Posttransplant cyclophosphamide (PTCy) is a promising graft-versus-host disease (GVHD) prophylaxis in haploidentical and matched unrelated donor hematopoietic stem cell transplantation (HSCT), but its role in matched sibling donor (MSD) transplants remains unclear. We conducted a retrospective study of 413 MSD-HSCT patients receiving peripheral blood stem cell (PBSC) grafts from January 2010 to January 2023. Patients were categorized into 4 groups: group I (calcineurin inhibitor [CNI] + methotrexate [MTX] or mycophenolate mofetil [MMF]), group II (CNI + MTX or MMF + antithymocyte globulin [ATG]), group III (PTCy + ATG + CNI), and group IV (PTCy + CNI + MMF). PTCy was associated with a significant reduction in grade 2- 4 and grade 3-4 acute GVHD and moderate-to-severe chronic GVHD compared with CNI + MTX (or MMF)–containing regimens. PTCy did not increase relapse risk; PTCy reduced nonrelapse mortality, leading to improved GVHD-free/relapse-free survival (GRFS; Hazard Ratio, 0.4; P < .001). PTCy was also associated with improved overall survival. Bloodstream infections were increased with PTCy. The addition of ATG to PTCy did not further improve GRFS and was associated with a higher incidence of clinically significant cytomegalovirus (csCMV) and Epstein-Barr virus (csEBV) reactivation and a numerical increase in NRM. PTCy significantly appeared to improve GRFS in the MSD setting using PBSC grafts. The addition of ATG to PTCy increases csCMV and csEBV reactivation without further improving GRFS. Prospective trials and PTCy dose optimization are warranted.

Interleukin-10 (IL-10)-producing group 2 innate lymphoid cells (ILC2₁₀) regulate inflammatory immune responses, yet their therapeutic potential remains largely unexplored. Here, we demonstrate that cell therapy with human ILC2₁₀ inhibits pathogenic T cell responses in humanized mouse models of graft-versus-host disease (GVHD), resulting in reduced GVHD severity and improved overall survival without limiting the graft-versus-leukemia effect. ILC2₁₀ conferred superior protection from GVHD than IL-10^-/low ILC2s, and blocking IL-10 and IL-4 abrogated ILC2₁₀ protective effects, indicating that these cytokines are important for the protective effects of ILC2₁₀. Notably, ILC2₁₀ provided comparable protection from GVHD to regulatory T cells without impairing T cell engraftment, instead decreasing intestinal T cell infiltration and suppressing CD4⁺ Th1 and CD8⁺ Tc1 cells. CITE-seq of expanded ILC2s revealed CD49d and CD86 are markers that allow for enrichment of ILC2₁₀ from conventional ILC2s and tracking of ILC2₁₀ in patient studies. Altogether, these findings demonstrate the potential of ILC2₁₀ in cell therapies for GVHD and other immune-mediated diseases.

Natural killer (NK) cell activity is influenced by cytokines and microenvironment factors, resulting in remarkably diverse functions, by contributing to inflammatory responses or serving as rheostats of adaptive immunity. Using single cell RNA sequencing (scRNA-seq), we identified a TGFβ1^highCD56^brightNK cell population associated with hematopoietic stem cell transplant recipients protected from acute graft-versus-host disease (GVHD). We further define a role for the combination of interleukin-2 (IL-2) and transforming growth factor β1 (TGF-β1) in promoting a regulatory phenotype in NK cells. “Induced” regulatory NK cells produce high amounts of TGF-β1, inhibited T cells, could promote naive T cells differentiation into regulatory T cells, and exhibited a unique transcriptional program that includes expression of IKZF2 (HELIOS) and ZNF683 (HOBIT). This phenotype was not stable, and “induced” regulatory NK cells lost the ability to secrete TGF-β1 upon exposure to different cytokines. These findings define protective CD56^brightNK cells post-hematopoietic stem cell transplantation, and demonstrate the combination of IL-2 and TGF-β1 promotes regulatory activity in NK cells.