Antigen-specific cytolysis of infected cells in murine candidiasis

Immune L3T4+ and Lyt-2+ lymphocytes play an important role in the acquired resistance of mice to challenge with virulent Candida albicans, and release macrophage-activating cytokines in response to yeast cells in vitro. To determine whether antigen (Ag)-specific cytotoxic T lymphocytes are generated during fungal infection, purified L3T4+ and Lyt-2+ lymphocytes from immunized mice were cultured in the presence of syngeneic accessory cells, Candida Ag, and IL-2. Yeast-infected bone marrow macrophages and peritoneal exudate neutrophils were used as target cells in a standard ⁵¹Cr release assay. Ag-specific, MHC-unrestricted lysis of infected macrophages was evident with immune Lyt-2+ cells after 5–10 days in culture. Under the same experimental conditions, the cytotoxic activity of L3T4+ cells was negligible, but its expression could be induced by the addition of anti-CD3 antibody.Culturing immune Lyt-2+ cells for shorter periods of time (1–2 days) resulted in preferential lysis of infected neutrophils. In addition, at limiting effector cell numbers, Ag-specific MHC-restricted lymphocytes with cytotoxic activity to infected macrophages could be identified. We suggest that C. albicans infection stimulates multiple cytotoxic T-cell precursors with varying recognition stringency, wich may have an important role in antifungal resistance in vivo.     

Epitope Context and Reshaping of Activated T Helper Cell Repertoire

In recent years, a growing interest in the study of peptide antigenicity in relation to the role of flanking sequences and protein topology in processing, presentation, and recognition has been observed. However, the information available on the antigenicity of recombinant fusion proteins and their effect on the selection of antigen receptor repertoires is limited. To analyze the role of molecular topology of T epitopes in a system relevant to human pathology, we have used the bacterially expressed Schistosoma japonicum glutathione S transferase (GST) to construct recombinant antigens containing HIV-1 derived T cell determinants, and human T cell clones specific for these determinants. We found that antigenicity of a given GST—peptide combination was not the same when T cells and antigen presenting cells from different individuals were tested. Our results show that differences in processing and presentation of chimeric proteins are not dictated by the use of diverse restriction elements. We also found that the context in which an antigenic peptide is delivered affects the recruited repertoire as defined according to T cell receptor Vβ usage and fine specificities of selected T cells.     

Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA-A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA-A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368–376. Both peptides contain consensus motifs of HLA-A2 binding peptides.     

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.     

Epitope specificity is critical for high and moderate avidity cytotoxic T lymphocytes associated with control of viral load and clinical disease in horses with equine infectious anemia virus

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infections in horses. We hypothesized that high-avidity CTL specific for nonvariable epitopes might be associated with low viral load and minimal disease in EIAV-infected horses. To test this hypothesis, memory CTL (CTLm) responses were analyzed in two infected horses with high plasma viral loads and recurrent disease (progressors), and in two infected horses with low-to-undetectable viral loads and mild disease (nonprogressors). High-avidity CTLm in one progressor recognized an envelope gp90 epitope, and the data documented for the first time in EIAV that viral variation led to CTL escape. Each of the nonprogressors had high-to-moderate avidity CTLm directed against epitopes within Rev, including the nuclear export and nuclear localization domains. These results suggested that the epitope specificity of high- and moderate-avidity CTLm was an important determinant for disease outcome in the EIAV-infected horses examined.     

High responsiveness of HLA-B57-restricted Gag-specific CD8+ T cells in vitro may contribute to the protective effect of HLA-B57 in HIV-infection

HLA-B57 has been shown to be associated with long-term asymptomatic HIV-1 infection. To investigate the biological mechanism by which the HLA-B57 allele could protect from HIV-1 disease, we studied both the number of CD8(+) T cells as well as CD8(+) T cell responsiveness directed to different HIV-1 Gag peptides presented by HLA-A2, -B8 or -B57. T cells specific for the HLA-B57 peptide KAFSPEVIPMF responded more readily and to a higher extend to antigenic stimulation in vitro than T cells specific for the HLA-A2 peptide SLYNTVATL or the HLA-B8 peptide EIYKRWII. This phenomenon was reproducible with T cells from individuals expressing HLA-B57 in combination with one or both of the other alleles and was persistent during long-term follow-up. Lower reactivity of A2- and B8-restricted T cells was not explained by mutations in the B8- or A2-restricted Gag-peptides. Moreover, no correlation between peptide mutation frequency and IFN-gamma production by the corresponding Gag-specific T cells was observed. In conclusion, functional differences were observed between T cells specific for HIV epitopes derived from the same protein presented by different HLA molecules. B57-restricted KAFSPEVIPMF-specific CD8(+) T cells have relatively high responsiveness, which could contribute to the protective effect of HLA-B57 in HIV infection.     

Identification of a novel gp100/pMel17 peptide presented by HLA-A*6801 and recognized on human melanoma by cytolytic T cell clones

Melanoma-associated peptides recognized by cytolytic T lymphocytes (CTL) in the context of several histocompatibility leukocyte antigens (HLA) are required for the development of specific immunotherapies. Using a transient transfection assay into COS-7 cells, we identified the gp100/pMel17 melanosomal protein as the shared antigen recognized by three independent CD8+ CTL clones in HLA-A*6801-restricted fashion. This finding was confirmed by the correlation between lack of gp100/pMel17 protein in a number of HLA-A*6801-positive melanomas and their resistance to lysis/cytokine production by the specific effectors. The gp100/pMel17 antigenic epitope was identified based on recognition of subfragments and on a computer-based prediction algorithm. Among a panel of gp100/pMel17-derived synthetic peptides only the 10-mer HTMEVTVYHR (gp100/pMel17182-191) induced tumor necrosis factor (TNF) release by CTL clones when pulsed on suitable target cells whereas both the 10-mer and the shorter 9-mer gp100/pMel17183-191 sensitized the same antigen-pulsed cells to lysis. In conclusion, the identification of the HTMEVTVYHR peptide will extend to HLA-A*6801 melanoma patients the possibility to exploit gp100/pMel17 melanosomal protein for experimental and clinical studies.     

An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--II. Evidence that the epitope recognized is involved in a late step in the cytolytic mechanism

A monoclonal antibody (RH1-38) which blocks multiple systems of cell-mediated cytotoxicity was functionally characterized. RH1-38 specifically blocks, in the absence of complement, natural killer (NK) activity (K562 targets) without any effect on NK-K562 conjugate formation. Kinetic studies suggested that the antibody blocks a step that occurs 30-120 min after effector populations are mixed with target cells. Single-cell cytotoxicity assays in agarose, combined with standard 51Cr release assays and Michaelis-Menten analysis revealed that RH1-38 markedly decreases Vmax and the number of active NK cells, again without any effect on the number of target-binding cells. The maximum recycling capacity was usually decreased, but in some experiments unchanged, in the presence of the monoclonal antibody. RH1-38 inhibited equally well whole peripheral blood mononuclear leukocytes (PBML), Percoll-fractionated lymphocytes enriched for NK activity, and interferon (IFN)-boosted NK activity. PBML exposed to RH1-38 and then washed mediated depressed NK activity which was partially reversed by subsequent treatment with IFN. These studies are most consistent with the hypothesis that RH1-38 inhibits a step late in the NK cytolytic mechanism rather than through an effect on conjugate formation. The primary effect is probably not on the IFN-generating or boosting mechanism, but a secondary effect on IFN-related mechanisms cannot be ruled out. Inhibition through an effect on a small lymphocyte modulator of NK activity is also unlikely but not rigorously excluded. Thus, RH1-38 appears to inhibit NK activity through a direct effect on NK effector cells, probably by interfering with a cell-surface molecule which is important in the expression of NK activity. The companion paper demonstrates that this monoclonal antibody immunoprecipitates a molecule which is very similar or identical to the LFA-1 antigen. Thus, RH1-38 recognizes either a novel epitope on the LFA-1 molecule or alternatively a distinct, functional killer cell surface molecule. The epitope appears to be involved in a late step in the cytolytic mechanism, possibly part of the effector cell lytic machinery.     

EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。