Impact of ATLG on CD4+ T-cell reconstitution after HSCT in children: a detailed immune profiling study

Introduction: Graft versus host disease (GvHD) prophylaxis impacts on post–hematopoietic stem cell transplantation (HSCT) immune reconstitution (IR). Specifically, anti-T lymphocyte globulin (ATLG) delays CD4+ T cell reconstitution. However, the mechanism by which ATLG alters the composition of CD4+ T-cell compartments remains elusive. Aims: Dissect the impact of ATLG on CD4+ T cells by integrating advanced and highly standardized flow cytometric panels, unsupervised clustering analysis, machine learning approaches, and molecular biology techniques. Results: We analyzed post-HSCT IR in 94 pediatric patients with a machine learning approach. ATLG exposure resulted in the most relevant variable affecting CD4+ T-cell counts. Severe combined immunodeficiency/recent thymic emigrant (SCID/RTE) and CD4 PERISCOPE EuroFlow antibody panels were applied to a sub-cohort of patients with acute lymphoblastic leukemia. Unsupervised clustering analysis showed that in the first months, post-HSCT CD4+ naïve T cells and RTEs were significantly reduced in ATLG-treated compared to no-ATLG patients, including haploidentical graft recipients receiving post-transplant cyclophosphamide. In no-ATLG patients, there was a significant contribution of residual recipient-derived cells to the RTE compartment, whereas in ATLG-treated patients RTEs were of donor origin. In addition, in no-ATLG patients, early RTEs and CD4+ T-cell counts correlated with infused CD3+ T cells/kg. Bone marrow and T-cell receptor excision circle (TREC) analysis showed that ATLG treatment does not affect early T-cell maturation. In vitro testing showed increased differentiation of naïve/RTE CD4+ into effector subsets upon ATLG exposure, confirmed by flow cytometry and RNA sequencing. Discussion: Our data show that ATLG induces concomitant differentiation and depletion of the peripheral CD4+ naïve/RTE T-cell compartment. Together, these findings suggest that naïve T-cell reconstitution after ATLG is delayed since it only relies on thymic maturation, differently from no-ATLG treated patients.