IFN-γ Expression In CD4 Research Articles

Loss of Mitochondrial Tusc2/Fus1 Triggers a Brain Pro-Inflammatory Microenvironment and Early Spatial Memory Impairment.

Brain pathological changes impair cognition early in disease etiology. There is an urgent need to understand aging-linked mechanisms of early memory loss to develop therapeutic strategies and prevent the development of cognitive impairment. Tusc2 is a mitochondrial-resident protein regulating Ca2+ fluxes to and from mitochondria impacting overall health. We previously reported that Tusc2-/- female mice develop chronic inflammation and age prematurely, causing age- and sex-dependent spatial memory deficits at 5 months old. Therefore, we investigated Tusc2-dependent mechanisms of memory impairment in 4-month-old mice, comparing changes in resident and brain-infiltrating immune cells. Interestingly, Tusc2-/- female mice demonstrated a pro-inflammatory increase in astrocytes, expression of IFN-γ in CD4+ T cells and Granzyme-B in CD8+T cells. We also found fewer FOXP3+ T-regulatory cells and Ly49G+ NK and Ly49G+ NKT cells in female Tusc2-/- brains, suggesting a dampened anti-inflammatory response. Moreover, Tusc2-/- hippocampi exhibited Tusc2- and sex-specific protein changes associated with brain plasticity, including mTOR activation, and Calbindin and CamKII dysregulation affecting intracellular Ca2+ dynamics. Overall, the data suggest that dysregulation of Ca2+-dependent processes and a heightened pro-inflammatory brain microenvironment in Tusc2-/- mice could underlie cognitive impairment. Thus, strategies to modulate the mitochondrial Tusc2- and Ca2+- signaling pathways in the brain should be explored to improve cognitive health.

Human T-cell activation withToxoplasma gondiiantigens loaded in maltodextrin nanoparticles

Toxoplasmosis is the most prevalent parasitic zoonosis worldwide, causing ocular and neurological diseases. No vaccine has been approved for human use. We evaluated the response of peripheral blood mononuclear cells (PBMCs) to a novel construct ofToxoplasma gondiitotal antigen in maltodextrin nanoparticles (NP/TE) in individuals with varying infectious statuses (uninfected, chronic asymptomatic, or ocular toxoplasmosis). We analyzed the concentration of IFN-γ after NP/TE ex vivo stimulation using ELISA and the immunophenotypes of CD4+and CD8+cell populations using flow cytometry. In addition, serotyping of individuals with toxoplasmosis was performed by ELISA using GRA6-derived polypeptides. Low doses of NP/TE stimulation (0.9 μg NP/0.3 μg TE) achieved IFN-γ–specific production in previously exposed human PBMCs without significant differences in the infecting serotype. Increased IFN-γ expression in CD4+effector memory cell subsets was found in patients with ocular toxoplasmosis with NP/TE but not with TE alone. This is the first study to show how T-cell subsets respond to ex vivo stimulation with a vaccine candidate for human toxoplasmosis, providing crucial insights for future clinical trials.

Recombinant antigen P29 of Echinococcus granulosus induces Th1, Tc1, and Th17 cell immune responses in sheep.

Echinococcosis is a common human and animal parasitic disease that seriously endangers human health and animal husbandry. Although studies have been conducted on vaccines for echinococcosis, to date, there is no human vaccine available for use. One of the main reasons for this is the lack of in-depth research on basic immunization with vaccines. Our previous results confirmed that recombinant antigen P29 (rEg.P29) induced more than 90% immune protection in both mice and sheep, but data on its induction of sheep-associated cellular immune responses are lacking. In this study, we investigated the changes in CD4+ T cells, CD8+ T cells, and antigen-specific cytokines IFN-γ, IL-4, and IL-17A after rEg.P29 immunization using enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and flow cytometry to investigate the cellular immune response induced by rEg.P29 in sheep. It was found that rEg.P29 immunization did not affect the percentage of CD4+ and CD8+ T cells in peripheral blood mononuclear cells (PBMCs), and was able to stimulate the proliferation of CD4+ and CD8+ T cells after immunization in vitro. Importantly, the results of both ELISPOT and ELISA showed that rEg.P29 can induce the production of the specific cytokines IFN-γ and IL-17A, and flow cytometry verified that rEg.P29 can induce the expression of IFN-γ in CD4+ and CD8+ T cells and IL-17A in CD4+ T cells; however, no IL-4 expression was observed. These results indicate that rEg.P29 can induce Th1, Th17, and Tc1 cellular immune responses in sheep against echinococcosis infection, providing theoretical support for the translation of rEg.P29 vaccine applications.

HTLV-1 p12 modulates the levels of prion protein (PrPC) in CD4+ T cells.

Infection with human T cell lymphotropic virus type 1 (HTLV-1) is endemic in Brazil and is linked with pro-inflammatory conditions including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a chronic neuroinflammatory incapacitating disease that culminates in loss of motor functions. The mechanisms underlying the onset and progression of HAM/TSP are incompletely understood. Previous studies have demonstrated that inflammation and infectious agents can affect the expression of cellular prion protein (PrPC) in immune cells. Here, we investigated whether HTLV-1 infection affected PrPC content in cell lines and primary CD4+cells in vitro using flow cytometry and western blot assays. We found that HTLV-1 infection decreased the expression levels of PrPC and HTLV-1 Orf I encoded p12, an endoplasmic reticulum resident protein also known to affect post-transcriptionally cellular proteins such as MHC-class I and the IL-2 receptor. In addition, we observed a reduced percentage of CD4+ T cells from infected individuals expressing PrPC, which was reflected by IFN type II but not IL-17 expression. These results suggested that PrPC downregulation, linked to both HTLV-1 p12 and IFN-γ expression in CD4+ cells, may play a role in the neuropathogenesis of HTLV-1 infection.

MicroRNA Let-7i Regulates Innate TLR4 Pathways in Peripheral Blood Mononuclear Cells of Patients with Ankylosing Spondylitis.

This study aimed to compare the changes in the expression of microRNA Let-7i in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS) and the correlation between Let-7i and innate pro-inflammatory factors. It is necessary to search for a new biomarker to guide the prognosis of AS. A total of 10 patients with AS and 10 healthy volunteers were selected as AS and control groups, respectively. The expression levels of Let-7i, Toll-like receptor 4 (TLR4), nuclear factor-κB (NF-κB), and interferon-gamma (IFN-γ) in PBMCs were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) to explore the relationship between Let-7i and pro-inflammatory factors. Furthermore, the relationship between Let-7i and TLR4 was determined by the luciferase reporter technology. The expression level of Let-7i in PBMCs of patients with AS was significantly lower than that of healthy control. The expression levels of TLR4, NF-κB, and IFN-γ in PBMCs derived from patients with AS were significantly higher than those of healthy control. The results show that Let-7i manipulation can regulate lipopolysaccharide (LPS)-induced TLR4 and IFN-γ expression in CD4+ T cells of patients with AS. The overexpression of Let-7i in T cells of patients with AS can suppress TLR4 and IFN-γ LPS-induced expression levels of cellular mRNA and protein. Let-7i can directly interfere TLR4-3'untranslated region (UTR) sequence and regulate the expression of the TLR4 gene in Jurkat T cells. Let-7i may be involved in the pathogenesis of AS, and Let-7i expression in PBMCs may be helpful for the diagnosis and treatment of AS in the future.

The implication of interferon-γ-producing immunocompetent cells for evaluating disease activity and severity in adult-onset Still's disease.

To investigate the relationship between interferon-γ (IFN-γ), IFN-γ-producing immunocompetent cells, their related cytokines, and the clinical features in adult-onset Still's disease (AOSD). Twenty-five patients with AOSD before initiating treatment (acute AOSD), 9 patients after remission (remission AOSD), and 12 healthy controls (HC) were included. Circulating IFN-γ-producing CD4+ and CD8+ cells, natural killer (NK) cells, and IFN-γ production in NK cells were evaluated by flow cytometry. Serum levels of IFN-γ, interleukin (IL)-6, IL-12, IL-15, and IL-18 were also measured. The obtained results were statistically analyzed with clinical findings. Serum levels of IFN-γ, IL-6, IL-12, IL-18, intracellular expression of IFN-γ in CD4+, CD8+, and NK cells were significantly higher in acute AOSD than in HC. The proportion of NK cells was significantly lower in acute AOSD than in HC. Serum levels of IFN-γ and IFN-γ expression in CD4+ cells were significantly correlated with serum ferritin levels. The proportion of NK cells had a significant inverse correlation with serum IFN-γ levels. A lower proportion of NK cells was significantly noted in patients refractory to initial immunosuppressive treatment. In remission AOSD, serum levels of IL-6, IL-12, and IL-18 were significantly higher than in HC. Increased serum levels of IFN-γ, increased expression of IFN-γ in CD4+ cells, and decreased NK cell proportion correlate with disease activity in AOSD. Moreover, a lower proportion of NK cells may be useful for predicting a refractory clinical course. Meanwhile, increased serum levels of IL-6, IL-12, and IL-18 may persist after clinical remission.

Abstract 1569: Role of NK and CD8+T cells in the antitumor effect elicited by combined treatment with radiation, tumor-specific antibody, and IL2 in a syngeneic murine model of head and neck squamous cell carcinoma

Abstract Background: Radiation therapy (RT) has been shown to activate an in situ vaccine (ISV) effects. Recently, we reported that combining tumor-antigen-specific monoclonal antibody (mAb), RT, and intratumoral (IT) injection of IL2 activates a much greater ISV response compared to RT alone. In this combination, RT can enhance the mAb-dependent cell-mediated cytotoxicity response by NK cells when delivered with a tumor-specific mAb. Here we evaluated the role of NK cells in activating the CD8+ T cell-dependent ISV effect following RT, mAb, and IT-IL2. Methods: MOC2 murine head and neck squamous cell carcinoma (HNSCC) cells expressing human epidermal growth factor receptor (MOC2-huEGFR+) were engrafted on the right flank of C57BL/6 mice. Tumors were treated with RT, the anti-huEGFR mAb cetuximab, and IT-IL2. In mice rendered tumor-free, immune memory was tested by re-engraftment with MOC2 wild-type tumor cells on the left flank. Splenic cells were isolated from a cohort of treated mice and cultured with MOC2 cells. These were analyzed by flow cytometry to quantify IFNγ expression. To test ISV efficacy in metastatic settings, mice were engrafted with MOC2-huEGFR+ right flank tumors and MOC2 wild-type left flank tumors. After treatment, immune infiltration was analyzed in both tumors, blood, and spleen. To assess the necessity of immune cells, CD4+, CD8+, or NK1.1+ cells were depleted. Results: Combining RT and IT cetuximab and IL2 resulted in greater tumor regression and overall survival compared to single or dual treatments in mice with MOC2-huEGFR+ tumors. Six of 7 disease-free mice exhibited immune memory. Coculture of splenocytes from ISV treated mice with MOC2-huEGFR cells in vitro showed increased IFNγ expression in CD4+, CD8+, and NK cells. However, when splenocytes were first sorted and then co-cultured, only CD8+ T cells showed increased IFNγ expression after coculture with MOC2-huEGFR+. In a two-tumor model, delivering this ISV regimen to a right flank MOC2-huEGFR+ tumor increased CD8+ T cell infiltration in a distant left flank MOC2 wild-type tumor without affecting the number of, or IFNγ expression in, CD4+ or NK cells. Depletion of CD8+ cells eliminated antitumor response at both the right and left flank tumors in this model. Interestingly, NK cells, while not necessary for tumor response at the ISV-targeted right flank, were essential to the activation of a systemic CD8+ T cell response at the distant left flank tumor. Conclusions: In a syngeneic murine model of HNSCC, the local combination of RT, cetuximab, and IL2 destroys the targeted tumor and enables this site to serve as a potent immune stimulus and personalized source of antigenicity for activation of adaptive T cell immunity. NK cells are necessary for the activation of this ISV effect and the resulting CD8+ T cell-dependent systemic antitumor response. Citation Format: Wonjong Jin. Role of NK and CD8+T cells in the antitumor effect elicited by combined treatment with radiation, tumor-specific antibody, and IL2 in a syngeneic murine model of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1569.

Impaired Cellular Immunity to SARS-CoV-2 in Severe COVID-19 Patients.

The high infection rate and rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) make it a world-wide pandemic. Individuals infected by the virus exhibited different degrees of symptoms, and most convalescent individuals have been shown to develop both cellular and humoral immune responses. However, virus-specific adaptive immune responses in severe patients during acute phase have not been thoroughly studied. Here, we found that in a group of COVID-19 patients with acute respiratory distress syndrome (ARDS) during hospitalization, most of them mounted SARS-CoV-2-specific antibody responses, including neutralizing antibodies. However, compared to healthy controls, the percentages and absolute numbers of both NK cells and CD8+ T cells were significantly reduced, with decreased IFNγ expression in CD4+ T cells in peripheral blood from severe patients. Most notably, their peripheral blood lymphocytes failed in producing IFNγ against viral proteins. Thus, severe COVID-19 patients at acute infection stage developed SARS-CoV-2-specific antibody responses but were impaired in cellular immunity, which emphasizes on the role of cellular immunity in COVID-19.

SARS-CoV-2: a storm is raging.

The pandemic coronavirus infectious disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is rapidly spreading across the globe. In this issue of the JCI, Chen and colleagues compared the clinical and immunological characteristics between moderate and severe COVID-19. The authors found that respiratory distress on admission is associated with unfavorable outcomes. Increased cytokine levels (IL-6, IL-10, and TNF-α), lymphopenia (in CD4+ and CD8+ T cells), and decreased IFN-γ expression in CD4+ T cells are associated with severe COVID-19. Overall, this study characterized the cytokine storm in severe COVID-19 and provides insights into immune therapeutics and vaccine design.

707 Responsiveness of the Adenosine A2A receptor (A2AR) regulates immune and keratinocyte responses in psoriasis

Psoriasis is a chronic inflammatory disease of the skin and joints associated with both innate and adaptive immunity. An imbalance between the activators and the modulators of immunity and their interactions with keratinocytes leads to the chronic inflammatory hallmarks of the disease. The A2AR is a key modulator of both immunity and inflammation. We have described a means to enhance A2AR function through a small molecule that acts as a positive allosteric modulator (PAM; AEA061). The PAM has no intrinsic biologic activity at the A2AR but enhances adenosine-mediated A2AR function. In a mouse model of TLR 7 agonist imiquimod (IMQ)-induced psoriasis-like dermatitis, the oral administration of the PAM reduced ear thickness, skin erythema, scale formation and inflammatory cytokine expression in the skin, ear tissue and in plasma. Transcriptome analysis of the skin indicated differential expression of 111 genes upon PAM treatment. Ex-vivo and in culture, the PAM decreased IMQ-induced expression of IFN-α in pDCs and IL-23, IL-36α and IL-36β in cDCs. Moreover, the PAM inhibited TCR-mediated IL-17 expression in γδT cells and IFN-γ expression in CD4+ T cells. In human epidermal keratinocytes, the PAM attenuated IFN-α-mediated IL-22R expression, reducing their proliferative response to IL-22. We have extended our investigation into the effects of the PAM on human immune cell subsets that are central to the initiation and maintenance of psoriasis in man. We observed attenuation of mRNA expression of IFN-α, IFN-β, and IL-23 in human pDCs and IL-17F in human γδT cells. The PAM also inhibited the production of cytokines by Th cells that are implicated in psoriasis, including IL-2, IFN-γ, TNF-α, sCD40, GM-CSF, CXCL10, MIP-1α, MIP-1β, RANTES and eotaxin. Thus, the enhancement of A2AR responsiveness to endogenous adenosine, targeted through positive allosteric modulation, alters innate and adaptive immune responses towards a homeostatic state and reduces disease expression.

Detection of T lymphocyte subsets and related functional molecules in follicular fluid of patients with polycystic ovary syndrome

Immune responses play an important role in the pathogenesis of polycystic ovary syndrome (PCOS). However, the characteristics of T lymphocyte subsets in PCOS remain insufficiently understood. In this study, lymphocytes of follicular fluid (FF) were obtained from oocyte retrieval before in-vitro fertilization (IVF) in infertile women with or without PCOS. The levels of cluster of differentiation 25 (CD25), CD69, programmed death 1 (PD-1), interferon-γ (IFN-γ), interleukin 17A (IL-17A) and IL-10 in T lymphocytes were determined by flow cytometry. Our results showed that the percentage of FF CD8+ T cells was significantly decreased in infertile patients with PCOS (P < 0.05). Furthermore, the levels of CD69 and IFN-γ were significantly decreased and the level of PD-1 was increased in both CD4+ and CD8+ T cells from infertile patients with PCOS (P < 0.05). Moreover, the expression of PD-1 on CD4+ or CD8+ T cells was positively correlated with the estradiol (E2) levels in the serum and reversely correlated with the expression of IFN-γ in CD4+ or CD8+ T cells in infertile patients with PCOS. These results suggested that T cell dysfunction may be involved in the pathogenesis of PCOS.

Pharmacological inhibition of USP7 promotes antitumor immunity and contributes to colon cancer therapy.

BackgroundEffectiveness of clinical therapy such as chemotherapy for solid tumors is limited by acquired drug resistance and side effects. Available antitumor immunity methods showed promising prospect of cancer therapy. However, more drug targets for boosting antitumor immunity still need to be explored and selective and effective compounds are yet to be developed.PurposeTo study the effect and possible mechanism of compound P5091, a selective USP7 inhibitor, on CT26 xenografts growth in mice.Materials and methodsCT26 xenografts model was employed to examine the anti-tumor effect of P5091. RT-PCR and ELISA analysis were used to detect the level of IFN-γ, TNF-α and IL-10 in tumor tissue and serum, respectively. IFN-γ expression in CD4+ and CD8+ T cells was analyzed by intracellular stain. The level of FOXP3 in Treg cells was confirmed by intracellular stain and western blotting.ResultsCompound P5091, a selective USP7 inhibitor, was found to inhibit CT26 xenografts growth in mice, which is comparable to the effect of Anti-PD-1 antibody. RT-PCR analysis showed that P5091 treatment decreased IL-10 mRNA level in tumor tissue while elevated mRNA level of IFN-γ and TNF-α. Moreover, ELISA analysis manifested decreased of IL-10 and elevation of IFN-γ and TNF-α in serum from tumor bearing mice. Intracellular stain showed increased IFN-g expression both in CD4+ and CD8+ T cells after P5091 treatment. Furthermore, P5091 treatment caused FOXP3 loss in Treg cells decreased the proportion of Treg cells in tumor bearing mice.ConclusionOur study here showed that P5091 may be a candidate for cancer immunotherapy.

Activation-induced cytidine deaminase deficiency accelerates autoimmune diabetes in NOD mice.

B cells play an important role in type 1 diabetes (T1D) development. However, the role of B cell activation-induced cytidine deaminase (AID) in diabetes development is not clear. We hypothesized that AID is important in the immunopathogenesis of T1D. To test this hypothesis, we generated AID-deficient (AID-/-) NOD mice. We found that AID-/-NOD mice developed accelerated T1D, with worse insulitis and high levels of anti-insulin autoantibody in the circulation. Interestingly, neither maternal IgG transferred through placenta, nor IgA transferred through milk affected the accelerated diabetes development. AID-/-NOD mice showed increased activation and proliferation of B and T cells. We found enhanced T-B cell interactions in AID-/-NOD mice, with increased T-bet and IFN-γ expression in CD4+ T cells in the presence of AID-/- B cells. Moreover, excessive lymphoid expansion was observed in AID-/-NOD mice. Importantly, antigen-specific BDC2.5 CD4+ T cells caused more rapid onset of diabetes when cotransferred with AID-/- B cells than when cotransferred with AID+/+ B cells. Thus, our study provides insights into the role of AID in T1D. Our data also suggest that AID is a negative regulator of immune tolerance and ablation of AID can lead to exacerbated islet autoimmunity and accelerated T1D development.

Ethyl Caffeate Ameliorates Collagen-Induced Arthritis by Suppressing Th1 Immune Response.

The present study was designed to assess the antiarthritic potential of ECF in collagen-induced arthritis (CIA) and explore its underlying mechanism. Methods. In vitro, lymphocyte proliferation assay was measured by CCK-8 kit. In vivo, the therapeutic potential of ECF on CIA was investigated; surface marker, Treg cell, and intracellular cytokines (IL-17A and IFN-γ) were detected by flow cytometry. Th1 cell differentiation assay was performed, and mRNA expression in interferon-γ-related signaling was examined by q-PCR analysis. Results. In vitro, ECF markedly inhibited the proliferation of splenocytes in response to ConA and anti-CD3. In vivo, ECF treatment reduced the severity of CIA, inhibited IFN-γ and IL-6 secretion, and decreased the proportion of CD11b+Gr-1+ splenic neutrophil. Meanwhile, ECF treatment significantly inhibited the IFN-γ expression in CD4+T cell without obviously influencing the development of Th17 cells and T regulatory cells. In vitro, ECF suppressed the differentiation of naive CD4+ T cells into Th1. Furthermore, ECF intensely blocked the transcriptional expression in interferon-γ-related signaling, including IFN-γ, T-bet, STAT1, and STAT4. Conclusion. Our results indicated that ECF exerted antiarthritic potential in collagen-induced arthritis by suppressing Th1 immune response and interferon-γ-related signaling.

Mycobacterium tuberculosis multi-drug-resistant strain M induces IL-17+IFNγ– CD4+ T cell expansion through an IL-23 and TGF-β-dependent mechanism in patients with MDR-TB tuberculosis

We have reported previously that T cells from patients with multi-drug-resistant tuberculosis (MDR-TB) express high levels of interleukin (IL)-17 in response to the MDR strain M (Haarlem family) of Mycobacterium tuberculosis (M. tuberculosis). Herein, we explore the pathways involved in the induction of Th17 cells in MDR-TB patients and healthy tuberculin reactors [purified protein derivative healthy donors (PPD+ HD)] by the M strain and the laboratory strain H37Rv. Our results show that IL-1β and IL-6 are crucial for the H37Rv and M-induced expansion of IL-17+ interferon (IFN)-γ- and IL-17+ IFN-γ+ in CD4+ T cells from MDR-TB and PPD+ HD. IL-23 plays an ambiguous role in T helper type 1 (Th1) and Th17 profiles: alone, IL-23 is responsible for M. tuberculosis-induced IL-17 and IFN-γ expression in CD4+ T cells from PPD+ HD whereas, together with transforming growth factor (TGF-β), it promotes IL-17+ IFN-γ- expansion in MDR-TB. In fact, spontaneous and M. tuberculosis-induced TGF-β secretion is increased in cells from MDR-TB, the M strain being the highest inducer. Interestingly, Toll-like receptor (TLR)-2 signalling mediates the expansion of IL-17+ IFN-γ- cells and the enhancement of latency-associated protein (LAP) expression in CD14+ and CD4+ T cells from MDR-TB, which suggests that the M strain promotes IL-17+ IFN-γ- T cells through a strong TLR-2-dependent TGF-β production by antigen-presenting cells and CD4+ T cells. Finally, CD4+ T cells from MDR-TB patients infected with MDR Haarlem strains show higher IL-17+ IFN-γ- and lower IL-17+ IFN-γ+ levels than LAM-infected patients. The present findings deepen our understanding of the role of IL-17 in MDR-TB and highlight the influence of the genetic background of the infecting M. tuberculosis strain on the ex-vivo Th17 response.

Non-Esterified Fatty Acids Profiling in Rheumatoid Arthritis: Associations with Clinical Features and Th1 Response.

ObjectivesSince lipid compounds are known to modulate the function of CD4+ T-cells and macrophages, we hypothesize that altered levels of serum non-esterified fatty acids (NEFA) may underlie rheumatoid arthritis (RA) pathogenesis.MethodsSerum levels of NEFA (palmitic, stearic, palmitoleic, oleic, linoleic, γ-linoleic, arachidonic –AA–, linolenic, eicosapentaenoic –EPA– and docosahexaenoic –DHA–) were quantified by LC-MS/MS after methyl-tert-butylether (MTBE)-extraction in 124 RA patients and 56 healthy controls (HC). CD4+ phenotype was studied by flow cytometry. TNFα, IL-8, VEGF, GM-CSF, IFNγ, IL-17, CCL2, CXCL10, leptin and resistin serum levels were quantified by immunoassays. The effect of FA on IFNγ production by PBMC was evaluated in vitro.ResultsLower levels of palmitic (p<0.0001), palmitoleic (p = 0.002), oleic (p = 0.010), arachidonic (p = 0.027), EPA (p<0.0001) and DHA (p<0.0001) were found in RA patients, some NEFA being altered at onset. Cluster analysis identified a NEFA profile (hallmarked by increased stearic and decreased EPA and DHA) overrepresented in RA patients compared to HC (p = 0.002), being associated with clinical features (RF, shared epitope and erosions), increased IFNγ expression in CD4+ T-cells (p = 0.002) and a Th1-enriched serum milieu (IFNγ, CCL2 and CXCL10, all p<0.005). In vitro assays demonstrated that imbalanced FA could underlie IFNγ production by CD4+ T-cells. Finally, changes on NEFA levels were associated with clinical response upon TNFα-blockade.ConclusionAn altered NEFA profile can be found in RA patients associated with clinical characteristics of aggressive disease and enhanced Th1 response. These results support the relevance of lipidomic studies in RA and provide a rationale for new therapeutic targets.

Opposing roles of STAT1 and STAT3 in IL-21 function in CD4+ T cells

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4(+) T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4(+) T cells. RNA-Seq analysis of CD4(+) T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4(+) T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.

Pediatric liver transplant recipients with operational tolerance exhibit features of immune activation and exhaustion (TRAN3P.869)

Abstract Experimental models describe immune exhaustion in liver transplant tolerance. It is not known if immune exhaustion is also occurs in clinical operational tolerance. We tested this in a cross-sectional cohort of pediatric liver transplant recipients with stable allograft function in the absence of immunosuppression (Operationally Tolerant, OT) in comparison to those Weaned off Immunosuppression (WI), continued on Maintenance Immunosuppression (MI) and Healthy Controls (HC). Results: In OT recipients, B cell frequencies (p &amp;lt;0.05) were increased and were enriched in both unswitched (p&amp;lt;0.005) and switched memory B cells (CD19+ IgM+ CD27+ and CD19+ IgG+ CD27+) (p&amp;lt;0.05) compard to MI. CD8 T cells in OT showed an increase in effector memory compared to MI (p&amp;lt;0.05). CD4 Treg frequencies were increased in OT compared to MI (p&amp;lt;0.0005). Both CD4 and CD8 T cells in OT showed an increase in cells expressing CD57, indicative of replicative senescence and these were enriched within CD4 central memory and CD8 effector memory when compared to MI (p&amp;lt;0.05). CD57+ T cells upregulated PD1 and 2B4, markers of exhaustion in OT compared to MI (p&amp;lt;0.05). Upon stimulation, IFNγ expression in CD4 T cells and IL-2 expression in CD8 T cells in OT were decreased compared to MI consistent with exhaustion. Moreover, OT showed decreased CD8 frequencies (p&amp;lt;0.05) and high PDL1 expression on monocytes (p&amp;lt;0.0005) suggesting that this population might contribute to T cell exhaustion in these recipients.

Inflammation Enhances IL-2 Driven Differentiation of Cytolytic CD4 T Cells

Cytolytic CD4 T cells (CD4 CTL) have been identified in vivo in response to viral infections; however, the factors necessary for driving the cytolytic phenotype have not been fully elucidated. Our previously published work suggests IL-2 may be the master regulator of perforin-mediated cytotoxicity in CD4 effectors. To further dissect the role of IL-2 in CD4 CTL generation, T cell receptor transgenic mice deficient in the ability to produce IL-2 or the high affinity IL-2 receptor (IL-2Rα, CD25) were used. Increasing concentrations of IL-2 were necessary to drive perforin (Prf) expression and maximal cytotoxicity. Granzyme B (GrB) expression and killing correlated with STAT5 activation and CD25 expression in vitro, suggesting that signaling through the high affinity IL-2R is critical for full cytotoxicity. IL-2 signaling was also necessary in vivo for inducing the Th1 phenotype and IFN-γ expression in CD4 T cells during influenza A (IAV) infection. In addition, GrB expression, as measured by mean fluorescent intensity, was decreased in CD25 deficient cells; however, the frequency of CD4 cells expressing GrB was unchanged. Similarly, analysis of cytolytic markers such as CD107a/b and Eomesodermin indicate high IL-2Rα expression is not necessary to drive the CD4 CTL phenotype during IAV infection. Thus, inflammatory signals induced by viral infection may overcome the need for strong IL-2 signals in driving cytotoxicity in CD4 cells.

High frequency but impaired function of Tregs in active CD patients

Objectives: Coeliac disease (CD) is an immune-mediated systemic disorder elicited by gluten and characterized by a small intestinal enteropathy. Besides active CD, we recognize an early stage of CD characterized by a normal small intestinal mucosa and positive CD serology. The aim of the study was to investigate the immunoregulatory mechanisms acting in the gut of active and potential CD patients. We evaluated the expression of IFN-γ and IL-10 in CD4+ T cells and the frequency of Foxp3+ regulatory T cells (Tregs) in duodenal biopsies. Finally, we investigated the functional properties of intestinal Foxp3+ Tregs from both groups of patients and the effects of IL-15 on their suppressive activity. Methods: CD4+ T cells were isolated from duodenal biopsies of 11 controls, 13 active CD and 15 potential CD patients (7 Marsh 0 (M0) and 8 Marsh 1 (M1)). IFN-γ and IL-10 expression were assessed by flow cytometry. The frequency of CD4+ CD25+ Foxp3+ Tregs frequency and function were investigated. Intestinal Tregs suppressive function was assessed on autologous peripheral blood responder CD4+ CD25− T cells (Tresp), in the presence of a polyclonal stimulus and with or without IL-15. Results: CD4+ IFN-γ+ T cell frequency was higher in duodenal biopsies of active (mean ± sd: 12.9 ± 3.2) and M1 potential CD (17.8 ± 5.9) than controls (4.9 ± 0.7; p < 0.05 and p < 0.05 respectively). On the contrary the highest number of CD4+ IL10+ T cells was observed in M0 potential CD patients (17 ± 5.8) compared to active (6.7 ± 1.7, p < 0.05) and control patients (6.1 ± 0.6; p < 0.05). The percentage of CD4+ CD25+ Foxp3+ T cells was significantly increased in active (14.1 ± 2.7%) and potential CD (9.9 ± 3.2%) if compared to controls (6.5 ± 3.1%; p < 0.01 and p < 0.05 respectively). In particular, in potential M1 CD it was significantly higher (10.9 ± 3.1%; p < 0.05). Intestinal Tregs from both active and potential CD exerted significant suppressive effects on Tresp cells (p < 0.05) in in vitro suppressive assay. Interestingly IL-15 was able to counteract their suppressive activity in active CD but not in potential CD. Conclusions: IFN-γ expression in CD4+ T cells correlates with CD stage, as it is increased in M1 potential and active patients; on the contrary in early CD (M0) a high frequency of IL-10 producing CD4+ T cells was found. This might prevent the progression towards mucosal damage down-regulating IFN-γ production and promoting Foxp3+ Tregs. These cells are significantly increased in the small intestinal mucosa of CD patients and are functionally active; however their suppressive activity is impaired by IL-15 only in active CD patients, suggesting a relation between high IL-15 expression, an impaired Tregs function and villous atrophy in active CD.