Influence of DM-sensitivity on immunogenicity of MHC class II restricted antigens

Background Graft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted antigens depending on their behavior towards HLA-DM. While DM-resistant antigens are presented in the presence of HLA-DM, DM-sensitive antigens rely on the expression of HLA-DO-the natural inhibitor of HLA-DM. Since expression of HLA-DO is not upregulated by inflammatory cytokines, DM-sensitive antigens cannot be presented on non-hematopoietic tissues even under inflammatory conditions. Therefore, usage of CD4+ T cells directed against DM-sensitive antigens might allow induction of graft-versus-leukemia effect without GvHD. As DM-sensitivity is likely linked to low affinity peptides, it remains elusive whether DM-sensitive antigens are inferior in their immunogenicity. Methods We created an in vivo system using a DM-sensitive and a DM-resistant variant of the same antigen. First, we generated murine cell lines overexpressing either H2-M or H2-O (murine HLA-DM and HLA-DO) to assign the two model antigens ovalbumin (OVA) and DBY to their category. Further, we introduced mutations within the two T-cell epitopes and tested the effect on DM-sensitivity or DM-resistance. Furthermore, we vaccinated C57BL/6 mice with either variant of the epitope and measured expansion and reactivity of OVA-specific and DBY-specific CD4+ T cells. Results By testing T-cell recognition of OVA and DBY on a murine B-cell line overexpressing H2-M and H2-O, respectively, we showed that OVA leads to a stronger T-cell activation in the presence of H2-O demonstrating its DM-sensitivity. In contrast, the DBY epitope does not rely on H2-O for T-cell activation indicating DM-resistance. By introducing mutations within the T-cell epitopes we could generate one further DM-sensitive variant of OVA and two DM-resistant counterparts. Likewise, we designed DM-resistant and DM-sensitive variants of DBY. On vaccination of C57BL/6 mice with either epitope variant we measured comparable expansion and reactivity of OVA-specific and DBY-specific T-cells both in vivo and ex vivo. By generating T-cell lines and clones of healthy human donors we showed that DM-sensitive antigens are targeted by the natural T-cell repertoire. Conclusion We successfully generated DM-sensitive and DM-resistant variants for two model antigens. Thereby, we demonstrated that DM-sensitive antigens are not inferior to their DM-resistant counterpart and are therefore interesting tools for immunotherapy after allogeneic stem cell transplantation.

[1]  J. Falkenburg,et al.  CD4 Donor Lymphocyte Infusion Can Cause Conversion of Chimerism Without GVHD by Inducing Immune Responses Targeting Minor Histocompatibility Antigens in HLA Class II , 2018, Front. Immunol..

[2]  A. Mackensen,et al.  Clinical-grade generation of peptide-stimulated CMV/EBV-specific T cells from G-CSF mobilized stem cell grafts , 2018, Journal of Translational Medicine.

[3]  B. Dörken,et al.  A Transgenic Dual-Luciferase Reporter Mouse for Longitudinal and Functional Monitoring of T Cells In Vivo , 2017, Cancer Immunology Research.

[4]  J. Goeman,et al.  Human leukocyte antigen-DO regulates surface presentation of human leukocyte antigen class II-restricted antigens on B cell malignancies. , 2014, Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation.

[5]  J. Falkenburg,et al.  HLA class II upregulation during viral infection leads to HLA-DP-directed graft-versus-host disease after CD4+ donor lymphocyte infusion. , 2013, Blood.

[6]  J. Goeman,et al.  Endogenous HLA class II epitopes that are immunogenic in vivo show distinct behavior toward HLA-DM and its natural inhibitor HLA-DO. , 2012, Blood.

[7]  C. Schmitt,et al.  Stromal Interferon-γ Signaling and Cross-Presentation Are Required to Eliminate Antigen-Loss Variants of B Cell Lymphomas in Mice , 2012, PloS one.

[8]  K. Wucherpfennig,et al.  HLA-DM Captures Partially Empty HLA-DR Molecules for Catalyzed Peptide Removal , 2010, Nature Immunology.

[9]  H. Schreiber,et al.  Bystander killing of cancer requires the cooperation of CD4+ and CD8+ T cells during the effector phase , 2010, The Journal of experimental medicine.

[10]  L. Denzin,et al.  H2-O, a MHC class II-like protein, sets a threshold for B-cell entry into germinal centers , 2010, Proceedings of the National Academy of Sciences.

[11]  J. Gorski,et al.  Peptide-MHC Class II Complex Stability Governs CD4 T Cell Clonal Selection , 2009, The Journal of Immunology.

[12]  J. Falkenburg,et al.  Identification of 4 new HLA-DR-restricted minor histocompatibility antigens as hematopoietic targets in antitumor immunity. , 2009, Blood.

[13]  F. Sallusto,et al.  Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells , 2009, The Journal of experimental medicine.

[14]  R. Hagedoorn,et al.  Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application , 2008, Haematologica.

[15]  J. Falkenburg,et al.  Identification of phosphatidylinositol 4-kinase type II β as HLA class II-restricted target in graft versus leukemia reactivity , 2008, Proceedings of the National Academy of Sciences.

[16]  Francisco A. Chaves,et al.  The impact of DM on MHC class II–restricted antigen presentation can be altered by manipulation of MHC–peptide kinetic stability , 2006, The Journal of experimental medicine.

[17]  Francisco A. Chaves,et al.  The relationship between immunodominance, DM editing, and the kinetic stability of MHC class II:peptide complexes , 2005, Immunological reviews.

[18]  Francisco A. Chaves,et al.  The kinetic stability of MHC class II:peptide complexes is a key parameter that dictates immunodominance. , 2005, Immunity.

[19]  L. Karlsson,et al.  Analysis of H2-O Influence on Antigen Presentation by B Cells , 2003, The Journal of Immunology.

[20]  P. Tiberghien,et al.  A novel ‘sort-suicide’ fusion gene vector for T cell manipulation , 2002, Gene Therapy.

[21]  Catherine J. Wu,et al.  Randomized trial of CD8+ T-cell depletion in the prevention of graft-versus-host disease associated with donor lymphocyte infusion. , 2002, Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation.

[22]  P. Marrack,et al.  Alternate interactions define the binding of peptides to the MHC molecule IAb , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[24]  E. Ward,et al.  Negative Selection during the Peripheral Immune Response to Antigen , 2001, The Journal of experimental medicine.

[25]  E. Schmitt,et al.  H2-M, a facilitator of MHC class II peptide loading, and its negative modulator H2-O are differentially expressed in response to proinflammatory cytokines , 2000, Immunogenetics.

[26]  O. Lantz,et al.  γ chain required for naïve CD4+ T cell survival but not for antigen proliferation , 2000, Nature Immunology.

[27]  Forest M. White,et al.  Immunodominance Among EBV-Derived Epitopes Restricted by HLA-B27 Does Not Correlate with Epitope Abundance in EBV-Transformed B-Lymphoblastoid Cell Lines1 , 2000, The Journal of Immunology.

[28]  M. Mitchell,et al.  Dendritic cells permit identification of genes encoding MHC class II-restricted epitopes of transplantation antigens. , 2000, Immunity.

[29]  B. Evavold,et al.  DO11.10 and OT-II T Cells Recognize a C-Terminal Ovalbumin 323–339 Epitope1 , 2000, The Journal of Immunology.

[30]  P. Cresswell,et al.  Negative regulation by HLA-DO of MHC class II-restricted antigen processing. , 1997, Science.

[31]  C. Huber,et al.  Prophylactic transfer of CD8-depleted donor lymphocytes after T-cell-depleted reduced-intensity transplantation. , 2007, Blood.

[32]  B. Evavold,et al.  DO 11 . 10 and OT-II T Cells Recognize a C-Terminal Ovalbumin 323 – 339 Epitope 1 , 2000 .