Gene Expression Profiling and Signaling Pathway Analysis of Tolerant T cells Circulating in Adult Patients after PTCy Haplotransplantation

Abstract The mechanisms for the signaling pathways of T cell-dependent tolerance after haploidentical hematopoietic stem cell transplantation (haplo-HSCT), are incompletely understood. We tested 8 patients (Pt) with leukemia or myelodysplastic syndromes (median age:51-yrs old) following IRB approved consenting. All received Flu+TBI (+/− Cy) pre-conditioning and haplo-HSCT with post-transplant Cytoxan (PTCy) and FK506 as GvHD prophylaxis. The circulating T cells were hyporeactive in mixed lymphocyte reaction (MLR) assay to recipient DC in Pts at 1yr (or 2yrs in 1 Pt) post-haplo-HSCT (~6m post-ISD withdrawal), while these T cells responded vigorously to 3rd party APC. Neither depletion of Tregs, nor blockade of IL10R or PD1 could reverse T-cell hyporeactivity. Low-dose IL2 triggered mild anti-host responses. Cytokine profiling further confirmed the MLR responses. ImmunoSEQ™ revealed the disappearance of alloreactive T-cell clones from peripheral pool with time, even though a few alloreactive T clones remained. Low dose IL2 had minimal impact to reverse the diminishment. Unlike graft T-cell responses against host DC, tolerant T cells in circulation against hDC showed relative downregulation of allograft rejection, T-helper response, co-stimulation, and cell proliferation pathway, that was paired with upregulation of co-inhibition, PD1-PDL1, Ferroptosis and CREB pathways. In summary, hyporeactivity of donor-derived circulating T cells to recipient DC were evident. We found Treg, Tr1 activity to be insignificant in maintaining long-term tolerance. T-cell clonal deletion appears to be the dominant mechanism, along with systemically altered signaling pathways in tolerant T-cells.