Background/Purpose:   Inflammatory infiltrates of muscles in polymyositis are dominated by CD4+CD28null and CD8+CD28null T cells. In contrast to conventional CD28+ T cells, these cells have a restricted T cell-receptor repertoire, rapidly release large amounts of IFN-γ and TNF, and can kill tissue cells by granzyme-B and perforin upon activation. Although their presence in myositis muscles have been demonstrated recently, their effect on muscle cells has not been clarified. Aim of the study was to investigate the potential myotoxic effect of CD28null T cells on muscle cells from patients with polymyositis, in a fully autologous co-culture system.

Methods: Muscle stem cells were extracted from biopsies from 6 patients with polymyositis (classified according to Bohan and Peter criteria) and differentiated into myotubes. T cell subsets from the same patients were isolated from peripheral blood by flow-cytometry. Co-cultures of autologous muscle cells were performed with un-stimulated or stimulated T cell subsets for 24-36 hours. Dead myotubes were quantified by calcein release assay and flow-cytometry. Blocking experiments were performed using relevant blocking antibodies to the co-cultures.

Results: CD28null T cells were spontaneously cytotoxic to autologous muscle cells in culture. The median cytotoxicity (induced muscle cell death) at 5:1 T cells-muscle cells ratio was 23.5% for CD4+CD28null and 22.3% for CD8+CD28null T cell subsets (n=4). In comparison, conventional CD4+ and CD8+ T cells displayed lower cytotoxicity (median CD4+CD28+ 10.3%, CD8+CD28+ 11.4%, n=4). Upon co-culture in stimulatory environment, both CD28null T cell subsets significantly induced more cell death in autologous muscle cells than CD28+ counterparts (n=5). Not only CD8+CD28null but also CD4+CD28null T cells displayed perforin polarization towards muscle cells and secreted higher levels of granzyme-B and IFN-γ in co-culture than CD28+ subsets (n=4). By blocking perforin, the myotoxicity was reduced by a median of 52% with CD4+CD28null T cells and by 56% in cultures with CD8+CD28null T-cells (n=3). TNF or IFN-γ did not induce death of muscle cells in the absence of T cells, but did upregulate HLA class I and II on muscle cells (n=3-5). Blockade of IFN-γ and TNF in co-cultures with T cells reduced the level of dead muscle cells by 46% (CD4+CD28null) and 53% (CD8+CD28null) (n=3). The reduced myotoxicity is likely attributed to reduced HLA expression, as HLA blockade resulted in a comparable reduction in myotoxicity, 61% (CD4+CD28null) and 55% (CD8+CD28null) (n=5).

Conclusion:  The myotoxic effect by CD28null T cells in polymyositis may be mediated by perforin-dependent killing and regulated by IFN-γ-induced HLA expression by muscle cells. Together, this suggests that CD28null T cells are key effector cells directly contributing to the muscle cell damage in polymyositis, hence represent target candidate for future therapies

ACR Meeting Abstracts - https://acrabstracts.org/abstract/cd28null-t-cells-from-polymyositis-patients-are-cytotoxic-to-autologous-muscle-cells-in-vitro-via-perforin-dependent-mechanisms/