CD4 IL-17 Research Articles

Human Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Loaded Mir-29-3p Targets AhR to Improve Juvenile Idiopathic Arthritis via Inhibiting the Expression of IL-22 in CD4+ T Cell.

Juvenile idiopathic arthritis (JIA) is one of the most common chronic inflammatory rheumatic diseases in children. Human umbilical cord mesenchymal stem cells (HUCMSCs)-derived exosomes (HUCMSCs-Exos) are involved in autoimmune diseases. This study investigates the mechanism of HUCMSC-Exos in improving JIA by targeting AhR through delivery of miR-29-3p to inhibit IL-22 expression in CD4+ T cells. Collagen induced arthritis (CIA) mouse model was established, and mice were treated with HUCMSCs-Exos and miR-29-3p antagomir, respectively. CD4+ T cells from JIA patients were used for cell experiments. The mechanism was elucidated by histopathological staining, transmission electron microscopy (TEM), immunohistochemistry, CCK-8 assay, flow cytometry, Western blotting, real-time PCR, and enzyme-linked immunosorbent assay (ELISA), laser confocal microscopy, and luciferase assay. JIA-CD4+ T cells showed higher expression of IL-22 and lower the levels of miR-29-3p, while HUCMSCs-Exos significantly inhibited the expression of IL-22 and increased the levels of miR-29a-3p, miR-29b-3p, and miR-29c-3p in CD4+ T cells from JIA patients. The expression of miR-29a-3p, miR-29b-3p, miR-29c-3p, AhR, and IL-22 in CD4+ T cells was significantly reversed when co-cultured with HUCMSCs transfected with miR-29-3p mimic or miR-29-3p inhibitor. In vivo experiment, HUCMSCs-Exos ameliorated CIA mice by delivering miR-29-3p to inhibit AhR, IL-22, IL-22R1, MMP3, and MMP13 expression. Furthermore, HUCMSCs-Exos also deliver miR-29-3p targeting AhR expression to inhibit IL-22 in JIA-CD4 + T cells through alleviating arthritic synovial fibroblast activation. HUCMSCs-Exos loaded miR-29-3p targets AhR to improve JIA via inhibiting the expression of IL-22 in CD4+ T cell, which provides a scientific basis for the treatment of JIA.

The role of polyreactive memory B cells in systemic lupus erythematosus.

In systemic lupus erythematosus (SLE), the production of autoantibodies is a crucial characteristic, and B cells play a significant role in its pathogenesis. B cells are the immune cells most associated with the genetic predispositions of SLE, and recent clinical studies showing that anti-CD19 CAR-T cell therapy induces drug-free remission have underscored the importance of B cells in SLE. Meanwhile, various B cell subsets exist across different stages of differentiation, from naive B cells to plasma-cells, and identifying the important subpopulations within SLE remains a critical future challenge. Years of B cell repertoire analyses have revealed the importance of polyreactive B cell receptors (BCRs) and autoantibodies that react to a various self-antigens and microbial antigens. Particularly, memory B cells with polyreactive BCRs, which play a crucial role in biological defense during the fetal stage, are characteristically differentiated in SLE. Type I interferon-mediated expression of CXCL13 and IL21 in CD4+ T cells is associated with the development of polyreactive memory B cells. The expansion of the polyreactive B cell repertoire, vital for defending against infections such as viruses, may exert an intrinsic function in SLE.

Mechanism of PD-1/PD-L1 in Regulating cTfr/cTfh Balance in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is frequent, an imbalance between helper cells (Th) and regulatory T cells (Treg) is the fundamental immunological cause of RA. This study investigates how recombinant human programmed cell death 1 (PD-L1) protein affects circulating T follicular helper (cTfh), circulating T follicular regulatory (cTfr), and their equilibrium. Magnetic bead sorting was used to select CD4+CXCR5+T cells from RA patients' and healthy individuals' peripheral blood mononuclear cells for in vitro growth. Recombinant human PD-L1 protein stimulated CD4+CXCR5+T cells. Cell counting kit 8 (CCK-8), flow cytometry surface labeling, ELISA, and RT-PCR were used to measure CD4+CXCR5+T cell proliferation inhibition, cTfh and cTfr frequencies, IL-21 expression, and PI3K, AKT, Bcl-6, and Blimp-1 mRNA levels. The recombinant human PD-L1 protein dose-dependently inhibited the proliferation of CD4+CXCR5+T cells in active RA peripheral blood. However, it has a weaker inhibitory effect on healthy peripheral blood CD4+CXCR5+T cells. PD-L1 protein decreased cTfh in active RA peripheral blood CD4+CXCR5+T overall cultured cells but did not affect cTfr; The cTfr/cTfh ratio increased but did not affect the frequency of cTfh and cTfr in healthy persons' cultured CD4+CXCR5+T cells. PD-L1 protein reduced IL-21 in CD4+CXCR5+T cell culture supernatant from active RA peripheral blood. Recombinant human PD-L1 protein lowered PI3K, AKT, and Bcl-6 mRNA in active RA peripheral blood CD4+CXCR5+T cell culture, including significant differences. But Blinmp-1 mRNA variations were neither substantial nor statistically different. PD-1/PD-L1 limits cTfh proliferation, differentiation, and activation via the PI3K/AKT signaling pathway regulates its immunological balance with cTfr, and corrects the cTfr/cTfh imbalance by controlling their interaction.

Fibroblast activation protein-targeted near-infrared photoimmunotherapy depletes immunosuppressive cancer-associated fibroblasts and remodels local tumor immunity.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) play a critical role in tumor immunosuppression. However, targeted depletion of CAFs is difficult due to their diverse cells of origin and the resulting lack of specific surface markers. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment that leads to rapid cell membrane damage. In this study, we used anti-mouse fibroblast activation protein (FAP) antibody to target FAP+ CAFs (FAP-targeted NIR-PIT) and investigated whether this therapy could suppress tumor progression and improve tumor immunity. FAP-targeted NIR-PIT induced specific cell death in CAFs without damaging adjacent normal cells. Furthermore, FAP-targeted NIR-PIT treated mice showed significant tumor regression in the CAF-rich tumor model accompanied by an increase in CD8+ tumor infiltrating lymphocytes (TILs). Moreover, treated tumors showed increased levels of IFN-γ, TNF-α, and IL-2 in CD8+ TILs compared with non-treated tumors, suggesting enhanced antitumor immunity. Cancers with FAP-positive CAFs in their TME grow rapidly and FAP-targeted NIR-PIT not only suppresses their growth but improves tumor immunosuppression. Thus, FAP-targeted NIR-PIT is a potential therapeutic strategy for selectively targeting the TME of CAF+ tumors.

Abstract 2660: CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment

Abstract Background: Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only about 20% of patients with a variety of cancers. Literature has demonstrated that “hot tumor” which contains high T lymphocytes in tumor microenvironment exhibits a better response to immunotherapies than “cold tumor”. Therefore, we hypothesize that T lymphocyte-recruited factors play the roles determining the immunotherapeutic efficacy, including CXCL9, 10, and 11, which are located in human chromosome 4 with consistent co-expression and protein structure. Aims: The aim of this study is to investigate whether CXCL9/10 is critical in determining the anti-PD-(L)1 immunotherapeutic efficacy. Methods: GEPIA, cBioPortal, and Kaplan-Meier Plotter were used to investigate the gene expression, co-expression, and immunotherapeutic association for CD274 (PD-L1), ICAM1, and CXCL9/10 in lung, colon, and liver cancers. qPCR and flow cytometry were used to detect the gene expression in tumor cells and T lymphocytes, particularly comparing the differences between LL/2 and CT26 cells whereas LL/2 is immunotherapy-resistant and CT26 is immunotherapy-sensitive. CD8+ T cell transwell assay and qPCR were used to measure the function of CXCL10 on CD8+ T cell migration and activation, whereas IFNγ and IL-2 as the activation markers. Results: We found that CXCL9/10 overexpressed in a variety of tumors, including lung, colon, and liver tumors with a correlation with PD-L1. However, PD-L1 was not overexpressed in most tumors. High PD-L1 and CXCL10 are basically associated with better survival rates in tumor patients receiving immunotherapies. Correlation analysis revealed that IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression in lung cancer. Since literature has indicated that LL/2-derived syngeneic tumor exhibits immunotherapy-resistant and CT26 exhibits immunotherapy-sensitive, we further compared the gene expression between them. We found that IFNA, IFNG, CXCL10, and PD-L1 all expressed higher levels in CT26 cells than LL/2. Moreover, we found that T lymphocytes specifically expressed high CXCR3, a receptor of CXCL9/10, including CD4+ T and CD8+ T cells by flow cytometry. As expected, CD8+ T cells can be recruited in a transwell assay by IFNα and CXCL9. In addition, we found that IFNα induced CXCL10 expression in monocytes and CXCL10 increased activation markers IFNγ and IL-2 in CD4+ T and CD8+ T cells. Conclusions: This study revealed that CXCL9/10 expression is correlated with PD-L1 in tumors, resulting in better immunotherapeutic efficacy. The evidence demonstrated that CT26 intrinsically exhibited higher IFNs and CXCL9/10 compared to LL/2. Therefore, CXCL9/10 potentially recruits and activates CD8+ T cells to tumor microenvironment, transferring “cold tumor” to “hot tumor”. This study indicates that CXCL9/10 is critical for immunotherapeutic efficacy. Citation Format: Chun-Chia Cheng, Jungshan Chang, Zong-Lin Sie, Ai-Sheng Ho, Cheng-Liang Peng, Chun-Chao Chang. CXCL9/10 is critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2660.

Actives from the Micro-Immunotherapy Medicine 2LMIREG® Reduce the Expression of Cytokines and Immune-Related Markers Including Interleukin-2 and HLA-II While Modulating Oxidative Stress and Mitochondrial Function.

Micro-immunotherapy (MI) is a therapeutic option employing low doses (LD) and ultra-low doses (ULD) of cytokines and immune factors to help the organism at modulating the immune responses. In an overpowering inflammatory context, this strategy may support the restoration of the body's homeostasis, as the active ingredients of MI medicines' (MIM) could boost or slow down the physiological functions of the immune cells. The aim of the study is to evaluate for the first time the in vitro anti-inflammatory properties of some actives employed by the MIM of interest in several human immune cell models. In the first part of the study, the effects of the actives from the MIM of interest were assessed from a molecular standpoint: the expression of HLA-II, interleukin (IL)-2, and the secretion of several other cytokines were evaluated. In addition, as mitochondrial metabolism is also involved in the inflammatory processes, the second part of the study aimed at assessing the effects of these actives on the mitochondrial reactive oxygen species (ROS) production and on the mitochondrial membrane potential. We showed that the tested actives decreased the expression of HLA-DR and HLA-DP in IFN-γ-stimulated endothelial cells and in LPS-treated-M1-macrophages. The tested MIM slightly reduced the intracellular expression of IL-2 in CD4+ and CD8+ T-cells isolated from PMA/Iono-stimulated human PBMCs. Additionally, while the secretion of IL-2, IL-10, and IFN-γ was diminished, the treatment increased IL-6, IL-9, and IL-17A, which may correspond to a "Th17-like" secretory pattern. Interestingly, in PMA/Iono-treated PBMCs, we reported that the treatment reduced the ROS production in B-cells. Finally, in PMA/Iono-treated human macrophages, we showed that the treatment slightly protected the cells from early cell death/apoptosis. Overall, these results provide data about the molecular and functional anti-inflammatory effects of several actives contained in the tested MIM in immune-related cells, and their impact on two mitochondria-related processes.

P029 Loss of adipose tissue protects mice from intestinal inflammation

Abstract Background Crohn’s disease (CD) is associated with creeping fat, characterized by hyperplasia of mesenteric adipocytes and increased secretion of various adipokines, including leptin. Leptin can promote intestinal inflammation by inducing pro-inflammatory cytokines such as TNFα in murine and human lymphocytes. Recent meta-analyses have shown that increased body mass index (BMI) increases the risk of developing CD. However, whether the surgical removal of mesenteric fat could improve the outcome of CD patients and the impact of fat-derived signals on intestinal autoimmunity remain elusive. Methods To decipher the role of adipose tissue in the regulation of intestinal autoimmunity, we investigated here how the absence of adipose tissue affects epithelial barrier functions and immune cell homeostasis. Therefore, we studied lipoatrophic Ppargfl/fl-Adipoq-Cre mice, that completely lack adipose tissue, under steady-state conditions and after induction of chronic dextran sodium sulfate (DSS)-mediated inflammation, as well as in allogeneic fat transplantation models. We characterized our models using flow cytometry, RNA sequencing, immunohistochemistry, western blot analyses, ELISA and calcium measurements. Results At steady state, lipoatrophic mice showed a decrease in CD4 IL-17+ T cells in the ileum, and lower frequencies of CD8 T cells and CD4 FoxP3+ cells in the colon, suggesting that fat-derived signals are required for proper T cell homeostasis in mice. To understand how the absence of adipose tissue affects the severity of intestinal inflammation, we challenged lipoatrophic or WT littermates with 3 cycles of 1.5% DSS, which induced weight loss in both WT and lipoatrophic mice. However, only fat-proficient WT mice developed severe colitis, whereas lipoatrophic animals had significantly reduced intestinal inflammation and displayed disturbances in the differentiation and function of pro-inflammatory T cells including Th1 and Th17 cells. Importantly, these defects in T cell function could be rescued by allogeneic transplantation of adipose tissue from lean wild-type mice but not by adipose tissue from leptin-deficient ob/ob mice, highlighting that the role of the adiopokine leptin as an important pro-inflammatory factor in IBD. Conclusion Taken together, our data demonstrate that adipose tissue acts as an important regulator of intestinal autoimmunity by controlling the differentiation and function of pro-inflammatory T cells through the secretion of adipokines including leptin. We believe that these observations may help explain why obese individuals with higher BMI may be at a higher risk of developing intestinal inflammation and Crohn’s disease.

Immunomodulatory effect of propolis Trigona spp. in mice infected with Toxoplasma gondii.

Toxoplasmosis is a zoonotic disease caused by Toxoplasma gondii. Infection with virulent strains of T. gondii induces excessive production of pro-inflammatory cytokines and increases fatality. Propolis has potential as an anti-inflammatory and immunomodulator. This study aimed to evaluate the pro-inflammatory cytokines response of Interleukin-2 (IL-2) and IL-17, and the anti-inflammatory cytokine IL-10 in mice infected with T. gondii RH strain and treated with ethanol extract of Trigona bee propolis (EEP) from Lombok, West Nusa Tenggara (NTB), Indonesia. A total of 36 mice were divided into six groups with six mice per group. Group 1 was considered as a negative control; mice were not infected and not treated while in groups 2 to 6, mice were infected with 100 T. gondii (tachyzoites). Mice in group 2 were infected but not treated whereas in groups 3, 4, 5, and 6, respectively, were treated orally with Cotrimoxazole 40 mg/kg body weight, EEP 1 mg, EEP 2 mg, and EEP 4 mg, the day after infection for 3 days. All mice under study were euthanized following ethical considerations with prior approvals, on the fifth day post-infection and spleens were taken to observe cytokines using flow cytometry. Statistical analysis was performed with one-way ANOVA and Duncan Multiple Range Test (you may add any statistical tool/software if used?? Like R version?, SPSS, version? If you used any of these/others)? EEP (1-4 mg) increased CD4+ T cells, CD8+ T cells, and CD11b+ cells in infected mice, though not significantly (p > 0.05), unlike cotrimoxazole, which showed significant increases (p < 0.05). EEP at 2 mg and 4 mg (G5 and G6) reduced IL-2 and IL-17 in CD4+ T cells, and at 4 mg (G6) in CD8+ T cells. IL-10 expression increased in CD11b+ cells at 1 mg and in CD4+ T cells at 4 mg (G6). Propolis ethanol extract from Trigona spp. bees acts as an immunomodulator and anti-inflammatory in T. gondii-infected mice, supporting its potential as an anti-toxoplasmosis agent.

CD4+ T cell-derived IL-22 enhances liver metastasis by promoting angiogenesis

ABSTRACT Metastasis is a cancer-related systemic disease and is responsible for the greatest mortality rate among cancer patients. Interestingly, the interaction between the immune system and cancer cells seems to play a key role in metastasis formation in the target organ. However, this complex network is only partially understood. We previously found that IL-22 produced by tissue resident iNKT17 cells promotes cancer cell extravasation, the early step of metastasis. Based on these data, we aimed here to decipher the role of IL-22 in the last step of metastasis formation. We found that IL-22 levels were increased in established metastatic sites in both human and mouse. We also found that Th22 cells were the key source of IL-22 in established metastasis sites, and that deletion of IL-22 in CD4+ T cells was protective in liver metastasis formation. Accordingly, the administration of a murine IL-22 neutralizing antibody in the establishment of metastasis formation significantly reduced the metastatic burden in a mouse model. Mechanistically, IL-22-producing Th22 cells promoted angiogenesis in established metastasis sites. In conclusion, our findings highlight that IL-22 is equally as important in contributing to metastasis formation at late metastatic stages, and thus, identify it as a novel therapeutic target in established metastasis.

Crosstalk within peripheral blood mononuclear cells mediates anti-inflammatory effects of n-3 PUFA-rich lipid emulsions in parenteral nutrition

Parenteral nutrition (PN) rich in n-6 and n-3 long-chain fatty acids is used in clinical practice for nourishing patients who are unable to receive adequate nutrition through their digestive systems. In this study, we compare the effect on inflammation of the commonly used lipid emulsions Omegaven (n-3-rich) and Intralipid (n-6-rich) in human peripheral blood mononuclear cells (PBMCs). PBMCs were treated with different doses of n-3-rich Omegaven and n-6-rich Intralipid and the immune cells were characterized by flow cytometry. We show that incubation of PBMCs with n-3-rich Omegaven leads to an increase in expression of CD1d and CD86 in CD14+monocytes. At the same time, an increased number of NKT cells expressing cytotoxic T cell antigen 4 is observed, suggesting immunological synapse formation. Both CD14+monocytes and NKT cells showed an increase in IL-10 production and a reduction in the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-4, which led to an increase in the number of FOXP3+T regulatory cells. In addition, we show that n-3-rich Omegaven reduces the expression of TNFα, IFNγ and IL-4 in CD4+T and CD8+T cells independent of the presented interaction between CD14+monocytes and NKT cells. The described mechanism of n-3 rich lipid emulsions was confirmed in PBMCs from patients with inflammatory bowel disease but not in colorectal cancer patients which seem to lack the interaction between CD14+monocytes and NKT cells. These results show a mechanism for the beneficial effect of the n-3-rich Omegaven in patients with inflammatory conditions but questions its use in patients with cancer. Hence, our results may assist in choosing the best lipid emulsion for patients who require PN.

Human placental mesenchymal stromal cells modulate IFN-γ and IL-10 secretion by CD4+T cells via CD73, and alleviate intestinal damage in mice with graft-versus-host disease

BackgroundIntestinal inflammatory damage is an important factor in the development of graft-versus-host disease (GVHD). IFN-γ and IL-10 play key roles in gastrointestinal inflammation, and human placental mesenchymal stromal cells (hPMSCs) can alleviate inflammatory damage during GVHD. CD73 is highly expressed by hPMSCs. We aimed to study whether hPMSCs could alleviate intestinal damage in GVHD mice by modulating IFN-γ and IL-10 in CD4+T cells by CD73. MethodsA GVHD mouse model was induced using 8-week-old C57BL/6J and BALB/c mice, which were treated with regular hPMSCs (hPMSCs) or hPMSCs expressing low level of CD73 (shCD73). Then, the levels of IFN-γ and IL-10 in CD4+T cells were determined using flow cytometry. Transmission electron microscopy, western blotting, and morphological staining were employed to observe the intestinal damage. ResultshPMSCs ameliorated pathological damage and inhibited the reduction of the tight junction molecules occludin and ZO-1. They also downregulated IFN-γ and upregulated IL-10 secretion in CD4+T cells via CD73. Moreover, IL-10 mitigated the inhibitory effects of IFN-γ on the expression of occludin in both Caco-2 and NCM460 cells in vitro, but did not affect ZO-1. In addition, hPMSCs upregulated the level of AMPK phosphorylation in CD4+T cells by CD73, which is positively associated with the proportion of CD4+IFN-γ+IL-10+T, and CD4+IFN-γ-IL-10+T cells. ConclusionsOur findings suggested that hPMSCs may balance the levels of IFN-γ and IL-10 in CD4+T cells by promoting the phosphorylation of AMPK via CD73, which alleviates the loss of occludin and ZO-1 in intestinal epithelial cells and, in turn, reduces inflammatory injury in GVHD mice.

Th17/Treg balance is regulated by myeloid-derived suppressor cells in experimental autoimmune myocarditis.

Autoimmune myocarditis is caused by both innate and adaptive immune responses. Many studies have found that myeloid-derived suppressor cells (MDSCs) suppress T-cell responses and reduce immune tolerance, while MDSCs may serve as a key player in inflammatory responses and pathogenesis in variety of autoimmune diseases. However, research into the role of MDSCs in experimental autoimmune myocarditis (EAM) remains lacking. We discovered that the expansion of MDSCs in EAM was closely related to the severity of myocardial inflammation. At an early stage of EAM, both adoptive transfer (AT) and selective depletion of MDSCs could inhibit the expression of IL-17 in CD4+ cells and downregulate the Th17/Treg ratio, alleviating excessive inflammation of EAM myocarditis. In another experiment, in addition, MDSCs transferred after selective depletion could increase IL-17 and Foxp3 expressions in CD4+ cells, as well as the Th17/Treg ratio, contributing to the aggravation of myocardial inflammation. MDSCs promoted the Th17 cell induction under Th17-polarizing conditions in vitro but suppressed Treg expansion. These findings suggest that MDSCs play a plastic role in sustaining mild inflammation in EAM by shifting Th17/Treg balance.

Rab4A controls the overexpression of CD38 and depletion of IL-2 in CD4+ T cells: Potential involvement in proinflammatory lineage development in systemic lupus erythematosus

Abstract Rab4A is a GTPase that is overexpressed in systemic lupus erythematosus (SLE) patients’ T cells and enhances cell surface receptor recycling. Increased expression of CD38 and loss of IL-2 production have independently been associated with proinflammatory T cell development in SLE. We investigated the impact of Rab4A on the expression of CD38 and the production of IL-2. Our lab created Rab4A-mutant Jurkat cell lines, containing GFP-expressing vector alone (control), doxycycline-inducible vectors that overexpress Rab4A (Rab4A hi) or the dominant-negative mutant Rab4A S27N(Rab4A DN). They were co-stimulated with anti-CD3 antibody and PMA. After 24 hours, cell surface markers and cytokines were analyzed by flow cytometry and protein levels by western blot. NAD +levels were measured by LC-MS/MS. In the Rab4A hicells compared to the control, CD38 expression was 2-fold upregulated (p=2.49×10 −13), intracellular production and secretion of IL-2 were significantly decreased (fold change=−0.566, p=1.16×10 −7and fold change= −0.481, p=0.0401, respectively), NAD +concentration was decreased (fold change=0.647, p=0.0039), while pSTAT3 levels were 2-fold increased (p=0.0318). CD38 is an NAD +hydrolase, which regulates Sirtuin-1 activity, a NAD +-dependent histone deacetylase that suppresses STAT3 activity. Elevated pSTAT3 levels may underlie diminished IL-2 production by binding to the promoter of FoxO1, which inhibits IL-2 production. The overexpression of Rab4A and CD38, increased pSTAT3, and diminished IL-2 production reflect changes observed in SLE patients. Our results suggest that increased expression of Rab4A may underlie the overexpression of CD38 and diminished secretion of IL-2 in SLE.

Kv1.3 blockade by ShK186 modulates CD4+ effector memory T-cell activity of patients with granulomatosis with polyangiitis.

Granulomatosis with polyangiitis (GPA) is a chronic relapsing systemic autoimmune vasculitis. Current treatment of GPA is unsatisfactory, as it relies on strong immunosuppressive regimens, with either CYC or rituximab, which reduce the immunogenicity of several vaccines and are risk factors for a severe form of COVID-19. This emphasizes the need to identify new drug targets and to develop treatment strategies with less harmful side effects. Since CD4+ effector memory T cells (TEM) play a key role in the pathogenesis of GPA, we aimed in this study to modulate CD4+TEM cell activity via Kv1.3 blockade using the specific peptide inhibiter, ShK-186. Peripheral blood samples from 27 patients with GPA in remission and 16 age- and sex-matched healthy controls (HCs) were pre-incubated in vitro in the presence or absence of ShK-186, followed by stimulation with phorbol myristate acetate, calcium ionophore and brefeldin-A. The effect of ShK-186 on the cytokine production (IFNγ, TNFα, IL-4, IL-17, IL-21) within total and subsets of CD4+ T helper (CD4+TH) cells were assessed using flow cytometry. ShK-186 reduced the expression level of IFNγ, TNFα, IL-4, IL-17 and IL-21 in CD4+TH cells from patients with GPA in vitro. Further analysis performed on sorted CD4+T cell subsets, revealed that ShK-186 predominantly inhibited the cytokine production of CD4+TEM cells. ShK-186 treatment reduced the production of the pro-inflammatory cytokines to the level seen in CD4+ TH cells from HCs. Modulation of cellular effector function by ShK-186 may constitute a novel treatment strategy for GPA with high specificity and less harmful side effects.

A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus.

Assessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources. We optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients. In conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level.

Antitumor impact of amygdalin on adaptive immune response in BALB/c mice with breast cancer

Amygdalin is a potential therapeutically target in cancer. The main purpose of this experimental study was to evaluate the therapeutic effect of amygdalin in the mice model of breast cancer. The percentages of CD4, CD8 T lymphocyte, intracellular IFN-γ, and Granzyme B were assessed in spleen cells of tumorized mice treated with 50 and 150 mg/kg of amygdalin (AG 50 and AG 150 ). The expression of caspase 3 and p53 , tumor size, and survival rate of Balb/c mice was determined in tumor tissue after amygdalin administration. No significant difference was observed in the frequency of CD4+ and CD8+ T cells in the three study groups. However, a significantly increased level of granzyme B in CD8+ T cells, as well as a significant decrease in the level of IL-10 in CD4+ T cells was detected in the AG 50 group compared to the AG 150 . There was no significant difference in the expression of caspase 3 and P53 between the two groups. A significant change was seen in tumor size and survival rate of AG 50 and AG 150 groups compared to the controls. Our findings indicated that antitumor effect of amygdalin in vivo was probably due to stimulating the effective immune response, not apoptotic genes induction.

Blimp1 suppressed CD4+ T cells-induced activation of fibroblast-like synoviocytes by upregulating IL-10 via the rho pathway.

B lymphocyte-induced maturation protein 1 (Blimp1) is a risk allele for rheumatoid arthritis (RA), but its functional mechanism in RA remains to be further explored. Flow cytometry was performed to detect CD4+ T cell differentiation. ELISA was used to measure inflammatory factor secretion. Lentivirus mediated Blimp1 overexpression vector (LV-Blimp1) or short hairpin RNA (sh-Blimp1) were used to infect CD4+ T cells stimulated by anti-CD28 and anti-CD3 mAbs. RA fibroblast-like synoviocytes (FLSs) were co-cultured with CD4+ T cells or T cell conditioned medium (CD4CM), and cell proliferation, invasion, and expression of adhesion molecules and cytokines in FLSs were evaluated. Mice were injected intradermally with type II collagen to establish a collagen-induced arthritis (CIA) mouse model, and the severity of CIA was evaluated with H&E and Safranin-O staining. Blimp1 knockdown increased pro-inflammatory factor secretion, but downregulated IL-10 concentration in activated CD4+ T cells. Blimp1 overexpression promoted regulatory T cells (Treg) CD4+ T cell differentiation and hindered T helper 1 (Th1) and T helper 17 (Th17) CD4+ T cell differentiation. Blimp1 overexpression suppressed the expression of pro-inflammatory factors and adhesion molecules in CD4+ T cells by upregulating IL-10. Moreover, Blimp1 overexpression impeded the enhanced effect of CD4+ T cells/CD4CM on cell adhesion, inflammation, proliferation, invasion and RhoA and Rac1 activities in FLSs by upregulating IL-10. Additionally, administration with LV-Blimp1 alleviated the severity of CIA. Blimp1 restrained CD4+ T cells-induced activation of FLSs by promoting the secretion of IL-10 in CD4+ T cells via the Rho signaling pathway.

Levels of IL-10 in CD4+ and CD8+ in patients with Type 2 diabetes mellitus and their correlation with mediterranean diet and anthropometric variables

Levels of IL-10 in CD4+ and CD8+ in patients with Type 2 diabetes mellitus and their correlation with mediterranean diet and anthropometric variables

Evidence for a Role of CCR6+ T Cells in Chronic Thromboembolic Pulmonary Hypertension.

IntroductionPrevious studies have shown an increase of T cells and chemokines in vascular lesions of patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, detailed characterization of these T cells is still lacking, nor have treatment effects been evaluated.MethodsWe included 41 treatment-naive CTEPH patients at diagnosis, 22 patients at 1-year follow-up, and 17 healthy controls (HCs). Peripheral blood T cells were characterized by flow cytometry for subset distribution, cytokine expression and activation marker profile. We used multiplex immunofluorescence to identify CCR6+ T cells in endarterectomy tissue from 25 patients.ResultsAt diagnosis, proportions of CCR6+ CD4+ T cells were increased in CTEPH patients compared with HCs. Patients displayed a significantly reduced production capacity of several cytokines including TNFα, IFNγ, GM-CSF and IL-4 in CD4+ T cells, and TNFα and IFNγ in CD8+ T cells. CD4+ and CD8+ T cells showed increased expression of the immune checkpoint protein CTLA4. Multivariate analysis separated CTEPH patients from HCs, based on CCR6 and CTLA4 expression. At 1-year follow-up, proportions of CCR6+CD4+ T cells were further increased, IFNγ and IL-17 production capacity of CD4+ T cells was restored. In nearly all vascular lesions we found substantial numbers of CCR6+ T cells.ConclusionThe observed increase of CCR6+ T cells and modulation of the IFNγ and IL-17 production capacity of circulating CD4+ T cells at diagnosis and 1-year follow-up – together with the presence of CCR6+ T cells in vascular lesions - support the involvement of the Th17-associated CCR6+ T cell subset in CTEPH.

Hepatitis B Virus-Specific Cellular Immunity Contributes to the Outcome of Occult Hepatitis B Virus Infection.

There is little known of immunologic factors leading to the occurrence of occult HBV infection (OBI). Specific cellular immune response to hepatitis B virus (HBV) core/pol peptides was compared between blood donor populations, including 37 OBIs, 53 chronic HBV infections (CHB), 47 resolved infections, and 56 non-infected controls, respectively. The rate of CD4+/CD8+ T cell proliferation in OBI or CHB carriers was higher than in HBV resolved and non-infected individuals (P < 0.05). The intensity of IFN-γ-secretion T-cell response of OBI carriers was highest, followed by CHB and resolved infections, and non-infected individuals (P < 0.05). The frequency of intracellular IFN-γ and IL-17A CD4+/CD8+ and IL-21 CD4+ T-cell responses was significantly higher in resolved infections than in OBI or CHB carriers (P < 0.05), while the level of extracellular IL-17A of peripheral blood mononuclear cells (PBMCs) was higher in OBI and CHB carriers than in resolved infections (P < 0.01). The frequency of intracellular IL-10 CD4+ T-cell response in CHB, OBI, and resolved infections was higher than in HBV non-infected individuals (P < 0.01). Intracellular IL-10 CD8+ T cell and extracellular IL-10 T-cell responses were higher in CHB than in OBI (P = 0.012) or HBV resolved infections (P < 0.01). In conclusion, the higher level of effective T-cell response with IFN-γ, IL-17A, and IL-21 contributes to resolved infection outcome, while higher levels of suppressive T-cell response with IL-10 result in HBV chronicity. OBI is an intermediary status between HBV resolved and chronic infections, in which IL-21 effector and IL-10 suppressor T-cell responses play an important role in directing the outcome of HBV infection.