Existence of MHC class I-restricted alloreactive CD4+ T cells reacting with peptide transporter-deficient cells

It is generally accepted that as the result of positive thymic selection, CD8-expressing T cells recognize peptide antigens presented in the context of MHC class I molecules and CD4-expressing T cells interact with peptide antigens presented by MHC class II molecules. Here we report the generation of TCRalpha/beta(+), CD3(+), CD4(+), CD8(-), MHC class I-restricted alloreactive T-cell clones which were induced using peripheral blood mononuclear cells from healthy individuals following in vitro stimulation with transporter associated with antigen processing (TAP)-deficient cell lines T2. The CD4(+) T-cell clones showed an HLA-A2.1-specific proliferative response against T2 cells which was inhibited by anti-CD3 and anti-CD4 monoclonal antibodies. These results suggest that interaction of the TCR with peptide-bound HLA class I molecules contributes to antigen-specific activation of these co-receptor-mismatched T-cell clones. Antigen recognition by alloreactive MHC class I-restricted CD4(+) T cells was inhibited by removing peptides bound to HLA molecules on T2 cells suggesting that the alloreactive CD4(+) T cells recognize peptides that bind in a TAP-independent manner to HLA-A2 molecules. The existence of such MHC class I-restricted CD4(+) T cells which can recognize HLA-A2 molecules in the absence of TAP function may provide a basis for the development of immunotherapy against TAP-deficient tumor variants which would be tolerant to immunosurveillance by conventional MHC class I-restricted cytotoxic lymphocytes.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

CD8 T cell responses to myelin oligodendrocyte glycoprotein-derived peptides in humanized HLA-A*0201-transgenic mice

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS. Though originally believed to be CD4-mediated, additional immune effector mechanisms, including myelin-specific CD8(+) T cells, are now proposed to participate in the pathophysiology of MS. To study the immunologic and encephalitogenic behavior of HLA-A*0201-binding myelin-derived epitopes in vivo, we used a humanized HLA-A*0201-transgenic mouse model. Eight HLA-A*0201-binding peptides derived from myelin oligodendrocyte glycoprotein (MOG), an immunodominant myelin self-Ag, were identified in silico. After establishing their relative affinity for HLA-A*0201 and their capacity to form stable complexes with HLA-A*0201 in vitro, their immunological characteristics were studied in HLA-A*0201-transgenic mice. Five MOG peptides, which bound stably to HLA-A*0201 exhibited strong immunogenicity by inducing a sizeable MOG-specific HLA-A*0201-restricted CD8(+) T cell response in vivo. Of these five candidate epitopes, four were processed by MOG-transfected RMA target cells and two peptides proved immunodominant in vivo in response to a plasmid-encoding native full-length MOG. One of the immunodominant MOG peptides (MOG(181)) generated a cytotoxic CD8(+) T cell response able to aggravate CD4(+)-mediated EAE. Therefore, this detailed in vivo characterization provides a hierarchy of candidate epitopes for MOG-specific CD8(+) T cell responses in HLA-A*0201 MS patients identifying the encephalitogenic MOG(181) epitope as a primary candidate.     

Different development of myelin basic protein agonist- and antagonist-specific human TCR transgenic T cells in the thymus and periphery

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111-129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111-129 of MBP (MBP(111-129)) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that approximately 50% of human MBP(111-129) (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP(111-129) (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP(111-129) (MBP122:Lys). We found that MBP(111-129) (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP(111-129) TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8(+) T cells develop along with MS2-3C8 TCR Tg CD4(+) T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4(+) and CD8(+) T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.     

Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.     

CD8-independent tumor cell recognition is a property of the T cell receptor and not the T cell

The CD8 coreceptor enhances T cell function by stabilizing the TCR/peptide/MHC complex and/or increasing T cell avidity via interactions with the intracellular kinases Lck and LAT. We previously reported a CD4(+) T cell (TIL 1383I), which recognizes the tumor-associated Ag tyrosinase in the context of HLA-A2. To determine whether CD8 independent tumor cell recognition is a property of the TCR, we used retroviral transduction to express the TIL 1383I TCR in the CD8(-) murine lymphoma, 58 alpha(-)/beta(-). Immunofluorescent staining of TCR-transduced cells with human TCR V beta subfamily-specific and mouse CD3-specific Abs confirmed surface expression of the transferred TCR and coexpression of mouse CD3. Transduced effector cells secreted significant amounts of IL-2 following Ag presentation by tyrosinase peptide-pulsed T2 cells as well as stimulation with HLA-A2(+) melanoma lines compared with T2 cells alone or HLA-A2(-) melanoma cells. Further analysis of TCR-transduced clones demonstrated a correlation between T cell avidity and cell surface expression of the TCR. Therefore, the TIL 1383I TCR has sufficient affinity to mediate recognition of the physiologic levels of Ag expressed by tumor cells in the absence of CD8 expression.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

Opposing effects of HLA class I molecules in tuning autoreactive CD8+ T cells in multiple sclerosis

The major known genetic risk factors in multiple sclerosis reside in the major histocompatibility complex (MHC) region. Although there is strong evidence implicating MHC class II alleles and CD4(+) T cells in multiple sclerosis pathogenesis, possible contributions from MHC class I genes and CD8(+) T cells are controversial. We have generated humanized mice expressing the multiple sclerosis-associated MHC class I alleles HLA-A(*)0301 (encoding human leukocyte antigen-A3 (HLA-A3)) and HLA-A(*)0201 (encoding HLA-A2) and a myelin-specific autoreactive T cell receptor (TCR) derived from a CD8(+) T cell clone from an individual with multiple sclerosis to study mechanisms of disease susceptibility. We demonstrate roles for HLA-A3-restricted CD8(+) T cells in induction of multiple sclerosis-like disease and for CD4(+) T cells in its progression, and we also define a possible mechanism for HLA-A(*)0201-mediated protection. To our knowledge, these data provide the first direct evidence incriminating MHC class I genes and CD8(+) T cells in the pathogenesis of human multiple sclerosis and reveal a network of MHC interactions that shape the risk of multiple sclerosis.     

Cutting edge: myelin basic protein-specific cytotoxic T cell tolerance is maintained in vivo by a single dominant epitope in H-2k mice.

Multiple sclerosis (MS) is believed to be an autoimmune disease mediated by T cells specific for CNS Ags. MS lesions contain both CD4+ and CD8+ T lymphocytes. The contribution of CD4+ T cells to CNS autoimmune disease has been extensively studied in an animal model of MS, experimental autoimmune encephalomyelitis. However, little is known about the role of autoreactive CD8+ cytotoxic T cells in MS or experimental autoimmune encephalomyelitis. We demonstrate here that myelin basic protein (MBP) is processed in vivo by the MHC class I pathway leading to a MBP79-87/Kk complex. The recognition of this complex by MBP-specific cytotoxic T cells leads to a high degree of tolerance in vivo. This study is the first to show that the pool of self-reactive lymphocytes specific for MBP contain MHC class I-restricted T cells whose response is regulated in vivo by the induction of tolerance.     

Amino-terminal extended peptide single-chain trimers are potent synthetic agonists for memory human CD8+ T cells

Upon Ag exposure, most memory T cells undergo restimulation-induced cell death. In this article, we describe a novel synthetic agonist, an N-terminal extended decamer peptide expressed as a single-chain trimer, the amino-terminal extended peptide MHC class I single-chain trimer (AT-SCT), which preferentially promotes the growth of memory human CD8(+) T cells with minimal restimulation-induced cell death. Using CMV pp65 and melanoma gp100 Ags, we observe the in vitro numerical expansion of a clonally diverse polyfunctional population of Ag-specific CD8(+) T cells from healthy individuals and vaccinated melanoma patients, respectively. Memory CD8(+) T cells stimulated with AT-SCT presented on MHC class I/II-null cells show reduced cytokine production, slower kinetics of TCR downregulation, and decreased cell death compared with native nonamer MHC class I single-chain trimer (SCT)-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT- and SCT-activated T cells. Probing of SCT and AT-SCT peptide-MHC complexes using fluorochrome-conjugated TCR multimers suggests that nonamer- and decamer-linked peptides may be anchored differently to the HLA-A2 peptide-binding groove. Our findings demonstrate that modified peptide-MHC structures, such as AT-SCT, can be engineered as T cell agonists to promote the growth and expansion of memory human CD8(+) T cells.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.     

Identification of HLA-A∗02:01-restricted CTL epitopes in Trypanosoma cruzi heat shock protein-70 recognized by Chagas disease patients

CD8(+) cytotoxic T lymphocyte (CTL) response is critical for controlling the infection of the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease. Since only a few CD8 antigens have been described in Chagas disease patients, the identification of new class I-restricted epitopes is urgently needed for the development of immunotherapies against T. cruzi infection. In this study, bioinformatic methods were used to predict HLA-A?02:01-binders, and 30 peptides were selected, synthesized and tested for HLA-A?02:01 binding. Among them, sixteen peptides with medium-to-high affinity were assayed for their recognition by CTL from HSP70-immunized or T. cruzi-infected transgenic B6-A2/K(b) mice. Our results show that four immunodominant epitopes (HSP70(210-8), HSP70(255-63), HSP70(316-24) and HSP70(345-53)) are contained in the T. cruzi HSP70 antigen. Indeed two of them (HSP70(210-8) and HSP70(316-24)) were also recognized by CTL of HLA-A?02:01(+) Chagas disease patients, indicating that these peptides are processed and displayed as MHC class I epitopes during the natural history of T. cruzi infection. The HLA-A?02:01 restriction was evidenced using peptide-pulsed K562-A2 cells as antigen-presenting cells. Both cytotoxic and cytokine-secreting activities were detected in response to the former two peptides and, moreover, 10/12 patients (83%) recognized at least one of these two HSP70-derived CD8(+) epitopes.     

Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide

Class I major histocompatibility complex (MHC) molecules bind short peptides derived from proteins synthesized within the cell. These complexes of peptide and class I MHC (pMHC) are transported from the endoplasmic reticulum to the cell surface. If a clonotypic T cell receptor expressed on a circulating T cell binds to the pMHC complex, the cell presenting the pMHC is killed. In this manner, some tumor cells expressing aberrant proteins are recognized and removed by the immune system. However, not all tumors are recognized efficiently. One reason hypothesized for poor T cell recognition of tumor-associated peptides is poor binding of those peptides to class I MHC molecules. Many peptides, derived from the proto-oncogene HER-2/neu have been shown to be recognized by cytotoxic T cells derived from HLA-A2(+) patients with breast cancer and other adenocarcinomas. Seven of these peptides were found to bind with intermediate to poor affinity. In particular, GP2 (HER-2/neu residues 654-662) binds very poorly even though it is predicted to bind well based upon the presence of the correct HLA-A2.1 peptide-binding motif. Altering the anchor residues to those most favored by HLA-A2.1 did not significantly improve binding affinity. The crystallographic structure shows that unlike other class I-peptide structures, the center of the peptide does not assume one specific conformation and does not make stabilizing contacts with the peptide-binding cleft.     

Hybrids of dendritic cells and tumor cells generated by electrofusion simultaneously present immunodominant epitopes from multiple human tumor-associated antigens in the context of MHC class I and class II molecules

Hybrid cells generated by fusing dendritic cells with tumor cells (DC-TC) are currently being evaluated as cancer vaccines in preclinical models and human immunization trials. In this study, we evaluated the production of human DC-TC hybrids using an electrofusion protocol previously defined for murine cells. Human DCs were electrically fused with allogeneic melanoma cells (888mel) and were subsequently analyzed for coexpression of unique DC and TC markers using FACS and fluorescence microscopy. Dually fluorescent cells were clearly observed using both techniques after staining with Abs against distinct surface molecules suggesting that true cell fusion had occurred. We also evaluated the ability of human DC-TC hybrids to present tumor-associated epitopes in the context of both MHC class I and class II molecules. Allogeneic DCs expressing HLA-A*0201, HLA-DR beta 1*0401, and HLA-DR beta 1*0701 were fused with 888mel cells that do not express any of these MHC molecules, but do express multiple melanoma-associated Ags. DC-888mel hybrids efficiently presented HLA-A*0201-restricted epitopes from the melanoma Ags MART-1, gp100, tyrosinase, and tyrosinase-related protein 2 as evaluated by specific cytokine secretion from six distinct CTL lines. In contrast, DCs could not cross-present MHC class I-restricted epitopes after exogenously loading with gp100 protein. DC-888mel hybrids also presented HLA-DR beta 1*0401- and HLA-DR beta 1*0701-restricted peptides from gp100 to CD4(+) T cell populations. Therefore, fusions of DCs and tumor cells express both MHC class I- and class II-restricted tumor-associated epitopes and may be useful for the induction of tumor-reactive CD8(+) and CD4(+) T cells in vitro and in human vaccination trials.     

Tapasin is a facilitator, not an editor, of class I MHC peptide binding

Tapasin has been proposed to function as a peptide editor to displace lower affinity peptides and/or to favor the binding of high affinity peptides. Consistent with this, cell surface HLA-B8 molecules in tapasin-deficient cells were less stable and the peptide repertoire was substantially altered. However, the binding affinities of peptides expressed in the absence of tapasin were unexpectedly higher, not lower. The peptide repertoire from cells expressing soluble tapasin was similar in both appearance and affinity to that presented in the presence of full-length tapasin, but the HLA-B8 molecules showed altered cell surface stability characteristics. Similarly, the binding affinities of HLA-A*0201-associated peptides from tapasin(+) and tapasin(-) cells were equivalent, although steady state HLA-A*0201 cell surface expression was decreased and the molecules demonstrated reduced cell surface stability on tapasin(-) cells. These data are inconsistent with a role for tapasin as a peptide editor. Instead, we propose that tapasin acts as a peptide facilitator. In this role, it stabilizes the peptide-free conformation of class I MHC molecules in the endoplasmic reticulum and thus increases the number and variety of peptides bound to class I MHC. Full-length tapasin then confers additional stability on class I MHC molecules that are already associated with peptides.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Lack of tumor recognition by cytolytic T lymphocyte clones recognizing peptide 195–203 encoded by gene <Emphasis Type="Italic">MAGE-A3</Emphasis> and presented by HLA-A24 molecules

Gene MAGE-A3 encodes tumor-specific antigenic peptides recognized by T cells on many tumors. MAGE-A3 peptides presented by HLA class I molecules have been identified using CD8 lymphocytes stimulated with cells that either expressed gene MAGE-A3 or were pulsed with candidate peptides. One antigen identified with the latter method is peptide MAGE-A3(195-203) IMPKAGLLI, presented by HLA-A24 molecules. It has been used to vaccinate advanced cancer patients. Here, we have used HLA/peptide tetramers to detect T cells recognizing this peptide. Their frequency was estimated to be 2 x 10(-8) of the blood CD8 cells in non-cancerous HLA-A24(+) individuals, which is tenfold lower than the reported frequencies of T cells against other MAGE peptides. In the blood of a patient vaccinated with MAGE-A3, the estimated frequency was 5 x 10(-7). Anti-MAGE-3.A24 cytolytic T cell clones were derived, that lysed peptide-pulsed cells with half-maximal effect at the low concentration of 500 pM. However, these CTL did not recognize a panel of HLA-A24(+) tumor cells that expressed MAGE-A3 at levels similar to those found in HLA-A1(+) tumor cells recognized by anti-MAGE-3.A1 CTLs. Furthermore, 293-EBNA cells transfected with MAGE-A3 and HLA-A24 constructs were hardly recognized by the anti-MAGE-3.A24 CTL clones. These results suggest that peptide MAGE-A3(195-203) is poorly processed and is not an appropriate target for cancer immunotherapy.     

Quantitation of CD8+ T cell expansion,memory, and protective immunity after immunization with peptide-coated dendritic cells

Dendritic cells (DCs) are potent APCs for naive CD8(+) T cells and are being investigated as vaccine delivery vehicles. In this study, we examine the CD8(+) T cell response to defined peptides from Listeria monocytogenes (LM), lymphocytic choriomeningitis virus, and murine CMV coated singly and in combination onto mature bone marrow-derived DCs (BMDCs). We show that immunization of mice with 2 x 10(5) mature BMDCs coated with multiple MHC class I peptides generates a significant Ag-specific CD8(+) T cell response in both the spleen and nonlymphoid organs. This immunization resulted in a peptide-specific hierarchy in the magnitude of CD8(+) T cell priming and noncoordinate kinetics in response to different peptide epitopes. Kinetics were not exclusively due to specific characteristics of the MHC class I molecule, and were not altered in an Ag-independent manner by concurrent LM infection. Mice immunized with listeriolysin O 91-99-coated BMDCs are protected against high dose challenge with virulent LM. This protection was enhanced by diversifying the memory CD8(+) T cell compartment, even in the absence of a large increase in Ag-specific CD8(+) memory T cells.     

HLA-B7-restricted EBV-specific CD8+ T cells are dysregulated in multiple sclerosis

It was hypothesized that the EBV-specific CD8(+) T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8(+) T cell response in greater detail, we analyzed the HLA-A2-, HLA-B7-, and HLA-B8-restricted EBV- and CMV-specific CD8(+) T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8(+) T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7(+) MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBV(RPP)-specific CD8(+) T cells ex vivo. However, the magnitude of the HLA-B*0702/EBV(RPP)-specific and HLA-B*0702/CMV(TPR)-specific CD8(+) T cell response (i.e., the percentage of tetramer(+) CD8(+) T cells in a study subject harboring CD8(+) T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBV(RPP) peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBV(RPP)-specific CD8(+) T cells were decreased. Altogether, our findings suggest that the HLA-B*0702-restricted viral (in particular the EBV one)-specific CD8(+) T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.