Restriction of Epstein-Barr virus-specific cytotoxic T cells by HLA-A, -B, and -C molecules

HLA-loss variants of an Epstein-Barr virus-transformed B-lymphoblastoid cell line (EBV-LCL) 721 were used as target cells to identify HLA molecules utilized by EBV-LCL-specific cytotoxic T cells. Split culture analysis of cytotoxic T cells plated at limiting dilution showed killing of HLA-loss variants bearing either HLA-A2 or -B5 molecules, with 10 times higher frequency of cytotoxic T cells restricted by the HLA-B5 molecule. Clonal analysis confirmed the restriction by HLA-A2 or -B5 of some cytotoxic T-cell clones and identified cytotoxic T-cell clones cytolytic for target cells which do not express HLA-A or -B but do express the HLA-C determinant. Thus, our results show immunodominance of the HLA-B5 restriction determinant for EBV-induced antigens in the donor of the HLA-loss variants and provide evidence that the HLA-C molecule can also serve as restriction determinant for EBV-LCL-specific cytotoxic T cells.     

HLA-restricted T lymphocyte-mediated cytotoxicity against herpes simplex virus-infected cells in humans.

Cytotoxic T lymphocytes (CTL) against herpes simplex virus (HSV) were induced in vitro from human peripheral blood lymphocytes by stimulation with HSV antigen. CTL generated by HSV type 1 (HSV-1) antigen stimulation killed not only HSV-1-infected target cells but also HSV type 2 (HSV-2)-infected target cells, though at a lower level. This evidence suggests that CTL against HSV recognize the HSV type-specific and type-common determinants on HSV-infected target cells. These CTL were generated from high responders against HSV-1 antigen as measured by antigen-specific T lymphocyte proliferation in vitro, but not to such an efficient degree from low responders. The cytotoxic activities of CTL against the allogeneic HSV-infected target cells were high when at least one of the HLA-A or -B antigens was shared. However, the HLA-A and -B nonidentical target cells were not killed effectively. The data presented here suggest the possibility of HLA restriction of HSV-specific CTL in humans.     

Human mumps virus-specific cytotoxic T lymphocytes: quantitative analysis of HLA restriction

To obtain quantitative information about the use of HLA antigens as restriction element by antiviral cytotoxic T lymphocytes (CTL), we have analyzed precursors of human mumps virus-specific CTL by limiting dilution. CTL generated by restimulation of peripheral blood T lymphocytes with autologous mumps virus (MV)-infected stimulator cells were restricted by autologous HLA class I antigens, and derived from the T4-8+ population. They were specific for MV and did not lyse autologous target cells infected with other viruses. Frequencies of MV-specific CTL precursors ranged from 1/500 to 1/8000. HLA restriction was analyzed by split-well analysis of individual CTL colonies. CTL recognizing HLA-A or B antigens were unequally distributed: HLA-B7, -B13, and -B27 were found to function as predominant, in some cases as exclusive, restriction elements, whereas other antigens such as HLA-A24 were never or rarely used. In several combinations, there was no evidence for antigenic variants of HLA molecules as reason for the failure to be recognized. The proportion of CTL precursors recognizing HLA-A2 and -B8 seemed to be dependent on the presence or absence of "dominant" restriction elements. We conclude that CTL precursors recognizing certain virus-HLA combinations are preferentially expanded during an infection, but that low responsiveness to a given combination is not necessarily absolute.     

HIV-1 diversity versus HLA class I polymorphism

HIV-1 is rapidly diversifying in African, Asian and Caucasoid populations, which in parallel display extensive polymorphism of genes encoding class I human leukocyte antigens (HLA). Immune responses mediated by HLA class I molecules are imprinting mutations in HIV-1, which in turn affects HIV-1 diversity. Intra- and inter-ethnic studies have shown reproducible HLA class I allele, haplotype and supertype associations with HIV-1 infection and the development of AIDS (HIV/AIDS). In Caucasoids and Africans, HLA-B57 and related alleles of the B58 supertype associate with low viraemia, delayed onset of AIDS and, possibly, cytotoxic T lymphocyte (CTL)-driven attenuation of HIV-1. In HIV-1-exposed but uninfected Southeast Asians, HLA-A11 has been associated with CTL responses directed against HIV-1 Nef. HLA-A11 displays unique peptide-binding properties and is recognized by natural killer cells utilizing the inhibitory killer Ig-like receptor 3DL2 in a peptide-dependent manner.     

Selective reactivation of Epstein-Barr virus-specific cytotoxic T cells by stimulation in vitro with allogeneic virus-transformed HLA-homozygous typing cells

Epstein-Barr (EB) virus-specific cytotoxic T-cell preparations, produced by stimulation in vitro of peripheral blood lymphocytes with the autologous virus-transformed cell line, are HLA-A and B antigen-restricted and, with some donors, show preferential restriction through one or two of the four relevant antigens of the donor's HLA type. It has now been demonstrated that such EB virus-specific cytotoxic T cells may also be reactivated by stimulation with allogeneic virus-transformed cells provided that there is no mismatch of the HLA-A and B antigens between the responder and stimulator cell donors. In particular, virus-transformed cell lines from HLA-homozygous donors HLA-A and B antigen-matched to one of the haplotypes of an HLA-heterozygous responder were shown to reactivate selectively only those EB virus-specific cytotoxic T cells restricted through the HLA-A and B antigens present on the allogeneic stimulating cells. In addition to confirming the polyclonal nature of the HLA-restricted EB virus-specific cytotoxic T-cell response, this new experimental procedure has allowed the production, and subsequent expansion as cell lines dependent upon T-cell growth factor, of those effector cells restricted through the "nonpreferred" HLA antigens that are poorly represented in the response induced by stimulation with autologous virus-transformed cells.     

Structure, Function, and Biosynthesis of the Major Human Histocompatibility Antigens (HLA-A and HLA-B)

Summary
In summary, the complete primary structure of one HLA antigen (HLA-B7) has now been determined, and sufficient information is available about other HLA-A and -B and H-2 molecules to begin to assess the location of the variable regions within these molecules. It is these regions of the molecules which have evolved to give rise to the extensive polymorphism in human populations, and they most likely define functionally important regions of the molecule. Thus, this information may help us to elucidate the natural biological function of these molecules, as, for example, in the recognition of allogeneic or virally infected syngeneic cells by cytotoxic T cells. Some functional studies, using purified HLA-A and -B antigens inserted in liposomes, have been initiated, and studies of the biosynthesis of these antigens are of great interest in their own right. Further, using cDNA probes, they are being extended to permit us ultimately to underScand the organization of the genes on the sixth human chromosome which encode HLA-A and -B antigens. Finally, such probes may permit the rapid accumulation of sequence information, which would allow us to underScand more completely the structure of these extremely important cell surface molecules.     

Killing of measles virus-infected cells by human cytotoxic T cells.

Lymphocytes from normal individuals were tested for the capacity to generate measles virus-specific cytotoxic T-cell responses after in vitro stimulation with measles virus. Approximately 12% (5 of 40) of the normal adults tested produced significant cytotoxic responses. The cytotoxic response was measles virus specific both at the level of stimulation and at the effector level. Studies of the specificity of cytotoxic effectors from five normal donors by direct lysis or cold target inhibition or both indicated that most, if not all, of the virus-specific activity was self-specific. A detailed analysis of one donor (W6) indicated that measles-specific cytotoxicity was largely HLA-A and -B restricted; unexplained cross-reactive lysis was observed with some targets, but this lysis appeared to be HLA related, since all of the targets expressed HLA-B12. An analysis of the cellular requirements for the production of measles-immune cytotoxic T lymphocytes demonstrated that T cells and macrophages (depleted of natural killer and K cells) were sufficient for the generation of killer cells. Most of the cytotoxic effector activity was mediated by OKT3+ OKT4- OKT8+ cells.     

Human T-cell hybrids with HLA-restricted proliferative response to Epstein-Barr virus-infected B cells.

Human T-cell hybrids were constructed from an HGPRT-negative mutant of the acute lymphoblastoid leukaemia cell-line CEM and an uncloned population of T cells from donor SW (SW-T; partner cell) known to have a strong specificity for the autologous Epstein-Barr virus (EBV)-transformed B cell, SWEBV. The resulting hybrids, 1A9, 1D12 and 2C8, were shown not to be cytotoxic to SWEBV, nor did they have natural killer-like (NK) activity. However, when presented with the target SWEBV in a mixed lymphocyte reaction (MLR), all of the hybrids rapidly increased their rate of proliferation by up to a factor of seven. Hybrid 1D12 also produced interleukin-2-like material (IL2) under these conditions. The hybrids did not react with the autologous PHA-blasts (SWPHA), nor with various unrelated targets. When tested against a bank of EBV-transformed B-cell targets, it was observed that the human T-cell hybrids 1A9 and 2C8 responded only to those targets bearing the antigen HLA Bw35. This response could be blocked by treating the target with the monoclonal antibody W6/32, specific for a shared determinant of the HLA-A, -B and -C antigens. Similarly, the human T-cell hybrid 1D12 reacted only against those targets bearing the antigen HLA DrW2, and this activity could be blocked by the monoclonal antibody DA6.231, specific for a common region of the HLA-DR and SB antigens. Thus, human T-cell hybrids can be produced which exhibit HLA-restricted responses to antigenic stimulation.     

Possible association between HLA antigens and the response to interferon in Japanese patients with chronic hepatitis C

Correlation between the major histocompatibility complex class I antigens (HLA-A, -B and -C) and the elimination from serum of hepatitis C virus in patients with chronic hepatitis C has not been understood. We analyzed HLA phenotypes and their relationship to the efficacy of interferon treatment. Of the 172 patients who were treated with 9 million units of interferon-α2a three times a week for 6 months, 54 patients were responders and 118 patients were non-responders. No significant difference was observed between the 172 patients and 199 healthy subjects with regard to the frequencies of HLA-A, -B and -C antigen phenotypes. However, HLA-B55, B62, CW3 and CW4 frequencies were significantly higher in responders than in non-responders to the interferon treatment. CW4 was found to link with B62, but other phenotypes were independent each other. Patients with HLA B55, B62 and CW3 had a significantly lower viral load, and showed a better response to interferon. These results suggest that HLA system does not have an influence on the evolution towards chronicity of the disease due to hepatitis C virus, but HLA B55, B62 or CW4, and CW3 may be a virus quantity-regulating factors which then affect to response to the interferon treatment, indicating that these HLA antigens in conjunction with a viral peptide is a key target antigen for cytotoxic T lymphocytes in patients with chronic hepatitis C.     

Major histocompatibility complex class I-restricted alloreactive CD4+ T cells

Although it is well established that CD4+ T cells generally recognize major histocompatibility complex (MHC) class II molecules, MHC class I-reactive CD4+ T cells have occasionally been reported. Here we describe the isolation and characterization of six MHC class I-reactive CD4+ T-cell lines, obtained by co-culture of CD4+ peripheral blood T cells with the MHC class II-negative, transporter associated with antigen processing (TAP)-negative cell line, T2, transfected with human leucocyte antigen (HLA)-B27. Responses were inhibited by the MHC class I-specific monoclonal antibody (mAb), W6/32, demonstrating the direct recognition of MHC class I molecules. In four cases, the restriction element was positively identified as HLA-A2, as responses by these clones were completely inhibited by MA2.1, an HLA-A2-specific mAb. Interestingly, three of the CD4+ T-cell lines only responded to cells expressing HLA-B27, irrespective of their restricting allele, implicating HLA-B27 as a possible source of peptides presented by the stimulatory MHC class I alleles. In addition, these CD4+ MHC class I alloreactive T-cell lines could recognize TAP-deficient cells and therefore may have particular clinical relevance to situations where the expression of TAP molecules is decreased, such as viral infection and transformation of cells.     

MHC-I-restricted epitopes conserved among variola and other related orthopoxviruses are recognized by T cells 30 years after vaccination

It is many years since the general population has been vaccinated against smallpox virus. Here, we report that human leukocyte antigen (HLA) class I restricted T cell epitopes can be recognized more than 30 years after vaccination. Using bioinformatic methods, we predicted 177 potential cytotoxic T lymphocyte epitopes. Eight epitopes were confirmed to stimulate IFN-γ release by T cells in smallpox-vaccinated subjects. The epitopes were restricted by five supertypes (HLA-A1, -A2, -A24 -A26 and -B44). Significant T cell responses were detected against 8 of 45 peptides with an HLA class I affinity of K _D less than or equal to 5 nM, whereas no T cell responses were detected against 60 peptides with an HLA affinity of K _D more than 5 nM. All epitopes were fully conserved in seven variola, vaccinia and cowpox strains. Knowledge of the long-term response to smallpox vaccination may lead to a better understanding of poxvirus immunity and may aid in the development of new improved vaccines and diagnostic tools. S. T. Tang and M. Wang contributed equally to this work.     

Characteristics of graft-infiltrating lymphocytes after human heart transplantation HLA mismatches and the cellular immune response within the transplanted heart

The influence of HLA mismatches between donor and recipient on the phenotypes, function, and specificity of T-lymphocyte cultures derived from endomyocardial biopsies was studied in 118 heart transplant recipients. In case of HLA-DR mismatches, the majority of the EMB-derived cultures were dominated by CD4⁺ T cells while, in patients with HLA-A and -B mismatches but without DR mismatches, CD8⁺ T cells comprised the predominant T-cell subset. Cytotoxicity against donor antigens was observed in 75% of the cultures. A significantly (p < 0.005) lower proportion of the cultures showed cytotoxicity against HLA-A antigens (36%) when compared with HLA-B (53%) or HLA-DR (49%). An HLA-A2 mismatch elicited a cytotoxic response that was comparable to that found against HLA-B and -DR antigens: 62% of the cultures from HLA-A2 mismatched donor-recipient combinations was reactive against A2. A higher number of A, B, or DR mismatches resulted in a higher number of cytotoxic cultures directed against these antigens. A higher number of HLA-B and -DR mismatches was associated with a lower freedom from rejection. Our data indicate that, despite the use of adequate immunosuppressive therapy, the degree of HLA matching plays a crucial role in the immune response against a transplanted heart, resulting in a significant effect on freedom from rejection.     

Human Coronavirus OC43 Interacts with Major Histocompatibility Complex Class I Molecules at the Cell Surface to Establish Infection

Human coronaviruses have been associated with common colds, diarrhea and enterocolitis, and have been implicated in multiple sclerosis. HLA class I molecules may play a critical role as receptor for OC43 because monoclonal antibody (mAb)W6/32 to HLA-A, -B and -C specificities completely blocks infectivity in human rhabdomyosarcoma (RD) cells. The role of HLA class 1 antigen as the virus receptor was examined using HLA-A3.1 stably transfected human plasma cells and untransfected HMY.C1R cells which do not express HLA-A and -B molecules. When the cells (5x10⁶) were infected at a multiplicity of one, the HLA.A3 transfected cells produced 10⁸ PFU of virus whereas no replication occurred in the HMY.C1R cells mAb W6/32 reduced the virus yield by 99.9% Cell membranes from HMY.C1R, HMY.A3 cells and chicken erythrocytes were biotinylated as live cells. Immunoprecipitation with polyclonal antiviral antibody to detect binding of biotinylated cell membranes to virus revealed that biotinylated HMY.A3 membranes co-precipitated with virus-antibody complexes when the immunoprecipitates were electrophoresed on SDS-PAGE gel, electroblotted and stained with Avidin-horseradish peroxidase. The results provide direct evidence that OC43 virus can recognize HLA class I as receptor on the cell surface.     

Two cytotoxic human-human hybridoma antibodies to HLA: TrAH10 (anti A3.1) and TrAG2 (anti B7,Bw42)

Two cytotoxic human-human hybridoma IgM antibodies to HLA were generated by EBV transformation of PBMC from multiparous women and fusion of EBV transformed cells with the human fusion partners KR4 or KR12. Both mAbs required the sensitive immunomagnetic cytotoxicity method to display killing of freshly prepared PBMC. One mAb (TrAH10) was specific for HLA-A3. Strikingly, TrAH10 reacted much more strongly with lymphoblastoid cell lines of HLA-A3.1 than of the rare variant HLA-A3.2, previously detected by cytotoxic T cells. Thus, in the microcytotoxicity test, the titer of concentrated TrAH10 was approximately 2000 times higher for A3.1 as compared to A3.2, and a clear difference was also observed in radioimmunoassay. Since the two HLA-A3 variants differ by only two amino acids at positions 152 and 156 of the alpha 2-domain's alpha-helix, the epitopes defined by the mAb TrAH10 and HLA-A3.1 specific cytotoxic T cells must be closely related. The observations with TrAH10 suggest that the HLA polymorphism detected by human mAbs may turn out to be as extensive as the T-cell defined HLA polymorphism. The other mAb (TrAG2) bound B7 and Bw42 with equal strength, and in addition bound weakly to some cells that were Bw22 or B39. Magnetic polymerbeads coated with affinity purified human mAbs TrAH10 or TrAG2 formed rosettes with EBV transformed cells carrying relevant HLA antigens; however, rosette formation with freshly isolated PBMC was very weak and unsuitable as a typing assay.     

Prediction of HLA class I-restricted T-cell epitopes of islet autoantigen combined with binding and dissociation assays

Identification of cognate peptides recognized by human leucocyte antigen (HLA)/T cell receptor (TCR) complex provides insight into the pathogenic process of type 1 diabetes (T1D). We hypothesize that HLA-binding assays alone are inadequate metrics for the affinity of peptides. Zinc transporter-8 (ZnT8) has emerged in recent years as a novel, major, human autoantigen. Therefore, we aim to identify the HLA-A2-restricted ZnT8 epitopes using both binding and dissociation assays. HLA class I peptide affinity algorithms were used to predict candidate ZnT8 peptides that bind to HLA-A2. We analyzed 15 reported epitopes of seven β-cell candidate autoantigens and eight predicted candidate ZnT8 peptides using binding and dissociation assays. Using IFN-γ ELISpot assay, we tested peripheral blood mononuclear cells (PBMCs) from recent-onset T1D patients and healthy controls for reactivity to seven reported epitopes and eight candidate ZnT8 peptides directly ex vivo. We found five of seven recently reported epitopes in Chinese T1D patients. Of the eight predicted ZnT8 peptides, ZnT8_153–161 had a strong binding affinity and the lowest dissociation rate to HLA-A*0201. We identified it as a novel HLA-A*0201-restricted T-cell epitope in three of eight T1D patients. We conclude that ZnT8_153–161 is a novel HLA-A*0201-restricted T-cell epitope. We did not observe a significant correlation (P = 0.3, R = ? 0.5) between cytotoxic T cell (CTL) response and peptide/HLA*0201 complex stability. However, selection of peptides based on affinity and their dissociation rate may be helpful for the identification of candidate CTL epitopes. Thus, we can minimize the number of experiments for the identification of T-cell epitopes from interesting antigens.     

Soluble HLA-A,-B,-C and -G molecules induce apoptosis in T and NK CD8+ cells and inhibit cytotoxic T cell activity through CD8 ligation

There is convincing evidence that soluble HLA-A,-B,-C (sHLA-A,-B,-C) and soluble HLA-G (sHLA-G) antigens can induce apoptosis in CD8(+) activated T cells although there is scanty and conflicting information about the mechanism(s) by which sHLA-A,-B,-C antigens and sHLA-G antigens induce apoptosis. In this study we have compared the apoptosis-inducing ability of sHLA-A,-B,-C antigens with that of sHLA-G1 antigens in CD8(+) T lymphocytes and CD8(+) NK cells. Furthermore we have compared the inhibitory effect of sHLA-A,-B,-C antigens and of sHLA-G1 antigens on the activity of EBV-specific CD8(+) cytotoxic T lymphocytes (CTL). sHLA molecules were purified from serum and from the supernatant of HLA class I-negative cells transfected with one gene encoding either classical or non-classical HLA class I antigens. Both classical and non-classical sHLA class I molecules trigger apoptosis in CD8(+) T lymphocytes and in CD8(+) NK cells, which lack the T cell receptor, and their apoptotic potency is comparable. The binding of sHLA-A,-B,-C and sHLA-G1 molecules to CD8 leads to Fas ligand (FasL) up-regulation, soluble FasL (sFasL) secretion and CD8(+) cell apoptosis by Fas/sFasL interaction. Moreover, classical and non-classical sHLA class I molecules inhibit the cytotoxic activity of EBV-specific CD8(+) CTL. As the amount ofsHLA-G molecules detectable in normal serum is significantly lower than that of sHLA-A,-B,-C molecules, the immunomodulatory effects of sHLA class I molecules purified from serum are likely to be mainly attributable to classical HLA class I antigens. As far as the potential in vivo relevance of these findings is concerned, we suggest that classical sHLA class I molecules may play a major immunoregulatory role in clinical situations characterized by activation of the immune system and elevated sHLA-A,-B,-C serum levels. In contrast, non-classical HLA class I molecules may exert immunomodulatory effects in particular conditions characterized by elevated sHLA-G levels such as pregnancy and some neoplastic diseases.     

Human T-cell leukemia virus type-I (HTLV-I)-specific T-cell responses detected using three-divided glutathione-S-transferase (GST)-Tax fusion proteins

Insufficient T-cell response to human T-cell leukemia virus type-I (HTLV-I) is a potential risk factor in adult T-cell leukemia (ATL). We established an assay system for detecting HTLV-I-specific T-cell response by using recombinant glutathione-S-transferase (GST) proteins fused with HTLV-I Tax protein that was divided into three portions, Tax-A, -B, and -C, corresponding to the N-terminal, central and C-terminal regions, respectively. When splenocytes from rats immunized with plasmids encoding Tax cDNA were incubated with these recombinant proteins, strong interferon gamma (IFN-gamma-producing responses occurred against GST-Tax proteins but not against control GST proteins. No such Tax-specific responses were observed in splenocytes from naive rats. Cocktails of oligopeptides corresponding to the Tax-A, -B, and -C regions also induced IFN-gamma-producing responses when incubated with splenocytes from immunized rats, but required higher amounts of antigens and there were a shorter periods of sustained T-cell responses than with GST-Tax protein-based assay. Although splenocytes from immunized rats predominantly reacted against GST-Tax-B protein, they failed to react with peptide cocktails corresponding to the Tax-B region, likely because the major epitope was interrupted in the initially prepared series of peptides. Using a newly prepared peptide series we found that splenocytes predominantly reacted with a peptide located in the Tax-B region that overlaps with a previously identified cytotoxic T lymphocytes (CTL) epitope of this rat strain. Using this system, we examined peripheral blood mononuclear cells (PBMC) from an ATL patient who underwent complete remission following hematopoietic stem cell transplantation (HSCT). PBMC from this patient produced a significant Tax-specific T-cell response predominantly against GST-Tax-A protein. This is consistent with the previous finding that this patient exhibited a strong HLA-A2-restricted CTL response to Tax 11-19 epitope, which is located in the Tax-A region. This study provides a diagnostic tool, useful for monitoring HTLV-I-specific T-cell immunity in patients and for surveying HTLV-I-carriers to identify an immunological group at high risk for ATL development, regardless of their human leukocyte antigen (HLA) types. It is also useful for predicting the location of T-cell epitopes, which may be applicable in future vaccine strategies.     

Variations in the T-cell repertoire against HLA antigens in humans.

Allospecific anti-HLA class I antigen cytotoxic T-lymphocyte precursor frequencies (CTLpf) have been estimated in peripheral blood of healthy blood donors with responder stimulator combinations mismatched for one HLA-A,B antigen. The CTLpf ranged from 1 in 400 to 1 in 10,000, with most frequent values of 1 in 600 to 4000. The following observations were made: (1) CTLpf against the same HLA antigen vary among different responders; (2) CTLpf of one responder against various HLA antigens may be different; (3) "narrow" responders produce cytotoxic T lymphocytes that recognize only the private (stimulator) alloantigen, while "broad" responders produce mainly broadly cross-reactive cytotoxic T lymphocytes with public specificity. Split-well analysis shows that very few cytotoxic T lymphocytes of "broad" responders recognize the private alloantigen only. These individual variations are not dependent on the HLA phenotype, because they also occurred in unrelated HLA-identical responders stimulated against the same mismatched stimulator cells.     

A comparison of gene transfer and antigen-loaded dendritic cells for the generation of CD4+ and CD8+ cytomegalovirus-specific T cells in HLA-A2+ and HLA-A2- donors.

Dendritic cells have been used effectively to select for human cytomegalovirus (CMV)-specific T cells for immunotherapy applications. The ability to process and present relevant major histocompatibility complex class I and II peptides to T cells makes them ideal for selecting CD4+ and CD8+ T cells regardless of HLA tissue type. This study compared the generation of CMV-specific T cells by using dendritic cells loaded with either CMV pp65495-503 peptide or CMV lysate or transduced with adenovirus encoding the pp65 gene (Ad5pp65GFP) for the generation of CD4+ and CD8+ CMV-specific T cells in HLA-A2+ and HLA-A2 - donors. In HLA-A2+ donors, CD8+ tetramer+ T cells increased with all antigens but were greatest in peptide- and Ad5pp65GFP-stimulated T cells. The CD4+ /CD8+ ratio in the stimulated T-cell cultures proved to be dependent on the antigen used. CMV lysate-stimulated cells were primarily CD4+, whereas peptide- and Ad5pp65GFP-stimulated cultures were mostly CD8+. Analysis of cells from lysate-stimulated or gene-transduced-stimulated cultures showed expansion of CMV-specific CD4+ T cells, indicating that major histocompatibility complex class II peptides were present in both antigens. Furthermore, CMV-specific T cells were generated from HLA-A2 - donors by using Ad5pp65GFP transduction or CMV lysate stimulation and were able to recognize a pp65 peptide restricted to the HLA-B35 allele. These data indicate that either CMV lysate or adenovirus encoding CMV antigenic genes may be useful for the generation of both CD4+ and CD8+ CMV-specific T cells in donors irrespective of HLA tissue type and may be applicable to clinical immunotherapy.     

Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210^bcr-abl fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210^bcr-abl is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210^bcr-abl breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.