A major barrier to successful use of allogeneic hematopoietic cell transplantation is acute graft-versus-host disease (aGVHD), a devastating condition that arises when donor T cells attack host tissues. With current technologies, aGVHD diagnosis is typically made after end-organ injury and often requires invasive tests and tissue biopsies. This impacts patient prognosis as treatments are dramatically less effective at late disease stages. Here we show that a novel positron emission tomography (PET) radiotracer, 2′-deoxy-2′-[18F]fluoro-9-β-D-arabinofuranosylguanine ([18F]F-AraG), targeted towards two salvage kinase pathways preferentially accumulates in activated primary T cells. [18F]F-AraG PET imaging of a murine aGVHD model enabled visualization of secondary lymphoid organs harboring activated donor T cells prior to clinical symptoms. Tracer biodistribution in healthy humans showed favorable kinetics. This new PET strategy has great potential for early aGVHD diagnosis, enabling timely treatments and improved patient outcomes. [18F]F-AraG may be useful for imaging activated T cells in various biomedical applications.

Adoptive transfer of freshly isolated natural occurring CD4+CD25+FoxP3+ regulatory T cells (Treg) prevents graft versus host disease (GvHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor derived Treg have been mainly used as they share the same MHC with conventional CD4+ and CD8+ T cells (Tcon) that are primarily responsible for GvHD. Third-party derived Treg are a promising alternative for cellular therapy as they can be prepared in advance, screened for pathogens and activity and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GvHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, while both donor and third-party but not host Treg protect from GvHD in allogeneic HCT with donor Treg being the most effective. In a MHC minor mismatched transplantation model (C57BL/6 → BALB/b) donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first two days after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg mediated protection from GvHD and support for the use of third-party Treg in clinical trials.

Our results have identified the microbiota as a potent therapeutic target that can be recruited to significantly reduce GVHD. Approaches to potentially translate these findings include investigating the safety of introduction of Blautia to allo BMT recipients, or alternatively developing nutritional strategies to support endogenous Blautia during the transplantation process.

CD4+ Foxp3+ regulatory T cells (Treg) are a subpopulation of T cells which regulate the immune system, maintain the tolerance of self-antigens and enhance immune tolerance after transplantation. It was also reported that recipient derived Treg could provide immune privilege to allogeneic stem cells (HSC) after transplantation. However, the precise interaction with Treg and HSC has not been fully elucidated. In this study, we investigated the role of recipient derived Treg in the engraftment and immune reconstitution following transplantation of purified allogeneic HSC and the effectiveness of Treg expansion following activation of DR3 (Death receptor 3, also called as TNFRSF25) signaling in this model. 　We first tested the effect of Treg depletion using Foxp3-DTR mice in allogeneic HSC transplantation. In this system, FACS-sorted purified HSC (Lin-cKit+Sca1+ population) derived from WT-FVB mice (CD45.1+/H2kq+) were injected into lethally irradiated B6-Foxp3-DTR mice (CD45.2+/H2kb+) with or without pre-treatment of diphtheria toxin (DT). On day 0 and day 28 after transplantation decreased frequencies of Foxp3+ cells in residual recipient derived CD4+ T cells were observed in peripheral blood from the DT treated mice (P<0.001 on day 0, P<0.002 on day 28). Although total myeloid chimerism was comparable between control and DT-treated mice, the frequency of donor derived immune cells including CD4+ T cells (P<0.01 on day 56), CD8+ T cells (P<0.01 on day 56), and B220+ B cells (P<0.001 on day 56) was significantly decreased in DT-treated mice. These data suggested that recipient derived Treg play an important role in allogeneic immune reconstitution after transplantation. DR3 is a member of the TNF receptor superfamily and we previously reported the expansion of Treg by the activation of this signaling pathway (Kim et al, ASH abstract 2013). We next tested whether activation of DR3 signaling by its agonistic antibody would affect the donor immune reconstitution after allogeneic HSC transplantation. The frequencies of Foxp3+ cells in CD4+ T cells were significantly increased in thymus, spleen, peripheral blood, and bone marrow 4 days after antibody injection (P<0.01). Isolated Treg derived from antibody treated mice showed stronger suppressive function in the mixed lymphoid reaction compared with those from isotype antibody treated mice. The mice treated with antibody on day -4 were transplanted with purified allogeneic HSC on day 0. Antibody treated mice showed a higher frequency of donor derived CD4+ T cells (P<0.001 on day 28), CD8+ T cells (P<0.05 on day 28), and B220+ B cells (P<0.05 on day 28) in this allogeneic HSC transplantation model. In summary, our data suggest that recipient derived CD4+Foxp3+ Treg play an important role in donor immune reconstitution and the activation of DR3 signaling in recipient mice enhances donor immune reconstitution by expansion of recipient derived Treg. H.N and BS-K contributed equally to this work. Disclosures No relevant conflicts of interest to declare.