Circulating Th17 Cells Research Articles

Upregulated IL-1 Receptor-associated Kinase 1 (IRAK1) in Systemic Lupus Erythematosus: IRAK1 Inhibition Represses Th17 Differentiation with Therapeutic Potential

ABSTRACTSystemic lupus erythematosus (SLE) is a typical autoimmune disease. Genome-wide analyses have revealed that interleukin-1 receptor-associated kinase 1 (IRAK1) is associated with susceptibility to SLE. Our previous study investigated the role of IRAK1 in nuclear factor-κB (NF-κB)-related pathways in a mouse model of lupus. In this study, we aimed to further explore the etiological role of IRAK1. The gene expression and phosphorylation of IRAK1 in CD4+ T cells from lupus patients and healthy controls were examined by quantitative reverse transcription-polymerase chain reaction and western blotting, respectively. The percentage of circulating Th17 cells and plasma IL-17A levels were evaluated by flow cytometry and enzyme-linked immunosorbent assay, respectively. The influence of IRAK1 suppression on Th17 development was assessed using an IRAK1 inhibitor and small interfering RNA. We found that IRAK1 transcript levels in CD4+ T cells were significantly upregulated in SLE patients in comparison to controls and were positively correlated with disease activity. In vitro experiments showed that lupus CD4+ T cells had more pronounced IRAK1 phosphorylation at threonine-209 upon IL-1β stimulation than did control cells. Moreover, IRAK1 expression was positively associated with Th17/IL-17A in patients. When naïve CD4+ T cells were polarized toward the Th17 subset, IRAK1 inhibition significantly repressed IL-17A production and the gene expression of Th17 markers, namely, retinoic acid receptor-related orphan receptor c, IL-23 receptor and IL-17A. In summary, IRAK1 is overexpressed and hyperactivated in CD4+ T cells from SLE patients. IRAK1 inhibition attenuates Th17 differentiation in the context of human SLE, suggesting a therapeutic opportunity.

Placental Ischemia‐Stimulated TH17 Cells Induce Preeclampsia‐Associated Cytolytic Natural Killer Cells During Pregnancy

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many of the characteristics of preeclampsia (PE) including hypertension, intrauterine growth restriction, and increases in circulating TH17 cells (TH17s), IL‐17 and placental cytolytic natural killer cells (cNKs). Although the stimulus for cNKs in PE is currently unknown, recent in vitro studies suggest that IL‐17 induces proliferation and cytolytic activity in human NK cells. We have previously demonstrated that placental ischemia (PI)‐stimulated TH17s mediate pathophysiology in pregnant rats similar to that seen in PE. In this study we tested the hypothesis that PI‐stimulated TH17s induce cNKs and their associated cytokines (IFNγ, TNFα) and cytolytic proteins (perforin, granzymes). We further hypothesized that inhibition of oxidative stress (ROS) with Tempol (Tp) would attenuate NK cytolytic activation. PI‐stimulated TH17s were isolated from RUPP and injected i.p. into normal pregnant (NP) rats on gestation day (GD) 12 with or without Tp administration ad libitum in drinking water (GD 12–19). Circulating TH17s and total NK cells (tNKs) and cNKs in placentas were quantified via flow cytometry in NP, NP+TH17, and NP+TH17+Tp rats. MAP, pup weight, placental ROS, cytokines, perforin and granzymes were measured. Circulating TH17s and IL‐17 were significantly increased after TH17 adoptive transfer. Circulating TH17s (% gated): NP (n=7) − 1.2±0.3%, NP+TH17 (n=7) − 4.2±0.8%, NP+TH17+Tp (n=7) − 0.8±0.2% (p<0.05 vs NP+TH17). MAP increased from 96.3±4.7 mmHg in NP to 117.9±2.2 mmHg in NP+TH17, and was normalized to 102±2.6 mmHg in NP+TH17+Tp (p<0.05 vs NP+TH17s). Placental tNKs (% gated) were significantly increased in NP+TH17 (19.7±4.6%) compared to NP (5.4±2.0%) and NP+TH17+Tp (3.1±1.3%; p<0.05 vs NP+TH17). Importantly, placental cNKs were significantly increased in response to TH17s (NP: 2.9±0.9% vs NP+TH17: 14.9 ±4.0%) and were inhibited with Tp treatment (2.8±1.0%; p<0.05 vs NP+TH17). Pup weight decreased from 2.4±0.04 mg in NP to 1.9±0.1 mg in NP+TH17 and increased to 2.3±0.04 mg in NP+TH17+Tp (p<0.05 vs NP+TH17). Placental ROS increased from 223.5±63 RLU in NP to 574.3±95.3 RLU in NP+TH17 and decreased to 247.8±71.5 RLU in NP+TH17+Tp (p<0.05 vs NP+TH17). Placental inflammation was significantly increased in response to PI‐stimulated TH17s and was attenuated with Tp treatment. Placental IFNγ: NP − 127.4±4.0 pg/mg, NP+TH17 − 153±16.6 pg/mg, NP+TH17+Tp − 116±4.0 pg/mg (p<0.05 vs NP+TH17); Placental TNFα: NP − 76.3±9.2 pg/mg, NP+TH17 − 129.8±11.1 pg/mg, NP+TH17+Tp − 61.5±21.1 pg/mg (p< 0.05 vs NP+TH17). Placental NK cytolytic proteins were significantly increased in response to PI‐stimulated TH17s. Placental perforin: NP − 0.3±0.2 pg/mg NP+TH17 − 1.6±0.2 pg/mg, NP+TH17+Tp − 1.2±0.7 pg/mg in (p<0.05 vs NP); Placental Granzyme A: NP − 91.6±18 pg/mg, NP+TH17 − 180.6±26.6 pg/mg, NP+TH17+Tp − 399.7±54.7 mg/pg (p<0.05 vs NP). These data suggest a role for PI‐stimulated TH17s to induce a cytotoxic phenotype in NKs during pregnancy and identifies stimulation of cNKs as a new mechanism by which TH17s may mediate PE pathophysiology.Support or Funding InformationNIH: HL130456, HD067541This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Catecholamines on Th17 Cells in Multiple Sclerosis

Objective. To study the possibly associations between the clinical features of multiple sclerosis(MS) and quantitative and the qualitative properties of Th17 cells and the dopamine (DA) and serum noradrenaline (NA) concentrations in patients with MS. Materials and methods. Complex neurological and immunological investigations were carried out on 43 patients with remitting MS. All patients were underwent neurological investigations including assessment of levels of disability on the EDSS scale. Serum DA and NA concentrations were determined by immunoenzyme analysis. The proportions of circulating Th-17 cells were assessed by multicolor flow cytometry. The functional activity of Th17- and Th-1 cells was assessed in terms of IL-17 and γ-interferon production by peripheral blood mononuclear cells stimulated with magnetic particles coated with anti-CD3/anti-CD28 antibodies. Results. The proportion of Th-17 cells and cytokine production in the group of MS patients in exacerbation were significantly greater than in remission and in the control group, while the DA level was lower. NA levels in MS patients were identical in exacerbation and remission but were significantly lower than in the control group. Conclusions. These data suggest that catecholamines have an inhibitory effect on Th17 cells in MS.

Effect of induction therapy on circulating T-helper 17 and T-regulatory cells in active proliferative lupus nephritis.

T-helper 17 (Th17) cells and T-regulatory (Treg) cells have been suggested to play pathogenic roles in lupus nephritis. The in vivo effects of current therapies for lupus nephritis (LN) on these cells have not been adequately studied. We conducted a prospective observational study among patients with active proliferative lupus nephritis (LN) who received Eurolupus induction therapy and assessed them as per the European League Against Rheumatism criteria for renal response. Peripheral circulatory Th17 and Treg cell numbers were enumerated at start of therapy, at 3 and 6 months follow-up periods using flow cytometry. Baseline values were compared with inactive lupus patients (iSLE) and healthy controls (HC). Thirty patients with LN, 20 iSLE and 22 HC were enrolled into the study. In LN, Th17 frequency was significantly higher compared to HC, and Treg frequency significantly lower compared to both iSLE and HC. Nineteen patients fulfilled criteria for response (partial or complete) at 6 months. Responder group showed a significant decline in Th17 frequency and an increasing trend in Treg frequency compared to baseline after 6 months of therapy. Circulating Th17 cells were significantly raised in patients with active proliferative LN and showed a significant reduction in responder patients following therapy.

Dynamic changes of T-cell subsets and their relation with tumor recurrence after microwave ablation in patients with hepatocellular carcinoma.

Thermal ablation can evoke an immune response, which may have effects on the prognosis of patients with hepatocellular carcinoma (HCC). Our aim is to investigate the changes of circulating T-cell subsets after microwave ablation (MWA) and to explore the risk factors of tumor recurrence in patients with hepatitis B virus (HBV)-related HCC. Thirty patients with HBV-related HCC were enrolled in this study. The blood samples were collected both before and after MWA (24 h, 72 h, and 1 month after MWA). The distributions of Th17 cells, regulatory Treg-cells, CD4+ T-cells, CD8+ T-cells, and CD3+ T-cells were determined by flow cytometer. The potential-related factors of tumor recurrence were analyzed by logistic regression. The levels of circulating T-cell subsets, except for Th17 cells, were relatively stable after MWA. The frequency of Th17 cells increased from 3.98% ± 2.40% before treatment to 5.53% ±3.27% 24 h after treatment. Eight of 30 patients had a tumor recurrence. The results of logistic regression suggested that among 11 candidates, only the level of Th17 cells was the risk factor of tumor recurrence. To remove the interference from other factors, seven patients with tumor(s) >3 cm or alpha-fetoprotein >400 ng/mL were excluded in another parallel logistic regression. The results of such regression clearly demonstrated that circulating Th17 cells is indeed a related factor of tumor recurrence. Thermal ablation may evoke a transitional immune response by increasing the frequency of Th17 cells. Patients with high levels of baseline circulating Th17 cells, instead of the transient elevation of Th17 cells induced by MWA, are at the risk of tumor recurrence.

Cutaneous toxicities of epidermal growth factor receptor inhibitors: A prospective study in 60 Asian patients.

Several prospective studies have been conducted in epidermal growth factor receptor (EGFR) inhibitor-related cutaneous reactions in Caucasian patients, but prospective studies in Asian populations are scarce. To investigate the cutaneous side effects of EGFR inhibitors in Asian cancer patients and to assess tumor response to dermatologic manifestations. Sixty patients with lung or colorectal cancer who were receiving EGFR inhibitors were prospectively followed for at least one year by oncologists and dermatologists. Of 60 patients (33 males, 27 females), 46 lung cancer patients received erlotinib (n=29) and gefitinib (n=17). Cetuximab was prescribed in 14 colorectal cancer patients. Fifty-eight patients (58/60, 96.7%) developed cutaneous reactions. The most common reactions were xerosis (82.8%), acne (79.3%), and skin desquamation (62.1%). Most reactions were mild and well-tolerated. Of 14 patients who had severe reactions, temporary treatment interruption was necessary in 3 patients and a decreasing dose was required in another 3 patients. There were no statistically significant differences in type, severity, or number of cutaneous reactions between responders (29/58) and non-responders (29/58) to EGFR inhibitors. At median follow-up time of 11.92±1.08 months, no patient died from cutaneous toxicities. Nine patients died from cancer and 11 patients lost to follow-up. In this Asian population, almost all patients (96.7%) developed cutaneous toxicities of EGFR inhibitors. Xerosis, acne, and desquamation were common in Asian cancer patients. Most reactions were mild and well tolerated. Due to limited number of patients, this study did not show significant associations between cutaneous toxicities and tumor response.

Longitudinal Antibody Signatures Following FVIII Replacement Therapy in Previously Untreated Patients with Severe Hemophilia Α- New Insights from the Hemophilia Inhibitor PUP Study (HIPS)

Background and objectivesThe Hemophilia Inhibitor PUP Study (HIPS, clinicaltrials.gov NCT01652027) is a prospective multicenter observational study of PUPs with severe hemophilia A during their first 50 exposure days (EDs) to recombinant Factor VIII (FVIII, Advate TM). The objective is to elucidate immune system changes and identify potential early biomarkers of inhibitor development in PUPs. Previously we reported that the appearance of high-affinity anti-FVIII antibodies preceded clinical diagnosis of FVIII inhibitors in patients (Reipert 2016). Here we present longitudinal antibody data including titers of FVIII-specific IgM, IgG1-4, IgA, apparent affinities of antibodies and titers of FVIII inhibitors for the first 15 patients who completed the antibody analysis of the HIPS study. Moreover, we present longitudinal monitoring data for key circulating immune cells such as FoxP3+ T cells (Tregs), pro-inflammatory Th17 cells, total T cells and granulysin+ NK cells.MethodsFVIII inhibitor testing (Nijmegen methodology, performed in the central laboratory Medical College of Vienna) and antibody analytics were done prior to first exposure, 7-9 days after ED 1 and 5-7 days after EDs 5, 10, 20, 30, 40, and 50. FVIII-specific antibody analytics, including specifications of isotypes, IgG subclasses and apparent affinities were done using methodology described by Whelan 2013 and Hofbauer 2015. Circulating immune cells including FoxP3+ T cells (Tregs), pro-inflammatory Th17 cells, total T cells and granulysin+ NK cells were monitored by epigenetic cell counting in whole blood using quantitative PCR-based methylation assays (www.epiontis.com)Results25 subjects have been enrolled among 15 study sites. Data for 15 subjects who had completed 50 exposure days were available for analysis. Among these 15 subjects, 4 developed FVIII inhibitors by study criteria (> 0.6 B.U. x 2 measurements in central laboratory) at exposure days 6, 10 and 20. All inhibitors were associated with the development of high affinity FVIII-specific antibodies. High-affinity IgG1 antibodies were detected first and subsequently switched to IgG3 and IgG4, switching to IgG2 was observed only in one of the 4 patients. Two of the 4 inhibitor patients initially developed low affinity IgG1 antibodies which were subsequently accompanied by high affinity IgG1 before switching to high-affinity IgG3 and IgG4. One of the 4 inhibitor patients expressed non-neutralizing FVIII-specific IgG1 antibodies prior to first FVIII infusion already and developed a high-titer inhibitor as early as ED6Six of the 15 patients who had completed 50 exposure days developed non-neutralizing antibodies (detected on at least 2 time points). Five of them expressed low affinity IgG1 which was never accompanied by high affinity IgG1 or by any other IgG subclass. One of the 6 patients initially expressed low affinity IgG1 which was later accompanied by non-neutralizing high affinity IgG1. Switching to other IgG subclasses was not observed.Three patients had no antibodies detected and 2 had non-neutralizing antibodies detected at a single time point only.Levels of circulating FoxP3+ Tregs in the infant patients were in the same range as in healthy adults with some variation between individuals. In contrast, circulating TH17 cells were barely detectable. The levels of granulysin+ NK cells showed substantial variations between patients as well as during longitudinal monitoring in the same patient.ConclusionAntibody data obtained from the first 15 patients who completed 50 exposure days in the HIPS study confirms that FVIII inhibitor development is preceded by the detection of high-affinity FVIII-specific antibodies. High affinity IgG1 antibodies appeared first and subsequently switched to IgG3 and IgG4 which indicates a T-cell dependent pathway in FVIII inhibitor development. Switching to IgG2 was only seen in 1 of the 4 inhibitor patients. In contrast, patients with non-neutralizing antibodies only expressed IgG1 antibodies and never switched to another IgG subclass. 5 of the 6 patients with non-neutralizing antibodies only expressed low affinity IgG1 which might reflect a T-cell independent pathway of antibody development.In summary, our data indicate that comprehensive analysis of FVIII-specific antibodies might provide early biomarkers which indicate evolving FVIII inhibitors. DisclosuresGangadharan:Shire: Employment. Reipert:Shire: Employment. Scheiflinger:Shire: Employment. Bowen:Shire: Research Funding. Fijn van Draat:ZonMW,CSL Behring: Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer, Novo Nordisk: Other: Lectures. Gruppo:Shire: Other: Honorarium for participation on physician advisory board. Male:Bayer, Baxalta, Biotest, CSL Behring, Novo Nordisk, Pfizer: Other: Speaker Honoraria and/Travel support. McGuinn:Shire/Baxalta: Consultancy; Shire / Baxalta, Spark, Biogen, Roche/Genetehc: Research Funding. Recht:Baxalta, Novo Nordisk, Biogen, Pfizer: Research Funding; Biogen , Kediron: Consultancy. Ragni:A Anylam,Biomarin,Tecere, Benitec: Honoraria; Alnylam, CSL Behring, Dimensions, Genetech/Roche,Pfizer, Shire, SPARK: Research Funding. Yaish:Baxalta, Bayer and Octapharma, CSL behring: Consultancy, Membership on an entity's Board of Directors or advisory committees; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees. Santagostino:: Bayer, Shire, Pfizer, Novo Nordisk, Kedrion, Roche, Sobi, Bioverativ, CSL Behring, Grifols, Octapharma: Speakers Bureau; Bayer, Shire, Pfizer, Novo Nordisk, Kedrion, Roche, Sobi, Bioverativ: Other: Advisory board. Brown:Shire: Research Funding.

TH17 Cell Frequency in Peripheral Blood Is Elevated in Overweight Children without Chronic Inflammatory Diseases.

The prevalence of obesity has dramatically increased in children in the last few decades and is associated with chronic inflammatory diseases. Fat tissue produces IL-6 and TNF-α, which are stimuli for TH17 cell differentiation. These cells are characterized by expression of the transcription factor receptor-related orphan receptor C (RORC) and by IL-17A production. In murine models, obesity has been linked with elevated TH17 cell frequencies. The aim of this study was to explore whether being overweight was associated with an elevated frequency of circulating TH17 cells or elevated messenger RNA (mRNA)-levels of IL-17A and RORC in children without chronic inflammatory diseases. We studied peripheral blood samples from 15 overweight and 50 non-overweight children without a history of autoimmune diseases, asthma, atopic dermatitis or allergic rhinoconjunctivitis. TH17 cells were quantified in Ionomycin stimulated peripheral blood mononuclear cells by flow cytometry using intracellular IL-17A staining. RORC- and IL-17A expressions were measured by real-time PCR. We found significantly elevated TH cell frequencies in overweight children compared then on-overweight controls with 34.7 ± 1.5% of CD3+CD4+ cells versus 25.4 ± 2.4% (mean ± SEM, p = 0.0023), respectively. Moreover, TH cell frequencies correlated positively with body mass index (r = 0.42, p = 0.0005, respectively). The relative mRNA expression of RORC (p = 0.013) and IL-17A (p = 0.014) were upregulated in overweight compared to non-overweight children. Childhood obesity is an independent factor that is associated with an elevated frequency of circulating TH17 cells and higher expression of RORC- and IL-17A-mRNA after in vitro stimulation with Ionomycin. This might be due to the inflammatory activity of the fat tissue. Studies on TH17 immunity should not only be adjusted for acute and chronic inflammatory diseases but also for overweight.

Th17/Treg Ratio in Serum Predicts Onset of Postoperative Atrial Fibrillation After Off-Pump Coronary Artery Bypass Graft Surgery

The aim of this study is to identify whether the balance between T helper 17 (Th17) cells and CD4+CD25+Foxp3+ regulatory T (Treg) cells could predict the postoperative atrial fibrillation (POAF) after coronary artery bypass graft surgery (CABG). We enrolled 88 patients from Xinhua Hospital who received off-pump CABG (OPCABG) surgery. The baseline characteristics of patients were recorded. The preoperative variables C-reactive protein (CRP) level, left atrial (LA) volume, EuroSCORE I score, CHADS2 score, and CHA2DS2-VASc score were calculated at enrolment. Circulating Th17 and Treg cell frequencies were determined by flow cytometry, and expressions of Th17- and Treg-related cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Compared to patients without POAF, the CRP level and peripheral circulating Th17 cell were significantly increased in POAF patients. Th17/Treg ratio was positively correlated with CRP level, LA volume, CHADS2 score, and CHA2DS2-VASc score. The areas under the receiver-operating characteristic (AUC) curves of Th17/Treg ratio for predicting POAF occurrence was higher than that of CRP level, LA volume, CHADS2 score and CHA2DS2-VASc score. Th17/Treg ratio combined with CRP level has the highest AUC and a greater balance between sensitivity and specificity for predicting POAF. Our data suggest that a Th17/Treg imbalance due to a Th17 shift, representing a pro-inflammatory tendency, participates in the development of POAF. Combining the Th17/Treg ratio with CRP level may provide a more accurate, sensitive, and specific indicator for prediction of POAF.

CD161 identifies polyfunctional Th1/Th17 cells in the genital mucosa that are depleted in HIV-infected female sex workers from Nairobi, Kenya

CD161 identifies a subset of circulating Th17 cells that are depleted in the blood and gut of HIV-infected individuals. In the female reproductive tract (FRT), the pattern of CD161 expression on CD4+ cells remains unknown. Here, we characterized CD161 expression in the FRT of Kenyan female sex workers (FSW). Compared to the blood, CD161+CD4+ T cells were enriched in the FRT of uninfected FSWs. These cells were depleted in FRT of HIV-infected FSWs. Cervical CD161+ cells harboured an activated phenotype (CD69, CD95, HLA-DR) with elevated expression of tissue-homing markers (CCR6, β7 integrin) and HIV co-receptor (CCR5). Mitogen-stimulated production of IL-17 confirmed the Th17 commitment of CD161+CD4+ T cells in the FRT with a predominance of polyfunctional Th1/Th17 cells. Here, we showed that the expression of CD161 on CD4+T cells is modulated at the FRT, but still identified a highly activated cellular subset, which differentiates into pro-inflammatory Th1/Th17 cells, expresses multiple HIV susceptibility markers and are depleted in HIV-infected individuals. The use of CD161 as a biomarker of HIV targets in the FRT reduces the need for functional assessment of cells and could have important implications in better understanding HIV pathogenesis and Th17 fate in the FRT of high-risk women.

Phenotypic and Functional Characteristics of Th17 (CD4+IL17A+) Cells in Human Oral Squamous Cell Carcinoma and Its Clinical Relevance

ABSTRACTRecent studies have suggested an important role of T helper 17 (Th17) cells in tumor biology however, their phenotypic and functional aspects are poorly understood in context with oral cancer. We therefore, investigated the various phenotypic and functional markers of Th17 cells elucidating their relevance in oral squamous cell carcinoma (OSCC). Multi-color flow cytometry (FACs) was used to analyze the frequency and different markers of circulating Th17 cells ex vivo in peripheral blood mono-nuclear cells (PBMCs) from 69 OSCC patients and 35 healthy controls. Percent Mean ± SEM of different types of cells were compared between the two groups using Mann–Whitney U test. We found significantly (p < 0.0001) increased frequency of Th17 cells in patients as compared to controls. These cells were found to express CCR6 profoundly but not CXCR4, CD62L, and CCR7 as chemokine receptors. Additionally, it expressed HLA-DR, CD69, and CD25 moderately but CD28 and CD161 highly. The cytokine profiling revealed 3 subsets namely Th17/1 (IL17A+IFNγ+), Th17/inflammatory (IL17A+IL8+), and Th17/2 (IL17A+IL4+) which were found to be elevated in patients as compared to controls. The early stage patients had a shift toward Th17/1 type and vice versa. Our results suggest that Th17 cells may have effector immune functions in oral cancer immunity through CCR6, CD161, HLA-DR, CD69, CD28 receptors and inducing Th17/1 type of cells expressing polyfunctional antitumor IFNγ cytokine. Thus, novel immune-boosting regimens based on enhancement of Th17 cells in oral cancer patients may provide therapeutic benefits in them.

Abstract P271: Interleukin-4 Supplementation Improves Chronic Inflammation and Hypertension in Response to Placental Ischemia During Pregnancy

Preeclampsia (PE), new onset hypertension in response to placental ischemia during pregnancy, is associated with chronic inflammation characterized by elevated tumor necrosis factor (TNF-α), interleukin-6 (IL-6), agonistic autoantibody to the Ang II type 1 receptor (AT1-AA). In addition, evidence for a shift towards proinflammatory CD4+TH1 vs CD4+TH2 profile exist in PE. Therefore we tested the hypothesis that IL-4, a cytokine essential for TH2 differentiation and proliferation, would improve pregnancy outcomes in response to placental ischemia (RUPP) by shifting the immune profile toward CD4+TH2. Interleukin-4, 600 ng/day, was administered via a mini-osmotic pump on day 14 of gestation into normal pregnant (NP) rats and into RUPP rats immediately following the reduced uterine perfusion pressure (RUPP) procedure and carotid catheters were inserted on gestation day 18. Blood pressure (MAP), TNF-α, IL-6, AT1-AA and circulating TH2 cells were measured on GD 19. MAP in NP rats (n=10) was 96 + 2, 101 + 2 in NP+IL-4 rats (n=14), 130 + 4 in RUPP rats (n=6), which improved to 108 + 2 mmHg in RUPP+IL-4 rats (n=15), p&lt; 0.05. Plasma levels of TNF-α and IL-6 were 25 + 6, 29 + 3 in NP rats (n=7-8/group), 116 + 30, 224 + 62 in RUPP rats (n=9-10/group), which decreased to 23 + 5 and 135 + 48 pg/mL in RUPP+IL-4 rats (n=5-7/group), p&lt;0.05. Importantly, plasma levels of AT1-AA were 0.3 + 0.4 in NP rats (n=8), 0.3 + 0.2 in NP+IL-4 rats (n=5), 18 + 0.3 in RUPP rats (n=9), which were blunted to 4 + 1 bpm in RUPP+IL-4 rats (n=14), p&lt;0.05. Circulating TH2 cells were 17.3 ± 2.7 in NP rats (n=3), 9.0 ± 2.0 in RUPP rats (n=5), which improved to 13.4 ± 3.2 % gated in RUPP+IL-4 rats (n=9). This study illustrates that administration of IL-4 decreases TNF-α, IL-6, AT1-AA, possibly by increasing TH2/IL-4 in RUPP rats, which ultimately led to a reduction in hypertension in response to placental ischemia of pregnancy.

Abstract P260: Placental Ischemia-stimulated TH17 Cells Induce Preeclampsia-associated Cytolytic Natural Killer Cells During Pregnancy

Preeclampsia (PE), a hypertensive disorder of pregnancy, is associated with increased circulating T H 17 cells (T H 17s), IL-17 and cytolytic Natural Killer cells (cNKs). Although the stimulus for cNKs in PE is currently unknown, recent in vitro studies suggest that IL-17 induces proliferation and cytotoxic activity in human NK cells. We have previously demonstrated that placental ischemia (PI)-stimulated T H 17s mediate pathophysiology in pregnant rats similar to that seen in PE. In this study we tested the hypothesis that PI-stimulated T H 17s induce cNKs and their associated cytokines, IFNγ and TNFα, and cytolytic proteins, perforin and granzyme B. T H 17s were injected i.p. into NP rats on gestation day (GD) 12. Total NK cells (tNKs) and cNKs in blood and placental lymphocytes were quantified via flow cytometry. MAP, pup and placental weight, tissue ROS, cytokines, perforin and granzyme B were measured. As previously shown circulating T H 17s and IL-17, MAP, IUGR, and placental and renal ROS were significantly increased after T H 17 adoptive transfer. MAP was 100.6±1.7 mmHg (n=7) in NP rats and 119.4±2.6 mmHg (n=7) in NP recipients of T H 17s (NP+T H 17, p&lt; 0.05). Placental tNKs (% gated) were significantly increased in NP+T H 17 (19.7±4.6%) compared to NP rats (5.4±2.0%; p&lt; 0.05). Importantly, placental cNKs were significantly increased in response to T H 17s (NP: 2.9±0.9% vs NP+T H 17: 14.9 ±4.0%, p&lt; 0.05). Serum and placental IFNγ significantly increased from 5.8±3.3 pg/mL and 1.1±0.6 pg/mL/mg, respectively in NP to 193±26.12 pg/mL and 3.9±0.6 pg/mL/mg in NP+T H 17 (p&lt; 0.05). Serum and placental TNFα increased from 0.9 pg/mL and 0.1 pg/mL/mg in NP to 4.5 pg/mL and 0.2 pg/mL/mg in NP+T H 17 (p&lt; 0.05). Placental perforin was significantly increased from 0.3 pg/mL/mg in NP to 1.6 pg/mL/mg in NP+T H 17 (p&lt; 0.05). Serum perforin, and serum and placental granzyme B were comparable between NP vs NP+T H 17. These data suggest a role for PI-stimulated T H 17s to induce a cytotoxic phenotype in NKs during pregnancy and identifies stimulation of cNKs as a new mechanism by which the T H 17/IL-17 pathway may mediate pathophysiology during PE. Further investigation into this innovative pathway may identify novel targets with therapeutic potential to improve outcomes associated with PE.

IL-22-producing CD4+T cells in the treatment response of rheumatoid arthritis to combination therapy with methotrexate and leflunomide

T cells are key players in immune-mediated rheumatoid arthritis (RA). We previously reported that interleukin (IL)-22+CD4+T helper (IL-22+ Th) cells and IL-22 critically control the pathogenesis of RA. Here we monitored circulating levels of different IL-22+ Th cell subsets and measured plasma levels of IL-22, IL-17, and interferon (IFN)-γ in 60 patients with active RA following 12-week combination methotrexate (MTX) and leflunomide (LEF) therapy (MTX+LEF) and 20 healthy individuals. We found the frequencies of circulating IFN-γ−IL-17−IL-22+ (Th22), IFN-γ−IL-17+ (total Th17), IFN-γ+IL-17−IL-22+ (IL-22+Th1) cells, and IFN-γ−IL-17+IL-22+ (IL-22+Th17) cells, as well as the plasma levels of IL-22, IL-17 and IFN-γ to be significantly reduced in RA patients that responded to treatment, but not in non-responders. Reductions in plasma IL-22 level significantly correlated with percentage of circulating Th22 cells and the decrease of plasma IL-22 level correlated with the reduction of DAS28 in responders. Our data suggests that circulating Th22 cells and plasma IL-22 level play a detrimental role in RA. The combination MTX+LEF therapy, by targeting Th22 cells and reducing IL-22 level, relieves the immune defects and ameliorates symptoms of RA. This study provides novel mechanistic understanding of the pathogenesis of RA, which may promote a design of better therapies for RA.

Role of T helper 22 and 17 cells in pathogenesis of severe preeclampsia

Objective To investigate the role of T helper (Th) 22 and Th17 cells in the pathogenesis of severe preeclampsia. Methods Thirty women with severe preeclampsia who delivered in the Third Affiliated Hospital of Zhengzhou University from October 2014 to February 2016 were enrolled in the study. Thirty healthy pregnant women matched for age and gestational weeks were recruited as the control group. The frequencies of Th22 and Th17 cells in peripheral whole blood were determined by flow cytometry. The concentrations of interleukin (IL)-22 and IL-17A in plasma were detected by enzyme-linked immunosorbent assay. Independent two samples t-test, non-parametric test and Spearman correlation analysis were used for statistical analysis. Results The percentage of Th22 and Th17 cells in the severe preeclampsia group were significantly higher than those in the control group, respectively[Th22 cells: 0.59% (0.39%-1.13%) vs 0.40% (0.23%-0.57%), Z=2.530, P=0.010; Th17 cells: 3.24% (3.02%-3.97%) vs 1.87% (1.53%-2.64%), Z=5.046, P=0.000]. So were the plasma levels of IL-22 and IL-17A[IL-22: 285.72 (247.63-306.69) vs 233.85 (184.92-258.38) pg/ml, Z=4.341, P=0.001; IL-17A: 27.53 (23.84-32.78) vs 17.36 (15.58-19.13) pg/ml, Z=4.924, P=0.000]. There was a positive correlation between circulating Th22 and Th17 cells in the severe preeclampsia group (r=0.534, P=0.015), while no correlation was found in the control group (r=0.345, P=0.136). Positive correlation was found in plasma level of IL-22 with Th22 cells (r=0.600, P=0.005), but not with Th17 cells (r=0.398, P=0.082) in the severe preeclampsia group. Conclusions Increased Th22 cells and high IL-22 concentrations in the peripheral blood of severe preeclampsia patients may indicate a self-defense mechanism in the maternal body. Th22 cells and Th17 cells may interact with each other. Key words: Pre-eclampsia; T-lymphocytes, helper-inducer; Interleukins

A Study of the Changes of T Helper 17 Cells and Regulatory T Cells in Herpes Zoster.

BackgroundImmunosuppression and age-related deficiencies in cell-mediated immunity are important factors for the reactivation of latent varicella-zoster virus (VZV). CD4+CD25+Foxp3+ regulatory T (Treg) cells and T helper 17 (Th17) cells are closely associated with various viral infections.ObjectiveWe analyzed Treg cells and Th17 cells in patients with herpes zoster and investigated their relationship with the reactivation of latent VZV.MethodsTreg and Th17 cells in peripheral blood and the ratio of Th17 to Treg cells were examined in patients with herpes zoster and healthy controls. Changes between pre-treatment and post-treatment estimates of Treg and Th17 cells and clinical parameters in patients with herpes zoster were also analyzed.ResultsThe proportion of circulating Th17 cells and the Th17/Treg cell ratio were significantly higher in patients with herpes zoster than controls (p=0.012, 0.013), but there was no significant difference in the proportion of Treg cells between groups. There was no significant difference in the proportions of Treg and Th17 cells and the Th17/Treg cell ratio before and after treatment and between the non-postherpetic neuralgia and postherpetic neuralgia groups. Changes in Treg and Th17 cells and the Th17/Treg cell ratio were not significantly correlated with changes in the visual analog scale. Body surface area was significantly correlated with Treg cells, Th17 cells, and the Th17/Treg cell ratio (p=0.022, 0.002, 0.004).ConclusionAn imbalance between Th17 and Treg cells is associated with the reactivation of VZV, which may contribute to pathogenesis of herpes zoster.

Changes of circulating Th22 cells in children with hand, foot, and mouth disease caused by enterovirus 71 infection.

Interleukin (IL)-22+CD4+T (Th22) cells play crucial roles in the pathogenesis of autoimmune diseases and infectious diseases, although the role of Th22 cells remains largely unclear in children with hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71). This study aims to explore the role of circulating IL-22+IL-17A−CD4+T (cTh22) cells in children with EV71-associated HFMD. We found that during the acute stage of illness, the frequencies of cTh22 and circulating IL-22+IL-17A+CD4+T (IL-22+cTh17) cells in CD4+T cells infrom affected patients, and especially in severely affected patients, were significantly higher than in healthy controls (HC). The major source of IL-22 production was cTh22 cells, partially from cTh17 cells. Moreover, the protein and mRNA levels of IL-22, IL-17A, IL-23, IL-6, and TNF-α were significantly different among the mild patients, severe patients and HC, as well as AHR and RORγt mRNA levels. A positive correlation was found between plasma IL-22 levels and cTh22 cell frequencies, and cTh17 cell and IL-22+ cTh17 cell frequencies. Furthermore, the frequencies of cTh22 were significantly decreased in the convalescent patients. Our findings indicated that cTh22 cells could play critical roles in the pathogenesis of EV71 infection, and are potential therapeutic targets for patients with EV71-associated HFMD.

Abstract B90: PFK-158 is a first-in-human inhibitor of PFKFB3 that selectively suppresses glucose metabolism of cancer cells and inhibits the immunosuppressive Th17 cells and MDSCs in advanced cancer patients

Abstract Background: 6-phosphofructo-2-kinase (PFKFB3) is an enzyme that controls the intracellular concentration of fructose-2,6-bisphosphate which is an allosteric activator of 6-phosphofructo-1-kinase (PFK-1), a key enzyme of glycolysis. PFK-1 is tightly controlled by multiple metabolic feedback mechanisms and dictates the overall rate of glycolytic flux to lactate and the TCA cycle. In human cancers, several oncogenic proteins (e.g. HIF-1α, PTEN, and AKT) converge to increase the expression and activity of PFKFB3, leading to the high glycolytic rates typically observed in cancer cells. In addition to being a promising cancer metabolism target, PFKFB3 is required for the differentiation and tumor-promoting functions of the immunomodulatory Th17 cells and myeloid derived suppressor cells (MDSCs), which are attractive cellular targets to induce tumor immunity and potentially mediate intrinsic resistance to immune checkpoint inhibitors. PFK-158 is a potent selective small molecule inhibitor of PFKFB3 that displays broad anti-tumor activity causing significant growth inhibition in human and syngeneic preclinical models. As resistance mechanisms frequently activate pathways that result in up-regulation of glycolysis and PFKFB3, combination treatments of PFK-158 with cytotoxic and targeted agents have resulted in increased efficacy and tumor regression. Results: PFK-158 is a potent selective small molecule inhibitor of PFKFB3 that displays broad anti-tumor activity and causes significant growth inhibition in multiple human and syngeneic preclinical models. The tolerability and potential clinical benefit of PFK-158 are being investigated in advanced cancer patients with solid malignancies in a Phase 1 dose-escalation, multi-center clinical trial (clinicaltrials.gov # NCT02044861). The final cohort (650 mg/m2) has been open for enrollment and PFK-158 has been well tolerated to date. Secondary end-points have been incorporated to assess peripheral F2,6BP levels and immunosuppressive and effector cells populations. Of the 15 patients evaluable for response assessment at the end of two months of treatment, 6 patients have experienced a clinical benefit associated with PFK-158 administration, including a late stage pancreatic cancer patient that had a 75% reduction in her CA19-9 levels after 1 month, a renal cell carcinoma patient currently in month 9 and an adenocystic carcinoma patient in month 12. In addition, we examined the immunomodulatory effects of PFK-158 on Th17 cells and MDSCs in vitro, in B16 melanoma-bearing mice and in advanced cancer patients and found that PFK-158: (i) suppresses human Th17 cell and MDSC differentiation in vitro; (ii) decreases splenic and tumor-infiltrating Th17 cells, γδ T17 cells and MDSCs, and increases CD4+ and CD8+ T cells in the tumors of B16-F10 melanoma-bearing mice; and (iii) decreases peripheral blood Th17 cells, γδ T17 cells and MDSCs and increases activated effector CD4+ and CD8+ T cells in advanced cancer patients. Interestingly, we are discerning a correlation between the initial level of circulating Th17 cells and clinical responses to PFK-158. Conclusion: PFK-158 is the first-in-human and first-in-class PFKFB3 inhibitor that is currently under clinical development. To date, PFK-158 has been well tolerated and shows signs of clinical activity. In addition to controlling glycolysis, over expression of PFKFB3 in key immunesuppressive cells also leads to an immunomodulatory mechanism of action, suggesting that additional clinical benefit could result from combining PFK-158 with targeted agents as well as with immunotherapeutic agents. Note: This abstract was not presented at the conference. Citation Format: Sucheta Telang, Kavitha Yaddanapudi, Jaspreet Grewal, Rebecca Redman, Siqing Fu, Paula Pohlmann, Devalingam Mahalingam, Michael Kurman, Gilles Tapolsky, Jason Chesney.{Authors}. PFK-158 is a first-in-human inhibitor of PFKFB3 that selectively suppresses glucose metabolism of cancer cells and inhibits the immunosuppressive Th17 cells and MDSCs in advanced cancer patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B90.

Gene Networks and Canonical Pathways Analysis in Classical Hodgkin Lymphoma Patients

Introduction: Molecular alterations involved in development of classical Hodgkin lymphoma (cHL) are only partially known. Genetic alterations in NFkB pathway and the imbalance of T regulatory (Treg) and TH17 lymphocytes has been recognized as critical pathogenetic mechanism involved in immune scape and blockade of apoptosis in Reed-Sternberg cells. Emerging evidences suggests that NFkB activation can promote lymphocyte proliferation and survival, and therapeutic inhibition of the NFkB pathway have been proposed. Recently, our group showed an increase of circulating Treg cells in patients with cHL at diagnosis, however, after therapy, elevated levels of circulating TH17 cells was observed. Increased frequencies of Treg lymphocytes in the tumor microenvironment and peripheral blood have been proposed as one of the mechanisms for this anergic state. TH17 exhibit effector functions in immune system and tumor-infiltrating TH17 cells are associated with better prognosis in human.Objectives: In this study we aimed to evaluate the immune gene expression profile in whole blood of cHL patients at diagnosis and after treatment, and evaluate pathways and gene interaction networks.Methods: This is an open multicenter study and, so far, we included 51 patients consecutively from February 2011 to November 2015. Twenty consecutively diagnosed cHL patients, with whole blood RNA extracted at diagnosis and after treatment, were recruited for this study and prospectively evaluated. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. The general expression of 96 messengers RNAs present in the peripheral blood and involved in immune response was performed by a customized quantitative real-time PCR array (TaqMan¨ Low Density Array). The data was normalized with B2M mRNAs levels and relative gene expression was calculated by the 2^DDCt method, considering Wilcoxon test and Benjamini-Hochberg adjustment to correct p-values. The differentially expressed genes were used to conduct functional/pathway enrichment analysis and construct gene interaction networks. Functional annotation networks were generated using Ingenuity Pathway Analysis (IPA; Ingenuity Systems) software.Results: From the 20 patients included for this study, 12 (60%) were male, 5 (31%) had Epstein Barr virus related cHL, 18 (90%) patients presented with B symptoms, 19 (95%) patients had advanced diseases at diagnosis (stage IIBX, III and IV) and 10% of patients relapsed. We considered paired samples from 15 patients before and after treatment. At diagnosis we observed that cHL patients presented higher expression of CD274 (2 fold, p=0.018), CD28 (1.5 fold, p=0.041), CTLA-4(1.5 fold, p=0.004), FAS (1.4 fold, p=0.041), ICOS (2.1 fold, p=0.015) and IL-10 (2.7 fold, p=0.002), and decreased expression of CCL2 (-2.7 fold, p=0.026), CCL5 (-1.6 fold, p=0.012), CD40 (-1.9 fold, p=0.003), CSF2 (-1.9 fold, p=0.012). We found no association between clinical and epidemiological characteristics with immune gene expression profile. We have found that after treatment, patients displayed a specific gene expression profile related to the top 5 canonical pathways summarized in following figure:We also built a biological network to investigate the connection between the differentially expressed genes and to predict the status (activation/inhibition) of the other connected genes (nodes). The in silico analysis revealed that NFkB, a node which its signaling activation is predicted to be activated before treatment and inhibited after (following figures). The network also showed different central node molecules (molecules connected with multiple other nodes from the network), that are indirectly related to Hodgkin lymphoma and should be further investigated as potential drugable targets.Conclusions: In this study, we showed that, at diagnosis, cHL patients presented an inflammatory gene expression profile in blood that changes after treatment to an effector/immunological one. NFkB is predicted to be activated before treatment and inhibited after ABVD chemotherapy and radiotherapy. This kind of system biology approach helped us to understand that cHL associated immunosuppression and the immune reconstitution after treatment maybe the key to develop new prognostic factors and treatment strategies as well as identify drugable targets. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Increase in circulating Th17 cells during anti-TNF therapy is associated with ultrasonographic improvement of synovitis in rheumatoid arthritis.

BackgroundAnti-TNF agents have revolutionised rheumatoid arthritis (RA) treatment; however, a third of patients fail to achieve therapeutic responses. Unexpectedly, studies in murine and human arthritis have indicated that anti-TNF treatment can increase circulating T helper 17 (Th17) cells, but the relationship to treatment response is unclear. To identify immune correlates of anti-TNF treatment response, we conducted a longitudinal study using clinical, ultrasound and T cell assessments.MethodsPatients with RA (n = 25) were studied at protocol visits during the initial 12 weeks of anti-TNF treatment. Improvement in the disease activity score of 28 joints (DAS28) >1.2 defined treatment responders (n = 16) and non-responders (n = 9). Changes in synovial thickening and vascularity of 10 metacarpophalangeal joints were quantitatively assessed by grey scale and power Doppler ultrasound. The frequency of circulating Th17 cells was determined by IL17 enzyme-linked immunospot assay (Elispot) and flow cytometry (fluorescence-activated cell sorting (FACS)).ResultsThe frequency of circulating IL17-producing cells increased significantly 12 weeks after anti-TNF initiation (Elispot median (range) specific spot forming cells (spSFC)/106 360 (280–645) vs 632 (367 − 1167), p = 0.003). The increase in CD4 + IL17+ cells at 12 weeks was confirmed by FACS (median (range) %, 0.7 (0.5–0.9) vs 1.05 (0.6–1.3); p = 0.01). The increase in circulating Th17 cells inversely correlated with reduction in synovial vascularity (r = -0.68, p = 0.007) and thickening (r = -0.39; p = 0.04). Higher frequencies of circulating Th17 cells at baseline were associated with poorer anti-TNF treatment response defined by ultrasonographic measures.ConclusionsThese results demonstrate a link between changes in circulating Th17 cells with resolution of ultrasonographic features of synovial inflammation and vascularity during anti-TNF treatment. The findings may reflect redistribution of Th17 cells from inflamed joints or TNF-driven regulation of Th17 cell production.Trial RegistrationClinicalTrials.gov: NCT01060098. Registered 29 January 2010.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1197-5) contains supplementary material, which is available to authorized users.