Davies, J.; (2005) Selective depletion of alloreactive T cells to reduce graft-versus-host disease and enhance immune reconstitution post allogeneic haematopoietic stem cell transplantation. Doctoral thesis , University of London.

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Abstract

Non-selective T cell depletion reduces the incidence of severe graft-versus-host disease after allogeneic haematopoietic stem cell transplantation but the cost is delayed and disordered antigen-specific immune reconstitution and increased infection. A method of selective depletion of alloreactive donor T cells expressing the activation marker CD69 after co-culture with recipient stimulator cells in a mixed lymphocyte reaction has previously been shown to reduce alloreactivity, whilst retaining third party responses in vitro and in a mismatched murine model led to donor T cell engraftment with a virtual absence of GvHD and significantly increased survival. We have further developed this technique by comparing two methods of potentiating allostimulation in the HLA-matched setting. Cytokine pre-treated recipient stimulator cells led to successful allostimulation of a minority of HLA-matched responder cells whereas the strategy of pre-treatment of recipient stimulator cells with OKT3 led to effective allostimulation in all pairs tested and led to more efficient selective abrogation of alloresponses after depletion of responder alloreactive cells. The retention of donor antiviral T cell frequencies were compared after selective HLA matched allodepletion using both techniques of potentiating allostimulation. Using both techniques the majority of CMV-specific T cells (quantified by HLA Class I tetramer assay and IFN-y ELISpot) and EBV-specific T cells (quantified by IFN-gamma ELISpot) were retained in the selectively allodepleted T cell pool. Preservation of antiviral CTLs in selectively albdepleted stem cell grafts would lead to improved antiviral immunity post transplant. The phenotypic characteristics of the alloreactive and non-alloreactive T cells within the donor pool were examined. 0069"* alloreactive T cells were found to consist of both naive and memory T cells and to exhibit significant skewing of TCR Vp sub-family distribution in both the HLA-mismatched and HLA-matched setting. The technique of selective allodepletion based on CD69 expression was found to retain functional CD4+CD25+ T regulatory cells The retention of immunosuppressive CD4+CD25+ T regulatory cells could lead to more ordered immune reconstitution and further suppress alloreactive responses post transplant. Direct stimulation of donor T cells with CMV peptide led to up regulation of CD69 on CMV-specific T cells and these cells exhibited TCR Vp sub-family overuse consistent with previously published data. Homology of TCR Vp sub-family overuse in HLA A*0201+ donor T cells following CMV peptide stimulation and HLA-matched allostimulation was demonstrated in some individuals suggesting supporting the existence of donor T cells possessing TCRs with affinity for CMV and minor histocompatability antigens. Sequential selective allodepletion and CMV antigen stimulation of donor T cells might lead to production of CMV-specific non-alloreactive donor T cell pools suitable for use as adoptive immunotherapy post-allogeneic haematopoietic stem cell transplantation. The techniques for allostimulation and selective allodepletion at a clinical scale and under sterile conditions have been developed in order to test the safety and the efficacy of this technique in a clinical pilot study of HLA-matched sibling donor allogeneic haematopoietic stem cell transplantation in adults with acute myeloid leukaemia.

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