An influenza A virus-specific and HLA-DRw8-restricted T cell clone cross-reacting with a transcomplementation product of the HLA-DR2 and DR4 haplotypes

The clone TA10 is a T3+ T4+ T8- proliferative and cytolytic human T cell clone. This clone has been shown to be specific for the hemagglutinin of influenza A Texas virus and restricted by an HLA class II molecule associated with the DRw8-Dw8.1 phenotype. Here we show that TA10 and all of its subclones can also react with eight HLA-DRw8 negative, Epstein-Barr virus (EBV)-transformed cell lines or phytohemagglutinin blasts in the absence of influenza antigens. All of these cell lines are HLA-DR2/DR4 with a classic DR2 long haplotype. The only nonreactive HLA-DR2/DR4 cell line observed bears a DR2 short haplotype. Only heterozygous HLA-DR2/DR4 but not parental DR2 or DR4 EBV-transformed cell lines can be recognized by TA10, indicating that the cross-reacting determinant is a transcomplementation product between HLA-DR2 and HLA-DR4 haplotypes. DR-specific, but not DQ- or DP-specific monoclonal antibodies, inhibit in the proliferation assay and in the chromium release test both the DRw8-Dw8.1-restricted and the anti-DR2/DR4 reactions. These results show that HLA-DR-restricted, anti-viral human T cell clone can evidence cross-reactivity for allospecific class II molecules of the major histocompatibility complex, and human CTL can recognize transcomplementation products of class II HLA genes. In addition, the results suggest that a beta-chain coded for by an HLA-DR gene and associated with an alpha-chain coded for by a still unidentified but possibly HLA-DQ gene constitute this functional transcomplementation product.     

HLA-binding regions of HIV-1 proteins. I. Detection of seven HLA binding regions in the HIV-1 Nef protein.

The physical association of HLA class I or H-2 molecules with 36 HIV-1 Nef synthetic peptides was studied using a direct peptide binding assay (PBA) in solid phase. To assess the functional significance of the PBA results, the Nef peptides were also tested for their ability to inhibit the lytic activity of human or murine CTL. The PBA results showed that seven partly overlapping regions of the Nef protein contained MHC binding peptides (4-18, 46-67, 73-94, 100-128, 126-155, 182-198, and 192-206). Five of these seven regions included all the already described epitopes recognized by CD8+ human CTL. The two other regions, 4-18 and 46-67, are not yet described as antigenic for human CD8+ cells but they are located in the N-terminal part of Nef that was previously described as being stimulator for rat or chimpanzee CD4+ cells. Altogether, it can be concluded that 1) In virtually 100% of the cases, the PBA is capable to detect known antigenic peptides recognized by CTL. 2) The PBA and the functional inhibition assay provide similar results, supporting the functional significance of PBA results. 3) The PBA is easy to handle on a large scale, using multiple peptide and several MHC molecules, so that it can be used as a routine method for prevision of possibly epitopic sequences. 4) Systematic studies of peptides issued from the whole sequence of a given protein allow to map polyepitopic areas that are probably the most interesting parts of proteins for a vaccine purpose.     

A study of functionally active amino acids involved in the interaction of HLA-A2 or HLA-A3 molecules with cytolytic T lymphocytes

A large series of HLA-A2/HLA-A3 recombinant genes were generated by using the in vivo recombination technique. These genes have each been modified in the last two-thirds of the third exon such that one or several HLA-A2-specific substitutions have been made in the HLA-A3 gene and vice versa. The recombinant genes were transfected into the murine cell line P815 and the transfectants were used as targets for a series of 20 human CTL lines or clones specific for HLA-A2 or HLA-A3, or restricted by HLA-A2 and specific for influenza A. Several patterns of anti-HLA-A2, anti-HLA-A3, and HLA-A2-restricted anti-influenza CTL activity were observed and when uncloned cell lines were studied, a progressive selection of some clones with a similar pattern of activity was regularly found. From the comparison of these different patterns the following conclusions can be drawn: 1) In most but not all cases both domains of the class I molecule were essential for CTL recognition, but residue 152 was critically important for the majority of CTL tested; 2) amino acids 114/116 were also critical in most cases, and their position close to amino acid 152 in the tertiary structure of the molecule may have some functional significance; and 3) amino acid 161, although highly conserved, plays an unexpected but very important role in CTL function.     

Two epitopes and one agretope map to a single HLA-A2 peptide recognized by H-2-restricted T cells

Mice immunized with syngeneic cells transfected with cloned genes coding for HLA class I molecules could recognize the human MHC Ag in the context of their own H-2 molecules. We obtained CTL clones from DBA/2 mice (H-2d) which had been immunized with P815 cells (a mastocytoma of DBA/2 origin) expressing either HLA-A2 or HLA-A3 or two different molecules containing recombined sequences of HLA-A2 and HLA-A3. Fourteen of these clones recognized a synthetic peptide corresponding to the region 170-185 of HLA-A2 in the context of H-2Kd. Moreover, from their activity on P815 cells expressing HLA-Cw3, two subpatterns could be distinguished: subpattern Cw3+, defined by those clones which lysed P815-Cw3, and subpattern Cw3- defined by those clones which did not lyse P815-Cw3. By testing the activity of clones of each subpattern on a series of modified synthetic peptides, we were able to define two epitopes on the same 170-185 peptide of HLA-A2. One of them was dependent on amino acids at positions 173 and 177, whereas the other was dependent on amino acid 177 alone. By using competition experiments, we were also able to define an agretopic region strongly dependent on the amino acid at position 178. Furthermore, experiments with L cells expressing molecules containing recombined sequences between H-2Kd and H-2Dd demonstrated the determinant role of residues 152, 155, and 156 from H-2Kd in the presentation to murine T cells of the 170-185 peptide of HLA-A2.     

Identification of Tenrec ecaudatus,a Wild Mammal Introduced to Mayotte Island,as a Reservoir of the Newly Identified Human Pathogenic Leptospira mayottensis

Leptospirosis is a bacterial zoonosis of major concern on tropical islands. Human populations on western Indian Ocean islands are strongly affected by the disease although each archipelago shows contrasting epidemiology. For instance, Mayotte, part of the Comoros Archipelago, differs from the other neighbouring islands by a high diversity of Leptospira species infecting humans that includes Leptospira mayottensis, a species thought to be unique to this island. Using bacterial culture, molecular detection and typing, the present study explored the wild and domestic local mammalian fauna for renal carriage of leptospires and addressed the genetic relationships of the infecting strains with local isolates obtained from acute human cases and with Leptospira strains hosted by mammal species endemic to nearby Madagascar. Tenrec (Tenrec ecaudatus, Family Tenrecidae), a terrestrial mammal introduced from Madagascar, is identified as a reservoir of L. mayottensis. All isolated L. mayottensis sequence types form a monophyletic clade that includes Leptospira strains infecting humans and tenrecs on Mayotte, as well as two other Malagasy endemic tenrecid species of the genus Microgale. The lower diversity of L. mayottensis in tenrecs from Mayotte, compared to that occurring in Madagascar, suggests that L. mayottensis has indeed a Malagasy origin. This study also showed that introduced rats (Rattus rattus) and dogs are probably the main reservoirs of Leptospira borgpetersenii and Leptospira kirschneri, both bacteria being prevalent in local clinical cases. Data emphasize the epidemiological link between the two neighbouring islands and the role of introduced small mammals in shaping the local epidemiology of leptospirosis.     

Cross-Reactions between the Cytotoxic T-Lymphocyte Responses of Human Immunodeficiency Virus-Infected African and European Patients

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.     

A role for the granzyme B inhibitor serine protease inhibitor 6 in CD8+ memory cell homeostasis

Generation and maintenance of protective immunological memory is the goal of vaccination programs. It has recently become clear that CD8+ memory T cells are derived directly from CTLs. The mechanisms underlying this transformation and the subsequent survival of memory cells are not completely understood. However, some effector molecules required by CTLs to eliminate infected cells have also been shown to control the number of Ag-specific cells. We report that memory cells express high levels of serine protease inhibitor (Spi) 6, an inhibitor of the effector molecule granzyme B, and that Spi6 can protect T cells from granzyme B-mediated apoptosis. In mouse models, both elevated expression of Spi6 and the complete absence of granzyme B in CD8+ T cells led to an increase in memory cells after infection with lymphocytic choriomeningitis virus. This was not the result of increased levels of antilymphocytic choriomeningitis virus CD8+ T cells during the expansion or contraction phases, but rather transgenic Spi6 directly influenced the survival of CD8+ memory T cells. We propose that expression of protective molecules, like Spi6, serves to shield metabolically active CD8+ memory T cells from their own effector molecules.     

Natural suppressor cell inhibiting T killer responses against retroviruses: a model for self tolerance

We previously reported the characterization of a spontaneous suppressor T cell population (NSC) present in naive mice and able to suppress the cytotoxic response (CTL) against tumor cells induced only by endogenous Gross virus (GLV). In this study we demonstrate the existence of such NSC inhibiting the CTL activity against tumor cells induced by the normally exogenous Moloney virus (M-MLV) in mice of the Mov-13 (V+) strain in which the M-MLV has been artificially endogenized and which express the virus during the embryonal life. These NSC are not found in other Mov strains in which the endogenized M-MLV is not expressed during fetal life. The implication of these data in the mechanism of self tolerance is discussed.     

Accessory cells in the in vitro generation of type C virus-specific T killer lymphocytes. II. Demonstration of a T helper cell in the spleen of in vivo primed mice

The existence of a helper T cell cooperating with cytolytic T lymphocytes (CTL) in cell-mediated anti-tumor responses specific for the virus-induced FMR antigens can be demonstrated by using unprimed thymocytes as CTL precursors and in vivo primed irradiated spleen cells as helper. The helper T cells express Thy-1.2 and Lyt-1.2 antigens at their surface, but not Lyt-2.2. The helper function required the presence of macrophages to be detected, is antigen specific, and appears unusually radiosensitive compared with previously described helper T cell function.