IFN-γ Producing Th1 Cells Research Articles

Microsomal Prostaglandin E Synthase-1 Controls Colonic Prostaglandin E2 Production and Exerts a Protective Effect on Colitis Induced by Trinitrobenzene Sulfonic Acid in Mice.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an isozyme of the prostaglandin (PG) E synthase that acts downstream of cyclooxygenase and catalyzes the conversion of PGH2 to PGE2. The impact of genetic deletion of mPGES-1 on the development of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, a well-established model of inflammatory bowel disease (IBD), was investigated in this study. After administration of TNBS, mice deficient in mPGES-1 (mPGES-1-/- mice) showed more severe colitis than did wild-type (WT) mice. Histological examination revealed that mPGES-1-/- mice had markedly exacerbated symptoms of colitis. mPGES-1 expression was detectable in the colons of WT mice at both the mRNA and protein levels. Lack of mPGES-1 resulted in marked reduction of colonic PGE2 production. Our study also showed a significant increase in colonic expression of interleukin-17A (IL-17A), as well as interferon γ (IFNγ) and tumor necrosis factor α, during colitis in mPGES-1-/- mice compared with that in WT mice. Furthermore, loss of mPGES-1 increased the populations of IL-17A-producing T-helper (Th) 17 and IFNγ-producing Th1 cells in mesenteric lymph nodes. These results suggest that mPGES-1 is the main enzyme responsible for colonic PGE2 production and deficiency of mPGES-1 facilitates the development of colitis and T-cell-mediated immunity. mPGES-1 might, therefore, impact T-cell-related immune response associated with IBD.

TRAF6 signaling in dendritic cells plays protective role against infectious colitis by limiting C. rodentium infection through the induction of Th1 and Th17 responses

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role in the induction of inflammatory responses not only in innate immune cells but also in non-immune cells, leading to the activation of adaptive immunity. Signal transduction mediated by TRAF6, along with its upstream molecule MyD88 in intestinal epithelial cells (IECs) is crucial for the maintenance of mucosal homeostasis following inflammatory insult. The IEC-specific TRAF6-deficient (TRAF6ΔIEC) and MyD88-deficient (MyD88ΔIEC) mice exhibit increased susceptibility to DSS-induced colitis, emphasizing the critical role of this pathway. Moreover, MyD88 also plays a protective role in Citrobacter rodentium (C. rodentium) infection-induced colitis. However, its pathological role of TRAF6 in infectious colitis remains unclear. To investigate the site-specific roles of TRAF6 in response to enteric bacterial pathogens, we infected TRAF6ΔIEC and dendritic cell (DC)-specific TRAF6-deficient (TRAF6ΔDC) mice with C. rodentium and found that the pathology of infectious colitis was exacerbated with significantly decreased survival rates in TRAF6ΔDC mice, but not in TRAF6ΔIEC mice, compared to those in control mice. TRAF6ΔDC mice showed increased bacterial burdens, marked disruption of epithelial and mucosal structures with increased infiltration of neutrophils and macrophages, and elevated cytokine levels in the colon at the late stages of infection. The frequencies of IFN-γ producing Th1 cells and IL-17A producing Th17 cells in the colonic lamina propria were significantly reduced in TRAF6ΔDC mice. Finally, we demonstrated that TRAF6-deficient DCs failed to produce IL-12 and IL-23 in response to C. rodentium stimulation, and to induce both Th1 and Th17 cells in vitro. Thus, TRAF6 signaling in DCs, but not in IECs, protects against colitis induced by C. rodentium infection by producing IL-12 and IL-23 that induce Th1 and Th17 responses in the gut.

Effective clearance of Chlamydia from the murine female reproductive requires IL-12p40, but not T-bet driven Th1 responses

Abstract Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted IFN-γ+ Th1 responses are thought to be vital for bacterial clearance, but the Th1 master transcription factor T-bet had not yet been fully interrogated in primary infection. Here, we investigated T-bet and IFN-γ involvement in clearance of primary infection from the FRT. Surprisingly, IFN-γ producing CD4+ T cells from the FRT expressed low levels of T-bet, indicating that Chlamydia-specific CD4 T cells are not classical T-bet+ Th1 cells. Additionally, mice deficient in T-bet competently cleared infection with wild-type kinetics and showed significant skewing towards Th17 responses, indicating compensation pathways through alternate Th fates. On the other hand, IFN-γ-, and IFN-γR-deficient mice resolved much of FRT infection, but displayed systemic dissemination and 100% mortality. Thus, IFN-γ protects mice from fatal systemic disease, but classical T-bet+ Th1 cells are not required for primary clearance. In investigating alternative Th fates, RORγt mutant mice deficient for Th17 responses exhibited a short delay in clearance of FRT infection, demonstrating an unanticipated role for Th17 cells in Chlamydia clearance. However, mice deficient in the cytokine subunit IL-12p40 that promotes both Th1 and Th17 cells displayed a prolonged delay in FRT clearance despite CD4 T cells retaining the capacity for IFN-γ production. In summary, the type 1 cytokine IFN-γ plays an essential role in systemic control of Chlamydia, while clearance of primary infection within the FRT is largely dependent on an IL-12p40 driven mechanism that does not require T-bet or classical IFN-γ producing Th1 cells. Supported by grants from NIH (RO1 A1103422, T32 AI060555)

Pulmonary and blood dendritic cells from sarcoidosis patients more potently induce IFNγ-producing Th1 cells compared with monocytes.

Sarcoidosis is a systemic inflammatory disease mainly affecting the lungs. The hallmark of sarcoidosis are granulomas that are surrounded by activated T cells, likely targeting the disease-inducing antigen. IFNγ-producing Th1 and Th17.1 T cells are elevated in sarcoidosis and associate with disease progression. Monocytes and dendritic cells (DCs) are antigen-presenting cells (APCs) and required for T cell activation. Several subsets of monocytes and DCs with different functions were identified in sarcoidosis. However, to what extent different monocyte and DC subsets can support activation and skewing of T cells in sarcoidosis is still unclear. In this study, we performed a transcriptional and functional side-by-side comparison of sorted monocytes and DCs from matched blood and bronchoalveolar lavage (BAL) fluid of sarcoidosis patients. Transcriptomic analysis of all subsets showed upregulation of genes related to T cell activation and antigen presentation in DCs compared with monocytes. Allogeneic T cell proliferation was higher after coculture with monocytes and DCs from blood compared with BAL and DCs induced more T cell proliferation compared with monocytes. After coculture, proliferating T cells showed high expression of the transcription factor Tbet and IFNγ production. We also identified Tbet and RORγt coexpressing T cells that mainly produced IFNγ. Our data show that DCs rather than monocytes from sarcoidosis patients have the ability to activate and polarize T cells towards Th1 and Th17.1 cells. This study provides a useful in vitro tool to better understand the contribution of monocytes and DCs to T cell activation and immunopathology in sarcoidosis.

Quantitative and Functional Alterations of 6-Sulfo Lacnac Dendritic Cells in Multiple Myeloma

Multiple myeloma (MM) is characterized by expansion of abnormal plasma cells in the bone marrow (BM). Monocytes and macrophages have been shown to accumulate in the microenvironment / BM of MM patients while promoting MM cells survival and proliferation. Recently, another population of myeloid cells expressing the M-DC8 antigen containing a 6-sulfo LacNAc structure, called 6-sulfo LacNac dendritic cells (Slan-DCs) has been described. Functionally, Slan-DCs have potent pro-inflammatory properties based on their capacity to produce large amounts of Tumor Necrosis Factor-α and Interleukin (IL) 12p70 upon stimulation with Toll-like receptor ligands such as R848. They also express C-X-C chemokine receptor type 4 which is important in homing to the BM. Nevertheless, little is known about microenvironmental interactions between MM cells and Slan-DCs.To gain insights on the Slan-DCs' potential role in MM pathophysiology, we evaluated their phenotype and functions in the BM and blood of patients with newly diagnosed symptomatic MM. Using multicolor flow cytometry, we performed quantitative and functional analyses. Compared to healthy donors (n=11), the percentages and absolute numbers of circulating HLA-DR+ M-DC8+ Slan-DCs were significantly decreased in the blood of MM patients (n=23) (for absolute number: 20.64 vs 7.61 Slan-DCs/μL, p=0.0015 and for percentage: 1.06 vs 0.49%, p=0.029). In contrast, there were no significant difference in the frequencies of Slan-DCs in the patients BM.We next addressed their functional properties, in particular cytokine production. We observed a significant decrease in IL-12-producing Slan-DCs from MM patients as compared to healthy donors both in blood (25.88% versus 56.86% respectively, p<0.0001) and BM (25.11% versus 48.83% respectively, p=0.016). In order to investigate whether the decrease in IL-12 secretion was due to the MM cells, circulating Slan-DCs from healthy controls were stimulated with R848 in the presence of 4 different MM cell lines. The capacity of Slan-DCs from healthy controls to secrete IL-12 under this stimulation was measured by ELISA. We observed that the RPMI-8226, KMS-12-PE and LP-1 MM cell lines inhibited R848-induced IL-12 secretion by Slan-DCs. Furthermore, we could show that primary MM cells from patients at diagnosis (magnetically sorted CD138+ cells from patients) were able to inhibit IL-12 secretion by Slan-DCs from healthy donors (n=5).Since MM cells inhibited Slan-DCs secretion of IL-12 and given the pivotal role of IL-12 in Th1 polarization, we investigated whether MM cells could modulate the capacity of Slan-DCs to induce T cell proliferation and Th1 differentiation. To this aim, we co-cultured T cells and Slan-DCs (n=5). Here, we found that normal Slan-DCs can efficiently induce the proliferation of T cells. In addition, their ability to promote T cell proliferation was more pronounced after R848 priming. However, after co-culture with RPMI-8226 MM cell line, the fraction of proliferating T cells at day 7 was significantly decreased (58.14% versus 28.37% ; p=0.03). Moreover, RPMI-8226 MM cell line markedly impaired the capacity of Slan-DCs to induce the differentiation of naïve CD4+ T cells into IFNγ-producing Th1 cells (34.59% versus 7.89% ; p=0.04).Based on the above results, we hypothesized that Slan-DCs' pro-inflammatory phenotype could also be impacted by MM cells. Thus, freshly-sorted Slan-DCs were cultured with or without MM cell lines and their phenotype was analyzed by flow cytometry (n=9). When Slan-DCs were cultured in the presence of MM cells, expression of CD14 and CD16, but not CD11b, was induced, in line with a phenotype of an “intermediate” monocytes population. Moreover, in the presence of MM cells, the expression of the costimulatory molecule CD86 was weakly induced on Slan-DCs (Mean of Fluorescence Intensity = 102049 versus 199470, p=0.004).In conclusion, these results provide evidence that MM cells are able to significantly impair the capacity of Slan-DCs to secrete IL-12 and stimulate CD3+ T cell proliferation and to polarize naïve CD4+ T cell into Th1 cells. The latter suggests that MM cells are able to strongly suppress the pro-inflammatory function of Slan-DCs, directing them towards a more tolerogenic profile. These findings reveal a novel mechanism of immune escape in MM, and may pave the way for newer immune-intervention strategies in combination with other currently available therapies. DisclosuresGarderet:Amgen: Honoraria; Takeda: Honoraria. Mohty:Sanofi: Honoraria, Speakers Bureau.

Anti-SARS-CoV-2 T-cell Responses After mRNA Vaccination in Belatacept-treated Renal Transplant Patients.

Dear Editor, We read with interest the recent letter by Chavarot et al1 on the humoral and T-cell responses after 2 injections of mRNA vaccine in kidney transplant recipients. The authors reported a low sero-conversion rate at day 60 and a T-cell response in only 30.4% of the patients measured. Importantly, the T-cell response was measured by IFNγ-EliSpot after stimulation with overlapping peptide pools for SARS-CoV-2 spike protein. We would like to further discuss the determination of the T-cell response, as it has important implications for the interpretation of the findings. The safety and efficacy of mRNA vaccines have been demonstrated in numerous clinical studies, and, in most of these studies, vaccine-specific T-cell responses were reported.2-4 In these studies, next to IFNγ EliSpot, T cells were detected in addition by flow cytometry to characterize the cytokine profile. Sahin et al3 found in a phase I/II study that BNT161b1 elicited a strong Th1 response. In most of the experimental groups, IL-2 producing T-cells dominated over IFNγ producing T-cells or even IFNγ+IL-2+ T cells. A similar observation was made in a phase I/II trial on BNT162b2 as reported in preprint by Sahin et al.4 In a phase I study on the mRNA-1273 vaccine, Anderson et al2 demonstrated that vaccine-specific T-helper-cell responses measured on day 43 were of the Th1 type. However, the IL-2 response by T helper cells was greater than the IFNγ response but lower than the TNFα response.2 This effect was consistent over 2 different age groups ranging from 56 to 70 y of age and ≥71 y of age. Thus, vaccine-specific T-cell responses are not restricted to IFNγ producing Th1 cells. We would like to point out that the lack of T-cell response reported by Chavarot et al may not give a comprehensive information on the overall T-cell response to vaccination in renal transplant patients as an IFNγ EliSpot was the only readout.1 IL-2 or TNFα single positive T helper cells are not detected in this assay but usually evolve after vaccination in healthy people. Thus, renal transplant patients may well harbor vaccine-specific T-cell immunity upon vaccination. Additionally, it remains unclear whether the impaired Th1 response is a mainly belatacept-dependent effect as suggested by the authors. From our point of view, this is not exclusively related to the inhibition of costimulation but to the high-grade immunosuppressive regimen after kidney transplantation. In summary, the measurement of vaccine-specific T-cell responses is complex and a comprehensive characterization of vaccine-specific T-cell responses is necessary to draw conclusions on the immunogenicity of BNT162b2 in renal transplant patients. Nevertheless, the present study provides substantial evidence that vaccine responses in kidney transplant patients should be monitored and individualized vaccination strategies might be needed in this vulnerable cohort.

SND1 promotes Th1/17 immunity against chlamydial lung infection through enhancing dendritic cell function.

To date, no reports have linked the multifunctional protein, staphylococcal nuclease domain-containing protein 1 (SND1), to host defense against intracellular infections. In this study, we investigated the role and mechanisms of SND1, by using SND1 knockout (SND1-/-) mice, in host defense against the lung infection of Chlamydia muridarum, an obligate intracellular bacterium. Our data showed that SND1-/- mice exhibited significantly greater body weight loss, higher organism growth, and more severe pathological changes compared with wild-type mice following the infection. Further analysis showed significantly reduced Chlamydia-specific Th1/17 immune responses in SND1-/- mice after infection. Interestingly, the dendritic cells (DCs) isolated from SND1-/- mice showed lower costimulatory molecules expression and IL-12 production, but higher IL-10 production compared with those from wild-type control mice. In the DC-T cell co-culture system, DCs isolated from SND1-/- infected mice showed significantly reduced ability to promote Chlamydia-specific IFN-γ producing Th1 cells but enhanced capacity to induce CD4+T cells into Foxp3+ Treg cells. Adoptive transfer of DCs isolated from SND1-/- mice, unlike those from wild-type control mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that SND1 plays an important role in host defense against intracellular bacterial infection, and suggest that SND1 can promote Th1/17 immunity and inhibit the expansion of Treg cells through modulation of the function of DCs.

Neutrophils mediate Th17 promotion in COVID-19 patients.

From the beginning of 2020, an urgent need to understand the pathophysiology of SARS‐CoV‐2 disease (COVID‐19), much of which is due to dysbalanced immune responses, resonates across the world. COVID‐19‐associated neutrophilia, increased neutrophil‐to‐lymphocyte ratio, aberrant neutrophil activation, and infiltration of neutrophils into lungs suggest that neutrophils are important players in the disease immunopathology. The main objective of this study was to assess the phenotypic and functional characteristics of neutrophils in COVID‐19 patients, with particular focus on the interaction between neutrophils and T cells. We hypothesize that the altered functional characteristics of COVID‐19 patient‐derived neutrophils result in skewed Th1/Th17 adaptive immune response, thus contributing to disease pathology. The expansion of G‐MDSC and immature forms of neutrophils was shown in the COVID‐19 patients. In the COVID‐19 neutrophil/T cell cocultures, neutrophils caused a strong polarity shift toward Th17, and, conversely, a reduction of IFNγ‐producing Th1 cells. The Th17 promotion was NOS dependent. Neutrophils, the known modulators of adaptive immunity, skew the polarization of T cells toward the Th17 promotion and Th1 suppression in COVID‐19 patients, contributing to the discoordinated orchestration of immune response against SARS‐CoV‐2. As IL‐17 and other Th17‐related cytokines have previously been shown to correlate with the disease severity, we suggest that targeting neutrophils and/or Th17 represents a potentially beneficial therapeutic strategy for severe COVID‐19 patients.

Functional characteristics of Th1, Th17, and ex-Th17 cells in EAE revealed by intravital two-photon microscopy

BackgroundT helper (Th) 17 cells are a highly plastic subset of T cells, which in the context of neuroinflammation, are able to acquire pathogenic features originally attributed to Th1 cells (resulting in so called ex-Th17 cells). Thus, a strict separation between the two T cell subsets in the context of experimental autoimmune encephalomyelitis (EAE) is difficult. High variability in culture and EAE induction protocols contributed to previous conflicting results concerning the differential contribution of Th1 and Th17 cells in EAE. Here, we systematically evaluate the role of different T cell differentiation and transfer protocols for EAE disease development and investigate the functional dynamics of encephalitogenic T cells directly within the inflamed central nervous system (CNS) tissue.MethodsWe compiled the currently used EAE induction protocols reported in literature and investigated the influence of the different Th1 and Th17 differentiation protocols as well as EAE induction protocols on the EAE disease course. Moreover, we assessed the cytokine profile and functional dynamics of both encephalitogenic Th1 and Th17 cells in the inflamed CNS using flow cytometry and intravital two-photon laser scanning microscopy. Lastly, we used astrocyte culture and adoptive transfer EAE to evaluate the impact of Th1 and Th17 cells on astrocyte adhesion molecule expression in vitro and in vivo.ResultsWe show that EAE courses are highly dependent on in vitro differentiation and transfer protocols. Moreover, using genetically encoded reporter mice (B6.IL17A-EGFP.acRFP x 2d2/2d2.RFP), we show that the motility of interferon (IFN)γ-producing ex-Th17 cells more closely resembles Th1 cells than Th17 cells in transfer EAE. Mechanistically, IFNγ-producing Th1 cells selectively induce the expression of cellular adhesion molecules I-CAM1 while Th1 as well as ex-Th17 induce V-CAM1 on astrocytes.ConclusionsThe behavior of ex-Th17 cells in EAE lesions in vivo resembles Th1 rather than Th17 cells, underlining that their change in cytokine production is associated with functional phenotype alterations of these cells.

Abstract B86: Treatment of glioma cells with IGF-1R antisense and irradiation induces the production of antigens that stimulate IFNγ production by tumor-specific CD4 T cells

Abstract We describe here a novel process for the production by glioma cells of cell-free, tumor-specific antigens that supplant the need for intact tumor cells in the induction of immunity against autologous tumor antigen. Radiation is thought to trigger the release of potentially immunostimulatory tumor antigens. However, a variety of tumor cells, including human glioblastoma multiforme (GBM) and mouse GL261 glioma cells, express high levels of insulin-like growth factor-1 receptor (IGF-1R), which has been associated with radioresistance. To determine if the combination of targeting IGF-1R with an antisense oligodeoxynucleotide (AS) and radiation causes the release of glioma antigens, we encapsulated GL261 cells in biodiffusion chambers with different doses of IGF-1R AS, subjected the chambers to 5Gy radiation, and cultured the chambers for approximately 18 hours. Bone marrow-derived dendritic cells (BMDCs) from naive mice were pulsed with the cell-free chamber contents and cultured with CD4 T cells from mice protected against the growth of GL261 cells implanted in the cerebral cortex by immunization with a GL261 cell-based vaccine. The numbers of T cells producing IFNγ in an ELISPOT assay were used as an index of antigen recognition. The contents of chambers that were incubated with viable GL261 or were irradiated but cultured without IGF-1R AS stimulated only weak T-cell responses. Substantial T-cell reactivity was only seen with BMDCs pulsed with the cell-free contents of irradiated chambers containing GL261 cells and IGF-1R AS. Overnight treatment of GL261 cells with IGF-1R AS prior to encapsulation and irradiation also promoted antigen release but was most effective only when a small amount of IGF-1R AS was also added to the chambers. Fully formulated chambers contained tumor tissues that had been cultured overnight with the IGF1-R antisense, encapsulated with additional antisense and irradiated. Chambers were incubated in PBS or implanted in the flanks of naive C57BL/6 mice overnight, then contents were retrieved and used to pulse naive BMDCs. CD4 T cells recovered from GL261-immune C57BL/6 mice were used to detect tumor antigenic determinants presented by the BMDCs with the readout being the number of cells producing IFNγ. We conclude that the treatment of IGF-1R-positive glioma cells with IGF-1R AS and sublethal irradiation stimulates the cells to release antigens capable of stimulating the IFNγ-producing CD4 Th1 cells that have been associated with therapeutic immunity against glioma cells in the GL261 model. When performed in a biodiffusion chamber, this process leads to the accumulation of immunostimulatory antigens in the absence of tumor cell products known to have inhibitory properties for Th1 cells. Immunization via the implantation of irradiated biodiffusion chambers containing autologous tumor cells recovered at surgery and IGF-1R AS is currently under investigation in a clinical trial for patients with GBM (IND 14379; ClinicalTrials.gov, NCT01550523). Citation Format: Samantha Garcia, Rhonda Kean, Aurore Lebrun, Emily Bongiorno, David W. Andrews, D. Craig Hooper. Treatment of glioma cells with IGF-1R antisense and irradiation induces the production of antigens that stimulate IFNγ production by tumor-specific CD4 T cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B86.

T Cell-Specific Overexpression of Acid Sphingomyelinase Results in Elevated T Cell Activation and Reduced Parasitemia During Plasmodium yoelii Infection.

The enzyme acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and is thereby involved in several cellular processes such as differentiation, proliferation, and apoptosis in different cell types. However, the function of ASM in T cells is still not well characterized. Here, we used T cell-specific ASM overexpressing mice (t-ASM/CD4cre) to clarify the impact of cell-intrinsic ASM activity on T cell function in vitro and in vivo. We showed that t-ASM/CD4cre mice exhibit decreased frequencies of Foxp3+ T regulatory cells (Tregs) within the spleen. Enforced T cell-specific ASM expression resulted in less efficient induction of Tregs and promoted differentiation of CD4+CD25− naïve T cells into IFN-γ producing Th1 cells in vitro. Further analysis revealed that ASM-overexpressing T cells from t-ASM/CD4cre mice show elevated T cell receptor (TCR) signaling activity accompanied with increased proliferation upon stimulation in vitro. Plasmodium yoelii infection of t-ASM/CD4cre mice resulted in enhanced T cell activation and was associated with reduced parasitemia in comparison to infected control mice. Hence, our results provide evidence that ASM activity modulates T cell function in vitro and in vivo.

PSORI-CM02 alleviates IMQ-induced mouse dermatitis via differentially regulating pro- and anti-inflammatory cytokines targeting of Th2 specific transcript factor GATA3

The IL-17-producing CD4+ T cell and γδT cells play critical roles in the pathogenesis of psoriasis (PS). PSORI-CM02 is a representative herbal formula for the treatment for PS in South China. It was confirmed to improve PS without obvious side effects in the clinic. Here we sought to clarify whether and how PSORI-CM02 regulates T cell differentiation and functions in IMQ-induced psoriasis-like BALB/c mouse model. Mice pre-treated 3 days with PSORI-CM02 significantly alleviated skin inflammation, as reduced in PASI score and classic psoriatic characteristics in pathological sections. CD3 and CD4 positive T cells were also fewer in the skin lesions of PSORI-CM02 groups, comparing to control group. PSORI-CM02 also decreased pro-inflammatory IFNγ mRNA and IL-17 A mRNA, while increased IL-4 mRNA in mouse skin lesions. In skin draining lymph nodes (DLN), PSORI-CM02 reduced the ratio of γδT cells and inhibited their function of producing IL-17 A. Nevertheless PSORI-CM02 had no effects on the ratio of total TCRβ+T cells and CD4 + T cells. But it regulated CD4 + T helper cells differentiation, and resulted in the decreasing percentage of IFNγ producing Th1 cells and IL-17 A producing Th17 cells, while increasing the ratio of IL-4 producing Th2 cells in DLN. Further data showed that PSORI-CM02 promote expression of Th2 specific transcript factor GATA3, but had no effects on T-bet and RORγ. Thus, we tentatively interpret that PSORI-CM02 impairs IMQ-induced psoriasis by promoting Th2 cell response targeting of GATA3.

IFN-γ Producing Th1 Cells Induce Different Transcriptional Profiles in Microglia and Astrocytes.

Autoreactive T cells that infiltrate into the central nervous system (CNS) are believed to have a significant role in mediating the pathology of neuroinflammatory diseases like multiple sclerosis. Their interaction with microglia and astrocytes in the CNS is crucial for the regulation of neuroinflammatory processes. Our previous work demonstrated that effectors secreted by Th1 and Th17 cells have different capacities to influence the phenotype and function of glial cells. We have shown that Th1-derived effectors altered the phenotype and function of both microglia and astrocytes whereas Th17-derived effectors induced direct effects only on astrocytes but not on microglia. Here we investigated if effector molecules associated with IFN-γ producing Th1 cells induced different gene expression profiles in microglia and astrocytes. We performed a microarray analysis of RNA isolated from microglia and astrocytes treated with medium and Th-derived culture supernatants and compared the gene expression data. By using the criteria of 2-fold change and a false discovery rate of 0.01 (corrected p < 0.01), we demonstrated that a total of 2,106 and 1,594 genes were differentially regulated in microglia and astrocytes, respectively, in response to Th1-derived factors. We observed that Th1-derived effectors induce distinct transcriptional changes in microglia and astrocytes in addition to commonly regulated transcripts. These distinct transcriptional changes regulate peculiar physiological functions, and this knowledge can help to better understand T cell mediated neuropathologies.

P.1.g.035 - Effects of antipsychotic drugs on the epigenetic modification of BDNF gene expression in the hippocampus of chronic restraint-stressed rats

Multiple sclerosis is a T cell mediated chronic demyelinating disease of the central nervous system. Although currently available therapies reduce relapses, they do not facilitate tolerization of myelin antigen-specific T lymphocytes to ensure prolonged protection against multiple sclerosis. Here, we show that treatment of NOD mice with the histone deacetylase inhibitor, Trichostatin A affords robust protection against myelin peptide induced experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Protection was accompanied by histone hyperacetylation, and reduced inflammation and axonal damage in the spinal cord. Drug treatment diminished the generation of CD4+ memory T cells and induced tolerance in CD4+ T cells recognizing the immunizing myelin peptide. During the early immunization period, CD4+ T cells producing GM-CSF + IFN-γ, GM-CSF + IL-17A, as well as those expressing both IL-17A + IFN-γ (double-producers) were detected in the secondary lymphoid organs followed by the appearance of cells producing IFN-γ and GM-CSF. On the other hand, IFN-γ producing Th1 cells appear first in the spinal cord followed by cells producing IL-17A and GM-CSF. Treatment with Trichostatin A substantially reduced the frequencies of all T cells secreting various lymphokines both in the periphery and in the spinal cord. These data indicate that epigenetic modifications induced by histone hyperacetylation facilitates T cell tolerance induction in the periphery leading to reduced migration of T cells to the spinal cord and mitigation of neuronal damage and improved clinical outcome. These results suggest that epigenetic modulation of the genome may similarly offer benefits to multiple sclerosis patients via abrogating the function of encephalitogenic T lymphocytes without exerting severe side effects associated with currently used disease-modifying therapies.

The epigenetic drug Trichostatin A ameliorates experimental autoimmune encephalomyelitis via T cell tolerance induction and impaired influx of T cells into the spinal cord

Multiple sclerosis is a T cell mediated chronic demyelinating disease of the central nervous system. Although currently available therapies reduce relapses, they do not facilitate tolerization of myelin antigen-specific T lymphocytes to ensure prolonged protection against multiple sclerosis. Here, we show that treatment of NOD mice with the histone deacetylase inhibitor, Trichostatin A affords robust protection against myelin peptide induced experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Protection was accompanied by histone hyperacetylation, and reduced inflammation and axonal damage in the spinal cord. Drug treatment diminished the generation of CD4+ memory T cells and induced tolerance in CD4+ T cells recognizing the immunizing myelin peptide. During the early immunization period, CD4+ T cells producing GM-CSF+IFN-γ, GM-CSF+IL-17A, as well as those expressing both IL-17A+IFN-γ (double-producers) were detected in the secondary lymphoid organs followed by the appearance of cells producing IFN-γ and GM-CSF. On the other hand, IFN-γ producing Th1 cells appear first in the spinal cord followed by cells producing IL-17A and GM-CSF. Treatment with Trichostatin A substantially reduced the frequencies of all T cells secreting various lymphokines both in the periphery and in the spinal cord. These data indicate that epigenetic modifications induced by histone hyperacetylation facilitates T cell tolerance induction in the periphery leading to reduced migration of T cells to the spinal cord and mitigation of neuronal damage and improved clinical outcome. These results suggest that epigenetic modulation of the genome may similarly offer benefits to multiple sclerosis patients via abrogating the function of encephalitogenic T lymphocytes without exerting severe side effects associated with currently used disease-modifying therapies.

Adipocyte adaptive immunity mediates diet-induced adipose inflammation and insulin resistance by decreasing adipose Treg cells

Obesity leads to a switch in subsets of CD4+ T cell in adipose tissue, characterized by an increase in IFNγ producing Th1 cells and a decrease in anti-inflammatory regulatory T (Treg) cells, which impairs systemic insulin sensitivity. What signals these changes is unknown. Herein we demonstrate that genetic deficiency of adipocyte MHCII decreases adipose IFNγ expression and increases adipose Treg abundance in obese mice, leading to reduced obesity-induced adipose inflammation and reduced insulin resistance without affecting weight gain. The preserved insulin sensitivity of high fat diet (HFD)-fed adipocyte-specific MHCII knockout (aMHCII−/−) mice was substantially attenuated by adipose-specific Treg ablation. Adipocytes of aMHCII−/− mice exhibit decreased capacity to stimulate IFNγ production in Th1 cells, whereas HFD-fed IFNγR1−/− mice were more insulin sensitive and had similarly high levels of Tregs in adipose tissue as aMHCII−/− mice. We further show that IFNγ strongly inhibits IL-33 effects to promote adipose Treg proliferation. Our results identify MHCII in adipocyte as a critical determinant of the obesity-induced adipose T cell subset switch and insulin resistance.

Adoptive Transfer of Tumor-Specific Th2 Cells Eradicates Tumors by Triggering an In Situ Inflammatory Immune Response.

Adoptive cell therapy (ACT) trials to date have focused on transfer of autologous tumor-specific cytotoxic CD8+ T cells; however, the potential of CD4+ T helper (Th) cells for ACT is gaining interest. While encouraging results have been reported with IFNγ-producing Th1 cells, tumor-specific Th2 cells have been largely neglected for ACT due to their reported tumor-promoting properties. In this study, we tested the efficacy of idiotype-specific Th2 cells for the treatment of mice with MHC class II-negative myeloma. Th2 ACT efficiently eradicated subcutaneous myeloma in an antigen-specific fashion. Transferred Th2 cells persisted in vivo and conferred long-lasting immunity. Cancer eradication mediated by tumor-specific Th2 cells did not require B cells, natural killer T cells, CD8+ T cells, or IFNγ. Th2 ACT was also curative against B-cell lymphoma. Upon transfer, Th2 cells induced a type II inflammation at the tumor site with massive infiltration of M2-type macrophages producing arginase. In vivo blockade of arginase strongly inhibited Th2 ACT, consistent with a key role of arginase and M2 macrophages in myeloma elimination by Th2 cells. These results illustrate that cancer eradication may be achieved by induction of a tumor-specific Th2 inflammatory immune response at the tumor site. Thus, ACT with tumor-specific Th2 cells may represent a highly efficient immunotherapy protocol against cancer. Cancer Res; 76(23); 6864-76. ©2016 AACR.

Infusion of Ex Vivo Activated Donor Type 2 Innate Lymphoid Cells Inhibit Th1 Activity and Reduces Acute Graft-Versus-Host Disease

▪The particular subsets of T cells that mediates aGvHD is not clear. Several studies have implicated Th1 and/or Th17 T cells in the pathogenesis of aGvHD. Pre-clinical data suggest that IFN-γ producing Th1 cells are perhaps more important for GI tract aGvHD whereas IL-17 producing Th17 cells are more important for skin and lung pathology. However, both murine models and clinical data indicate that suppression of Th1 and Th17 responses by shifting to a Th2 T cell phenotype limits aGvHD. One potential mechanism for the enhanced Th1 tropism of the lower GI tract is the role of conditioning therapy on the release of cytokines and bacterial products that may polarize naïve T cells toward Th1 cells.The explosion of data surrounding innate immune cells, such as type 2 innate lymphoid cells (ILC2) has increased the possibility of discovering additional candidate suppressive cells. In patients undergoing allo-HCT, peripheral blood ILCs slowly recover and activated ILCs are associated with reduced GVHD lethality. Since ILC2 cells may drive donor Th2 responses that reduce GVHD lethality and hence their deficiency may predispose to Th1 mediated GVHD, we quantified ILC2 numbers in the GI tract and secondary lymphoid organs after radiation. B6D2 mice were given a lethal dose of radiation and ILCs enumerated using flow cytometry. As shown by others, ILC3 cells in the lamina propria (LP) of the colon are resistant to radiation but CD4+ T cells in the LP, spleen and MLN are sensitive to radiation showing significant reduction of total cells within 24 hrs. In striking contrast to ILC3 cells, ILC2 cells from the LP of the colon and spleen were highly sensitivite to radiation. Thus, the absence of recipient ILC2 cells in the colon and MLN, may critically lead to alterations in the microenvironment leading to Th1 polarization in the lower GI tract induced after allo-SCT.Given ILC2 production of Th2 cytokines and the data indicating that shifting the cytokine micro-environment away from Th1/Th17 to Th2 reduces the severity of aGVHD, we hypothesized that co-transplantation of ILC2 cells would reduce aGvHD and increase survival after allo-SCT. ILC2 cells can be indentified as lin- and ICOS+, CD127+ and ST2+. ILC2 cells were expanded for 6 days in the presence of IL-7 and IL-33 which resulted in increased percentages of IL-4, IL-5 and IL-13 expressing cells with no evidence of expansion of other ILC groups based upon IFN-γ expression for ILC1 or IL-22 expression for ILC3 cells.To test the efficacy of restoring ILC2 cells after radiation, we performed survival studies in two distinct aGVHD models. In the B6 to BALB/c models of aGvHD after allo-SCT, co-transplantation of one dose of ILC2 cells significantly improved survival after allo-SCT (p<0.0001 log rank) (Figure 1A) with an increase in median survival of 12 days and a reduction in clinical GvHD score (Day 20 score, 6.5 ± 0.3 vs. 3.5 ± 0.4 SEM), beginning at day 10 in a very stringent MHC completely mismatched model. An even greater improvement in survival was seen with ILC2 infusion at the time of transplant in B6D2F1 recipients of B6 bone marrow and T cells. In the B6 to BALB/c model, analysis of donor T cells cytokine production by flow cytometry showed a significant reduction in the frequency of IFNγ producing CD4+ T cells in the LP of recipients of ILC2 cells (Figure 1B). Interestingly, we found that ILC2 cells were equally effective in the complete mismatch model when given on the day of transplant or seven days after transplant.In summary, we have shown that ILC2 cells are radiation sensitive and that co-infusion of a single dose of ILC2 can increase survival, reduce clinical signs of aGvHD and reduced Th1 cytokine production by donor T cells. These data suggest that the Th1 skewed microenvironment in target organs of GvHD seen after conditioning therapy may be due to reduce ILC2 numbers and replacing the ILC2 population with donor-derived cells in the transplant recipient returns the immune microenvironment to a more desirable, tolerogenic status. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anthriscus sylvestris-derived extract induces Th1 and Th17 cell differentiation via the upregulation of IL12 and IL23 production

We have previously shown that root extracts from Anthriscus sylvestris (AS) can stimulate antigen-presenting cells such as dendritic cells and macrophages, which implies that AS extract can be useful as an immune adjuvant to promote the immune response. Here, we investigated the in vivo effect of AS extract on CD4+ T cell differentiation using the DO11.10 ovalbumin (OVA)-specific TCR transgenic mouse model. We found that the repeated injection of AS extract plus OVA antigens resulted in increases in both the Th1-polarizing IL12 and Th17-polarizing IL23 cytokines on macrophages. Consistent with the upregulation of IL12 and IL23, both IFNγ-producing Th1 and IL17-producing Th17 cells specific for OVA antigens were significantly induced upon AS stimulation. In addition, natural killer T (NKT) cells and neutrophils were activated to produce IL17 in AS extract-treated mice. Furthermore, in vivo treatment with AS extract significantly decreased the frequency of Foxp3+CD25+ inducible Treg cells compared to the vehicle-treated control group. Taken together, our findings suggest that in vivo treatment with AS extract induces proinflammatory cytokine production by macrophages, leading to the activation of IL17-producing innate immune cells such as NKT cells and neutrophils, presumably at early time points and later, as well as the induction of IFNγ- or IL17-producing adaptive immune cells such as Th1 and Th17 cells with the concomitant downregulation of Treg cells. These findings imply that AS-derived extract might be useful as an adjuvant to treat Th2-biased immune diseases such as atopic dermatitis.

Streptococcus pneumoniae specific Th17 memory immunity provides cross protection against invasive pneumococcal diseases in mice. (IRC9P.701)

Abstract Pneumonia caused by Streptococcus pneumoniae (Sp) remains a leading cause of serious illness and numerous deaths in children and elderly worldwide. Current pneumococcal vaccine is effective in preventing colonization by inducing serotype-specific antibodies. However, there is an increasing prevalence of infection by serotype strains not included in the vaccine; this highlights the need for a universal vaccine that protects against all serotypes. In current study, we found that mice intranasal immunized with Sp were protected against challenge with a different serotype Sp strain and cleared bacteria from the lung by day 2 post-challenge, and exhibited less apoptosis and tissue damage in lung. Sp infection in lung resulted in a tremendous CD4+T cell expansion and activation that consisted of mostly IL-17 producing Th17 cells but also IFNγ producing Th1 cells. Adoptive transfer of T cells from Sp immunized mice provided cross protection with a dominant Th17 recall response in the lung from donor CD4+ memory T cells. Furthermore, adoptive transfer of Sp specific CD4+ memory T cells provided cross protection against pneumonia and bacteremia, which can be blocked by IL-17 neutralizing antibody and transfer Sp memory CD4+ T cells from IL-17 knockout mice. Our results suggest that Sp memory Th17 cells played a key role in providing broad protective immunity against invasive Sp infection in a serotype independent manner.