Diverse Patterns of T Cell Reactivity Directed against Multiple Y Chromosome-Derived, HLA a*0201 Restricted, Peptides in Patients with chronic Gvhd
Authors: Ofran Yishay , Brusic Vladimir , Kim H. T , Soiffer Robert J. , Antin J. H , Ritz Jerome 
Published: April 2020

Abstract
Minor histocompatibility antigens (mHA) are targets of graft versus host disease (GVHD) and graft versus leukemia (GVL) responses. In male patients with female donors, Y chromosome-encoded mHA are known to be highly immunogenic but few MHC class I presented epitopes have been identified and T cell responses to HY mHA have not been examined in large cohorts of patients. We recently developed a novel method for rapid identification of putative mHA based on in-silico prediction of potential MHC class I restricted peptides. Criteria for HY mHA prediction included the following: Y chromosome encoded gene sequence; high affinity binding to HLA A*0201 (≤100nM); lack of identical sequence in homologous X gene; validated protein expression not restricted to male-specific tissues; and amino acid disparities between Y and X chromosome homologues predicted to be immunogenic. In this study, 43 peptide epitopes (9–10 amino acids) representing 5 Y-encoded proteins (DBY, SMCY, UTY, PCDH11Y, USP9Y) were identified. The only previously known Y-encoded (SMCY) HLA A*0201 restricted mHA (FIDSYICQV) ranked first in the list of predicted epitopes. Two peptides failed synthesis and 41 HY peptides were tested for T cell reactivity in post-transplant samples from 21 male patients with female donors (M-F), 9 male patients with male donors (M-M) and 19 healthy donors (7 males; 12 females). All patients and donors were HLA A*0201+ and all patients had chronic GVHD. T cell reactivity was determined by ELISPOT. Peripheral blood mononuclear cells were stimulated with individual peptides for 7 days. IFN-g secreting T cells were enumerated after 18 hour restimulation with HLA A*0201 T2 cells pulsed with the same peptide. Known HLA A*0201 restricted peptides from EBV, CMV, HIV and HCV were used as positive and negative controls. T cell responses to individual peptides could be blocked by anti human HLA class I antibodies but not by anti HLA class II antibodies. Using the recursive partitioning method, a minimum of 20 spots/2×105 PBMC above background level was identified as an optimal cut-off point to classify positive responses for the majority of peptides. T cell responses to some HY peptides were also detected in M-M patients and healthy donors, but responses were most frequent and of greater magnitude in M-F patients. The median number of positive peptide responses per patient was 8 in the M-F patient group, 1 in M-M patients and 2 in healthy donors (p=0.002 for M-F vs M-M, p=0.02 for M-F vs healthy donors). All M-F patients responded to at least 1 HY peptide whereas 3/9 (33%) M-M patients and 7/19 (37%) healthy donors did not respond to any peptides. 18/21 (86%) M-F patients responded to 3 or more peptides but this level of reactivity was only detected in 2/9 (22%) M-M patients and 7/19 (37%) healthy donors. High frequency responses (&gt;50 spots/2×105 PBMC) were detected in 19/21 (91%) M-F patients but only in 2/9 (22%) M-M patients, 1/7 (14%) healthy males and 4/12 (33%) healthy females. T cell responses were detected in at least 1 M-F patient for 36 of 41 peptides and responses were detected against all 5 Y-encoded proteins. However, a subset of 20 peptides appeared to be highly immunogenic with T cells responses detected in &gt;25% of M-F patients. No single HY peptide elicited responses in all M-F patients including the previously known HY mHa, which was only positive in 40% of M-F patients. In fact 9 other peptides derived from DBY (1), SMCY (3), UTY (3), PCDH11Y (1) and USP9Y (1) elicited responses in 40–71% of M-F patients. Each of these 10 peptides elicited high frequency responses (&gt;50 spots/2×105 PBMC) in at least 3 M-F patients but not in any M-M patients or healthy males. Within the M-F patients, the frequency of response was not associated with severity of cGVHD, underlying hematologic disease, age, stem cell source, transplant conditioning regimen or donor type. There was a correlation between time post transplant and the number of HY peptide responses (r=0.53, p=0.002 for all F-M and M-M patients combined). The functional application of bioinformatic models represents a new approach for identifying large numbers of novel HY peptides and assessing T cell responses after transplantation. These studies demonstrate a highly diverse T cell response despite identical mismatch and HLA type. Extending this method to other HLA alleles and to autosomal genetic disparities will improve our understanding of the role of mHA in GVL and GVHD after allogeneic transplantation.