IFN-gamma and donor leukocyte infusions for relapsed myeloblastic malignancies after allogeneic hematopoietic stem cell transplantation
We conducted a phase I trial (NCT04628338) of IFN-? combined with donor leukocyte infusions (DLI) in myeloblastic malignancies that relapsed post-HLA-matched allogeneic stem cell transplantation (alloSCT). Patients self-administered IFN-? for 1-4 weeks, followed by DLI and concurrent IFN-? for 12 weeks. IFN-? monotherapy was well tolerated by all subjects (n=7). Four of 6 DLI recipients achieved minimal residual disease-negative complete remissions and full donor hematopoietic recovery. Median overall survival was 579 days (range, 97-906) in responders. Single cell RNA sequencing (scRNAseq) was performed on bone marrow (BM) samples collected before IFN-? treatment and 48-60 hours after the 1st or 2nd dose of IFN-? from three patients: one responder (Patient 1) and two non-responders (Patients 5 and 7). Recipient-derived cells were distinguished from donor-derived cells based on single nucleotide polymorphisms (SNP) in expressed genes. As expected, most immature pluripotent and myeloid lineage cells (HSC, myeloid progenitor cells, early erythroid cells) at relapse were of recipient genotypic origin, while lymphoid progenitors were mainly donor derived. Genomic copy number in single cells showed that most HSCs or myeloid progenitor cells harbored recurrent chromosome abnormalities known to each patient. To investigate the effects of in vivo IFN-? within the major lineage clusters, we performed single-cell pathway analyses (SCPA). Transcription changes in the post-IFN-? specimens were predominantly found in the non-lymphoid subsets (HSC-like cells, myeloid progenitors, CD14 or CD16 monocytes, and erythroid cells). IFN-? response pathway was activated in HSC-like cells (Patients 1 and 7) and myeloid progenitor cells (Patients 1, 5, 7) in the post-IFN-? samples. Post-IFN-? samples also had evidence of activation of “TNF-alpha signaling via NFkb” in HSC-like and myeloid progenitor cell populations. “IL2 STAT5 signaling” and “inflammatory response” pathways were also engaged in post-IFN-? myeloid populations. These scRNAseq analysis confirmed in vivo IFN-? action on the dominant malignant myeloid population. Overall design: scRNAseq was performed on BM from Patients 1, 5 and 7 collected before and 48-72 hours after the 1st or 2nd dose of IFN-?. For Patient 1, we prepared bulk, unenriched bone marrow (BM) as well as BM enriched in CD34+ BM myeloblasts by immunomagnetic selection; for Patients 5 and 7, only bulk BM cells were analyzed. Samples were captured with Chromium Controller (10x Genomics) and libraries were generated using the Chromium Next GEM Single Cell 5' v2 (Dual Index) kit (10x Genomics) according to the manufacturer's instructions. Libraries were sequenced using a NextSeq 2000 in 1 run (Illumina P3 kit), with a target of 20,000 reads per cell for gene expression.