In agreement with the expression of T-bet, both human DN B cells and mouse ABCs secrete anti-ds-DNA (our results herein) or anti-chromatin (55) autoimmune IgG antibodies. of DN B cells from the peripheral B cell pool significantly decreases secretion of anti-self IgG antibodies. These results altogether confirm the crucial role of DN B cells in the secretion of anti-self IgG antibodies in individuals with obesity. Keywords: aging, B cells, obesity, inflammation, antibody responses Introduction Obesity, defined as body-mass index (BMI) 30 kg/m2 by the Centers for Disease Control and Prevention and the World Health Organization, is usually a condition associated with chronic low-grade systemic inflammation, known as inflammaging (1). Inflammaging has been shown to induce chronic immune activation (IA), which contributes to functional impairment of immune cells and decreased immunity. Obesity and associated inflammation lead to several debilitating chronic diseases such as type-2 diabetes, cancer, atherosclerosis, and inflammatory bowel disease (2C9). We have previously shown that obesity is associated with decreased antibody responses to the influenza vaccine and decreased B cell function (10), measured by activation-induced cytidine deaminase (AID) after or stimulation with mitogens, antigens and vaccines. AID is the enzyme that regulates Ig class switch recombination (CSR) and somatic hypermutation (SHM) (11), two processes leading to the generation of high affinity protective antibodies (12C14). The reduced CCNG1 B cell resposes in individuals with obesity are likely due to the fact that B cells from obese individuals, as compared to those from lean individuals, are enriched in memory B cells, and in particular in the subset of Double Unfavorable (DN) B cells, which is the most pro-inflammatory B cell subset (10, 15), reported to be increased in the blood of individuals with inflammatory conditions and diseases. These include aging (16C18), autoimmune diseases such as Rheumatoid Arthritis (19), Systemic Lupus Erythematosus (SLE) (20, 21), Multiple Sclerosis (22), Alzheimers disease (23), Sjogrens disease (24) and pemphigus (25). DN B cells have also been reported to be increased in the blood of patients affected by chronic infectious diseases such as HIV (26), Hepatitis C (27) and Malaria (28). These results have suggested that these cells likely expand after chronic exposure to autoantigens or pathogen-derived antigens, leading to the production of autoimmune or protective antibodies, respectively. DN B cells are also expanded in the blood of COVID-19 patients and associated with anti-viral antibody responses and poor clinical outcomes, as recently shown (29). In this paper, we show that this plasma of individuals with obesity is usually enriched in Insulin levels modulator anti-self IgG antibodies and we tested three different antigenic specificities: double strand (ds)DNA, malondihyldehyde (MDA) and adipocyte-derived antigens. We Insulin levels modulator selected these antigenic specificities because obesity is associated with increased DNA damage (measured by dsDNA) (30), increased oxidative stress and lipid peroxidation (measured by MDA) (31, 32), and increased excess fat mass (measured by adipocyte-associated antigens released by the adipose tissue) (33). Plasma levels of these anti-self IgG antibodies are positively associated with blood frequencies of DN B cells. We confirmed our previous findings that this frequencies of DN B cells are increased in the blood of obese versus lean individuals. Moreover, we found that DN B cells show higher expression of IA markers and of the transcription factor T-bet associated with autoimmunity. The removal of DN B cells from the total B cell pool significantly reduced Insulin levels modulator secretion of anti-self IgG antibodies. These results reveal a critical role for DN B cells in the secretion of anti-self IgG antibodies in individuals with obesity. Materials and Methods Subjects Experiments were performed using blood isolated from lean (n=20, 30C54 years) and obese (n=20, 27C55 years) adult female individuals, with average body Mass Index (BMI, kg/m2) 21 1 and 42 3, respectively. The individuals participating in the study were screened for diseases known to alter the immune response or for consumption of medications that could alter the immune response. We excluded subjects with autoimmune diseases, congestive heart failure, cardiovascular disease, chronic renal failure, malignancies, renal or hepatic diseases, infectious disease, trauma or surgery, pregnancy, or documented current material and/or alcohol abuse. Study participants provided written informed consent. The study was reviewed and approved by our Institutional Review Board (IRB, protocols 20070481 and 20160542), which reviews all human research conducted under the auspices of the University of Miami. PBMC Collection PBMC were collected using Vacutainer CPT tubes (BD 362761) and cryopreserved. PBMC (1×106/ml) were thawed and cultured in complete medium (c-RPMI, RPMI Insulin levels modulator 1640, supplemented with 10% FCS, 10 g/ml Pen-Strep, 1 mM Sodium Pyruvate, and 2 x 10C5 M 2-ME and 2 mM L-glutamine). Flow Cytometry After thawing, PBMC (2 x 106/ml) were stained for 20?min at room heat with the following antibodies: anti-CD45.