Samuel Lundin

Large-scale identification of genes implicated in kidney glomerulus development and function

To advance our understanding of development, function and diseases in the kidney glomerulus, we have established and large‐scale sequenced cDNA libraries from mouse glomeruli at different stages of development, resulting in a catalogue of 6053 different genes. The glomerular cDNA clones were arrayed and hybridized against a series of labeled targets from isolated glomeruli, non‐glomerular kidney tissue, FACS‐sorted podocytes and brain capillaries, which identified over 300 glomerular cell‐enriched transcripts, some of which were further sublocalized to podocytes, mesangial cells and juxtaglomerular cells by in situ hybridization. For the earliest podocyte marker identified, Foxc2, knockout mice were used to analyze the role of this protein during glomerular development. We show that Foxc2 controls the expression of a distinct set of podocyte genes involved in podocyte differentiation and glomerular basement membrane maturation. The primary podocyte defects also cause abnormal differentiation and organization of the glomerular vascular cells. We surmise that studies on the other novel glomerulus‐enriched transcripts identified in this study will provide new insight into glomerular development and pathomechanisms of disease.

The Transfer of Immunity from Mother to Child

Abstract: The newborns immune system grows fast from a small size at birth by exposure primarily to the intestinal microflora normally obtained from the mother at and after birth. While building up its immune system, the infant is supported by the transplacental IgG antibodies, which also contain anti‐idiotypic antibodies, possibly also actively priming the offspring. The second mode of transfer of immunity occurs via the milk. Numerous major protective components, including secretory IgA (SIgA) antibodies and lactoferrin, are present.

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.

Oral Tolerance Induction with Antigen Conjugated to Cholera Toxin B Subunit Generates Both Foxp3+CD25+ and Foxp3−CD25− CD4+ Regulatory T Cells

Oral administration of Ag coupled to cholera toxin B subunit (CTB) efficiently induces peripheral immunological tolerance. We investigated the extent to which this oral tolerance is mediated by CD25+CD4+ regulatory T cells (Treg). We found that total Treg, KJ1–26+ Treg and CTLA-4+ Treg were all increased in Peyer’s patches, mesenteric lymph nodes, and, to a lesser extent, in spleen of mice after intragastric administration of OVA/CTB conjugate, which also increased TGF-β in serum. This could be abolished by coadministering cholera toxin or by treatment with anti-TGF-β mAb. CD25+ Treg, but also CD25−CD4+ T cells from OVA/CTB-treated BALB/c or DO11.10 mice efficiently suppressed effector T cell proliferation and IL-2 production in vitro. Following adoptive transfer, both T cell populations also suppressed OVA-specific T cell and delayed-type hypersensitivity responses in vivo. Foxp3 was strongly expressed by CD25+ Treg from OVA/CTB-treated mice, and treatment also markedly expanded CD25+Foxp3+ Treg. Furthermore, in Rag1−/− mice that had adoptively received highly purified Foxp3−CD25−CD4+ OT-II T cells OVA/CTB feeding efficiently induced CD25+ Treg cells, which expressed Foxp3 more strongly than naturally developing Treg and also had stronger ability to suppress effector OT-II T cell proliferation. A remaining CD25− T cell population, which also became suppressive in response to OVA/CTB treatment, did not express Foxp3. Our results demonstrate that oral tolerance induced by CTB-conjugated Ag is associated with increase in TGF-β and in both the frequency and suppressive capacity of Foxp3+ and CTLA-4+ CD25+ Treg together with the generation of both Foxp3+ and Foxp3−CD25− CD4+ Treg.

Oral tolerization with peptide 336–351 linked to cholera toxin B subunit in preventing relapses of uveitis in Behcet's disease

Behcets disease (BD) specific peptide (p336–351) was identified within the human 60 kD heat shock protein (HSP60). Oral p336–351 induced uveitis in rats which was prevented by oral tolerization with the peptide linked to recombinant cholera toxin B subunit (CTB). This strategy was adopted in a phase I/II clinical trial by oral administration of p336–351‐CTB, 3 times weekly, followed by gradual withdrawal of all immunosuppressive drugs used to control the disease in 8 patients with BD. The patients were monitored by clinical and ophthalmological examination, as well as extensive immunological investigations. Oral administration of p336–351‐CTB had no adverse effect and withdrawal of the immunosuppressive drugs showed no relapse of uveitis in 5 of 8 patients or 5 of 6 selected patients who were free of disease activity prior to initiating the tolerization regimen. After tolerization was discontinued, 3 of 5 patients remained free of relapsing uveitis for 10–18 months after cessation of all treatment. Control of uveitis and extra‐ocular manifestations of BD was associated with a lack of peptide‐specific CD4+ T cell proliferation, a decrease in expression of TH1 type cells (CCR5, CXCR3), IFN‐γ and TNF‐α production, CCR7+ T cells and costimulatory molecules (CD40 and CD28), as compared with an increase in these parameters in patients in whom uveitis had relapsed. The efficacy of oral peptide‐CTB tolerization will need to be confirmed in a phase III trial, but this novel strategy in humans might be applicable generally to autoimmune diseases in which specific antigens have been identified.

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.

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.

Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

Accumulation of CCR4+ CTLA-4hi FOXP3+CD25hi Regulatory T Cells in Colon Adenocarcinomas Correlate to Reduced Activation of Conventional T Cells

Background Colorectal cancer usually gives rise to a specific anti-tumor immune response, but for unknown reasons the resulting immunity is not able to clear the tumor. Recruitment of activated effector lymphocytes to the tumor is important for efficient anti-tumor responses, while the presence of regulatory T cells (Treg) down-modulate tumor-specific immunity. We therefore aimed to determine homing mechanisms and activation stage of Treg and effector T cell infiltrating colon tumors compared to cells from the unaffected mucosa in patients suffering from colon adenocarcinoma. Methodology/Principal Findings Lymphocytes were isolated from unaffected and tumor mucosa from patients with colon adenocarcinoma, and flow cytometry, immunohistochemistry, and quantitative PCR was used to investigate the homing mechanisms and activation stage of infiltrating Treg and conventional lymphocytes. We detected significantly higher frequencies of CD25highFOXP3+CD127low putative Treg in tumors than unaffected mucosa, which had a complete demethylation in the FOXP3 promotor. Tumor-associated Treg had a high expression of CTLA-4, and some appeared to be antigen experienced effector/memory cells based on their expression of αEβ7 (CD103). There were also significantly fewer activated T cells and more CTLA-4+ conventional T cells susceptible to immune regulation in the tumor-associated mucosa. In contrast, CD8+granzyme B+ putative cytotoxic cells were efficiently recruited to the tumors. The frequencies of cells expressing α4β7 and the Th1 associated chemokine receptor CXCR3 were significantly decreased among CD4+ T cells in the tumor, while frequencies of CD4+CCR4+ lymphocytes were significantly increased. Conclusions/Significance This study shows that CCR4+CTLA4hi Treg accumulate in colon tumors, while the frequencies of activated conventional Th1 type T cells are decreased. The altered lymphocyte composition in colon tumors will probably diminish the ability of the immune system to effectively attack tumor cells, and reducing the Treg activity is an important challenge for future immunotherapy protocols.

Human IgA‐secreting cells induced by intestinal, but not systemic, immunization respond to CCL25 (TECK) and CCL28 (MEC)

Organ‐specific homing of lymphoid cells depends on the expression of tissue‐specific adhesion molecules and production of specific chemokines. CCL25 (TECK) and CCL28 (MEC) have been reported to direct circulating memory/effector B cells to mucosal tissues. Here, we examined if differential responsiveness to mucosal and systemic chemokines could explain the differential migration pattern of circulating human antibody‐secreting cells (ASC), induced by mucosal and systemic immunization. There was a robust migration of specific IgA‐ and IgM‐ASC induced by Salmonella vaccination toward the mucosal chemokines CCL25 and CCL28. In contrast, tetanus‐specific ASC migrated to the systemic chemokine CXCL12 (SDF‐1α) and showed no response to CCL25 or CCL28, not even tetanus‐specific IgA‐ASC. Cell sorting experiments demonstrated that Salmonella‐specific ASC co‐expressed CCR9 and CCR10. Our results show that induction site, rather than isotype commitment, determines the chemokine responsiveness and migration pattern of human effector B cells.