In Vivo Demonstration of Donor Idiotype Specific T Cells That Kill Primary CLL Cells Post Allogeneic Transplant.

Following allogeneic stem cell transplantation there is evidence of a graft versus leukemia effect (GVL). Whether this is mediated by an immune response against histocompatibility antigens also expressed on the tumor cells, i.e. by graft versus host disease (GVHD) or might be contributed to by response against specific tumor associated antigens (TAA) is currently under intense investigation. If the latter is the case, then it may be possible to generate tumor specific immune responses, thereby decreasing tumor relapse and minimizing GVHD. We and others have previously demonstrated that it is possible ex vivo to characterize CD8+ T cell responses against idiotype (Id) determinants and that these T cells can be quantified and further characterized by tetramer and ELISPOT assays. We now sought to determine whether it was possible to demonstrate evidence of in vivo immune responses against Id in patients with chronic lymphocytic leukemia (CLL) after reduced intensity conditioning (RIC) allogeneic transplant from HLA matched donors. To be included in this study, patients had to express HLA-A*0101, HLA-A*0201 or HLA-A*0301 for which tetramer constructs were available, have an IgH sequence containing Id determinants that could bind to the appropriate HLA-Class I and have serial PB and BM samples after RIC transplantation available for analysis. Of 36 patients who had undergone transplant, 21 fulfilled these criteria. No Id specific T cells could be detected in PB or BM samples from any of these patients before transplantation, although it was possible to generate autologous Id specific T cells ex vivo. Id specific cells could be detected by tetramer staining at some point post transplant in 17 of these 21 patients (80%), with a frequency ranging from 0.2 to 2.9% of CD8+ T cells. In all cases these cells were of donor origin, demonstrated either by microchimerism, or in the case of sex mismatched donors, by FISH. The earliest appearance of these specific T cells was from 80–120 (median 100) days post RIC transplant, and of note an increased frequency of Id specific cells was often co-incident with subsequent development of chronic GVHD. In some cases, the Id specific cells remained detectable for up to one year post transplant, but the detection of these cells appeared to require persistence of tumor in vivo, and once patients no longer had PCR detectable disease, Id specific T cells could no longer be detected. Id specific T cells could also be further amplified ex vivo using peptide pulsed antigen presenting cells and cytokines. In all cases we were able to demonstrate that the tetramer sorted T cells could kill the patients’ primary CLL cells in vitro, but we have no direct evidence that this was occurring in vivo. Indeed it is unlikely that these Id specific T cell responses could solely be responsible for the anti-tumour activity, and although we did not examine specific immune responses against minor histocompatibility antigens, these would undoubtedly be present since the majority of these patients developed clinically evident GVHD. However, the finding that we are able to demonstrate in vivo donor specific immune responses against TAAs such as Id, that are capable of killing primary tumor cells, provides the rational for the development of clinical programs aimed at maximizing specific immune responses. We are currently performing pre-clinical studies aimed at generating TAA specific T cells for subsequent infusion to patients as an alternative to non-specific donor lymphocyte infusions.