Alloreactivity Directed Against the Widely Distributed HY Antigen Impairs Antitumor Immunity and Results in T-Cell Dysfunction
Authors: Shand Jessica C , Capitini Christian M. , Qin Haiying , Nasholm Nicole , Duncan Brynn B , Fry Terry J 
Published: October 2019

Abstract
Abstract 2964

INTRODUCTION:
The curative potential of allogeneic transplant for high-risk malignancy is based on the observation that alloreactivity can result in a clinically significant graft-versus-tumor (GVT) effect. However, we have observed that alloreactivity directed against non-tumor restricted miHA's reduces quantitative responses to vaccines targeting tumor-specific antigens. The relative impact of the GVHD-mediating antigen on the potency of the GVT response when the antigen is shared has not been well studied.

METHODS:
A murine allotransplant system in which the clinically relevant GVHD antigen HY drives both graft-versus-host disease and the antitumor response was utilized. Following lethal irradiation, combinations of B6 male (HY-expressing) and female (HY-naïve) donors and recipients were used in T-cell depleted bone marrow transplants to control for HY expression in hematopoetic and non-hematopoetic compartments. Delayed donor lymphocyte infusion (DLI) with female HY-specific transgenic T-cells was then performed which allowed tracking of antigen-specific cells. Mice were subsequently challenged with an immunogenic HY-expressing tumor (MB49). In tumor protection studies, transplant recipients received a male dendritic cell vaccine at the time of DLI. Recipients were monitored for clinical GVHD scoring, weight loss, tumor-free and overall survival. Surface phenotyping of HY-specific CD8 T cells from recipient bone marrow, tumor-draining lymph node (LN) and spleen was performed serially by flow cytometry using congenic markers. Statistical analyses were performed using paired Student t-tests and Kaplan-Meier survival estimates.

RESULTS:
Transplantation of female marrow and HY-specific T cells into male recipients produces a mild HY-targeted GVHD, indicated by weight loss and skin GVHD scores. Female recipients of female marrow and HY-specific DLI had 100% survival following HY-expressing tumor challenge. In contrast, male recipients had only 20 +/− 4.7% tumor-free survival (p<0.0001), despite receiving HY-reactive female marrow and HY-specific DLI. Administration of an HY-expressing male dendritic cell vaccine did not improve either tumor growth velocity or tumor-free survival in male recipients. Despite a poor antitumor response in males, expression of HY on nonhematopoetic tissues produced a significant expansion of HY specific T-cells following DLI, regardless of tumor-bearing status (30.5 −77.4% total CD8 from spleen, draining LN and marrow, vs 0.01–1% from female recipient controls, p<0.0001). This suggested that impaired tumor control was due to dysfunction, rather than deletion, of HY-specific T cells. Indeed, nearly 100% of HY-specific CD8 isolated from the spleen, tumor-draining lymph node, and bone marrow of male recipients expressed high levels of PD-1, a phenomenon observed at all time points in tumor-bearing and non tumor-bearing male recipients with HY-directed GVHD. Non-HY specific CD8 cells did not express PD-1 (p<0.0001). Further, HY-specific CD8 from spleen and tumor-draining LN of male recipients display a significantly increased percentage of CD44+CD62L- effector memory (72.4 +/− 17.2%) vs. CD44+CD62L+ central memory (15.9 +/− 9.7%, p= 0.006) cells compared to non-HY specific CD8 cells (26.5% +/− 2.8 % vs. 28.2 +/− 12.7%, p= 0.52) from male and female recipient controls.

CONCLUSIONS:
In an experimental system where HY is expressed on both recipient nonhematopoetic tissue and tumor, HY-directed alloreactivity impairs the antitumor response despite antigen-specific DLI and effective vaccination. Characterization of alloreactive CD8 T cells in this setting reveals a persistence of effector memory and high levels of PD-1 expression, which suggest T-cell dysfunction as a possible mechanism. Further studies of T-cell dysfunction in this model may identify targets for therapeutic blockade following adoptive immunotherapy with particular relevance to those clinical situations where GVHD does not enhance GVT.

Disclosures:
No relevant conflicts of interest to declare.