Abstract
MHC molecules carrying selected peptides will bind specifically to their cognate T cell receptor on individual clones of reactive T cells. Fluorescently labeled, tetrameric MHC-peptide complexes have been widely used to detect and quantitate antigen specific T cell populations via flow cytometry. We hypothesized that such MHC-peptide tetramers could also be used to selectively deplete unique reactive T cell populations, while leaving the remaining T cell repertoire and immune response intact. We show in an MHC-matched, minor antigen disparate, murine BMT model (BALB.B → C57BL/6), MHC-peptide-tetramers can be used to deplete the T cells responsible for Graft-Versus-Host-Disease (GVHD), while leaving the remaining immune response intact, as demonstrated by the retention of Graft-Versus-Tumor (GVT) activity. Using PE-labeled tetramers, anti-PE microbeads and an autoMACs separation system, we successfully depleted donor splenocytes of alloantigen specific T cells prior to transplantation. We demonstrated the specificity of the depletion by showing loss of the tetramer reactivity after depletion, whereas no changes were observed in the Vβ repertoire and the percentage of T cells, B cells, NK cells, monocyte/macrophages and granulocytes between pre- and post-depletion samples. When analyzed 6 days after transplantation, mice receiving specifically-depleted splenocytes had <0.5% of their CD8+ T cells reactive against the alloantigen (tetramer +) as compared to >8.5% of the CD8+ T cells in mice that received control-depleted splenocytes. A nearly 50% decrease in in vivo proliferation of donor splenocytes, assessed by CFSE dilution, was seen 3 days after transplant in recipients of specifically-depleted splenocytes, as compared to mice receiving control-depleted splenocytes. However, pre- and post-depletion splenocytes (specific and control) were equally capable of mounting an immune response against third party cells as demonstrated by mixed lymphocyte reaction. In a series of bone marrow transplants designed to assess GVHD and GVT, mice receiving specifically-depleted splenocytes had a nearly 4-fold increased median survival due to significant decreases in GVHD morbidity and mortality compared to recipients of control-depleted splenocytes. All mice receiving splenocytes (tetramer-depleted or not) showed equal GVT activity. Finally, we were able to demonstrate the simultaneous abrogation of GVHD and the retention of GVT in a single bone marrow transplant. In recipients of specifically-depleted splenocytes, there was a 33% long-term survival and significant increases in median survival, as compared to recipients of non-depleted splenocytes, control-depleted splenocytes or bone marrow only; all of these latter groups succumbed to GVHD or tumor. This method also provides the proof-of-concept for similar strategies to selectively remove other unwanted T cell clones, which could result in novel therapies for certain autoimmune disorders, T cell malignancies and solid organ graft rejection.
Graft-Versus-Host Disease Amelioration by Human Bone Marrow Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles Is Associated with Peripheral Preservation of Naive T Cell Populations