Assessment of Longevity of the Direct and Indirect CD4 T Cell Allorecognition Response

Abstract

Introduction: Recipient CD4 T cells can recognise alloantigen ‘directly’, intact on donor antigen presenting cells (APCs), and ‘indirectly’, as self-restricted processed allopeptide. The relative duration of each pathway remains unclear, and further complicated by our recent description that the longevity of indirect responses differed according to target alloantigen. This work however examined different CD4 T cell responses in different murine models, and to control for differences in the immunogenicity of each, we developed a new model of chronic allograft vasculopathy that would allow definitive examination of all CD4 T cell allorecognition pathways operating in each recipient. Methods: Several mouse strains were intercrossed to generate a novel strain, bm12.Kd.IE, which differs from wild-type B6 by possessing additional MHC class I (H-2Kd) and MHC class II (IAbm12 and IE) molecules, but lacks the IAb MHC class II antigen. Heterotopic cardiac allografts were performed using bm12.Kd.IE donors into B6 recipients, and the model characterised by measuring rejection kinetics, degree of allograft vasculopathy, and production of alloantibody and autoantibody. The relative duration of direct and indirect allorecognition of different alloantigens was assessed by comparing division of monoclonal populations of alloreactive, CFSE-labelled TCR-transgenic CD4 T cells that were adoptively transferred either on day 0 or 28 post transplantation. Indirect allorecognition of MHC I was assessed using TCR75 CD4 T cells (I-Ab-restricted, H2-Kd peptide-specific); class II using TEa CD4 T cells (I-Ab-restricted, I-E peptide-specific) and minor antigen using Mar CD4 T cells (I-Ab-restricted, H-Y peptide-specific). Direct allorecognition of I-Abm12 MHC II was assessed using ABM CD4 T cells. Results: Bm12.Kd.IE hearts survived >50 days in B6 recipients, with histological evidence of chronic allograft vasculopathy and production of anti-Kd and anti-I-E alloantibody. Autoantibody, assessed by indirect immunfluosrescent binding to nuclear-antigen-expressing HEp-2 cells, also developed. Flow cytometric analysis of CFSE labelled transgenic CD4 T cells confirmed that direct pathway responses were robust immediately after transplant, with extensive division of the transferred ABM CD4 T cells, but were minimal four weeks later. Similarly, strong indirect pathway responses against MHC II alloantigen occurred immediately after transplant, but were surprisingly undetectable at later time points. In contrast, indirect alloresponses against MHC I alloantigen were equally strong at early and late times after transplant. Comparable results were obtained when indirect alloresponses against minor H-Y antigen were studied, although division at five weeks was less marked than immediately after transplant. Conclusions: Our results provide experimental evidence to support the paradigm that direct pathway CD4 T cells responses are short-lived, but also highlight that the duration of indirect pathway responses varies according to target alloantigen. The transient nature of anti-class II responses presumably reflects rapid loss of donor APC after transplantation.