26 In vitro Cultured Allogeneic Cytotoxic T Cells Mediate GVL Without Systemic GVHD Despite Expression of Functional LPAM.

Abstract

Background: Previous studies have shown that in vitro cultured alloreactive T cells had comparable GVL activity but decreased GVHD as compared to naïve cells. It has been shown that expression of LPAM on CD8 T cells is important for gut homing specificity in GVHD. Our preliminary data showed that the expression of LPAM is decreased upon in vitro culture of CTLs. Recently, retinoic acid (RA) has been shown to up regulate LPAM expression on naive T cells. We hypothesize that in vitro cultured CTLs without retinoic acid lack the ability to cause GVHD in part due to deficient LPAM expression.Methods: B6PL spleen/lymph node cells were stimulated against DBA splenocytes with IL-2 & IL-7 ± 100 nm RA. Day 14 CTL & CTLRA were infused into allogeneic recipients &compared to naïve T cells.Results: CTL & CTLRA showed CD8 LPAM expression of 0.7% & 61% (p<.01). Both groups had comparable in vitro migration towards to SDF, but CTLRA had ↑ migration towards TECK; 17.3% vs. 4.6% (p<.01). Homing analysis revealed ↑ migration of CTLRA to PP & MLN [Homing index (CTLRA/CTL) 2.3 & 2.5 respectively]. 600 cGy irradiated B6D2F1 mice were given 0.5x106 P815 murine mastocytoma cells on day 0 followed by B6 BM cells with either 5x106 CTLs or 10x106 naïve lymphocytes on day 1. CTLs and naïve lymphocytes mediated a potent GVL effect in spleen & BM. Recipients of naïve lymphocytes developed lethal GVHD with high clinical GVHD scores (median survival 17 days; =14) where as, CTL group had significantly improved survival due to attenuated GVHD (median 60 days; =10, p<0.001). In pure GVHD experiments, CTL, CTLRA & naive lymphocytes induced lethal BM aplasia (n= 10, mean Hb 3g% in all) in the absence of BM rescue at d12 as compared to rad controls (n= 8, mean Hb 7.5g%) without systemic GVHD in any group. With BM rescue, naïve cells resulted in lethal systemic GVHD at d20 (=8; Serum ALT 188 IU/L, clinical & histological GVHD scores of 8 & 11.5 respectively, p<.01). Where as recipients of CTL or CTLRA were alive >d150 (=8; ALT 60 IU/L, clinical GVHD score of 2) despite ↑ migration of CTLRA cells to gut in contrast to CTL without RA (p<.01).Conclusions: We demonstrate that in vitro cultured alloreactive CTLs mediate a rapid GVL effect that was equivalent to the infusion of naïve T cells but with attenuated sub clinical GVHD. We also showed that in vitro cultured CTLs have defective gut homing which could be corrected by up regulating LPAM expression with the addition of RA. Thus defective gut homing does not explain the lack of lethal GVHD seen with in vitro cultured CTL.