CD62L expression marks SARS-CoV-2 memory B cell subset with preference for neutralizing epitopes.

Severe acute respiratory syndrome coronavirus 2-neutralizing antibodies primarily target the spike receptor binding domain (RBD). However, B cell antigen receptors (BCRs) on RBD-binding memory B (Bmem) cells have variation in the neutralizing activities. Here, by combining single Bmem cell profiling with antibody functional assessment, we dissected the phenotype of Bmem cell harboring the potently neutralizing antibodies in coronavirus disease 2019 (COVID-19)-convalescent individuals. The neutralizing subset was marked by an elevated CD62L expression and characterized by distinct epitope preference and usage of convergent VH (variable region of immunoglobulin heavy chain) genes, accounting for the neutralizing activities. Concordantly, the correlation was observed between neutralizing antibody titers in blood and CD62L+ subset, despite the equivalent RBD binding of CD62L+ and CD62L- subset. Furthermore, the kinetics of CD62L+ subset differed between the patients who recovered from different COVID-19 severities. Our Bmem cell profiling reveals the unique phenotype of Bmem cell subset that harbors potently neutralizing BCRs, advancing our understanding of humoral protection.

Orally administered Lactiplantibacillus plantarum OLL2712 decreased intestinal permeability, especially in the ileum: Ingested lactic acid bacteria alleviated obesity-induced inflammation by collaborating with gut microbiota.

Introduction: Chronic inflammation caused by dietary obesity has been considered to induce lifestyle-related diseases and functional ingredients with anti-inflammatory effects are attracting attention. Although multiple studies on obesity had proved the anti-inflammatory effects of ingestion of lactic acid bacteria (LAB) and other functional ingredients on adipose tissue, the precise effects on the intestine, especially on the individual intestinal segments have not been made clear. In this study, we elucidated the mechanisms of Lactiplantibacillus plantarum (basonym: Lactobacillus plantarum) OLL2712 in suppressing obesity-induced inflammation using high fat diet (HFD)-fed mice obesity model. Methods: We orally administered heat-treated LAB to HFD-fed mice model, and investigated the inflammatory changes in adipose tissue and intestinal immune cells. We also analyzed gut microbiota, and evaluated the inflammation and permeability of the duodenum, jejunum, ileum and colon; four intestinal segments differing in gut bacteria composition and immune response. Results: After 3-week LAB administration, the gene expression levels of proinflammatory cytokines were downregulated in adipose tissue, colon, and Peyer's patches (PP)-derived F4/80+ cells. The LAB treatment alleviated obesity-related gut microbiota imbalance. L. plantarum OLL2712 treatment helps maintain intestinal barrier function, especially in the ileum, possibly by preventing ZO-1 and Occludin downregulation. Discussion: Our results suggest that the oral administration of the LAB strain regulated the gut microbiota, suppressed intestinal inflammation, and improved the gut barrier, which could inhibit the products of obesity-induced gut dysbiosis from translocating into the bloodstream and the adipose tissue, through which the LAB finally alleviated the inflammation caused by dietary obesity. Barrier improvement was observed, especially in the ileum, suggesting collaborative modulation of the intestinal immune responses by ingested LAB and microbiota.

Heterologous SARS-CoV-2 spike protein booster elicits durable and broad antibody responses against the receptor-binding domain.

The immunogenicity of mRNA vaccines has not been well studied when compared to different vaccine modalities in the context of additional boosters. Here we show that longitudinal analysis reveals more sustained SARS-CoV-2 spike receptor-binding domain (RBD)-binding IgG titers with the breadth to antigenically distinct variants by the S-268019-b spike protein booster compared to the BNT162b2 mRNA homologous booster. The durability and breadth of RBD-angiotensin-converting enzyme 2 (ACE2) binding inhibitory antibodies are pronounced in the group without systemic adverse events (AEs) after the S-268019-b booster, leading to the elevated neutralizing activities against Omicron BA.1 and BA.5 variants in the stratified group. In contrast, BNT162b2 homologous booster elicited antibodies to spike N-terminal domain in proportion to the AE scores. High-dimensional immune profiling identifies early CD16+ natural killer cell dynamics with CCR3 upregulation, as one of the correlates for the distinct anti-RBD antibody responses by the S-268019-b booster. Our results illustrate the combinational effects of heterologous booster on the immune dynamics and the durability and breadth of recalled anti-RBD antibody responses against emerging virus variants.

Immune responses related to the immunogenicity and reactogenicity of COVID-19 mRNA vaccines.

Vaccination for the prevention of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is considered the most promising approach to control the pandemic of coronavirus disease 2019 (COVID-19). Although various COVID-19 vaccines have been developed worldwide using several modalities, the vaccines that have shown the highest efficacy to date are mRNA vaccines. Despite their extensive usage, the mechanisms that stimulate the immune responses associated with their immunogenicity and reactogenicity remain largely unknown. In this review, we summarize and discuss current knowledge on immune responses to COVID-19 mRNA vaccines, including potential immune responses and correlating factors underlying the immunogenicity and reactogenicity of mRNA vaccines. We also describe recent trends in the optimization of lipid nanoparticles and vaccination routes. Further understanding of vaccine-elicited immune responses will guide the development of more effective and safe vaccines.

Validating and automating learning of cardiometabolic polygenic risk scores from direct-to-consumer genetic and phenotypic data: implications for scaling precision health research.

INTRODUCTION: A major challenge to enabling precision health at a global scale is the bias between those who enroll in state sponsored genomic research and those suffering from chronic disease. More than 30 million people have been genotyped by direct-to-consumer (DTC) companies such as 23andMe, Ancestry DNA, and MyHeritage, providing a potential mechanism for democratizing access to medical interventions and thus catalyzing improvements in patient outcomes as the cost of data acquisition drops. However, much of these data are sequestered in the initial provider network, without the ability for the scientific community to either access or validate. Here, we present a novel geno-pheno platform that integrates heterogeneous data sources and applies learnings to common chronic disease conditions including Type 2 diabetes (T2D) and hypertension. METHODS: We collected genotyped data from a novel DTC platform where participants upload their genotype data files and were invited to answer general health questionnaires regarding cardiometabolic traits over a period of 6 months. Quality control, imputation, and genome-wide association studies were performed on this dataset, and polygenic risk scores were built in a case-control setting using the BASIL algorithm. RESULTS: We collected data on N = 4,550 (389 cases / 4,161 controls) who reported being affected or previously affected for T2D and N = 4,528 (1,027 cases / 3,501 controls) for hypertension. We identified 164 out of 272 variants showing identical effect direction to previously reported genome-significant findings in Europeans. Performance metric of the PRS models was AUC = 0.68, which is comparable to previously published PRS models obtained with larger datasets including clinical biomarkers. DISCUSSION: DTC platforms have the potential of inverting research models of genome sequencing and phenotypic data acquisition. Quality control (QC) mechanisms proved to successfully enable traditional GWAS and PRS analyses. The direct participation of individuals has shown the potential to generate rich datasets enabling the creation of PRS cardiometabolic models. More importantly, federated learning of PRS from reuse of DTC data provides a mechanism for scaling precision health care delivery beyond the small number of countries who can afford to finance these efforts directly. CONCLUSIONS: The genetics of T2D and hypertension have been studied extensively in controlled datasets, and various polygenic risk scores (PRS) have been developed. We developed predictive tools for both phenotypes trained with heterogeneous genotypic and phenotypic data generated outside of the clinical environment and show that our methods can recapitulate prior findings with fidelity. From these observations, we conclude that it is possible to leverage DTC genetic repositories to identify individuals at risk of debilitating diseases based on their unique genetic landscape so that informed, timely clinical interventions can be incorporated.

SARS-CoV-2-specific CD4+ T cell longevity correlates with Th17-like phenotype.

Determinants of memory T cell longevity following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain unknown. In addition, phenotypes associated with memory T cell longevity, antibody titers, and disease severity are incompletely understood. Here, we longitudinally analyzed SARS-CoV-2-specific T cell and antibody responses of a unique cohort with similar numbers of mild, moderate, and severe coronavirus disease 2019 cases. The half-lives of CD4+ and CD8+ T cells were longer than those of antibody titers and showed no clear correlation with disease severity. When CD4+ T cells were divided into Th1-, Th2-, Th17-, and Tfh-like subsets, the Th17-like subset showed a longer half-life than other subsets, indicating that Th17-like cells are most closely correlated with T cell longevity. In contrast, Th2- and Tfh-like T cells were more closely correlated with antibody titers than other subsets. These results suggest that distinct CD4+ T cell subsets are associated with longevity and antibody responses.

Distinct immune cell dynamics correlate with the immunogenicity and reactogenicity of SARS-CoV-2 mRNA vaccine.

Two doses of Pfizer/BioNTech BNT162b2 mRNA vaccine elicit robust severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibodies with frequent adverse events. Here, by applying a high-dimensional immune profiling on 92 vaccinees, we identify six vaccine-induced immune dynamics that correlate with the amounts of neutralizing antibodies, the severity of adverse events, or both. The early dynamics of natural killer (NK)/monocyte subsets (CD16+ NK cells, CD56high NK cells, and non-classical monocytes), dendritic cell (DC) subsets (DC3s and CD11c- Axl+ Siglec-6+ [AS]-DCs), and NKT-like cells are revealed as the distinct cell correlates for neutralizing-antibody titers, severity of adverse events, and both, respectively. The cell correlates for neutralizing antibodies or adverse events are consistently associated with elevation of interferon gamma (IFN-γ)-inducible chemokines, but the chemokine receptors CCR2 and CXCR3 are expressed in distinct manners between the two correlates: vaccine-induced expression on the neutralizing-antibody correlate and constitutive expression on the adverse-event correlate. The finding may guide vaccine strategies that balance immunogenicity and reactogenicity.

ß-elemene regulates M1-M2 macrophage balance through the ERK/JNK/P38 MAPK signaling pathway.

Macrophages are classified into classically activated M1 macrophages and alternatively activated M2 macrophages, and the two phenotypes of macrophages are present during the development of various chronic diseases, including obesity-induced inflammation. In the present study, ß-elemene, which is contained in various plant substances, is predicted to treat high-fat diet (HFD)-induced macrophage dysfunction based on the Gene Expression Omnibus (GEO) database and experimental validation. ß-elemene impacts the imbalance of M1-M2 macrophages by regulating pro-inflammatory cytokines in mouse white adipose tissue both in vitro and in vivo. In addition, the RAW 264 cell line, which are macrophages from mouse ascites, is used to identify the effects of ß-elemene on inhibiting bacterial endotoxin lipopolysaccharide (LPS)-induced phosphorylation of mitogen-activated protein kinase (MAPK) pathways. These pathways both induce and are activated by pro-inflammatory cytokines, and they also participate in the process of obesity-induced inflammation. The results highlight that ß-elemene may represent a possible macrophage-mediated therapeutic medicine.

SARS-CoV-2 Omicron-neutralizing memory B cells are elicited by two doses of BNT162b2 mRNA vaccine.

Multiple SARS-CoV-2 variants have mutations in the spike receptor binding domain (RBD) with potential to evade neutralizing antibody. In particular, the Beta and Omicron variants escape from antibody neutralizing activity in those who received two doses of BNT162b2 mRNA vaccine. Nonetheless, boosting with a third vaccine dose or by breakthrough infection improves the overall breadth of the neutralizing antibodies, but the mechanism remains unclear. Here, we longitudinally profiled the cellular composition of RBD-binding memory B cell subsets and their antibody binding and neutralizing activity against SARS-CoV-2 variants after the second dose of mRNA vaccine. Two doses of the mRNA vaccine elicited plasma neutralizing antibodies with a limited activity against Beta and Omicron but induced an expanded antibody breadth overtime, up to 4.9 months after vaccination. In contrast, more than one-third of RBD-binding IgG+ memory B cells with a resting phenotype initially bound the Beta and Omicron variants and steadily increased the B cell receptor breadth overtime. As a result, a fraction of the resting memory B cell subset secreted Beta and Omicron-neutralizing antibody when stimulated in vitro. The neutralizing breadth of the resting memory B cell subset helps us understand the prominent recall of Omicron-neutralizing antibodies after an additional booster or breakthrough infection in fully vaccinated individuals.

ß-Elemene Suppresses Obesity-Induced Imbalance in the Microbiota-Gut-Brain Axis.

As a kind of metabolically triggered inflammation, obesity influences the interplay between the central nervous system and the enteral environment. The present study showed that ß-elemene, which is contained in various plant substances, had effects on recovering the changes in metabolites occurring in high-fat diet (HFD)-induced obese C57BL/6 male mice brains, especially in the prefrontal cortex (PFC) and hippocampus (HIP). ß-elemene also partially reversed HFD-induced changes in the composition and contents of mouse gut bacteria. Furthermore, we evaluated the interaction between cerebral metabolites and intestinal microbiota via Pearson correlations. The prediction results suggested that Firmicutes were possibly controlled by neuron integrity, cerebral inflammation, and neurotransmitters, and Bacteroidetes in mouse intestines might be related to cerebral aerobic respiration and the glucose cycle. Such results also implied that Actinobacteria probably affected cerebral energy metabolism. These findings suggested that ß-elemene has regulatory effects on the imbalanced microbiota-gut-brain axis caused by obesity and, therefore, would contribute to the future study in on the interplay between cerebral metabolites from different brain regions and the intestinal microbiota of mice.

Temporal maturation of neutralizing antibodies in COVID-19 convalescent individuals improves potency and breadth to circulating SARS-CoV-2 variants.

Antibody titers against SARS-CoV-2 slowly wane over time. Here, we examined how time affects antibody potency. To assess the impact of antibody maturation on durable neutralizing activity against original SARS-CoV-2 and emerging variants of concern (VOCs), we analyzed receptor binding domain (RBD)-specific IgG antibodies in convalescent plasma taken 1-10 months after SARS-CoV-2 infection. Longitudinal evaluation of total RBD IgG and neutralizing antibody revealed declining total antibody titers but improved neutralization potency per antibody to original SARS-CoV-2, indicative of antibody response maturation. Neutralization assays with authentic viruses revealed that early antibodies capable of neutralizing original SARS-CoV-2 had limited reactivity toward B.1.351 (501Y.V2) and P.1 (501Y.V3) variants. Antibodies from late convalescents exhibited increased neutralization potency to VOCs, suggesting persistence of cross-neutralizing antibodies in plasma. Thus, maturation of the antibody response to SARS-CoV-2 potentiates cross-neutralizing ability to circulating variants, suggesting that declining antibody titers may not be indicative of declining protection.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

A technique for creating an experimental type Ia endoleak model in the thoracic aorta of swine.

PURPOSE: This study aimed to create an animal model of type Ia endoleak that creates persistent problems after thoracic endovascular aortic repair. MATERIALS AND METHODS: In six swine, thoracic aortic aneurysms were created using the harvested jugular vein. We created a type Ia endoleak using a composite stent-graft comprising the first stent-graft (reverse-tapered: thicker part, 16 mm; thinner part, 10 mm) and the second stent-graft (tapered: thicker part, 18-20 mm; thinner part, 16 mm). This double-component stent-graft was deployed in the abdominal aorta and then moved upward to the proximal entry site of the thoracic aneurysm using the inflated balloon for precise positioning. After the surgical procedure and on postoperative day 8, aortography was performed to detect residual endoleak, and then the swine were euthanized. RESULTS: A stable aneurysm (mean size of all aneurysms, 16.8 ± 1.72 mm × 11.8 ± 2.32 mm) and type Ia endoleak were successfully observed in all swine. A single stent-graft was sufficient in one of the six swine. CONCLUSION: A novel technique to create a type Ia endoleak model can be successfully developed in swine.

Association of HLA-DRB1*09:01 with severe COVID-19.

HLA-A, -C, -B, and -DRB1 genotypes were analyzed in 178 Japanese COVID-19 patients to investigate the association of HLA with severe COVID-19. Analysis of 32 common HLA alleles at four loci revealed a significant association between HLA-DRB1*09:01 and severe COVID-19 (odds ratio [OR], 3.62; 95% CI, 1.57-8.35; p = 0.00251 [permutation p value = 0.0418]) when age, sex, and other common HLA alleles at the DRB1 locus were adjusted. The DRB1*09:01 allele was more significantly associated with risk for severe COVID-19 compared to preexisting medical conditions such as hypertension, diabetes, and cardiovascular diseases. These results indicate a potential role for HLA in predisposition to severe COVID-19.

Outpatient Cardiac Rehabilitation Suppresses Deterioration of Renal Function in Patients ≥75 Years of Age With Heart Disease.

BACKGROUND: This study investigated the effect of outpatient cardiac rehabilitation (OCR) and physical activity on the estimated glomerular filtration rate based on serum cystatin C (eGFRcys) in patients with heart disease (HD) aged ≥75 years.Methods and Results:This non-randomized prospective intervention study involved 136 patients (non-OCR group, n=66; OCR group, n=70), 55 of whom were aged ≥75 years (non-OCR group, n=29; OCR group, n=26). Renal function (eGFRcys) was evaluated at discharge and 3 months thereafter. A linear mixed model (LMM) was used to assess changes in renal function over time. The hospital readmission rate within 3 months after discharge was also evaluated. LMM analysis showed that the change in eGFRcys was -2.27 and +0.48 mL/min/1.73 m2in the non-OCR and OCR groups, respectively (F=2.960, P=0.022). Further, among patients aged ≥75 years in the non-OCR and OCR groups, the change in eGFRcys was -3.83 and -1.08 mL/min/1.73 m2, respectively (F=2.719, P=0.039). The proportion of patients aged ≥75 years who were rehospitalized due to exacerbation of HD was 16.9% (n=10) and 6.7% (n=2) in the non-OCR and OCR groups, respectively. CONCLUSIONS: Among patients with HD aged ≥75 years, participation in OCR reduces the decline in renal function and hospital readmission rates.

Myeloid cell dynamics correlating with clinical outcomes of severe COVID-19 in Japan.

An expanded myeloid cell compartment is a hallmark of severe coronavirus disease 2019 (COVID-19). However, data regarding myeloid cell expansion have been collected in Europe, where the mortality rate by COVID-19 is greater than those in other regions including Japan. Thus, characteristics of COVID-19-induced myeloid cell subsets remain largely unknown in the regions with low mortality rates. Here, we analyzed cellular dynamics of myeloid-derived suppressor cell (MDSC) subsets and examined whether any of them correlate with disease severity and prognosis, using blood samples from Japanese COVID-19 patients. We observed that polymorphonuclear (PMN)-MDSCs, but not other MDSC subsets, transiently expanded in severe cases but not in mild or moderate cases. Contrary to previous studies in Europe, this subset selectively expanded in survivors of severe cases and subsided before discharge, but such transient expansion was not observed in non-survivors in Japanese cohort. Analysis of plasma cytokine/chemokine levels revealed positive correlation of PMN-MDSC frequencies with IL-8 levels, indicating the involvement of IL-8 on recruitment of PMN-MDSCs to peripheral blood following the onset of severe COVID-19. Our data indicate that transient expansion of the PMN-MDSC subset results in improved clinical outcome. Thus, this myeloid cell subset may be a predictor of prognosis in cases of severe COVID-19 in Japan.

Reflected wave intensity increases based on aortic diameter after endovascular aortic therapy in a goat model.

Reflected wave increases after endovascular aortic repair (EVAR) in patients with aortic aneurysm. This affects the left ventricular (LV) diastolic function and leads to a poor prognosis. This study aimed to evaluate the relationship between increased reflected wave amplitude and aortic diameter after EVAR. EVAR was performed in seven healthy goats. We assessed wave intensity (WI), aortic diameter, and stiffness parameter ß. Moreover, we evaluated the relationship between negative reflected wave (NW, reflected waves toward the heart from the periphery by WI) and other parameters after EVAR. Results showed an increase in stiffness parameter ß (3.5 ± 0.3 vs 15.9 ± 4.7, p = 0.018) and a decrease in the change of aortic diameter (6.9 ± 0.7 vs 2.7 ± 0.4%, p = 0.018) after EVAR. The NW was significantly amplified after EVAR from baseline (-589.8 ± 143.4 to - 1192.3 ± 303.7 mmHg-m/sec3, p = 0.043). The NW showed a significant correlation with maximum aortic diameter (R = 0.707, p = 0.038) and minimum aortic diameter (R = 0.724, p = 0.033). The reflected wave was enhanced after EVAR and was correlated to the aortic diameter at the stent-graft site. It is important to consider that patients with smaller aortic diameters in landing zone who undergo EVAR may develop LV dysfunction.

Intestinal regulatory T cell induction by ß-elemene alleviates the formation of fat tissue-related inflammation.

The role of the intestinal immune system in the inhibition of fat tissue-related inflammation by dietary material is yet to be elucidated. Oral administration of ß-elemene, contained in various foodstuffs, downregulated expressions of inflammatory cytokines and increased Foxp3+CD4+ T cells in adipose tissue of obese mice. However, ß-elemene did not affect the inflammatory response of adipose tissue in vitro, suggesting that the inhibition observed in vivo was not due to direct interactions of adipose tissue with ß-elemene. Instead, ß-elemene increased Foxp3+CD4+ T cell population enhancing gene expressions of transforming growth factor ß 1, retinaldehyde dehydrogenase 2, integrin αvß8, and interleukin-10 in intestinal dendritic cells (DCs) in vivo and in vitro. Taken together, this study suggested the therapeutic effects of ß-elemene on treating experimental obesity-induced chronic inflammation by adjusting the balance of immune cell populations in fat tissue through the generation of regulatory T cells in the intestinal immune system by modulating DC function.

Age-Dependent Decrease in the Induction of Regulatory T Cells Is Associated With Decreased Expression of RALDH2 in Mesenteric Lymph Node Dendritic Cells.

A decline in immune function with aging has been reported. Regulatory T cell (Treg) induction is known to decrease with age, and elucidating the underlying mechanism is important for preventing age-related diseases due to age-related chronic inflammation. In the intestine, dendritic cells (DCs) play an important role in inducing Tregs specific to oral antigens, and they efficiently induce Tregs via production of retinoic acid (RA), a vitamin A metabolite, catalyzed by the enzyme retinaldehyde dehydrogenase 2 (RALDH2). We have previously reported that in the mesenteric lymph node (MLN), a secondary lymphoid tissue in which immune responses to oral antigens are induced, four DC subsets express different levels of CD11b, CD103, and PD-L1, and we have reported that the CD11b-CD103+PD-L1high subset expresses the highest levels of the RALDH2 gene and induces Tregs in vitro. We examined Treg induction in young and aged mice using a Treg induction model by administering a food antigen, and we found that antigen-specific Treg induction was decreased in aged mice. We further investigated the MLN DCs, and a significant decrease in RALDH2 gene expression was observed in MLN DCs from aged mice. As factors, we found that the proportion of the CD11b-CD103+PD-L1high subset was decreased in aged mice compared with that in young mice and that RALDH enzyme activity was decreased in the CD11b-CD103+PD-L1high and CD11b+CD103+PD-L1high subsets. Furthermore, analysis of the methylation of the RALDH2 gene promoter region revealed that CpG motifs were more methylated in the MLN DCs of aged mice, suggesting that RALDH2 expression was suppressed by epigenetic changes. Finally, we found that RA treatment tended to increase Treg induction. These results suggest that the regulation of RA production may be involved in the age-related decrease in antigen-specific Treg induction.