Approaches toward the investigation of T cell specificities and antigenic determinants in an MHC-matched mouse GVHD model
C57BL/6J→ C.B10-H2b (B6→BALB.B) bone marrow transplantation (BMT) provides a major histocompatibility complex (MHC)-matched (H2b) mouse model for the study of graft versus host disease (GVHD). Two of the major goals in establishing an MHC-matched GVHD model, are the discovery of the T cell specificities involved in mediating disease and the identification of the antigenic determinants that drive the anti-host reactivity. This work describes two novel approaches toward these goals in the B6→BALB.B BMT model. Previous experiments utilizing T cell receptor (TCR) spectratype-sequencing, have shown a dominant TCR Vβ14-DJβ2.4 coding sequence prevalent in the thoracic duct lymphocyte (TDL) population of irradiated BALB.B mice undergoing B6 CD8+ T cell mediated GVHD. In order to investigate the alloreactive potential of T cells expressing this particular isolated TCRβ sequence, we retrovirally transduced B6-TCRβ-/- bone marrow cells with a construct containing the cDNA for Green Fluorescent Protein (GFP) and for the specific Vβ14-DJβ2.4 TCR sequence. The marrow was then used to reconstitute irradiated B6 mice. Surprisingly, the emergent T cells in the peripheral blood overwhelmingly (> 95%) displayed a CD4+ phenotype. Strong CD4+ phenotype preference (> 85%) was also seen in T cells emerging from bone marrow transduced with a control Vβ14-DJβ2.3 sequence. The results of subsequent work described in this thesis demonstrate that this CD4+ phenotype preference of the Vβ14+ cells is likely to be at least partially due to the selecting MHC (H2b) background, and that while the TCR on the transduced cells predisposes their development to the CD4 lineage, it does not absolutely preclude CD8 lineage differentiation. Importantly, GFP+ cells from BALB.B-primed B6 mice, which had been administered bone marrow cells transduced with the Vβ14-DJβ2.4 TCR construct, exhibited anti-BALB.B reactivity in an IFNγ ELISPOT system. On the other hand, GFP+Vβ14+ T cells from primed mice receiving marrow cells transduced with a control vector expressing Vβ14-DJβ2.3 TCR, did not show anti-BALB.B reactivity. This indicates that T cells harboring a TCRβ sequence that is associated with B6→BALB.B GVHD, can actually mediate in vitro reactivity. This work also describes a “reverse immunology” approach toward the identification of BALB.B antigenic determinants. We queried the NCBI Single Nucleotide Polymorphism (SNP) database at http://www.ncbi.nlm.nih.auc/projects/SNP/MouseSNP.cgi for nonsynonymous coding SNP varying between the C57BL/6J and the BALB/c (representative of BALB.B) mouse strains. The Syfpeithi MHC peptide binding prediction program at http://www.syfpeithi.de was used to evaluate the binding affinity of the resulting peptide sequences to MHC H2Kb and H2Db molecules. Overall, 20 nonsynonymous coding SNP were identified which map to genes with expected expression in GVHD target tissue, and fall within coding sequence for putative epitopes on H2Db or H2Kb MHC molecules. Full validation of this approach will require a demonstration that in vivo GVHD pathology is at least partially driven by T cell immunoreactivity toward the identified antigenic determinants. However, we have demonstrated the feasibility of utilizing coding SNP data to identify candidate antigenic determinants in a mouse MHC-matched transplantation model. The approaches described in this work may be utilizable not only in the context of investigation into MHC-matched BMT, but also in other areas of immunological research.
Appel, Michael Jay, "Approaches toward the investigation of T cell specificities and antigenic determinants in an MHC-matched mouse GVHD model" (2008). ETD Collection for Thomas Jefferson University. AAI3442423.