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Graft-versus-host-disease (GVHD) is a major cause of morbidity and mortality in stem cell transplantation (SCT). Acute GVHD is thought to be mediated by donor T cells that target and destroy host tissues. Studies into the host immune system are limited, as it is assumed that host T cells in peripheral tissues such as skin are depleted by conditioning regimens in parallel to T cells in lymphoid organs and blood. However, it is recently appreciated that a high number of memory T cells reside long-term within the organs most affected by GVHD. We investigated whether host skin T cells survive SCT conditioning and are present during acute GVHD. To do so, excess skin biopsy tissue was obtained from patients with acute skin GVHD who had undergone myeloablative or non-myeloablative conditioning and allogeneic SCT with sex-mismatched donor cells. Tissue was stained for T cell markers via immunofluorescence concurrently with in situ hybridization for the X and Y chromosomes. All seventeen patients studied demonstrated persistence of host T cells in skin, often in equal or greater numbers than donor T cells. Host skin T cells survived regardless of myeloablative or non-myeloablative conditioning, of patient age, and of day post-SCT (samples ranged from 12–213 days post-SCT). Comparatively, blood T cell chimerism was 100% donor in nearly all samples, indicating diverging effects of conditioning on T cell compartments. Skin host T cells demonstrated pro-inflammatory cytokine production. These results provide a novel avenue of research into GVHD pathobiology and suggest that alternative conditioning regimens should be employed if host skin T cells are to be targeted.

Exosomes are nanovesicles generated in multivesicular endosomes that are released to the extracellular space by different cell types. Since DC-derived exosomes (dexosomes) express MHC-peptide and costimulatory molecules, we investigated the role of dexosomes in elicitation of the anti-donor response in transplantation. Dexosomes were generated from bone marrow-derived DC and labeled with PKH67 for traffic studies. CD4+ TCRtg T cells (Thy1.1+) specific for the IEα52-68 (BALB/c)-IAb (B10) complex were CFSE-labeled and transferred i.v. to host Thy1.2+ B6 mice. Graft infiltrating DC were isolated from BALB/c (CD45.2+) cardiac grafts 3 days after transplantation in B6 (CD45.2+) recipients and genetically-engineered ex vivo to release exosomes expressing the reporter marker eGFP, and then transferred i.v. into host CD45.1+ B6 mice. We demonstrated that although graft-infiltrating leukocytes release exosomes ex vivo, they do not secrete enough exosomes in circulation to stimulate donor-reactive T-cells in lymphoid organs. Instead, migrating DCs (generated in vitro or isolated from allografts), once they home in the spleen, they transfer exosomes expressing eGFP to spleen-resident DCs. Thus, exchange of exosomes between DCs in lymphoid organs constitutes a mechanism by which passenger leukocytes transfer alloAg to recipient's APC and amplify generation of donor-reactive T-cells.

Current dogma in transplantation presumes that therapeutic maturation-resistant dendritic cells (MR-DC) prevent allograft rejection by directly interacting with donor-reactive T-cells in vivo. However, this assumption remains untested. Our study therefore aimed to elucidate the mechanism by which donor MR-DC down-regulate the anti-donor response to prolong allograft survival in mice. We employed as prototypic donor MR-DC, bone marrow-derived DC treated with the active form of vitamin D3, 1α,25(OH)2D3. We demonstrate that donor-derived MR-DC prolonged cardiac allograft survival similarly as donor apoptotic or viable splenocytes, suggesting that different donor-derived cellular therapies share a common mechanism of action. We determined that once injected i.v., MR-DC were short-lived and failed to directly tolerize donor-reactive T-cells. Rather, apoptotic MR-DC were internalized and re-processed by quiescent recipient splenic CD11chiCD8α+ and CD11chiCD8- DC, which in turn induced deletion of effector T-cells and outgrowth of regulatory T-cells via the indirect pathway. Importantly, activated recipient DC induced robust T-cell activation via indirect presentation. Our data suggest that therapeutic DC, like other more practical cellular therapies, serve as a source of alloantigen for recipient quiescent DC that in turn down-regulate the anti-donor response to prolong allograft survival.