Nanna Ny Kristensen

High numbers of IL-2-producing CD8+ T cells during viral infection: correlation with stable memory development

Using infections with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus in mice as model systems, we have investigated the ability of antigen-primed CD8+ T cells generated in the context of viral infections to produce IL-2. Our results indicate that acute immunizing infection normally leads to generation of high numbers of IL-2-producing antigen-specific CD8+ T cells. By costaining for IL-2 and IFN-gamma intracellularly, we found that IL-2-producing cells predominantly constitute a subset of cells also producing IFN-gamma. Comparison of the kinetics of generation revealed that IL-2-producing cells appear slightly delayed compared with the majority of IFN-gamma producing cells, and the relative frequency of the IL-2-producing subset increases with transition into the memory phase. In contrast to acute immunizing infection, few IL-2-producing cells are generated during chronic LCMV infection. Furthermore, in MHC class II-deficient mice, which only transiently control LCMV infection, IL-2-producing CD8+ T cells are initially generated, but by 4 weeks after infection this subset has nearly disappeared. Eventually the capacity to produce IFN-gamma also becomes impaired, while cell numbers are maintained at a level similar to those in wild-type mice controlling the infection. Taken together, these findings indicate that phenotyping of T cell populations based on capacity to produce cytokines, and especially IL-2, can provide important information as to the functional status of the analysed cell subset. Specifically, combined analysis of the capacity to produce IL-2 and IFN-gamma can be used as a predictor for loss of function within the CD8+ T cell compartment.

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.

TH17 cell induction and effects of IL-17A and IL-17F blockade in experimental colitis.

Background: T helper (TH) 17 cells are believed to play a pivotal role in development of inflammatory bowel disease, and their contribution to intestinal inflammation has been studied in various models of colitis. TH17 cells produce a range of cytokines, some of which are potential targets for immunotherapy. However, blockade of IL-17A alone with secukinumab was not effective in Crohn’s disease. In this regard, the pathogenic impact of IL-17A versus IL-17F during intestinal inflammation is still unresolved. Methods: Development of IFN-&ggr;–producing, IL-17A–producing, and IL-17F–producing CD4+ T cells was analyzed in the CD4+CD25− T-cell transfer model of colitis at varying degrees of colitis. The pathogenic roles of IL-17A and IL-17F were investigated by treating colitic mice with neutralizing antibodies against these 2 cytokines. Results: We found that colitis development was associated with an increase in IL-17A–producing TH17 cells in spleen, mesenteric lymph nodes, and lamina propria. In contrast, the relative abundance of IFN-&ggr;–producing TH1 cell was stable in all 3 organs during progression of colitis, and the frequency of IFN-&ggr;+IL-17A+ double-positive cells declined in spleen and mesenteric lymph node but not in lamina propria. IL-17F was coexpressed in TH17 cells and IFN-&ggr;+IL-17A+ double positive but not in TH1 cells and its expression inversely correlated with colitis development. In vivo neutralization of both IL-17A and IL-17F ameliorated colitis in particular at early administration, whereas neutralization of IL-17A or IL-17F alone was inefficient. Conclusions: TH17 cell development correlates with colitis progression, and concurrent neutralization of their cytokine products IL-17A and IL-17F ameliorates intestinal inflammation. These findings suggest combined IL-17A and IL-17F blockade as a potential strategy in inflammatory bowel disease therapy.

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.

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.

Consumption of Probiotics Increases the Effect of Regulatory T Cells in Transfer Colitis

Background: Probiotics may alter immune regulation. Recently, we showed that the probiotic bacteria Lactobacillus acidophilus NCFM™ influenced the activity of regulatory T cells (Tregs) in vitro. The aim of the present work was to demonstrate if L. acidophilus NCFM™ also affects the function of Tregs in vivo. Methods: Development of colitis after transfer of CD4+CD25− T cells and protection from colitis by Tregs was studied in immunodeficient SCID mice which were simultaneously tube‐fed with L. acidophilus NCFM™ or L. salivarius Ls‐33 for 5 weeks. Results: Probiotic‐fed SCID mice transplanted with low numbers of Tregs in addition to the disease‐inducing T cells were completely protected from colitis. This was in contrast to the control group, which showed intermediate levels of inflammation. In addition, feeding with probiotics lowered serum levels of inflammatory cytokines in both colitic mice and in mice protected from colitis by Tregs. Gene expression patterns of rectum samples of protected mice that receive either one of the probiotics showed a closer resemblance to naïve SCID mice than did patterns of the control group. The mechanism of action of the probiotics appears to be an indirect effect by inducing a Treg‐favorable environment rather than a direct effect on the Tregs. Conclusions: L. acidophilus NCFM™ and L. salivarius Ls‐33 feeding of SCID mice increases the in vivo effect of Tregs, resulting in a gene expression pattern in the rectum resembling that of the naïve SCID mouse. (Inflamm Bowel Dis 2011;)

B cells exposed to enterobacterial components suppress development of experimental colitis

Background: B cells positively contribute to immunity by antigen presentation to CD4+ T cells, cytokine production, and differentiation into antibody secreting plasma cells. Accumulating evidence implies that B cells also possess immunoregulatory functions closely linked to their capability of IL‐10 secretion. Methods: Colitis development was followed in CD4+CD25− T cell transplanted SCID mice co‐transferred with B cells exposed to an enterobacterial extract (ebx‐B cells). B and T cell cytokine expression was measured by flow cytometry and enzyme‐linked immunosorbent assay (ELISA). Results: We demonstrate that splenic B cells exposed to ebx produce large amounts of IL‐10 in vitro and express CD1d and CD5 previously known to be associated with regulatory B cells. In SCID mice transplanted with colitogenic CD4+CD25− T cells, co‐transfer of ebx‐B cells significantly suppressed development of colitis. Suppression was dependent on B cell‐derived IL‐10, as co‐transfer of IL‐10 knockout ebx‐B cells failed to suppress colitis. Ebx‐B cell‐mediated suppression of colitis was associated with a decrease in interferon gamma (IFN‐&ggr;)‐producing TH1 cells and increased frequencies of Foxp3‐expressing T cells. Conclusions: These data demonstrate that splenic B cells exposed to enterobacterial components acquire immunosuppressive functions by which they can suppress development of experimental T cell‐mediated colitis in an IL‐10‐dependent way. (Inflamm Bowel Dis 2011;)

CXC chemokine receptor 3 expression increases the disease-inducing potential of CD4+ CD25- T cells in adoptive transfer colitis.

Background CD4+CD25− T cells induce severe colitis when injected into immunodeficient recipients. The migration of disease‐inducing cells to the bowel is controlled by adhesion molecules and chemotactic proteins. Chemokine receptors expressed on the T cells are therefore potential targets for anti‐inflammatory therapy in inflammatory bowel disease. In this study, we have investigated the role of the chemokine receptor CXCR3 in the development of chronic colitis in a murine model. Method Expression of CXCR3 on CD4+ T cell from normal and colitic mice was assessed by flow cytometry. Development of colitis was followed after transfer of either normal or CXCR3−CD4+CD25−T cell into immunodeficient host. In addition, the ability of regulatory T cell to function in vivo in the absence of CXCR3 was tested. Results We find CXCR3 to be expressed on 80% to 90% of CD4+ T cells isolated from colitic mice compared with only 4% to 10% of CD4+ T cells in normal naïve mice. Injecting CD4+CXCR3−CD25− T cells into immunodeficient hosts results in an ameliorated form of colitis with a lack of clinical symptoms, suggesting that CXCR3 expression is important for enteroantigen priming of CD4+ T cells and/or subsequent migration into the gut wall. In contrast, CXCR3 expression does not affect the function of regulatory T cells because CXCR3−/− regulatory T cells are just as capable as their wild‐type counterpart of controlling disease development. The diminished disease‐inducing capability of CXCR3−/− T cells is not caused by the absence of enteroantigen specificity; we also tested the enteroantigen‐specific proliferative ability of CD4+CD25− T cells from CXCR3−/− mice in vitro and found that they respond even more strongly than wild‐type cells. Conclusions The present data indicate that CXCR3 plays an important role in controlling the migration of disease‐inducing CD4+CD25− T cells into the gut wall. In contrast, lack of CXCR3 expression by regulatory T cells does not compromise their function in this model of colitis.