Allo-HLA Reactive CD8 T-Cells May Recognize Tissue Specific Peptides Explaining Tissue Restricted GVHD after HLA Mismatched SCT.
Authors: Amir Avital , Griffioen Marieke , Kester Michel D.G. , Honders M. Willy , Willemze Roelof , Falkenburg J.H. Frederik , Heemskerk Mirjam 
Published: November 2019

Abstract
HLA disparity between patient and donor increases the risk of GVHD after allogeneic stem cell transplantation (SCT). However, similar to fully HLA matched SCT, the clinical manifestation of GVHD after HLA mismatched SCT is frequently restricted to skin, gut and liver. Based on the frequency of allo-HLA reactive T-cells, which is about a 1000-fold higher than the frequency of minor histocompatibility antigen specific T-cells, the immune response after HLA mismatched SCT is expected to be mediated by allo-HLA reactive T-cells. Theoretically allo-HLA reactive T-cells can exert three different types of recognition. They may directly recognize the mismatched HLA molecule, independent of the peptide it presents. Alternatively they could be peptide dependent but promiscuous in their peptide recognition, or peptide specific like in normal T-cell recognition. Previous studies all showed peptide independent recognition by at least part of the studied T-cells. Since HLA class I is expressed by all nucleated cells, these data would imply that after HLA mismatched SCT alloreactive T-cells would be able to equally damage all tissues. Whereas previous reports mainly studied in vitro generated allo-HLA reactive T-cells, we characterized the pattern of peptide recognition of a large number of different T-cells retrieved from a patient suffering from GVHD after HLA-A2 mismatched donor lymphocyte infusion. Activated CD8+ T-cells were single cell sorted based on HLA-DR expression, expanded and tested for alloreactivity and HLA restriction. 46 of the 56 generated CD8 T-cells clones were alloreactive and allo-HLA-A2 restricted. The alloreactive CD8 clones showed usage of different T-cell receptor Vβ chains with different CDR3 sequences, illustrating polyclonality. To evaluate the tissue recognition, the clones were tested against different HLA-A2 positive target cells derived from various hematopoietic and non-hematopoietic tissues. The clones showed major differential recognition of the different tissue target cells, varying from lack of recognition to strong recognition of the same target. To analyze the peptide recognition pattern, the clones were tested against TAP deficient T2 cells loaded with HPLC fractions of peptides eluted from HLA-A2. The different clones recognized different HPLC fractions, but each clone recognized of only one single fraction, indicating peptide specificity. Three clones recognized non-pulsed T2 cells, which was interpreted in previous studies to demonstrate peptide independence. T2 cells, however, do present peptides which are independent on TAP to enter the ER. By testing reactivity against TAP independent peptides, one clone showed recognition of a peptide derived from the signal sequence of TRAPα, which is positioned at the ER membrane, and is therefore TAP independent. This indicates that recognition of T2 cells by the allo-HLA reactive T-cells was also based on peptide specific recognition. Finally, two clones were screened against COS cells expressing HLA-A2 transfected with a c-DNA library constructed from an EBV-LCL. Each clone showed recognition of only one (different) gene out of 40.000 c-DNAs. These results show that all allo-HLA reactive T-cells exerted peptide specific recognition. Differential expression of tissue specific peptides that can be recognized in the context of allo-HLA molecules may explain tissue specific peptides as a cause for tissue restriction in GVHD after HLA mismatched SCT.