Susan Masewicz

Selective T-cell-receptor gene usage in allorecognition and graft-versus-host disease

Immune recognition of foreign HLA molecules is initiated by T cell recognition mediated by alloreactive T cell receptor (TCR) molecules. We analyzed the diversity of TCR expression in the clinical setting of allorecognition in a patient with acute graft-versus-host disease following bone marrow transplantation. Nearly 200 TCR transcripts from peripheral blood lymphocytes were cloned and sequenced at two time points during GVHD. HLA genes in the transplant donor and the recipient were mismatched for a very specific HLA-DR subtype: HLA-DRB1 genes in the donor (DR4/Dw4) and the recipient (DR4/Dw14) encode HLA molecules that differ at only two amino acids, providing a very restricted target for allorecognition. We also studied TCR genes from five T cell clones derived in vitro from mixed lymphocyte cultures between Dw4-positive responder and Dw14-positive stimulator cells. Comparisons of the derived TCR sequences implicate nonrandom patterns of TCR selection both in vivo and in vitro.

T-cell receptor Vβ selectivity in T-cell clones alloreactive to HLA-Dw14

The HLA-DR4 subtypes Dw14 and Dw4 are T-cell-defined allospecificities encoded by the DRB1*0404 and DRB1*0401 genes, respectively. Although these allelic subtypes differ in only two amino acids, allorecognition between Dw14 and Dw4-positive individuals is brisk. This provides an opportunity to analyze T-cell receptor (TCR) usage in a very limited and specifically targeted case, namely the Dw4 anti-Dw14 allogeneic T-cell response. The variable (V), diversity (D), and joining (J) region sequences of the TCR beta chain from two different Dw14-specific alloreactive T-cell clones derived from a Dw4 donor were examined. Clone EMO25 recognized the Dw14.1, Dw14.2, and Dw15 subtypes, which share a DRB1 polymorphism at codon 71 on a DR4 background, while clone EMO36 reacted with only the Dw14.1 subtype associated with polymorphisms at codons 71 and 86. TCR beta cDNA from each clone was amplified using an anchored polymerase chain reaction (PCR) and subsequently expanded with V beta- and C beta-specific primers for asymmetric PCR and direct DNA sequencing. Both clones were found to express the same TCR V beta 8.2 gene segment; however, they have several different residues within the V beta-D beta-J beta junctional regions. V beta 8 usage was also enriched in polyclonal cells obtained from mixed lymphocyte cultures performed between the Dw4 and Dw14 responder-stimulator combination from which EMO25 and EMO36 were derived.

Human T-cell receptor TCRAV, TCRBV, and TCRAJ sequences newly found in T-cell clones reactive with allogeneic HLA class II antigens

1 Department of Immunology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan 2 Human Immunogenetics Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98 104, USA 3 ARC Epidemiology Research Unit, University of Manchester, Manchester M13 9PT, England 4 Section for Transplantation Immunology and IIImlunohaematology, Medizinische Universit~itsklinik, W-7400 Ttibingen, Germany 5 Laboratorio di Immunogenetica, Istituto Nazionale per la Ricerca sul Cancro, 16 132 Genova, Italy

Complexity of Human Immune Response Profiles for CD4+ T Cell Epitopes from the Diabetes Autoantigen GAD65

Complex protein antigens contain multiple potential T cell recognition epitopes, which are generated through a processing pathway involving partial antigen degradation via proteases, binding to MHC molecules, and display on the APC surface, followed by recognition via the T cell receptor. We have investigated recognition of the GAD65 protein, one of the well-characterized autoantigens in type I diabetes, among individuals carrying the HLA-DR4 haplo-types characteristic of susceptibility to IDDM. Using sets of 20-mer peptides spanning the GAD65 molecule, multiple immunostimulatory epitopes were identified, with diverse class II DR molecules functioning as the restriction element. The majority of T cell responses were restricted by DRB1 molecules; however, DRB4 restricted responses were also observed. Antigen-specific T cell clones and lines were derived from peripheral blood samples of pre-diabetic and IDDM patients and T cell recognition and response were measured. Highly variable proliferative and cytokine release profiles were observed, even among T cells specific for a single GAD65 epitope.

Inhibition of allostimulated HLA-DQ and DP-specific T cells by staphylococcal enterotoxin A

Bacterial superantigens have two immunologically important features. They bind MHC class II molecules and stimulate T cells bearing certain V beta TCR phenotypes. Superantigens such as SEA, SEB, and TSST bind to each of the three HLA class II isotypes (DR, DQ, and DP). Allotypic variation seems to play an important role in superantigen binding to class II molecules, but the functional implications of these differences remain largely unknown. In the present investigation, we studied the effects of SEA, SEB, and TSST on allostimulation of HLA-DR-, DQ-, and DP-allospecific T-cell clones. To avoid direct stimulation of T-cell responses by the superantigens, SEA and/or SEB nonresponsive T-cell clones were selected. We show that SEA strongly inhibited DQ- and DP-specific T-cell responses. In contrast, SEB and TSST had only weak inhibitory effects. DR-specific T-cell responses were unaffected or only weakly inhibited by the superantigens tested. The inhibition appeared not to be due to induction of cytotoxicity or suppression of either T cells or EBV-LCLs by SEA. In conclusion, the bacterial superantigen SEA can block alloantigen-specific stimulation of T clones in vitro. These results suggest that SEA binds to certain MHC class II molecules in a way that prevents MHC-TCR interactions.

Alloreactive T-cell clones identify multiple HLA-DQw3 variants

HLA-DQw3 is a broadly defined alloantigen that has been subdivided by serological, biochemical, and molecular methods into three distinct specificities: DQw7, DQw8, and DQw9. In order to characterize functionally relevant structural polymorphisms within this family of alloantigens, we generated a series of DQw3-reactive T-cell clones that together recognize six different variants of DQw3. T-cell clones IG11 and IG9 were found to recognize three distinct functional variants associated with a majority of DQw3+ cells, while clones 21J, IE6, 64B, and IC3 recognized four more narrowly distributed functional variants associated with unique DQw7, DQw8, and DQw9 subsets. Comparison of known DQB gene sequences suggested candidate recognition sites for clones IG11 and 64B in the region of amino acid residues 66 to 71 and residue 57 of the DQ beta chain. In contrast, no unique DQB or DQA sequences were found that individually corresponded to the reactivity patterns of clones 21J, IE6, IG9, or IC3, suggesting that an interaction between DQ alpha and DQ beta chains determines allo-recognition. These data are consistent with the hypothesis that T cells recognize specific alloepitopes on HLA class II molecules, either as distinct structural elements that trigger an alloresponse or, more indirectly, as contact elements that influence alloreactivity by governing the binding of foreign peptide. The results illustrate the diversity of possible T cell responses directed toward HLA-DQ molecules and suggest that T cell recognition of the DQ heterodimer alone, or a peptide antigen bound to the DQ heterodimer, can be affected either by the individual DQ alpha and beta chains, or by a more complex interaction between the two.