Sudipto Ganguly

Aldehyde Dehydrogenase Expression Drives Human Regulatory T Cell Resistance to Posttransplantation Cyclophosphamide

Regulatory T cells become resistant to cyclophosphamide after allogeneic stimulation, protecting against GVHD. Taming Graft-Versus-Host Disease Allogeneic bone marrow transplant is a curative therapy for many different diseases of the blood. Yet, the transplanted cells have a wild side—they sometimes can attack the recipient, causing graft-versus-host disease (GVHD). One way of preventing GVHD is with posttransplant cyclophosphamide, but what exactly cyclophosphamide does to the reactive T cells remains unclear. Now, Kanakry et al. report that regulatory T cells (Tregs) may play a critical role in this process. The authors observed that although cyclophosphamide treatment decreased conventional T cell numbers in patients, memory Tregs were relatively preserved. Tregs recovered faster than conventional T cells after therapy, in part due to the protective expression of aldehyde dehydrogenase (ALDH). Indeed, blocking ALDH in cultured cells sensitized Tregs to cyclophosphamide treatment. Moreover, in a xenograft model, removing Tregs prevented the protective effect of cyclophosphamide after transplant. These data provide insight into the mechanistic underpinnings of Tregs in transplant recipients. High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4+ T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4+ T cells with relative preservation of memory CD4+Foxp3+ T cells. In particular, CD4+CD45RA−Foxp3+hi effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4+Foxp3+ T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4+ T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy’s GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD.

Donor CD4+ Foxp3+ regulatory T cells are necessary for posttransplantation cyclophosphamide-mediated protection against GVHD in mice

Posttransplantation cyclophosphamide (PTCy) is an effective prophylaxis against graft-versus-host disease (GVHD). However, it is unknown whether PTCy works singularly by eliminating alloreactive T cells via DNA alkylation or also by restoring the conventional (Tcon)/regulatory (Treg) T-cell balance. We studied the role of Tregs in PTCy-mediated GVHD prophylaxis in murine models of allogeneic blood or marrow transplantation (alloBMT). In 2 distinct MHC-matched alloBMT models, infusing Treg-depleted allografts abrogated the GVHD-prophylactic activity of PTCy. Using allografts in which Foxp3(+) Tregs could be selectively depleted in vivo, either pre- or post-PTCy ablation of donor thymus-derived Tregs (tTregs) abolished PTCy protection against GVHD. PTCy treatment was associated with relative preservation of donor Tregs. Experiments using combinations of Foxp3(-) Tcons and Foxp3(+) Tregs sorted from different Foxp3 reporter mice indicated that donor Treg persistence after PTCy treatment was predominantly caused by survival of functional tTregs that retained Treg-specific demethylation and also induction of peripherally derived Tregs. Finally, adoptive transfer of tTregs retrieved from PTCy-treated chimeras rescued PTCy-treated, Treg-depleted recipients from lethal GVHD. Our findings indicate that PTCy-mediated protection against GVHD is not singularly dependent on depletion of donor alloreactive T cells but also requires rapidly recovering donor Tregs to initiate and maintain alloimmune regulation.

Post-kala-azar dermal leishmaniasis - an overview.

Post‐kala‐azar dermal leishmaniasis (PKDL) is a dermal sequela of visceral leishmaniasis (VL), reported mainly from two regions – Sudan in eastern Africa and the Indian subcontinent, with incidences of 50–60% and 5–10%, respectively. Importantly, patients with PKDL are considered as reservoirs of VL, linking its eradication to effective control of PKDL. The etiopathogenesis of PKDL is presumably due to an immunological assault on latent dermal parasites. Immunological markers include IL‐10, whose expression in skin and plasma of Sudanese patients with VL predicted onset of PKDL. Cell‐mediated immune responses, notably restoration of IFN‐γ production by antigen‐stimulated lymphocytes are well documented in Sudanese PKDL, but remain ambiguous in the Indian form; recently, antigen‐specific IL‐10‐producing CD8+ lymphocytes have been implicated in pathogenesis. In Indian PKDL, upregulation of intralesional IFN‐γ and TNF‐α is counterbalanced by IL‐10 and TGF‐β together with downregulated IFN‐γ R1. Although IL‐10 curtails excessive IFN‐γ‐mediated reactivity and ensures parasite survival, its cellular source remains to be confirmed, with infiltrating regulatory T cells (Tregs) being a likely candidate. Future functional investigations on Tregs and their interaction with lesional effector lymphocytes would be indispensable for development of immunomodulatory therapies against Leishmania infection.

Human Mesenchymal Stromal Cells Attenuate Graft‐Versus‐Host Disease and Maintain Graft‐Versus‐Leukemia Activity Following Experimental Allogeneic Bone Marrow Transplantation

We sought to define the effects and underlying mechanisms of human, marrow‐derived mesenchymal stromal cells (hMSCs) on graft‐versus‐host disease (GvHD) and graft‐versus‐leukemia (GvL) activity. Irradiated B6D2F1 mice given C57BL/6 BM and splenic T cells and treated with hMSCs had reduced systemic GvHD, donor T‐cell expansion, and serum TNFα and IFNγ levels. Bioluminescence imaging demonstrated that hMSCs redistributed from lungs to abdominal organs within 72 hours, and target tissues harvested from hMSC‐treated allogeneic BMT (alloBMT) mice had less GvHD than untreated controls. Cryoimaging more precisely revealed that hMSCs preferentially distributed to splenic marginal zones and regulated T‐cell expansion in the white pulp. Importantly, hMSCs had no effect on in vitro cytotoxic T‐cell activity and preserved potent GvL effects in vivo. Mixed leukocyte cultures containing hMSCs exhibited decreased T‐cell proliferation, reduced TNFα, IFNγ, and IL‐10 but increased PGE2 levels. Indomethacin and E‐prostanoid 2 (EP2) receptor antagonisms both reversed while EP2 agonism restored hMSC‐mediated in vitro T‐cell suppression, confirming the role for PGE2. Furthermore, cyclo‐oxygenase inhibition following alloBMT abrogated the protective effects of hMSCs. Together, our data show that hMSCs preserve GvL activity and attenuate GvHD and reveal that hMSC biodistribute to secondary lymphoid organs wherein they attenuate alloreactive T‐cell proliferation likely through PGE2 induction. Stem Cells 2015;33:601–614

Redundant Innate and Adaptive Sources of IL17 Production Drive Colon Tumorigenesis

IL17-producing Th17 cells, generated through a STAT3-dependent mechanism, have been shown to promote carcinogenesis in many systems, including microbe-driven colon cancer. Additional sources of IL17, such as γδ T cells, become available under inflammatory conditions, but their contributions to cancer development are unclear. In this study, we modeled Th17-driven colon tumorigenesis by colonizing Min(Ap) (c+/-) mice with the human gut bacterium, enterotoxigenic Bacteroides fragilis (ETBF), to investigate the link between inflammation and colorectal cancer. We found that ablating Th17 cells by knocking out Stat3 in CD4(+) T cells delayed tumorigenesis, but failed to suppress the eventual formation of colonic tumors. However, IL17 blockade significantly attenuated tumor formation, indicating a critical requirement for IL17 in tumorigenesis, but from a source other than Th17 cells. Notably, genetic ablation of γδ T cells in ETBF-colonized Th17-deficient Min mice prevented the late emergence of colonic tumors. Taken together, these findings support a redundant role for adaptive Th17 cell- and innate γδT17 cell-derived IL17 in bacteria-induced colon carcinogenesis, stressing the importance of therapeutically targeting the cytokine itself rather than its cellular sources. Cancer Res; 76(8); 2115-24. ©2016 AACR.

The myeloid immune signature of enterotoxigenic Bacteroides fragilis-induced murine colon tumorigenesis.

Enterotoxigenic Bacteroides fragilis (ETBF), a human commensal and candidate pathogen in colorectal cancer (CRC), is a potent initiator of interleukin-17 (IL-17)-dependent colon tumorigenesis in MinApc+/− mice. We examined the role of IL-17 and ETBF on the differentiation of myeloid cells into myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages, which are known to promote tumorigenesis. The myeloid compartment associated with ETBF-induced colon tumorigenesis in Min mice was defined using flow cytometry and gene expression profiling. Cell-sorted immature myeloid cells were functionally assayed for inhibition of T-cell proliferation and inducible nitric oxide synthase expression to delineate MDSC populations. A comparison of ETBF infection with that of other oncogenic bacteria (Fusobacterium nucleatum or pks+Escherichia coli) revealed a specific, ETBF-associated colonic immune infiltrate. ETBF-triggered colon tumorigenesis is associated with an IL-17-driven myeloid signature characterized by subversion of steady-state myelopoiesis in favor of the generation of protumoral monocytic-MDSCs (MO-MDSCs). Combined action of the B. fragilis enterotoxin BFT and IL-17 on colonic epithelial cells promoted the differentiation of MO-MDSCs, which selectively upregulated Arg1 and Nos2, produced NO, and suppressed T-cell proliferation. Evidence of a pathogenic inflammatory signature in humans colonized with ETBF may allow for the identification of populations at risk for developing colon cancer.

Iron enhances generation of free radicals by Artemisinin causing a caspase-independent, apoptotic death in Leishmania donovani promastigotes

Abstract An increasing incidence of unresponsiveness to antimonials in Leishmaniasis has led to identification of plant-derived anti-leishmanial compounds like Artemisinin. Since iron-mediated generation of free radicals sustains the anti-malarial activity of Artemisinin, this study investigated whether similar mechanisms accounted for its activity in Leishmania promastigotes. Artemisinin effectively disrupted the redox potential via an increased generation of free radicals along with a decrease in levels of non-protein thiols. Attenuation of its IC50 by a free radical scavenger N-acetyl l-cysteine and an iron chelator desferoxamine established the pivotal role of free radicals and of the potentiating effect of iron. An enhanced Fluo-4 fluorescence reflected Artemisinin-induced mobilization of intracellular calcium, which triggered apoptosis. However, the absence of any detectable caspase activity indicated that the leishmanicidal activity of Artemisinin is mediated by an iron-dependent generation of reactive intermediates, terminating in a caspase-independent, apoptotic mode of cell death.

Situational aldehyde dehydrogenase expression by regulatory T cells may explain the contextual duality of cyclophosphamide as both a pro-inflammatory and tolerogenic agent

In two recent publications, we demonstrated that after allogeneic stimulation, regulatory T cells (Tregs) increase expression of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of cyclophosphamide detoxification, thereby becoming cyclophosphamide resistant. Differential ALDH expression may explain why cyclophosphamide has pro- and anti-inflammatory effects that are temporally and contextually dependent.

Dendritic cell activation enhances anti-PD-1 mediated immunotherapy against glioblastoma

Introduction The glioblastoma (GBM) immune microenvironment is highly suppressive as it targets and hinders multiple components of the immune system. Checkpoint blockade (CB) is being evaluated for GBM patients. However, biomarker analyses suggest that CB monotherapy may be effective only in a small fraction of GBM patients. We hypothesized that activation of antigen presentation would increase the therapeutic response to PD-1 blockade. Results We show that activating DCs through TLR3 agonists enhances the anti-tumor immune response to CB and increases survival in GBM. Mice treated with TLR3 agonist poly(I:C) and anti-PD-1 demonstrated increased DC activation and increased T cell proliferation in tumor draining lymph nodes. We show that DCs are necessary for the improved anti-tumor immune response. Conclusions This study suggests that augmenting antigen presentation is an effective multimodal immunotherapy strategy that intensifies anti-tumor responses in GBM. Specifically, these data represent an expanded role for TLR3 agonists as adjuvants to CB. Methods Using a preclinical model of GBM, we tested the efficacy of combinatorial immunotherapy with anti-PD-1 and TLR3 agonist, poly(I:C). Characterization of the immune response in tumor infiltrating immune cells and in secondary lymphoid organs was performed. Additionally, dendritic cell (DC) depletion experiments were performed.

CD8+IL-17+ T cells mediate neutrophilic airway obliteration in T-bet-deficient mouse lung allograft recipients

Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet(-/-) recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet(-/-) recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8(+) T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ-dominant responses in WT mice. CD4(+) T cells produced IL-17 but not IFN-γ responses in T-bet(-/-) recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8(+)IFN-γ(+) responses in both T-bet(-/-) and WT mice but had no attenuating effect on lung rejection pathology in T-bet(-/-) recipients or on the development of obliterative airway inflammation that occurred only in T-bet(-/-) recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade-resistant rejection pathology and airway inflammation in T-bet(-/-) recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet(-/-) allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet-deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade-resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8(+)IL-17(+) T cells. Our data support T-bet-deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation.