Raghavan Chinnadurai

IDO-Independent Suppression of T Cell Effector Function by IFN-γ–Licensed Human Mesenchymal Stromal Cells

Human bone marrow–derived mesenchymal stromal cells (MSCs) inhibit proliferation of activated T cells, and IFN-γ plays an important role in this process. This IFN-γ–licensed veto property is IDO-dependent. To further decipher the mechanistic underpinnings of MSC veto function on T cells, we investigated the effect of MSCs and IFN-γ–licensed MSCs on T cell effector function as assayed by cytokine secretion of T cells. Although MSCs and IFN-γ–licensed MSCs inhibit T cell proliferation, only IFN-γ–licensed MSCs significantly inhibit Th1 cytokine (IFN-γ, TNF-α, and IL-2) production by T cells. Additionally, IFN-γ–licensed MSCs inhibit T cell degranulation as well as single, double, and triple cytokine–producing T cells. Although IFN-γ–licensed MSCs upregulate their IDO activity, we found that MSC IDO catalytic function is dispensable with regard to MSC-driven inhibition of T cell effector function. Novel flow cytometry based functional screening of MSC-expressed, IFN-γ–licensed inhibitory molecules identified B7H1 and B7DC/PD1 pathways as essential effectors in blocking T cell function. Small interfering RNA–mediated blocking of B7H1 and B7DC reverses the inhibitory potential of IFN-γ–licensed MSCs on T cell effector function. Mechanistic analysis revealed that clustering of MHC and coinhibitory molecules are indispensable for the inhibitory effect of IFN-γ MSCs. Although exogenous IL-2 reverses B7H1-Ig–mediated inhibition of T cell proliferation, it does not affect the veto function of IFN-γ MSCs on both T cell proliferation and effector function. Our results reveal a new immunosuppressive property of IFN-γ–licensed MSCs that inhibits T cell effector function independent of IDO but through the ligands for PD1.

Actin Cytoskeletal Disruption following Cryopreservation Alters the Biodistribution of Human Mesenchymal Stromal Cells In Vivo

Summary Mesenchymal stromal cells have shown clinical promise; however, variations in treatment responses are an ongoing concern. We previously demonstrated that MSCs are functionally stunned after thawing. Here, we investigated whether this cryopreservation/thawing defect also impacts the postinfusion biodistribution properties of MSCs. Under both static and physiologic flow, compared with live MSCs in active culture, MSCs thawed from cryopreservation bound poorly to fibronectin (40% reduction) and human endothelial cells (80% reduction), respectively. This reduction correlated with a reduced cytoskeletal F-actin content in post-thaw MSCs (60% reduction). In vivo, live human MSCs could be detected in murine lung tissues for up to 24 hr, whereas thawed MSCs were undetectable. Similarly, live MSCs whose actin cytoskeleton was chemically disrupted were undetectable at 24 hr postinfusion. Our data suggest that post-thaw cryopreserved MSCs are distinct from live MSCs. This distinction could significantly affect the utility of MSCs as a cellular therapeutic.

B7-H4 mediates inhibition of T cell responses by activated murine hepatic stellate cells†

Liver fibrosis is mediated by the transformation of hepatic stellate cells (HSC) from a quiescent to an activated state. To understand the role of HSC in liver immunity, we investigated the effect of this transition on T cell stimulation in vitro. Unlike quiescent HSC, activated HSC did not induce proliferation of antigen‐specific T cells. Phenotypic analysis of quiescent and activated HSC revealed that activated HSC expressed the coinhibitory molecule B7‐H4. Silencing B7‐H4 by small interfering RNA (siRNA) in activated HSC restored the ability of T cells to proliferate, differentiate, and regain effector recall responses. Furthermore, expression of B7‐H4 on HSC inhibits early T cell activation and addition of exogenous interleukin (IL)‐2 reversed the T cell anergy induced by activated HSC. Conclusion: These studies reveal a novel role for activated HSC in the attenuation of intrahepatic T cell responses by way of expression of the coinhibitory molecule B7‐H4, and may provide fundamental insight into intrahepatic immunity during liver fibrogenesis. (HEPATOLOGY 2010)

Cryopreserved Mesenchymal Stromal Cells Are Susceptible to T-Cell Mediated Apoptosis Which Is Partly Rescued by IFNγ Licensing.

We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSCs fitness post freeze‐thaw. We have observed that thawed MSC can suppress T‐cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T‐cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO‐free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1‐methyl Tryptophan, an Indoleamine 2,3‐dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw‐induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T‐cell cytolysis and affect MSCs cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429–2442

The safety of autologous and metabolically fit bone marrow mesenchymal stromal cells in medically refractory Crohn's disease - a phase 1 trial with three doses.

Mesenchymal stromal cells ability to reset immune functionalities may be useful in Crohns disease.

Hepatic Transplant and HCV: A New Playground for an Old Virus

Hepatitis C virus (HCV) infection is a major global health problem affecting 170 million people worldwide. The majority of infected individuals fail to resolve their infection, with a significant number developing chronic, progressive HCV‐related liver disease. HCV infection is the leading indication for liver transplantation and unfortunately, all patients with detectable viral load before transplantation will have rapid, recurrent infection. What remain to be determined are factors contributing to the severity of HCV recurrence. Such factors are unique to the posttransplant setting and include: viral genetic diversity and composition, immunosuppression, donor/recipient age and sex, genetic factors and the liver microenvironment. Importantly, the possibility that the severity of HCV recurrence might be also influenced by factors related to the primary course of disease (i.e. viral set point, previously acquired adaptations of the virus) must be further evaluated. In this sense, recurrent HCV infection should not be regarded merely as another acute infection, but rather, it should be cautioned that problems first arising during the primary course of disease may be accentuated during recurrence. Development of novel therapeutic approaches will require a thorough understanding of viral and host determinants of infection resolution and how these factors may change in the posttransplant setting.

Cryopreserved MSCs are Susceptible to T‐cell Mediated Apoptosis which is partly Rescued by IFNγ Licensing

We have previously demonstrated that cryopreservation and thawing lead to altered Mesenchymal stromal cells (MSC) functionalities. Here, we further analyzed MSCs fitness post freeze‐thaw. We have observed that thawed MSC can suppress T‐cell proliferation when separated from them by transwell membrane and the effect is lost in a MSC:T‐cell coculture system. Unlike actively growing MSCs, thawed MSCs were lysed upon coculture with activated autologous Peripheral Blood Mononuclear Cells (PBMCs) and the lysing effect was further enhanced with allogeneic PBMCs. The use of DMSO‐free cryoprotectants or substitution of Human Serum Albumin (HSA) with human platelet lysate in freezing media and use of autophagy or caspase inhibitors did not prevent thaw defects. We tested the hypothesis that IFNγ prelicensing before cryobanking can enhance MSC fitness post thaw. Post thawing, IFNγ licensed MSCs inhibit T cell proliferation as well as fresh MSCs and this effect can be blocked by 1‐methyl Tryptophan, an Indoleamine 2,3‐dioxygenase (IDO) inhibitor. In addition, IFNγ prelicensed thawed MSCs inhibit the degranulation of cytotoxic T cells while IFNγ unlicensed thawed MSCs failed to do so. However, IFNγ prelicensed thawed MSCs do not deploy lung tropism in vivo following intravenous injection as well as fresh MSCs suggesting that IFNγ prelicensing does not fully rescue thaw‐induced lung homing defect. We identified reversible and irreversible cryoinjury mechanisms that result in susceptibility to host T‐cell cytolysis and affect MSCs cell survival and tissue distribution. The susceptibility of MSC to negative effects of cryopreservation and the potential to mitigate the effects with IFNγ prelicensing may inform strategies to enhance the therapeutic efficacy of MSC in clinical use. Stem Cells 2016;34:2429–2442

Challenges in animal modelling of mesenchymal stromal cell therapy for inflammatory bowel disease

Utilization of mesenchymal stromal cells (MSCs) for the treatment of Crohns disease and ulcerative colitis is of translational interest. Safety of MSC therapy has been well demonstrated in early phase clinical trials but efficacy in randomized clinical trials needs to be demonstrated. Understanding MSC mechanisms of action to reduce gut injury and inflammation is necessary to improve current ongoing and future clinical trials. However, two major hurdles impede the direct translation of data derived from animal experiments to the clinical situation: (1) limitations of the currently available animal models of colitis that reflect human inflammatory bowel diseases (IBD). The etiology and progression of human IBD are multifactorial and hence a challenge to mimic in animal models; and (2) Species specific differences in the functionality of MSCs derived from mice versus humans. MSCs derived from mice and humans are not identical in their mechanisms of action in suppressing inflammation. Thus, preclinical animal studies with murine derived MSCs cannot be considered as an exact replica of human MSC based clinical trials. In the present review, we discuss the therapeutic properties of MSCs in preclinical and clinical studies of IBD. We also discuss the challenges and approaches of using appropriate animal models of colitis, not only to study putative MSC therapeutic efficacy and their mechanisms of action, but also the suitability of translating findings derived from such studies to the clinic.

Mesenchymal Stromal Cells Derived From Crohn's Patients Deploy Indoleamine 2,3-dioxygenase-mediated Immune Suppression, Independent of Autophagy.

Autologous bone marrow-derived mesenchymal stromal cells (MSCs) for adoptive cell therapy of luminal Crohns disease (CD) are being tested in clinical trials. However, CD is associated with dysregulation of autophagy and its effect on MSCs immunobiology is unknown. Here, we demonstrate no quantitative difference in phenotype, in vitro growth kinetics and molecular signatures to IFNγ between MSCs derived from CD and healthy individuals. CD MSCs were indistinguishable from those derived from healthy controls at inhibiting T-cell proliferation through an indoleamine 2,3-dioxygenase (IDO)-dependent mechanism. Upon IFNγ prelicensing, both MSC populations inhibit T-cell effector functions. Neither a single-nucleotide polymorphism (SNP) rs7820268 in the IDO gene, nor a widely reported CD predisposing SNP ATG16L1rs2241880 modulated the suppressive function of MSCs carrying these haplotypes. IFNγ stimulation or coculture with activated T cells upregulated the expression of autophagy genes and/or vacuoles on MSCs. Pharmacological blockade of autophagy pathway did not reverse the immunosuppressive properties and IFNγ responsiveness of MSCs confirming the absence of a functional link between these two cell biochemical properties. We conclude that autophagy, but not IDO and IFNγ responsiveness, is dispensable for MSCs immunosuppressive properties. MSCs from CD subjects are functionally analogous to those of healthy individuals.

Liver fibrosis occurs through dysregulation of MyD88‐dependent innate B‐cell activity

Chronic liver disease mediated by activation of hepatic stellate cells (HSCs) leads to liver fibrosis. Here, we postulated that the immune regulatory properties of HSCs might promote the profibrogenic activity of B cells. Fibrosis is completely attenuated in carbon tetrachloride–treated, B cell–deficient µMT mice, showing that B cells are required. The retinoic acid produced by HSCs augmented B‐cell survival, plasma cell marker CD138 expression, and immunoglobulin G production. These activities were reversed following addition of the retinoic acid inhibitor LE540. Transcriptional profiling of fibrotic liver B cells revealed increased expression of genes related to activation of nuclear factor κ light chain enhancer of activated B cells, proinflammatory cytokine production, and CD40 signaling, suggesting that these B cells are activated and may be acting as inflammatory cells. Biological validation experiments also revealed increased activation (CD44 and CD86 expression), constitutive immunoglobulin G production, and secretion of the proinflammatory cytokines tumor necrosis factor‐α, monocyte chemoattractant protein‐1, and macrophage inflammatory protein‐1α. Likewise, targeted deletion of B‐cell‐intrinsic myeloid differentiation primary response gene 88 signaling, an innate adaptor with involvement in retinoic acid signaling, resulted in reduced infiltration of migratory CD11c+ dendritic cells and Ly6C++ monocytes and, hence, reduced liver pathology. Conclusion: Liver fibrosis occurs through a mechanism of HSC‐mediated augmentation of innate B‐cell activity. These findings highlight B cells as important “first responders” of the intrahepatic immune environment. (Hepatology 2015;61:2067‐2079)