Tumor-Associated CD19+CD39- B Regulatory Cells Deregulate Class-Switch Recombination to Suppress Antibody Responses.

B cells are an essential component of humoral immunity. Their primary function is to mount antigen-specific antibody responses to eliminate pathogens. Despite an increase in B-cell number, we found that serum-IgG levels were low in patients with breast cancer. To solve this conundrum, we used high-dimensional flow cytometry to analyze the heterogeneity of B-cell populations and identified a tumor-specific CD19+CD24hiCD38hi IL10-producing B regulatory (Breg)-cell subset. Although IL10 is a Breg-cell marker, being an intracellular protein, it is of limited value for Breg-cell isolation. Highly expressed Breg-cell surface proteins CD24 and CD38 also impede the isolation of viable Breg cells. These are hurdles that limit understanding of Breg-cell functions. Our transcriptomic analysis identified, CD39-negativity as an exclusive, sorting-friendly surface marker for tumor-associated Breg cells. We found that the identified CD19+CD39‒IL10+ B-cell population was suppressive in nature as it limited T helper-cell proliferation, type-1 cytokine production, and T effector-cell survival, and augmented CD4+FOXP3+ regulatory T-cell generation. These tumor-associated Breg cells were also found to restrict autologous T follicular helper-cell expansion and IL21 secretion, thereby inhibiting germinal transcript formation and activation-induced cytidine deaminase expression involved in H-chain class-switch recombination (CSR). This isotype-switching abnormality was shown to hinder B-cell differentiation into class-switched memory B cells and subsequent high-affinity antibody-producing plasma B cells, which collectively led to the dampening of IgG-mediated antibody responses in patients with cancer. As low IgG is associated with poor prognosis in patients with cancer, Breg-cell depletion could be a promising future therapy for boosting plasma B cell-mediated antibody responses.

FOXP3/HAT1 Axis Controls Treg Infiltration in the Tumor Microenvironment by Inducing CCR4 Expression in Breast Cancer.

Infiltrating T-regulatory cells in the tumor microenvironment is a key impediment to immunotherapy and is linked to a poor prognosis. We found that tumor-infiltrating Tregs express a higher expression of the chemokine receptor CCR4 than peripheral Tregs in breast cancer patients. CCL22 and CCL17 are released by tumor cells and tumor-associated macrophages, attracting CCR4+ Tregs to the tumor site. The Treg lineage-specific transcription factor FOXP3 changes the CCR4 promoter epigenetically in conjunction with HAT1 to provide a space for FOXP3 binding and activation of the CCR4 gene. To increase CCR4 expression in Tregs, the FOXP3/HAT1 axis is required for permissive (K23 and K27) or repressive (K14 and K18) acetylation of histone-3. In murine breast and melanoma tumor models, genetic ablation of FOXP3 reduced CCR4+ Treg infiltration and tumor size while also restoring anti-tumor immunity. Overexpression of FOXP3, on the other hand, increased CCR4+ Treg infiltration, resulting in a decreased anti-tumor immune response and tumor progression. These findings point to FOXP3 playing a new role in the tumor microenvironment as a transcriptional activator of CCR4 and a regulator of Treg infiltration.

GFI1/HDAC1-axis differentially regulates immunosuppressive CD73 in human tumor-associated FOXP3+ Th17 and inflammation-linked Th17 cells.

Plasticity between Th17 and Treg cells is regarded as a crucial determinant of tumor-associated immunosuppression. Classically Th17 cells mediate inflammatory responses through production of cytokine IL17. Recently, Th17 cells have also been shown to acquire suppressive phenotypes in tumor microenvironment. However, the mechanism by which they acquire such immunosuppressive properties is still elusive. Here, we report that in tumor microenvironment Th17 cell acquires immunosuppressive properties by expressing Treg lineage-specific transcription factor FOXP3 and ectonucleotidase CD73. We designate this cell as Th17reg cell and perceive that such immunosuppressive property is dependent on CD73. It was observed that in classical Th17 cell, GFI1 recruits HDAC1 to change the euchromatin into tightly-packed heterochromatin at the proximal-promoter region of CD73 to repress its expression. Whereas in Th17reg cells GFI1 cannot get access to CD73-promoter due to heterochromatin state at its binding site and, thus, cannot recruit HDAC1, failing to suppress the expression of CD73.

Regulatory lymphocytes: the dice that resolve the tumor endgame

A large number of cancer patients relapse after chemotherapeutic treatment. The immune system is capable of identifying and destroying cancer cells, so recent studies have highlighted the growing importance of using combinatorial chemotherapy and immunotherapy. However, many patients have innate or acquired resistance to immunotherapies. Long-term follow-up in a pooled meta-analysis exhibited long-term survival in approximately 20% of patients treated with immune checkpoint inhibitors or the adoptive transfer of chimeric T cells. It has been reported that high levels of immunoregulatory cells in cancer patients contribute to immunotherapy resistance via immunosuppression. Among the most important regulatory cell subtypes are the CD4+ T-regulatory cells (Tregs), identified by their expression of the well-characterized, lineage-specific transcription factor FOXP3. In addition to CD4+ Tregs, other regulatory cells present in the tumor microenvironment, namely CD8+ Tregs and IL10-producing B-regulatory cells (Bregs) that also modulate the immune response in solid and lymphoid tumors. These cells together have detrimental effects on tumor immune surveillance and anti-tumor immunity. Therefore, targeting these regulatory lymphocytes will be crucial in improving treatment outcomes for immunotherapy.

Incidence and effect of insulin resistance on progression of atherosclerosis in rheumatoid arthritis patients of long disease duration.

BACKGROUND: The continued atherosclerotic risk in rheumatoid arthritis (RA) has been inadequately explained by conventional factors. Chronic inflammation and endothelial activation seems responsible for developing insulin resistance (IR). The study was aimed to assess the role of inflammation and endothelial activation causing IR in long term RA patients leading to increased atherosclerotic risk. METHODS: Fifty (25 long-duration and 25 short-duration) RA patients and twenty-three healthy controls were recruited excluding potential confounding co-morbidities. Fasting insulin, proinflammatory cytokines, endothelial stress markers and adipokines were quantified by ELISA. Homeostasis Model Assessment (HOMA)-IR calculated using glucose and insulin values. Atherosclerotic indices were measured using ultrasound. RESULTS: Lipid profile was comparable among groups. Mean carotid intima media thickness (cIMT) was significantly higher in both RA groups (p = 0.0062) compared to controls. HOMA-IR was significantly higher in long-duration RA (p = 0.005); it showed significant associations with DAS 28 (p = 0.01) and hsCRP (p = 0.03) in this subset. Mean cIMT for short-duration RA (p = 0.02) and long-duration RA (p = 0.0006) respectively was also significantly associated with HOMA-IR. Pro-inflammatory markers like TNF-α, resistin and leptin were highest in long-duration RA, higher in short-duration RA when compared to control group respectively. HOMA-IR was significantly dependent on TNF-α (p = 0.008), resistin (p = 0.031), leptin (p = 0.0054). Mean cIMT showed association with all parameters mainly with TNF-α (p = 0.001), iNOS (p = 0.001), resistin (p = 0.008) and leptin (p = 0.04). CONCLUSIONS: Persistent inflammation leads to altered adipokine secretion promoting IR in RA patients with long disease duration. Treatment with conventional disease modifying anti-rheumatic drugs (DMARDs) is incomplete to control chronic inflammation and limit progression of atherosclerosis.

Association of Angiogenic and Inflammatory Markers with Power Doppler Ultrasound Vascularity Grade and DAS28-CRP in Early Rheumatoid Arthritis: A Comparative Analysis.

OBJECTIVE: Upregulation of various proinflammatory and angiogenic mediators orchestrates the typical pathological synovial alterations in rheumatoid arthritis (RA). DAS28-CRP is commonly used for assessment of RA disease activity and power Doppler ultrasonography (PDUS) is an important modality for assessing synovial vascularity. This study was intended to look for the association of various inflammatory and angiogenic mediators, with respect to different PDUS vascularity grades and disease activity status, in early RA patients. METHODS: 50 early RA patients (<6 months disease duration) with either moderate or high disease activity and 30 healthy controls were included in this study. RA patients were subcategorized based on PDUS vascularity grading of wrist joints. Serum levels of proinflammatory cytokines [tumor necrosis factor-α (TNF- α); interleukin-6(IL-6)] and angiogenic markers [angiopoietin-1 and 2 (Ang-1 and Ang-2); vascular endothelial growth factor (VEGF) ] were measured and compared among different patient subgroups. RESULTS: Among 50 patients, 22 and 28 patients had moderate and high DAS28-CRP score, respectively. Patients with grade 3 PDUS score, even with moderate DAS value, showed a significant rise in Ang-1 (p<0.02), VEGF (p<0.008), Ang-2 (p <0.001), and TNF-α (p<0.005) level compared to grade 2 PDUS patients with high DAS values. CONCLUSION: Higher serum level of angiogenic and inflammatory markers was noted among patients with moderate disease activity but with advanced PDUS vascularity (grade 3) in comparison to high disease activity group with less severe PDUS vascularity (grade 2). PDUS vascularity grading better reflects some markers of angiogenesis and inflammation, than composite disease activity index.

Effects of disease modifying anti-rheumatic drugs on subclinical atherosclerosis and endothelial dysfunction which has been detected in early rheumatoid arthritis: 1-year follow-up study.

OBJECTIVE: The study was designed to explore the effect of disease modifying anti-rheumatic drugs (DMARDs) on synovial inflammation as well as on atherosclerotic indices in patients with early rheumatoid arthritis (RA). METHODS: The study included 35 early RA patients (disease duration <12 months). Inflammatory variables, like erythrocyte sedimentation rate (ESR) and high sensitivity C-reactive protein (hsCRP) were measured. Carotid intima-media thickness (cIMT) and endothelial dependent flow-mediated vasodilatation (ED-FMD) were measured by high-resolution ultrasonography. Disease activity of RA was assessed by disease activity score (DAS28) and quality of life was determined by Health Assessment Questionnaire-Disability Index (HAQ-DI) Score. All the above parameters were assessed both at baseline and follow-up after 1 year. Patients were treated with methotrexate (MTX), hydroxycholoroquine (HCQ) and sulfasalazine (SSZ) depending on their disease activity. RESULTS: After a year of treatment, variables like ESR, hsCRP, DAS28 and HAQ-DI showed significant improvement (p < 0.0001 for each variable). However, there was no such significant change observed in the lipid profile after 1 year from the baseline. Average body mass index (BMI) of patients remained same at the one year follow-up. The cIMT values after 1 year decreased significantly [0.43 ± 0.08mm] from the baseline [0.50 ± 0.16mm] [p = 0.002]. Similarly, in case of FMD%, the post-1-year treatment values [7.57 (4.04-13.03)] improved significantly from the baseline [5.26 (2.9-10.6)] [p = 0.041]. CONCLUSION: Subclinical atherosclerosis and endothelial dysfunction are demonstrable features even in early RA which improved after therapy. Early intervention of RA with DMARDs not only controls the disease but also retards the atherosclerotic progression.

Subclinical atherosclerosis and endothelial dysfunction in patients with early rheumatoid arthritis as evidenced by measurement of carotid intima-media thickness and flow-mediated vasodilatation: an observational study.

OBJECTIVE: In this study, we aimed to investigate the frequency of endothelial dysfunction and subclinical atherosclerosis in early rheumatoid arthritis (RA) patients by carotid intima-media thickness (cIMT) and endothelial-dependent flow mediated vasodilatation (ED-FMD) as compared with healthy controls. METHODS: The study included 35 early RA patients (disease duration <12 months) and 35 healthy controls. Intima-media thickness of common carotid artery and ED-FMD of brachial artery were measured by high-resolution ultrasonography. Disease activity of RA was assessed by Disease Activity Score and activities of daily living were determined by Health Assessment Questionnaire-Disability Index Score. RESULTS: RA patients (age 38.3 ± 10.6 years) had average disease duration of 0.46 ± 0.28 years and 22 patients (62.9%) were rheumatoid factor (RF) positive (RF titer >9.56 IU/mL). There were no significant differences between age, sex, and lipid profiles of patient and control group. cIMT was significantly higher in RA patients (0.50 ± 0.16 mm) than in controls (0.44 ± 0.09 mm) (P = 0.007). Similarly, FMD% was significantly lower in RA patients [5.26 (2.9-10.6)] as compared with controls [10.34 (7.4-14.3)] (P = 0.004). Age, systolic blood pressure, tender joint count, and swollen joint count had significant correlations with patient cIMT. RF titer came out to be the major risk factor for increased cIMT of the patients. CONCLUSIONS: Compared with controls, early RA patients have higher cIMT and lower FMD%, denoting premature atherosclerosis. Our data suggest that early determination of FMD% and cIMT may be useful tools to assess cardiovascular risk even in early RA patients.