Leukemic Blasts Acting as Host Antigen Presenting Cells Trigger a Combined CD4 and CD8 Allo-Immune Response Directed against Mismatched HLA Class I
Authors: Amir Avital L. , Hagedoorn Renate S. , Marijt W.A. Eric , Willemze Roel , Falkenburg J.H. Frederik , Heemskerk Mirjam H.M. 
Published: April 2020

Abstract
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.