Preferential escape of subdominant CD8+ T cells during negative selection results in an altered antiviral T cell hierarchy.

Negative selection is designed to purge the immune system of high-avidity, self-reactive T cells and thereby protect the host from overt autoimmunity. In this in vivo viral infection model, we show that there is a previously unappreciated dichotomy involved in negative selection in which high-avidity CD8(+) T cells specific for a dominant epitope are eliminated, whereas T cells specific for a subdominant epitope on the same protein preferentially escape deletion. Although this resulted in significant skewing of immunodominance and a substantial depletion of the most promiscuous T cells, thymic and/or peripheral deletion of high-avidity CD8(+) T cells was not accompanied by any major change in the TCR V beta gene family usage or an absolute deletion of a single preferred complementarity-determining region 3 length polymorphism.

Estimating the precursor frequency of naive antigen-specific CD8 T cells.

The constraint of fitting a diverse repertoire of antigen specificities in a limited total population of lymphocytes results in the frequency of naive cells specific for any given antigen (defined as the precursor frequency) being below the limit of detection by direct measurement. We have estimated this precursor frequency by titrating a known quantity of antigen-specific cells into naive recipients. Adoptive transfer of naive antigen-specific T cell receptor transgenic cells into syngeneic nontransgenic recipients, followed by stimulation with specific antigen, results in activation and expansion of both donor and endogenous antigen-specific cells in a dose-dependent manner.

Evolution of the T cell repertoire during primary, memory, and recall responses to viral infection.

Many viral infections induce a broad repertoire of CD8(+) T cell responses that initiate recognition and elimination of infected cells by interaction of TCRs with viral peptides presented on infected cells by MHC class I proteins. Following clearance of the infection, >90% of activated CD8(+) T cells die, leaving behind a stable pool of memory CD8(+) T cells capable of responding to subsequent infections with enhanced kinetics. To probe the mechanisms involved in the generation of T cell memory, we compared primary, memory, and secondary challenge virus-specific T cell repertoires using a combination of costaining with MHC class I tetramers and a panel of anti-Vss Abs, as well as complementarity-determining region 3 length distribution analysis of TCR Vss transcripts from cells sorted according to tetramer binding.

Impaired anti-viral T cell responses due to expression of the Ly49A inhibitory receptor.

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells.

In vivo dynamics of anti-viral CD8 T cell responses to different epitopes. An evaluation of bystander activation in primary and secondary responses to viral infection.

Viral infections induce extensive T cell proliferation in vivo. However, only a small fraction (1-5%) of the activated T cells have been shown to be virus specific leading to the prevailing notion that most of the T cell expansion represents cytokine-mediated by-stander activation and/or cross reactive stimulation of non specific cells. To re-examine this issue we quantitated antigen specific CD8 T cells during acute LCMV infection of mice using three sensitive techniques: (i) intracellular cytokine production, (ii) single cell ELISPOT and (iii) direct visualization of antigen specific CD8 T cells by staining with MHC class I tetramers + peptide.