Graft Versus Leukemia Separates From Graft Versus Host Disease By Magnitude and Avidity Of The Allo-Reactive T Cell Response After Allogeneic Stem Cell Transplantation and Donor Lymphocyte Infusion

Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can be a curative treatment for patients with hematological malignancies due to the capacity of allo-reactive donor derived T cells to mediate a curative potent graft versus leukemia (GVL) effect. However, associated acute graft versus host disease (GVHD) remains a major risk. To study the role of CD8+ T cells in GVL reactivity and GVHD, we selected patients who responded to DLI (without preceding cytoreductive treatment) for recurrent disease or incomplete donor chimerism after alloSCT. The patients were grouped according to absence (7 patients) or presence (6 patients) of GVHD. To quantify the number of circulating activated CD8+ T cells before DLI and at the time of disease regression or conversion to full donor chimerism we measured the frequencies of CD8+ HLA-DR+ T cells in peripheral blood samples by flowcytometry. Before DLI, highly variable numbers of CD8+ HLA-DR+ T cells were found (37.8 ± 42.9 x10⁶/L), that significantly increased after DLI (309±473 x10⁶/L, p<0.005), demonstrating involvement of CD8+ HLA-DR+ T cells in immune responses after DLI. To determine the specificity and functional avidity of the CD8+ HLA-DR+ T cells, these cells were isolated using flowcytometric cell sorting and clonally expanded. From a total of 30 samples, on average 225 T cell clones per sample were obtained and tested for recognition of patient and donor derived EBV-LCL, CD40L stimulated B cells (CD40L-B cells) and monocyte derived dendritic cells (monoDC). Surprisingly, in many samples from both patient cohorts high percentages of clones recognizing EBV-LCL derived from both patient and donor but not recognizing CD40L-B cells and monoDC were found. These T cells may be involved in anti-EBV responses irrespective of the presence of a GVL effect or GVHD. To investigate whether the magnitude of the allo-immune response was different in patients with or without GVHD coinciding the GVL effect, we compared the frequencies of allo-reactive T cell clones in samples from both patient groups. Significantly lower percentages of allo-reactive T cell clones were found in patients without GVHD as compared to patients with GVHD (5.1 ± 7.0% versus 32.5 ± 20.0% respectively, p<0.01), showing that coinciding GVHD is associated with an increased magnitude of the allo-reactive T cell response. Per patient, we determined the number of unique antigens targeted by the isolated T cell clones by characterizing the targeted MiHA using whole genome association scanning. In line with the lower total number of allo-reactive T cells, a lower number of unique MiHA was targeted in patients without GVHD (2.7±3.5) as compared to patients with GVHD (10.2±5.8, p=0.015). To determine whether occurrence of GVHD could be explained by the tissue specificity and functional avidity of the allo-reactive T cell response after DLI, we tested the T cell clones obtained from both patient cohorts for recognition of fibroblasts (FB) derived from skin biopsies of the patient. To mimic pro-inflammatory conditions, FB were pretreated for 4 days with 100 IU/ml IFN-γ. Recognition of untreated FB was exclusively mediated by T cell clones obtained from patients with GVHD, whereas recognition of IFN-γ pretreated FB was found for clones isolated from patients with or without coinciding GVHD. In addition, several T cell clones isolated from patients without GVHD were found to be directed against MiHA encoded by genes with a broad expression profile in non-hematopoietic cells comprising FB, despite absence of FB recognition under non-inflammatory conditions. This suggests that in addition to the tissue expression profile of the MiHA other factors, comprising the local inflammatory milieu, play a role in the risk of developing GVHD. In conclusion, our data show a strong correlation between the magnitude and the functional avidity of the allo-reactive CD8+ T cell response and the occurrence of GVHD after DLI. We hypothesize that the limited production of pro-inflammatory cytokines due to the moderate magnitude of the immune response in patients mounting a GVL response without coinciding GVHD reactivity may have prevented the induction of GVHD by the lower avidity allo-reactive T cells, that under pro inflammatory conditions can mediate GVHD by recognition of normal non-hematopoietic cells of the patient.