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In the present study, we demonstrate that the non-classical MHC class I molecule HLA-G impairs specific cytolytic T cell functions in addition to its well-established inhibition of NK lysis. The antigen-specific cytotoxic T lymphocyte (CTL) response analyzed was mediated by CD8(+) T cells specific for the influenza virus matrix epitope, M58-66, presented by HLA-A2. The transfection of HLA-G1 cDNA in target cells carrying the M58-66 epitope reduced their lysis by these virus-specific CTL. This HLA-G-mediated inhibition of antigen-specific CTL lysis was (i) peptide dose dependent, (ii) reversed by blocking HLA-G with a specific mAb and (iii) still observed despite the blockade of HLA-E/CD94/NKG2A interaction. By inhibiting both CTL and NK functions, HLA-G appears to have an extensive role in immune tolerance.

CD8+ T lymphocytes are involved in protective immune responses to infected or tumor cells. In this report, we examined the regulation of antigen-specific CD8+ T cell frequency and avidity by distinct Th cell subsets. Peptide-specific CD8+ T cells were induced by immunization of mice with a MHC class I-restricted epitope, co-injected with a MHC class II-restricted epitope to recruit Th cells. CD8+ T cell responses were assessed directly ex vivo for lytic activity and IFN-gamma secretion using the enzyme-linked immunospot (ELISPOT) assay. Co-immunization in incomplete Freund's adjuvant (IFA) with three different helper peptides induced IFN-gamma- and IL-2-secreting Th cells, in the absence of IL-4 secretion, suggesting preferential Th1 profiles. Such immunization resulted in the increase of antigen-specific CD8+ T cell frequency, which was detected in blood as efficiently as in lymph nodes and spleen, and elicited high-avidity CD8+ T cells. We investigated whether these effects were dependent upon a particular Th profile. When alum was used instead of IFA, the production of IL-2 by Th cells was still significant, while the production of IFN-gamma was undetectable. Such Th cell activation failed to support an increase of antigen-specific CD8+ T cell frequency. Altogether, these results document in vivo the regulatory role played by Th cells in CD8+ T cell activation and may be relevant for the design of efficient vaccination schedules.

Tumor cells can have different morphologic or metabolic phenotypes and display genetic instability. Thus they could also vary in their ability to present epitopes to the immune system. We have analyzed the presentation of H-2 Kb- and Db-restricted cytotoxic T lymphocyte (CTL) epitopes of a tumor-associated antigen by three cell lines derived from hepatocarcinomas developed in vivo by mice transgenic for SV40 T targeted to the liver. SV40 T is the obvious tumor-specific antigen and epitopes derived from this antigen were therefore studied. The study included four already known epitopes that can be presented by SV40-transformed kidney cells and two new CTL epitopes that were identified in the present work. CTL lines specific for each epitope were obtained from C57BL/6 mice and were used to map the presentation of SV40 T peptides by the hepatocarcinoma cells. These tumor cells were derived from the same tissue, induced by the same agent and all naturally presented peptide p232-240 from p53. Despite these common features, they all had different patterns of spontaneous presentation of SV40 T CTL epitopes. The mechanisms underlying this disparity are discussed, together with the possible consequences for establishing immunotherapeutic strategies.

There is now good evidence that cytokines contribute to the regulation of tumor growth. The cytokine-driven modulation of tumor growth was investigated during the progression of a hepatocellular carcinoma (HCC) in SV40 large T tumor antigen transgenic mice. In vivo, an increased rate of liver growth correlated with increased transforming growth factor (TGF)-beta 1 mRNA expression, while the greatest amounts of tumor necrosis factor (TNF)-alpha mRNA were detected earlier during tumor development. Conversely, no particular alteration of IL-1 alpha, IL-1 beta, IL-6, IL-2, IL-4 and IFN-gamma mRNA production could be reported. In vitro, hepatocyte-like tumor cell lines established at two stages, either before or after HCC differentiation, were characterized. The early-stage-derived cell line produced TNF-alpha mRNA, but had barely detectable expression of TGF-beta 1 mRNA, while later-stage-derived cell lines showed the reciprocal pattern. All cell lines displayed a lack of sensitivity to TNF-alpha, although some degree of sensitivity to TNF-alpha could be observed in the presence of actinomycin-D or after treatment with IFN-gamma. The early-stage-derived cell line was sensitive to the growth inhibitory effects of TGF-beta 1, but late-stage-derived tumor cell lines displayed a loss of sensitivity to TGF-beta 1 which correlated with the increased expression of TGF-beta 1 mRNA. Altogether, this suggests that tumor cells contribute to the discrete TNF-alpha and TGF-beta 1 expression patterns during HCC progression. This model of HCC could be of valuable interest to assess the impact of various immunotherapeutic strategies on modulation of tumor growth.

Free unmodified peptides are poor immunogens for cytotoxic T lymphocytes (CTL) in mice, unless they are injected with adjuvant. Although it is generally accepted that CD4+ Th cells are essential for CTL priming with peptides, it is not clear how long sequences devoid of any CD4 epitope, or strict CD8 epitopic sequences too short to be presented in a MHC class II-restricted fashion, can generate such responses. We thus have examined the extent to which the immunization protocol affects the need for a CD4 epitope. Since peptides are potentially important for vaccination, we also examined the duration of the CTL responses using a set of peptides that contained a CD4 epitope, or a CD8 epitope, or both epitopes in the same sequence, and compared immunization protocols previously found to induce CTL responses with peptides. The in vivo injection protocol had a marked effect, since the same CD8 sequence could generate a CTL response in a Th-dependent or Th-independent fashion, depending on the protocol used. The T cell help provided by natural CD4-CD8 sequences was inefficient in Th-dependent CTL priming and CTL generation required help from a stronger exogenous CD4 peptide. The peptides could be injected either as a single tandem CD8-CD4 peptide or as a mixture of two separate peptides. Th-independent CTL responses primed in other conditions proved to be as strong as Th-dependent responses, at least when the animals were killed shortly after the last injection. However, only CTL responses generated together with specific Th cells persisted for several months. Moreover, the efficacy of CTL persistence seemed to be correlated with the strength of the CD4 epitope for priming. This has important implications for the design of peptide vaccines.

We have investigated the influence of mutations in the Ras 61 codon on the immunogenicity of synthetic peptides and H-Ras p21 proteins. H-2k mice produced Th responses when immunized with mutated peptides in which the Gln at position 61 in the wild type sequence was replaced by Leu (L) or His (H). T cell hybridomas specific for the 61L and 61H peptides were then produced. The responses of both were I-Ak restricted. Competition experiments indicated that the wild type peptide did not bind to the I-Ak molecule whereas the two mutations generated a site on the peptides that was agretopic for the I-Ak molecule. Nevertheless the recognition of the corresponding Ras proteins was highly dependent upon the nature of the substitution. The H-Ras p21 protein with the 61L mutation (61L) was processed by syngeneic splenocytes and the epitope dependent on 61L was recognized as efficiently as the corresponding peptide by the T cell hybridoma specific for 61L. In contrast, the processing of H-Ras p21 with the 61H mutation (61H) was probably inefficient in producing the epitope recognized by the hybridoma specific for 61H. Furthermore, immunization studies with the two mutated H-Ras p21 proteins suggest that only the 61L substitution can be exploited for immunotherapy. Thus this work demonstrates that any peptide immunotherapy must be undertaken with the reservation that not all oncogenic mutations at codon 61 will be amenable to immune therapy.