Monika Gad

Dendritic cells in peripheral tolerance and immunity

Dendritic cells capable of influencing immunity exist as functionally distinct subsets, T cell‐tolerizing and T cell‐immunizing subsets. The present paper reviews how these subsets of DCs develop, differentiate and function in vivo and in vitro at the cellular and molecular level. In particular, the role of DCs in the generation of regulatory T cells is highlighted.

Phenotypic and Functional Characterization of Clinical Grade Dendritic Cells Generated from Patients with Advanced Breast Cancer for Therapeutic Vaccination

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro‐generated monocyte‐derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte‐derived DC were generated in vitro utilizing granulocyte macrophage colony‐stimulating factor and rh‐interleukin‐4 (IL‐4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor‐alpha (TNF‐α) induced significant IL‐12 p70 secretion, which was increased after addition of a decoy IL‐10 receptor. The lymph node homing chemokine receptor CCR‐7 expression was induced by TNF‐α + IL‐1β + IL‐6 + prostaglandin E2 but was not induced by Poly I:C + TNF‐α. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient‐derived DC for antigen‐specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T‐cell stimulation, whereas the allostimulatory capacity of patient‐derived DC was significantly decreased. These data suggest that patient‐derived DC are more differentiated but are less sensitive to maturation‐inducing agents than DC obtained from healthy individuals.

Demonstration of strong enterobacterial reactivity of CD4+CD25– T cells from conventional and germ-free mice which is counter-regulated by CD4+CD25+ T cells

Unfractionated CD4+ T cells from the gut‐associated lymphoid tissue (GALT) and peripheral lymph nodes are unresponsive when exposed to enterobacterial antigens in vitro. Under similar conditions, CD4+ T cells depleted in vivo or in vitro of CD4+CD25+ T cells proliferate extensively. The CD4+CD25– T cell reactivity depends on MHC class II presentation, specific TCR stimulation, CD4 ligation, and antigen processing by antigen‐presenting cells. The CD4+CD25– T cells respond to autologousand heterologous enterobacterial antigens, but not to antigens from the feces of germ‐free mice. Surprisingly, CD4+CD25– T cells obtained from the GALT of germ‐free mice also proliferate when exposed to enterobacterial antigens, and adding back the conventional or germ‐free CD4+CD25+ T cells to the enteroantigen‐stimulated CD4+CD25– T cells abolishes proliferation. As judged from carboxyfluorescein diacetate succinimidyl ester‐labeling experiments, 4–5% of the CD4+CD25– T cells respond to enteroantigen. The data show for the first time that CD4+CD25– T cells with reactivity towards the enterobacterial flora and regulatory CD4+CD25+ T cells are present in both conventional and germ‐free mice. The data suggest that a significant proportion of the peripheral pool of CD4+CD25– T cells express anti‐enterobacterial reactivity, which, due to the presence of regulatory CD4+CD25+ T cells, is kept in a quiescent state.

Dexamethasone/1α-25-dihydroxyvitamin D3-treated dendritic cells suppress colitis in the SCID T-cell transfer model

Autoantigen‐presenting immunomodulatory dendritic cells (DCs) that are used for adoptive transfer have been shown to be a promising therapy for a number of autoimmune diseases. We have previously demonstrated that enteroantigen‐pulsed DCs treated with interleukin‐10 (IL‐10) can partly protect severe combined immunodeficient (SCID) mice adoptively transferred with CD4+ CD25− T cells from the development of wasting disease and colitis. We therefore established an in vitro test that could predict the in vivo function of DCs and improve strategies for the preparation of immunomodulatory DCs in this model. Based on these in vitro findings, we here evaluate three methods for DC generation including short‐term and long‐term IL‐10 exposure or DC exposure to dexamethasone in combination with vitamin D3 (Dex/D3). All DCs resulted in lower CD4+ CD25− T‐cell enteroantigen‐specific responses in vitro, but Dex/D3 DCs had the most prominent effect on T‐cell cytokine secretion. In vivo, Dex/D3 DCs most efficiently prevented weight loss and gut pathology upon CD4+ CD25− T‐cell transfer in SCID mice, although the effect on gut pathology was antigen independent. Our data in the SCID T‐cell transfer model illustrate some correlation between in vitro and in vivo DC function and document that prevention of experimental inflammatory bowel disease by transfer of immunosuppressive DCs is possible.

Blockage of the neurokinin 1 receptor and capsaicin-induced ablation of the enteric afferent nerves protect SCID mice against T-cell-induced chronic colitis

Background: The neurotransmitter substance P (SP) released by, and the transient receptor potential vanilloid (TRPV1), expressed by afferent nerves, have been implicated in mucosal neuro‐immune‐regulation. To test if enteric afferent nerves are of importance for the development of chronic colitis, we examined antagonists for the high‐affinity neurokinin 1 (NK‐1) SP receptor and the TRPV1 receptor agonist capsaicin in a T‐cell transfer model for chronic colitis. Methods: Chronic colitis was induced in SCID mice by injection of CD4+CD25− T cells. The importance of NK‐1 signaling and TRPV1 expressing afferent nerves for disease development was studied in recipient SCID mice systemically treated with either high‐affinity NK‐1 receptor antagonists or neurotoxic doses of capsaicin. In addition, we studied the colitis‐inducing effect of NK‐1 receptor deleted CD4+CD25− T cells. Results: Treatment with the NK‐1 receptor antagonist CAM 4092 reduced the severity of colitis, but colitis was induced by NK‐1 receptor‐deleted T cells, suggesting that SP in colitis targets the recipient mouse cells and not the colitogenic donor T cells. Capsaicin‐induced depletion of nociceptive afferent nerves prior to CD4+CD25− T‐cell transfer completely inhibited the development of colitis. Conclusions: Our data demonstrate the importance of an intact enteric afferent nerve system and NK‐1 signaling in mucosal inflammation and may suggest new treatment modalities for patients suffering from inflammatory bowel disease. (Inflamm Bowel Dis 2009)

Colitic scid mice fed Lactobacillus spp. show an ameliorated gut histopathology and an altered cytokine profile by local T cells

Background: Scid mice transplanted with CD4+ T blast cells develop colitis. We investigated if the disease was influenced in colitic mice treated with antibiotic and fed Lactobacillus spp. Methods: Colitic scid mice were treated for 1 week with antibiotics (vancomycin/meropenem) followed or not followed by a 3‐week administration of Lactobacillus reuteri DSM‐12246 and Lactobacillus rhamnosus 19070‐2 at 2 × 109 live bacteria/mouse/24 hours. After 12 weeks, the rectums were removed for histology, and CD4+ T cells from the mesenteric lymph nodes (MLN) were polyclonally activated for cytokine measurements. Results: Irrespective of no treatment or treatments with antibiotics and probiotics, all mice transplanted with T cell blasts lost 10% of their body weight during the 12‐week experimental period, whereas the nontransplanted mice had a 10% weight increase (P < 0.001). All mice treated with antibiotics but not fed probiotics showed severe gut inflammation, whereas only 2 of the 7 mice fed probiotics showed signs of severe colitis (P < 0.05). MLN‐derived CD4+ T cells from this latter group of mice showed lower levels of interleukin‐4 secretion (P < 0.05) and a tendency to higher interferon‐&ggr; production than mice not fed probiotics. Conclusions: Our data suggest that probiotics added to the drinking water may ameliorate local histopathological changes and influence local cytokine levels in colitic mice but not alter the colitis‐associated weight loss.

Characterization of T-regulatory cells, induced by immature dendritic cells, which inhibit enteroantigen-reactive colitis-inducing T-cell responses in vitro and in vivo

Regulatory T (Treg) cells, derived from co‐cultures of unfractionated CD4+ T cells and immature dendritic cells (DC), suppress enteroantigen‐induced proliferation of CD4+ CD25− T cells. The DC‐induced Treg cells are a mixture of CD25+ (10–20%) and CD25− (80–90%) T cells. However, all the suppressor activity in vitro and in vivo resides in the CD25+ T‐cell subset. The CD25+ DC‐induced Treg cells can inhibit enteroantigen‐induced proliferation in vitro through a transwell membrane, and their function does not appear to depend on previous activation. DC‐induced CD25+ Treg cells display a naïve phenotype, expressing high levels of CD45RB and l‐selectin (CD62L). In addition, the DC‐induced Treg cells mediate a stronger suppressive activity than prototype CD25+ regulatory T cells. The DC‐induced Treg cells, and hereof purified CD25+ and CD25− T‐cell fractions, were co‐injected into severe combined immunodeficiency (SCID) mice with colitis‐inducing CD4+ CD25− T cells. Both unfractionated CD4+ and purified CD25+ Treg cells fully protected the recipients against the development of colitis. In contrast, co‐transfer of fractionated CD25− T cells offered no protection against disease development. Enterobacterial antigen‐exposed CD4+ T cells of the protected mice secreted higher levels of interleukin‐10 and lower levels of interferon‐γ than the unprotected mice. The present data demonstrate DC‐induced CD4+ CD25+ Treg cells, which phenotypically and functionally differ from the generally accepted prototype of CD25+ Treg cells. These data may initiate new procedures for the expansion of Treg cells for clinical use.

Immunobiological Effects of Glucosamine In Vitro

Glucosamine (GlcN) and N‐acetyl‐d‐glucosamine (GlcNAc) were assayed in vitro for their effects on proliferation, cytotoxicity and cytokine secretion in primary and secondary mixed lymphocyte cultures (MLCs). In addition, we studied the effect of GlcN and GlcNAc on the proliferation of purified CD4+ T cells exposed to immobilized anti‐CD3 antibody. The present data show that GlcN, but not GlcNAc, inhibits CD4+ T‐cell proliferation, the generation of alloreactive cytotoxic T lymphocytes (CTLs) and the secretion of interferon‐γ (IFN‐γ) and interleukin‐5 (IL‐5) in primary MLC. In secondary T helper‐2 (Th2)‐polarized MLC, GlcN, but not GlcNAc, inhibits IL‐4 and IL‐5 secretion, whereas no effect was found on IFN‐γ secretion in Th1‐polarized MLC. Dendritic cells treated with GlcN showed a 75–80% decreased capacity for antigen cross‐presentation and allostimulation. In cellular bioassays, GlcN was shown to inhibit the stimulatory activity of IL‐4 and IL‐2, as well as the cytotoxic activity of tumour necrosis factor‐α (TNF‐α). In conclusion, GlcN suppresses unprimed T‐cell responses by interfering with antigen‐presenting cell functions and by a direct inhibitory effect on T‐cell proliferation. In addition, GlcN inhibits the secretion of cytokines in antigen‐stimulated unprimed T cells and primed Th2‐polarized cells.

Tolerogenic dendritic cells pulsed with enterobacterial extract suppress development of colitis in the severe combined immunodeficiency transfer model

Immunomodulatory dendritic cells (DCs) that induce antigen‐specific T‐cell tolerance upon in vivo adoptive transfer are promising candidates for immunotherapy of autoimmune diseases. The feasibility of such a strategy has recently proved its efficacy in animal models of allotransplantation and experimental allergic encephalitis, but the effect in inflammatory bowel disease has not yet been demonstrated. In severe combined immunodeficient (SCID) mice, adoptively transferred CD4+ CD25– T cells repopulate the lymphoid tissues and lead to development of chronic colitis characterized by CD4+ T‐cell proliferation against enterobacterial extract in vitro. In this model, we adoptively transferred in‐vitro‐generated bone‐marrow‐derived DCs exposed to interleukin‐10 (IL‐10) and an enterobacterial extract. We show that these cells are CD11c positive with intermediate expression of CD40, CD80 and CD86 and have a diminished secretion of IL‐6, IL‐12 p40/70, tumour necrosis factor‐α and keratinocyte‐derived chemokine (KC) compared to DCs treated with enterobacterial extract alone. In vivo, these cells prevented weight loss in SCID mice adoptively transferred with CD4+ CD25– T cells, resulted in a lower histopathology colitis score and tended to result in higher serum levels of IL‐1α, IL‐10, IL‐12, IL‐13, IL‐17, KC and monokine induced by interferon‐gamma (MIG). These data underscore the potential of using immunomodulatory DCs to control inflammatory bowel disease and demonstrate its potential use in future human therapeutic settings.

Characterization of monocyte-derived dendritic cells maturated with IFN-α

Dendritic cells (DC) are promising candidates for cancer immunotherapy. These cells can be generated from peripheral blood monocytes cultured with granulocyte macrophage‐colony stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4). In order to obtain full functional capacity, maturation is required, but the most potent reagents such as LPS or polyriboinosinic polyribocytidylic acid (Poly I:C) are not approved for clinical use. We tested the ability of type I interferon (IFN) to induce such maturation. We found that 24‐h IFN‐α co‐culture of day 7 monocyte‐derived DC generated with GM‐CSF and IL‐4 induces increased numbers of DC positive for CD54 and CD40 together with the co‐stimulatory molecule CD80 but not the activation marker CD83. Also, IFN‐α maturation leads to an increase in IP‐10 and MCP‐1 chemokine secretion, but only a minor increase in IL‐12p40 secretion. In line with this, maturation with IFN‐α has only a small effect on induction of autologous T‐cell stimulatory capacity of the DC. However, an increase in DC allogeneic T‐cell stimulatory capacity was observed. These data suggest that IFN‐α has a potential as a maturation agent used in DC‐based cancer vaccine trials, but not as a single reagent.