Research Article

Immunology and Cell Biology (1996) 74, 96–104; doi:10.1038/icb.1996.13

Antigen-Specific apoptosis in immortalized T cells by soluble MHC class II-peptide complexes

Subhashini Arimilli1, John B Mumm1 and Bishwajit Nag1

1Anergen Inc., 301 Penobscot Drive, Redwood City, California, USA

Correspondence: Bishwajit Nag, Anergen Inc, 301 Penobscot Drive, Redwood City, CA 94063, USA.

Received 29 September 1995; Accepted 16 November 1995.

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Abstract

The recognition of T cell receptors (TCR) by purified major histocompatibility complex (MHC) class II-peptide complexes in the absence of costimulatory signals leads to the induction of T cell nonresponsiveness or anergy. In a recent study using human T cell clones, it was observed that prolonged incubation of resting T cells with soluble MHC II-peptide complexes appears to result in T cell apoptosis. The present study shows that the engagement of TCR by soluble MHC II-peptide complexes also results in antigen-specific apoptosis in immortalized T cells. Apoptosis was demonstrated in a herpes saimiri virus (HSV) transformed human T cell clone (SS8T) restricted for HLA-DR2 in association with an epitope from the myelin basic protein [MBP(84–102)]. A dose- and time-dependent T cell death was observed upon incubation of SS8T cloned T cells with purified complexes of native human HLA-DR2 and MBP(83–102)Y83 peptide. The specificity of T cell apoptosis was demonstrated by exposing SS8T cells with DR2 alone and DR2 bound to another high affinity epitope [MBP(124–143)] from the same MBP. Recently, we have shown that the complexes of HLA-DR2 and [MBP(83–102)Y83] can be reconstituted by refolding Escherichia coli expressed individual DR2 a and beta (B5*010l) polypeptide chains lacking the transmembrane region. When SS8T cloned T cells were exposed to purified reconstituted rDR2.MBP(83–102)Y83 complexes, similar apoptosis of T cells was observed. Agarose gel analysis of T cells incubated with complexes showed a degradation of celluar deoxyribonucleic acid (DNA) to oligonucleosomal bands, a characteristic of apoptosis. The quantitative detection of DNA strand breaks was performed by pulsing T cells with 5-bromo-2'-deoxyuridine(BrdU) followed by the detection of BrdU-labelled DNA fragments using an antibody sandwich enzyme-linked immuno assay (ELISA). The fragmentation of DNA was also measured by double fluorescence flow cytometry by 3' end labelling of fragmented DNA with biotinylated-deoxyuridine triphosphate (dUTP) in the presence of terminal deoxynucleotide transferase (TdT) enzyme. The expression of the bel-2 protein in SS8T cells following TCR engagement by soluble MHC II-peptide complexes was monitored by chemiluminescence blot analysis using anti-bel-2 monoclonal antibody. Finally, the nucleosomal condensation of T cells following complex treatment, characteristics of typical apoptosis, was demonstrated by transmission electron microscopy. These results suggest that the binding of soluble MHC class II-peptide complexes to TCR induces antigen-specific apoptosis in transformed CD4 positive T cells in vitro. Such induction of apoptosis by soluble MHC II-peptide complexes may provide a novel therapeutic strategy to delete autoreactive T cells in various autoimmune diseases.

Keywords:

reconstituted MHC-peptide complexes, soluble MHC class II-peptide complex, T cell apoptosis

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