Generation of autologous immunity to acute myeloid leukaemia and maintenance of complete remission following interferon-alpha treatment.

Interferon-alpha (IFN-alpha) is established as part of the treatment for chronic myeloid leukaemia, although its precise mode of action remains largely unknown. Its use in acute myeloid leukaemia (AML) has been limited. We have previously documented autologous cytolytic activity against AML blasts in patients after autologous bone marrow transplantation. Here we present a patient with poor-risk AML who relapsed from first complete remission (CR) and was unwilling to undergo high-dose chemotherapy with stem cell rescue. In second chemotherapy-induced CR, the patient had no evidence of antileukaemia cytolytic activity in an in vitro assay, and she commenced IFN-alpha (Roferon). She subsequently developed high levels of leukaemia-specific cytotoxicity, and has remained in second CR for two years. These findings support the use of IFN-alpha in patients with poor-risk AML, and suggest that one mechanism of action may be immunological.

The effect of T cell depletion with Campath-1M on immune reconstitution after chemotherapy and allogeneic bone marrow transplant as treatment for leukaemia.

The prophylactic use of T cell depletion (TCD) strategies for the prevention of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation remains widespread. Initial reports of high incidence of graft rejection after TCD BMT led to a move away from this approach but improved conditioning regimens have reduced this risk substantially. The use of TCD has also been associated with higher relapse risk post-BMT although the success of donor leukocyte infusion (DLI) as treatment for relapse has reduced this problem, especially in chronic myeloid leukaemia (CML). Currently the use of TCD BMT is increasing particularly due to the relative increase in BMT from non-related donors for whom TCD is the optimal GVHD prophylaxis. However, doubts remain over the long-term effect on the reconstituted immune system of recipients of TCD BMT, particularly in adult recipients. In this study we have undertaken a detailed sequential analysis in 23 patients who received allo-grafts from HLA-identical sibling donors after high-dose chemo/radiotherapy for acute or chronic leukaemia. Of these patients, 11 received non-manipulated grafts, five received 'partially TCD' (PTCD) and a further seven received 'fully TCD' (FTCD) bone marrow. T cell depletion was performed ex vivo by Campath-1M plus autologous serum as a source of complement. Partial TCD describes grafts with a T cell reduction of 1-2 log. Full TCD refers to grafts with a reduction of >2.5 log. The decision regarding the optimal degree of TCD was clinical and was based upon the perceived relative risk of relapse based upon the disease and remission status. All patients were monitored for up to 12 months post-BMT with regard to reconstitution of T and NK cell subsets. T cell depletion at either level was associated with a slower recovery of CD4 cells. This was most marked in the FTCD recipients and lasted throughout the period of study. CD8 cell recovery was also slower in the TCD recipients but this normalised throughout the 12 months post-BMT. The ratio of CD45RA+:CD45RO+ increased in all recipients after month 3. This suggests that a degree of extra-thymic T cell maturation can occur in recipients of allogeneic BMT. NK cell recovery was more rapid in the TCD recipients and these differences were maintained throughout the first year.

The in vitro detection of anti-leukaemia-specific cytotoxicity after autologous bone marrow transplantation for acute leukaemia.

Anti-leukaemia activity after allogeneic bone marrow transplantation has been studied extensively but its antigen specificity and effector cell phenotype remain unknown. Here we report a study in three recipients of autologous bone marrow transplantation done as part of the treatment for acute leukaemia, in whom we were able to detect innate specific anti-leukaemia activity post-transplant. One patient maintained selective cell-mediated cytolytic activity against her autologous leukaemic cells in the absence of cytolysis of her normal bone marrow mononuclear cells (BMMC). She remains in complete remission 3 years after ABMT for acute myeloid leukaemia (M5). A second patient was transplanted for acute lymphoblastic leukaemia and had detectable anti-leukaemia activity up to 20 weeks post-ABMT. At this point anti-leukaemia activity could no longer be demonstrated and the patient suffered a relapse 2 weeks later. A third patient was transplanted for AML (M4 Eo) and lacked detectable leukaemia-specific immune reactivity at 1, 3 and 6 months post-ABMT. She relapsed 6 months after her ABMT and returned to complete remission after further chemotherapy. She commenced treatment with alpha interferon and regained NK function. Furthermore, she developed high level cytolytic activity against her autologous leukaemic cells in the absence of activity against her remission bone marrow samples. She remains in complete remission 17 months after her initial relapse. This is the first report of an apparent association between in vitro leukaemia-specific cytolytic activity in individual patients after ABMT and clinical outcome. It encourages the theory that autologous immunomodulation may be useful in the future treatment of leukaemia.

Understanding the Graft-Versus-Leukaemia Reaction.

The role of the immune response in control and erradication of leukaemia remains controversial but there is increasing evidence that the principal mechanism of cure after intensive chemotherapy and allogeneic BMT is immune mediated. This is evidenced by the observations of a reduced risk of leukaemia relapse after allogeneic BMT when compared with autologous or syngeneic BMT. Furthermore, an increased incidence of leukaemic relapse has been reported after aggressive GvHD prophylaxis with cyclosporin or lymphocyte depletion. The association between GvHD and the graft-versus-leukaemia activity of allogeneic BMT is strong and has led to a number of workers concluding that GvL might be inseparable from GvHD although there is increasing evidence that this is not so. The lymphocyte subsets responsible for GvHD and GvL remain to be elucidated in man despite intensive efforts. Certainly T cells, natural killer cells and a population of in vivo activated killer cells are involved in GvL and an effective immune response probably requires a combined approach. The target antigens of GvL are also controversial. The majority of GvL studies have been conducted in the allogeneic transplant setting in which activity to leukaemia-specific peptides is easily masked by reactivity to undetected MHC mismatches and to minor histocompatibility antigens. Despite this the search for leukaemia-specific peptides has been fruitful in the case of the product of the BCR/ABL translocation in CML. The ultimate aim of a number of groups in all aspects of oncology is the development of effective and specific immunotherapy. A variety of approaches have been attempted over the last 80 years, including vaccination with irradiated leukaemic blasts and numerous trials of interleukin-2. We now know more about the mechanisms of induction of immunity than ever before and this knowledge, combined with sophisticated molecular biology and virology, promises to revolutionise the immunotherapy of leukaemia over the next ten years.

Temporal dynamics of CD69 expression on lymphoid cells.

Lymphocyte activation remains an area of intense interest to immunologists and cell biologists and the dynamics of expression of surface molecules during the process are widely studied. The CD69 C-type lectin is reportedly the earliest activation antigen on lymphocytes and can be detected within hours of mitogenic stimulation. Recently reports have described differential activation dynamics with respect to different antigenic or mitogenic stimuli. This study has investigated the dynamics of CD69 expression over time after mitogenic, allogeneic, cytokine and target cell mediated activation of T-cell and NK cell subsets. It is apparent that the dynamics of CD69 expression differ with respect to the cell type and the method of stimulation. Mitogenic stimulation resulted in the most rapid expression of CD69 on both T- and NK cells while alloantigen stimulation induced a far slower response. Target cell stimulation of NK cells gave paradoxical results in that the CD69 + ve subset increased as a proportion of the total NK cells but did not increase in number. This was due to the selective binding of CD69 - ve NK cells to the target cells and their subsequent loss from the lymphoid gate. We confirmed this by showing that CD69 + ve NK cells do not lyse K562 target cells. This observation demonstrates the caution needed in the analysis of flow cytometric data based solely upon relative proportions of cells within discrete subsets.