Mega-scale single-cell profiling reveals novel biomarkers associated with acute GvHD after allogeneic hematopoietic stem cell transplantation

Alloreactive T cells mediate graft-versus-leukemia (GvL) reactions and acute graft-versus-host disease (aGvHD) in AML patients following allogeneic hematopoietic stem cell transplantation. To investigate biomarkers that identify alloreactive T cells associated with either beneficial GvL or detrimental aGvHD, we collected graft samples and two post-transplant follow-up blood samples (day 30 and day 100) of ten AML patients undergoing hematopoietic stem cell transplantation and profiled over 777,000 CD45+ leukocytes in total by combinatorial barcoding-based mega-scale single-cell RNA sequencing. Using immune receptor sequences as intrinsic clonal barcodes, we observed that especially CD8+ graft-derived T cells persisted and displayed enhanced proliferation, clonal expansion, and likely alloreactivity. Notably, patient-derived peripheral leukocytes that survived the conditioning, as identified by sex-chromosome-related genes, were primarily CD4+ T helper cells. MDGA1 expression on T cells and NK cells emerged as a novel biomarker potentially associated with aGvHD. Additionally, we observed a significant deficiency of ADGRG1 expression, a marker of alloreactive cytotoxic T cells, by aß and ?d T cells from relapsed patients. In conclusion, mega-scale single-cell monitoring of graft and hematopoietic immune cell reconstitution allowed us to demonstrate that MDGA1 and ADGRG1 may function as complementary biomarkers expressed by distinct circulating T cells that are associated with divergent outcomes in AML patients, enabling precise risk stratification of alloHSCT outcomes and presenting potential therapeutic targets. Overall design: Ten adult ( > 18y) patients with AML in complete remission who underwent alloHSCT with peripheral blood stem cell grafts (matched unrelated, n = 8; matched related, n = 2) in the Department for Stem Cell Transplantation of University Cancer Center Hamburg-Eppendorf from December 2021 to May 2022 were included in this study (Table 1). The study was approved by ethics commission of the Medical Chamber of Hamburg and was performed according to the approved study protocol (version 1.1 from 01.11.2021). Written informed consent was obtained from all participants. The majority of patients received myeloablative conditioning (n = 7) with ATG-based immunoablation (n = 9). After a median follow-up of 20 months (18–22), three patients died (relapse, n = 2; NRM, n = 1). The relapses occurred on days +149 and +622. Five patients developed acute GvHD (II-IV) at a median of 57 days (19–136). The eight patients remained alive in complete remission at the last follow-up. Peripheral blood stem cell graft samples were collected at the time of transplantation and were cryopreserved on the next day. The PBMC samples of recipients were collected at day +30 (D30) and day +100 (D100) and were cryopreserved on the same or the following day. Mononuclear cells were isolated using a Ficoll gradient and cryopreserved in DMSO until they were used for single cell analysis. All DAPI- (Thermo Fisher Scientific Cat# D1306) (RRID:AB_2629482) live cells were sorted from frozen PBMC samples at the flow cytometry core facility of University Medical Center Hamburg-Eppendorf (RRID:SCR_017156). The sorted cells were fixed and permeabilized using Evercode™ Cell Fixation v2 (Parse Biosciences, Cat. ECF2001) following the manufacturer's protocol. Fixed cells were stored at -80 °C. All samples were then processed together to prepare single-cell libraries using one Evercode™ WT Mega v2 Kit (Parse Biosciences, Cat. ECW02050), according to the manufacturer's instructions. Fifteen sub-libraries were sequenced on the Illumina NovaSeq 6000 S4 flow cell platform (RRID:SCR_016387) using PE150 chemistry with a sequencing depth of 25,000 reads per cell.