Immunogenetics

The research in this laboratory has centered on investigations into the regulation of the cell response to minor histocompatibility antigens (HA) in mice. These studies are particularly relevant to the field of transplantation since this family of antigens stimulates rejection of allografts transplanted to recipients matched for the major histocompatibility complex (MHC). Minor HA are presented as peptides by MHC class I and class II molecules to cytolytic T lymphocytes (CTL) and T helper lymphocytes, respectively. Immunodominant minor HA are of particular interest in that they preferentially stimulate cytolytic T lymphocyte (CTL) regardless of the number of additional minor HA barriers. A principal focus has been the understanding of the molecular basis for minor HA peptide binding to MHC class I molecules and the recognition of these complexes by antigen-specific T cell receptors (TcRs). Toward this end, we have studied the dominant mouse H4 minor HA peptide through the selection of mimotopes for this peptide as well as sequencing the alpha and beta chains of H4-specific TcRs. The results of these experiments indicated that the H4 peptide includes an Arg/His amino acid at position 7 and the CDR3 regions of H4-specific beta chains are characterized by net negative charges. These and other observations have steered our research program toward the test of the hypothesis that dominant minor HA peptides include positive charges in their carboxy termini and these positive charges play a prominent role in recognition by specific TcRs through the formation of salt bridges with negatively charged residues in beta CDR3 regions.

The recognition of minor HA peptides by TcRs occurs in the context of in vivo systems that process peptides and mediate allograft rejection. We have investigated the mechanism of skin allograft rejection through the quantitation of transcription of cytokine genes expressed by lymphocytes infiltrating skin allografts from the time of transplantation through to rejection. The results of these experiments suggest the importance of tumor necrosis factor-a expression in both the wound-healing and rejection processes. These initial observations have been followed by an investigation into transcriptional regulation during the wound healing process in order to achieve a greater understanding of the molecular bases for this process and their potential effects on subsequent T cell responses to allografts.

An important emphasis in this laboratory is maintaining a balance between the focused studies and experiments that are simply aimed at interesting and intriguing questions in biology. In this regard, we have collaborated with Drs. Frances Berdan and Jack States to combine techniques in anthropology and molecular biology to decipher the origins of an Anasazi feathered artifact. More recently, we have completed the sequencing of mitochondrial DNA from three tassel-eared squirrels (Sciurus berti); phylogenetic comparisons of these sequences with those obtained from other mammalian species support the proposed paraphyly of rodents.

Specific Research Topics

The principal focus of the laboratory has been and will continue to be the investigation of the genetic regulation of the T cell response to minor histocompatibility antigens (HA) in mice. Minor HA are encoded by extensive families of autosomal and sex-linked genes in mice and humans and are recognized as short peptides bound to class I and class II major histocompatibility complex (MHC) gene products. As such, the MHC matching of donors and recipients establishes optimal conditions for the T cell response to minor HA peptides present in donor tissues/cells and absent in recipients. The recognition of these peptides requires both the binding of the peptides to MHC molecules as well as recognition of these complexes by peptide-specific T cell receptors (TcRs).

A principal objective of this laboratory?s research is the understanding of the molecular basis for the binding of one mouse minor HA peptide, H4, to class I Kb molecules and the recognition of these H4:Kb complexes by H4-specific TcRs. The H4 peptide plays a prominent role in stimulating CTLs since it is a dominant peptide that stimulates a CTL response regardless of additional incompatibilities involving multiple minor HA peptides. Complementary experiments were performed to (1) identify mimotopes for the H4 peptide that cross-reacted with the natural H4 peptide and (2) sequence the alpha and beta transcripts encoding H4?specific TcRs. We selected a panel of mimotopes derived from a library of random octamer peptides with fixed binding motifs; a striking characteristic of all selected H4 mimotopes was the inclusion of Arg/His at the seventh position. This positive charge appeared to be complemented by opposing negative charges in the beta CDR3 regions of H4?specific TcRs that are predicted to interact directly with the carboxy end of class I-bound peptides. The beta CDR3 regions of TcRs expressed by both in vitro-selected H4?specific CTL clones as well as CTLs infiltrating H4?incompatible skin grafts at the time of rejection were characterized by net negative charges. These and other observations have steered our research program toward the test of the hypothesis that dominant minor HA peptides include positive charges in their carboxy termini and these positive charges play a prominent role in recognition by specific TcRs through the formation of salt bridges with negatively charged residues in beta CDR3 regions.

These studies of the specificity of minor HA peptide recognition are balanced by investigations into the mechanism of rejection of minor HA-incompatible skin allografts. We have probed skin allografts from the time of transplantation through to rejection with quantitative-RT-PCR to estimate the levels of transcription of gene products predicted to mediate allograft rejection. These gene products included perforin, Fas ligand, and tumor necrosis factor-a (TNFa). Only perforin expression correlated with rejection. Interestingly, TNFa was expressed during this entire time period with the highest levels occurring 24 hours after transplantation in both allografts and autografts. These results point toward a role for the pleiotropic TNFa cytokine in both the beneficial process of wound healing and the detrimental process of rejection.

The final area of investigation involves experiments that are simply aimed at answering what we consider to be interesting questions in biology. This approach recently led us to the analysis of an ancient scarlet macaw feather garment ascribed to the Anasazi culture. We sequenced the full-length cytochrome b gene from the tassel-eared squirrel (Sciurus aberti) pelt that comprises part of what is believed to be the belt of this artifact and compared the sequence to those we have shown to be expressed by present-day tassel-eared squirrels. This molecular approach was coupled to an anthropological analysis of materials and construction techniques to formulate the hypothesis that this artifact was constructed in the southwest U.S. using native materials (including the pelt) and scarlet macaw feathers imported from Mexico.