Host Treg Research Articles

Tregs in transplantation tolerance: role and therapeutic potential.

CD4+ Foxp3+ regulatory T cells (Tregs) are indispensable for preventing autoimmunity, and they play a role in cancer and transplantation settings by restraining immune responses. In this review, we describe evidence for the importance of Tregs in the induction versus maintenance of transplantation tolerance, discussing insights into mechanisms of Treg control of the alloimmune response. Further, we address the therapeutic potential of Tregs as a clinical intervention after transplantation, highlighting engineered CAR-Tregs as well as expansion of donor and host Tregs.

HBV immune tolerance of HBs-transgenic mice observed through parabiosis with WT mice.

It was extensively recognized that central tolerance to HBV exists in HBs-transgenic (Tg) mice, however, the immune response to HBV vaccine may be inspired in adult HBs-Tg mice after boosting with potent adjuvants, leaving a mystery to explore its immune tolerance. Here, WT-HBs-Tg parabiotic mice model was generated by conjoining WT (donor) and HBs-Tg (host) mouse via parabiotic surgery, in order to see how immunocompetent WT mice naturally respond to HBV, and how tolerant HBs-Tg mice influence the anti-HBV immunity from WT mice. It was found that WT CD8+ T cells markedly accumulated into the liver of HBs-Tg parabionts, and importantly, almost all HBsAg-specific CD8+ T cells derived from WT but not HBs-Tg mice, making a clear separation of a normal immune response from WT donor and a tolerant response by recipient host. Further, in the absence of host but not donor spleen, HBsAg-specific CD8+ T cells disappeared, indicating that host spleen was the indispensable site for donor HBsAg-specific CD8+ T cell priming though its mechanisms need further study. We found that donor CD4+ T helper cells were necessary for donor HBsAg-specific CD8+ T cell response by CD4-deficiency in WT or in HBs-Tg mice, indicating that an immune response was elicited between CD4+ T helper cells and CD8+ cytotoxic T cells of donor in the host but not donor spleen. It was noted that compared to donor CD4+ T cells, host CD4+ T cells were characterized with more tolerant features by harboring more CD25+Foxp3+ Tregs with higher expression of PD-1 and TIGIT in the spleen of HBs-Tg parabionts, which exhibited suppressive function on CD8+ T cells directly. Moreover, the Th1/Treg ratio was enhanced after parabiosis, suggesting that donor T helper cells may overcome the negative regulation of host Tregs in host spleen. In conclusion, both incompetent anti-HBV CD8+ T cells and insufficient help from CD4+ T cells are the major mechanisms underlying immune tolerance in HBs-Tg mice which helps explain HBV persistence.

MTORC1 Inhibition Protects Human Regulatory T Cells From Granzyme-B-Induced Apoptosis.

Regulatory T cells (Tregs) have shown great promise as a means of cellular therapy in a multitude of allo- and auto-immune diseases—due in part to their immunosuppressive potency. Nevertheless, the clinical efficacy of human Tregs in patients has been limited by their poor in vivo homeostasis. To avert apoptosis, Tregs require stable antigenic (CD3ζ/T-cell-receptor-mediated), co-stimulatory (CD28-driven), and cytokine (IL-2-dependent) signaling. Notably, this sequence of signals supports an activated Treg phenotype that includes a high expression of granzymes, particularly granzyme B (GrB). Previously, we have shown that aside from the functional effects of GrB in lysing target cells to modulate allo-immunity, GrB can leak out of the intracellular lysosomal granules of host Tregs, initiating pro-apoptotic pathways. Here, we assessed the role of inhibiting mechanistic target of rapamycin complex 1 (mTORC1), a recently favored drug target in the transplant field, in regulating human Treg apoptosis via GrB. Using ex vivo models of human Treg culture and a humanized mouse model of human skin allotransplantation, we found that by inhibiting mTORC1 using rapamycin, intracytoplasmic expression and functionality of GrB diminished in host Tregs; lowering human Treg apoptosis by in part decreasing the phosphorylation of S6K and c-Jun. These findings support the already clinically validated effects of mTORC1 inhibition in patients, most notably their stabilization of Treg bioactivity and in vivo homeostasis.

Experimental colonization with Blastocystis ST4 is associated with protective immune responses and modulation of gut microbiome in a DSS-induced colitis mouse model

BackgroundBlastocystis is a common gut protistan parasite in humans and animals worldwide, but its interrelationship with the host gut microbiota and mucosal immune responses remains poorly understood. Different murine models of Blastocystis colonization were used to examine the effect of a common Blastocystis subtype (ST4) on host gut microbial community and adaptive immune system.ResultsBlastocystis ST4-colonized normal healthy mice and Rag1−/− mice asymptomatically and was able to alter the microbial community composition, mainly leading to increases in the proportion of Clostridia vadinBB60 group and Lachnospiraceae NK4A136 group, respectively. Blastocystis ST4 colonization promoted T helper 2 (Th2) response defined by interleukin (IL)-5 and IL-13 cytokine production, and T regulatory (Treg) induction from colonic lamina propria in normal healthy mice. Additionally, we observed that Blastocystis ST4 colonization can maintain the stability of bacterial community composition and induce Th2 and Treg immune responses to promote faster recovery from experimentally induced colitis. Furthermore, fecal microbiota transplantation of Blastocystis ST4-altered gut microbiome to colitis mice reduced the severity of colitis, which was associated with increased production of short-chain fat acids (SCFAs) and anti-inflammatory cytokine IL-10.ConclusionsThe data confirm our hypothesis that Blastocystis ST4 is a beneficial commensal, and the beneficial effects of Blastocystis ST4 colonization is mediated through modulating of the host gut bacterial composition, SCFAs production, and Th2 and Treg responses in different murine colonization models.

357 - Recipient Pre-HSCT Treatment with TNFRSF25 and CD25 Agonists Ameliorates Gvhd in a Host Treg Dependent Manner Following Murine MHC-Mismatched Transplantation

357 - Recipient Pre-HSCT Treatment with TNFRSF25 and CD25 Agonists Ameliorates Gvhd in a Host Treg Dependent Manner Following Murine MHC-Mismatched Transplantation

AML-260: Influence of Chimerism in T-Regulatory Cells on Relapse Rate in Acute Leukemia Patients after Allo-HSCT

<h3>Introduction:</h3> T-regulatory cells (Treg) prevent the development of graft-versus-host disease (GVHD) after allo-HSCT (Beres & Drobyski, 2013). At the same time, this cell population can limit the anti-tumor response (graft-versus-leukemia, GVL), which can cause relapses. We report data of chimerism in a Treg population and the frequency of relapses in acute leukemia patients (ALL and AML) after allo-HSCT. <h3>Objective:</h3> To evaluate a possible relationship between mixed chimerism in Treg cells and the rate of relapses in acute leukemia patients after allo-HSCT. <h3>Methods:</h3> The study included 31 patients after allo-HSCT (ALL n=6, AML n=25). The median age was 38 years (19–66). Peripheral blood samples for analysis were taken on day +30 after transplantation. Immunomagnetic separation (Miltenyi Biotec, Germany) was used to isolate cell population with CD4+CD25+ phenotype, which is predominantly associated with Treg cells. Extraction of DNA was performed from the obtained cells. Chimerism in DNA samples was determined using the STR-PCR method. The percentage of donor chimerism was calculated using standard procedures (Nollet <i>et al.,</i> 2001). Statistical analysis of the data was carried out using SPSS ver 23 (IBM, Chicago, IL, USA). Fisher's exact test was used to analyze the 2 × 2 contingency tables. <h3>Results:</h3> In the group of patients with less than 11% of cells with host genotype (more than 89% of cells of donor origin), the relapse rate was significantly higher: 52.6% (10 of 19) than in the other group of patients with 11% and more cells with host genotype: 8.3% (1 of 12; p=0.02). At the same time, donor chimerism in the unselected bone marrow did not differ significantly from the groups (p = 0.36) and reached 100% (75–100%) and 97.5% (90–100%). The study groups were balanced for all other factors that could affect the relapse rate: disease status, graft source, and GVHD. <h3>Conclusions:</h3> According to our data, we proposed that host Treg cells are not capable to suppress GVL, which explains significant differences in relapse frequencies in patients with acute leukemia after allo-HSCT. Predominance of host Treg cells may be a favorable prognostic sign, but this hypothesis needs to be confirmed on the larger cohort of patients.

The Role and Function of Regulatory T Cells in Toxoplasma gondii-Induced Adverse Pregnancy Outcomes.

Infection with Toxoplasma gondii (T. gondii) during the pregnant period and its potentially miserable outcomes for the fetus, newborn, and even adult offspring continuously occur worldwide. People acquire infection through the consumption of infected and undercooked meat or contaminated food or water. T. gondii infection in pregnant women primarily during the gestation causes microcephaly, mental and psychomotor retardation, or death. Abnormal pregnancy outcomes are mainly associated with regulatory T cell (Treg) dysfunction. Tregs, a special subpopulation of T cells, function as a vital regulator in maintaining immune homeostasis. Tregs exert a critical effect on forming and maintaining maternal-fetal tolerance and promoting fetal development during the pregnancy period. Forkhead box P3 (Foxp3), a significant functional factor of Tregs, determines the status of Tregs. In this review, we summarize the effects of T. gondii infection on host Tregs and its critical transcriptional factor, Foxp3.

Human cord blood-derived regulatory T-cell therapy modulates the central and peripheral immune response after traumatic brain injury.

Traumatic brain injury (TBI) causes a profound inflammatory response within the central nervous system and peripheral immune system, which contributes to secondary brain injury and further morbidity and mortality. Preclinical investigations have demonstrated that treatments that downregulate microglia activation and polarize them toward a reparative/anti‐inflammatory phenotype have improved outcomes in preclinical models. However, no therapy to date has translated into proven benefits in human patients. Regulatory T cells (Treg) have been shown to downregulate pathologic immune responses of the innate and adaptive immune system across a variety of pathologies. Furthermore, cellular therapy has been shown to augment host Treg responses in preclinical models; yet, studies investigating the use of Treg as a therapeutic for TBI are lacking. In a rodent TBI model, we demonstrate that human umbilical cord blood Treg modulate the central and peripheral immune response after injury in vitro and in vivo.

IL-6 Receptor Blockade but Not IL-2 Treatment Contributes to Long Term NHP Cardiac Allograft Survival in Transient Mixed Chimeras

Long term tolerance of cardiac allografts has been achieved in non-human primates (NHPs) using a mixed chimerism protocol and by co-transplanting a kidney from the same heart and bone marrow donor. In stark contrast, heart alone recipients rejected in 150-180 days. T cell subset analyses showed a greater expansion of regulatory T cells (Treg) in heart/kidney recipients compared to heart alone controls. To achieve tolerance of isolated heart allografts, we added IL-2 and/or anti-IL6R therapy to enhance host Treg activity. Twelve cynomolgus heart alone transplant recipients underwent donor bone marrow transplantation with mixed chimerism conditioning that included non-myeloablative total body irradiation, thymic irradiation, horse anti-thymocyte globulin, anti-CD154 mAb, and cyclosporine until POD 28. Five animals in Group A received low dose IL-2 (1MIU/m2 SC from POD -6 to 5) and IL-6 receptor blockade with tocilizumab (10 mg/kg IV on POD 0, 7, 14, 21, 28, 56, 84, and 112). Seven recipients in Group B, received tocilizumab alone (10 mg/kg IV on POD 0, 7, 14, 21, 28, 56, and 84). In Group A, 3/5 animals rejected their allografts at days 127, 198, and 254 post-transplant with evidence of acute cellular rejection (ACR), antibody mediated rejection (AMR), and donor specific antibodies (DSA). Two animals died on days 7 and 11 post-transplant from sepsis and pancytopenia, respectively. In Group B, 3/7 recipients are ongoing (POD 111 and 62) with one animal over 509 days with no evidence of ACR. 1/7 rejected at day 169 post-transplant with ACR 3R, pAMR3, and DSA and 3/7 animals died on days 5, 9, and 65 post-transplant from a technical failure, sedation complication, and pancytopenia, respectively. Recipients in Group A exhibited an average 15-fold expansion in the percent CD25+FoxP3+ of CD3+CD4+ cells in the periphery compared to an average 3-fold expansion in Group B recipients. Adding IL-6 receptor blockade to a mixed chimerism protocol has achieved tolerance of an isolated heart allograft for the first time. This protocol or a version thereof has the potential to be trialed in humans. The surprising deleterious effects of low dose IL-2 may be related to its ability to activate alloreactive T cells despite augmenting Tregs.

Immunomodulation Followed by Antigen-Specific Treg Infusion Controls Islet Autoimmunity.

Optimal immune-based therapies for type 1 diabetes (T1D) should restore self-tolerance without inducing chronic immunosuppression. CD4+Foxp3+ regulatory T cells (Tregs) are a key cell population capable of facilitating durable immune tolerance. However, clinical trials with expanded Tregs in T1D and solid-organ transplant recipients are limited by poor Treg engraftment without host manipulation. We showed that Treg engraftment and therapeutic benefit in nonautoimmune models required ablative host conditioning. Here, we evaluated Treg engraftment and therapeutic efficacy in the nonobese diabetic (NOD) mouse model of autoimmune diabetes using nonablative, combinatorial regimens involving the anti-CD3 (αCD3), cyclophosphamide (CyP), and IAC (IL-2/JES6-1) antibody complex. We demonstrate that αCD3 alone induced substantial T-cell depletion, impacting both conventional T cells (Tconv) and Tregs, subsequently followed by more rapid rebound of Tregs Despite robust depletion of host Tconv and host Tregs, donor Tregs failed to engraft even with interleukin-2 (IL-2) support. A single dose of CyP after αCD3 depleted rebounding host Tregs and resulted in a 43-fold increase in donor Treg engraftment, yet polyclonal donor Tregs failed to reverse diabetes. However, infusion of autoantigen-specific Tregs after αCD3 alone resulted in robust Treg engraftment within the islets and induced remission in all mice. This novel combinatorial therapy promotes engraftment of autoantigen-specific donor Tregs and controls islet autoimmunity without long-term immunosuppression.

Trichinella spiralis Excretory–Secretory Products Stimulate Host Regulatory T Cell Differentiation through Activating Dendritic Cells

Trichinella spiralis maintains chronic infections within its host, involving a variety of immunomodulatory properties, the mechanisms of which have not been completely elucidated. In this study, we found that T. spiralis infection induced strong regulatory T cell responses through parasite excretory–secretory (ES) products, characterized by increase of CD4+CD25+Foxp3+ and CD4+CD25−Foxp3+ Treg cells accompanied by high levels of IL-10 and TGF-β. T. spiralis adult worm excretory–secretory products (AES) and muscle larvae excretory–secretory products (MES) were both able to activate BMDCs in vitro to facilitate their maturation and to create regulatory cytokines IL-10 and TGF-β. The T. spiralis AES- and MES-pulsed dendritic cells (DCs) possessed abilities not only to present antigens to sensitized CD4+ T cell to stimulate their proliferation but also to induce naive CD4+ T cells to differentiate to Treg cells secreting IL-10 and TGF-β. The passive transfer of T. spiralis AES- and MES-pulsed bone marrow-derived dendritic cells (BMDCs) conferred the naive mice to acquire the differentiation of Treg cells. T. spiralis AES possesses a better ability to induce Treg cells than did MES, although the latter has the ability to induce CD4+CD25−Foxp3+ Treg cells. The results obtained in this study suggested that T. spiralis ES products stimulate the differentiation of host Treg cells possibly through activating dendritic cells to create a regulatory environment that benefits the survival of the parasite in the host.

Association of Regulatory T-Cell Expansion With Progression of Amyotrophic Lateral Sclerosis

Neuroinflammation appears to be a key modulator of disease progression in amyotrophic lateral sclerosis (ALS) and thereby a promising therapeutic target. The CD4+Foxp3+ regulatory T-cells (Tregs) infiltrating into the central nervous system suppress neuroinflammation and promote the activation of neuroprotective microglia in mouse models of ALS. To our knowledge, the therapeutic association of host Treg expansion with ALS progression has not been studied in vivo. To assess the role of Tregs in regulating the pathophysiology of ALS in humans and the therapeutic outcome of increasing Treg activity in a mouse model of the disease. This prospective multicenter human and animal study was performed in hospitals, outpatient clinics, and research institutes. Clinical and function assessment, as well as immunological studies, were undertaken in 33 patients with sporadic ALS, and results were compared with 38 healthy control participants who were consecutively recruited from the multidisciplinary ALS clinic at Westmead Hospital between February 1, 2013, and December 31, 2014. All data analysis on patients with ALS was undertaken between January 2015 and December 2016. Subsequently, we implemented a novel approach to amplify the endogenous Treg population using peripheral injections of interleukin 2/interleukin 2 monoclonal antibody complexes (IL-2c) in transgenic mice that expressed mutant superoxide dismutase 1 (SOD1), a gene associated with motor neuron degeneration. In patients with ALS, Treg levels were determined and then correlated with disease progression. Circulating T-cell populations, motor neuron size, glial cell activation, and T-cell and microglial gene expression in spinal cords were determined in SOD1G93A mice, as well as the association of Treg amplification with disease onset and survival time in mice. The cohort of patients with ALS included 24 male patients and 9 female patients (mean [SD] age at assessment, 58.9 [10.9] years). There was an inverse correlation between total Treg levels (including the effector CD45RO+ subset) and rate of disease progression (R = -0.40, P = .002). Expansion of the effector Treg population in the SOD1G93A mice was associated with a significant slowing of disease progression, which was accompanied by an increase in survival time (IL-2c-treated mice: mean [SD], 160.6 [10.8] days; control mice: mean [SD], 144.9 [10.6] days; P = .003). Importantly, Treg expansion was associated with preserved motor neuron soma size and marked suppression of astrocytic and microglial immunoreactivity in the spinal cords of SOD1G93A mice, as well as elevated neurotrophic factor gene expression in spinal cord and peripheral nerves. These findings establish a neuroprotective effect of Tregs, possibly mediated by suppression of toxic neuroinflammation in the central nervous system. Strategies aimed at enhancing the Treg population and neuroprotective activity from the periphery may prove therapeutically useful for patients with ALS.

Utility of IL-2 Complexes in Promoting the Survival of Murine Orthotopic Forelimb Vascularized Composite Allografts.

Vascularized composite allografts (VCA) are novel, life-enhancing forms of transplantation (Tx). However, host immune responses to the various VCA components, especially those involving skin, are complex and make selection of appropriate therapy challenging. Although the interplay between Foxp3+ T regulatory (Treg) cells and CD4 and CD8 effector T cells is of central importance in determining the acceptance or rejection of solid organ allografts, there is little information available concerning the contribution of Treg cells to VCA survival. In addition, the effects of therapeutic expansion in vivo of host Treg cell populations on VCA survival are unknown. We established a fully major histocompatibility complex-disparate (BALB/c- > C57BL/6) murine orthotopic forelimb Tx model to explore the benefits of pre- and post-Tx IL-2/anti-IL-2 monoclonal antibody complex (IL-2C) administration to expand the host Treg cell population and thereby attempt to promote Treg cell-dependent VCA survival. Both strategies expanded the Treg cell population in vivo and prolonged VCA survival (P < 0.001), but IL-2C administration pre-Tx led to significantly longer survival compared with IL-2C administration post-Tx (P < 0.01). In addition, compared with post-Tx therapy, pre-Tx therapy resulted in an increased ratio of Treg cells to CD8+ T cells (P < 0.001), reduced proliferation of CD4 and CD8 effector T cells, and reduced production of IFN-γ. Optimal effects were seen when combined with rapamycin therapy, whereas the combination of IL-2C therapy plus calcineurin inhibitor was counterproductive. Our studies involving different IL-2C-mediated Treg cell expansion strategies demonstrate that pre-Tx IL-2C therapy may be a useful component for developing strategies to promote VCA survival.

A structurally distinct TGF-\u03b2 mimic from an intestinal helminth parasite potently induces regulatory T cells

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-β signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β, including binding to mammalian TGF-β receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-β or other members of the TGF-β family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.

Abstract 962: Novel targeting of tumor-infiltrating TNFR2 Tregs: removing suppression with microenvironment-specific antibodies

Abstract Background: A recognized and potent subtype of regulatory T cells (Tregs) expresses tumor necrosis factor receptor 2 (TNFR2), a member of the tumor necrosis factor (TNF) superfamily. TNFR2 expressing Tregs are overabundant in human and murine tumors and are the most potent suppressors of host immune responses. In addition, recent data in diverse human tumors show that TNFR2-expressing host T cells are the dominant Tregs in the tumor microenvironment, expressed far in excess of OX40 and GITR. Unlike many tumor markers, TNFR2 has limited expression in the immune system and almost no expression on parenchymal cells. This makes TNFR2 Tregs an attractive target for anticancer therapy. Methods: We produced monoclonal antibodies to human TNFR2 with the goal of inactivating the potent host Tregs that express TNFR2, competing with the agonism of TNF and inactivating infiltating Tregs of human tumors. Tregs from fresh ovarian ascites fluid were compared to Tregs from normal human blood donors for antibody potency. Results: Using a 48-hr cell-based Treg assay, we showed that TNFR2 antagonistic antibodies can inhibit Treg proliferation, suppress soluble TNFR2 secretion on normal cells and allow T effector expansion. The antagonism is dominant, succeeding even in the presence of TNF, which is a TNFR2 agonist. Furthermore, the structural biology of dominant TNFR2 receptor antagonism uncovers a unique conformation that equally inhibits downstream NFkB signaling. We also showed that dominant TNFR2 antibodies in a dose dependent fashion kill Tregs in ovarian cancer infiltrates more strongly than Tregs from healthy donors. Treg killing in the cancer microenvironment was dependent on cell proliferation and turnover the Tregs. Conclusion: In sum, blocking TNFR2 signaling on the most potent Tregs of the tumor microenvironment with a dominant antagonist antibody selectively heightens death of the immunosuppressive Tregs in the tumor microenvironment, at least in ovarian cancer. With the expanding knowledge that TNFR2 Tregs are the most potent host cells of the immune response, their dominance in diverse human tumor infiltrates and the natural low expression of this marker on normal lymphoid cells makes targeting of TNFR2 in the tumor microenvironment attractive. Citation Format: Heather Torrey, John Butterworth, Toshiyuki Mera, Yoshiaki Okubo, Limei Wang, Danielle Baum, Audrey Defusco, Sara Plager, Sarah Warden, Daniel Huang, Eva Vanamee, Rosemary Foster, Denise L. Faustman. Novel targeting of tumor-infiltrating TNFR2 Tregs: removing suppression with microenvironment-specific antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 962. doi:10.1158/1538-7445.AM2017-962

Effect of Ex Vivo-Expanded Recipient Regulatory T Cells on Hematopoietic Chimerism and Kidney Allograft Tolerance Across MHC Barriers in Cynomolgus Macaques.

Infusion of recipient regulatory T (Treg) cells promotes durable mixed hematopoietic chimerism and allograft tolerance in mice receiving allogeneic bone marrow transplant (BMT) with minimal conditioning. We applied this strategy in a Cynomolgus macaque model. CD4 CD25 Treg cells that were polyclonally expanded in culture were highly suppressive in vitro and maintained high expression of FoxP3. Eight monkeys underwent nonmyeloablative conditioning and major histocompatibility complex mismatched BMT with or without Treg cell infusion. Renal transplantation (from the same BMT donor) was performed 4 months post-BMT without immunosuppression to assess for robust donor-specific tolerance. Transient mixed chimerism, without significant T cell chimerism, was achieved in the animals that received BMT without Treg cells (N = 3). In contrast, 2 of 5 recipients of Treg cell BMT that were evaluable displayed chimerism in all lineages, including T cells, for up to 335 days post-BMT. Importantly, in the animal that survived long-term, greater than 90% of donor T cells were CD45RA CD31, suggesting they were new thymic emigrants. In this animal, the delayed (to 4 months) donor kidney graft was accepted more than 294 days without immunosuppression, whereas non-Treg cell BMT recipients rejected delayed donor kidneys within 3 to 4 weeks. Early CMV reactivation and treatment was associated with early failure of chimerism, regardless of Treg cell administration. Our studies provide proof-of-principle that, in the absence of early CMV reactivation (and BM-toxic antiviral therapy), cotransplantation of host Treg cell can promote prolonged and high levels of multilineage allogeneic chimerism and robust tolerance to the donor.

Microbiota and Lipotoxicity.

Obesity and metabolic syndrome is a multisystemic disorder, that is characterized by excess caloric intake and spillover lipotoxicity caused by ectopic lipid accumulation in non-adipose tissues. Low grade chronic inflammation and insulin resistance are the hallmarks of the disorder, which further aggravate the condition. Gut microbiota constitutes an indispensible part of human superorganism's energy harvesting apparatus. The dynamic composition of microbiota changes with age, life style and host metabolic background. The wealth of genetic repertoire provided by these microorganism enables to extend host's substrate processing and harvesting capability. Some of these compounds including short chain fatty acids and indole act as signalling molecules on mammalian cells and modulate their behaviour. Nonetheless, this symbiotic style of interaction is restrained by immune system. The role of chronic low grade inflammation in metabolic syndrome is well established. Treg cells are the key players that sense and reshape the composition of microbiota. In this regard, any disturbance in Treg functionality may aggravate the inflammation and shift the symbiotic balance towards dysbiosis, which is characterized by autoimmunity and insulin resistance. Thus, immune system is responsible for the modulation of host and microbiota metabolisms and Treg cells act as a bridge in between.

MTOR Signaling Regulates Protective Activity of Transferred CD4+Foxp3+ T Cells in Repair of Acute Kidney Injury.

CD4+Foxp3+ regulatory T cells (Tregs) are required for normal immune homeostasis. Recent studies suggested that Treg transfer facilitates recovery from acute kidney injury (AKI), but the molecular events that maintain Treg function after adoptive transfer remain unclear. This study aimed to investigate the regulation of mammalian target of rapamycin (mTOR) signaling in the Treg-mediated therapeutic effect on ischemic AKI. We noted significant Treg expansion in C57BL/6 mouse kidney, with enhanced immunosuppressive capacity after renal ischemia/reperfusion. mTOR inhibition significantly increased the frequency of Tregs in cultured CD4+ T cells, with enhanced production of anti-inflammatory cytokines, which, conversely, was reduced by mTOR activation. Rapamycin, an inhibitor of mTOR, was transiently administered to C57BL/6 mice before ischemia/reperfusion surgery. No beneficial effect of rapamycin treatment was seen in the early recovery of AKI as a result of its inhibitory effect on tubular regeneration. However, rapamycin markedly enhanced the expansion of kidney Tregs, with increased mRNA expression of anti-inflammatory cytokines. Adoptive transfer of rapamycin-treated Tregs markedly suppressed conventional T cells, responder myeloid cells, and reactive myofibroblasts; however, it promoted host Tregs and alternative macrophages, leading to better renal function and less kidney fibrosis. Taken together, Treg transfer with mTOR inhibition markedly improves outcomes of ischemic AKI. These findings reveal an important role for mTOR signaling in maintaining Treg activity after adoptive transfer and highlight the therapeutic potential of targeting Tregs in acute and chronic kidney disease.

IL-33 mediated expansion of host ST2+ Treg prior to allogeneic hematopoietic cell transplantation decreases macrophage and effector T cell activation reducing acute GVHD

Abstract Graft-versus-host disease (GVHD) is the leading complication and cause of mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Recently, we reported that IL-33 released from tissue damaged during allo-HCT conditioning mediates pro-inflammatory responses by Teffector cells resulting in greater acute GVHD lethality and clinical symptoms in mice. However, IL-33 is a pleiotropic cytokine that mediates both pro-inflammatory and anti-inflammatory responses. We hypothesized that the beneficial effects mediated by IL-33 could be harnessed if IL-33 was present prior to inflammatory mediators released during conditioning. Here, we demonstrate that exogenous IL-33 given peri allo-HCT (days −10 to 4) to recipients of MHC-disparate allo-HCT asserts its immunoregulatory properties reduced GVHD lethality and clinical symptoms compared to non-treated controls. IL-33 treatment peri allo-HCT resulted in the expansion of recipient Treg and suppressive myeloid cells that persisted post irradiation. Host Treg depletion in C57Bl/6 FoxP3-DTR mice by diphtheria toxin given concurrently with peri allo-HCT IL-33 treatment accelerated GVHD symptoms and lethality compared to peri allo-HCT IL-33 treatment alone, demonstrating that host Treg were necessary for GVHD protection. Transfer of Treg deficient in ST2, the IL-33 receptor, afforded less GVHD protection than wild-type Treg. ST2+ Treg controlled macrophage activation and prevented accumulation of Teffector cells in GVHD target tissue. Thus, we demonstrate that peri allo-HCT IL-33 expanded and activated host Treg have potential as a therapeutic modality to prevent and treat GVHD.

Toll-like receptor agonist therapy augments the antitumor activity of adoptively transferred CD8+ T cells without host preconditioning

Abstract Lymphodepletion enhances adoptive T cell transfer (ACT) therapy by activating the innate immune system via microbes released from the radiation-injured gut. Microbial LPS is a key mediator of lymphodepletion enhancement, but our ability to use these TLR agonists to bolster the potency of T cell-based cancer therapies remains elusive. Herein, we used LPS as a tool to address how and when to use TLR agonists to improve cancer immunotherapy. We utilized the pmel-1 melanoma mouse model. B16F10-bearing mice were lymphodepleted with 5Gy total body irradiation (TBI) and given a tripartite ACT therapy (consisting of transferred pmel-1 CD8+ T cells, vaccination with fowlpox encoding gp100, and IL-2) along with TLR4 agonist LPS. The timing of LPS administration and the requirement of individual components of the tripartite therapy were evaluated. We discovered that while exogenous administration of LPS was able to enhance CD8+ T cells’ tumor destruction, LPS treatment alone did not replace individual components of the tripartite regimen. Interestingly, administering LPS one day before ACT compromised tumor regression. Conversely, administering LPS after ACT potentiated the antitumor effectiveness of the regimen, thereby supporting the expansion of transferred CD8+ T cells over host Treg cells. Non-toxic TLR agonists MPL and CpG also improved ACT therapy. Finally, TBI preconditioning was no longer needed to regress tumors in mice depleted of host CD4+ T cells, given a tripartite ACT regimen and then treated with a TLR agonist. Collectively, our results identify how and when to administer TLR agonists to augment ACT in the absence of host preconditioning. These findings have implications for the design of next generation T cell therapies.