Minnelide suppresses GVHD and enhances survival while maintaining GVT responses.

Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent antiinflammatory and antitumor activity in several preclinical models and has proven both safe and efficacious in clinical trials for advanced gastrointestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared with posttransplant cyclophosphamide (PTCy). Strikingly, we found Minnelide improved survival, weight loss, and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score, and colonic length. Notably, in addition to decreased donor T cell infiltration early after aHSCT, several regulatory cell populations, including Tregs, ILC2s, and myeloid-derived stem cells in the colon were increased, which together may account for Minnelide's GVHD suppression after aHSCT. Importantly, Minnelide's GVHD prevention was accompanied by preservation of graft-versus-tumor activity. As Minnelide possesses anti-acute myeloid leukemia (anti-AML) activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for patients with AML undergoing aHSCT.

Regulatory T Cell Amelioration of Graft-versus-Host Disease following Allogeneic/Xenogeneic Hematopoietic Stem Cell Transplantation Using Mobilized Mouse and Human Peripheral Blood Donors.

The present studies examined experimental transplant outcomes using mobilized peripheral blood from mice and humans together with FoxP3+Treg cells. Donor mice were treated with filgrastim and / or plerixafor and their peripheral blood (PB) displayed significant elevations in hematopoietic stem and progenitor populations. Some of these PB donors were concurrently administered a Treg expansion strategy consisting of a TL1A-Ig fusion protein low dose rIL-2. A significant increase (4-5x) in the frequency Tregs occurred during mobilization. C3H.SW PB was collected from mobilized and Treg unexpanded ("TrUM") or mobilized and Treg expanded ("TrEM") donors and transplanted into MHC-matched B6 (H2b) recipients. Recipients of TrEM, exhibited significantly reduced weight loss and clinical GVHD scores compared to recipients of TrUM. Notably, recipients of TrEM exhibited comparable GVL activity to TrUM recipients against leukemia levels. Next, huTregs (CD4+CD25+CD127lo) from a healthy human PB mobilized donor were expanded ex-vivo prior to transplant into NSG/ NOD-scid IL2Rgammanull mice. We found that treatment with ex-vivo expanded huTregs resulted in significant reduction of lethality and clinical xGVHD scores. Notably, post-transplant, PB huTregs levels remained elevated and the frequency of huCD4+Tconv and CD8+ cells was diminished supporting the improved xGVHD outcomes. These findings demonstrated that the use of mPB containing elevated Treg levels significantly reduced GVHD following "MUD" and MHC-mismatched mouse HSCT without loss of GVL activity. Moreover, utilizing ex-vivo expanded huTregs from a mobilized PB donor and added back to donor PB ameliorated xGVHD. In total, these studies support the notion that in vivo or ex-vivo manipulation of donor Tregs together with mobilized peripheral blood could provide therapeutic approaches to improve aHSCT outcomes.

Recipient Tregs: Can They Be Exploited for Successful Hematopoietic Stem Cell Transplant Outcomes?

Human and mouse CD4+FoxP3+ T cells (Tregs) comprise non-redundant regulatory compartments which maintain self-tolerance and have been found to be of potential therapeutic usefulness in autoimmune disorders and transplants including allogeneic hematopoietic stem cell transplantation (allo-HSCT). There is substantial literature interrogating the application of donor derived Tregs for the prevention of graft versus host disease (GVHD). This Mini-Review will focus on the recipient's Tregs which persist post-transplant. Although treatment in patients with low dose IL-2 months post-HSCT are encouraging, manipulating Tregs in recipients early post-transplant is challenging, in part likely an indirect consequence of damage to the microenvironment required to support Treg expansion of which little is understood. This review will discuss the potential for manipulating recipient Tregs in vivo prior to and after HSCT (fusion proteins, mAbs). Strategies that would circumvent donor/recipient peripheral blood harvest, cell culture and ex-vivo Treg expansion will be considered for the translational application of Tregs to improve HSCT outcomes.

Improved NK Cell Recovery Following Use of PTCy or Treg Expanded Donors in Experimental MHC-Matched Allogeneic HSCT.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is complicated by graft- versus-host disease (GVHD), which causes immune dysfunction and further delays immune reconstitution through its effects on primary and secondary lymphoid organs. Treatments to prevent GVHD and improve immune recovery following allo-HSCT are needed. Post-transplantation cyclophosphamide (PTCy) is a well-established and clinically widely used method for GVHD prophylaxis after HLA-matched as well as haploidentical allo-HSCT, as well as a promising strategy in the setting of mismatched unrelated donor allo-HSCT. Recently, regulatory T cells (Tregs), a critical subset for immune homeostasis and tolerance induction, have been evaluated for use as GVHD prophylaxis in experimental models and clinical trials. Natural killer (NK) cells are one of the first lymphoid populations to reconstitute following allo-HSCT and are important mediators of protective immunity against pathogens, and are also critical for limiting post-transplantation relapse of hematologic cancers. Several reports have noted that a delay in NK cell recovery may occur following experimental mouse allo-HSCT as well as after clinical allo-HSCT. Here we examined how 2 treatment strategies, PTCy and donor expanded Tregs (TrED), in experimental MHC-matched allo-HSCT affect NK recovery. Our experiments show that both strategies improved NK cell numbers, with PTCy slightly better than TrED, early after allo-HSCT (1 month) compared with untreated allo-HSCT recipients. Importantly, NK cell IFN-γ production and cytotoxic function, as reflected by CD107 expression as well as in vivo killing of NK-sensitive tumor cells, were improved using either PTCy or TrED versus control allo-HSCT recipients. In conclusion, both prophylactic treatments were found to be beneficial for NK recovery and NK cell function following MHC-matched minor antigen-mismatched experimental allo-HSCT. Improved NK recovery could help provide early immunity toward tumors and pathogens in these transplant recipients.

Use of Post-transplant Cyclophosphamide Treatment to Build a Tolerance Platform to Prevent Liquid and Solid Organ Allograft Rejection.

Corneal transplantation (CT) is the most frequent type of solid organ transplant (SOT) performed worldwide. Unfortunately, immunological rejection is the primary cause of graft failure for CT and therefore advances in immune regulation to induce tolerance remains an unmet medical need. Recently, our work and others in pre-clinical studies found that cyclophosphamide (Cy) administered after ("post-transplant," PTCy) hematopoietic stem cell transplantation (HSCT), i.e., liquid transplants is effective for graft vs. host disease prophylaxis and enhances overall survival. Importantly, within the past 10 years, PTCy has been widely adopted for clinical HSCT and the results at many centers have been extremely encouraging. The present studies found that Cy can be effectively employed to prolong the survival of SOT, specifically mouse corneal allografts. The results demonstrated that the timing of PTCy administration is critical for these CT and distinct from the kinetics employed following allogeneic HSCT. PTCy was observed to interfere with neovascularization, a process critically associated with immune rejection of corneal tissue that ensues following the loss of ocular "immune privilege." PTCy has the potential to delete or directly suppress allo-reactive T cells and treatment here was shown to diminish T cell rejection responses. These PTCy doses were observed to spare significant levels of CD4+ FoxP3+ (Tregs) which were found to be functional and could readily receive stimulating signals leading to their in vivo expansion via TNFRSF25 and CD25 agonists. In total, we posit future studies can take advantage of Cy based platforms to generate combinatorial strategies for long-term tolerance induction.

Analyses and Correlation of Pathologic and Ocular Cutaneous Changes in Murine Graft versus Host Disease.

Graft versus host disease (GVHD) is initiated by donor allo-reactive T cells activated against recipient antigens. Chronic GVHD (cGVHD) is characterized by immune responses that may resemble autoimmune features present in the scleroderma and Sjogren's syndrome. Unfortunately, ocular involvement occurs in approximately 60-90% of patients with cGVHD following allo-hematopoietic stem cell transplants (aHSCT). Ocular GVHD (oGVHD) may affect vision due to ocular adnexa damage leading to dry eye and keratopathy. Several other compartments including the skin are major targets of GVHD effector pathways. Using mouse aHSCT models, the objective was to characterize cGVHD associated alterations in the eye and skin to assess for correlations between these two organs. The examination of multiple models of MHC-matched and MHC-mismatched aHSCT identified a correlation between ocular and cutaneous involvement accompanying cGVHD. Studies detected a "positive" correlation, i.e., when cGVHD-induced ocular alterations were observed, cutaneous compartment alterations were also observed. When no or minimal ocular signs were detected, no or minimal skin changes were observed. In total, these findings suggest underlying cGVHD-inducing pathological immune mechanisms may be shared between the eye and skin. Based on the present observations, we posit that when skin involvement is present in aHSCT patients with cGVHD, the evaluation of the ocular surface by an ophthalmologist could potentially be of value.

STING differentially regulates experimental GVHD mediated by CD8 versus CD4 T cell subsets.

The stimulator of interferon genes (STING) pathway has been proposed as a key regulator of gastrointestinal homeostasis and inflammatory responses. Although STING reportedly protects against gut barrier damage and graft-versus-host disease (GVHD) after major histocompatibility complex (MHC)-mismatched allogeneic hematopoietic stem cell transplantation (aHSCT), its effect in clinically relevant MHC-matched aHSCT is unknown. Studies here demonstrate that STING signaling in nonhematopoietic cells promoted MHC-matched aHSCT-induced GVHD and that STING agonists increased type I interferon and MHC I expression in nonhematopoietic mouse intestinal organoid cultures. Moreover, mice expressing a human STING allele containing three single-nucleotide polymorphisms associated with decreased STING activity also developed reduced MHC-matched GVHD, demonstrating STING's potential clinical importance. STING-/- recipients experienced reduced GVHD with transplant of purified donor CD8+ T cells in both MHC-matched and MHC-mismatched models, reconciling the seemingly disparate results. Further examination revealed that STING deficiency reduced the activation of donor CD8+ T cells early after transplant and promoted recipient MHC class II+ antigen-presenting cell (APC) survival. Therefore, APC persistence in STING pathway absence may account for the increased GVHD mediated by CD4+ T cells in completely mismatched recipients. In total, our findings have important implications for regulating clinical GVHD by targeting STING early after aHSCT and demonstrate that an innate immune pathway has opposing effects on the outcome of aHSCT, depending on the donor/recipient MHC disparity.

Superior immune reconstitution using Treg-expanded donor cells versus PTCy treatment in preclinical HSCT models.

Posttransplant cyclophosphamide (PTCy) has been found to be effective in ameliorating acute graft-versus-host disease (GVHD) in patients following allogeneic hematopoietic stem cell transplantation (aHSCT). Adoptive transfer of high numbers of donor Tregs in experimental aHSCT has shown promise as a therapeutic modality for GVHD regulation. We recently described a strategy for in vivo Treg expansion targeting two receptors: TNFRSF25 and CD25. To date, there have been no direct comparisons between the use of PTCy and Tregs regarding outcome and immune reconstitution within identical groups of transplanted mice. Here, we assessed these two strategies and found both decreased clinical GVHD and improved survival long term. However, recipients transplanted with Treg-expanded donor cells (TrED) exhibited less weight loss early after HSCT. Additionally, TrED recipients demonstrated less thymic damage, significantly more recent thymic emigrants, and more rapid lymphoid engraftment. Three months after HSCT, PTCy-treated and TrED recipients showed tolerance to F1 skin allografts and comparable immune function. Overall, TrED was found superior to PTCy with regard to weight loss early after transplant and initial lymphoid engraftment. Based on these findings, we speculate that morbidity and mortality after transplant could be diminished following TrED transplant into aHSCT recipients, and, therefore, that TrED could provide a promising clinical strategy for GVHD prophylaxis.

Very Low Numbers of CD4+ FoxP3+ Tregs Expanded in Donors via TL1A-Ig and Low-Dose IL-2 Exhibit a Distinct Activation/Functional Profile and Suppress GVHD in a Preclinical Model.

Regulatory T cells (Tregs) are essential for the maintenance of tolerance and immune homeostasis. In allogeneic hematopoietic stem cell transplantation (aHSCT), transfer of appropriate Treg numbers is a promising therapy for the prevention of graft-versus-host disease (GVHD). We have recently reported a novel approach that induces the marked expansion and selective activation of Tregs in vivo by targeting tumor necrosis factor receptor superfamily 25 (TNFRSF25) and CD25. A potential advance to promote clinical application of Tregs to ameliorate GVHD and other disorders would be the generation of more potent Treg populations. Here we wanted to determine if very low doses of Tregs generated using the "2-pathway" stimulation protocol via TL1A-Ig fusion protein and low-dose IL-2 (targeting TNFRSF25 and CD25, respectively) could be used to regulate preclinical GVHD. Analysis of such 2-pathway expanded Tregs identified higher levels of activation and functional molecules (CD103, ICOS-1, Nrp-1, CD39, CD73, il-10, and tgfb1) versus unexpanded Tregs. Additionally, in vitro assessment of 2-pathway stimulated Tregs indicated enhanced suppressor activity. Notably, transplant of extremely low numbers of these Tregs (1:6 expanded Tregs/conventional T cells) suppressed GVHD after an MHC-mismatched aHSCT. Overall, these results demonstrate that 2-pathway stimulated CD4+ FoxP3+ Tregs were quantitatively and qualitatively more functionally effective than unexpanded Tregs. In total, the findings in this study support the notion that such 2-pathway stimulated Tregs may be useful for prevention of GVHD and ultimately promote more widespread application of aHSCT in the clinic.

BET Bromodomain Inhibitors Which Permit Treg Function Enable a Combinatorial Strategy to Suppress GVHD in Pre-clinical Allogeneic HSCT.

A recent approach for limiting production of pro-inflammatory cytokines has been to target bromodomain and extra-terminal (BET) proteins. These epigenetic readers of histone acetylation regulate transcription of genes involved in inflammation, cardiovascular disease, and cancer. Development of BET inhibitors (BETi) has generated enormous interest for their therapeutic potential. Because inflammatory signals and donor T cells promote graft-versus-host disease (GVHD), regulating both pathways could be effective to abrogate this disorder. The objective of the present study was to identify a BETi which did not interfere in vivo with CD4+FoxP3+ regulatory T cell (Treg) expansion and function to utilize together with Tregs following allogeneic hematopoietic stem cell transplantation (aHSCT) to ameliorate GVHD. We have reported that Tregs can be markedly expanded and selectively activated with increased functional capacity by targeting TNFRSF25 and CD25 with TL1A-Ig and low dose IL-2, respectively. Here, mice were treated over 7 days (TL1A-Ig + IL-2) together with BETi. We found that the BETi EP11313 did not decrease frequency/numbers or phenotype of expanded Tregs as well as effector molecules, such as IL-10 and TGF-ß. However, BETi JQ1 interfered with Treg expansion and altered subset distribution and phenotype. Notably, in Treg expanded mice, EP11313 diminished tnfa and ifng but not il-2 RNA levels. Remarkably, Treg pSTAT5 expression was not affected by EP11313 supporting the notion that Treg IL-2 signaling remained intact. MHC-mismatched aHSCT (B6 → BALB/c) was performed using in vivo expanded donor Tregs with or without EP11313 short-term treatment in the recipient. Early post-transplant, improvement in the splenic and LN CD4/CD8 ratio along with fewer effector cells and high Treg levels in aHSCT recipients treated with expanded Tregs + EP11313 was detected. Interestingly, this group exhibited a significant diminution of GVHD clinical score with less skin and ocular involvement. Finally, using low numbers of highly purified expanded Tregs, improved clinical GVHD scores were observed in EP11313 treated recipients. In total, we conclude that use of this novel combinatorial strategy can suppress pre-clinical GVHD and posit, in vivo EP11313 treatment might be useful combined with Treg expansion therapy for treatment of diseases involving inflammatory responses.

Marked in Vivo Donor Regulatory T Cell Expansion via Interleukin-2 and TL1A-Ig Stimulation Ameliorates Graft-versus-Host Disease but Preserves Graft-versus-Leukemia in Recipients after Hematopoietic Stem Cell Transplantation.

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.

Novel Scoring Criteria for the Evaluation of Ocular Graft-versus-Host Disease in a Preclinical Allogeneic Hematopoietic Stem Cell Transplantation Animal Model.

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.

Recruitment of Donor T Cells to the Eyes During Ocular GVHD in Recipients of MHC-Matched Allogeneic Hematopoietic Stem Cell Transplants.

PURPOSE: The primary objective of the present study was to identify the kinetics and origin of ocular infiltrating T cells in a preclinical model of graft-versus-host disease (GVHD) that induces eye tissue damage. METHODS: Graft-versus-host disease was induced using an major histocompatibility complex (MHC)-matched, minor histocompatibility-mismatched hematopoietic stem cell transplant (HSCT) model. This approach, which utilized congenic and EGFP-labeled donor populations, mimics a matched, clinically unrelated donor (MUD) cell transplant. Systemic and ocular GVHD were assessed at varying time points using clinical examination, intravital microscopy, immune phenotype via flow cytometric analyses, and immunohistochemical staining. RESULTS: Following transplant, we observed characteristic changes in GVHD-associated immune phenotype as well as clinical signs present in recipients post transplant. Notably, the kinetics of the systemic changes and the ocular damage paralleled what is observed clinically, including damage to the cornea as well as the conjunctiva and lacrimal gland. Importantly, the infiltrate contained predominantly donor CD4 as well as CD8 T cells with an activated phenotype and macrophages together with effector cytokines consistent with the presence of a TH1 alloreactive population. CONCLUSIONS: Overall, the findings here unequivocally demonstrated that donor T cells compose part of the corneal and ocular adnexa infiltrate in animals undergoing ocular GVHD. In total, the results describe a novel and promising preclinical model characterized by both systemic and ocular changes as detected in significant numbers of patients undergoing GVHD following allo-HSCT, which can help facilitate dissecting the underlying immune mechanisms leading to damage associated with ocular GVHD.

The allure and peril of hematopoietic stem cell transplantation: overcoming immune challenges to improve success.

Since its inception in the mid-twentieth century, the complication limiting the application and utility of allogeneic hematopoietic stem cell transplantation (allo-HSCT) to treat patients with hematopoietic cancer is the development of graft-versus-host disease (GVHD). Ironically, GVHD is induced by the cells (T lymphocytes) transplanted for the purpose of eliminating the malignancy. Damage ensuing to multiple tissues, e.g., skin, GI, liver, and others including the eye, provides the challenge of regulating systemic and organ-specific GVH responses. Because the immune system is also targeted by GVHD, this both: (a) impairs reconstitution of immunity post-transplant resulting in patient susceptibility to lethal infection and (b) markedly diminishes the individual's capacity to generate anti-cancer immunity--the raison d'etre for undergoing allo-HSCT. We hypothesize that deleting alloreactive T cells ex vivo using a new strategy involving antigen stimulation and alkylation will prevent systemic GVHD thereby providing a platform for the generation of anti-tumor immunity. Relapse also remains the major complication following autologous HSCT (auto-HSCT). While GVHD does not complicate auto-HSCT, its absence removes significant grant anti-tumor responses (GVL) and raises the challenge of generating rapid and effective anti-tumor immunity early post-transplant prior to immune reconstitution. We hypothesize that effective vaccine usage to stimulate tumor-specific T cells followed by their amplification using targeted IL-2 can be effective in both the autologous and allogeneic HSCT setting. Lastly, our findings support the notion that the ocular compartment can be locally targeted to regulate visual complications of GVHD which may involve both alloreactive and self-reactive (i.e., autoimmune) responses.

High-content analysis of proapoptotic EphA4 dependence receptor functions using small-molecule libraries.

Small-molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of proapoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of an SMC library was performed using stable EphA4-expressing NIH 3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.