POSTER TEMPLATE BY: www.PosterPresentations.com Identification of novel MHC class II-restricted male-specific mHAg encoded by SMCY (JARID1D) Nobuharu Fujii, MD, PhD, Kellie V. Rosinski, Paulo V Campregher, MD and Edus H Warren III, MD, PhD Program in Immunology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA Background - 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. - To date, however, only two MHC class I-restricted, and no MHC class II-restricted, H-Y antigens encoded by SMCY have been characterized. SMCY encodes more 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 (Fig. 1 and Fig. 2), 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 (HLA-A2/A3, HLA-B7/- , HLA-C7/-, HLA-DQ 6WG/- , HLA- DR*1501/ -) , and were stimulated in vitro with dendritic cells derived from his pretransplant PBMC that had been pulsed with the SMCY pentadecapeptides (Fig. 3). 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 (Fig. 4 and Fig. 5). 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, ELKKLQIYGP GPKMM, presented by HLA-DRB1*1501, and has a CD3 + , CD4 + , CD8  , CD45RA  , CD45RO + surface phenotype (Fig. 6, Fig. 7 and Fig. 8). 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 h 1 cytokines (IFN- and IL-2), but the clone also produced low to moderate levels of the T h 2 cytokines IL-4, IL-10, and TGF-β (Fig. 9). 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 ELKKLQIYGA GPKMM peptide was not recognized, demonstrating that the clone is SMCY-specific (Fig. 10). 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 (Fig. 11). These results suggest that CD4 + T cell responses against the SMCY 232-246 epitope could potentially contribute to GVL activity after F→M HCT. Hypothesis Results Future Directions Fig 1. Alignment of the amino terminal segments of the SMCY and SMCX proteins along with the tiling pattern of the 36 overlapping pentadecapeptides 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. Supported by National Blood Foundation and ASH Travel Awards. Conclusion 1. SMCY-specific CD4 positive T cell clone 13H3 could be generated from a male patient transplanted from HLA identical female donor using overlapping peptides. 2. 13H3 clone was HLA-DR*1501 restricted CD4 positive T cells. 3. 13H3 clone recognized primary leukemia cells in a HLA- DR*1501 restricted fashion. Materials and Methods IYPYE FQSGANHVQCNTHPF NEVKDKEYKPHSIPL YSRRAKRLQPDPEPT ELKKLQIYG IYPYEMFQS ANHVQCNTHPFDNEV LRQSVQPSKFSSYSR PEPTEEDIEKHPELK QIYG IYPYEMFQSGANH QCNTHPFDNEVKDKE VQPSKFSSYSRRAKR EEDIEKHPELKKLQI PYEMFQSGANHVQCN HPFDNEVKDKEYKPH KFSSYSRRAKRLQPD EKHPELKKLQIYG SMCY 161 IYPYEMFQSGANHVQCNTHPFDNEVKDKEYKPHSIPLRQSVQPSKFSSYSRRAKRLQPDPEPTEEDIEKHPELKKLQIYG SMCX 161 VYPYEMYQSGANLVQCNTRPFDNEEKDKEYKPHSIPLRQSVQPSKFNSYGRRAKRLQPDPEPTEEDIEKNPELKKLQIYG PG GLMAKDKDKTVHKKV CPPTVTVKDEQSGGG VSSTLLKQHLSLEPC KTTMQLRKNHSSAQ PGPKMM KDKDKTVHKKVTCPP VTVKDEQSGGGNVSS LLKQHLSLEPCTKTT QLRKNHSSAQ PGPKMMGLGLM KTVHKKVTCPPTVTV DEQSGGGNVSSTLLK HLS---------LEPCTKTTMQLR NHSSAQ MMGLGLMAKDKDKTV KKVTCPPTVTVKDEQ GGGNVSSTLLKQHLS EPCTKTTMQLRKNHS AQ SMCY 241 PGPKMMGLGLMAKDKDK--TVHKKVTCPPTVTVKDEQSGGGNVSSTLLKQHLS--------LEPCTKTTMQLRKNHSSAQ SMCX 241 AGPKMMGLGLMAKDKTLRKKDKEGPECPPTVVVKEELGGDVKVESTSPKTFLESKEELSHSPEPCTKMTMRLRRNHSNAQ F DSYICQVCSRGDEDD PEIPRGIWRCPKCIL ECKQPPEAFGFEQAT EYSLQSFGEM FIDSY CQVCSRGDEDDKLLF RGIWRCPKCILAECK PPEAFGFEQATQEYS QSFGEM FIDSYICQV SRGDEDDKLLFCDGC RCPKCILAECKQPPE FGFEQATQEYSLQSF EM FIDSYICQVCSRG DKLLFCDGCDDNYHI CILAECKQPPEAFGF QATQEYSLQSFGEM SMCY 311 FIDSYICQVCSRGDEDDKLLFCDGCDDNYHIFCLLPPLPEIPRGIWRCPKCILAECKQPPEAFGFEQATQEYSLQSFGEM SMCX 321 FIESYVCRMCSRGDEDDKLLLCDGCDDNYHIFCLLPPLPEIPKGVWRCPKCVMAECKRPPEAFGFEQATREYTLQSFGEM Fig 2. SIx pools of 36 overlapping peptides for T cell culture Fig 3. Method of SMCY-specificT cell generation Previously reported HLA-A2 restricted epitope Peptide pool No. 0 100 200 300 400 500 600 1 2 3 4 5 6 Spots / 5x10^4 cells Pep mix + Pep mix - 0 10 2 10 3 10 4 10 5 PE-A 0 10 2 10 3 10 4 10 5 APC-A 0.0 72 23. 6 53. 4 22. 9 CD8-PE FIDSYICQV Pentamer-APC #25 IFN-γ ELISPOT assay 0 10 20 30 40 50 60 70 #25 #26 #27 #28 #29 #30 pool5 No pep Spots/1x10^4 cells NHSS AQFID SYI CQV Fig 5. T cells in pool No.5 was specific for previously reported HLA-A2 restricted SMCY epitope FIDSYICQV. Fig 4. 1 st screening IFN-γ ELISPOT assay using peptide pools. SMCY -specific T cell responses were detected in the wells with peptide pool No.2 and No.5. 0 10 20 30 40 50 60 70 80 90 30 10 3 1 0.3 E/T ratio % Specific lysis Pt (M)-LCL Donor (F) -LCL Donor (F) -LCL + ELKKLQIYGP GPKMM Donor (F) -LCL + irrelevant peptide CD8-FITC IFN-gamma-PE 3.25% Sorting and cloning by limiting dilution IFN-γ secretion assay CD3 CD4 CD8 CD45RA CD45RO Phenotype of clone 13H3 13H3 clone 0 100 200 300 400 Pep#8+ Spots/1x10^3 cells 0 10 2 10 3 10 4 10 5 FITC-A 0 20 40 60 80 100 % of Max 0 10 2 10 3 10 4 10 5 FITC-A 0 20 40 60 80 100 % of Max 0 10 2 10 3 10 4 10 5 FITC-A 0 20 40 60 80 100 % of Max 0 10 2 10 3 10 4 10 5 PE-Cy7-A 0 20 40 60 80 100 % of Max 0 10 2 10 3 10 4 10 5 APC-A 0 20 40 60 80 100 % of Max Fig 6. Isolation of 13H3 clone specific for ELKKLQIYGP GPKMM from pool No 2. The phenotype of 13H3 clone was memory type CD4 T cell. Fig 7. Cytotoxicity of 13H3 clone. 13H3 clone killed ELKKLQIYGP GPKMM loaded donor (female) LCL. However, 13H3 clone did not kill patient (male) LCL. Fig 8. HLA restriction of 13H3 clone. 13H3 clone recognized peptide loaded HLA-DR*1501+ LCLs. Fig 9. Cytokine profile of 13H3 clone. 13H3 clone produced mainly Th1 cytokines, but also produced Th2 cytokines and TGF-β. IFN-gamma ELISA 0 500 1000 1500 2000 2500 3000 IFN-gamma (pg/ml) pcDNA3.1-DRA1 pcDNA3.1-DRB1*1501 pcDNA3.1-SMCX(ELKKLQIYGP GPKMM) or pcDNA3.1-SMCY( ELKKLQIYGA GPKMM) COS7 13H3 clone Coculture 0 10 20 30 40 50 60 70 GIM WY TAM TDG EBC PKJ Do (F) - LCL Pt (M) -LCL only T cell clone DR1501+ / male DR1501/ female not DR1501 / male APC (primary leukemia cells) IFN-gamma (pg/ml) Fig 10. 13H3 clone recognized endogenously expressed SMCY epitope ELKKLQIYGA GPKMM. The clone did not recognize corresponding SMCX epitope ELKKLQIYGA GPKMM. Fig 11. 13H3 clone recognized three of five primary leukemia cells derived from HLA-DR*1501 positive male patients. (Representative two of three are shown here.) pool 2 0 100 200 300 400 500 600 700 800 #7 #8 #9 #10 #11 #12 Pool 2 No pep Peptide Spots/1x10^4 cells ELKKLQIYGP GPKMM * * Pentamer assay No pep DRB1*1501/1104, DQ*01/*03 DRB1*1406/1501, DQ*03/*05 DRB1*1301/*0301 DQB1*0602/*0603 DRB1*0101/*1301 DQ*05/*06 -5 0 5 10 15 20 25 30 Pt (UPN25071) DRB1 *1501/- DQB1 *06WG/- (06WG: *0602 or *0611) % Specific lysis (E/T ratio=5) Target cells LCL#1 LCL#2 LCL#3 LCL#4 Minigenes Transfection DR tected. F1000 Poster Bank. Copyrigh Copyright protected. F1000 Poster Bank. Copyright protected. F Poster Bank. Copyright protected. F1000 Poster Bank. Copyright protected. F1000 Poster ected. F1000 Poster Bank. Copyright protected. F1000 Poster Bank. Copyright protected. F1000 Poster Bank. Copyrig Poster Bank. Copyright protected. F1000 Poster Bank. Copyright protected. F1000 Poster Bank. Copyright protected. Copyright protected. F1000 Poster Bank. Copyright protected. F1000 Poster Bank. 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