A quantitative, single-cell PCR analysis of an antigen-specific TCR repertoire selected during an in vivo CD8 response: direct evidence for a wide range of clone sizes with uniform tissue distribution

The development of T cell effector and memory responses against foreign ant igens (Ags) involves the activation, differentiation and proliferation of n aive T cells expressing distinct Ag-specific TCRs, Understanding the comple xity of Ag-selected TCR repertoires in individual responders in terms of th e sequences selected and their relative frequencies may provide indications about how a repertoire is established and suggest ways to influence the ou tcome of an immune response. Most methods of repertoire analysis are unsuit able for calculating the relative in vivo frequencies of Ag-specific clones (expressing distinct TCRs) selected during an immune response, whereas seq uence data obtained by single-cell PCR analysis directly reflect cell frequ encies if a sufficiently large number of cells is sampled. Using a CD8 T ce ll response in normal mice in which AE selected cells are identified by cel l surface phenotype and rearranged TCRBV sequences are determined by PCR am plification of genomic DNA directly from single cells, we have analyzed a l arge number (> 200 per animal) of structurally-related Ag-specific TCRs to calculate the frequencies of distinct TCRs selected by individual mice. We found that each responder selects a unique Ag-specific TCR repertoire in wh ich the various TCRBV sequences are present in a wide range of frequencies. However: the overall distribution of sequences is quite similar for differ ent responder animals. Moreover, an individuals selected TCR repertoire is uniformly represented among Ag-specific CD8 cells circulating in the blood or localized in the spleen or liver. Relatively few sequences make up the b ulk of the repertoire and account for the oligoclonality observed in earlie r studies. We discuss various models that could account for this skewed dis tribution of an Ag-selected TCR repertoire. (C) 1999 Elsevier science Ltd. All rights reserved.