Targeted Immune Depletion: Several factors influence the engraftment of allogeneic hematopoietic stem cells including host immune status, histocompatibility, hematopoietic """"""""space"""""""", stem cell dose, and subsets and doses of T cells within the allograft. Host immune status has not been emphasized, relative to allogeneic stem cell engraftment, when myeloablative conditioning regimens are utilized for allogeneic HSCT, as they markedly attenuate the HVG response that mediates graft rejection. However, when non-myeloablative and reduced-intensity conditioning regimens are utilized prior to allogeneic HSCT, it has been both observed in murine models and inferred from clinical trials data that engraftment is inversely correlated with host immune status. As there can be significant variability in the immune status of patients being considered for alloHSCT, we surmised that individual patients required different levels of host immune depletion in order to achieve the rapid and complete engraftment of allogeneic hematopoietic stem cells, especially if they were from someone other than an HLA-matched sibling. To address the problem of host immune status variability, we developed a novel treatment approach, referred to as targeted immune-depletion(TID), which utilizes an induction regimen consisting of repetitive cycles of disease-specific immunosuppressive chemotherapy at conventional doses that is administered until a pre-determined level of host immune depletion, as determined by circulating T cell numbers, has been achieved. The targeted immune depletion approach has been implemented through a systematic, step-wise progression of studies in clinical settings in which one of the barriers to engraftment (e.g. T cell depletion, HLA-disparity) was increased, while other engraftment variables were kept constant. In our first trial, 99-C-0143, we demonstrated that targeted immune depletion could result in rapid and sustained complete donor engraftment in patients receiving TCR allografts from an HLA matched sibling. However, a significant proportion of patients treated on this study experienced relatively high rates of acute GVHD associated with cyclosporine monotherapy for GVHD prevention. The use of cyclosporine monotherapy in CC 99-C-0143 was based upon the early belief that the incidence of GVHD might be lower following a reduced-intensity conditioning regimen.20 Our observations in CC 99-C-0143 suggested that cyclosporine monotherapy is inadequate immunosuppression in the face of relatively rapid and complete engraftment of donor lymphoid cells. We therefore designed a study of TCR allografts from HLA-matched related donors using targeted immune depletion and a standard dual-agent GVHD regimen, cyclosporine plus methotrexate. The primary aim of this study, 03-C-0077, was to determine the impact of the addition of methotrexate on hematopoietic recovery, donor/recipient chimerism, and the incidence of acute GVHD. An additional aim was to evaluate the relationships between interleukin-7 (IL-7) and interleukin 15 (IL-15) with immune depletion, engraftment kinetics, GVHD, and immune reconstitution. IL-7 is a major cytokine regulator of B-cell development and T-lymphocyte homeostasis. IL-15 is an important cytokine in the differentiation, maintenance and expansion of NK and CD8+ T cells. Consistent with their homeostatic effects, we observed that IL-7 and IL-15 levels rose from baseline as transplant conditioning induced lymphopenia, and then decreased with lymphocyte recovery. These inverse correlations were strongest within 1 month post-alloHSCT. IL-7 and IL-15 levels varied inversely according to lymphocyte counts after transplant, consistent with homeostatic response. The IL-7 levels at day +7 (P = 0.01) and day+14 (P = 0.00003) post-transplant were associated with the subsequent development of acute GVHD (Figure 5C). Among patients who developed acute GVHD, median IL-7 levels were 25.7 and 20.9 pg/ml at days +7 and +14, respectively. Patients who did not develop acute GVHD had median IL-7 levels of 11.8 and 8.22 pg/ml at days +7 and +14, respectively. Higher IL-7 levels at day+14 were strongly associated with more severe grades of acute GVHD (p less than 0.0001) Logistic regression analysis confirmed that the IL-7 level at day+14 was the strongest single parameter associated with acute GVHD. Using a cutoff value of 13 pg/ml, the IL-7 level at day+14 predicted the subsequent development of acute GVHD with a sensitivity of 85.7% and specificity of 88.2%. The positive and negative predictive values for this single-variable model were 85.7% and 88.2%, respectively. Based on the above results, we have initiated a clinical trial, 07-C-0195 utilizing TCR allografts from HLA-matched HLA-matched unrelated donors (URD) and the targeted immune depletion approach to study its effect upon engraftment. The use of stem cells from URD increases the availability of allogeneic cellular therapy to an additional 30-50% of patients; however, reduced-intensity alloHSCT from HLA-matched URD are associated with higher degrees of graft rejection and mixed chimerism due to differences in minor histocompatibility antigens between donor and recipient. A clearly superior GVHD prophylaxis regimen has not been established in the URD setting. Two GVHD prophylaxis regimens, which have been reported to have relatively good results in regard to acute GVHD in the URD setting, are alemtuzumab with cyclosporine31 and the combination of tacrolimus, methotrexate, and sirolimus.32 The two regimens work through biologically distinct mechanisms, and their effects upon immune reconstitution, which have not previously been well studied with either regimen, are predicted to be markedly different, regardless of the presence or absence of acute GVHD. This study design provides a unique opportunity to investigate and compare in a preliminary fashion post-transplant immune reconstitution related to each of these GVHD prophylaxis regimens, which have distinct biologic properties relative to their ability to prevent GVHD.
The specific aims of this protocol are extensive and detailed studies of post-transplant immune constitution. These studies are a natural extension of our prior work and take advantage of the unique expertise and resources within the laboratories of the ETIB, intramural programs of the NCI, and expertise on CDR3 spectratyping at Hackensack University with whom we have formed a formal collaboration. This study will provide preliminary data relative to multiple aspects of immune reconstitution, which is important and essential to the understanding of this biology and development of strategies to improve outcomes related to GVHD and immune dysregulation. The results of this pilot trial will be used for the development of subsequent studies related to alloHSCT from URD incorporating techniques and approaches that are currently under investigation in the ETIB and POB. The URD transplant initiative is an inter-institutional (NCI, NHLBI, and NIAID), interagency (NIH and DOD) and extramural (National Marrow Donor Program) collaboration.
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