CD52/GPI- T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation

Alemtuzumab is a CD52-specific lympho-depleting antibody. CD52- T cells emerge under alemtuzumab selection pressure. We sought to investigate the phenotype and function of the CD52- T cell fraction and related their presence to clinical outcome. We obtained longitudinal peripheral blood samples from 67 consecutive patients undergoing allo-HSCT between 2013-2016. Forty-seven patients (70%) had a myeloid disease (acute myelogenous leukemia or myelodysplastic syndrome) whereas 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10 mg/d from day -5 for 5 days) as part of their conditioning protocol. Sixty-three (94%) received reduced-intensity conditioning chemotherapy, whereas 4 (6%) received a myeloablative regimen. All patients received post-transplantation cyclosporine A for graft-versus-host disease (GVHD) prophylaxis. Six (9%) also received methotrexate, whereas 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68%, and relapse-free survival was 48%. Twenty-none percent of patients experienced acute GVHD (grade 2 or above), and 15% developed chronic GVHD. CD52- T cells were detectable in 66 of 67 consecutive patients. CD52- T cells demonstrated low binding of fluorescent aerolysin, indicating downregulation of the glycophosphatidylinositol anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with paroxysmal nocturnal hemoglobinuria. CD52- T cells were almost exclusively CD4⁺ and exhibited a dominant memory phenotype with only small numbers of CD25⁺ CD127^low Foxp3⁺ regulatory T cells. CD52- T cells exhibited alloreactive specificity in vitro and have a distinct TCR repertoire to CD52+ T cells. Early after allo-hematopoietic stem cell transplantation, the presence of a significant population of CD52- T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that the CD52- T cell fraction may represent a residual "footprint" of an early CD4+ T cell alloreactive response and may have been rescued from alemtuzumab-mediated lysis by antigen engagement in vivo. These data help to delineate the nature of T cell escape from alemtuzumab surveillance and contribute to increasing interest in the importance of CD4+ T cells in alloreactive immune responses, which could help inform immunotherapy protocols.