Rheumatoid arthritis patients treated with Janus kinase inhibitors show reduced humoral immune responses following BNT162b2 vaccination.

OBJECTIVES: The mRNA-based COVID-19 vaccines are now employed globally and have shown high efficacy in preventing SARS-CoV-2 infection. However, less is known about the vaccine efficacy in immune-suppressed individuals. This study sought to explore whether humoral immunity to the COVID-19 vaccine BNT162b2 is altered in RA patients treated with Janus kinase inhibitors by analysing their antibodies titre, neutralization activity and B cell responses. METHODS: We collected plasma samples from 12 RA patients who were treated with Janus kinase inhibitors and received two doses of the BNT162b2 vaccine, as well as 26 healthy individuals who were vaccinated with the same vaccine. We analysed the quantity of the anti-spike IgG and IgA antibodies that were elicited following the BNT162b2 vaccination, the plasma neutralization capacity and the responsiveness of the B-lymphocytes. We used ELISA to quantify the antibody titres, and a plasma neutralization assay was used to determine the virus neutralization capacity. Alteration in expression of the genes that are associated with B cell activation and the germinal centre response were analysed by quantitative PCR. RESULTS: Reduced levels of anti-spike IgG antibodies and neutralization capacity were seen in the RA patients who were treated with JAK inhibitors in comparison with healthy individuals. Furthermore, B cell responsiveness to the SARS-CoV-2 spike protein was reduced in the RA patients. CONCLUSION: RA patients who are treated with JAK inhibitors show a suppressed humoral response following BNT162b2 vaccination, as revealed by the quantity and quality of the anti-spike antibodies.

The association between elevated serum interleukin-22 and the clinical diagnosis of axial spondyloarthritis: A retrospective study.

OBJECTIVE: There is an unmet need for a reliable biomarker for the differentiation of axial spondyloarthritis (AxSpA) from its mimickers. Serum levels of interleukin-22 (IL-22) have previously been found to be significantly elevated in patients with AxSpA compared with healthy individuals or persons with osteoarthritis. METHODS: Consecutive patients with established or suspected AxSpA were enrolled. The clinical data, as well as results of laboratory and imaging studies, were acquired from patients' charts. The final diagnosis of definite or probable SpA, or an alternative diagnosis, was determined, and the serum levels of IL-22 were examined by enzyme-linked immunosorbent immunoassay. RESULTS: Interleukin-22 levels were significantly higher in patients with definite AxSpA (29 patients) compared with patients with alternative diagnoses (14 patients) and healthy volunteers (16 individuals; P < 0.001 for both comparisons). The sensitivity and specificity of the serum IL-22 for the AxSpA diagnosis were 0.68 (95% CI 0.49-0.84) and 0.86 (95% CI 0.68-0.95), respectively, for the cut-off value of 5 pg/mL. In patients with AxSpA, serum IL-22 levels did not correlate with modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Ankylosing Spondylitis Disease Activity Score (ASDAS), or serum C-reactive protein. CONCLUSION: Serum IL-22 levels are elevated in patients with the clinical diagnosis of AxSpA and can potentially serve as an independent biomarker for the differentiation of AxSpA from its non-inflammatory mimickers.

Ultrafine particles in airways: a novel marker of COPD exacerbation risk and inflammatory status.

PURPOSE: Ultrafine particles (UFP) are toxic due to their small size and penetration into deeper lung compartments. We aimed to evaluate the exhaled breath condensate (EBC)-UFP content as a reflection of inflammation and oxidative stress status in COPD patients and as an exacerbation risk marker. METHODS: EBC was collected by conventional methods. Particles were analyzed with NanoSight LM20. EBC carbonyl and 8-hydroxydeoxyguanosine (8-OHdG) levels were measured using ELISA kits. Study population (58 COPD patients and 40 healthy smoker and non-smoker controls) underwent spirometry, diffusion capacity, EBC testing, and blood sampling. RESULTS: Absolute eosinophil count, C-reactive protein (CRP), and lactate dehydrogenase in serum were elevated in the COPD group compared with the controls (224 U/L, 5 mg/L, and 391 U/L vs 154 U/L, 3 mg/L, and 330 U/L, P=0.009, P=0.05, and P=0.004, respectively). COPD patients had lower UFP concentrations in EBC compared with controls (0.24 E8/mL vs 0.51 E8/mL, P≤0.001). A mirror image was detected in serum: COPD patients had higher UFP concentrations compared with controls (9.8 E8/mL vs 6.7 E8/mL, respectively, P=0.03). EBC carbonyl and 8-OHdG levels were higher among COPD patients compared with controls (5.1 per 1 µg/mL protein and 0.036 ng/mL vs 0.41 per 1 µg/mL protein and 0.003 ng/mL, P=0.001 and P≤0.001, respectively). EBC UFP concentrations were negatively correlated with pack years (R=-0.44, P ≤0.001) and positively correlated with FEV1 and diffusing lung capacity for carbon monoxide (R=0.46, 0.23, P ≤0.001 and P=0.04, respectively). Low EBC UFP concentrations (≤0.18 E8/mL) and CRP levels ≥5 mg/L were independent predictors of the frequent exacerbator phenotype (OR 3.6; 95% CI: 1.06-7.97; P=0.04 and OR 4.4; 95% CI: 1.24-10.2; P=0.02, respectively). CONCLUSION: UFP content in EBC reflects the inflammatory state of airways. Low UFP concentrations in EBC and high in serum of COPD patients support our hypothesis that increased epithelial permeability could be the mechanism behind those findings.

The association between semaphorin 3A levels and gluten-free diet in patients with celiac disease.

Celiac disease (CD) is an inflammatory disease affecting the small intestine. We aim to assess serum level and expression of semaphorin 3A (Sema3A) on T regulatory (Treg) cells in CD patients. Twenty-six newly diagnosed celiac patients, 13 celiac patients on a gluten-free diet and 16 healthy controls included in the study. Sema3A protein level in the serum of celiac patients was significantly higher compared to healthy group (7.17±1.8ng/ml vs. 5.67±1.5ng/ml, p=0.012). Sema3A expression on Treg cells was statistically lower in celiac patients compared to healthy subjects (p=0.009) and significantly lower in celiac patients compared to celiac patients on gluten free diet (p=0.04). Negative correlation was found between Sema3A on Teg cells and the level of IgA anti-tTG antibodies (r=-0.346, p<0.01) and anti-DGP (r=-0.448, p<0.01). This study suggests involvement of the Sema3A in the pathogenesis of CD.

The Expansion of CD25 high IL-10 high FoxP3 high B Regulatory Cells Is in Association with SLE Disease Activity.

B regulatory cells (Bregs) belong to a subgroup of activated B cells tasked with maintaining self-tolerance and preventing autoimmunity. While sharing similar regulatory mechanisms such as IL-10 dependency, they also defer in exhibiting their suppressive effects by expressing Fas-Ligand, TGF-beta, and PDL-1. In this study we show, for the first time, the expansion of CD25(high)FoxP3(high) Bregs in systemic lupus erythematosus (SLE) patients compared to healthy individuals (18.5 ± 3.052% versus 11.0 ± 1.654%, p < 0.001, resp.). This expansion was also shown to correlate with SLE disease activity (r = 0.75). In addition, CD25(high)FoxP3(high) Bregs were also IL-10(high) expressing and further expanded when stimulated with semaphorin 3A. In sum we show that CD25(high)FoxP3(high) are an additional subtype of Bregs, involved in regulating SLE disease activity. Being IL-10 expressing, we may assume that they are one of the sources of increased serum IL-10 in SLE patients. Further studies are required in order to assess the relation between high serum IL-10 and CD25(high)FoxP3(high) Breg cells.

Immunization of pregnant women against pertussis: the effect of timing on antibody avidity.

BACKGROUND: The Centers for Disease Control and Prevention recommend tetanus-diphteria-acellular pertussis (Tdap) immunization during pregnancy, preferably at 27-36 weeks gestation. AIMS: First, to assess the relative avidity index (RAI) of umbilical cord immunoglobulin G (IgG) to pertussis toxin (PT) for newborns of women immunized with Tdap during late pregnancy as compared to unimmunized women. Second, to assess whether there is a preferential period of gestational Tdap immunization that provides the highest RAI of umbilical cord IgG to PT. METHODS: RAI of IgG to PT was assessed via an adapted ELISA using NH4SCN as a dissociating agent. RESULTS: We found that newborns of women immunized with Tdap during late pregnancy (n=52) had higher mean RAI of umbilical cord IgG to PT than those of unimmunized women (n=8), 73.77%±12.08 (95% CI, 70.41-77.13) vs. 50.23%±21.32 (95% CI, 32.41-68.06), p<0.001. Further, the RAI of umbilical cord IgG to PT was significantly higher in newborns of women immunized at 27-30(+6) weeks gestation (n=20) when compared with newborns of women immunized at 31-36 weeks (n=22) and >36 weeks (n=7), 79.53%±5.61 (95% CI, 76.91-82.16) vs. 71.56%±12.58 (95% CI, 65.98-77.14) vs. 63.93%±17.98 (95% CI, 47.31-80.56), p<0.03. CONCLUSION: Gestational Tdap immunization between 27 and 30(+6) weeks resulted in the highest avidity of IgG to PT conveyed at delivery as compared with immunization beyond 31 weeks gestation. Future studies should be conducted to confirm our findings to optimize pertussis-controlling strategies.

The induction of breast milk pertussis specific antibodies following gestational tetanus-diphtheria-acellular pertussis vaccination.

BACKGROUND: Center for Disease Control and Prevention recommends vaccination of pregnant women with tetanus-diphtheria-acellular pertussis (Tdap). AIM: To measure pertussis specific antibodies, total protein and their ratio in breast milk following gestational Tdap vaccination. METHODS: Women who received Tdap after the 20th week of pregnancy were recruited and unvaccinated women served as controls. Breast milk total protein, immunoglobulin A (IgA) to pertussis toxin (PT), filamentous hemagglutinin (FHA) and immunoglobulin G (IgG) to PT, FHA and pertactin (PRN) were measured. To overcome the dilution that occurs in the transition from colostrum to mature breast milk, we calculated pertussis specific antibody to total protein ratio. RESULTS: Pertussis specific IgA was the predominant pertussis immunoglobulin in the colostrum of Tdap vaccinated women with the geometric mean concentrations (GMCs) of IgA to FHA higher than for IgA to PT, 24.12 ELISA units/milliliter (EU/mL) vs. 8.18EU/mL, respectively, p<0.004. There were differences between the vaccinated women and controls in the GMCs of IgA to FHA and IgG to PRN in the colostrum, 24.12EU/mL vs. 6.52EU/mL, p=0.01 and 2.46EU/mL vs. <0.6EU/mL, p=0.03, respectively. The GMCs of total protein showed significant decline over 8 weeks in the vaccinated women and controls, p<0.004. Among vaccinated women, there was significant decline in the GMCs of IgA to PT and FHA over 8 weeks, p<0.001. The geometric mean ratio of IgA to FHA to total protein also declined significantly over 8 weeks in the vaccinated women, p<0.01, demonstrating a true decrease, however, pertussis IgA was measurable at 8 weeks. CONCLUSIONS: Select colostrum pertussis antibody levels were significantly higher among women vaccinated with Tdap during pregnancy compared with unvaccinated women. Among vaccinated women, maximal levels of pertussis specific IgA were in the colostrum but still detected at 8 weeks. Lactation may augment infant's protection against pertussis.

A regulatory role for CD72 expression on B cells in systemic lupus erythematosus.

BACKGROUND: B regulatory cells and their regulatory products/markers, such us semaphorin 3A (sema3A) and its receptor NP-1, FcγIIB, IL-10, and others, act at the very base of self-tolerance, maintenance, and prevention of autoimmune disease development. OBJECTIVES: The aim of the present study was to assess the involvement of CD72, a regulatory receptor on B cells, in systemic lupus erythematosus (SLE). In addition, the potential of soluble sema3A in enhancing the expression of CD72 on B cells of SLE patients was investigated. RESULTS: CD72 expression on activated B cells of SLE patients was significantly lower than that of normal controls. This lower expression of CD72 in SLE patients correlated inversely with SLE disease activity and was associated with lupus nephritis, the presence of anti-dsDNA antibodies, and low levels of complement. Co-culture of purified B cells from healthy controls with condition-media containing recombinant sema3A resulted in significant enhancement of CD72. Similar enhancement of CD72 on activated B cells from SLE patients, though significant, was still lower than in normal individuals. CONCLUSIONS: The lower expression of CD72 on activated B cells from SLE patients correlates with SLE disease activity, lupus nephritis, the presence of anti-dsDNA antibodies, and low levels of complement. The improvement of CD72 expression following the addition of soluble semaphorin 3A suggests that CD72 may be useful as a biomarker to be followed during the treatment of SLE.

Increased Toll-like receptor 9 expression by B cells from inflammatory bowel disease patients.

BACKGROUND: Toll-like receptors (TLRs) are important mediators of the innate immune response. Our aim was to evaluate TLR9 expression in peripheral B cells, taken from inflammatory bowel disease (IBD) patients before and after anti-inflammatory treatment. Nineteen patients with IBD (12-crohn's disease, 7-ulcerative colitis) and 18 healthy controls were included in the study. Disease severity was assessed using the Pediatric/Adults crohn's disease activity index and the ulcerative colitis activity index as needed. Accordingly, patients were classified as mild, moderate or severe disease. Peripheral B cells isolated from IBD patients, before and after anti-inflammatory treatment, and from the control group, were cultured for 24 h with and without CpG oligodeoxynucleotides (ODN-CpG) 0.5 µM. TLR9 expression by memory B cells (CD19+CD27+) was assessed by flow cytometry. RESULTS: We found that TLR9 expression by peripheral B cells was significantly higher in IBD patients than that in healthy controls (12.42 ± 9.5 MFI vs. 6.0 ± 2.6 MFI p = 0.02). The addition of ODN-CpG to B cells resulted in a significantly increase of TLR9 expression in B cells from healthy controls (6.5 ± 3.2 MFI vs. 8.8 ± 4.2 MFI p = 0.007). On the contrary, B cells from IBD patients only partly respond to the addition of ODN-CpG after anti-inflammatory treatment (6.3 ± 3.8 vs. 7.3 ± 3.7, p = 0.1). TLR9 expression was positively correlated with IBD disease severity (r = 0.681, p < 0.0001). CONCLUSIONS: TLR9 expression in memory B from IBD patients is elevated and associated with disease severity.

Higher expression of latency-associated peptide on the surface of peripheral blood monocytes in patients with rheumatoid arthritis may be protective against articular erosions.

Latency-associated peptide (LAP) forms small latent complexes with transforming growth factor beta 1 (TGF-ß1). TGF-ß-LAP complexes can be detected on the surfaces of immune cells and have been recently shown to play a role in immune regulation through TGF-ß1-mediated functions. A study was undertaken to investigate the correlation of LAP expression on the surface of immune cells and presence of articular erosions in patients with rheumatoid arthritis (RA). Venous blood was obtained from patients with severe RA as well as from healthy control subjects. Surface expression of LAP on peripheral blood mononuclear cells was analyzed by flow cytometry, measured as flow cytometric intensity separately on CD14(+) and CD14(-) cells, and compared between RA patients and healthy subjects. Patients with RA demonstrated higher surface expression of LAP on both CD14(+) and CD14(-) mononuclear cells than healthy individuals. Patients with erosive RA had significantly reduced intensity of anti-LAP staining on the CD14(+) cells when compared to RA patients without erosions (p = 0.01). The intensity of anti-LAP staining on CD14(-) cells was not different between groups of RA patients. Higher expression of LAP on the surface of the cells of monocyte lineage may be protective of formation of articular erosions in RA. Further studies are needed to elaborate the mechanism of this phenomenon.

Elevated serum B-cell activating factor in patients with chronic urticaria.

The B and T cells abnormalities that have been described among CU patients, lend support to its regard as an autoimmune disease. In this study we compared serum B-cell activation factor (BAFF) levels in 46 CU patients to 24 healthy controls and evaluated a possible association between elevated serum BAFF in CU patients and the presence of autologous serum skin test, anti-thyroid antibodies, antinuclear antibodies, high total IGE levels, and urticaria disease severity. We found that serum BAFF levels is elevated statistically significant in CU patients compared to healthy control (1228±342 pg/ml vs. 758±313 pg/ml, P<0.0001). CU patients with severe disease activity had significantly higher serum BAFF levels compared to patients with mild CU (1394.6±299.6 vs. 1097.6±221.3, P=0.0008). No association was found between the presence of positive autologous serum skin test, anti-thyroid antibodies or antinuclear antibodies or high levels of total IgE and serum BAFF levels in CU Patients. We conclude that CU patients have higher levels of serum BAFF which associate with disease severity.

Changes in immunomodulatory constituents of human milk in response to active infection in the nursing infant.

INTRODUCTION: To investigate whether immunologic factors in breast milk change in response to nursing infants' infection. RESULTS: Total CD45 leukocyte count dropped from 5,655 (median and interquartile range: 1,911; 16,871) in the acute phase to 2,122 (672; 6,819) cells/ml milk after recovery with macrophage count decreasing from 1,220 (236; 3,973) to 300 (122; 945) cells/ml. Tumor necrosis factor-α (TNFα) levels decreased from 3.66 ± 1.68 to 2.91 ± 1.51 pg/ml. The decrease in lactoferrin levels was of borderline statistical significance. Such differences were not recorded in samples of the controls. Interleukin-10 levels decreased in the sick infants' breast milk after recovery, but also in the healthy controls, requiring further investigation. Secretory immunoglobulin A levels did not change significantly in the study or control group. DISCUSSION: During active infection in nursing infants, the total number of white blood cells, specifically the number of macrophages, and TNFα levels increase in their mothers' breast milk. These results may support the dynamic nature of the immune defense provided by breastfeeding sick infants. METHODS: Breast milk from mothers of 31 infants, up to 3 months of age, who were hospitalized with fever, was sampled during active illness and recovery. Milk from mothers of 20 healthy infants served as controls.

Human CD19(+)CD25(high) B regulatory cells suppress proliferation of CD4(+) T cells and enhance Foxp3 and CTLA-4 expression in T-regulatory cells.

Studies in both animal models and humans have shown a subset of B cells behaving as immuno-regulatory cells, being a source of inhibitory cytokines such as IL-10 and TGF-ß. Our aims were to establish the presence of human B regulatory (Breg) cells and to assess their ability to suppress proliferation of CD4(+) T cells and to mediate T regulatory (Treg) cells' properties. For this purpose, human Breg, CD4(+) T and Treg cells were purified using magnetic microbeads. CFSE-labeled CD4(+) T cells were stimulated and cultured alone or with Breg cells. Their proliferative response was determined 72 hours later based on the CFSE staining. In parallel, Treg cells were cultured alone or with Breg cells in different conditions for 24 hours, and then stained and analyzed for Foxp3 and CTLA-4 expression. We found that, the co-culture of Breg cells (defined as CD25(high) CD27(high) CD86(high) CD1d(high) IL-10(high) TGF-ß(high)) with autologous stimulated CD4(+) T cells decreased significantly (in a dose-dependent way) the proliferative capacity of CD4(+) T cells. Furthermore, Foxp3 and CTLA-4 expression in Treg cells were enhanced by non-stimulated and further by ODN-CD40L stimulated Breg cells. The regulatory function of Breg cells on Treg cells was mainly dependent on a direct contact between Breg and Treg cells, but was also TGF-ß but not IL-10 dependent. In conclusion, human Breg cells decrease the proliferation of CD4(+) T cells and also enhance the expression of Foxp3 and CTLA-4 in Treg cells by cell-to-cell contact.

IVIg attenuates TLR-9 activation in B cells from SLE patients.

INTRODUCTION: Toll-like receptor-9 (TLR-9) plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). The aim of this study was to evaluate the influence of intravenous immunoglobulin (IVIg) on CpG oligodeoxynucleotides (ODN-CpG) activated B cells from SLE patients. METHODS: Peripheral blood B cells were isolated from 16 SLE patients and 21 healthy age-matched controls. B cells were cultured with ODN-CpG 1µM alone or IVIg (10mg/ml) together with ODN-CpG. After 24-h incubation, B cells and supernatants were collected and analyzed for interleukin (IL)-10, IL-6 secretion, and TLR-9 expression. RESULTS: IVIg decreased the secretion of IL-10 from ODN-CpG-activated B cells isolated from both SLE patients and healthy controls (194 ± 46.2 to 103.2 ± 27.13 pg/ml, p < 0.016, 153.2 ± 19 vs 84.6 ± 7.5, p < 0.0001, respectively). Similarly, IVIg decreased the secretion of IL-6 from ODN-CpG-activated B cell isolated from both SLE patients and healthy controls (431.2 ± 83 to 307.6 ± 94.3 pg/ml, p < 0.0008, 319.5 ± 31 vs 193.3 ± 22.8, p < 0.0001, respectively). The decrement of IL-10 and IL-6 secretion was associated with a significant decrease in TLR-9 expression in memory B cells from SLE patients and healthy controls (11.47 ± 1.2 vs 13.29 ± 1.2, p = 0.005, 11 ± 0.8 vs 12.8 ± 0.98, p = 0.0016, respectively). CONCLUSIONS: IVIg attenuates the activation of TLR-9 in B cells from SLE patients, suggesting a novel additional mechanism of IVIg mode of action in these patients.

Regulatory T cells (CD4(+)CD25(bright)FoxP3(+)) expansion in systemic sclerosis correlates with disease activity and severity.

BACKGROUND: The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc). METHODS: Ten patients with SSc donated 20ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4(+) cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-beta and IL-10 by activated CD4(+) cells was measured by ELISA in culture supernatants. RESULTS: The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r=0.71, p=0.034 and r=0.67, p=0.044, respectively). ELISA-measured production of TGF-beta and IL-10 by CD4(+) cells was similar in patients and controls. CONCLUSIONS: Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-beta or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.

Etanercept impairs maturation of human monocyte-derived dendritic cells by inhibiting the autocrine TNFalpha-mediated signaling.

The success of anti-tumor necrosis factor alpha (TNFalpha) therapies has led to increased interest as to the mechanisms and consequences of TNFalpha blockade. The aim of the study was to examine the effects of TNFalpha blockade by etanercept on lipopolysaccharide (LPS) or peptidoglycan (PG)-induced maturation of human monocyte-derived dendritic cells (MDDCs). MDDCs grown from peripheral blood of healthy donors were stimulated by LPS or PG with/without the presence of etanercept. Concentrations of TNFalpha in cell supernatants were assessed by ELISA, while the cells were stained with monoclonal antibodies to CD83, CD80, CD86, CD11c, CD40, HLA-DR, and annexin-V and acquired using a flow cytometer. Etanercept significantly decreased the stimulated cell surface expression of HLA-DR, CD80, CD86, CD40 and CD83 on MDDCs in all examined samples. Etanercept in the same dose, but denatured to loss of specificity for TNFalpha, failed to change any of the aforementioned markers. In the presence of etanercept, concentrations of TNFalpha in cell supernatants were decreased by 53% on average, with a range of 25%-87%. Etanercept impaired the stimulated maturation of MDDCs by neutralizing the induced TNFalpha, produced by the same MDDCs after antigenic stimulation. The reported data confirms that TNFalpha blockade may have a direct effect on DCs, with a wide spectrum of potential secondary effects downstream. The data also suggests the presence of TNFalpha-mediated autocrine signaling, serving to accelerate or catalyze the maturation process of MDDCs.

Intravenous immunoglobulin therapy affects T regulatory cells by increasing their suppressive function.

Intravenous Ig therapy (IVIg) is reported to be a useful regimen in treating autoimmune diseases. In this study, we asked whether IVIg (in vitro) could increase the expression of TGF-beta, IL-10, and the transcription factor FoxP3 in T regulatory (Treg) cells, and the idea that IVIg could enhance suppressive properties of these cells. CD4(+) T cells from 12 healthy individuals were cultured in the presence or absence of IVIg vs human control IgG during 16, 24, and 36 h. Using FACS analysis and gating on CD4(+)CD25(high) Treg cells, we assessed the expression of intracellular TGF-beta, IL-10, and FoxP3. In addition, the production of TNF-alpha by stimulated CD4(+) T cells alone or in culture with CD25(+) by itself or together with IVIg was also assessed. The presence of IVIg with Treg cells in culture significantly increased the intracellular expression of TGF-beta (17.7 +/- 8.5% vs 29.8 +/- 13%; p = 0.02), IL-10 (20.7 +/- 4.7% vs 34.2 +/- 5.2%; p = 0.008) and FoxP3 (20.8 +/- 5.2% vs 33.7 +/- 5.9%; p = 0.0006) when compared with cells cultured alone or with control human IgG. The suppressive effect of CD4(+)CD25(+) T cells presented as the decrease of TNF-alpha production by stimulated CD4(+)CD25(-) (effector T cells) was further increased by adding IVIg to cell culture. We hereby demonstrate an additional mechanism by which IVIg could maintain self-tolerance and decrease immune-mediated inflammation.