Administration of cyclophosphamide after transplantation (post-transplantation cyclophosphamide, PTC) has shown promise in the clinic as a prophylactic agent against graft-versus-host disease (GVHD). An important issue with regard to recipient immune function and reconstitution after PTC is the extent to which, in addition to diminution of antihost allo-reactive donor T cells, the remainder of the nonhost allo-reactive donor T cell pool may be affected. To investigate PTC's effects on nonhost reactive donor CD8 T cells, ova-specific (OT-I) and gp100-specific Pmel-1 T cells were labeled with proliferation dyes and transplanted into syngeneic and allogeneic recipients. Notably, an intermediate dose (66 mg/kg) of PTC, which abrogated GVHD after allogeneic HSCT, did not significantly diminish these peptide-specific donor T cell populations. Analysis of the rate of proliferation after transplantation illustrated that lymphopenic-driven, donor nonhost reactive TCR Tg T cells in syngeneic recipients underwent slow division, resulting in significant sparing of these donor populations. In contrast, after exposure to specific antigens at the time of transplantation, these same T cells were significantly depleted by PTC, demonstrating the global susceptibility of rapidly dividing T cells after an encounter with cognate antigen. In total, our results, employing both syngeneic and allogeneic minor antigen-mismatched T cell replete models of transplantation, demonstrate a concentration of PTC that abrogates GVHD can preserve most cells that are dividing because of the accompanying lymphopenia after exposure. These findings have important implications with regard to immune function and reconstitution in recipients after allogeneic hematopoietic stem cell transplantation.

Potential applications of neural stem cells (NSCs) for transplantation requires understanding myosin heavy chain (MHC) expression and the ability of T cells and natural killer (NK) cells to recognize this progenitor population. Cells from the cortices of day-13 embryonic (E13) B6 (H-2(b)) mice were explanted and cultured to expand NSCs. Analysis of P2-P17-cultured cells using anti-MHC class I/II monoclonal antibodies (mAbs) showed marginal expression of both products. Although recombinant murine interferon-gamma (rmIFN gamma) exposure did not alter the multipotential capacity of these stem cells, titration of mrIFN gamma NSC cultures demonstrated that MHC molecules could be strongly upregulated after addition of 3 ng/ml rmIFN gamma for 60 hours. To assess the susceptibility of NSCs with low or absent versus high levels of MHC expression to lysis by cytotoxic T lymphocyte (CTL) and NK populations, untreated and rmIFN gamma-treated NSC target cells were examined. Untreated NSCs were not recognized by BALB/c (H-2(d)) allospecific anti-H-2(b) CTL, consistent with the mAb findings; however, upregulation of MHC products on both early and later passaged NSCs resulted in their efficient lysis by CTL. NK cells were prepared from syngeneic B6 or allogeneic BALB/c mice. Although NK cells effectively killed control YAC-1 target cells, these effectors did not kill MHC-deficient (or expressing) NSC targets. Thus, similar to hematopoietic, embryonic, and mesenchymal stem cell populations, unmanipulated NSCs are not readily killed by T and NK cells. These findings suggest that following transplant into syngeneic or allogeneic recipients, NSCs may exhibit diminished susceptibility to clearance by host T- and NK-cell populations.

Regulatory T cells (Tregs) are critical for self-tolerance. Although adoptive transfer of expanded Tregs limits graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT), ex vivo generation of large numbers of functional Tregs remains difficult. Here, we demonstrate that in vivo targeting of the TNF superfamily receptor TNFRSF25 using the TL1A-Ig fusion protein, along with IL-2, resulted in transient but massive Treg expansion in donor mice, which peaked within days and was nontoxic. Tregs increased in multiple compartments, including blood, lymph nodes, spleen, and colon (GVHD target tissue). Tregs did not expand in bone marrow, a critical site for graft-versus-malignancy responses. Adoptive transfer of in vivo-expanded Tregs in the setting of MHC-mismatched or MHC-matched allogeneic HSCT significantly ameliorated GVHD. Critically, transplantation of Treg-expanded donor cells facilitated transplant tolerance without GVHD, with complete sparing of graft-versus-malignancy. This approach may prove valuable as a therapeutic strategy promoting transplantation tolerance.

Allogeneic bone marrow transplantation is an effective treatment for a number of malignant and nonmalignant diseases (Applebaum. 2001. Nature. 411: 385–389 and Copelan. 2006. N Engl J Med. 354: 1813–1826). However, the application of this therapeutic modality has been impeded by a number of confounding side effects, the most frequent and severe of which is the development of graft-versus-host disease (GVHD) (Copelan. 2006. N Engl J Med. 354: 1813–1826 and Blazar and Murphy. 2005. Philos Trans R Soc Lond B Biol Sci. 360: 1747–1767). Alloreactive donor T cells are critical for causing GVHD (Fowler. 2006. Crit Rev Oncol Hematol. 57: 225–244 and Ferrara and Reddy. 2006. Semin Hematol. 43: 3–10), whereas recent data demonstrated a significant role for the naturally occurring thymic-derived donor CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) (Bluestone and Abbas. 2003. Nat Rev Immunol. 3: 253–257 and Shevach. 2006. Immunity. 25: 195–201) in suppressing experimental GVHD after bone marrow transplantation (Blazar and Taylor. 2005. Biol Blood Marrow Transpl. 11: 46–49 and Joffe and van Meerwijk. 2006. Semin Immunol. 18: 128–135) . Host APCs are required for induction of GVHD by the conventional donor T cells. However, it is not known whether they are also obligatory for donor Treg-mediated suppression of GVHD. Using multiple clinically relevant MHC-matched and -mismatched murine models of GVHD, we investigated the role of host APCs in the suppression of GVHD by donor Tregs. We found that alloantigen expression by the host APCs is necessary and sufficient for induction of GVHD protection by donor Tregs. This requirement was independent of their effect on the maintenance of Treg numbers and the production of IL-10 or IDO by the host APCs.

Ocular complications occur after transplantation in 60% to 90% of chronic graft-versus-host disease (GVHD) patients and significantly impair vision-related quality of life. Ocular surface inflammation and dry eye disease are the most common manifestations of ocular GVHD. Ocular GVHD can be viewed as an excellent preclinical model that can be studied to understand the immune pathogenesis of this common and debilitating disease. A limitation of this is that only a few experimental models mimic the ocular complications after hematopoietic stem cell transplantation (HSCT) and have focused on the acute GVHD process. To address this issue, we used a preclinical animal model developed by our group where ocular involvement was preceded by systemic GVHD to gain insight regarding the contributing immune mechanisms. Employing this "matched unrelated donor" model enabled the development of clinical scoring criteria, which readily identified different degrees of ocular pathology at both the ocular surface and adnexa, dependent on the level of conditioning before HSCT. As far as we are aware, we report for the first time that these clinical and immune responses occur not only on the ocular surface, but they also heavily involve the lid margin region. In total, the present study reports a preclinical scoring model that can be applied to animal models as investigators look to further explore GVHD's immunologic effects at the level of the ocular surface and eyelid adnexa compartments. We speculate that future studies will use this clinical scoring index in combination with what is recognized histologically and correlated with serum biomarkers identified in chronic/ocular GVHD.

Engraftment failure following allogeneic bone marrow (BM) transplantation is of clinical concern particularly involving T-cell–depleted inoculum and transplantations for aplastic anemia. Immune resistance by lymphoid and natural killer (NK) populations with “barrier” function is well established. Major histocompatibility complex (MHC)–identical marrow allografts were examined to investigate effector pathways in non-NK–mediated resistance. Barrier function was examined in cytotoxic normal and deficient B6 (H-2^b) recipients primed to donor minor histocompatibility antigen (MiHA) prior to BM transplantation. Host resistance was sensitively evaluated by colony-forming unit (CFU) assays to directly assess for donor progenitor cell (PC) and peripheral chimerism. B6 host CD8⁺ T cells but not CD4⁺ or NK1.1⁺ cells effected rejection of primitive (CFU-HPP [high-proliferative potential]) and lineage-committed (CFU-IL3/GM [interleukin 3/granulocyte macrophage]) allogeneic donor progenitors. To address complementation by the cytotoxic pathways existing in singly deficient (perforin or FasL) recipients, cytotoxically double (perforin plus FasL) deficient (cdd) recipients were used. Resistance in B6-cdd recipients was comparable to that of wild-type B6 recipients and was also dependent on CD8⁺ T cells. A “triple” cytotoxic deficient model, involving transplantation of TNFR1^−/− (tumor necrosis factor receptor 1) progenitor grafts did not diminish the ability of B6-cdd recipients to reject allografts. Finally, injection of anti-TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) monoclonal antibody (mAb) in B6-cdd recipients also failed to inhibit rejection of TNFR1^−/− marrow grafts. In total, these studies demonstrate that CD8⁺ host T cells can effectively resist MHC-matched MiHA-mismatched donor PCs via alternative effector pathway(s) independent of perforin-, FasL-, TNFR-1–, and TRAIL-dependent cytotoxicity. Therefore, inhibition of these effector pathways in sensitized recipients is unlikely to result in stem cell engraftment following PC allografts.