Abstract
In allogeneic hematopoietic stem cell transplantation (HSCT), devising new strategies to separate GVHD and GVL responses is of critical importance. However, this is a difficult task, as GVHD and GVL rely on the same recognition of allogeneic MHC by donor-derived T cells. CD8+ T cells are key effector cells that mediate both GVHD and GVL. In mouse models of allogeneic HSCT, the infusion of donor-derived CD8+ T cells eliminates tumor growth but also causes severe GVHD. The activation of CD8+ T cells can be potentially manipulated by perturbing the signaling pathways downstream of the T cell receptor (TCR). TCR signaling depends on the formation of a proximal multimolecular complex, which is nucleated by adaptor proteins such as SLP-76. The phosphorylation of the Y145 residue of SLP-76 is critical for activation of the downstream enzyme PLCg1. As such, a YàF mutation at Y145 of SLP-76 (Y145F) causes decreased TCR-mediated signaling and attenuated T cell function. Here, we investigated how the SLP-76 Y145F mutation in CD8+T cells may impact GVHD and GVL responses in a mouse model of allogeneic HSCT.
We employed a major MHC-mismatch mouse model of GVHD involving the transplantation of C57BL/6 (B6)-derived bone marrow (BM) into lethally irradiated Balb/c mice (B6àBalb/c). BM-transplanted mice were also injected with FACS-sorted CD8+ T cells either B6 wildtype (WT) mice or Y145F mice. Recipients of Y145F CD8+ T cells showed significantly (p<0.001) less weight loss, lower clinical score, and improved survival compared to mice injected with WT CD8+ T cells. Next, to determine whether the Y145F CD8+ T cells could mediate GVL effects, BM-transplanted Balb/c mice were additionally challenged intravenously with 1 x 105 luciferase-positive A20 leukemia cells. As expected, BM-transplanted Balb/c mice succumbed from A20 tumor growth, whereas mice injected with WT CD8+ T cells cleared the tumor but developed GVHD. Surprisingly, mice receiving Y145F CD8+ T cells eradicated the leukemic cells but did not develop GVHD. These data suggest that the Y145F mutation in CD8+T cells may be able to separate GVHD from GVL effects.
In addition to defective TCR signaling observed in peripheral T cells of Y145F mice, a majority of Y145F KI CD8+ T cells adopt a memory-like CD44hi phenotype through exposure to high levels of IL-4 produced in the thymus of these mice. To test whether the CD44hi CD8+ T cell phenotype was necessary and/or sufficient for the separation of GVHD and GVL effects, BM-transplanted Balb/c mice were injected with FACS-sorted CD44hi or CD44lo CD8+ T cells from WT or Y145F KI mice and challenged with A20 leukemia cells. While BM-transplanted mice receiving CD44hi CD8+ T cells from Y145F mice displayed intact GVL responses without causing GVHD, mice injected with CD44lo CD8+ T cells from Y145F mice displayed impaired ability to clear the tumor cells. Moreover, recipients of CD44hi or CD44lo CD8+ T cells from WT mice cleared the tumor but exhibited severe GVHD. These findings were corroborated with data obtained with an inducible system, whereby CD8+ T cells are affected by the Y145F mutation only after full maturation and thus do not display a CD44hi phenotype (Y145F conditional knock-in mice). Bone marrow-transplanted recipients receiving Y145F conditional knock-in CD8+ T cells developed GVHD and exhibited an attenuated GVL response, suggesting that the Y145F mutation needed to be present during T cell development. Together, these data suggest that either the Y145F mutation or CD44hi phenotype alone in CD8+T cells is insufficient to separate GVHD from GVT.
Our data demonstrate that perturbation of the TCR signaling pathway downstream of Y145 of SLP-76 in CD8+ T cells results in separation of GVHD from GVL effects. Experiments to mechanistically test how the Y145F signaling mutation synergizes with the CD44hi phenotype of CD8+ T cells to allow for the separation of the GVHD and GVL effects are currently underway. Our novel and unexpected finding could lead to a novel therapeutic strategy for treatment of acute GVHD after allogeneic HSCT.
Disclosures
No relevant conflicts of interest to declare.
An Inducible Transgenic Mouse Model for Immune Mediated Hepatitis Showing Clearance of Antigen Expressing Hepatocytes by CD8+ T Cells