In Vitro Cultured Allogeneic Cytotoxic T Cells Mediate Hematopoietic GVHD without Gut GVHD Despite Expression of Functional LPAM (α4β7 Integrin).
Authors: Kalpatthi Ram , Nicol Kathleen , Hendey Lindsay , Boyer Michael W. 
Published: April 2020

Abstract
Graft-versus-host disease (GVHD) remains a significant complication of allogeneic bone marrow transplantation. We and others have shown that in vitro cultured CD8 cytotoxic T cells (CTL) have attenuated GVHD compared to naïve cells, while retaining GVL activity. It has been shown that expression of α4β7 integrin on CD8 T cells is important for gut homing specificity in GVHD. Recently, retinoic acid (RA) has been shown to upregulate α4β7 expression on naïve T cells. Thus, we hypothesize that in vitro cultured CTL without RA lack the ability to cause GVHD in part due to deficient α4β7 expression. We used an established murine GVHD model in which spleen and lymph node cells from B6SJL mice were stimulated with splenocytes from DBA mice and were restimulated on day 7. Cultures were supplemented with IL-2 & IL-7 with and without addition of RA (100nm) on day 2. Day 14 comparison of CTL with and without RA revealed comparable CD4 (1.7% vs 0.7% respectively) and CD8 populations (96% vs 97% respectively). Phenotyping of CTL with and without RA showed CD8 α4β7 expression of 58% and 0.8% and CD8 CCR9 53% and 10% respectively. In vitro cytotoxicity was comparable between CTL with and without RA: 51% vs 41% at an effector target ratio of 10:1 (n=3, p=0.3). Both CTL groups had comparable in vitro migration towards to SDF, IP-10 & MIP-3α (p= ns). However RA treated CTL had increased migration towards TECK (chemokine expressed in small intestine); 17.3% vs 4.6% (n=4, p=0.01) and decreased migration towards TARC (chemokine expressed in skin); 2% vs 13% (n=4, p=0.03). For in vivo homing, 107 labeled cells from each CTL with (CFSE) and without RA (TRITC) were coinjected intravenously and mice were sacrificed 16 hours later for analysis. RA treated CTL had increased homing to peyer’s patch and MLN compared to CTL without RA. [Homing index (CTLRA/CTL) 2.3 and 2.5 respectively]. This finding is exaggerated in the radiated host [Homing index (CTLRA/CTL) 15 for PP and 11 for MLN]. CTL generated with or without RA (5x106 cells each) were injected intravenously in to irradiated (600 Rads) B6D2F1 recipients (3 groups; Radiation control, CTL with and without RA). Mice were followed for clinical GVHD scores and sacrificed when moribund. CBC and histopathologic GVHD scores (Liver, skin, lung, small and large intestines) were obtained. Both CTL groups developed lethal bone marrow (BM) aplasia around day 24 as compared to radiation control group; however, clinical and histopathologic GVHD scores were similar in all groups (Table 1). Our data demonstrate that both CTL with and without RA cause a lethal hematopoietic graft versus host reaction. Despite high α4β7 and CCR9 expression, significant in vitro migration to TECK and in vivo homing to gut associated lymphoid tissues, RA treated CTL did not cause significant GVHD in gut, liver or skin. This suggests that defective gut homing alone may not be sufficient to explain the attenuated GVHD from cultured CTL. Future studies are planned to confirm these findings in other GVHD models and to elucidate the mechanisms of attenuated GVHD from cultured CTL.
Table 1 (Data from Day 24 sacrifice) Parameter (Mean) Radiation control CTL CTLRA p value* * CTL or CTLRA vs Radiation control group Hb (g/dL) 12.2 3.8 3.1 0.0001 WBC x106/L 1000 300 300 0.07 Platelets x103/L 667,200 50,200 29,500 0.003 BM cells from 2 femur (106) 15.3 0.9 1.1 0.0006 Combined GVHD histology score 4.9 4.4 4.2 0.3