T-Cell Reconstitution Can be a Predictive Biomarker for Severe Acute GvHD in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation with a Treosulfan/Fludarabine Conditioning Regimen

Immune Reconstitution (IR) following allogeneic HCT is a dynamic and multifaceted process that significantly impacts HCT outcomes and could serve as a predictive biomarker. Treosulfan is being increasingly used in HCT as a safer alternative to busulfan. We have previously reported the pattern of early IR post-HSCT in patients with thalassemia receiving a Treo-containing conditioning regimen (Pai et al. TCT 2023). Here, we studied the immune reconstitution pattern in HCT with treosulfan based conditioning and its correlation with the HCT outcomes.Ninety-three consecutive patients with Treo/Flu as conditioning regimen at our center between October 2021 and September 2023 (Table) were enrolled in this study. Peripheral blood samples were collected pre-conditioning, end of conditioning, third consecutive day of neutrophil engraftment (Absolute Neutrophil Count, ANC>500 cells/µl), Days +28, +60 and +90 post-HCT. Multicolor flow cytometry-based Immunophenotyping was done to enumerate the levels of Lymphocytes and their subsets – T cells, B cells, and NK cells and the Myeloid lineage at all time points. Helper T cells (Th) and Cytotoxic T cells (Tc) cells and their stages of differentiation [Central memory (CM), Effector memory (EM), Naïve, and Terminally differentiated (TD)] were also ascertained. Clinical outcomes such as engraftment, chimerism, rejection, and acute and chronic Graft versus Host Disease status (aGvHD, cGvHD) were documented prospectively. Patients with Gr.II-IV aGvHD were categorized as severe, and those with Gr. I and those who did not develop GvHD were classified as no GvHD group.Flow cytometry analysis showed almost complete lymphodepletion [3.83 cells/ µl (0-30.73) vs. 1490.32 cells/ µl (72.76-9084.5)] post-conditioning compared to pre-conditioning levels. NK cells recovered faster when compared to T cells and B cells, showing a gradual increase from ANC but did not reach pre-transplant levels until D+90. Thirty-seven out of eighty-three patients (45%) evaluable at D+28 achieved normal counts of NK cells [Ref range 253 (82-594) cells/ µl]. Further analysis with clinical outcomes revealed that at engraftment, T cells (CD3+) were lower in patients who developed severe aGvHD versus patients in no GvHD group [62.68 (1.4-385) Vs. 160.8 (2.05-840.6) cells/µl, p=0.026] while at D+28, TD Tc were lower [8.51(0-39.89) Vs. 22.72 (0.15-333.6) cells/µl, p=0.0007]. Interestingly, lower counts of TD Th cells at day+28 were associated with severe aGvHD versus patients with no GvHD [0.6 (0-18.28) Vs. 2.6 (0-40.52) cells/µl, p=0.042], Figure. Our results suggest that a lower number of T-cells and their subsets (TD Tc/ Th cells) could be a biomarker in predicting the occurrence of severe aGvHD. We are attempting to define a cut-off for predicting severe aGvHD in a prospective cohort.