Cord blood Streptococcus pneumoniae‐specific cellular immune responses predict early pneumococcal carriage in high‐risk infants in Papua New Guinea

In areas where Streptococcus pneumoniae is highly endemic, infants experience very early pneumococcal colonization of the upper respiratory tract, with carriage often persisting into adulthood. We aimed to explore whether newborns in high‐risk areas have pre‐existing pneumococcal‐specific cellular immune responses that may affect early pneumococcal acquisition. Cord blood mononuclear cells (CBMC) of 84 Papua New Guinean (PNG; high endemic) and 33 Australian (AUS; low endemic) newborns were stimulated in vitro with detoxified pneumolysin (dPly) or pneumococcal surface protein A (PspA; families 1 and 2) and compared for cytokine responses. Within the PNG cohort, associations between CBMC dPly and PspA‐induced responses and pneumococcal colonization within the first month of life were studied. Significantly higher PspA‐specific interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α, interleukin (IL)‐5, IL‐6, IL‐10 and IL‐13 responses, and lower dPly‐IL‐6 responses were produced in CBMC cultures of PNG compared to AUS newborns. Higher CBMC PspA‐IL‐5 and PspA‐IL‐13 responses correlated with a higher proportion of cord CD4 T cells, and higher dPly‐IL‐6 responses with a higher frequency of cord antigen‐presenting cells. In the PNG cohort, higher PspA‐specific IL‐5 and IL‐6 CBMC responses were associated independently and significantly with increased risk of earlier pneumococcal colonization, while a significant protective effect was found for higher PspA‐IL‐10 CBMC responses. Pneumococcus‐specific cellular immune responses differ between children born in pneumococcal high versus low endemic settings, which may contribute to the higher risk of infants in high endemic settings for early pneumococcal colonization, and hence disease.     

Impaired cytokine responses in patients with cryopyrin‐associated periodic syndrome (CAPS)

Cryopyrin‐associated periodic syndrome (CAPS) is characterized by dysregulated inflammation with excessive interleukin (IL)‐1β activation and secretion. Neonatal‐onset multi‐system inflammatory disease (NOMID) is the most severe form. We explored cytokine responses in 32 CAPS patients before and after IL‐1β blocking therapy. We measured cytokines produced by activated peripheral blood monuclear cells (PBMCs) from treated and untreated CAPS patients after stimulation for 48 h with phytohaemagglutinin (PHA), PHA plus IL‐12, lipopolysaccharide (LPS) or LPS plus interferon (IFN)‐γ. We measured IL‐1β, IL‐6, IL‐10, tumour necrosis factor (TNF), IL‐12p70 and IFN‐γ in the supernatants. PBMCs from three untreated CAPS patients were cultured in the presence of the IL‐1β blocker Anakinra. Fifty healthy individuals served as controls. CAPS patients had high spontaneous production of IL‐1β, IL‐6, TNF and IFN‐γ by unstimulated cells. However, stimulation indexes (SIs, ratio of stimulated to unstimulated production) of these cytokines to PHA and LPS were low in NOMID patients compared to controls. Unstimulated IL‐10 and IL‐12p70 production was normal, but up‐regulation after PHA and LPS was also low. LPS plus IFN‐γ inadequately up‐regulated the production of IL‐1β, IL‐6, TNF and IL‐10 in CAPS patients. In‐vitro but not in‐vivo treatment with Anakinra improved SIs by lowering spontaneous cytokine production. However, in‐vitro treatment did not improve the low stimulated cytokine levels. Activating mutations in NLRP3 in CAPS are correlated with poor SIs to PHA, LPS and IFN‐γ. The impairment in stimulated cytokine responses in spite of IL‐1β blocking therapy suggests a broader intrinsic defect in CAPS patients, which is not corrected by targeting IL‐1β.     

Pregnancy IFN-gamma responses to foetal alloantigens are altered by maternal allergy and gravidity status

Background: During pregnancy, variations in maternal–foetal cellular interactions may influence immune programming. This study was carried out to determine if maternal responses to foetal alloantigens are altered by maternal allergic disease and/or previous pregnancies. Methods: For this cohort study, peripheral blood was collected from allergic (n = 69) and nonallergic (n = 63) pregnant women at 20, 30, 36‐week gestation and 6‐week postpartum (pp). Cord blood was collected at delivery. Mixed lymphocyte reactions were used to measure maternal cytokine responses [interleukin‐6 (IL‐6), IL‐10, IL‐13 and (interferon‐γ) IFN‐γ] at each time point towards foetal mononuclear cells. Results: Maternal cytokine responses during pregnancy (20, 30 and 36 weeks) were suppressed compared to the responses at 6‐week pp. The ratio of maternal IFN‐γ/IL‐13 and IFN‐γ/IL‐10 responses were lower during pregnancy. Allergic mothers had lower IFN‐γ responses at each time‐point during pregnancy with the greatest difference in responses observed at 36‐week gestation. When allergic and nonallergic women were further stratified by gravidity group, IFN‐γ responses of allergic multigravid mothers were significantly lower than nonallergic multigravid mothers during pregnancy. Conclusions: During normal pregnancy, peripheral T‐cell cytokine responses to foetal alloantigens may be altered by both allergic status of the mother and previous pregnancies. These factors could influence the cytokine milieu experienced by the foetus and will be further explored in the development of allergic disease during early life.     

ORIGINAL ARTICLE: Prenatal Priming of Cord Blood T Lymphocytes by Microbiota in the Maternal Vagina

Problem In the vagina of women at the reproductive age, more than 170 strains of bacteria and yeasts are found. The effect of vaginal flora on neonatal T cells is yet to be investigated. Method of study We analyzed CD45RA and CD45RO expression on neonatal CD4+ T cells and cytokine production in CBMC cultures (interferon‐γ (IFN‐γ ), interleukin‐4 (IL‐4) and IL‐12) related to vaginal bacteria isolated from a maternal vagina. We collected vaginal swabs from 36 women at the first stage of the delivery and cord blood from their newborns. IFN‐γ, IL‐4, and IL‐12 in stimulated CBMC were measured and the expression of CD45RA/CD45RO on CD4+ T cells was assessed. Results We noted the difference in CD45RO CD4+ expression and IL‐12 levels between the newborns whose mothers were or were not colonized with Lactobacillus in the vagina (newborns whose mothers were colonized with Lactobacillus: CD45RO‐10%±3; IL‐12‐0.2 pg/mL ± 0.05; newborns whose mothers were not colonized with Lactobacillus: CD45RO‐6%±3; IL‐12‐2.0 pg/mL ± 0.7). Conclusion Our results may indicate that lactobacilli in maternal vagina influence the development of neonatal immune system. Yet, more research is needed using specified bacterial antigens.     

Drug‐specific in vitro release of IL‐2, IL‐5, IL‐13 and IFN‐γ in patients with delayed‐type drug hypersensitivity

Background: The most prevalent drug hypersensitivity reactions are T‐cell mediated. The only established in vitro test for detecting T‐cell sensitization to drugs is the lymphocyte transformation test, which is of limited practicability. To find an alternative in vitro method to detect drug‐sensitized T cells, we screened the in vitro secretion of 17 cytokines/chemokines by peripheral blood mononuclear cells (PBMC) of patients with well‐documented drug allergies, in order to identify the most promising cytokines/chemokines for detection of T‐cell sensitization to drugs. Methods: Peripheral blood mononuclear cell of 10 patients, five allergic to β‐lactams and five to sulfanilamides, and of five healthy controls were incubated for 3 days with the drug antigen. Cytokine concentrations were measured in the supernatants using commercially available 17‐plex bead‐based immunoassay kits. Results: Among the 17 cytokines/chemokines analysed, interleukin‐2 (IL‐2), IL‐5, IL‐13 and interferon‐γ (IFN‐γ) secretion in response to the drugs were significantly increased in patients when compared with healthy controls. No difference in cytokine secretion patterns between sulfonamide‐ and β‐lactam‐reactive PBMC could be observed. The secretion of other cytokines/chemokines showed a high variability among patients. Conclusion: The measurement of IL‐2, IL‐5, IL‐13 or IFN‐γ or a combination thereof might be a useful in vitro tool for detection of T‐cell sensitization to drugs. Secretion of these cytokines seems independent of the type of drug antigen and the phenotype of the drug reaction. A study including a higher number of patients and controls will be needed to determine the exact sensitivity and specificity of this test.     

Plasmodium falciparum infection of the placenta impacts on the T helper type 1 (Th1)/Th2 balance of neonatal T cells through CD4+CD25+ forkhead box P3+ regulatory T cells and interleukin‐10

Placental malaria infection affects the T helper type 1 (Th1)/Th2 balance in neonatal children. We investigated a potential role of regulatory T cells in this balance by comparing T cell responses of cord blood mononuclear cells (CBMC) from parasitized and non‐parasitized placenta of Gambian women. CBMC were depleted of CD4⁺CD25⁺ forkhead box P3 (FoxP3)⁺ regulatory T cells and analysed in vitro for their ability to produce interferon (IFN)‐γ, sCD30 and interleukin (IL)‐10 in response to phytohaemagglutinin (PHA), live Plasmodium falciparum, schizont extracts and the recombinant P. falciparum blood stage antigen merozoite surface protein 1 (MSP1₁₉). As expected, lower IFN‐γ and higher sCD30 responses were observed for the cells from the parasitized group. In addition, higher IL‐10 levels were produced by CBMC from the parasitized group. Depletion of regulatory T cells decreased IL‐10 production, which resulted in a restoration of IFN‐γ expression in response to all stimuli. The Th2 marker sCD30 remained significantly higher in the parasitized group in response to malaria protein antigens while similar levels were recovered between both groups in response to live P. falciparum. Similar effects were observed by adding an antibody that blocks IL‐10 function. These results suggest that the impact of P. falciparum infection on Th1 differentiation of neonatal T cells can be ascribed to regulatory T cells through production of IL‐10.     

Cellular immune responses in multiple sclerosis patients treated with interferon‐beta

We investigated cellular immune responses at baseline in peripheral blood mononuclear cells (PBMC) of patients with multiple sclerosis (MS) treated with interferon (IFN)‐β and classified into responders and non‐responders according to clinical response criteria. Levels for IFN‐γ, interleukin (IL)‐17A, IL‐17F, IL‐10 and IL‐4 were determined in activated PBMC of 10 responders, 10 non‐responders and 10 healthy controls by cytometric bead arrays. Cytokine levels in cell culture supernatants were similar between responders and non‐responders, and comparable to those obtained in healthy controls. These findings do not support differential cellular immune responses in PBMC at baseline between IFN‐β responders and non‐responders.     

Correlation of Th1-type cytokine expression and induced proliferation to lipopolysaccharide

Recent evidence from mouse models indicates that neonatal exposure to lipopolysaccharide (LPS) can prevent experimentally induced allergic disease. Furthermore, we noted that human cord blood mononuclear cells (CBMC) have an increased proliferative response to LPS relative to their respective maternal peripheral blood mononuclear cells (PBMC). We sought, therefore, to examine the cytokine expression profile induced by LPS in CBMC and its relationship to the LPS-mediated proliferative response. CBMC and maternal PBMC were evaluated for IL-10, IL-4, IL-13, IL-12 alpha, and IFN-gamma expression after LPS stimulation by real-time PCR. IFN-gamma secretion was detected by enzyme-linked immunosorbent assay. LPS increased IFN-gamma and IL-13, but decreased IL-4 expression in CBMC (P < 0.024, P < 0.014, and P < 0.027, respectively). In PBMC, however, no significant changes in expression were noted after LPS stimulation. Stimulation by LPS significantly increased the secretion of IFN-gamma in CBMC compared with PBMC at the two concentrations analyzed (1 ng/ml, P < 0.048; 10,000 ng/ml, P < 0.003). The magnitude of the LPS-mediated proliferative effect in CBMC directly correlated to the level of induction of IFN-gamma (P < 0.01), but inversely correlated to the induced levels of IL-4 and IL-13 (P < 0.01 and P = 0.01, respectively). No association of the CBMC proliferative response to IL-12 alpha or IL-10 was noted. Thus, a high proliferative response to LPS in CBMC correlates with a change from a Th2- to Th1-induced cytokine expression profile. Since early exposure to LPS may protect against allergic disease, one may speculate that an aberrant response to LPS may increase the likelihood of developing overt disease in susceptible individuals.     

Relationship of CD146 expression to secretion of interleukin (IL)‐17, IL‐22 and interferon‐γ by CD4+ T cells in patients with inflammatory arthritis

Expression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4⁺ T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146⁺CD4⁺ and interleukin (IL)‐17⁺CD4⁺ T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146⁺CD4⁺ T cells were enriched for secretion of IL‐17 [alone or with IL‐22 or interferon (IFN)‐γ] and for some putative Th17‐associated surface markers (CD161 and CCR6), but not others (CD26 and IL‐23 receptor). CD4⁺ T cells producing IL‐22 or IFN‐γ without IL‐17 were also present in the CD146⁺ subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4⁺ helper T cells.     

Production of Cytokines During Interaction of Peripheral Blood Mononuclear Cells with Autologous Ovarian Cancer Cells or Benign Ovarian Tumour Cells

Cytokines produced by tumour and immune cells may play a significant role in a modulation of immune cells response against tumour. We investigated an ability of peripheral blood mononuclear cells (PBMC) of patients with early and advanced stages of ovarian cancer and from non‐cancer patients to produce various cytokines in the presence or absence of autologous ovarian cancer (OC) cells or benign ovarian tumour (BOT) cells. Activated PBMC of patients with advanced stage of cancer produced slight amount of interferon γ (IFN‐γ) and what’s more, the production of IFN‐γ was decreased in the presence of OC cells. PBMC of patients with ovarian cancer or benign ovarian tumour generated comparable amounts of interleukin 6 and 10 (IL‐6, IL‐10), and transforming growth factor β1 (TGF‐β1). PBMC of the patients with cancer produced higher amount of tumour necrosis factor α (TNF‐α) than PBMC of non‐cancer patients. We demonstrated here that the reciprocal contact of OC cells from advanced cancer with autologous PBMC altered the direction of produced cytokines and leads to the down‐regulation of IFN‐γ and TNF‐α as well as to up‐regulation of immunosuppressive (IL‐10, TGF‐β1) and pro‐inflammatory (IL‐6) cytokines production.     

Human polymorphonuclear leukocytes produce cytokines in response to Leishmania major promastigotes

Polymorphonuclear leukocytes (PMN) release cytokines that may influence the development of the subsequent adaptive immune response. Little is known about cytokines produced by human PMN in response to Leishmania (L.). In this study, mRNA expression of Interleukin (IL)‐12p40, IL‐12p35, Interferon (IFN)‐γ, transforming growth factor (TGF)‐β, IL‐1, and IL‐4 in PMN of volunteers stimulated with L. major promastigotes has been investigated by real‐time PCR and the results were confirmed by flow cytometer. The results showed that L. major did not induce mRNA expression of IL12p40, IL12p35, IFN‐γ, and TGF‐β in PMN, while IL‐1 and IL‐4 mRNA were induced. Flow cytometry results confirmed no IFN‐γ production by PMN with or without stimulation. IL‐12p70 was present in untreated and L. major‐treated PMN, and these cells release IL‐12 following incubation with L. major. Significant amount of IL‐1 even without treatment with promastigotes was detected in PMN. Moreover, the proportion of PMN, which produce IL‐1 in response to L. major, was increased compared with the percent of unstimulated IL‐1‐producing PMN. The results showed the accumulation of small amounts of IL‐4 in PMN after stimulation. In conclusion, our results indicate that IL‐12 and IL‐1 are pre‐stored in human PMN, nor L. major induces IL‐1 and IL‐4, but not IL‐12, IFN‐γ, nor TGF‐β expression in these cells.     

Allergen‐specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression

Background A sensitive measurement of low numbers of intracellular cytokine‐expressing antigen‐specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy. Objective To evaluate the applicability of this method for measuring changes in cytokine production by allergen‐specific T cells in a clinical trial setting. Methods Ex vivo ragweed‐specific CD154 and intracellular cytokine expression were evaluated using a subset of subjects in an environmental chamber study of allergic rhinitis immunotherapy. PBMC were collected and cryopreserved from Amb a 1‐immunostimulatory oligodeoxynucleotide conjugate (AIC)‐treated (n=17) and placebo‐treated (n=15) ragweed‐allergic subjects both after pre‐ and post‐treatment ragweed exposures. In vitro allergen‐stimulated CD3⁺CD4⁺CD154⁺ T cell intracellular IL‐4, IL‐5, IL‐13, and IFN‐γ expression were evaluated by flow cytometry. Results Compared with the T helper type 2 (Th2) cytokine expression measured after pre‐treatment ragweed exposures, placebo‐treated subjects demonstrated a significantly elevated ragweed‐ and Amb a 1‐specific T cell IL‐4 and IL‐13 co‐expression (P=0.005 and P=0.022, respectively) and a significantly elevated ragweed‐specific IL‐5 expression (P<0.001) following post‐treatment ragweed exposures. In contrast, AIC‐treated subjects demonstrated no increases in allergen‐specific Th2 cytokine expression following post‐treatment ragweed exposures. IFN‐γ expression remained low and un‐changed in both groups. Subject reported total nasal symptom scores demonstrated modest but significant correlations with Amb a 1‐ and ragweed‐stimulated intracellular Th2 cytokine responses. Conclusion Combined CD154 and intracellular cytokine staining in PBMC can be used to sensitively monitor changes in antigen‐specific T cell subset frequencies in clinical studies. Antigen‐specific cytokine expression moderately correlated with the reported levels of allergic symptoms. Trial Registration NCT00537355 Cite this as: J. D. Campbell, P. Buchmann, S. Kesting, C. R. Cunningham, R. L. Coffman and E. M. Hessel, Clinical & Experimental Allergy, 2010 (40) 1025–1035.     

Mycobacterial antigen‐induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non‐diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non‐diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell‐mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non‐diabetic TB patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen‐induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐β], and Th2 (IL‐4, IL‐5, IL‐10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1_pool stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1_pool in healthy subjects. In response to complex mycobacterial antigens, both IFN‐γ and TNF‐β were secreted by PBMC of all groups whereas diabetic TB patients secreted IL‐10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN‐γ and IL‐10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.     

Interleukin‐27 suppresses osteoclastogenesis via induction of interferon‐γ

Interleukin (IL)‐27 is a heterodimeric cytokine that is known to have both stimulatory and inhibitory functions during immune responses. We investigated the effects of IL‐27 on arthritis and bone erosion in the murine collagen‐induced arthritis (CIA) model. We demonstrate that the inhibitory effect of IL‐27 on osteoclastogenesis is associated with interferon‐γ (IFN‐γ) production by using an IFN‐γ knockout mouse model. The IL‐27‐Fc was injected into both CIA and IFN‐γ‐deficient mice. The effects of IL‐27‐Fc on osteoclast differentiation were evaluated both in vitro and in vivo. The IL‐27‐Fc‐injected mice showed significantly lower arthritis indices and fewer tartrate‐resistant acid‐phosphatase‐positive osteoclasts in their joint tissues than untreated mice. Interleukin‐27 inhibited osteoclastogenesis from bone marrow‐derived mononuclear cells in vitro, which was counteracted by the addition of anti‐IFN‐γ antibody. The IL‐27‐Fc did not affect arthritis in IFN‐γ knockout mice. Interleukin‐27 also suppressed osteoclast differentiation in human and intriguingly, it could promote the expression of IFN‐γ on priming osteoclasts. These results imply that IL‐27 suppressed the generation of CIA and osteoclastogenesis, which were mediated by the induction of IFN‐γ.     

Multi‐functional flow cytometry analysis of CD4+ T cells as an immune biomarker for latent tuberculosis status in patients treated with tumour necrosis factor (TNF) antagonists

Although monitoring tuberculosis (TB) infection during long‐term treatment with tumour necrosis factor (TNF) antagonists is of great importance, no monitoring strategy has yet proved successful. Indeed, even the newly proposed interferon‐gamma release assays (IGRAs) are known to produce dynamic changes in IFN‐γ plasma levels, making them unreliable indicators of patients' pathological/clinical status. We used intracellular cytokine flow cytometry (ICCFC) to investigate the performance of multi‐functional CD4⁺ T cells producing IFN‐γ, interleukin (IL)‐2 and/or TNF in response to Mycobacterium tuberculosis‐specific antigens in subjects treated with TNF antagonists. Patients were classified into three groups based on their TB status before commencement of treatment and on IFN‐γ level fluctuations evaluated by IGRA during a 36‐month follow‐up period. The cytokine profile of M. tuberculosis‐specific CD4⁺ T cells showed that latent tuberculosis infection (LTBI) subjects had a higher frequency of double‐positive IFN‐γ⁺ IL‐2⁺ CD4⁺ T cells and triple‐positive IFN‐γ⁺ IL‐2⁺ TNF⁺ CD4⁺ T cells compared to those without LTBI, who showed IFN‐γ‐level fluctuations over time. In contrast, this latter group of patients showed similar proportions of cells producing IFN‐γ alone, IL‐2 alone and IL‐2 in combination with TNF in response to M. tuberculosis‐specific antigens. It therefore appears that patients with and without LTBI infection are characterized by different intracellular cytokine profiles. This is the first study evaluating ICCFC in patients treated with TNF antagonists, and suggests that multi‐functional analysis of CD4⁺ T cells could be useful for ruling out TB infection in patients classified at screening as LTBI‐negative but who show IGRA fluctuations under long‐term TNF antagonist treatment.     

IFN‐γ stimulates CpG‐induced IL‐10 production in B cells via p38 and JNK signalling pathways

The production of IL‐10, a potent immunosuppressive cytokine, must be strictly regulated to ensure a balanced immune response. IFN‐γ, a key cytokine in multiple immune processes and pathologies, is known as an inhibitor of IL‐10 production by monocytes and macrophages, but also has some regulatory functions. In the present study, we explored the role of IFN‐γ on Toll‐like receptor (TLR)‐induced IL‐10 production in murine peritoneal and spleen cells and in human peripheral blood mononuclear cells. IFN‐γ inhibited IL‐10 production induced by TLR2, TLR3, TLR4 and TLR7/8 agonists, but stimulated IL‐10 production when cells were triggered with CpG oligodeoxynucleotides, a specific TLR9 agonist. The stimulatory effect of IFN‐γ on TLR9‐induced IL‐10 was restricted to B cells. In line with the increased IL‐10, B cells stimulated with CpG and IFN‐γ profoundly inhibited CD4 T cell proliferation. Further research into the mechanisms involved, revealed that the mitogen‐activated protein kinases p38 and JNK are essential players in this stimulatory effect, and that the phosphatase MKP1 – an inhibitor of p38 and JNK activity – is downregulated after combined stimulation with IFN‐γ and CpG. Our data may represent a novel immunoregulatory role of IFN‐γ in B cells after triggering of TLR9, by stimulating IL‐10 production.     

The self‐antigen,thyroglobulin, induces antigen‐experienced CD4+ T cells from healthy donors to proliferate and promote production of the regulatory cytokine,interleukin‐10, by monocytes

Thyroglobulin (TG), as autoantigen, induces in vitro proliferation of T and B cells from normal individuals, but the cytokine production differs from that in patients with autoimmune thyroid disease. Here, we investigate whether normal T cells responding to TG are naive, or have previously encountered TG in vivo, using their responses to classic primary and secondary antigens, keyhole limpet haemocyanin (KLH) and tetanus toxoid (TT), respectively, for comparison. While TG elicited T‐cell proliferation kinetics typical of a secondary response, the cytokine profile was distinct from that for TT. Whereas TT induced pro‐inflammatory cytokines [interleukin‐2 (IL‐2)/interferon‐γ (IFN‐γ)/IL‐4/IL‐5], TG evoked persistent release of the regulatory IL‐10. Some donors, however, also responded with late IFN‐γ production, suggesting that the regulation by IL‐10 could be overridden. Although monocytes were prime producers of IL‐10 in the early TG response, a few IL‐10‐secreting CD4⁺ T cells, primarily with CD45RO⁺ memory phenotype, were also detected. Furthermore, T‐cell depletion from the mononuclear cell preparation abrogated monocyte IL‐10 production. Our findings indicate active peripheral tolerance towards TG in the normal population, with aberrant balance between pro‐ and anti‐inflammatory cytokine responses for some donors. This observation has implications for autoantigen recognition in general, and provides a basis for investigating the dichotomy between physiological and pathological modes of auto‐recognition.     

Analysis of T-lymphocyte subsets after phytohemagglutinin stimulation in normal and type 1 diabetic mothers and their infants.

PROBLEM: Our aim was to investigate the immunological status of diabetic pregnancy, which is an overlap of diabetic immunity abnormalities and the immunological modifications normally occurring during pregnancy. METHOD: We studied lymphocyte subpopulations and lymphokine production, after 96 h of phytohemagglutinin (PHA) stimulation, from normal and Type I diabetic pregnant women at delivery time and from the respective cord blood. RESULTS: Peripheral blood mononuclear cells (PBMC) from both normal and Type I diabetic mothers showed an increase in CD8+ and a decrease in CD4+ cells compared to the respective cord blood mononuclear cells (CBMC). Moreover, Type I PBMC showed a lower number of "activated" CD3+ DR+ cells and a higher number of CD8+ CD25+ cells with respect to normal women, which may reflect the dysregulatory pattern due to the autoimmune condition. Type I CBMC showed a big increase in the number of CD4+ Leu8+ cells, a cell subpopulation characterized by inhibitory activity. Finally, as regards lymphokine release in culture supernatants, type I diabetes seemed to be associated with an over-production of IL1 and IL6, although the latter increase is less evident in CBMC cultures. CONCLUSIONS: The present study shows that diabetic pregnancy is associated with major alterations of cell-mediated immunity leading to a state of immunodepression. Moreover, our study suggests that the maternal immunological status influences fetal immunity, as demonstrated by the increase in the number of regulatory cells and by the altered pattern of lymphokine production (IL1 and IL6) by lymphocytes derived from diabetic CBMC. The latter phenomenon perfectly mirrors maternal PBMC characteristics.