Chemokine-guided CD4+ T cell help enhances generation of IL-6RalphahighIL-7Ralpha high prememory CD8+ T cells

CD4(+) T cells promote effective CD8(+) T cell-mediated immunity, but the timing and mechanistic details of such help remain controversial. Furthermore, the extent to which innate stimuli act independently of help in enhancing CD8(+) T cell responses is also unresolved. Using a noninfectious vaccine model in immunocompetent mice, we show that even in the presence of innate stimuli, CD4(+) T cell help early after priming is required for generating an optimal pool of functional memory CD8(+) T cells. CD4(+) T cell help increased the size of a previously unreported population of IL-6Ralpha(high)IL-7Ralpha(high) prememory CD8(+) T cells shortly after priming that showed a survival advantage in vivo and contributed to the majority of functional memory CD8(+) T cells after the contraction phase. In accord with our recent demonstration of chemokine-guided recruitment of naive CD8(+) T cells to sites of CD4(+) T cell-dendritic cell interactions, the generation of IL-6Ralpha(high)IL-7Ralpha(high) prememory as well as functional memory CD8(+) T cells depended on the early postvaccination action of the inflammatory chemokines CCL3 and CCL4. Together, these findings support a model of CD8(+) T cell memory cell differentiation involving the delivery of key signals early in the priming process based on chemokine-guided attraction of naive CD8(+) T cells to sites of Ag-driven interactions between TLR-activated dendritic cells and CD4(+) T cells. They also reveal that elevated IL-6Ralpha expression by a subset of CD8(+) T cells represents an early imprint of CD4(+) T cell helper function that actively contributes to the survival of activated CD8(+) T cells.     

Tec kinases Itk and Rlk are required for CD8+ T cell responses to virus infection independent of their role in CD4+ T cell help

Itk and Rlk are members of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. These proteins are involved in the regulation of signaling processes downstream of the TCR in CD4(+) T cells, particularly in the phosphorylation of phospholipase C-gamma1 after TCR activation; furthermore, both Itk and Rlk are important in CD4(+) T cell development, differentiation, function, and homeostasis. However, few studies have addressed the roles of these kinases in CD8(+) T cell signaling and function. Using Itk(-/-) and Itk(-/-)Rlk(-/-) mice, we examined the roles of these Tec family kinases in CD8(+) T cells, both in vitro and in vivo. These studies demonstrate that the loss of Itk and Rlk impairs TCR-dependent signaling, causing defects in phospholipase C-gamma1, p38, and ERK activation as well as defects in calcium flux and cytokine production in vitro and expansion and effector cytokine production by CD8(+) T cells in response to viral infection. These defects cannot be rescued by providing virus-specific CD4(+) T cell help, thereby substantiating the important role of Tec kinases in CD8(+) T cell signaling.     

Protective CD8 memory T cell responses to mouse melanoma are generated in the absence of CD4 T cell help

Background

We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma.

Methodology and Principal Findings

To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (T_reg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete T_reg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision.

Conclusions and Significance

This work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Rat peripheral CD4+CD8+ T lymphocytes are partially immunocompetent thymus-derived cells that undergo post-thymic maturation to become functionally mature CD4+ T lymphocytes

CD4+CD8+ double-positive (DP) T cells represent a minor subpopulation of T lymphocytes found in the periphery of adult rats. In this study, we show that peripheral DP T cells appear among the first T cells that colonize the peripheral lymphoid organs during fetal life, and represent approximately 40% of peripheral T cells during the perinatal period. Later their proportion decreases to reach the low values seen in adulthood. Most DP T cells are small size lymphocytes that do not exhibit an activated phenotype, and their proliferative rate is similar to that of the other peripheral T cell subpopulations. Only 30-40% of DP T cells expresses CD8beta chain, the remaining cells expressing CD8alphaalpha homodimers. However, both DP T cell subsets have an intrathymic origin since they appear in the recent thymic emigrant population after injection of FITC intrathymically. Functionally, although DP T cells are resistant to undergo apoptosis in response to glucocorticoids, they show poor proliferative responses upon CD3/TCR stimulation due to their inability to produce IL-2. A fraction of DP T cells are not actively synthesizing the CD8 coreceptor, and they gradually differentiate to the CD4 cell lineage in reaggregation cultures. Transfer of DP T lymphocytes into thymectomized SCID mice demonstrates that these cells undergo post-thymic maturation in the peripheral lymphoid organs and that their CD4 cell progeny is fully immunocompetent, as judged by its ability to survive and expand in peripheral lymphoid organs, to proliferate in response to CD3 ligation, and to produce IL-2 upon stimulation.     

Initial antigen encounter programs CD8+ T cells competent to develop into memory cells that are activated in an antigen-free, IL-7- and IL-15-rich environment

Although much is known concerning the immunobiology of CD8+ T memory cells, the initial events favoring the generation of CD8+ T memory cells remain poorly defined. Using a culture system that yields memory-like CD8+ T cells, we show that 1 day after Ag encounter, Ag-activated T cells developed into memory-like T cells, but this optimally occurred 3 days after Ag encounter. Key phenotypic, functional, and molecular properties that typify central memory T cells were expressed within 48 h when the activated CD8+ T cells were cultured with IL-7 or IL-15 in the absence of Ag or following transfer into normal mice. These data support a model whereby Ag activation of naive CD8+ T cells not only programs effector cell expansion and contraction but the potential to develop into a memory cell which ensues in an Ag-free environment containing IL-7 or IL-15.     

IL-2 and IL-15 regulate CD154 expression on activated CD4 T cells

The cellular and humoral immune system is critically dependent upon CD40-CD154 (CD40 ligand) interactions between CD40 expressed on B cells, macrophages, and dendritic cells, and CD154 expressed primarily on CD4 T cells. Previous studies have shown that CD154 is transiently expressed on CD4 T cells after T cell receptor engagement in vitro. However, we found that stimulation of PBLs with maximal CD28 costimulation, using beads coupled to Abs against CD3 and CD28, led to a very prolonged expression of CD154 on CD4 cells (>4 days) that was dependent upon autocrine IL-2 production. Previously activated CD4 T cells could respond to IL-2, or the related cytokine IL-15, by de novo CD154 production and expression without requiring an additional signal from CD3 and CD28. These results provide evidence that CD28 costimulation of CD4 T cells, through autocrine IL-2 production, maintains high levels of CD154 expression. This has significant impact on our understanding of the acquired immune response and may provide insight concerning the mechanisms underlying several immunological diseases.     

Frequency analysis of CD4+CD8+ T cells cloned with IL-4

The coexpression of both CD4 and CD8 molecules on T cells occurs in the peripheral blood at a low frequency and can be generated transiently on CD4+ peripheral blood T cells by treatment with lectin which induces CD8 biosynthesis and cell surface expression. We have cloned T cells in a nonselective fashion from normal subjects in the presence of either IL-2, rIL-4 and IL-2, or rIL-4 and have examined the phenotypic expression of CD4 and CD8. The addition of excess rIL-4 increased the expression of CD8 on the surface of CD4+ T cell clones but did not increase CD4 expression on CD8+ T cell clones. There were three patterns of CD4 and CD8 expression observed: high density CD8 with no CD4 expression; high density CD4 with low CD8 expression; or high density CD4 with higher cell surface CD8 expression which was regulated by the presence of rIL-4. CD4+ T cell clones originally cultured in IL-2 and rIL-4 and subsequently grown in IL-2 alone exhibited decreased expression of the CD8 molecule. The increased expression of CD8 did not correlate with NK activity or lectin-dependent cytotoxicity in an antigen independent system. In addition, rIL-4 alone or in combination with IL-2 appeared to accelerate the growth curve of T cell clones as compared to IL-2 alone. These results show that IL-4 can upregulate CD8 expression on CD4+ T cell clones while not effecting CD4 expression on CD8+ T cell clones. As class I MHC is the ligand for the CD8 molecule, expression of CD8 induced by IL-4 on CD4+ T cells may allow for increased nonspecific cell to cell contact during the course of an inflammatory response.     

The role of CD4 T cell help for CD8 CTL activation

CD4 T cells play an important role in the initiation and persistence of CD8 T cells responses. In this review, we report on and evaluate the mechanisms by which CD4 T cells contribute to activation of CD8 T cells and the signal pathways of the down-streaming events after CD4 T cell help.     

CD8 T cell-mediated killing of Cryptococcus neoformans requires granulysin and is dependent on CD4 T cells and IL-15

Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl(2,) or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.     

Cutting edge: Lipopolysaccharide induces IL-10-producing regulatory CD4+ T cells that suppress the CD8+ T cell response

TLR ligands are potent activators of dendritic cells and therefore function as adjuvants for the induction of immune responses. We analyzed the capacity of TLR ligands to enhance CD8+ T cell responses toward soluble protein Ag. Immunization with OVA together with LPS or poly(I:C) elicited weak CD8+ T cell responses in wild-type C57BL/6 mice. Surprisingly, these responses were greatly increased in mice lacking CD4+ T cells indicating the induction of regulatory CD4+ T cells. In vivo, neutralization of IL-10 completely restored CD8+ T cell responses in wild-type mice and OVA-specific IL-10 producing CD4+ T cells were detected after immunization with OVA plus LPS. Our study shows that TLR ligands not only activate the immune system but simultaneously induce Ag specific, IL-10-producing regulatory Tr1 cells that strongly suppress CD8+ T cell responses. In this way, excessive activation of the immune system may be prevented.     

Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection

CD4 T cells are known to assist the CD8 T cell response by activating APC via CD40-CD40 ligand (L) interactions. However, recent data have shown that bacterial products can directly activate APC through Toll-like receptors, resulting in up-regulation of costimulatory molecules necessary for the efficient priming of naive T cells. It remains unclear what role CD4 T cell help and various costimulation pathways play in the development of CD8 T cell responses during bacterial infection. In this study, we examined these questions using an intracellular bacterium, Listeria monocytogenes, as a model of infection. In CD4 T cell-depleted, CD4(-/-), and MHC class II(-/-) mice, L. monocytogenes infection induced CD8 T cell activation and primed epitope-specific CD8 T cells to levels commensurate with those in normal C57BL/6 mice. Furthermore, these epitope-specific CD8 T cells established long-term memory in CD4(-/-) mice that was capable of mounting a protective recall response. In vitro analysis showed that L. monocytogenes directly stimulated the activation and maturation of murine dendritic cells. The CD8 T cell response to L. monocytogenes was normal in CD40L(-/-) mice but defective in CD28(-/-) and CD137L(-/-) mice. These data show that in situations where infectious agents or immunogens can directly activate APC, CD8 T cell responses are less dependent on CD4 T cell help via the CD40-CD40L pathway but involve costimulation through CD137-CD137L and B7-CD28 interactions.     

Defective alloreactive CD8 T cell function and memory response in allograft recipients in the absence of CD4 help

We have shown that alloreactive CD8 T cell activation may proceed via CD4-dependent and CD4-independent pathways, and that CD8 T cell activation in Ag-primed animals is independent of CD154 costimulation. In this report, we further analyzed the activation and function of alloreactive CD8 CTL effectors in CD4 knockout (KO) skin/cardiac allograft recipients. FACS analysis showed that alloreactive CD8 T cells were activated at a significantly reduced level in CD4 KO mice. Importantly, these helpless CD8 T cells failed to develop CD154 blockade resistance following reactivation by the same alloantigen, indicative of defective memory formation. Only transient CD4 help was required, as short-term CD4 blockade at the time of first skin graft challenge only delayed alloreactive CD8 activation, without affecting the CD8 T cell memory response to a second skin graft. Moreover, postoperative CD4 blockade had no effect on alloreactive CD8 activation. Alloreactive CD8 cells generated in the absence of CD4 help exhibited decreased effector responses. Interestingly, intragraft induction of T cell-targeted chemokines early after transplant was also dependent on CD4 help, as the induction kinetics of CXCL9 and CCL5 in CD4 KO recipients was significantly delayed, coupled with similarly delayed infiltration by CD3/CD8 cells. Remarkably, helpless CD8 cells ultimately entering the graft still displayed significantly diminished T cell effector molecules (IFN-gamma, granzyme B). Thus, CD4 help is critical for alloreactive CD8 activation, function, and memory formation.     

Defective MHC class II presentation by dendritic cells limits CD4 T cell help for antitumor CD8 T cell responses

Cancer immunosurveillance failure is largely attributed to insufficient activation signals and dominant inhibitory stimuli for tumor Ag (TAg)-specific CD8 T cells. CD4 T cells have been shown to license dendritic cells (DC), thereby having the potential for converting CD8 T cell responses from tolerance to activation. To understand the potential cooperation of TAg-specific CD4 and CD8 T cells, we have characterized the responses of naive TCR transgenic CD8 and CD4 T cells to poorly immunogenic murine tumors. We found that whereas CD8 T cells sensed TAg and were tolerized, the CD4 T cells remained ignorant throughout tumor growth and did not provide help. This disparity in responses was due to normal TAg MHC class I cross-presentation by immature CD8alpha+ DC in the draining lymph node, but poor MHC class II presentation on all DC subsets due to selective inhibition by the tumor microenvironment. Thus, these results reveal a novel mechanism of cancer immunosubversion, in which inhibition of MHC-II TAg presentation on DC prevents CD4 T cell priming, thereby blocking any potential for licensing CD8alpha+ DC and helping tolerized CD8 T cells.     

Requirement for CD4 T cell help in maintenance of memory CD8 T cell responses is epitope dependent

CD4 Th cells play critical roles in stimulating Ab production and in generating primary or maintaining memory CTL. The requirement for CD4 help in generating and maintaining CTL responses has been reported to vary depending on the vector or method used for immunization. In this study, we examined the requirement for CD4 T cell help in generating and maintaining CTL responses to an experimental AIDS vaccine vector based on live recombinant vesicular stomatitis virus (VSV) expressing HIV Env protein. We found that primary CD8 T cell responses and short-term memory to HIV Env and VSV nucleocapsid (VSV N) proteins were largely intact in CD4 T cell-deficient mice. These responses were efficiently recalled at 30 days postinfection by boosting with vaccinia recombinants expressing HIV Env or VSV N. However, by 60 days postinfection, the memory/recall response to VSV N was lost in CD4-deficient mice, while the recall response HIV Env was partially maintained in the same animals for at least 90 days. This result indicates that there are epitope-specific requirements for CD4 help in the maintenance of memory CD8 T cell responses. Our results also suggest that choice of epitopes might be critical in an AIDS vaccine designed to protect against disease in the context of reduced or declining CD4 T cell help.     

Human bone marrow hosts polyfunctional memory CD4+ and CD8+ T cells with close contact to IL-15-producing cells

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4(+) and CD8(+) T cells have not yet been elucidated. In this study, we demonstrate that CD4(+) and CD8(+) effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4(+) and CD8(+) T cells are in the immediate vicinity of IL-15-producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4(+) and CD8(+) T cell subsets. Moreover, the IL-15-inducible molecules Bcl-x(L), MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15-producing cells, is important for the maintenance of a polyfunctional memory CD4(+) and CD8(+) T cell pool.     

Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells

In vivo electroporation dramatically enhances plasmid vaccine efficacy. This enhancement can be attributed to increased plasmid delivery and, possibly, to some undefined adjuvant properties. Previous reports have demonstrated CD8(+) T cell priming by plasmid vaccines is strongly dependent upon CD4(+) T cell help. Indeed, the efficacy of a plasmid vaccine expressing Escherichia coli beta-galactosidase was severely attenuated in MHC class II-deficient (C2D) mice. To determine whether electroporation could compensate for the absence of CD4(+) T cell help, C2D mice were immunized by a single administration of plasmid in combination with electroporation using two conditions which differed only by the duration of the pulse (20 or 50 msec). Both conditions elicited robust cellular and humoral responses in wild-type mice, as measured by IFN-gamma ELISPOT, anti-beta-galactosidase ELISA, and protection from virus challenge. In C2D mice, the cellular response produced by the vaccine combined with the 50-msec pulse, as measured by ELISPOT, was identical to the response in wild-type mice. The 20-msec pulse elicited a milder response that was approximately one-fifth that of the response elicited by the 50-msec pulse. By contrast, the 20-msec conditions provided comparable protection in both wild-type and C2D recipients whereas the protection elicited by the 50-msec conditions in C2D mice was weaker than in wild-type mice. Further investigation is required to understand the discordance between the ELISPOT results and outcome of virus challenge in the C2D mice. Nonetheless, using this technique to prime CD8(+) T cells using plasmid vaccines may prove extremely useful when immunizing hosts with limiting CD4(+) T cell function, such as AIDS patients.     

Clonality and longevity of CD4+CD28null T cells are associated with defects in apoptotic pathways

CD4(+)CD28(null) T cells are oligoclonal lymphocytes rarely found in healthy individuals younger than 40 yr, but are found in high frequencies in elderly individuals and in patients with chronic inflammatory diseases. Contrary to paradigm, they are functionally active and persist over many years. Such clonogenic potential and longevity suggest altered responses to apoptosis-inducing signals. In this study, we show that CD4(+)CD28(null) T cells are protected from undergoing activation-induced cell death. Whereas CD28(+) T cells underwent Fas-mediated apoptosis upon cross-linking of CD3, CD28(null) T cells were highly resistant. CD28(null) T cells were found to progress through the cell cycle, and cells at all stages of the cell cycle were resistant to apoptosis, unlike their CD28(+) counterparts. Neither the activation-induced up-regulation of the IL-2R alpha-chain (CD25) nor the addition of exogenous IL-2 renders them susceptible to Fas-mediated apoptosis. These properties of CD28(null) T cells were related to high levels of Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein, an inhibitor of Fas signaling that is normally degraded in T cells following activation in the presence of IL-2. Consistent with previous data showing protection of CD28(null) cells from spontaneous cell death, the present studies unequivocally show dysregulation of apoptotic pathways in CD4(+)CD28(null) T cells that favor their clonal outgrowth and maintenance in vivo.     

CD4 T cells are required for CD8 T cell survival during both primary and memory recall responses

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help dependent, it remains controversial when the help is needed. In this study, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to vaccinia virus infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to vaccinia virus infection and may help to design effective vaccine strategies.     

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.