ALBERT

Beschreibung: HLA disparity between patient and donor increases the risk and the severity of graft versus host disease (GVHD). In order to safely administer HLA mismatched stem cell transplantation (SCT) and donor lymphocyte infusion (DLI), it is important to understand the mechanisms underlying GVHD in HLA mismatched settings. The degree of patient chimerism, especially of the antigen presenting cells, at the time of DLI has been shown to influence the development of GVHD. This is suggested by the correlation between the time after SCT of DLI administration and the occurrence of GVHD. In mouse models the underlying role of the presence of host APCs in the occurrence of GVHD has been demonstrated in both MHC matched and mismatched models. In this study, the alloimmune response in a patient suffering from GVHD after an HLA class I mismatched DLI was characterized. The patient received a T-cell depleted SCT followed by two DLIs from the same donor. The SCT and first DLI, administered for mixed chimerism, did not result in any signs of GVHD. In contrast, the second DLI led to severe GVHD. Patient chimerism in different cell subsets present prior to the two DLIs showed no significant difference in patient T-cell chimerism and there were no patient B-cell, monocytes or dendritic cells present prior to each DLI. However, a significant difference in the presence of patient leukemic blasts was found at the time of the two DLIs. Prior to the first DLI no leukemic cells could be demonstrated, whereas 9% leukemic blasts were present in bone marrow prior to the second DLI. We showed that the leukemic blasts expressed HLA class II. Correlating with the absence of GVHD, the first DLI did not lead to activation of donor T-cells while the second DLI led to severe GVHD accompanied by a vigorous CD8 and CD4 T-cell response. Activated donor derived CD8 and CD4 T-cells from this response were isolated by single cell sort based on HLA-DR expression. Expanded clones were characterized for alloreactivity, HLA restriction, specificity and clonality. We showed that 50 of the 53 expanded CD8 T-cells were alloreactive and that all alloreactive CD8 T-cells were restricted to the mismatched HLA-A2. Additionally, by sequencing the T-cell receptor (TCR) Vβ CDR3 region, we showed that all alloreactive CD8 clones expressed a different CDR3 region, illustrating polyclonality. Of the 88 expanded CD4 clones, 21 were alloreactive and the majority of the alloreactive CD4 T-cell clones were directed against an HLA-A2 derived peptide presented in HLA-DR1. By Vβ analysis we showed that the CD4 T-cell response was also polyclonal. Finally, to investigate the role of the leukemic blasts in the initiation of the GVHD, the capacity of the different cell subsets present prior to DLI 1 or 2 to stimulate donor CD8 and CD4 T-cells was analyzed. This showed that only the leukemic blasts were able to highly stimulate both the CD8 and the CD4 T-cells. Our data indicate that the leukemic blasts by acting as host APCs triggered a combined CD4 and CD8 allo-immune response directed against the mismatched HLA, and thus initiated not only anti-leukemic reactivity but also lethal GVHD.

Beschreibung: Allogeneic stem cell transplantation (allo-SCT) is commonly used in the treatment of patients with hematological malignancies. However, the impaired immune reconstitution after allo-SCT is coincided by an increased incidence of viral complications in the first six months after the transplant, especially in seropositive patients transplanted with a stem cell graft from a seronegative donor. Infusions of unmodified donor T cells (DLI) can lead to the induction of anti-tumor immune responses caused by donor T cells recognizing minor histocompatibility antigens (mHag) on the recipient cells, but is often coincided by the induction of graft versus host disease. Moreover, the number of patients in which profound and sustained anti-tumor responses are induced is limited, most likely due to the inadequate induction of a primary immune response. A more effective and specific control of the malignant disorder or the infectious complications may be accomplished by the administration of in-vitro selected tumor-specific or virus-specific T cells. Although the in-vitro induction of primary immune responses against both leukemic cells and defined epitopes derived from mHag, tumor antigens (Ag), and viral Ag has been shown to be feasible by us and others, the robustness of the procedure remains limited, hampering large scale clinical application. We hypothesized that the major complication in the in-vitro induction of primary immune responses is the unfavorable balance between the number and activation of antigen-specific precursor T cells (Tprec), estimated to be

Beschreibung: The anti-tumor effect of donor lymphocyte infusions (DLI) after HLA-matched allogeneic stem cell transplantation (allo-SCT) can be mediated by donor T cells recognizing minor histocompatibility antigens (mHag) in the context of self HLA on the malignant cells of the recipient. However, T cells recognizing self antigens (Ag) that are overexpressed in malignant cells like the Wilms tumor protein (WT1) have also been proposed to contribute to the anti-tumor reactivity both after allo- and autologous SCT. WT1 is expressed in various types of cancer, including hematological malignancies at higher levels compared to their non-malignant counterparts. Different groups have isolated CD8+ HLA-A2 restricted WT1-specific T cells after HLA-matched transplantation and/or WT1 vaccination. Although these T cells stain with HLA-A2/WT1 peptide tetrameric complexes and are capable of recognizing peptide loaded HLA-A2+ target cells, recognition of primary leukemic cells could hardly be demonstrated. Stauss et al demonstrated recognition of endogenously processed Ag by WT1 specific T cells. However, these CD8 T cells were isolated in a situation where responder and stimulator cells were mismatched for HLA-A2. Based on these data we hypothesized that high avidity HLA-A2 restricted WT1 specific T cells may be deleted during thymic selection in HLA-A2+ donors. As a result, it is likely that only low avidity HLA-A2 restricted WT1 specific CD8+ T cells can be isolated from HLA-A2 positive donors, whereas it might be possible to isolate high avidity HLA-A2 restricted WT1 specific T cells from HLA-A2 negative donors due to the absence of the HLA restriction element in the thymus irrespective of the local WT1 expression. To test this hypothesis, we induced immune responses against the HLA-A2 binding WT1-derived peptide RMFPNAPYL using either HLA-A2+ or A2- donor T cells as responder cells. In case of HLA-A2+ donors (n=3) we stimulated CD45RO depleted donor T cells with autologous monocyte-derived antigen-presenting cells (APCs) loaded with 1E-6M WT1 peptide in the presence of 5ng/mL IL-7. At day 10 we either enriched tetramer-positive cells using immunomagnetic beads (MACS) or restimulated the responses with peptide-loaded autologous PBMC. At day 20 tetramer positive cells were isolated in bulk cultures and single cell/well by flowcytometric cell sorting. When using HLA-A2 negative donor T cells as responder cells, we used HLA-A2+ stimulator cells from an unrelated donor matched for all other class I alleles. The similar protocol was used for the induction of the immune response. At day 10 CD4+ T cells were depleted and the WT1 specific T cells were either enriched using tetramers and MACS or restimulated with peptide loaded HLA-A2+ PBMC. Using these approaches we were capable of isolating CD8+ T cell clones staining brightly with the A2/WT1 tetramer and expressing different T cell receptors (TCRs). HLA-A2+ WT1 specific T cells recognized only HLA-A2+ target cells loaded with 1E-8-1E-6M peptide both in IFNg release assays and cytotoxicity assays. Especially peptide-loaded target cells with an APC phenotype like EBVs were recognized efficiently, whereas normal peripheral blood cells were only recognized when loaded with 1E-6M peptide. In contrast, the HLA-A2 negative WT1-specific T cells efficiently recognized normal peripheral blood cells upon loading of