Anna Rudin

Characterization of human CD25+ CD4+ T cells in thymus, cord and adult blood

CD4+ CD25+ regulatory T cells prevent organ‐specific autoimmune diseases in various animal models. We analysed human lymphoid tissues to identify similar CD25+ regulatory T cells. Adult peripheral blood contained two populations of CD4+ T cells that expressed CD25 at different densities. The larger population (≈ 40%) expressed intermediate levels of CD25 (CD25+) and displayed a memory T‐cell phenotype (CD45RA−/RO+, CD45RBlow, CD95+, CD62Llow, CD38low). The smaller population of cells (≈ 2%) expressed very high levels of CD25 (CD25++). In addition to the activation/memory T‐cell antigens mentioned above they also expressed intracellular CD152 (CTLA‐4) as well as enhanced levels of cell‐surface CD122, similar to the murine CD4+ CD25+ regulatory counterpart. To exclude that the CD25++ cells had not been recently primed by external antigen we analysed cord blood and thymus. CD25++, CD152+ and CD122++ cells were present in paediatric thymus (10% of CD4+ CD8− thymocytes) expressing signs of recent selection (CD69+) and in cord blood (5% of CD4+ cells) where they showed a naive phenotype. In addition, cord blood contained a small population of CD25+ cells (≈ 2% of CD4 T cells) that were CD152− and CD122low and displayed signs of activation. Together with published data that CD25+ CD25++ cells from the thymus and peripheral blood are regulatory, our results suggest that regulatory CD25+ T cells leave the thymus in a naïve state and become activated in the periphery.

Mucosal FOXP3-expressing CD4+ CD25high regulatory T cells in Helicobacter pylori-infected patients

ABSTRACT Helicobacter pylori chronically colonizes the stomach and duodenum and causes peptic ulcers or gastric adenocarcinoma in 10 to 20% of infected individuals. We hypothesize that the inability of patients to clear H. pylori infections is a consequence of active suppression of the immune response. Here we show that H. pylori-infected individuals have increased frequencies of CD4+ CD25high T cells in both the stomach and duodenal mucosa compared to uninfected controls. These cells have the phenotype of regulatory T cells, as they express FOXP3, a key gene for the development and function of regulatory T cells, as well as high levels of the cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) protein. In contrast, mucosal CD4+ CD25low and CD4+ CD25− cells express little FOXP3 mRNA and low levels of the CTLA-4 protein. Mucosal CD4+ CD25high T cells are present in individuals with asymptomatic H. pylori infections as well as in duodenal ulcer patients. The frequencies of CD4+ CD25high cells are also increased in the stomachs of H. pylori-infected patients with gastric adenocarcinoma, particularly in cancer-affected tissues. These findings suggest that regulatory T cells may suppress mucosal immune responses and thereby contribute to the persistence of H. pylori infections.

Defective suppression of Th2 cytokines by CD4+CD25+regulatory T cells in birch allergics during birch pollen season

Background CD4+CD25+ regulatory T cells suppress proliferation and cytokine production by human T cells both to self‐antigens and exogenous antigens. Absence of these cells in human newborns leads to multiple autoimmune and inflammatory disorders together with elevated IgE levels. However, their role in human allergic disease is still unclear.

Innate Immune Responses of Human Neonatal Cells to Bacteria from the Normal Gastrointestinal Flora

ABSTRACT The hygiene hypothesis postulates that the prevalence of allergy has increased due to decreased microbial stimulation early in life, leading to delayed maturation of the immune system. The aim of this study was to examine the cytokine pattern produced from cord blood mononuclear cells relative to adult cells after stimulation with bacterial strains from the normal flora. Mononuclear cells from cord and adult blood samples were stimulated with the following bacteria: Bifidobacterium adolescentis, Enterococcus faecalis, Lactobacillus plantarum, Streptococcus mitis, Corynebacterium minutissimum, Clostridium perfringens, Bacteroides vulgatus, Escherichia coli, Pseudomonas aeruginosa, Veillonella parvula, and Neisseria sicca. The levels of interleukin 12 (IL-12), tumor necrosis factor alpha (TNF-α), IL-10, and IL-6 were measured by enzyme-linked immunosorbent assay. The TNF-α production was also analyzed after blocking CD14, Toll-like receptor 2 (TLR-2), and TLR-4 prior to stimulation with bacteria. The levels of IL-12 and TNF-α were similar in cord and adult cells. Gram-positive bacteria induced considerably higher levels of IL-12 and TNF-α than gram-negative bacteria in both cord and adult cells. The levels of IL-6 were significantly higher in newborns than in adults, whereas the levels of IL-10 were similar in newborns and adults. Gram-negative and gram-positive bacteria induced similar levels of IL-6 and IL-10 in cord cells. L. plantarum bound or signaled through CD14, TLR-2, and TLR-4, whereas E. coli acted mainly through CD14 and TLR-4. These results indicate that the innate immune response in newborns to commensal bacteria is strong and also suggest that different bacterial strains may have differential effects on the maturation of the immune system of infants.

Nasal and Vaginal Vaccinations Have Differential Effects on Antibody Responses in Vaginal and Cervical Secretions in Humans

ABSTRACT Sexually transmitted diseases are a major health problem worldwide, but there is still a lack of knowledge about how to induce an optimal immune response in the genital tract of humans. In this study we vaccinated 21 volunteers nasally or vaginally with the model mucosal antigen cholera toxin B subunit and determined the level of specific immunoglobulin A (IgA) and IgG antibodies in vaginal and cervical secretions as well as in serum. To assess the hormonal influence on the induction of antibody responses after vaginal vaccination, we administered the vaccine either independently of the stage in the menstrual cycle or on days 10 and 24 in the cycle in different groups of subjects. Vaginal and nasal vaccinations both resulted in significant IgA and IgG anti-cholera toxin B subunit responses in serum in the majority of the volunteers in the various vaccination groups. Only vaginal vaccination given on days 10 and 24 in the cycle induced strong specific antibody responses in the cervix with 58-fold IgA and 16-fold IgG increases. In contrast, modest responses were seen after nasal vaccination and in the other vaginally vaccinated group. Nasal vaccination was superior in inducing a specific IgA response in vaginal secretions, giving a 35-fold increase, while vaginal vaccination induced only a 5-fold IgA increase. We conclude that a combination of nasal and vaginal vaccination might be the best vaccination strategy for inducing protective antibody responses in both cervical and vaginal secretions, provided that the vaginal vaccination is given on optimal time points in the cycle.

Functional CD4+CD25high regulatory T cells are enriched in the colonic mucosa of patients with active ulcerative colitis and increase with disease activity

Background: Factors determining the extension and degree of inflammation in the colonic mucosa of patients with ulcerative colitis (UC) are largely unknown, but CD4+CD25high regulatory T cells (Tregs) have been implicated to play an important role in suppressing inflammation. Therefore, the aims of this study were to determine whether colonic Tregs have suppressive effects on colonic effector T cells in UC and to analyze the association between segmental colonic Treg distribution and disease activity. Materials and Methods: The suppressive activity of colonic CD4+CD25high Tregs from patients with active UC was determined in coculture assays measuring proliferation and cytokine production. The frequency of Tregs and the expression of the Treg marker FOXP3 were analyzed with flow cytometry and RT‐PCR in isolated cells and the whole mucosa from patients with active and inactive disease, as well as healthy mucosa. Results: Colonic CD4+CD25high T cells from patients with UC suppressed the proliferation and cytokine secretion of colonic effector CD4+ T cells. Healthy controls but not patients with UC had lower Treg frequencies in the sigmoid than in the ascending colon. Patients with UC with active disease had increased frequency of colonic Tregs. The frequency of Tregs was positively correlated with colonic disease activity and serum C‐reactive protein. Conclusions: Colonic CD4+CD25high Tregs are able to suppress colonic effector T cell activity in vitro, and the Treg frequency in the inflamed intestine increases with disease activity in patients with active UC. This suggests that Tregs may be outnumbered by other inflammatory cells or that their suppressive activity may be influenced by the in vivo environment.

Pattern of Cytokine Responses to Gram-Positive and Gram-Negative Commensal Bacteria Is Profoundly Changed when Monocytes Differentiate into Dendritic Cells

ABSTRACT The normal gastrointestinal bacterial flora is crucial for the maturation of acquired immunity via effects on antigen-presenting cells (APCs). Here we investigated how two types of APCs, monocytes and dendritic cells (DCs), react to different bacterial strains typical of the commensal intestinal microflora. Purified human monocytes and monocyte-derived DCs were stimulated with UV-inactivated gram-positive (Lactobacillus plantarum and Bifidobacterium adolescentis) and gram-negative (Escherichia coli and Veillonella parvula) bacterial strains. Monocytes produced higher levels of interleukin 12p70 (IL-12p70) and tumor necrosis factor (TNF), as detected by an enzyme-linked immunosorbent assay, in response to L. plantarum than in response to E. coli and V. parvula. In contrast, DCs secreted large amounts of IL-12p70, TNF, IL-6, and IL-10 in response to E. coli and V. parvula but were practically unresponsive to L. plantarum and B. adolescentis. The lack of a response to the gram-positive strains correlated with lower surface expression of Toll-like receptor 2 (TLR2) on DCs than on monocytes. The surface expression of TLR4 on DCs was undetectable when it was analyzed by flow cytometry, but blocking this receptor decreased the TNF production in response to V. parvula, indicating that TLR4 is expressed at a low density on DCs. Gamma interferon increased the expression of TLR4 on DCs and also potentiated the cytokine response to the gram-negative strains. Our results indicate that when monocytes differentiate into DCs, their ability to respond to different commensal bacteria dramatically changes, and they become unresponsive to probiotic gram-positive bacteria. These results may have important implications for the abilities of different groups of commensal bacteria to regulate mucosal and systemic immunity.

CD4 T cell activation by myelin oligodendrocyte glycoprotein is suppressed by adult but not cord blood CD25+ T cells.

Regulatory T cells expressing CD25 have been shown to protect rodents from organ‐specific autoimmune diseases. Similar CD25+ cells with a memory phenotype exerting suppressive function after polyclonal or allogeneic stimulation are also present in adult human blood. We demonstrate that adult human CD25+ cells regulate the response to myelin oligodendrocyte glycoprotein (MOG), as depletion of CD25+ cells increases responses of PBMC and the addition of purified CD25+ cells suppresses MOG‐specific proliferation and IFN‐γ production of CD4+CD25– T cells. In contrast, cord blood CD25+ cells do not inhibit responses to self antigens, and only a small subpopulation of cord CD25+ cells expresses the typical phenotype of adult regulatory T cells (CD45RA– and GITR+) enabling suppression of polyclonal responses. We conclude that activation of self‐reactive T cells in normal healthy individuals is prevented by the presence of self‐antigen‐specific CD25+ regulatory T cells and that the majority of these cells mature after birth.

Phase I Evaluation of Intranasal Trivalent Inactivated Influenza Vaccine with Nontoxigenic Escherichia coli Enterotoxin and Novel Biovector as Mucosal Adjuvants, Using Adult Volunteers

ABSTRACT Trivalent influenza virus A/Duck/Singapore (H5N3), A/Panama (H3N2), and B/Guandong vaccine preparations were used in a randomized, controlled, dose-ranging phase I study. The vaccines were prepared from highly purified hemagglutinin and neuraminidase from influenza viruses propagated in embryonated chicken eggs and inactivated with formaldehyde. We assigned 100 participants to six vaccine groups, as follows. Three intranasally vaccinated groups received 7.5-μg doses of hemagglutinin from each virus strain with either 3, 10, or 30 μg of heat-labile Escherichia coli enterotoxin (LTK63) and 990 μg of a supramolecular biovector; one intranasally vaccinated group was given 7.5-μg doses of hemagglutinin with 30 μg of LTK63 without the biovector; and another intranasally vaccinated group received saline solution as a placebo. The final group received an intramuscular vaccine containing 15 μg hemagglutinin from each strain with MF59 adjuvant. The immunogenicity of two intranasal doses, delivered by syringe as drops into both nostrils with an interval of 1 week between, was compared with that of two inoculations by intramuscular delivery 3 weeks apart. The intramuscular and intranasal vaccine formulations were both immunogenic but stimulated different limbs of the immune system. The largest increase in circulating antibodies occurred in response to intramuscular vaccination; the largest mucosal immunoglobulin A (IgA) response occurred in response to mucosal vaccination. Current licensing criteria for influenza vaccines in the European Union were satisfied by serum hemagglutination inhibition responses to A/Panama and B/Guandong hemagglutinins given with MF59 adjuvant by injection and to B/Guandong hemagglutinin given intranasally with the highest dose of LTK63 and the biovector. Geometric mean serum antibody titers by hemagglutination inhibition and microneutralization were significantly higher for each virus strain at 3 and 6 weeks in recipients of the intramuscular vaccine than in recipients of the intranasal vaccine. The immunogenicity of the intranasally delivered experimental vaccine varied by influenza virus strain. Mucosal IgA responses to A/Duck/Singapore (H5N3), A/Panama (H3N2), and B/Guandong were highest in participants given 30 μg LTK63 with the biovector, occurring in 7/15 (47%; P = 0.0103), 8/15 (53%; P = 0.0362), and 14/15 (93%; P = 0.0033) participants, respectively, compared to the placebo group. The addition of the biovector to the vaccine given with 30 μg LTK63 enhanced mucosal IgA responses to A/Duck/Singapore (H5N3) (P = 0.0491) and B/Guandong (P = 0.0028) but not to A/Panama (H3N2). All vaccines were well tolerated.

CD4 + CD25 + FOXP3 + regulatory T cells from human thymus and cord blood suppress antigen-specific T cell responses

Activation of self‐reactive T cells in healthy adults is prevented by the presence of autoantigen‐specific CD4+CD25+ regulatory T cells (CD25+ Treg). To explore the functional development of autoantigen‐reactive CD25+ Treg in humans we investigated if thymic CD25+ Treg from children aged 2 months to 11 years and cord blood CD25+ Treg are able to suppress proliferation and cytokine production induced by specific antigens. While CD4+CD25− thymocytes proliferated in response to myelin oligodendrocyte glycoprotein (MOG), tetanus toxoid and beta‐lactoglobulin, suppression of proliferation was not detected after the addition of thymic CD25+ Treg. However, CD25+ Treg inhibited interferon (IFN)‐γ production induced by MOG, which indicates that MOG‐reactive CD25+ Treg are present in the thymus. In contrast, cord blood CD25+ Treg suppressed both proliferation and cytokine production induced by MOG. Both cord blood and thymic CD25+ Treg expressed FOXP3 mRNA. However, FOXP3 expression was lower in cord blood than in thymic CD25+ T cells. Further characterization of cord blood CD25+ T cells revealed that FOXP3 was highly expressed by CD25+CD45RA+ cells while CD25+CD45RA− cells contained twofold less FOXP3, which may explain the lower expression level of FOXP3 in cord blood CD25+ T cells compared to thymic CD25+ T cells. In conclusion, our data demonstrate that low numbers of MOG‐reactive functional CD25+ Treg are present in normal thymus, but that the suppressive ability of the cells is broader in cord blood. This suggests that the CD25+ Treg may be further matured in the periphery after being exported from the thymus.