Endogenous-peptide-dependent alloreactivity: new scientific insights and clinical implications.

T-cell alloreactivity is generated via immune responsiveness directed against allogeneic (allo) human leucocyte antigen (HLA) molecules. Whilst the alloresponse is of extraordinary potency and frequency, it has often been assumed to be less peptide-specific than conventional T-cell reactivity. Recently, several human studies have shown that both alloreactive CD8(+) and CD4(+) T cells exhibit exquisite allo-HLA and endogenous peptide specificity that has also underpinned tissue-specific allorecognition. In this review, we summarize former and recent scientific evidence in support of endogenous peptide (self-peptide)-dependence of T-cell alloreactivity. The clinical implications of these findings will be discussed in the context of both solid organ transplantation and haematopoietic stem cell transplantation (HSCT). Insights into the understanding of the molecular basis of T-cell allorecognition will probably translate into improved allograft survival outcomes, lower frequencies of graft vs host disease and could potentially be exploited for selective graft vs leukaemia effect to improve clinical outcomes following HSCT.

TCR cross-reactivity and allorecognition: new insights into the immunogenetics of allorecognition.

Alloreactive T cells are core mediators of graft rejection and are a potent barrier to transplantation tolerance. It was previously unclear how T cells educated in the recipient thymus could recognize allogeneic HLA molecules. Recently it was shown that both naïve and memory CD4+ and CD8+ T cells are frequently cross-reactive against allogeneic HLA molecules and that this allorecognition exhibits exquisite peptide and HLA specificity and is dependent on both public and private specificities of the T cell receptor. In this review we highlight new insights gained into the immunogenetics of allorecognition, with particular emphasis on how viral infection and vaccination may specifically activate allo-HLA reactive T cells. We also briefly discuss the potential for virus-specific T cell infusions to produce GvHD. The progress made in understanding the molecular basis of allograft rejection will hopefully be translated into improved allograft function and/or survival, and eventually tolerance induction.

Alloreactivity from human viral specific memory T-cells.

The mechanisms by which alloreactive memory T-cells are generated in non-sensitized individuals have begun to be elucidated. It is generally accepted that a very high level of crossreactivity is an essential feature of the T-cell receptor. Indeed it has recently been shown that alloreactivity from viral specific memory T-cells is far more common than predicted, 45% of viral specific T-cell clones were found to be allo-HLA crossreactive. In this overview the evidence for crossreactive alloresponses from human viral specific memory T-cells is discussed with special emphasis on the unexpected high frequency of these crossreactive responses, the peptide and tissue specificity of the responses, and the mechanistic insights gleaned from the elucidation of the crystal structure of an allo-HLA crossreactive viral specific TCR. The possible implications for clinical solid organ and bone marrow transplantation and tolerance induction will be discussed.

The reliability of long and short cases undertaken as practice for a summative examination.

BACKGROUND: This study explores whether long and short cases performed in the workplace during training could be integrated into an overall summative assessment. Less examiner training and a less formalized structure might compromise reliability, but increased testing time might improve it. METHODS: Results of practice long and short cases, undertaken in preparation for the Royal Australasian College of Physicians clinical examination, were compared with actual examination results. The effects on reliability of the examination were compared by modelling varying combinations of practice and examination long and short cases. RESULTS: Fifty-nine candidates in two centres undertook 256 practice long cases and 154 practice short cases. Two practice long cases correlated with two examination long cases (r= 0.46). The reliability of a single long case was 0.22 under practice conditions and 0.36 under examination conditions. The reliability of a single short case was similar under either condition (0.18 vs 0.21). Reliability of over 0.80 could be achieved by assimilating two examination long cases and four examination short cases with varying combinations of seven practice cases. CONCLUSIONS: Long cases undertaken in the workplace are not as reliable those undertaken under examination conditions, but short cases have similar reliability under either condition.

New tools to monitor the impact of viral infection on the alloreactive T-cell repertoire.

Accumulating evidence suggests that alloreactive memory T-cells may be generated as a result of viral infection. So far, a suitable tool to define the individual human leukocyte antigen (HLA) cross-reactivity of virus-specific memory T-cells is not available. We therefore aimed to develop a novel system for the detection of cross-reactive alloresponses using single HLA antigen expressing cell lines (SALs) as stimulator. Herein, we generated Epstein-Barr Virus (EBV) EBNA3A specific CD8 memory T-cell clones (HLA-B*0801/FLRGRAYGL peptide restricted) and assayed for alloreactivity against a panel of SALs using interferon-gamma Elispot as readout. Generation of the T-cell clones was performed by single cell sorting based on staining with viral peptide/major histocompatibility complex-specific tetramer. Monoclonality of the T-cell clones was confirmed by T-cell receptor (TCR) polymerase chain reaction analysis. First, we confirmed the previously described alloreactivity of the EBV EBNA3A-specific T-cell clones against SAL-expressing HLA-B*4402. Further screening against the entire panel of SALs also showed additional cross-reactivity against SAL-expressing HLA-B*5501. Functionality of the cross-reactive T-cell clones was confirmed by chromium release assay using phytohemagglutinin blasts as targets. SALs are an effective tool to detect cross-reactivity of viral-specific CD8 memory T-cell clones against individual class I HLA molecules. This technique may have important implications for donor selection and monitoring of transplant recipients.