Minor Histocompatibility Antigen DDX3Y Induces HLA-DQ5-Restricted T Cell Responses with Limited TCR-Vβ Usage Both In Vivo and In Vitro  David Laurin, Eric Spierings, Lars T. van der Veken, Abdelbasset Hamrouni, J.H. Frederik Falkenburg, Gerard Souillet, Corine Vermeulen, Annie Farre, Claire Galambrun, Dominique Rigal, Yves Bertrand, Els Goulmy, Assia Eljaafari  Biology of Blood and Marrow Transplantation  Volume 12, Issue 11, Pages 1114-1124 (November 2006) DOI: 10.1016/j.bbmt.2006.07.012 Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Reactivity of H-Y minor H antigen-specific T cell clones toward HLA-DQB1*0501- and HLA-DQB1*0502- but not HLA-DQB1*0503- or HLA-DQB1*0504-expressing male EBV-B LCLs. Clones E6 (A) and L14 (B) were stimulated with various HLA-DQ5-expressing male EBV-B LCLs. Proliferation was measured by thymidine incorporation, as described in Methods. Biology of Blood and Marrow Transplantation 2006 12, 1114-1124DOI: (10.1016/j.bbmt.2006.07.012) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Proliferation of clones E6 and L14 in response to DBY-encoded epitopes. HLA-DQ5-expressing female EBV-B LCLs transduced with RPS4Y, TMSB4Y, EIF1AY, or DDX3Y genes were used to stimulate clone E6 (A) or clone L14 (B). HLA-identical male EBV-B LCLs were used as positive controls. Proliferation was measured by thymidine incorporation, as described in Methods. Biology of Blood and Marrow Transplantation 2006 12, 1114-1124DOI: (10.1016/j.bbmt.2006.07.012) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Proliferation of clones E6 and L14 in response to particular DBY-encoded peptides. A, C, Overlapping peptides from divergent region of DDX3Y were loaded onto autologous EBV-B LCLs. Mix1 contains 11 peptides, corresponding to amino acids 1-123 of DDX3Y DBY, Mix2 contains 11 peptides from regions 114-431, and Mix3 contains 10 peptides from regions 422-630. B, D, Peptides in Mix2 were also used individually (ppt 171-190 sequence is TGSNCPPHIENFSDIDMGEI). HLA-identical male EBV-B LCLs were used as positive controls. Clone E6 (A, B) and clone L14 (C, D) proliferation was measured by thymidine incorporation, as described in Methods. Biology of Blood and Marrow Transplantation 2006 12, 1114-1124DOI: (10.1016/j.bbmt.2006.07.012) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Proliferation of clones E6 and L14 in response to the previously identified minimal peptide for maximal recognition. The 20-amino acid long peptide TGSNCPPHIENFSDIDMGEI (171-190) and the 12-amino acid short peptide HIENFSDIDMGE were loaded onto autologous female EBV-B LCLs; HLA-identical male EBV-B LCLs were used as positive controls. Proliferation of clones E6 (A) and L14 (B) was measured by thymidine incorporation, as described in Methods. Biology of Blood and Marrow Transplantation 2006 12, 1114-1124DOI: (10.1016/j.bbmt.2006.07.012) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Only the P9 residue of HIENFSDIDMGE is critical for epitope recognition by L14 and E6 clones. Irradiated HLADQB1*0501 female EBV-B LCLs were primed for 1.30 hours with the indicated peptides at the concentration of 10 μg/mL or without a peptide as negative control. Then the L14 and E6 clones were added to wells at a ratio of 1:60 (clone:EBV-B LCL). After 3 days, 3H-thymidine (3HT) was added for 16 hours and proliferation was measured as indicated in Methods. Biology of Blood and Marrow Transplantation 2006 12, 1114-1124DOI: (10.1016/j.bbmt.2006.07.012) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions