Enhancement of antigen-specific activation of CD8+ memory cytotoxic T cells by B cell-derived factors.

Purified CD8+ T cells from influenza A/WSN-immune BALB/c (H-2d) mice respond with the generation of secondary A/WSN-specific Tc cells in vitro when stimulated with a synthetic peptide (NPP) with a sequence derived from influenza A virus nucleoprotein with high affinity for Kd class I MHC molecules. The process of the conversion of NPP-Kd-responding Tc cell precursors into effector Tc cells in a population of CD8+ T cells occurs with no demonstrable requirements for accessory cells or their lymphokine products. The addition of culture supernatants from several mouse and human B cell lymphomas and LPS-activated normal mouse B cells to the culture of NPP-stimulated immune CD8+ T cells enhanced the induction of secondary Ag-specific Tc cells. None of the tested supernatants in the absence of Ag (NPP) induced cytolytic Tc cells, indicating that B cell-derived secretory factors can exert their activity only on Ag-exposed CD8+ T cells. The augmentatory effect of these supernatants on Ag-specific activation of memory CD8+ T cells was attributed to the synergism between B cell-derived factors and IL-2 which is produced endogenously in cultures of NPP-stimulated D8+ T cells. The possible role of B cell-derived helper factors is discussed.     

Transfer of CD8+ T Cells into SCID Mice and Activation of Memory Virus-Specific Cytotoxic T Cells

The requirements for the activation of naive and memory CD8⁺ cytotoxic T(Tc) cells into effector virus-specific Tc cells after transferring them into SCID mice were investigated. SCID mice reconstituted with splenocytes or purified CDS⁺ T cells from naive or influenza-immune syngeneic mice and immunized with influenza virus generated effector Tc cells specific for influenza virus-infected target cells in vitro. The kinetics of Ihe response varied between those two populations. The generation of effector Tc cells after transfer of memory CD8⁺ T cells indicates that there exists no absolute requirement for “help” in the activation of memory virus-immune Tc cells. However, under the conditions described here the in vitro immunogenic peptide NPP derived from influenza nucleoprotein is not sufficient to elicit a response in vivo.     

Clearance of Sendai virus by CD8+ T cells requires direct targeting to virus-infected epithelium

Minimal numbers of CD8⁺ T cells are found in bronchoalveolar lavage (BAL) populations recovered from Sendai virus-infected mice that are homozygous (?/?) for β2-microglobulin (β2-m) gene disruption. The prevalence of the CD8⁺ set was substantially increased in the pneumonic lungs of 8?12-week radiation chimeras made using substantially class I major histocompatibility complex (MHC) glycoprotein-negative β2-m (?/?) recipients and normal β2-m (+/+) bone marrow. Even so, the CD8⁺ (but not the CD4⁺) lymphocyte counts were still much lower than in the (+/+)→(+/+) controls. The (+/+)→(+/+) and (+/+)→(?/?) chimeras cleared Sendai virus and potent virus-immune CD8⁺ cytotoxic T lymphocytes (CTL) specific for H-2K^b + viral nucleoprotein peptide were found in the BAL from both groups. However, following in vivo depletion of the CD4⁺ population, only the (+/+)→(+/+) mice were able to deal with the infection. Similarly, adoptively transferred, H-2K^b-restricted CD8⁺ T cells from previously-primed (+/+) mice also failed to clear virus from the lungs of (+/+)→(?/?) chimeras infected within 2 weeks of reconstitution with bone marrow, though they were effective in the (+/+)→(+/+) controls. Sendai virus-immune CD8⁺ T cells are thus unable to eliminate virus-infected β2-m (?/?) lung epithelial cells that might be thought to be expressing very small amounts of either isolated class I heavy chain, or class I MHC glycoprotein that has bound β2-m derived from β2-m (+/+) T cells or macrophages present in the pneumonic lung. Furthermore, the CD8⁺ CTL that are being exposed to β2-m (+/+) stimulators in the BAL population cannot operate in some bystander mode to clear virus from respiratory epithelium.     

Induction of primary anti-viral cytotoxic T cells by in vitro stimulation with short synthetic peptide and interleukin-7.

The present study investigated whether a short synthetic peptide NPP, with a modified sequence (147-158 R156-) derived from influenza A virus nucleoprotein with high affinity for Kd major histocompatibility complex class I molecules, could induce primary influenza virus-specific cytotoxic T (Tc) cells in vitro. Naive BALB/c (H-2d) splenocytes did not respond to the stimulation with only NPP with the generation of effector Tc cells specific for influenza A virus-infected target cells in vitro. However, they were able to do so if cultured with NPP in the presence of IL-7. IL-7 activity in this system differed significantly from IL-2 activity in the specificity of the effect. The use of exogenous IL-2, instead of IL-7, with NPP resulted in the induction of lytic cells that lysed both influenza virus-infected and uninfected syngeneic target cells. These results suggest that IL-7 is a potent regulatory cytokine in the antigen-specific activation of resting naive Tc cell precursors and may provide the necessary conditions for the induction of human primary Tc cells in vitro.     

Human keratinocyte induction of rapid effector function in antigen-specific memory CD4+ and CD8+ T cells

The ability of human keratinocytes to present antigen to T cells is controversial and, indeed, it has been suggested that keratinocytes may promote T cell hyporesponsiveness. Furthermore, it is unclear whether keratinocytes can process antigen prior to MHC class I and class II presentation. We tested the ability of keratinocytes to induce functional responses in epitope-specific CD4+ and CD8+ memory T cells using peptides, protein and recombinant expression vectors as sources of antigen. Keratinocytes were able to efficiently process and present protein antigen to CD4+ T cells, resulting in cytokine secretion (Th1 and Th2). This interaction was dependent on keratinocyte expression of HLA class II and ICAM-1, which could be induced by IFN-gamma. In addition, keratinocytes could present virally encoded or exogenous peptide to CD8+ T cells, resulting in T cell cytokine production and target cell lysis. Finally, T cell lines grown using keratinocytes as stimulators showed no loss of function. These findings demonstrate that keratinocytes are able to efficiently process and present antigen to CD4+ and CD8+ memory T cells and induce functional responses. The findings have broad implications for the pathogenesis of cutaneous disease and for transcutaneous drug or vaccine delivery.     

Minimal activation of memory CD8+ T cell by tissue-derived dendritic cells favors the stimulation of naive CD8+ T cells

Of the many dendritic cell (DC) subsets, DCs expressing the monomorphic coreceptor CD8 alpha-chain (CD8alpha) are localized permanently in lymphoid organs, whereas 'tissue-derived DCs' remain in nonlymphoid tissues until they 'capture' antigen and then move to local lymph nodes. Here we show that after lung infection, both naive and memory CD8+ 'killer' T cells responded to influenza virus antigens presented by lymph node-resident CD8alpha+ DCs, but only naive cells responded to antigens presented by lung-derived DCs. This difference provides a mechanism for priming naive T cell responses in conditions in which robust memory predominates. Our findings have implications for immunity to pathogens that can mutate their T cell epitopes, such as influenza virus and human immunodeficiency virus, and challenge the long-held view that memory T cells have less-stringent requirements for activation than naive T cells have.     

Age-associated decrease in virus-specific CD8+ T lymphocytes during primary influenza infection

The mechanism of the age-associated decrease in CD8+ T cell response of mice to virus infection was examined in young adult (6 months) and aged (22 months) C57BL/6 mice during primary pulmonary influenza A virus infection. A significant age-associated decrease in both the percentage (P<0.0001) and number (P<0.05) of CD8+ T cells binding MHC Class I tetramers containing influenza A nucleoprotein (NP) epitope and in virus-specific CTL activity (P<0.05) was observed with pulmonary lymphocytes. The percentage of NP+CD8+ cells of individual mice strongly correlated with NP-specific cytotoxic activity (r(2)=0.77, P<0.02) and with the percentage of CD8+ cells that produced interferon-gamma (r(2)=0.86, P<0.002) in both young and aged mice. Comparable expression of the CD28, CD25, and the memory CD44(hi)/CD62L(lo) phenotype was detected on NP+CD8+ lymphocytes from mice of both age groups. There was a delay in the maximal expansion of NP+CD8+ cells in aged compared to young mice that paralleled a delay in maximal cytotoxic activity and in virus clearance. These data suggest that the age-related impairment of CD8+ lymphocyte activity during a primary influenza A infection is due to a defect in the expansion, rather than in effector activity, of influenza-specific CD8+ T cells.     

Bacterial superantigens reactivate antigen-specific CD8+ memory T cells

Superantigens stimulate naive CD4+ and CD8+ T cells in a TCR V beta- specific manner. However, it has been reported that memory T cells are unresponsive to superantigen stimulation. In this study, we show that staphylococcal enterotoxins (SE) can activate influenza virus-specific CD8+ memory cytotoxic T cells. In vivo SEB challenge of mice that had recovered from influenza virus infection (memory mice) resulted in the generation of vigorous influenza-specific cytotoxic T lymphocyte (CTL) activity and in vitro SEA or SEB stimulation of splenic T cells from memory mice, but not naive mice, also induced influenza-specific CTL. Analysis of the mechanism of activation suggested that although there may be a component of cytokine-mediated bystander activation, the CTL activity is largely generated in response to direct TCR engagement by superantigen. Moreover, influenza-specific CTL could be generated from purified CD8+ CD62L loCD44hi (memory phenotype) T cells cultured in the presence of T cell-depleted splenic antigen-presenting cells and SE. Purified CD8+ memory T cells also secreted lymphokines and synthesized DNA in response to superantigen. These results definitively demonstrate that CD8+ memory T cells respond to SE stimulation by proliferating and developing appropriate effector function. Furthermore, the data raise the possibility that otherwise inconsequential exposure to bacterial superantigens may perturb the CD8+ T cell memory pool.      

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross-presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress-inducible Hsp70 to enhance activation and proliferation of human memory CD4+ T cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen-specific CD4+ T cells as confirmed by HLA-DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen-specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ T cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen-specific CD4+ T cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ T cells.     

CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells

We studied the contribution of CD4 T cell help to survival and competitive fitness of CD8 memory T cells specific for influenza virus nucleoprotein. In agreement with recent studies, the optimal generation of functional memory CD8 T cells required CD4 help, although long-term maintenance of resting CD8 memory T cells did not absolutely depend on the presence of CD4 T cells. Nonetheless, CD4 T cells were essential during differentiation of CD8 memory T cells to imprint on them the capacity to compete effectively with other memory T cells. CD8 memory cells generated with help survived better in secondary polyclonal hosts, and co-transfer into lymphopenic hosts together with "un-helped" CD8 memory cells showed improved homeostatic expansion of CD8 memory cells that had been generated with CD4 help. Therefore, the requirement for CD4 help in CD8 T cell memory extends to homeostatic parameters that ensure the maintenance and competitive fitness of memory clones.     

Thymic selection and peptide-induced activation of T cell receptor-transgenic CD8 T cells in interleukin-2-deficient mice

The requirement for interleukin-2 (IL-2) in repertoire selection and peripheral activation of CD8 T cells was tested in mice rendered IL-2 deficient by gene targeting and expressing a transgenic T cell receptor (TcR) (F5) specific for influenza nucleoprotein (NP) 366-374 + H-2D^b. Positive selection of the transgenic F5 TcR into the CD8 compartment proceeded normally. Both in vivo and in vitro, the antigenic peptide induced depletion of immature thymocytes and proliferation of mature CD8 T cells regardless of the presence of an intact IL-2 gene. In contrast, cytotoxic T lymphocyte (CTL) activity was only generated by T cells from IL-2⁺ F5 transgenic mice. Exogenous IL-2 was able to fully restore the CTL response of IL-2^?/? responder cells in vitro. Thus, both in vivo and in vitro, clonal expansion of CD8 T cells can proceed in the absence of IL-2, whereas in peptide-immunized F5 transgenic mice, induction of cytotoxic effector function is IL-2 dependent.     

Positive selection of V beta 2+ CD8+ T cells.

T cells bearing V beta 4, V beta 6, V beta 10, V beta 14, and V beta 17a are positively selected by MHC class I and/or class II molecules with poorly elucidated mechanisms. In this paper levels of V beta 2+ CD4+ and V beta 2+ CD8+ T cells from 33 inbred, five F1 hybrid, and 48 [(C58 x DBA/2)F1 x DBA/2] backcross mice have been examined. The results show that (i) V beta 2+ CD8+ T cells are positively selected by MHC class I H-2k molecules, (ii) this positive selection might be mediated by a non-H-2 ligand(s) in association with the Kk molecule, and (iii) inbred strains of mice, so far examined, do not have endogenous superantigens for deletion of V beta 2+ T cells.     

Inhibition of T-cell mediated cytotoxicity by Novobiocin suggests multiple pathways for both CD4+ and CD8+ cytotoxic T cells.

The effect of the topoisomerase II inhibitor Novobiocin on T-cell mediated cytotoxicity was tested under various assay conditions. When effector cells were class I major histocompatibility complex (MHC)-specific CD8+ cytotoxic T cells (Tc), Novobiocin caused a biphasic pattern of inhibition and the two components of the inhibition could be separated based on Ca2+ requirement. Unseparated populations of class II MHC specific Tc, containing CD4+ and CD8+ effectors gave the same pattern of inhibition. When CD8+ cells were depleted from the latter population of effectors, different patterns of inhibition from those obtained with CD8+ Tc were seen and furthermore the target affected the pattern of inhibition. Overall the results add further support to there being more than one pathway of CD8+ T-cell mediated cytotoxicity and further illustrate differences between CD4+ and CD8+ T-cell mediated cytotoxicity.     

An important role of Tyk2 in APC function of dendritic cells for priming CD8+ T cells producing IFN-gamma

Tyrosine kinase 2 (Tyk2) contributes to the signals triggered by IL-12 for IFN-gamma production by NK cells and T cells. We found in this study that Tyk2-deficient (-/-) mice showed increased susceptibility at the early stage after an i.p. infection with Listeria monocytogenes, accompanied by impaired IFN-gamma production. The numbers of both MHC class Ib (H2-M3)- or MHC class Ia (Kb)-restricted CD8+ T cells producing IFN-gamma and exhibiting cytotoxicity were significantly decreased in Tyk2-/- mice after infection with L. monocytogenes. Using an adoptive transfer system of OT-I cells expressing OVA(257-264)/Kb-specific TCR into Tyk2-/- mice followed by challenge with recombinant L. monocytogenes expressing OVA, we found that the defective Tyk2 signaling in the host environment was at least partially responsible for the impaired CD8+ T cytotoxic-1 (Tc1) cell responses in Tyk2-/- mice following the infection. Adoptive transfer with MHC class Ib- or MHC class Ia-binding peptide-pulsed BM-derived DC from Tyk2-/- mice induced lower levels of the Ag-specific CD8+ Tc1 cells producing IFN-gamma. These results suggest that Tyk2 signaling is also important for DC function in the induction of MHC class Ia- and class Ib-restricted CD8+ Tc1 cells following L. monocytogenes infection.     

Cytotoxic T lymphocyte precursor cells specific for the major histocompatibility complex class I-like antigen, Qa-2, require CD4+ T cells to become primed in vivo and to differentiate into effector cells in vitro.

Experiments were performed to determine whether CD4+ T cells are required for the generation of cytotoxic T lymphocytes (CTL) specific for the nonpolymorphic major histocompatibility complex (MHC) class I-like antigen, Qa-2. Splenic T cells from BALB/cBy (Qa-2b) mice that had been immunized with irradiated BALB/cJ (Qa-2a) splenocytes generated CTL following in vitro stimulation with BALB/cJ splenocytes. These CTL lysed all Qa-2+, but not Qa-2- targets, regardless of the H-2 haplotypes of target cells or their non-MHC backgrounds. This apparent MHC class I-unrestricted recognition of Qa-2 antigen was confirmed using Qa-2-specific CTL clones. The Qa-2-primed CTL precursor cells (CTLp) and CTL were found to be CD8+ T cells. Primed splenocytes depleted of CD4+ T cells prior to culture failed to generate CTL, but addition of lymphokines to the culture restored the CTL generation. Stimulation of primed splenic T cells with irradiated Qa-2+ T blast cells, instead of splenocytes or B blast cells, led to little to no CTL generation, suggesting that MHC class II molecules are involved in the presentation of Qa-2 antigen to CD4+ T cells. This was also supported by the results of experiments using Qa-2+, class II- thymoma cells of BALB/c origin. Stimulation of the thymoma-primed splenic T cells with the mitomycin C-treated thymoma cells resulted in no generation of anti-Qa-2 CTL, despite the fact that high levels of CTL specific for minor histocompatibility (H) antigens and H-2d were generated by immunizing the corresponding allogeneic hosts with the thymoma. However, the addition of lymphokines rendered thymoma-primed T cells capable of generating anti-Qa-2 CTL. Both CD4+ and CD8+ T cell populations, isolated from the BALB/cJ splenocyte-primed responder cells, proliferated in vitro in response to the Qa-2+ splenocytes, suggesting that Qa-2-reactive CD4+ T cells were present in the immunized mice. Depletion of CD4+ T cells from thymectomized BALB/cBy mice with anti-L3T4 monoclonal antibodies markedly reduced, but did not eliminate anti-Qa-2 CTL generation. In contrast, depletion of CD8+ T cells led to a complete abrogation of the CTL response. Addition of lymphokines to the culture of responder cells depleted of either T cell subset did not restore their reactivity. It is concluded that anti-Qa-2 CTLp need "help" from CD4+ T cells to become primed in vivo. Furthermore, primed CTLp also need "help" or lymphokines provided by CD4+ T cells to differentiate into effector CTL in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

Self peptide/MHC class I complexes have a negligible effect on the response of some CD8+ T cells to foreign antigen

MHC molecules loaded with self peptides do not trigger a T cell immune response but may deliver signals important for peripheral T cell survival and function. It is unclear if self peptide/MHC complexes on APC in addition can influence the T cell response to co-presented foreign ligands. To address this question, TAP-sufficient and TAP-deficient cells were loaded with ovalbumin peptide (pOVA) to generate APC that present pOVA/H-2Kb complexes in the context of high or low levels of self peptide-loaded MHC class I, respectively. The two cell types were then used to stimulate different CD8+ T cells specific for ovalbumin while the number of presented pOVA/H-2Kb complexes was independently assessed by staining with 25-D1, an antibody against pOVA/H-2Kb. In each case, T cell activation was independent of TAP expression by the APC and depended exclusively on the amount of 25-D1 staining. We conclude that the number of pOVA/Kb complexes and not their frequency relative to self peptide/MHC complexes determines the response of those T cells tested here. These results imply that the repertoire of self peptide/MHC class I complexes presented by APC has a negligible effect on the response of some CD8+ T cells to foreign ligands.     

Reduced lysis by CD8+ cytotoxic T cells in mixed lymphocyte reactions induced via CD4+ T cells exposed to chemically modified antigen presenting cells.

The resistance by T lymphocytes to activation by antigen (anergy) is well documented for CD4+ T-helper (Th) cells, although less is known about CD8+ cytotoxic T lymphocytes (CTL). One widely used method of inducing anergy of CD4+Th is presentation of antigen by ECDI (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide)-fixed antigen-presenting cells (APCs). We report here that in murine mixed lymphocyte reactions (MLRs), a marked reduction in detected cytotoxicity (which is mediated predominantly by CD8+ CTL) occurs on day 7 if the bulk cultures are restimulated 2 days previously with ECDI-fixed allogeneic splenocytes. No differences were seen between untreated cultures on days 5 and 7, or on day 7 of cultures to which were added unfixed allogeneic splenocytes, fixed or unfixed syngeneic splenocytes, or 'third-party' allogeneic splenocytes, 2 days previously. The effect is not mediated directly on CD8+ cells, since MLRs depleted of CD4+ cells immediately prior to exposure to fixed allogeneic splenocytes fail to show reduced lysis. On the other hand, reduced lysis did occur if CD4+ cells, purified from the MLRs on day 4, were exposed to ECDI-fixed allogeneic splenocytes and then returned to MLRs previously depleted of CD4+ cells. Moreover the effect is overcome using exogenous interleukin-2 (IL-2). We propose that CD4+ cells, restimulated by a regimen shown previously to induce their anergy, can cause a reduction in CD(8+)-mediated cytotoxicity in MLRs.     

The generation of memory antigen-specific cytotoxic T cell responses by CD28/CD80 interactions in the absence of antigen

The interaction of co-stimulatory molecules CD80/CD86 on antigen-presenting cells with CD28 on naive CD8⁺ cytotoxic T (Tc) cells is understood to be critical in the induction of Tc effectors. CD80 is capable of providing signal 2 for the activation of Tc cells, but has no effect if encountered in the absence of specific peptide/MHC complexes (signal 1). We have found that CD80 presented in vitro to resting memory viral-immune or alloimmune Tc cells can provide sufficient stimulus for the generation of effector Tc cells in the absence of specific antigen, the peptide/MHC class I complex. Effector Tc cells generated in vitro from influenza- or class I alloantigen-primed mice by co-stimulation in the absence of antigen require exogenous interleukin (IL)-2 signaling via the cell surface-expressed IL-2 receptor or, under conditions of IL-2 blockade, exogenous IL-7. Activation of memory Tc cells by signal 1 and 2 is independent of IL-2 and IL-7. Although memory influenza-immune Tc cells did respond to CD80 in the absence of antigen, the presence of antigen +CD80 enabled an earlier induction of these Tc cells and they retained their lytic activity in vitro over a longer time period. The capacity of memory Tc cells to be activated by signal 2 alone provides one explanation for the observed heterogeneity of phenotype of memory T cells in vivo and a possible mechanism for the maintenence of memory in the absence of persisting antigen.     

Memory CD8 T cells require CD8 coreceptor engagement for calcium mobilization and proliferation, but not cytokine production

Memory T-cell responses are faster and more robust than those of their naive counterparts. The mechanisms by which memory T cells respond better to subsequent antigenic exposure remain unresolved. A portion of the more rapid response is undoubtedly the result of the increased frequency of antigen-specific cells. In addition, there are also differences in the cells themselves with respect to their requirements for costimulation and the apparent avidity of the T cells. We used major histocompatibility complex (MHC) class I tetramers to stimulate T cells to focus on the interaction of T-cell receptor (TCR)/MHC and CD8 in the absence of other molecules that are present on cell surfaces and so contribute to the activation of T cells by undefined mechanisms. Mutated MHC class I tetramers that are unable to engage CD8 were used to investigate the role of CD8 engagement in memory cell activation. Either wild-type tetramers or tetramers carrying the mutation were used to stimulate both memory and naive TCR transgenic T cells in vitro. Surprisingly, like naive cells, memory CD8(+) T cells required CD8 engagement for calcium mobilization and optimum proliferation. In contrast, the requirements for cytokine production differed. Unlike naive cells, memory cells were able to produce cytokine in the absence of CD8 engagement. This suggests both a CD8-dependent pathway for early events and a CD8-independent pathway for cytokine production in memory cells.