Separation of Gvhd from GVL Responses By Modulation of TCR Signaling
Authors: Karimi Mobin , Jordon Martha , Kambayashi Taku 
Published: October 2019

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.