Splenic Treg Research Articles

Tolerogenic bone marrow-derived dendritic cells induce neuroprotective regulatory T cells in a model of Parkinson\u2019s disease

BackgroundAdministration of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases regulatory T cell (Treg) number and function with control of neuroinflammation and neuronal protection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease (PD). Recently, we demonstrated in an early phase 1 clinical trial that GM-CSF also improves motor skills in PD patients. However, the mechanisms of Treg induction and its effects on neuroprotective responses remain unknown. As GM-CSF induces tolerogenic dendritic cells (DCs) that in turn convert conventional T cells to Tregs, the pathways for DC induction of Tregs were assessed.MethodsFollowing differentiation, bone marrow-derived dendritic cells (BMDCs) were cultured in media or GM-CSF with or without post-culture stimulation with nitrated α-synuclein (N-α-Syn). Expression of cell surface co-stimulatory molecules and proinflammatory cytokines, and induction of Tregs were evaluated. The neuroprotective capacity of tolerogenic BMDCs was assessed by adoptive transfer to MPTP-intoxicated mice. The extent of neuroinflammation and numbers of surviving dopaminergic neurons were assessed in relation to Treg numbers.ResultsCo-culture of differentiated BMDCs with conventional T cells led to Treg induction. Stimulation of BMDCs with N-α-Syn increased expression of co-stimulatory molecules and proinflammatory cytokines, with modest increases in Treg numbers. In contrast, continued culture of BMDCs with GM-CSF modestly altered expression of co-stimulatory molecules and proinflammatory cytokines and chemokines, but decreased Treg induction. Continued culture in GM-CSF and combined stimulation with N-α-Syn reduced Treg induction to the lowest levels. Adoptive transfer of tolerogenic BMDCs to MPTP-intoxicated mice increased splenic Tregs, attenuated neuroinflammatory responses, and protected nigrostriatal dopaminergic neurons.ConclusionsGM-CSF acts broadly to differentiate DCs and affect immune transformation from effector to regulatory immune responses. DCs skew such immune responses by increasing Treg numbers and activities that serve to attenuate proinflammatory responses and augment neuroprotection.

Prenatal exposure to cadmium alters the transciptome of regulatory T cells in C57BL/6 mice and may contribute to immunological health of the offspring

Abstract Cadmium (Cd) is a carcinogen present at superfund sites, in battery manufacturing facilities, in and around zinc smelters, and in cigarettes (the most common exposure pathway). In humans, prenatal Cd exposure (preCd) is associated with differential DNA methylation patterns in blood leukocytes of mother-baby pairs. C57BL/6 mice chronically exposed to low doses of Cd in utero develop abnormal immune phenotypes including reduced regulatory T cell (Treg) numbers in the spleen, but not thymus, of adult animals. These data suggest that the probable immune consequences of preCd exposure are cause for alarm. To further define the mechanisms behind these phenotypic differences, we isolated Treg and CD4+ T conventional (Tconv) cells from the spleens of preCd offspring at 8 woa. Target cells were enriched using magnetic isolation prior to purification by flow cytometry. RNA-seq and Ingenuity Pathway analyses were conducted to define the Treg and Tconv transcriptomes and identify mechanisms dysregulated with preCd exposure. Treg cells exhibited more changes in gene expression than Tconv cells. In the splenic Tregs, there were 289 significantly changed genes (166 upregulated and 135 downregulated) in females and 135 (82 upregulated and 53 downregulated) in males. Of note, only 23 upregulated genes and 3 downregulated genes were shared between females and males. However, common pathways, including CREB1 signaling, were disrupted in both sexes. As CREB signaling plays an important role in TGFβ-mediated generation and maintenance of FoxP3 Tregs, inhibition of this pathway may promote Treg instability and contribute to the decrease in Treg numbers observed in the periphery of preCd-exposed mice.

Prostaglandin I2 promotes Treg function in vitro

Abstract Our laboratory has shown in recent years that prostaglandin I2 (PGI2) promotes immune tolerance through inhibition of T cell and dendritic cell function. Specifically, we have shown that PGI2 is able to restrain Th1 and Th2 immune responses, suggesting that it has immunosuppressive properties. However, PGI2’s impact on the functionality of T regulatory (Treg) cells, a T cell subtype important for the suppression/resolution of immune responses which express the canonical transcription factor forkhead box p3 (Foxp3), is unknown. Thus, we hypothesized that PGI2 promotes the function of Tregs. To test this hypothesis, we utilized mice deficient in IP (IP KO), the receptor for PGI2.. We found that splenic Tregs from IP KO mice have a significantly lower mean fluorescence intensity (MFI) of Foxp3 compared to wild-type (WT) Tregs. Furthermore, the percent suppression of CFSE-stained WT effector T cells by IP KO Tregs was significantly decreased compared to the percent suppression of CFSE-stained WT effector T cells by WT Tregs. Moreover, the percentage of induced Tregs (iTregs) generated from naïve T cells isolated from IP KO mice was significantly reduced compared to WT mice. As a whole, our preliminary data demonstrate the ability of PGI2 to promote Treg function, and thus suggest that PGI2 may serve as a novel treatment strategy for T cell mediated diseases.

Importance of B cells to development of regulatory T cells and prolongation of tissue allograft survival in recipients receiving autologous bone marrow transplantation.

We previously showed that congenic bone marrow transplantation (BMTx) post myeloablation augmented tissue allograft survival in association with increased regulatory T (Treg) cells of both host and bone marrow donor origin. Regulatory B (Breg) cells can also modulate T-cell immunity and B cells may be implicated in the development of Treg cells. Accordingly, we explored the effect of B-cell depletion invivo on augmented graft survival post BMTx. C57BL/6 mice received BALB/c skin allografts followed 7days later by myeloablation using cyclophosphamide and busulphan. Mice then received T-cell-depleted bone marrow from CD45.1 congenic donors, and ongoing immunosuppression with rapamycin (to day 28 after BMTx). Control mice received cyclophosphamide and busulphan followed by rapamycin, but not congenic bone marrow. At different times post BMTx, mice received B-cell-depleting antibody treatment, and the effect on both skin graft survival, and induction of Treg cells was assessed. BMTx resulted in significantly prolonged skin graft survival versus control mice, in association with attenuated donor-specific alloreactivity relative to controls, increased splenic Treg cells and significantly diminished anti-donor IgG. In mice receiving infusion of B-depleting antibodies for 12days from day 15 post BMTx, both graft survival and Treg cell activity were diminished, particularly for functional Treg cells of donor origin. Adoptive transfer of Breg cells from mice harvested at 15days post BMTx prolonged survival in naive transplanted mice and increased Treg cell levels. Thus, autologous BMTx augmentation of graft survival is dependent in part upon a population of Breg cells that can modulate the function of donor-derived Treg cells.

The effects of miR-10a on the immune function of splenic CD4 + CD25 + Treg cells in septic mice

Objective To investigate the role of miR-10a in CD4+CD25+Treg-mediated immunosuppression during sepsis and its potential role in immunotherapy for sepsis. Methods Sepsis mouse model was established by cecal ligation and puncture(CLP). Balb/c mice of clean grade were sacrificed 1, 3, 5, and 7 days after operation. Blood as well as spleen samples were harvested at given intervals. The splenic CD4+CD25+Treg cells and CD4+T cells were isolated by MACS microbeads. Cells were cultured, and phenotypes were analyzed by flow cytometry. The miR-10a expressed in Treg cells were detected by Real-time PCR. After administration of LV-mmu-miR-10a-5p-inhibition, the immunosuppressive function have been detected. Statistical analyses were performed using one-way analysis of variance (SPSS19.0, Chicago, USA) test followed by Dunnett-t test to compare among three or more groups or by Student’s t-test to compare between two groups. Results The percentages of splenic Tregs (CD4+CD25+/CD4+T) was (7.34±1.2)% in normal group, and the increase in percentage of Tregs in spleen has been observed in septic mice (P<0.05). The mean fluorescence intensity (MFI) of Foxp3+Treg was increased in septic mice compared with sham group (P<0.05). The expression of miR-10a was significantly elevated on CLP 1-7 day (P<0.05). After down-regulation of miR-10a in septic mice, the percentages of Tregs (CD4+CD25+/CD4+T) was significantly increased in septic mice (P<0.05), the MFI of Foxp3+Treg was increased in septic mice compared with control group (P<0.05). The CD4+T cell proliferative activity in CLP-induced mice was significantly suppressed on CLP 3 day compared with sham group (P<0.05).After down-regulation of miR-10a in septic mice, the CD4+T cell proliferative activity was significantly suppressed compared with control group (P<0.05). Conclusions Treg plays a critical role in immunosuppression in septic mice. Inhibition of miR-10a in vivo could enhence immunesuppression of CD4+CD25+Treg. Therefore miR-10a may participate in the regulation of CD4+CD25+Treg immunosuppression in sepsis and become the target for immunotherapy . Key words: Regulatory T cells; Sepsis; MicroRNA-10a; Forkhead/winged helix transcription factor p3; Immune function; Effector T cell; Proliferative activity; Mean fluorescence intensity

Fli1-haploinsufficient dermal fibroblasts promote skin-localized transdifferentiation of Th2-like regulatory T cells

BackgroundFriend leukemia virus integration 1 (Fli1) deficiency, a predisposing factor of systemic sclerosis (SSc), induces SSc-like phenotypes in various cell types. A recent study demonstrated the transdifferentiation of T helper type 2 cell (Th2)-like regulatory T cells (Tregs) in SSc lesional skin through interleukin (IL)-33 produced by fibroblasts. Therefore, we investigated the role of Fli1 deficiency in dermal fibroblast-mediated transdifferentiation of Tregs.MethodsCytokine expression was assessed in Tregs by flow cytometry and in skin samples and cultivated cells by immunostaining, immunoblotting, and/or qRT-PCR. Fli1 binding to the target gene promoters was examined by chromatin immunoprecipitation. Murine dermal fibroblasts and Tregs were cocultured with or without blocking antibodies against target cytokines.ResultsTh2- and Th17-like cell proportions in skin-homing Tregs were increased in bleomycin-treated Fli1+/− mice compared with bleomycin-treated wild-type mice, whereas Th1-, Th2-, and Th17-like cell proportions in splenic Tregs were comparable. Fli1+/− fibroblasts overproduced IL-33 and IL-6, in particular IL-33, and Fli1 occupied the IL33 and IL6 promoters in dermal fibroblasts. Importantly, the IL-4-producing cell proportion was significantly higher in wild-type Tregs cocultured with Fli1+/− fibroblasts than in those cocultured with wild-type fibroblasts, which were canceled by neutralizing anti-IL-33 antibody. Under the same coculture condition, an increased tendency of IL-17A-producing cell proportion, which was possibly mediated by IL-6, was evident.ConclusionsFli1 haploinsufficiency increases the proportions of Th2- and Th17-like Tregs in bleomycin-induced profibrotic skin conditions, in which IL-33-producing dermal fibroblasts contribute to Th2-like Treg transdifferentiation, suggesting a critical role of Fli1 deficiency in the interaction of dermal fibroblasts with immune cells in pathological skin fibrosis.

Connexin 43 in splenic lymphocytes is involved in the regulation of CD4+CD25+ T lymphocyte proliferation and cytokine production in hypertensive inflammation.

Chronic inflammation promotes the development of hypertension and is associated with increased T cell infiltration and cytokine production in impaired organs. Gap junction protein connexin 43 (Cx43), is ubiquitously expressed in immune cells and plays an important role in T cell proliferation and activation, and cytokine production. However, the correlation between Cx43 in T cells and the hypertensive inflammatory response remains unknown. Thus, in this study, we wished to examine this correlation. First, our results revealed that hypertension caused significant thickening of the vascular wall, inflammatory cell infiltration into part of the renal interstitium and glomerular atrophy, and it increased the tubular damage scores in the kidneys of spontaneously hypertensive rats (SHRs). Moreover, the SHRs exhibited stenosis in the central artery wall of the spleen with increased serum levels of interleukin (IL)-2 and IL-6 compared with normotensive Wistar-Kyoto (WKY) rats. The spleens of the SHRs exhibited a significantly decreased percentage of CD4+CD25+ (Treg) T cells. However, the percentages of CD3+, CD4+ and CD8+ T cell and the levels of CD4+Cx43 and CD8+Cx43 did not differ significantly between the SHRs and WKY rats. In cultured lymphocytes from the SHRs and WKY rats, low percentages of Treg cells and reduced cytokine (IL-2 and IL-6) mRNA expression levels were observed in the lymphocytes obtained from the SHRs and WKY rats treated with the connexin blocker, Gap27, or concanavalin A (ConA) plus Gap27. The effects of ConA and Gap27 differed between the SHRs and WKY rats. On the whole, our findings demonstrate that the splenic Treg cell-mediated suppression in SHRs may be involved in hypertensive inflammatory responses. Cx43 in the gap junctional channel may regulate lymphocyte activation and inflammatory cytokine production.

Is the adaptive immune response in murine Trypanosoma cruzi infection influenced by zinc supplementation?

Chagas disease afflicts 7 to 8 million people worldwide and congenital Chagas' disease usually leads to changes in the maternal environment, culminating in fetal adaptations. Several articles have described the importance of micronutrients on pregnancy, which is sensitive to infections. In Trypanosoma cruzi endemic regions, the Chagas disease is aggravated by the lack of micronutrients in an average diet, to which pregnant women are more susceptible. The aim of this study was to evaluate distinct T cells phenotypes and intracellular cytokines by flow cytometry in pregnant Wistar rats under zinc therapy during experimental Chagas' disease. Twenty female Wistar rats were infected with 1×105 blood trypomastigotes (Y strain) and 30days after infection the animals were mated and grouped: pregnant infected (PI–n=5), pregnant infected/zinc supplied (PIZ–n=5), pregnant control (PC–n=5), control/zinc supplied (PCZ–n=5). Zinc supplementation: 20mg of zinc/Kg/day (gavage) for 18days followed by euthanasia. The immune parameters showed: decreased percentages of CD62LlowCD44high surface marker for infected and treated group (PIZ) when compared to PI (p<0.05). Concerning to T regulatory cells (Treg cells), a significantly lower percentage of splenic Treg cells was found in the infected and treated group (PIZ) as compared to the PI group (p<0.05). The expression of the co-stimulatory molecule CD28+ displayed a significant reduced percentage in TCD8+ for infected and zinc treated group (PIZ) as compared to (PI). The percentages of CD4+/CD11a+ T cells subsets were lower on PIZ as compared to PI. Concerning to CD45RA+ (B lymphocytes) analysis, infected pregnant and treated group (PIZ) showed a significant decrease in CD45RA percentage when compared to (PI) (p<0.05). The intracellular cytokine profiles for TCD4+ and TCD8+ producing IL-4 and IFN-γ revealed that zinc treated and untreated infected pregnant group (PI and PIZ) displayed increased cytokines concentrations as compared to zinc treated and untreated pregnant controls (PC and PCZ). Our data revealed the involvement of zinc as a signaling molecule in the modulation of the inflammatory process and immune response which occurs during pregnancy of T. cruzi infected rats. Zinc acted in a dual fashion, modulating the host's immune response in a way to protect the organism against the deleterious effects of the infection and an overwhelming pro-inflammatory response during pregnancy.

Modulation of Th17 and regulatory T-cell responses during murine pregnancy contributes to increased maternal susceptibility to Salmonella Typhimurium infection.

Salmonella Typhimurium (S. Tm) infection in pregnant mice results in massive placental infection, fetal loss, and exacerbated systemic infection. The Th17 host response can aid control of S. Tm infection, whereas successful pregnancy correlates to a dampened inflammatory and enhanced regulatory T-cell (Treg) response. Mice were infected systemically with S. Tm and tissue bacterial burden, splenic Th17 and Treg cell numbers, and serum cytokines were analyzed. Splenic and/or placental mRNA expression of IL-17A, RORγ-t, IL-10, and TNF was determined. The effects of in vivo CD25+ cell depletion and TLR4 blockade on the course of S. Tm infection and Th17 response were determined. Enhanced S. Tm burden in pregnant mice was associated with time-dependent increased serum inflammatory cytokines. In vivo, TLR4 blockade reduced splenic S. Tm burden, suggesting detrimental TLR4-mediated inflammation. However, the splenic and placental Th17 response was reduced in S. Tm-infected pregnant mice relative to non-pregnant controls. Alternatively, there was an increase in splenic Treg frequency in pregnant mice and depletion of this subset reduced bacterial burden and increased the Th17 response. Downregulation of Th17 cell responses by Tregs during pregnancy potentially contributes to exacerbation of S. Tm infection in pregnant mice.

Human amylin induces CD4+Foxp3+ regulatory T cells in the protection from autoimmune diabetes.

Autoimmune diabetes is a disorder of immune homeostasis that leads to targeted insulin-secreting islet β cell destruction characterized by insulitis. Human amylin (hA) is an important neuroendocrine hormone co-secreted with insulin by pancreatic β cells. Here, we report hA immune-modulatory action through inducing regulatory T cells. We ex vivo-treated human peripheral blood mononuclear cells (hPBMCs) with hA for 24h and counted CD4+Foxp3+ regulatory T cells (Treg) using flow cytometry. Diabetic status was monitored and splenic Treg were measured in non-obese diabetic (NOD) male mice. NOD mice were intraperitoneally injected once daily with hA (n=25) or solvent for control (n=25) for 7months continuously. Spleen tissues were collected at the end of intervention and processed for flow cytometry and Western blot. We found a 2.9-fold (p<0.05) increase of CD4+Foxp3+ Treg in hPBMCs treated with 10nmol/L hA compared with negative control. Incidence of diabetes in hA-treated NOD mice decreased 44% (p=0.045) in the 6th month and 57% (p=0.0002) in the 7th month. Meanwhile, the hA treatment induced a 1.5-fold increase of CD4+Foxp3+ Treg from mouse splenocytes (p=0.0013). Expression of transforming growth factor-β (TGF-β) and toll-like receptor-4 (TLR-4) were upregulated in hA-treated mice. Human amylin might protect against autoimmune diabetes via the induction of CD4+Foxp3+ Treg, which suggests a novel approach to improve autoimmune conditions.

Effect of Halofuginone on the Pathogenesis of Autoimmune Thyroid Disease in Different Mice Models

Our objectives were to investigate the therapeutic effect of halofuginone (HF) in the treatment of autoimmune thyroid diseases (AITDs) and explore its underlying mechanism of action. The Graves' disease (GD) model was generated by immunizing female BALB/c mice with adenovirus expressing the TSH receptor A subunit (Ad-TSHR289). The Ad-TSHRA+HF and Ad- TSHRA+DMSO groups were injected intraperitoneally with HF or the vehicle control (DMSO), respectively. The autoimmune thyroiditis (AIT) group consisted of female NOD.H-2h4 mice that were administered NaI in the drinking water and intraperitoneally injected daily with the vehicle control (DMSO) during the study period. The AIT/HF group consisted of female NOD.H-2h4 mice that were administered NaI in the drinking water and intraperitoneally injected daily with HF. The frequencies of splenic Th17 cells, Tregs and Bregs were determined by flow cytometry. The mRNA levels of IL-17, forkhead box P3 (Foxp3), RORγt and IL-10 were determined by real-time PCR. In both Ad-TSHRA+DMSO and Ad-TSHRA+HF groups, 10 out of 15 mice displayed serum T4 and TSAb levels above 3 SD beyond the mean control levels. The number of CD4+CD25+Foxp3+ T lymphocytes in the GD model was significantly increased in the HF group compared with the DMSO group (P < 0.05). The mRNA level of Foxp3 was significantly increased in the Ad-TSHRA+HF group compared with the Ad-TSHRA+DMSO group (P < 0.05). However, neither the abundance of CD4+IL-17+ T cell subpopulation nor the mRNA expression level of RORγt differed significantly between the Ad-TSHRA+HF and Ad-TSHRA+DMSO groups (P > 0.05). The serum TgAb titer was significantly reduced in the AIT/HF group compared with the AIT group (P < 0.01). The differences in the number of CD4+CD25+Foxp3+ T lymphocytes and the mRNA levels of Foxp3 between the AIT/HF and AITgroups were not significant (P > 0.05). However, the number of CD4+IL-17+ T cells and the mRNA levels of IL-17 and RORγt were significantly increased in HF-treated mice compared with the non-treated AIT-induced mice (P < 0.05). Treatment with HF significantly decreased the incidence of AIT by decreasing the number of CD4+IL-17+ T cells.

Modulation of Th17 and Regulatory T cell responses during murine pregnancy contributes to increased maternal susceptibility to Salmonella Typhimurium infection

PROBLEM Salmonella Typhimurium (S. Tm) infection in pregnant mice results in massive placental infection, fetal loss, and exacerbated systemic infection. The Th17 host response can aid control of S. Tm infection, whereas successful pregnancy correlates to a dampened inflammatory and enhanced regulatory T-cell (Treg) response. METHOD OF STUDY Mice were infected systemically with S. Tm and tissue bacterial burden, splenic Th17 and Treg cell numbers, and serum cytokines were analyzed. Splenic and/or placental mRNA expression of IL-17A, RORγ-t, IL-10, and TNF was determined. The effects of in vivo CD25+ cell depletion and TLR4 blockade on the course of S. Tm infection and Th17 response were determined. RESULTS Enhanced S. Tm burden in pregnant mice was associated with time-dependent increased serum inflammatory cytokines. In vivo, TLR4 blockade reduced splenic S. Tm burden, suggesting detrimental TLR4-mediated inflammation. However, the splenic and placental Th17 response was reduced in S. Tm-infected pregnant mice relative to non-pregnant controls. Alternatively, there was an increase in splenic Treg frequency in pregnant mice and depletion of this subset reduced bacterial burden and increased the Th17 response. CONCLUSION Downregulation of Th17 cell responses by Tregs during pregnancy potentially contributes to exacerbation of S. Tm infection in pregnant mice.

Pre-transplant infusion of donor-derived dendritic cells maintained at the immature stage by sinomenine increases splenic Foxp3+ Tregs in recipient rats after renal allotransplantation

ObjectiveThe immunosuppressive mechanism of sinomenine in organ allotransplantation was investigated, especially its effect of blocking dendritic cell (DC) maturation, which might influence the frequency of regulatory T cells (Tregs). MethodsBone marrow cells from male donor Wistar rats were induced to differentiate into DCs in vitro in the presence or absence of sinomenine, and characterized by flow cytometry. These two groups of DCs were respectively injected into male recipient Sprague-Dawley rats via the tail vein, at both high and low doses. Sprague-Dawley rats receiving saline injection were used as controls. Seven days later, renal transplantation was performed from donor Wistar rats to the recipient Sprague-Dawley rats. Seven days after transplantation, spleens were collected from the recipients. The proportions of Tregs and Foxp3+ Tregs to CD4+ T cells were determined using flow cytometry. ResultsWith sinomenine treatment, the frequency of mature DCs was reduced, as indicated by lower expression of the surface markers CD80, CD86, and RT1B. In recipient Sprague-Dawley rats that received sinomenine-treated DCs before renal allotransplantation, the proportions of splenic Tregs and Foxp3+ Tregs were significantly higher than in control recipients receiving saline or DCs without sinomenine treatment (all p<0.05). A high dose of sinomenine-treated DCs (106 cells) had a more obvious effect in increasing Tregs than the low dose (105 cells) (p<0.05). ConclusionPre-transplant infusion of donor-derived sinomenine-induced maturation arrested DCs could result in the increase of Foxp3+ Tregs in the spleens of recipients after renal allotransplantation.

Abstract 167: Ibrutinib regulates tumor microenvironment and enhances response to everolimus in renal cell carcinoma mouse models

Abstract Introduction: Ibrutinib (ibr), a first-in-class, once-daily, oral inhibitor of Bruton’s tyrosine kinase (BTK), is indicated for the treatment of patients with CLL/SLL, MCL and WM. Ibr also inhibits EGFR/HER2 and has demonstrated efficacy against EGFR+ NSCLC and HER2+ breast cancer in vitro and in xenograft models (Gao 2014; Chen 2016). Further, ibr modulated host immunity and enhanced anti-PD-L1 activity in solid tumor models otherwise insensitive to BTK or HER kinase inhibition (Sagiv-Barfi 2015), suggesting that ibr may be active in renal cell carcinoma (RCC) via multiple mechanisms. Here we determined the impact of ibr alone and in combination with everolimus (eve) on tumor growth and the tumor microenvironment in syngeneic and xenograft RCC mouse models. Methods: Cell proliferation was determined with CellTiter-Glo using a 3-day treatment regimen. Subcutaneous implantation of 786-0 and Renca cells established xenograft and syngeneic models in nude and immune-competent mice. Treatment started when tumor volume reached 150 mm3 (786-0) or 55 mm3 (Renca) and was measured twice weekly. Immunophenotying of tumor infiltrates was determined by flow cytometry. For Treg differentiation, magnetically-sorted CD4+CD25- mouse splenic T cells were stimulated with anti-CD3/CD28 antibodies and TGF-β1 for 5 days. Results: Ibr significantly inhibited the growth of syngeneic Renca tumors (TGI%=32.4 at Day 19, p≤0.05) in Balb/c mice and enhanced the efficacy of eve, although Renca cells were not sensitive to ibr in vitro. The combination significantly reduced the numbers of splenic and tumor Tregs in addition to reducing PD-1 expression on tumor CD8+ T cells. Consistently, in vitro Treg differentiation assays revealed that, while the combination of ibr and eve significantly reduced Treg differentiation, either drug alone showed little effect. In 786-0 xenograft model, ibr alone slowed tumor growth in a subgroup of mice and significantly enhanced the effect of eve (p≤0.05 or 0.01). To identify whether BTK or EGFR/HER2 is the target, CGI-1746, a BTK inhibitor, and lapatinib, an EGFR/HER2 inhibitor, were tested. Surprisingly, both CGI-1746 and lapatinib were active in inhibiting tumor growth (TGI%=26.7 or 37.6 respectively, p≤0.05 or 0.01), and slightly potentiated the efficacy of eve. However, consistent with ibr results, neither CGI-1746 nor lapatinib showed any effect on the cell growth of 786-0 cells. Conclusion: This study suggests that ibr has antitumor activity against RCC alone and when combined with eve in animal models. This effect may be mediated by modulation of the tumor microenvironment, such as inhibiting Treg differentiation and suppressing PD-1 expression on CD8+ T cells, and both BTK and EGFR/HER2 are involved. Further investigation is needed to clarify the mechanism of action, but the results here provide a rationale for ibr as a novel agent for RCC in combination with mTOR inhibitors. Citation Format: Jun Chen, Chun-Te Chen, Jing Liu, Jeff Hsu, Taisei Kinoshita, Betty Y. Chang. Ibrutinib regulates tumor microenvironment and enhances response to everolimus in renal cell carcinoma mouse models [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 167. doi:10.1158/1538-7445.AM2017-167

Abstract LB-211: Phenotypic change specific to tumor-infiltrating regulatory T cells by LFPRLR knockdown

Abstract The clinical relevance and prognostic significance of tumor-infiltrating Tregs in different types of cancers makes them appealing therapeutic targets. However, targeting all Tregs initiates autoimmune disease. In the current study, we have found that knockdown of the long form of the prolactin receptor (LFPRLR) inhibits the function of tumor Tregs with no functional change in peripheral Tregs. Knockdown of the LFPRLR was by splice modulating oligomer (SMO), tested in the syngeneic 4T1-Balb/c immune-competent breast cancer model. Similar effects would be predicted to occur in prostate and ovarian cancer. Knockdown reduced tumor output of CCL17, a chemokine regulated by the major LFPRLR signaling Jak2/Stat5 pathway. Reduced CCL17 output in turn resulted in impaired Treg recruitment into tumors, allowing enhanced CD4+ and CD8+ T cell activities within the tumor and reducing metastasis to distal organs. Also observed was a reduction in Tregs at metastatic sites. RNAseq transcriptomic analysis of LFPRLR SMO-treated tumor Tregs revealed that among immune-suppressive cytokines, expression of IL-10 and TGF-β was not affected, but EBI3, a component of IL-35, became undetectable. Of cytokines known to promote metastasis, IL-6, IL23a and IL34 were reduced. When tumor Tregs, isolated from LFPRLR SMO-treated animals, were co-cultured with 4T1 breast cancer cells, they failed to drive epithelial to mesenchymal transition, while tumor Tregs isolated from control SMO-treated animals did transform 4T1 cells into a more mesenchymal phenotype. Although tumor T regs were reduced by treatment, other major components of the tumor microenvironment, including B cells, CD8+ T cells, macrophages, NK T cells, dendritic cells, myeloid-derived suppressor cells, and fibroblasts were not changed by LFPRLR knockdown, while effector CD4+ T cells were increased and tumor endothelial cells were decreased. Although the population of CD8+ T cells was not changed, their activity was enhanced, as determined by their production of IL-2 and IFN-γ when stimulated. Examination of splenic Tregs from animals treated with LFPRLR- or control SMO for 28 days showed normal suppressive activity of anti-CD3-stimulated T cell proliferation. In conclusion, systemic LFPRLR knockdown has direct and indirect effects on tumor Tregs that modify their cytokine profile and reduce their suppressive capability and ability to drive epithelial to mesenchymal transition of tumor cells. Citation Format: Kuan-Hui E. Chen, Ameae Walker. Phenotypic change specific to tumor-infiltrating regulatory T cells by LFPRLR knockdown [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 LB-211. doi:10.1158/1538-7445.AM2017-LB-211

The imbalance of Th17/Treg cells is involved in the progression of nonalcoholic fatty liver disease in mice

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a common, chronic liver disease worldwide. Recent studies have shown that T helper (Th) 17 and regulatory T (Treg) cells play critical roles in various disorders of liver inflammation. Here, we explored the value of polyene phosphatidylcholine capsules (PPC) for regulating the imbalance of Th17/Treg cells in the pathogenesis of mice with NAFLD.MethodsC57BL/6 mice were randomly divided into three groups as follows:normal diet (ND), high-fat diet (HF),and HF plus PPC(HF + PPC). The frequencies of splenic Th17 and Treg cells were measured by flow cytometry, and their related cytokines were analyzed by CBA and real-time PCR.ResultsAt the end of 24 weeks, mice in the HF group had a higher frequency of intrahepatic Th17 cells,and a lower proportion of Treg cells compared with the ND group. The levels of Th17 cell-related cytokines (IL-6, IL-17 and IL-23) in serum and in liver tisse were increased,and the hepatic mRNA levels of RORγt, STAT3 and IL-6 were also increased. By contrast,the FoxP3 mRNA level was decreased in the HF group. Moreover, significant pathological and biochemical changes in the liver, as well as serum biochemical changes, were found in mice with NAFLD. Interestingly, following treatment with PPC, the levels of liver inflammation,frequencies of Th17/Treg cells and associated cytokines,and biochemical data were significantly altered.ConclusionThese findings demonstrate a critical role for PPC in partially attenuating liver inflammatory responses in mice with NAFLD that involves the imbalance of Treg/Th17 cells and associated cytokines.

Human adipose-derived mesenchymal stem cells alleviate obliterative bronchiolitis in a murine model via IDO

BackgroundLong-term survival of lung transplantation is hindered by the development of obliterative bronchiolitis (OB). Adipose-derived stem cells (ASCs) were documented to have more potent immunosuppressive ability than mesenchymal stem cells (MSCs) from bone marrow and placenta. The goal of our study is to evaluate the effect of repeated administration of ASCs on OB and the involvement of indoleamine 2,3-dioxygenase (IDO) mediating the protective effect of ASCs in a heterotopic tracheal transplantation (HTT) model.MethodsFor studies in vitro, ASCs were treated with interferon-γ (IFN-γ). For in vivo study, tracheas from BALB/c or C57BL/6 donors were transplanted into C57BL/6 recipients to create a HTT model. On days 0, 1, 3, 5, 8, 12, 15, 20 and 25 post-transplant, the allogeneic recipient mice were administered intravenously with phosphate buffered saline, 1 × 106 human ASCs, or 1 × 106 human ASCs plus 1-methyltryptophan (1-MT), an IDO inhibitor. On days 3, 7, 14 and 28, serum, trachea and spleen samples were harvested for analysis.ResultsASCs homed to heterotopic tracheal grafts after infusion. Multiple doses of ASCs significantly increased tracheal IDO levels in allografts. There were significant increases in graft and serum IFN-γ levels in allografts compared with isografts. IFN-γ elevated IDO expression and activity in ASCs in vitro. ASCs alleviated OB in allografts as evidenced by reduced epithelial loss, epithelial apoptosis, and intraluminal obstruction. The effects of ASCs on OB were blocked by 1-MT. 1-MT also blocked the alterations in pro and anti-inflammatory cytokines as well as CD3+ T cell infiltration induced by ASCs. ASCs induced not only splenic levels of CD4+CD25+Foxp3+ regulatory T cells (Treg) but also IL-10 and TGF-β-producing Treg. Furthermore, IDO inhibition abolished the changes of splenic Treg induced by ASCs. In addition, Treg reduction by cyclophosphamide treatment did not alter the effects of ASCs on tracheal IDO expression in allografts confirming Treg induction is downstream of IDO.ConclusionsRepeated doses of ASCs are capable of ameliorating OB. ASCs act at least in part via elevating IDO expression. ASCs promote the generation of Treg and suppress T cell infiltration via an IDO-dependent mechanism.

IL233, A Novel IL-2 and IL-33 Hybrid Cytokine, Ameliorates Renal Injury.

CD4+Foxp3+ regulatory T cells (Tregs) protect the kidney during AKI. We previously found that IL-2, which is critical for Treg homeostasis, upregulates the IL-33 receptor (ST2) on CD4+ T cells, thus we hypothesized that IL-2 and IL-33 cooperate to enhance Treg function. We found that a major subset of Tregs in mice express ST2, and coinjection of IL-2 and IL-33 increased the number of Tregs in lymphoid organs and protected mice from ischemia-reperfusion injury (IRI) more efficiently than either cytokine alone. Accordingly, we generated a novel hybrid cytokine (IL233) bearing the activities of IL-2 and IL-33 for efficient targeting to Tregs. IL233 treatment increased the number of Tregs in blood and spleen and prevented IRI more efficiently than a mixture of IL-2 and IL-33. Injection of IL233 also increased the numbers of Tregs in renal compartments. Moreover, IL233-treated mice had fewer splenic Tregs and more Tregs in kidneys after IRI. In vitro, splenic Tregs from IL233-treated mice suppressed CD4+ T cell proliferation better than Tregs from saline-treated controls. IL233 treatment also improved the ability of isolated Tregs to inhibit IRI in adoptive transfer experiments and protected mice from cisplatin- and doxorubicin-induced nephrotoxic injury. Finally, treatment with IL233 increased the proportion of ST2-bearing innate lymphoid cells (ILC2) in blood and kidneys, and adoptive transfer of ILC2 also protected mice from IRI. Thus, the novel IL233 hybrid cytokine, which utilizes the cooperation of IL-2 and IL-33 to enhance Treg- and ILC2-mediated protection from AKI, bears strong therapeutic potential.

CD27 co-stimulation increases regulatory T cell survival and limits progression of atherosclerosis.

Abstract Co-stimulatory signals are important shaping adaptive immunity which is pivotal to all stages of atherosclerosis. The interaction of the co-stimulatory molecules CD27 and CD70 modulates Treg development. Furthermore, T cell activation, proliferation, and also differentiation are affected by CD27-CD70 interactions. Thus, we hypothesized that the deficiency of CD27 will lead to exacerbated atherosclerosis. Cd27−/− mice were crossed with Apoe−/− mice. Cd27−/−Apoe−/− and littermate controls (Cd27+/+Apoe−/−) were sacrificed at the age of 18 and 28 weeks. Bone marrow of Cd27−/−Apoe−/− and littermate controls was transplanted into Apoe−/− recipient mice. Lesion size, histology and cellular composition were analyzed in the ascending aorta. Human and murine T cells co-localize with CD27 in atherosclerotic lesions. At early stages of atherosclerosis, Cd27−/−Apoe−/− mice displayed bigger and more complex atherosclerotic lesions. Flow cytometry revealed a significant decrease in abundance of splenic and aortic Tregs and increased apoptosis of Treg in the thymus of Cd27−/−Apoe−/− mice. In contrast, at later stages of atherosclerosis (28 weeks), splenic and lesional Treg content and atherosclerotic burden were similar. Transplantation of Cd27−/−Apoe−/− bone marrow, in contrast to control, induced increased atherosclerotic plaque size and a profound pro-inflammatory plaque phenotype accompanied by reduced frequency of aortic and splenic Tregs. Furthermore, the number of circulating leukocytes was increased. Taken together, CD27 deficiency impairs thymic Treg development which exacerbates early atherogenesis. However, the role of CD27–CD70 interactions does not affect later stages of atherosclerosis.

Emergence of inflammatory Treg in premature fetal Rhesus macaques in a model of LPS-induced chorioamnionitis

Abstract Placental inflammation, termed chorioamnionitis (chorio) is one of the largest contributors to premature birth, and has been associated with several complications post-birth, mainly pulmonary and cerebral. However, a full understanding of the fetal responses to the inflammatory milieu created by chorio remains limited. To address this question, we developed an experimental model of chorio in which intra-amniotic (IA) injection of LPS in pregnant Rhesus macaques lead to inflammation of the placenta similar to what is described in human chorio. In this model of fetal inflammation, fetal splenic regulatory T cells (Treg) frequency decreased, but CD4+IL-17+ cells increased. IL-17 and other pro-inflammatory cytokines (IL-2, IL-8, IFNγ) were mainly expressed by a subset of FoxP3+ T cells that also had a low expression of Helios, thus exhibiting characteristics of “inflammatory Treg”. Several pro-inflammatory cytokines, notably TNFα and IL-1 are elevated in the cord blood and amniotic fluid of LPS exposed neonates. IL-1 receptor antagonist (IL-1RA) and anti-TNFα decreased the frequency of splenic inflammatory Treg; however, they did not normalize overall Treg frequency. Collectively, our results show that in-utero exposure to inflammation triggers a rapid pro-inflammatory response in the fetus, particularly affecting Treg frequency and phenotype, and cytokines like IL-1 and TNFα are important for many of these pathways. Due to the role of Treg in maintaining immune homeostasis, altered Treg frequency and phenotype in response to in-utero inflammation potentially contributes to the detrimental outcomes of these neonates.