Targeting Alpha 4 Integrin (CD49d) to Reduce GvHD

Acute graft versus host disease (aGvHD) is a life-threatening condition that complicates allogeneic hematopoietic cell transplantation (allo-HCT). Donor T cells recognize the recipientÕs tissues as foreign causing GvHD, however, these same donor T cells are also responsible for the beneficial graft versus leukemia (GVL). Distinguishing between GvHD and GvL is crucial for the development of safe and effective therapies following allogeneic bone marrow transplantation.

We have recently reported that the disruption of alloreactive donor T cell trafficking to the GvHD target organs significantly reduces GvHD in both an MHC fully-mismatched and a minor-mismatched allo-HCT models (Choi et al., Blood 2012). In this study, we examined if inhibition of α4, which is required for transendothelial migration and access to lymphoid system and GvHD target organs, could reduce GVHD. On T cells α4 integrin subunit can form a heterodimer with either β1 to form α4β1 integrin (VLA-4) or β7 to form α4β7 (LPAM-1). To genetically eliminate α4 from allogeneic donor T cells, we generated Tie-2 cre+ α4fl/fl mice (B6, H-2b, CD45.2+). Splenic pan T cells were isolated from these mice and T cell-depleted bone marrow cells (TCD BM) isolated from congenic B6 mice (CD45.1+). 5x10⁶ TCD BM and 5x10⁵ splenic pan T cells were transplanted into lethally irradiated (900 cGy) allogeneic Balb/c recipient mice (H-2d, CD45.2+). We found that α4 deficient T cells significantly reduced GvHD compared to Tie-2 cre+ control T cells (Fig. A). α4 deficient T cells had no significant effect on donor engraftment and achieved complete donor chimerism. In addition, recipients transplanted with α4 deficient T cells had significantly lower histopathology score (median score 3 vs. 6; p=0.0263). Similar results were observed when donor T cells were infused at day 11 post allo-HCT. To test whether α4 deficient T cells maintain GvL we performed bioluminescence imaging (BLI) using a systemic leukemia mouse model. CBRluc-expressing A20 leukemia cells (Balb/c-derived) were transplanted intravenously (1 × 10⁵ cells) along with TCD BM (B6, CD45.1⁺) into Balb/c recipients at day 0. Pan T cells (B6, CD45.2⁺) either from Tie-2 cre+ or α4 deficient mice were infused at day 11 to allow sufficient time for the leukemia cells to expand. After weekly BLI, we found α4 deficient T cells were able to control leukemia cells as effectively as Tie-2 cre+ Control T cells (p=0.3748). In addition, we performed BLI to track CBRluc-transduced pan T cells (2 × 10⁶ cells) after allo-HCT in vivo. We found a significant difference in the percentage of BLI signal intensity between control and α4 deficient T cells in spleen and gut at day 14 and 21 post allo-HCT. (Fig. B)

While we speculate defective T cell trafficking to GvHD target organs would be the primary reason for the reduced GvHD in the recipients of α4 deficient T cells, we examined other possible mechanisms such as T cell alloreactivity and proliferation. We found that α4 deficient T cells proliferated at the same rate as Tie-2 cre+ T cells in the presence of anti-CD3/CD28 antibody-coated beads. However, α4 deficient CD8 T cells proliferate slower in the presence of allogeneic antigen presenting cell compared to Tie-2 cre+ T cells. Interestingly, α4 deficient T cells significantly upregulate CTLA-4 and GZMB compared to Tie-2 cre+ control T cells. These data suggest that not only altered T cell trafficking to GvHD target organs but also altered T cell functions might be the reason for the reduced GvHD.

In conclusion, we propose that α4 represents a promising therapeutic target for future efforts to mitigate GvHD after allo-HCT. In addition, this strategy can be exploited in other diseases besides GvHD such as solid organ transplantation, chronic inflammatory diseases and autoimmune diseases.

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Figure 1.

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