Identification of Novel MHC Class II-Restricted Male-Specific mHAg Encoded bySMCY(JARID1D)..

Abstract 1344

Poster Board I-366

Male recipients of female hematopoietic cell grafts, when compared with all other donor/recipient gender combinations, have an increased risk for both acute and chronic GVHD, but also have a significantly decreased risk of posttransplant relapse. F→M HCT is also characterized at the cellular level by donor (female) T cell responses against male-specific minor histocompatibility (H-Y) antigens, which can contribute to both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) activity. SMCY is a Y-chromosome gene that has previously been shown to encode at least two distinct MHC class I-restricted H-Y antigens presented by HLA-A*0201 and HLA-B*0702, respectively. Also, association between CD8⁺ T cell responses specific for the SMCY_311-319 FIDSYICQV epitope and GVHD or GVL has been reported. A CD8⁺ FIDSYICQV-specific T cell clone was also reported to induce histological signs of GVHD reaction in an in vitro skin-explant assay. To date, however, only two MHC class I-restricted, and no MHC class II-restricted, H-Y antigens encoded by SMCY have been characterized. Given the large size of the SMCY and the homologous SMCX proteins and the fact that they are only 85% identical at the amino acid sequence level, we hypothesized that SMCY encodes other MHC class I- and class II-restricted H-Y antigens, and that T cell responses against these epitopes may likewise contribute to GVHD and GVL activity after F→M HCT. Arrays of pentadecapeptides with eleven-residue overlap were designed to tile regions of the SMCY protein that are non-identical to the corresponding regions of its X chromosome-encoded homologue SMCX, and then used to generate SMCY-specific T cell lines recognizing novel SMCY-encoded MHC class I- and class II-restricted H-Y antigens. Peripheral blood mononuclear cells (PBMC) were obtained on posttransplant day +126 from a 46 year-old male patient with monosomy 7 AML who had received a hematopoietic cell graft from his MHC-identical sister, and were stimulated in vitro with dendritic cells derived from his pretransplant PBMC that had been pulsed with the SMCY pentadecapeptides. After three stimulations, a SMCY peptide-specific CD4⁺ T cell line as well as a SMCY_311-319 (FIDSYICQV)-specific CD8⁺ T cell line were obtained. After cloning by limiting dilution, we further characterized the SMCY-specific CD4⁺ T cell clone, 13H3. The 13H3 T cell clone recognizes the SMCY_232-246 15-mer peptide, ELKKLQIYGPGPKMM, presented by HLA-DRB1*1501, and has a CD3⁺, CD4⁺, CD8⁻, CD45RA⁻, CD45RO⁺ surface phenotype. The cytokine release profile of this clone when assessed with SMCY_232-246-loaded donor-derived EBV-LCL, as measured by the Luminex assay, is characterized mainly by T_h1 cytokines (IFN-g and IL-2), but the clone also produced low to moderate levels of the T_h2 cytokines IL-4, IL-10, and TGF-β. A minigene encoding SMCY_232-246 was recognized by the 13H3 clone in a HLA-DRB1*1501-dependent fashion when transfected into COS-7 cells, but a minigene encoding the homologous SMCX-derived ELKKLQIYGAGPKMM peptide was not recognized, demonstrating that the clone is SMCY-specific. The 13H3 clone recognized 3 of 5 HLA-DRB1*1501⁺ male primary leukemia cells, but did not recognize either of 2 HLA-DRB1*1501⁻ male or either of 2 HLA-DRB1*1501⁺ female primary leukemia cells. These results suggest that CD4⁺ T cell responses against the SMCY_232-246 epitope could potentially contribute to GVL activity after F→M HCT. A SMCY_232-246/HLA-DRB1*1501 tetramer has been constructed which specifically marks the 13H3 T cell clone, and future studies will use this reagent to determine whether CD4⁺ T cells specific for this epitope can be detected directly ex vivo in posttransplant blood samples from HLA-DRB1*1501⁺ F→M HCT recipients.