Repertoire breadth of human CD4+ T cells specific for HIV gp120 and p66 (primary antigens) or for PPD and tetanus toxoid (secondary antigens)

Antigen derived peptides bound on MHC class II molecules on presenting cells stimulate specific CD4 lymphocytes that are in a naive state if antigen is given for the first time, or in a memory state if antigen has been previously encountered. In order to compare clonal heterogeneity of the human CD4+ T helper repertoire in primary vs. recall responses, we have generated T cell lines in vitro by repeated stimulation of peripheral lymphocytes with primary or with recall antigens. Clonal heterogeneity was broad in the case of recall response to tetanus toxoid or PPD, with a high frequency of specific precursors (> 100 cells/10(6) lymphocytes). In contrast, T cell lines responsive to primary antigens (HIV gp120 or HIV p66) were oligoclonal as defined by TCR V beta gene usage and by spectratyping, and the precursor frequency was low (< 2 cells/10(6) lymphocytes). Primary T cell lines generated from blood samples drawn at different times from the same donor showed that clones with identical TCR CDR3 region coding sequences were expanded, suggesting that in these individuals a large progeny derived from one single precursor is present, even though a previous encounter with the antigen was not documented. Assuming an even in vivo distribution of such cells, the presence of one precursor every 10(6) CD4 lymphocytes (within the CD4 T repertoire that comprises roughly 10(11) CD4 T cells) indicates that approximately 10(5) identical T cells from the same clonal precursor account for the primary response against the model antigens we have studied.

Human T leukaemia virus type 1 (HTLV-1) specific T-helper cell response: clonal fluctuations and repertoire heterogeneity.

The naive T-helper (Th) repertoire specific for HTLV-1 envelope (env) has been examined on antigen specific T-cell lines and clones from non-immune individuals. Clonal heterogeneity was determined by analysing the T-cell receptor (TCR) Vbeta gene usage and by sequencing the hypervariable regions of the TCR genes. Fluctuations in the V beta gene usage were determined by comparing the TCR Vbeta gene profiles of T-cell lines at different times. We found that a diverse repertoire for HTLV-1 env could be triggered in vitro. Diverse Vbeta genes were used by the same line tested at different times, suggesting that clonal composition of an antigen-specific T-cell line is not constant in vitro. Clones in fact may be up- and down-regulated and clonotypes undetectable at one time point can emerge upon subsequent restimulation. Therefore evaluation of the clonal composition of a T-cell line gives a snapshot of the dominant clones at the time of analysis, and does not tell the whole picture of the antigen-specific ensemble. Furthermore, by sequencing the TCR genes, we identified clones with identical Vbeta gene usage which differed in hypervariable regions (CDR3), indicating their derivation from independent precursors and contributing to overall clonal heterogeneity. If these data can be extended to HTLV-1-infected patients studied in vivo, the Th cell repertoire specific for HTLV-1 env may prove very heterogenous, with important implications for vaccine development.

Handling of retroviral antigens by human antigen-presenting cells.

Antigen-specific T helper cells play an important role in retroviral infections. Indeed, they provide help for B-cell activation and antibody production and for clonal expansion of cytolytic lymphocytes. Therefore, we used retrovirus-specific human T helper clones in order to define modes of antigen presentation, antigen-presenting cells and the molecular context of Th epitopes that could be exploited in the design of immunogens aimed at optimizing the Th cell response. In particular, we describe several mechanisms of receptor-mediated antigen uptake that enhance the stimulation of human T-cell clones specific for HIV and HTLV-1 antigens; we report on the differential recognition of Th epitopes depending on the molecular-viral context; we show that dendritic cells are the most efficient presenting cells and are essential for the induction of in vitro primary Th cell responses; and finally, we propose that Th cells specific for internal, conserved antigens of HIV such as reverse transcriptase, may be candidates for intrastructural help resulting in induction of envelope specific antibodies.