The implications of different IgG subclasses on graft rejection in sensitized individuals

The presence of memory B cells may contribute to graft damage. In fact, pre-transplant B cell sensitization in the absence of circulating antibody represents a risk factor, since grafts transplanted into recipients with historical positive but current negative direct crossmatch do less well than patients with no evidence of B cell sensitisation. This definition of ‘negative’ is based on conventional complement-dependent cytotoxicity (CDC) assays and, more recently, on flow cytometry. In this study, I have analysed the significance of antibodies undetectable by these conventional techniques but found to be present using the more sensitive novel Luminex technology which has now become available to detect class I and class II HLA-specific antibodies present in pre-transplant patients’ sera at the time of transplantation. Even in the absence of low level antibodies, there may still be undetected B cell sensitization. An ELISpot assay has previously been developed to quantitate B cell responses. In this study, I have developed the ELISpot assay in an attempt to determine B cell allosensitization. This project aims were: 1. To evaluate the relationship between renal transplant outcome and the presence of low grade HLA-specific IgG-antibodies pre-transplant, both non-donor-specific as well as donor-specific antibodies detected using Luminex technology. 2. To develop ELISpot assays that detect the potential responses of the various cells of the B cell compartment, reflecting naive allo-reactivity as well as that which has been induced by historical sensitisation events. 3. To detect the presence of post-transplant HLA-specific IgG antibodies (both donorspecific and non-donor specific) in tolerant drug-free patient and determine their significance in defining the state of tolerance. Summary of the results: 1. Luminex technology is a more sensitive technique than flow cytometry and CDC in detecting low-level donor-HLA-specific antibodies which were present in pre-transplant patients’ sera at the time of transplantation and is associated with a higher incidence of rejection episodes. 2. IgM- and IgG-detecting ELISpot assays were developed to determine the frequency of antibody-secreting cells. I have begun to apply these methods to determine the humoral reactivity against alloantigen in sensitised patients. 3. Tolerant drug-free patients had no detectable donor-specific antibodies (DSA). In the non-tolerant patients, DSA-positive patients had worse graft function than DSAnegative patients

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients