Martina A. Sherritt

Selection for T-cell receptor Vβ-Dβ-Jβ gene rearrangements with specificity for a myelin basic protein peptide in brain lesions of multiple sclerosis

MULTIPLE sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system in which a restricted cellular immune response has been observed. In order to establish whether such T cell responses are likely to be antigen-specific particularly with regard to myelin basic protein (MBP), we analysed T-cell receptor (TCR) gene rearrangements directly from MS brain plaques, using the polymerase chain reaction on reverse transcribed messenger RNA, and compared these with TCR of previously described MBP-specific T cell clones from MS and the rat model experimental allergic encephalomyelitis. Rearranged Vβ5.2 genes were detected in the brains of all patients who were HLA DRB1*1501, DQA1*0102, DQB1*0602, DPB1*0401. The Vβ5.2–Dβ–Jβ sequences in these MS brain plaques revealed five motifs. One of the common motifs was identical to that described for the VDJ region of a Vβ5.2 T-cell clone. This clone was from an MS patient who was HLA DRB1*1501, DQB1*0602, DPB1*0401, and it was cytotoxic towards targets containing the MBP peptide 89–106 (ref. 1). The deduced amino-acid sequence of this VDJ rearrangement, Leu-Arg-Gly, has also been described in rat T cells cloned from experimental allergic encephalomyelitis lesions, which are specific for MBP peptide 87–99 (ref. 2). VDJ sequences with specificity for this MBP epitope constitute a large fraction (40%) of the TCR Vβ5.2 N(D)N rearrangements in MS lesions. The capacity of rat T cells with these VDJ sequences to cause experimental allergic encephalomyelitis2 and the prevalence of such sequences in demyelinated human lesions indicate that T cells with this rearranged TCR may be critical in MS.

Reconstitution of the latent T-lymphocyte response to Epstein-barr virus is coincident with long-term recovery from posttransplant lymphoma after adoptive immunotherapy

Background. Adoptive transfer of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) has been used to treat EBV-induced posttransplant lymphoproliferative disease (PTLD) in solid-organ recipients. This study defines, in detail, the temporal relationship between adoptive transfer and the clinical response, EBV DNA load, and CTL response to EBV latent and lytic antigens in a patient with a subcutaneous PTLD presentation treated with adoptive transfer of autologous CTL. Methods. A heart transplant patient developed multiple subcutaneous PTLD deposits and was treated with a total of six doses (20 × 106 CTL per dose) of cultured autologous polyclonal EBV-specific CTL by adoptive transfer. Results. Complete regression occurred after the sixth CTL dose, and the patient has remained disease-free from 47 weeks to the present (136 weeks). Real-time polymerase chain reaction analysis showed a reduction in viral load after therapy. Enzyme-linked immunospot analysis using defined EBV CTL epitopes showed that the CTL precursor frequency (pCTL) toward a lytic antigen epitope was elevated early in the course of disease but tended to decrease to lower levels after long-term regression of PTLD. The most dramatic result was seen in relation to three latent CTL epitopes studied. Long-term regression of PTLD was characterized by high pCTL toward the latent antigens. Conclusions. Increased pCTL reactivity to latent EBV CTL epitopes is coincident with recovery from disease after adoptive transfer of autologous CTL. Furthermore, the results are compatible with the belief that activation of a sustained CTL response to EBV latent epitopes is protective and may be a characteristic of patients in long-term remission from PTLD.

Effect of pre-existing cytotoxic T lymphocytes on therapeutic vaccines

Therapeutic vaccines which aim to induce CD8+ cytotoxic T lymphocyte (CTL) responses will often be required to perform in the presence of pre‐existing CTL which recognize epitopes within the vaccine. Here we explore the ability of a viral vaccine vector presenting several co‐dominant CTL epitopes to prime CTL responses in animals that have a pre‐existing CTL response to one of the epitopes in the vaccine. The vaccine was usually capable of inducing multiple new responses, suggesting that immunodomination effects of pre‐existing CTL may generally be minimal following vaccination. However, when large numbers of pre‐existing CTL were present, a novel type of immune modulation was observed whereby (1) the vaccine failed to prime efficiently new CTL responses that were restricted by the same MHC gene as the pre‐existing responses, and (2) vaccine‐induced CTL responses restricted by other MHC genes were enhanced. These results may have implications for therapeutic multi‐epitope vaccines for diseases like HIV and melanoma, which aim to broaden CTL responses.

Contrasting Epstein-Barr virus-specific cytotoxic T cell responses to HLA A2-restricted epitopes in humans and HLA transgenic mice: implications for vaccine design

This study investigates the hierarchy of cytotoxic T cell (CTL) responses to twelve HLA A2-restricted epitopes from the latent, lytic and structural proteins of Epstein-Barr virus (EBV) in acute infectious mononucleosis and in healthy seropositive donors and the relative immunogenecity of these epitopes in transgenic mice. Responses to the lytic epitope were uniformly strong in all healthy seropositive individuals and acute infectious mononucleosis donors while moderate or low responses were observed to the latent and structural epitopes, respectively in both groups studied. In contrast, when HLA A2/Kb transgenic mice were immunised with these peptide epitopes, CTL responses were observed to all epitopes with a maximal response to the epitopes within the structural proteins and low to moderate responses to the latent epitopes. This hierarchy of CTL responses in mice was also reflected in an MHC stabilisation analysis. These contrasting CTL responses in humans following natural infection compared to the immunogenicity of these epitopes and their ability to stabilise MHC may need to be considered when designing an EBV vaccine.

Use of recombinant vaccinia to restimulate antigen specific human peripheral blood cytotoxic T lymphocytes

A simple method is described for using recombinant vaccinia virus for restimulating human peripheral blood mononuclear cells (PBMC) to generate antigen specific CD8 + alpha beta cytotoxic T cell (CTL) effector populations. Effector PBMC were restimulated in vitro with a subpopulation of PBMC, which had been infected with recombinant vaccinia at very low multiplicities of infection in the absence of serum. This protocol avoided the vaccinia mediated overt cytopathic effects on the effector PBMC and generated bulk CTL cultures, which could be used for the identification of epitopes recognised by antigen specific CTL.