Koh, Mickey Boon Chai; (2000) Alloantigen specific t cell depletion from haematopoietic stem cell grafts for the prevention of graft versus host disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Graft versus host disease (GvHD) is a multi-step immune process involving lymphocyte activation and proliferation. We have devised a novel method of selectively depleting alloreactive cells from haematopoietic stem cell grafts while retaining a pool of immunocompetent non alloreactive lymphocytes possessing antiviral and possibly anti-leukaemic activity and capable of hastening immune reconstitution. This method involved identifying the alloreactive cells thought to initiate GvHD by means of an activation marker, CD69 in an in-vitro system and depleting these cells by paramagnetic bead sorting. The temporal dynamics of CD69 expression as well as other activation markers in an allogeneic setting was first initially determined to determine the optimal marker and time for depletion. Using flow cytometric analysis and cell proliferation data, the engineered graft was shown to retain only 12% of its original alloreactivity but preserving 78% of its 3rd party reactivity. This system has been tested on mismatched and histocompatibility matched donor/patient pairs. A modified mixed lymphocyte culture system using cytokines has also been studied and this has proved to be of clinical significance in predicting GvHD. Cytokines, in particular, ?-interferon was shown to upregulate various cell surface molecules important in antigen presentation and may explain in part the crucial role of cytokines in GvHD. This depletion strategy was then tested in a NOD/SCID murine GvHD model. This involved comparing the intraperitoneal injection of 5-10x106 unmanipulated T cells from a CBA (H-2k) mouse into a non-lethally irradiated (250 cGy) NOD/SCID (H-2g7) recipient (positive control), with that of mice who received allo-depleted cells. This allodepletion strategy protected against death from lethal GvHD in a complete MHC mismatched setting (survival 12.5% in positive control versus 71.4% in allo-depleted group). In parallel, by using tetrameric HLA-peptide complexes and looking at CMV+, HLA-A2 individuals, it has been demonstrated that the non-alloreactive fraction using this strategy retained 90% of the specific anti-CMV activity, suggesting that these grafts would protect from CMV reactivation. Moreover, the alloreactive cells are easily recoverable in this selective T-cell depletion strategy for cryopreservation and ready for immediate access as therapeutic donor lymphocyte infusions in cases of frank relapse post-transplant.

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