but also for their self-renewing ability, both of which contribute to the homeostasis of memory CD8(+) T cells.     

OX40/OX40L costimulation affects induction of Foxp3+ regulatory T cells in part by expanding memory T cells in vivo

OX40 is a member of the TNFR superfamily and has potent T cell costimulatory activities. OX40 also inhibits the induction of Foxp3(+) regulatory T cells (Tregs) from T effector cells, but the precise mechanism of such inhibition remains unknown. In the present study, we found that CD4(+) T effector cells from OX40 ligand-transgenic (OX40Ltg) mice are highly resistant to TGF-beta mediated induction of Foxp3(+) Tregs, whereas wild-type B6 and OX40 knockout CD4(+) T effector cells can be readily converted to Foxp3(+) T cells. We also found that CD4(+) T effector cells from OX40Ltg mice are heterogeneous and contain a large population of CD44(high)CD62L(-) memory T cells. Analysis of purified OX40Ltg naive and memory CD4(+) T effector cells showed that memory CD4(+) T cells not only resist the induction of Foxp3(+) T cells but also actively suppress the conversion of naive CD4(+) T effector cells to Foxp3(+) Tregs. This suppression is mediated by the production of IFN-gamma by memory T cells but not by cell-cell contact and also involves the induction of T-bet. Importantly, memory CD4(+) T cells have a broad impact on the induction of Foxp3(+) Tregs regardless of their origins and Ag specificities. Our data suggest that one of the mechanisms by which OX40 inhibits the induction of Foxp3(+) Tregs is by inducing memory T cells in vivo. This finding may have important clinical implications in tolerance induction to transplanted tissues.     

Constitutive OX40/OX40 ligand interaction induces autoimmune-like diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4(+) T cells, but not CD8(+) T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4(+) T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4(+) T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.     

OX40 controls functionally different T cell subsets and their resistance to depletion therapy

T cell depletion is a widely used approach in clinical transplantation. However, not all T cells are equally sensitive to depletion therapies and a significant fraction of T cells persists even after aggressive treatment. The functional attributes of such T cells and the mechanisms responsible for their resistance to depletion are poorly studied. In the present study, we showed that CD4(+) T cells that are resistant to polyclonal anti-lymphocyte serum (ALS) mediated depletion exhibit phenotypic features of memory cells and uniformly express OX40 on the cell surface. Studies using the foxp3gfp knockin mice revealed that the remaining CD4(+)OX40(+) cells consist of Foxp3(+) Tregs and Foxp3(-) T effector/memory cells. The ALS-resistant CD4(+)OX40(+) cells failed to mediate skin allograft rejection upon adoptive transferring into congenic Rag(-/-) mice, but removal of Foxp3(+) Tregs from the OX40(+) cells resulted in prompt skin allograft rejection. Importantly, OX40 is critical to survival of both Foxp3(+) Tregs and T effector/memory cells. However, OX40 exhibits opposing effects on the functional status of Foxp3(+) Tregs and T effector/memory cells, as stimulation of OX40 on T effector cells induced amplified cell proliferation but stimulation of OX40 on the Foxp3(+) Tregs impaired their suppressor functions. Our study demonstrates that OX40 is a critical molecule in regulating survival and functions of depletion-resistant T cells; and these findings may have important clinical implications.     

IL-15 transpresentation augments CD8+ T cell activation and is required for optimal recall responses by central memory CD8+ T cells

Although the adaptive immune system has a remarkable ability to mount rapid recall responses to previously encountered pathogens, the cellular and molecular signals necessary for memory CD8(+) T cell reactivation are poorly defined. IL-15 plays a critical role in memory CD8(+) T cell survival; however, whether IL-15 is also involved in memory CD8(+) T cell reactivation is presently unclear. Using artificial Ag-presenting surfaces prepared on cell-sized microspheres, we specifically addressed the role of IL-15 transpresentation on mouse CD8(+) T cell activation in the complete absence of additional stimulatory signals. In this study we demonstrate that transpresented IL-15 is significantly more effective than soluble IL-15 in augmenting anti-CD3epsilon-induced proliferation and effector molecule expression by CD8(+) T cells. Importantly, IL-15 transpresentation and TCR ligation by anti-CD3epsilon or peptide MHC complexes exhibited synergism in stimulating CD8(+) T cell responses. In agreement with previous studies, we found that transpresented IL-15 preferentially stimulated memory phenotype CD8(+) T cells; however, in pursuing this further, we found that central memory (T(CM)) and effector memory (T(EM)) CD8(+) T cells responded differentially to transpresented IL-15. T(CM) CD8(+) T cells undergo Ag-independent proliferation in response to transpresented IL-15 alone, whereas T(EM) CD8(+) T cells are relatively unresponsive to transpresented IL-15. Furthermore, upon Ag-specific stimulation, T(CM) CD8(+) T cell responses are enhanced by IL-15 transpresentation, whereas T(EM) CD8(+) T cell responses are only slightly affected, both in vitro and in vivo. Thus, our findings distinguish the role of IL-15 transpresentation in the stimulation of distinct memory CD8(+) T cell subsets, and they also have implications for ex vivo reactivation and expansion of Ag-experienced CD8(+) T cells for immunotherapeutic approaches.     

A novel role of CD30/CD30 ligand signaling in the generation of long-lived memory CD8+ T cells

Memory CD8+ T cells can be divided into two subsets, central memory (T(CM)) and effector memory (T(EM)) CD8+ T cells. We found that CD30, a member of the TNFR-associated factor (TRAF)-linked TNFR superfamily, signaling is involved in differentiation of long-lived CD8+ T(CM) cells following Listeria monocytogenes infection. Although CD8+ T(EM) cells transiently accumulated in the nonlymphoid tissues of CD30 ligand (CD153-/-) mice after infection, long-lived memory CD8+ T(CM) cells were poorly generated in these mice. CCR7 mRNA expression was down-regulated in CD8+ T cells of the spleen of CD153-/- mice in vivo and the expression was up-regulated in CD8+ T(EM) cells by anti-CD30 mAb cross-linking in vitro. These results suggest that CD30/CD30 ligand signaling plays an important role in the generation of long-lived memory CD8+ T cells at least partly by triggering homing receptors for T(CM) cells.     

CD8+ T effector memory cells protect against liver-stage malaria

Identification of correlates of protection for infectious diseases including malaria is a major challenge and has become one of the main obstacles in developing effective vaccines. We investigated protection against liver-stage malaria conferred by vaccination with adenoviral (Ad) and modified vaccinia Ankara (MVA) vectors expressing pre-erythrocytic malaria Ags. By classifying CD8(+) T cells into effector, effector memory (T(EM)), and central memory subsets using CD62L and CD127 markers, we found striking differences in T cell memory generation. Although MVA induced accelerated central memory T cell generation, which could be efficiently boosted by subsequent Ad administration, it failed to protect against malaria. In contrast, Ad vectors, which permit persistent Ag delivery, elicit a prolonged effector T cell and T(EM) response that requires long intervals for an efficient boost. A preferential T(EM) phenotype was maintained in liver, blood, and spleen after Ad/MVA prime-boost regimens, and animals were protected against malaria sporozoite challenge. Blood CD8(+) T(EM) cells correlated with protection against malaria liver-stage infection, assessed by estimation of number of parasites emerging from the liver into the blood. The protective ability of Ag-specific T(EM) cells was confirmed by transfer experiments into naive recipient mice. Thus, we identify persistent CD8 T(EM) populations as essential for vaccine-induced pre-erythrocytic protection against malaria, a finding that has important implications for vaccine design.     

Critical synergy of CD30 and OX40 signals in CD4 T cell homeostasis and Th1 immunity to Salmonella

CD30 and OX40 (CD134) are members of the TNFR superfamily expressed on activated CD4 T cells, and mice deficient in both these molecules harbor a striking defect in the capacity to mount CD4 T cell-dependent memory Ab responses. This article shows that these mice also fail to control Salmonella infection because both CD30 and OX40 signals are required for the survival but not commitment of CD4 Th1 cells. These signals are also needed for the survival of CD4 T cells activated in a lymphopenic environment. Finally, Salmonella and lymphopenia are shown to act synergistically in selectively depleting CD4 T cells deficient in OX40 and CD30. Collectively these findings identify a novel mechanism by which Th1 responses are sustained.     

Mice deficient in OX40 and CD30 signals lack memory antibody responses because of deficient CD4 T cell memory

Recently, we reported that a CD4(+)CD3(-)CD11c(-) accessory cell provided OX40-dependent survival signals to follicular T cells. These accessory cells express both OX40 ligand and CD30 ligand, and the receptors, OX40 and CD30, are both expressed on Th2-primed CD4 T cells. OX40 and CD30 signals share common signaling pathways, suggesting that CD30 signals might substantially compensate in OX40-deficient mice. In this report we have dissected the signaling roles of CD30 alone and in combination with OX40. CD30-deficient mice showed an impaired capacity to sustain follicular germinal center responses, and recall memory Ab responses were substantially reduced. Deficiencies in OX40 and CD30 signals were additive; secondary Ab responses were ablated in double-deficient mice. Although the initial proliferation of OX40/CD30 double-knockout OTII transgenic T cells was comparable to that of their normal counterparts, they failed to survive in vivo, and this was associated with reduced T cell numbers associated with CD4(+)CD3(-) cells in B follicles. Finally, we show that OX40/CD30 double-knockout OTII transgenic T cells fail to survive compared with normal T cells when cocultured with CD4(+)CD3(-) cells in vitro.     

Strength of stimulus and clonal competition impact the rate of memory CD8 T cell differentiation

The developmental pathways of long-lived memory CD8 T cells and the lineage relationship between memory T cell subsets remain controversial. Although some studies indicate the two major memory T cell subsets, central memory T (T(CM)) and effector memory T (T(EM)), are related lineages, others suggest that these subsets arise and are maintained independently of one another. In this study, we have investigated this issue and examined the differentiation of memory CD8 T cell subsets by tracking the lineage relationships of both endogenous and TCR transgenic CD8 T cell responses after acute infection. Our data indicate that TCR transgenic as well as nontransgenic T(EM) differentiate into T(CM) in the absence of Ag. Moreover, the rate of memory CD8 T cell differentiation from T(EM) into the self-renewing and long-lived pool of T(CM) is influenced by signals received during priming, including Ag levels, clonal competition, and/or the duration of infection. Although some T(EM) appear to not progress to T(CM), the vast majority of T(CM) are derived from T(EM). Thus, long-lasting, Ag-independent CD8 T cell memory results from progressive differentiation of memory CD8 T cells, and the rate of memory T cell differentiation is governed by events occurring early during T cell priming.     

Persistence and function of central and effector memory CD4+ T cells following infection with a gastrointestinal helminth

Immunity in the gastrointestinal tract is important for resistance to many pathogens, but the memory T cells that mediate such immunity are poorly characterized. In this study, we show that following sterile cure of a primary infection with the gastrointestinal parasite Trichuris muris, memory CD4+ T cells persist in the draining mesenteric lymph node and protect mice against reinfection. The memory CD4+ T cells that developed were a heterogeneous population, consisting of both CD62L(high) central memory T cells (T(CM)) and CD62L(low) effector memory T cells (T(EM)) that were competent to produce the Th type 2 effector cytokine, IL-4. Unlike memory T cells that develop following exposure to several other pathogens, both CD4+ T(CM) and T(EM) populations persisted in the absence of chronic infection, and, critically, both populations were able to transfer protective immunity to naive recipients. CD62L(high)CD4+ T(CM) were not apparent early after infection, but emerged following clearance of primary infection, suggesting that they may be derived from CD4+ T(EM). Consistent with this theory, transfer of CD62L(low)CD4+ T(EM) into naive recipients resulted in the development of a population of protective CD62L(high)CD4+ T(CM). Taken together, these studies show that distinct subsets of memory CD4+ T cells develop after infection with Trichuris, persist in the GALT, and mediate protective immunity to rechallenge.     

The lipopolysaccharide adjuvant effect on T cells relies on nonoverlapping contributions from the MyD88 pathway and CD11c+ cells

Bacterial LPS is a natural adjuvant that induces profound effects on T cell clonal expansion, effector differentiation, and long-term T cell survival. In this study, we delineate the in vivo mechanism of LPS action by pinpointing a role for MyD88 and CD11c(+) cells. LPS induced long-term survival of superantigen-stimulated CD4 and CD8 T cells in a MyD88-dependent manner. By tracing peptide-stimulated CD4 T cells after adoptive transfer, we showed that for LPS to mediate T cell survival, the recipient mice were required to express MyD88. Even when peptide-specific CD4 T cell clonal expansion was dramatically boosted by enforced OX40 costimulation, OX40 only synergized with LPS to induce survival when the recipient mice expressed MyD88. Nevertheless, these activated, but moribund, T cells in the MyD88(-/-) mice acquired effector properties, such as the ability to synthesize IFN-gamma, demonstrating that effector differentiation is not automatically coupled to a survival program. We confirmed this notion in reverse fashion by showing that effector differentiation was not required for the induction of T cell survival. Hence, depletion of CD11c(+) cells did not affect LPS-driven specific T cell survival, but CD11c(+) cells were paramount for optimal effector T cell differentiation as measured by IFN-gamma potential. Thus, LPS adjuvanticity is based on MyD88 promoting T cell survival, while CD11c(+) cells support effector T cell differentiation.     

Antigen-specific central memory CD4+ T lymphocytes produce multiple cytokines and proliferate in vivo in humans

The function of Ag-specific central (T(CM)) and effector (T(EM)) memory CD4+ T lymphocytes remains poorly characterized in vivo in humans. Using CD154 as a marker of Ag-specific CD4+T cells, we studied the differentiation of memory subsets following anti-hepatitis B immunization. Hepatitis B surface Ag (HBs)-specific memory CD4+T cells were heterogeneous and included T(CM) (CCR7+CD27+) and T(EM) (CCR7(-)CD27(+/-)). HBs-specific T(CM) and T(EM) shared the capacity to produce multiple cytokines, including IL-2 and IFN-gamma. Several years postimmunization, approximately 10% of HBs-specific memory CD4+ T cells were in cycle (Ki67+) and the proliferating cells were CCR7+. These results suggest that the model of functional specialization of T(CM) and T(EM) cannot be applied to protein vaccine Ags and support the concept that T(CM) are capable of self-renewal and contribute to maintain the pool of memory cells.     

Human effector memory CD4+ T cells directly recognize allogeneic endothelial cells in vitro and in vivo

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (T(EM)) and central memory (T(CM)) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ T(EM) (CD45RO+ CCR7- CD62L-) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ T(CM) (CD45RO+ CCR7+ CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ T(EM) secrete more IFN-gamma within 48 h in response to allogeneic ECs than do T(CM). In contrast, T(EM) and T(CM) secrete comparable amounts of IFN-gamma in response to allogeneic monocytes (Mo). In the same cultures, both T(EM) and T(CM) produce IL-2 and proliferate in response to IFN-gamma-treated allogeneic human EC or Mo, but T(CM) respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-gamma secretion by CD4+ T(EM) cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-gamma production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic T(EM) production of IL-2 but not IFN-gamma. We conclude that human CD4+ T(EM) directly recognize and respond to allogeneic EC in vitro by secreting IFN-gamma and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ T(EM) can mediate allogeneic EC injury in vivo.     

Dynamic differentiation of activated human peripheral blood CD8+ and CD4+ effector memory T cells

Two functionally different memory T cell subsets were originally defined based on their different CCR7 expression profile, but the lineage relationship between these subsets referred to as central memory T cells (T(CM)) and effector memory T cells (T(EM)), is not resolved. A prevalent model proposes a linear progressive differentiation from T(CM) to T(EM). Our results demonstrate that on activation, human CCR7-CD62L- peripheral blood CD8+ and CD4+ T(EM) cells exhibit a dynamic differentiation, involving transient as well as stable changes to T(CM) phenotype and properties. Whereas the larger fraction of T(EM) cells increases expression of effector molecules, such as perforin or IFN-gamma, a smaller fraction first acquires CCR7 expression. We demonstrate that this acquisition of lymph node homing potential is associated with strong proliferation similar to that of activated T(CM) cells. After proliferation, most of these cells lose CCR7 expression again and acquire effector functions (e.g., perforin production). A small proportion (approximately 6%), however, maintain phenotypic and functional T(CM) properties over a long time interval. These results suggest that T(EM) cells provide immediate effector function by a fraction of cells as well as self-renewal by others through up-regulation of CCR7 followed by either secondary peripheral effector function or long term maintenance of T(CM)-like properties.     

Protective immunity induced with the RTS,S/AS vaccine is associated with IL-2 and TNF-α producing effector and central memory CD4 T cells

A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP)-specific CD4(+) T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (T(E/EM)) and/or central memory (T(CM)) CD4(+) T cells and whether one or both of these sub-populations is the primary source of cytokine production. We showed for the first time that PBMC from malaria-non-exposed RTS,S-immunized subjects contain both T(E/EM) and T(CM) cells that generate strong IL-2 responses following re-stimulation in vitro with CSP peptides. Moreover, both the frequencies and the total numbers of IL-2-producing CD4(+) T(E/EM) cells and of CD4(+) T(CM) cells from protected subjects were significantly higher than those from non-protected subjects. We also demonstrated for the first time that there is a strong association between the frequency of CSP peptide-reactive CD4(+) T cells producing IL-2 and the titers of CSP-specific antibodies in the same individual, suggesting that IL-2 may be acting as a growth factor for follicular Th cells and/or B cells. The frequencies of CSP peptide-reactive, TNF-α-producing CD4(+) T(E/EM) cells and of CD4(+) T(E/EM) cells secreting both IL-2 and TNF-α were also shown to be higher in protected vs. non-protected individuals. We have, therefore, demonstrated that in addition to TNF-α, IL-2 is also a significant contributing factor to RTS,S/AS vaccine induced immunity and that both T(E/EM) and T(CM) cells are major producers of IL-2.     

Pathogen-induced inflammatory environment controls effector and memory CD8+ T cell differentiation

In response to infection, CD8(+) T cells integrate multiple signals and undergo an exponential increase in cell numbers. Simultaneously, a dynamic differentiation process occurs, resulting in the formation of short-lived effector cells (SLECs; CD127(low)KLRG1(high)) and memory precursor effector cells (CD127(high)KLRG1(low)) from an early effector cell that is CD127(low)KLRG1(low) in phenotype. CD8(+) T cell differentiation during vesicular stomatitis virus infection differed significantly than during Listeria monocytogenes infection with a substantial reduction in early effector cell differentiation into SLECs. SLEC generation was dependent on Ebi3 expression. Furthermore, SLEC differentiation during vesicular stomatitis virus infection was enhanced by administration of CpG-DNA, through an IL-12-dependent mechanism. Moreover, CpG-DNA treatment enhanced effector CD8(+) T cell functionality and memory subset distribution, but in an IL-12-independent manner. Population dynamics were dramatically different during secondary CD8(+) T cell responses, with a much greater accumulation of SLECs and the appearance of a significant number of CD127(high)KLRG1(high) memory cells, both of which were intrinsic to the memory CD8(+) T cell. These subsets persisted for several months but were less effective in recall than memory precursor effector cells. Thus, our data shed light on how varying the context of T cell priming alters downstream effector and memory CD8(+) T cell differentiation.     

Molecular signatures distinguish human central memory from effector memory CD8 T cell subsets

Memory T cells are heterogeneous in terms of their phenotype and functional properties. We investigated the molecular profiles of human CD8 naive central memory (T(CM)), effector memory (T(EM)), and effector memory RA (T(EMRA)) T cells using gene expression microarrays and phospho-protein-specific intracellular flow cytometry. We demonstrate that T(CM) have a gene expression and cytokine signaling signature that lies between that of naive and T(EM) or T(EMRA) cells, whereas T(EM) and T(EMRA) are closely related. Our data define the molecular basis for the different functional properties of central and effector memory subsets. We show that T(EM) and T(EMRA) cells strongly express genes with known importance in CD8 T cell effector function. In contrast, T(CM) are characterized by high basal and cytokine-induced STAT5 phosphorylation, reflecting their capacity for self-renewal. Altogether, our results distinguish T(CM) and T(EM)/T(EMRA) at the molecular level and are consistent with the concept that T(CM) represent memory stem cells.