Identification of CD137+ IFN-g- CD4+ Alloreactive T Cells Post Myeloablative Double Cord Blood Transplant: A Synergistic Role in the Graft Verus Graft Interaction

Using flow cytometric techniques to directly analyze the developing reactivity of T cells isolated from the recipient after double cord blood transplant (dCBT) against cells (B-LCL) derived from each donor, we previously reported that naive CD8⁺ T cells in the engrafting unit are activated in vivo by antigens expressed by non-engrafting unit, which expand and differentiate into IFN-γ secreting CD8⁺ effector T cells. Surprisingly, our initial studies did not identify IFN-γ secreting alloreactive CD4⁺ T cells, despite the majority of CB units being mismatched at one or more class II alleles (Gutman et al. Blood 2010). However, these studies did not exclude the possibility that CD4⁺ T cells, especially the IFN-γ^- Th₂ and Th₁₇ cells, may be involved in the GvG interactions of dCBT. Herein, we now demonstrate the presence of alloreactive CD4⁺ T cells in dCBT recipients and investigate their role in the GvG interaction. Included are 13 patients who underwent a myeloablative dCBT on an IRB approved protocol  between Feb 2013 and June 2014. Post-transplant peripheral blood collection, donor B-LCL generation, and in vitro MLR with intracellular cytokines staining were performed as previously described (Gutman et al. Blood 2010) with small revision. Our current experimental approach includes the detection of surface marker CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNFR)-family, as a means to identify a more encompassing compartment of alloreactive T cells in addition to IFN-γ secretion. Consistent with our previous report, no significant CD4⁺ IFN-γ secreting population was detected in the PBMCs obtained from day 14 post-CBT patient blood after stimulation with LCLs from the non-engrafting unit (Fig 1). However, the frequency of CD4⁺ cells that express CD137 (median, 8.86%; range, 1.39-16.7%) was significantly higher than the frequency of IFN-γ secreting cells (median, 2%; range, 0.2-4.39%) when stimulated with LCLs from the non-engrafting unit (p<0.0001). Moreover, the CD137 expressing CD4⁺ T cell population was specific for allogeneic stimulation with significantly higher CD137 expression frequency when stimulated with LCLs from the non-engrafting unit as compared to stimulation with LCLs from the engrafting unit (p<0.0001, Fig 1). In summary, this study provides the first clear evidence that a distinct population of CD137⁺IFN-γ^- CD4⁺ alloreactive T cells is present in the peripheral blood of patients undergoing dCBT and are involved in the GvG effects observed after dCBT. Functional definition of this distinct population (Th₂ or Th₁₇) might disclose an important role of GvG interactions in the regulation of GVHD, the prevention of graft rejection, and the maintenance of GVL effects as hypothesized by Fig 2.