Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines

The antigen 85 (ag85) complex, composed of three proteins (ag85A, B and C), is a major protein component of the Mycobacterium tuberculosis cell wall. Each protein possesses a mycolyltransferase activity required for the biogenesis of trehalose dimycolate (cord factor), a dominant structure necessary for maintaining cell wall integrity. The crystal structure of recombinant ag85C from M. tuberculosis, refined to a resolution of 1.5 A, reveals an alpha/beta-hydrolase polypeptide fold, and a catalytic triad formed by Ser 124, Glu 228 and His 260. ag85C complexed with a covalent inhibitor implicates residues Leu 40 and Met 125 as components of the oxyanion hole. A hydrophobic pocket and tunnel extending 21 A into the core of the protein indicates the location of a probable trehalose monomycolate binding site. Also, a large region of conserved surface residues among ag85A, B and C is a probable site for the interaction of ag85 proteins with human fibronectin.     

Protein kinase Cdelta regulates antigen receptor-induced lytic granule polarization in mouse CD8+ CTL

Lytic granule exocytosis is the major pathway used by CD8+ CTL to kill virally infected and tumor cells. Despite the obvious importance of this pathway in adaptive T cell immunity, the molecular identity of enzymes involved in the regulation of this process is poorly characterized. One signal known to be critical for the regulation of granule exocytosis-mediated cytotoxicity in CD8+ T cells is Ag receptor-induced activation of protein kinase C (PKC). However, it is not known which step of the process is regulated by PKC. In addition, it has not been determined to date which of the PKC family members is required for the regulation of lytic granule exocytosis. By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we show that PKCdelta is required for granule exocytosis-mediated lytic function in mouse CD8+ T cells. Our studies demonstrate that PKCdelta is required for lytic granule exocytosis, but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation. Importantly, defective lytic function in PKCdelta-deficient cytotoxic lymphocytes is reversed by ectopic expression of PKCdelta. Finally, we show that PKCdelta is not involved in target cell-induced reorientation of the microtubule-organizing center, but is required for the subsequent exocytosis step, i.e., lytic granule polarization. Thus, our studies identify PKCdelta as a novel and selective regulator of Ag receptor-induced lytic granule polarization in mouse CD8+ T cells.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcgammaR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in "license to kill." These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.     

Affinity thresholds for naive CD8+ CTL activation by peptides and engineered influenza A viruses

High-avidity interactions between TCRs and peptide + class I MHC (pMHCI) epitopes drive CTL activation and expansion. Intriguing questions remain concerning the constraints determining optimal TCR/pMHCI binding. The present analysis uses the TCR transgenic OT-I model to assess how varying profiles of TCR/pMHCI avidity influence naive CTL proliferation and the acquisition of effector function following exposure to the cognate H-2K(b)/OVA(257-264) (SIINFEKL) epitope and to mutants provided as peptide or in engineered influenza A viruses. Stimulating naive OT-I CD8(+) T cells in vitro with SIINFEKL induced full CTL proliferation and differentiation that was largely independent of any need for costimulation. By contrast, in vitro activation with the low-affinity EIINFEKL or SIIGFEKL ligands depended on the provision of IL-2 and other costimulatory signals. Importantly, although they did generate potent endogenous responses, infection of mice with influenza A viruses expressing these same OVA(257) variants failed to induce the activation of adoptively transferred naive OT-I CTLps, an effect that was only partially overcome by priming with a lipopeptide vaccine. Subsequent structural and biophysical analysis of H2-K(b)OVA(257), H2-K(b)E1, and H2-K(b)G4 established that these variations introduce small changes at the pMHCI interface and decrease epitope stability in ways that would likely impact cell surface presentation and recognition. Overall, it seems that there is an activation threshold for naive CTLps, that minimal alterations in peptide sequence can have profound effects, and that the antigenic requirements for the in vitro and in vivo induction of CTL proliferation and effector function differ substantially.     

MHC recognition by hapten-specific HLA-A2-restricted CD8+ CTL

T cell recognition by peptide-specific alphabeta TCRs involves not only recognition of the peptide, but also recognition of multiple molecular features on the surface of the MHC molecule to which the peptide has been bound. We have previously shown that TCRs that are specific for five different peptides presented by HLA-A2 recognize similar molecular features on the surface of the alpha1 and alpha2 helices of the HLA-A2 molecule. We next asked whether these same molecular features of the HLA-A2 molecule would be recognized by hapten-specific HLA-A2-restricted TCRs, given that hapten-specific T cells frequently show reduced MHC dependence/restriction. The results show that a panel of CD8+ CTL that are specific for the hapten DNP bound to two different peptides presented by HLA-A2 do the following: 1) show stringent MHC restriction, and 2) are largely affected by the same mutations on the HLA-A2 molecule that affected recognition by peptide-specific CTL. A small subset of this panel of CD8+ CTL can recognize a mutant HLA-A2 molecule in the absence of hapten. These data suggest that TCR recognition of a divergent repertoire of ligands presented by HLA-A2 is largely dependent upon common structural elements in the central portion of the peptide-binding site.     

Soluble MHC-peptide complexes induce rapid death of CD8+ CTL

Soluble MHC-peptide (pMHC) complexes, commonly referred to as tetramers, are widely used to enumerate and to isolate Ag-specific CD8(+) CTL. It has been noted that such complexes, as well as microsphere- or cell-associated pMHC molecules compromise the functional integrity of CTL, e.g., by inducing apoptosis of CTL, which limits their usefulness for T cell sorting or cloning. By testing well-defined soluble pMHC complexes containing linkers of different length and valence, we find that complexes comprising short linkers (i.e., short pMHC-pMHC distances), but not those containing long linkers, induce rapid death of CTL. This cell death relies on CTL activation, the coreceptor CD8 and cytoskeleton integrity, but is not dependent on death receptors (i.e., Fas, TNFR1, and TRAILR2) or caspases. Within minutes of CTL exposure to pMHC complexes, reactive oxygen species emerged and mitochondrial membrane depolarized, which is reminiscent of caspase-independent T cell death. The morphological changes induced during this rapid CTL death are characteristic of programmed necrosis and not apoptosis. Thus, soluble pMHC complexes containing long linkers are recommended to prevent T cell death, whereas those containing short linkers can be used to eliminate Ag-specific CTL.     

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.     

Development of antigen-specific CD8+ CTL in MHC class I-deficient mice through CD4 to CD8 conversion

CD8+ CTL are the predominant tumoricidal effector cells. We find, however, that MHC class I-deficient mice depleted of CD8+ T cells are able to mount an effective antitumor immunity after immunization with fused dendritic/tumor cells. Such immunity appears to be mediated by the generation of phenotypic and functional CD8+ CTL through CD4+ to CD8+ conversion, which we have demonstrated at the single cell level. CD4+ to CD8+ conversion depends on effective in vivo activation and is promoted by CD4+ T cell proliferation. The effectiveness of this process is shown by the generation of antitumor immunity through adoptive transfer of primed CD4 T cells to provide protection against tumor cell challenge and to eliminate established pulmonary metastases.     

A melanoma multiepitope polypeptide induces specific CD8+ T-cell response

Strategies using epitope-based vaccination are being considered for melanoma immunotherapy, in an attempt to overcome failure of other modalities. In the present study, we designed and produced a multiepitope polypeptide for melanoma (MEP-mel), which contains three repeats of four antigenic epitopes (gp100: 209-217 (210M); gp100: 280-288 (288V); Mart1: 26-35 (27L); tyrosinase: 368-376 (370D). The peptides were attached to each other by linkers containing sequences recognized by the proteasome, to improve protein cleavage and antigen presentation. The results show that peptide-specific T cells produced IFN-gamma when stimulated with MEP-mel-transfected dendritic cells. The presentation of peptides by MEP-mel-transfected dendritic cells was proteasome-dependent and was more long-lasting than the presentation of exogenously delivered native peptides. When dendritic cells were loaded with MEP-mel protein, weak cross presentation was induced. The production of multiepitope molecules based on several peptides linked by sequences sensitive to proteasomal cleavage represents a promising new tool for the improvement of cancer immunotherapy.     

Human virus-specific CD8+ CTL clones revert from CD45ROhigh to CD45RAhigh in vivo: CD45RAhighCD8+ T cells comprise both naive and memory cells.

It has been generally believed that human CD8+ memory cells are principally found within the CD45ROhigh population. There are high frequencies of CD8+ memory CTL specific for the human CMV tegument phosphoprotein pp65 in PBMC of long-term virus carriers; the large population of memory CTL specific for a given pp65 peptide contains individual CTL clones that have greatly expanded. In this study, we found high frequencies of pp65 peptide-specific memory CTL precursors in the CD45ROhighCD45RA- population, but also appreciable frequencies in the CD45RAhigh subpopulation. Because the majority of CD8+ T cells in PBMC are CD45RAhigh, more of the total pp65-specific memory CTL pool is within the CD45RAhigh than in the CD45ROhigh compartment. Using clonotypic oligonucleotide probes to quantify the size of individual pp65-specific CTL clones in vivo, we found the CD45RAhigh population contributed 6- to 10-fold more than the CD45ROhigh population to the total virus-specific clone size in CD8+ cells. During primary CMV infection, an individual virus-specific CTL clone was initially CD45ROhigh, but after resolution of infection this clone was detected in both the CD45ROhigh and the CD45RAhigh populations. We conclude that CD45RA+ human CD8+ T cells do not solely comprise naive cells, but contain a very significant proportion of memory cells, which can revert from the CD45ROhigh to CD45RAhigh phenotype in vivo.     

Memory CD8+ T cells protect dendritic cells from CTL killing

CD8(+) T cells have been shown to be capable of either suppressing or promoting immune responses. To reconcile these contrasting regulatory functions, we compared the ability of human effector and memory CD8(+) T cells to regulate survival and functions of dendritic cells (DC). We report that, in sharp contrast to the effector cells (CTLs) that kill DCs in a granzyme B- and perforin-dependent mechanism, memory CD8(+) T cells enhance the ability of DCs to produce IL-12 and to induce functional Th1 and CTL responses in naive CD4(+) and CD8(+) T cell populations. Moreover, memory CD8(+) T cells that release the DC-activating factor TNF-alpha before the release of cytotoxic granules induce DC expression of an endogenous granzyme B inhibitor PI-9 and protect DCs from CTL killing with similar efficacy as CD4(+) Th cells. The currently identified DC-protective function of memory CD8(+) T cells helps to explain the phenomenon of CD8(+) T cell memory, reduced dependence of recall responses on CD4(+) T cell help, and the importance of delayed administration of booster doses of vaccines for the optimal outcome of immunization.     

Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.     

A model for CD8+ CTL tumor immunosurveillance and regulation of tumor escape by CD4 T cells through an effect on quality of CTL.

Understanding immune mechanisms influencing cancer regression, recurrence, and metastasis may be critical to developing effective immunotherapy. Using a tumor expressing HIV gp160 as a model viral tumor Ag, we found a growth-regression-recurrence pattern, and used this to investigate mechanisms of immunosurveillance. Regression was dependent on CD8 T cells, and recurrent tumors were resistant to CTL, had substantially reduced expression of epitope mRNA, but retained the gp160 gene, MHC, and processing apparatus. Increasing CTL numbers by advance priming with vaccinia virus expressing gp160 prevented only the initial tumor growth but not the later appearance of escape variants. Unexpectedly, CD4 cell depletion protected mice from tumor recurrence, whereas IL-4 knockout mice, deficient in Th2 cells, did not show this protection, and IFN-gamma knockout mice were more susceptible. Purified CD8 T cells from CD4-depleted mice following tumor regression had more IFN-gamma mRNA and lysed tumor cells without stimulation ex vivo, in contrast to CD4-intact mice. Thus, the quality as well as quantity of CD8+ CTL determines the completeness of immunosurveillance and is controlled by CD4 T cells but not solely Th2 cytokines. This model of immunosurveillance may indicate ways to enhance the efficacy of surveillance and improve immunotherapy.     

Antigenic epitopes regulate the phenotype of CD8+ CTL primed by exogenous antigens

We previously reported that insulin-specific, MHC class I-restricted CTL precursors can be primed by injecting C57BL/6 mice with bovine insulin in CFA. These bovine insulin-primed CTL displayed a type 0 CTL phenotype, producing IL-4, IL-5, IL-10, low levels of IFN-gamma, but no TNF-alpha. By contrast, CTL generated from C57BL/6 mice primed with OVA in CFA produced IFN-gamma and TNF-alpha but no IL-4, IL-5, or IL-10 and therefore were classified as type 1 CTL. Although CD4+ T cell subsets have been compared extensively in the literature, CTL subsets are less well characterized. Here, the phenotype, function, and requirements for the in vivo activation of type 1 and type 0 CTL cells were studied. Although both types of CTL express many of the same cell-surface Ags, OVA-specific CTL but not bovine insulin-primed CTL expressed CT-1, a carbohydrate epitope of CD45, and bovine insulin-primed CTL but not OVA-specific CTL expressed Fas constitutively. Priming of CTL was abrogated by depletion of phagocytic cells but not CD4+ T cells, whereas depletion of CD4+ T cells but not phagocytic cells inhibited Ab responses in the same mice. Neither endogenous IL-4 nor the dose of priming Ag altered the CTL phenotypes, but the antigenic peptides of OVA and bovine insulin were key to determining the differentiation of either type 1 or type 0 CTL. To our knowledge, this is the first time that antigenic epitopes have been demonstrated to influence the phenotype of Ag-specific CTL responses. These results may be relevant to the development of peptide vaccines in which a particular type of CTL response is desired.     

Induction of CD8+ T-lymphocyte responses to a secreted antigen of Mycobacterium tuberculosis by an attenuated vaccinia virus

Protective immunity against Mycobacterium tuberculosis infection requires the activation of mycobacterium-specific CD8+ T cells, as well as CD4+ T cells. Therefore, optimizing strategies that stimulate CD8+ T cells recognizing dominant mycobacterial antigens, including secreted proteins, may lead to the development of more effective vaccines against tuberculosis. To generate a viral vaccine that is safe in humans, the early secreted protein, MPT64, was expressed in the attenuated vaccinia virus (VV) strain, modified vaccinia virus Ankara (MVA-64). The immunogenicity of MVA-64 was compared with that of the Western Reserve strain of VV (VVWR-64). The replication-defective MVA-64 was as efficient as VVWR-64 in inducing specific antibodies and cytolytic T-cell responses to a defined H-2-Db-restricted epitope on MTP-64. In addition, priming with MPT64-expressing plasmid DNA (DNA-64), and boosting with either MVA-64 or VVWR-64, markedly enhanced MPT64-specific cytolytic and IFN-gamma-producing CD8+ T-cell responses. These findings suggest that MVA may be a suitable vaccine carrier for stimulating mycobacterium-specific CD8+ T-cell responses and may be particularly relevant for developing vaccines for use in regions endemic for tuberculosis and HIV infection.     

Ubiquitin-fusion degradation pathway: a new strategy for inducing CD8 cells specific for mycobacterial HSP65

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8⁺ T cells. In this study, we exploited UPS to induce CD8⁺ T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-γ-producing CD8⁺ T cells compared with those from the latter group of mice.     

A CD8+ T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of peptidomimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of an anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8+ CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization.     

Identification of murine poxvirus-specific CD8+ CTL epitopes with distinct functional profiles

Murine T cell epitopes against vaccinia virus (VV) have not been characterized to date in part due to the large and complex genome of VV. We have identified and characterized two CD8+ T cell epitopes on the A47L (modified VV Ankara strain (MVA)-029) and J6R (MVA-043) proteins of VV that are Db and Kb restricted, respectively. Following i.p. immunization with VV New York City Board of Health (NYCBH) strain, MVA-029 peptide-stimulated splenocytes secreted IFN-gamma from 7 days to 7 mo postimmunization, and virus-stimulated effectors were also able to lyse MVA-029-pulsed target cells at the same time points. In contrast, MVA-043 peptide-stimulated splenocytes secreted very low levels of IFN-gamma only at day 7 but maintained the ability to lyse target cells up to 2 mo postimmunization. Both MVA-029 and MVA-043 peptide-stimulated lymph node cells degranulated similarly as assessed by Ag-induced CD107 expression. T cell responses to whole-virus stimulation remained robust and steady during the acute and memory T cell response to VV. Identification of T cell epitopes on VV will enable further studies to increase our understanding of the role of CD8+ T cells in VV infection and assist in the design of new protective strategies.     

The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL

Stimulation of PBMC, in children recovering from acute measles, with autologous EBV-transformed and measles virus (MV)-infected lymphoblastoid cell lines (B-LCL) expanded primarily MV-specific CD8+ T cells. A large number of CD8+ T cell clones were obtained either by passaging of bulk cultures at limiting dilutions or by direct cloning of PBMC without previous stimulation in bulk culture. The MV-specific CD8+ T cell clones responding in a proliferative and a CTL assay were found to be class I MHC restricted. In contrast, CD4+ MV-specific T cell clones, which were generated by the same protocol, recognized MV in association with class II MHC molecules. Analysis of processing requirements for Ag presentation to CD8+ and CD4+ T cell clones, measured by the effect of chloroquine in a proliferative T cell response, revealed that both types of T cells recognized MV Ag processed via the endogenous/cytoplasmic pathway. Thus, these studies indicate that, as in most other viral infections and in contrast to previous suggestions, the class I MHC-restricted CTL response by CD8+ T cells may be an important factor in the control and elimination of MV infection. Therefore, the role proposed for CD4+ class II-restricted T cells in recovery from measles needs to be reevaluated.