Isolation of viable antigen-specific CD4 T cells by CD40L surface trapping.

A number of techniques have recently been developed for the identification of antigen-specific cells, yet the ability of these techniques to identify all subclasses of memory T cells has often been overlooked. Here we describe a novel approach for the isolation of live antigen-specific CD4 T cells using CD40L and CD69 surface staining and demonstrate its utility for isolating antigen-specific rhesus macaque CD4 T cells. Critical to the success of the technique was staining for CD40L concurrent with antigen stimulation. Isolation of CD4 T cells based on CD40L/CD69 surface marker upregulation identified both effector and central memory CD4 T cells. In contrast, the majority of central memory CD4 T cells did not secrete TNFalpha or IFNgamma and thus would not be identified by techniques based on their secretion. The methodology described here therefore complements existing approaches for isolating viable antigen-specific CD4 T cells, opens new avenues for investigating human diseases in nonhuman primate animal models and may prove beneficial in instances where the induced response is largely T cell central memory restricted.

Examination of the highly diverse CD4(+) T-cell repertoire directed against an influenza peptide: a step towards TCR proteomics.

We combined several recent technological advances in immunology and molecular biology to identify and sequence a large number of T-cell receptor (TCR) genes specific for a particular antigen. We utilized class II MHC tetramers and interferon-gamma surface capture to isolate from samples of peripheral blood the population of CD4(+) T cells responding to a peptide derived from influenza hemagglutinin and restricted by HLA-DR1. Detailed analysis of hundreds of clones from three different patients revealed an extremely diverse repertoire, with little overlap between patients. We observed no dominant usage of particular Vbeta segments nor any clear CDR3 sequence motif in the responding T cells, but most of the clones appear to utilize acidic residues in the CDR1 and CDR3 regions, presumably to interact with the exposed basic residues in the MHC-peptide complex. This methodology could be expanded to a large scale to identify the generalized rules governing TCR-MHC engagement and factors which shape the T-cell repertoire after vaccination and in autoimmune pathologies.

Clonotype tracking of TCR repertoires during chronic virus infections.

Human viral infections such as HIV and EBV typically evoke a strong and diverse CD8(+) T cell response. Relatively little is known about the extent to which TCR repertoire evolution occurs during viral infection or how repertoire evolution affects the efficacy of the CD8(+) T cell response. In this study we describe a general approach for tracking TCR repertoire evolution during viral infection. IFNgamma surface capture and MHC class I tetramer staining were independently used to isolate EBV-specific CD8(+) T cells from peripheral blood. Anchored RT-PCR and clonotype TCR repertoire analysis were performed immediately after isolating the cells. We find that the TCR repertoires of the IFNgamma-secreting and MHC class I tetramer staining populations were similar. In one subject a detailed analysis of the TCR repertoire during the first year of EBV infection was performed and over 600 TCR sequences targeting an EBV-immunodominant epitope were analyzed. Although some repertoire evolution occurred during the year, in general, the degree of repertoire drift was small. TCR repertoire analysis for an HIV-immunodominant epitope revealed a highly conserved amino acid motif in the Dbeta region of TCR that recognizes the epitope and suggested that T cell precursor frequency influences which epitopes are targeted early in HIV infection. This methodology, which allows one to sort antigen-specific T cells based on different functional assays and to obtain a snapshot of their TCR repertoire with relative ease, should lead to a richer understanding of the rules underlying antigen recognition and T cell evolution during viral infection.