Siplizumab selectively depletes effector memory T cells and promotes a relative expansion of alloreactive regulatory T cells in vitro

Siplizumab, a humanized anti-CD2 monoclonal antibody, has been used in conditioning regimens for hematopoietic cell transplantation and tolerance induction with combined kidney-bone marrow transplantation. Siplizumab-based tolerance induction regimens deplete T cells globally while enriching regulatory T cells (Tregs) early posttransplantation. Siplizumab inhibits allogeneic mixed-lymphocyte reactions (MLRs) in vitro. We compared the impact of siplizumab on Tregs versus other T cell subsets in HLA-mismatched allogeneic MLRs using PBMCs. Siplizumab predominantly reduced the percentage of CD4(+) and CD8(+) effector memory T cells, which express higher CD2 levels than naive T cells or resting Tregs. Conversely, siplizumab enriched proliferating CD45RA(-) FoxP3(HI) cells in MLRs. FoxP3 expression was stable over time in siplizumab-containing cultures, consistent with enrichment for bona fide Tregs. Consistently, high-throughput TCR beta CDR3 sequencing of sorted unstimulated and proliferating T cells in MLRs revealed selective expansion of donor-reactive Tregs along with depletion of donor-reactive CD4(+) effector/memory T cells in siplizumab-containing MLRs. These results indicate that siplizumab may have immunomodulatory functions that may contribute to its success in tolerance-inducing regimens. Our studies also confirm that naive in addition to effector/memory T cells contribute to the allogeneic MLR and mandate further investigation of the impact of siplizumab on alloreactive naive T cells.