Donor CD8+ T Cells Facilitate Graft-Versus-Tumor Effect Via Alloantigen Rather Than Tumor-Specific Antigen Recognition Following Murine Myeloablative BMT.
Authors: Pillai Asha B. , Teo Pearline A. , Mukhopadhyay Aditi , Strober Samuel 
Published: April 2020

Abstract
Previous data from our group and others has shown that donor CD8+ Tcells mediate graft-versus-tumor (GVT) function in murine myeloablative bone marrow transplantation (BMT) via Perforin/Fas ligand-dependent mechanisms. However, there has to date been no analysis of the mechanism of tumor recognition (i.e allo- versus tumor-specific antigen recognition) by donor CD8+ T cells following myeloablative MHC-mismatched BMT. In order to test the hypothesis that donor CD8 T-cells require allo-antigen recognition to maintain graft-versus-tumor effect, we developed stable full chimeras by transplanting T-cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) donor mice into myeloablated BALB/c (H-2d) hosts given 800 cGy total body irradiation (TBI) and evaluated the in vivo ability of CD8+ TCR+ splenocytes from these donors to kill the BCL1 tumor (a BALB/c-derived B-cell lymphoma carrying a detectable tumor-specific idiotype which can be monitored via peripheral blood flow cytometric analysis). These chimeras showed complete donor chimerism in myeloid, B- and T-lymphocytic lineages by day +100 following transplantation, and splenocytes from these chimeras exhibited tolerance to host-type but not third-party alloantigens. BALB/c hosts were given 800 cGy TBI on day -1, followed on day 0 by intravenous administration of 500 BCL1 tumor cells and infusion of 0.3x 106 CD8+ T cells of C57 origin (H-2b+) sorted by flow cytometric analysis from the either spleens of the chimeric mice or from the spleens of untreated wild-type C57BL/6 mice. All hosts were given 5 x 106 T cell-depleted wild-type C57 bone marrow cells. All mice were observed for clinical signs of graft-versus-host disease (GVHD) and mortality through day +100. Autopsy was performed at death to assess for sub-clinical target organ involvement with GVHD or tumor. Donor chimerism and BCL1 status was assessed at day +28 and day +100 by 3-color flow cytometric analysis for donor-specific MHC versus T-, B- and myeloid lineage markers as well as tumor-specific idiotype in the peripheral blood (or in the spleen at time of death for animals dying prior to day +100).
All animals receiving BCL1 tumor cells and sorted CD8+T cells from wild-type untreated C57 donors cleared tumor idiotype but succumbed to GVHD. All animals receiving tumor cells and sorted chimeric C57 CD8+ T cells remained free of clinical or pathologic evidence of GVHD, but died with tumor progression. Control myeloablated animals given C57 TCD BM alone with BCL1 tumor cells all succumbed to tumor, whereas those receiving C57 whole bone marrow with tumor demonstrated tumor survival without GVHD. The data indicate that chimeric donor peripheral CD8+ T cells, which lose their capacity to induce lethal GVHD in BALB/c hosts, also lose the capacity to eradicate BALB/c-type lymphoma cells. We conclude that CD8+ T cell-induced GVT effect in this model is dependent upon alloantigen rather than tumor-specific antigen recognition.