Intracellular Cytokine Production Research Articles

AB0034 IFN-Gamma-Producing VZV-Specific CD4 T-Cells in Patients with Rheumatoid Arthritis (RA)

BackgroundVaricella-zoster-virus (VZV) establishes a life-long latent infection with risk of reactivation in patients with autoimmune disorders receiving immunosuppressive/immunomodulatory therapy.ObjectivesThe study was aimed to evaluate the VZV-specific cellular immune response in...

OP0220 Pathogenic Role of IL-10 Producing Helper T Cells in Systemic Lupus Erythematosus

BackgroundIL-10 is a potent anti-inflammatory cytokine that contributes to the suppressive functions of regulatory T-cells, but it is also a well-known B cell growth and differentiation factor produced by CD4+...

AB0201 Cell Surface Expression of DNAM-1 (CD226) on CD8+ T Cells is Increased in Patients with Systemic Sclerosis

BackgroundDNAM-1 (CD226) is well known as a genetic risk factor of many autoimmune diseases such as systemic sclerosis (SSc). More recently, Avouac et al. have reported that mice deficient for...

Phosphorylation of glycogen synthase kinase-3β in metabolically abnormal obesity affects immune stimulation-induced cytokine production.

Obesity is a severe health problem worldwide, which leads to multiple comorbidities including type 2 diabetes mellitus and cardiovascular diseases. Inflammation has been found to be an important characteristic of adipose tissue in obese subjects. However, obesity is also associated with compromised immune responses to infections and the impact of obesity on immune function has not been fully understood. To clarify the role of obesity in the immune responses, we investigated the Toll-like receptor (TLR)-induced cytokine secretion by leukocytes from obese and lean subjects. We also investigated the relationship between insulin-induced intracellular signaling and cytokine production using peripheral blood mononuclear cells (PBMCs) and a monocytic cell line THP-1. We found decreased TLR-induced interferon-γ, interleukin-6 (IL-6) and tumor necrosis factor-α secretions and elevated IL-10 secretion by leukocytes from obese subjects when compared with lean controls. PBMCs from obese subjects showed enhanced basal Akt and glycogen synthase kinase-3β (GSK-3β) phosphorylation, which did not further increase with insulin and lipopolysaccharide (LPS) stimulation. We also found that LPS-induced IκBα degradation was inhibited in PBMCs from obese subjects. By using THP-1 cells with GSK-3β knockdown or cells treated with hyperinsulinemic and high-fatty acid conditions, we found that LPS-induced nuclear factor κB (NF-κB) activation was inhibited and cyclic adenosine monophosphate response element-binding protein (CREB) activation was enhanced. These findings indicate that GSK-3β is important in the regulation of NF-κB and CREB activation in leukocytes under the metabolic condition of obesity. Our study revealed a key mechanism through which metabolic abnormalities compromise leukocyte functions in people with obesity.

IFNγ drives human primary skin mast cells to function as antigen presenting cells. (INC6P.341)

Abstract We assessed the antigen presenting ability of mature primary human skin mast cells (MCs) in vitro. MCs do not express HLA II at baseline, but do so when stimulated with IFNγ. IFNγ-primed, but not un-primed, MCs facilitated activation of Jurkat T cells by superantigen. Unlike polyclonal primary CD4+ T cells, which harbor a subset of IFNγ producing cells, Jurkat cells do not spontaneously produce IFNγ. Co-culture of un-primed MCs with CD4+ T cells for 2 days upregulated HLA II expression on MCs and subsequent superantigen mediated T cell activation. MC HLA II upregulation is primarily due to the Th1 cell subset and is prevented by IFNγ neutralizing and/or receptor blocking antibody. Superantigen bypasses HLA II-loaded peptide:T cell receptor interactions; therefore, we assessed the ability of MCs to present cytomegalovirus antigen. MCs primed ±cytomegalovirus antigen exposure for 3 days then ±IFNγ for the last 2 of those days were washed thoroughly and co-cultured with HLA-matched CD4+ cells. After one week, CD4+ T cells from cytomegalovirus seropositive, but not seronegative, individuals showed proliferation (CFSE dilution), activation (CD25high and CD69high expression), and appropriate intracellular cytokine production (IFNγ and IL-4, but not IL-17). Thus, MCs can take up, process, and present antigen in an HLA II-restricted manner, causing antigen specific activation and proliferation of CD4+ T cells.

Laboratory Diagnosis of Primary Immunodeficiencies

Primary immune deficiency disorders represent a highly heterogeneous group of disorders with an increased propensity to infections and other immune complications. A careful history to delineate the pattern of infectious organisms and other complications is important to guide the workup of these patients, but a focused laboratory evaluation is essential to the diagnosis of an underlying primary immunodeficiency. Initial workup of suspected immune deficiencies should include complete blood counts and serologic tests of immunoglobulin levels, vaccine titers, and complement levels, but these tests are often insufficient to make a diagnosis. Recent advancements in the understanding of the immune system have led to the development of novel immunologic assays to aid in the diagnosis of these disorders. Classically utilized to enumerate lymphocyte subsets, flow cytometric-based assays are increasingly utilized to test immune cell function (e.g., neutrophil oxidative burst, NK cytotoxicity), intracellular cytokine production (e.g., TH17 production), cellular signaling pathways (e.g., phosphor-STAT analysis), and protein expression (e.g., BTK, Foxp3). Genetic testing has similarly expanded greatly as more primary immune deficiencies are defined, and the use of mass sequencing technologies is leading to the identification of novel disorders. In order to utilize these complex assays in clinical care, one must have a firm understanding of the immunologic assay, how the results are interpreted, pitfalls in the assays, and how the test affects treatment decisions. This article will provide a systematic approach of the evaluation of a suspected primary immunodeficiency, as well as provide a comprehensive list of testing options and their results in the context of various disease processes.

A8.9 Rheumatoid arthritis synovial IL-21 + CD4+ t cells specifically induce matrix metalloproteinase production by fibroblast-like synoviocytes

Background/PurposeIL-21 is a cytokine produced by activated CD4+ T cells and T follicular helper cells (TFh) that has been implicated in several autoimmune diseases including rheumatoid arthritis (RA). IL-21 regulates...

Peripheral and Site‐Specific CD4+CD28null T Cells from Rheumatoid Arthritis Patients Show Distinct Characteristics

Proinflammatory CD4(+) CD28(null) T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4(+) CD28(null) T cells from blood and synovial fluid in comparison with conventional CD28-expressing CD4(+) T cells. Forty-four patients with RA, displaying a distinct CD4(+) CD28(null) T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-γ, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4(+) CD28(null) T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4(+) CD28(+) T cells and produced higher levels of both IFN-γ and TNF after TCR cross-linking. CD4(+) CD28(null) T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4(+) CD28(null) cells from the blood, a significant production was observed in CD4(+) CD28(null) T cells from synovial fluid. CD4(+) CD28(null) T cells were not only found to differ from conventional CD4(+) CD28(+) T cells in the circulation, but we could also demonstrate that synovial CD4(+) CD28(null) T cells showed additional effector functions (IL-17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28(null) population.

Self-Engagement Of An NKT Cell Inhibitory Receptor During Development Alters Receptor Expression and Cellular Function

Invariant NKT (iNKT) cells have been shown to be pro-inflammatory or anti-inflammatory in many pathologic conditions including infections, cancer, autoimmune diseases and allergy. Additionally, early expansion of iNKT cells correlates with successful engraftment and low incidence of graft-versus host disease suggesting a role for iNKT cells in tolerance to allogeneic transplantation. iNKT cells recognize glycolipids bound to CD1d via their T cell receptor (TCR), but also express the inhibitory receptors for MHC class I ligands (MHC I.) Little is known regarding the importance of iNKT cell self-recognition of MHC I during development and the occurrence of autoimmunity, allergy or tolerance to in utero hematopoietic cellular transplantation (IUHCT). Given the importance of NK cell self-recognition during development, we hypothesized that, like NK cells, self-engagement of MHC I inhibitory receptors during development alters the phenotype and function of self-responsive iNKT cells. Prenatal allogeneic chimeras were established by in utero transplantation of Balb/c fetal liver light density cells at E14 into age-matched C57BL/6 fetal recipients. A kinetic evaluation of iNKT cell receptor expression and function was performed after birth. iNKT cells were evaluated for expression of activating (Ly49D) and inhibitory (Ly49A, F, G) receptors specific for the donor MHC I (H-2d.) The functional response of iNKT cell subsets was measured following in vivo activation with KRN7000, the ligand for iNKT cells. Intracellular cytokine production (IFN-gamma and IL-4) production was assessed in the alloresponsive Ly49A+ iNKT cell subset and compared to naïve controls and B10.D2 (H2d) mice. We found that a negligible fraction of iNKT cells expressed the activating Ly49D receptor in either chimera or controls (<1%.) In chimeric mice, the frequency of iNKT cells expressing Ly49A, F, or G did not change significantly. However, the levels of Ly49A expression were significantly reduced on iNKT cells from all organs tested suggesting developmental recognition of donor class I ligands had occurred. These reduced receptor levels did not appear to be due to sequestration as acid stripping did not alter the phenotype. Lastly, Ly49A+ but not Ly49A- iNKT cells in chimeric mice exhibited significantly increased production of IL-4 in response to TCR stimulation suggesting a shift toward a regulatory phenotype. Consistent with this finding, IFN-gamma levels in chimeras were minimally changed compared to controls. These findings indicate that inhibitory receptor self-engagement during development leads to: 1) alterations in receptor expression; 2) shift toward a tolerogenic phenotype in self-responsive iNKT cell phenotypes; and 3) no change in the receptor expression or function of non-responsive iNKT cell phenotypes. Further studies will directly evaluate the impact of self-recognition of iNKT cells in the pathogenesis of autoimmunity and allergy or tolerance following IUHCT. Disclosures: No relevant conflicts of interest to declare.

48: Immunophenotyping innate lymphoid and T helper cell populations in human asthma

Asthma is a heterogeneous disease driven by T-helper type 2 (Th2) cell-driven airway inflammation, due to the production of the pro-inflammatory cytokines IL-13, IL-5 and IL-4. Many cell types in the allergic lung produce IL-13, including T cells and type 2 innate lymphoid cells (ILC2). To identify the T helper cell subsets that contribute to disease, we immunophenotyped blood, bronchoalveolar lavage fluid (BAL) and induced sputum cells from asthmatic patients. Using 10–12 color flow cytometry, we characterized innate cells and T helper cell populations in two separate immunophenotyping panels, and correlated cell surface markers with intracellular cytokine production. To incorporate a larger number of parameters into a single immunophenotyping panel, we have started to use the cytometry by time-of-flight mass spectrometry (CyTOF) platform as an alternative for airway biospecimens. CyTOF allows us to look at upwards of 30–40 markers on every cell in each sample, including both cell surface and intracellular markers, and will help us determine the interplay among the different cell types that contribute to allergic airway disease.

NKp44L expression on CD4+ T cells is associated with impaired immunological recovery in HIV-infected patients under highly active antiretroviral therapy

HIV-infected immunological nonresponders (InRs) patients fail to show satisfactory CD4+ T-cell recovery despite virologically effective HAART. We propose that NKp44L, the cellular ligand of an activating natural killer (NK) receptor, expressed only on uninfected bystander CD4+ T cells from HIV-1 infected patients, could play a major role in this phenomenon by sensitizing these cells to NK killing. Phenotype and multifunctional status of CD4+ T cells, especially the subsets expressing and not expressing NKp44L, were characterized for HIV-infected patients receiving HAART for at least 2 years, during which their viral load remained less than 40 copies/ml; 53 were InRs (CD4 cell count always <350 cells/µl), and 82 immunological responders (CD4 cell count always ≥350 cells/µl). Flow cytometry determined NKp44L expression in association with specific markers of proliferation, maturation, activation, homeostasis, and intracellular cytokine production. Degranulation of NKp44+ determined the functional capacity of NK cells. InRs exhibited high levels of NKp44L+CD4+ T cells. Compared with NKp44L negative cells, the frequency of naive CD45RA+CCR7+ T cells expressing NKp44L fell (P < 0.001) and their proliferative capacity grew. Moreover, apoptosis and a unique ability to produce multiple cytokines (IL-2, IFN-γ, and TNF-α) without or after phytohemagglutinin or anti-CD3/CD28 stimulation distinguished NKp44L+ T cells. InR status is associated to a significant expansion of highly differentiated, multifunctional and apoptotic CD4+ T cells expressing NKp44L. This could explain a rapid CD4+ T-cell turnover in InR preventing immune recovery. These data suggest a new target for developing therapeutic strategies to prevent NKp44L expression and then stimulating immune recovery in InRs.

Booster Vaccinations against Cancer Are Critical in Prophylactic but Detrimental in Therapeutic Settings

Although cancer vaccines are in the clinic, several issues remain to be addressed to increase vaccine efficacy. In particular, whether how and how frequently a patient should be boosted remains to be defined. Here, we have assessed the ability of dendritic cell (DC)-based vaccines to induce a long-lasting tumor-specific CTL response in either prophylactic or therapeutic settings by taking advantage of transplantable and spontaneous mouse tumor models. Implementing a 24-hour ex vivo intracellular cytokine production assay, we have found that priming with a DC-based vaccine induced a long-lasting CTL response in wild-type mice, and homologous boosting better sustained the pool of central memory T cells, which associated with potent protection against B16F1 melanoma challenge. Appropriate timing of booster vaccination was also critical, as a tight boosting schedule hindered persistence of IFN-γ-competent memory CD8(+) T cells and mice survival in prophylactic settings. Conversely, prime/boost vaccination proved to be of no advantage or even detrimental in therapeutic settings in B16F1 and transgenic adenocarcinoma of the mouse prostate (TRAMP) models, respectively. Although DC priming was indeed needed for tumor shrinkage, restoration of immune competence, and prolonged survival of TRAMP mice, repeated boosting did not sustain the pool of central memory CTLs and was detrimental for mice overall survival. Thus, our results indicate that booster vaccinations impact antitumor immunity to different extents, depending on their prophylactic or therapeutic administration, and suggest evaluating the need for boosting in any given patient with cancer depending on the state of the disease.

PTH-106 Unravelling the Mechanism of Action of Infliximab in Crohn’S Disease and Healthy Controls Following in Vitro Culture with Blood-Enriched Dendritic Cells

IntroductionDendritic cells (DC) play a key role in discriminating between commensal microorganisms and harmful pathogens. DC phenotype and cytokine production determine the type of immune response elicited by T-cells following...

AB0148 T-cell phenotype and intracellular cytokine-production in t-cells in patients with systemic sclerosis

BackgroundSystemic Sclerosis (SSc) is a disease with mostly unknown etiology. Until now, various cytokines have been identified that may play a role in the pathogenesis of the disease.ObjectivesOur aim was...

Intracellular cytokine production and cognition in healthy older adults

Elevated concentrations of the pro-inflammatory cytokines IL-1β and IL-6 have been associated with impaired cognitive performance. There are, however, few studies that have examined the relationship between cytokine production and specific aspects of cognition in healthy older individuals. Two-colour flow cytometry was used to determine intracellular cytokine production by activated monocytes, and neuropsychological tests were performed using the Cambridge Neuropsychological Test Automated Battery (CANTAB) in 93 apparently healthy men and women aged 55-70 years. A series of hierarchical regression analyses was carried out to examine the contribution of IL-1β and IL-6 (% expression and production (antibody binding capacity (ABC))) to recognition, attention and working memory, after controlling for socio-demographic variables (age, sex and social class). IL-1β% expression and IL-6 production predicted aspects of working memory. Recognition memory was found to be sensitive to the affects of age and social class. The current study suggests that higher intracellular cytokine production by activated monocytes may be predictive of lower cognitive performance in working memory in healthy older individuals. These findings indicate that utilization of models for in vivo cytokine production upon immune challenge may be useful in studying specific aspects of memory affected during inflammatory responses, for example in individuals at risk for cognitive decline owing to age-related inflammatory disorders.

Inhibiting the Programmed Death 1 Pathway Rescues Mycobacterium tuberculosis–Specific Interferon γ–Producing T Cells From Apoptosis in Patients With Pulmonary Tuberculosis

Overexpression of programmed death 1 (PD-1) receptor is thought to inhibit the effector T-cell response in human tuberculosis. However, the precise mechanism of such inhibition remains unclear. The present study addresses the role of PD-1 in dampening host T-cell function among patients with pulmonary tuberculosis. Expression of PD-1 and its ligands (PD-L1/L2) on T cells, B cells, and monocytes was evaluated by flow cytometry (FACS). In vitro stimulation of peripheral blood mononuclear cells in the presence of Mycobacterium tuberculosis antigens was performed with and without blocking PD-1, and intracellular cytokine production was measured by FACS. We showed higher frequencies of T cells, monocytes, and B cells expressing PD-1 and its ligand(s) among patients with pulmonary tuberculosis. Infections with live M. tuberculosis upregulated PD-L1 expression on monocytes. In vitro PD-1 blocking rescued M. tuberculosis-specific interferon γ (IFN-γ)-producing T cells from undergoing apoptosis. The number of PD-1-expressing T cells decreased significantly during therapy and inversely correlated with IFN-γ-dominant T-cell response against M. tuberculosis. Manipulation of PD-1 signaling may restore the host T-cell response and thus may have therapeutic potential. PD-1 also may serve as a biomarker to monitor host immunity among patients with tuberculosis during therapy and vaccine studies.

Expression of TREM-1 is triggered by flaviviruses: possible role in virus-associated innate immune responses (P1381)

Abstract Activation of Triggering Receptor Expressed on Myeloid cells 1 (TREM-1), an immunoreceptor expressed mainly on immune cells results in a cascade of inflammatory effects including cytokine production. In addition, TREM-1 also modulates signaling cascades of other PRRs such as TLRs and NLRs. Engagement of the TREM-1 receptor via adaptor protein DAP12 leads to the activation of downstream kinases, mobilization of intracellular calcium and production of pro-inflammatory cytokines. Innate immune responses are essential for the clearance, dissemination and neurovirulence of West Nile virus (WNV), a neurotropic flavivirus, which has emerged in the U.S. as a significant cause of viral encephalitis in humans. However, the role of TREM-1 signaling pathway in immunity to flaviviruses is unclear. In this study we investigated the changes in the expression kinetics and activation of TREM-1 following WNV infection. Expression of TREM-1 was markedly increased in WNV-infected mouse immune cells and embryonic fibroblasts (MEFs) at 48 hrs after infection and in the mouse brain at day 8 after infection. Further, activation of TREM-1 using agonist antibody resulted in an increase in the levels of IFN-β and key cytokines in WNV-infected MEFs. Our results so far indicate a possible role of TREM-1 in the induction of inflammatory innate immune responses to WNV and further studies are ongoing to characterize unexplored mechanisms of TREM-1 associated with the pathogenesis of flaviviruses.

Tu1159 Unravelling the Mechanism of Action of Infliximab in Crohn's Disease and Healthy Controls Following In Vitro Culture With Blood-Enriched Dendritic Cells

Introduction Dendritic cells (DC) play a key role in discriminating between commensal microorganisms and harmful pathogens. DC phenotype and cytokine production determine the type of immune response elicited by T-cells following antigen presentation. DC also direct the T-cells to target tissues to perform their function via imprinting tissue-specific homing markers. In CD, dysregulation of the immune response to gut microbiota & aberrant immune cell trafficking play a role in disease pathogenesis. Infliximab (IFX) is an effective treatment for CD but its mechanism of action is unclear. In this study we investigated the in-vitro effect of IFX on phenotype & ongoing cytokine production of human blood-enriched DC from patients with active CD and healthy controls (HC) Methods Low density cells (LDC), enriched for DC, were obtained following Ficoll & Nycoprep separation of blood from patients with active CD (CDAI > 220) and HC. LDC were cultured (0.5x106cells/ml) with IFX (1.10&100μg/ml&basal) for 24hr. Activation marker (CD40, CD80, HLADR), TLR receptor (TLR2.4) and homing marker (CCR4.5.7.8.9.10, β7) expression was quantified by flow cytometry. Natural ongoing intracellular cytokine production (TNFα, TGFβ&IL-6.10.12.15) was assessed via intracellular staining and flow cytometry. Cytokine secretion was measured on cell-free culture supernatants via Multiplex. Unpaired t-test and one-way ANOVA statistical analyses were applied Results TNFα and IL-6 were increased in culture supernatants from CD although their intracellular ongoing cytokine production was decreased. LDC from CD had decreased β7 (gut-homing integrin) expression. Following IFX culture, LDC decreased β7 expression & CCR9 intensity ratio (dose-dependent). There was a trend towards reduction in TLR2 and 4 expression (not statistically significant). IL-12 production by LDC from HC was increased following IFX culture. There was a marked reduction in TNFα and IL-12 in cell supernatant following culture with IFX Conclusion Increased TNFα and IL-6 in culture supernatants from CD patients coupled with a decrease in ongoing production by DC suggests a negative cytokine feedback system. Reduced β7 expression on LDC from CD patients may suggest that DC have already been recruited to the site of mucosal inflammation. Further experiments are needed to confirm this. Reduced expression of CCR9 and elevated production of IL-12 in LDC cultured with IFX, shows reduced affinity for gut-homing and increased immunogenicity and may suggest a possible mechanism for IFX-induced paradoxical inflammation. The most dramatic effect of IFX was a reduction in TNFα in the supernatant, which is likely to represent neutralisation of the cytokine Disclosure of Interest None Declared.

Impact of oral typhoid vaccination on the human gut microbiota and correlations with s. Typhi-specific immunological responses.

The resident microbial consortia of the human gastrointestinal tract play an integral role in modulating immune responses both locally and systemically. However, detailed information regarding the effector immune responses after vaccine administration in relation to the gastrointestinal microbiota is absent. In this study, the licensed oral live-attenuated typhoid vaccine Ty21a was administered in a clinical study to investigate whether oral immunization resulted in alterations of the microbiota and to identify whether a given microbiota composition, or subsets of the community, are associated with defined S. Typhi-specific immunological responses. The fecal microbiota composition and temporal dynamics were characterized using bacterial 16S rRNA pyrosequencing from individuals who were either immunized with the Ty21a typhoid vaccine (n = 13) or served as unvaccinated controls (n = 4). The analysis revealed considerable inter- and intra-individual variability, yet no discernible perturbations of the bacterial assemblage related to vaccine administration were observed. S. Typhi-specific cell mediated immune (CMI) responses were evaluated by measurement of intracellular cytokine production using multiparametric flow cytometry, and humoral responses were evaluated by measurement of serum anti-LPS IgA and IgG titers. Volunteers were categorized according to the kinetics and magnitude of their responses. While differences in microbial composition, diversity, or temporal stability were not observed among individuals able to mount a positive humoral response, individuals displaying multiphasic CMI responses harbored more diverse, complex communities. In line with this preliminary observation, over two hundred operational taxonomic units (OTUs) were found to differentiate multiphasic and late CMI responders, the vast majority of which classified within the order Clostridiales. These results provide an unprecedented view into the dramatic temporal heterogeneity of both the gut microbiota and host immune responses.

B-cell linker protein expression contributes to controlling allergic and autoimmune diseases by mediating IL-10 production in regulatory B cells

Regulatory B cells that exhibit the cell-surface CD1d(hi)CD5(+) phenotype and produce IL-10 are termed B10 cells. Although B10 cells exert potent suppressive functions in patients with various allergic and autoimmunity disorders, the precise signaling mechanisms required for B10 cell functions remain unknown. B-cell linker protein (BLNK) is an essential component of the B-cell antigen receptor signaling pathway and is required for optimal B-cell development. We sought to elucidate the signaling pathways that are responsible for IL-10 production in B10 cells and in vivo mechanisms of how impaired B10 cell functions influence allergic and autoimmune responses. For in vitro assays, splenic CD1d(hi)CD5(+) B cells from BLNK-deficient (BLNK(-/-)) mice were analyzed for intracellular signaling pathways and cytokine production. Contact hypersensitivity (CHS) and experimental autoimmune encephalomyelitis were examined by using BLNK(-/-) mice. Although the CD1d(hi)CD5(+) B-cell population was present in BLNK(-/-) mice, IL-10 production was impaired both in vitro and in vivo. BLNK(-/-) mice had exaggerated CHS and experimental autoimmune encephalomyelitis responses, which were normalized by adoptive transfer of splenic CD1d(hi)CD5(+) B cells from wild-type mice. In mice with CHS, BLNK(-/-) mice exhibited decreased B-cell and regulatory T-cell percentages and increased CD8(+) T-cell percentages in the skin and lymph nodes. In vitro BLNK was required for LPS-induced signal transducer and activator of transcription 3 phosphorylation in CD1d(hi)CD5(+) B cells. Finally, secreted IL-10 leads to autocrine expansion of IL-10-producing B cells. BLNK serves as a critical signaling component for B10 cell function by mediating IL-10 production.