Targeting human CD2 by the monoclonal antibody CB.219 reduces intestinal inflammation in a humanized transfer colitis model.

The cell adhesion molecule CD2 facilitates antigen-independent T-cell activation and CD2 deficiency or blockade reduces intestinal inflammation in murine models. We here aimed to evaluate the therapeutic potential of monoclonal antibodies (mAb) specific for human CD2 in colitis treatment. Transfer colitis induced by naïve CD4(+) T cells expressing human CD2 was treated with anti-human CD2 mAb. The mAb CB.219 protected from severe colitis in a preventive treatment regimen, while therapeutic treatment ameliorated intestinal inflammation. Diminished intestinal tissue damage was paralleled by a profound suppression of lamina propria lymphocytes to produce pro-inflammatory cytokines and tumor necrosis factor α as well as the neutrophil chemoattractant CXC motif ligand 1 and the CC chemokine ligand 3. Furthermore, infiltration with macrophages and T cells was low. Thus, reduced intestinal inflammation in our humanized colitis model by targeting CD2 on T cells with the mAb CB.219 suggests a novel approach for colitis treatment.

Human peripheral gammadelta T cells possess regulatory potential.

Deficiency in gammadelta T cells aggravates colitis in animal models suggesting that gammadelta T cells have regulatory properties. Therefore, proliferation, suppression and cytokine secretion of human gammadelta T cells were determined in vitro. Human peripheral gammadelta T cells were isolated from the whole blood of healthy donors by magnetic antibody cell sorting technology. The proliferation after CD3/CD28 stimulation was measured by (3)[H]thymidine incorporation. Interferon-gamma (IFN-gamma), interleukin-2 (IL-2), transforming growth factor-beta (TGF-beta) and IL-10 concentrations were measured by enzyme-linked immunosorbent assay; TGF-beta messenger RNA was also measured by reverse transcription-polymerase chain reaction. The expression of latency associated peptide (LAP), a TGF-beta complex component, intracellular cytokine content and T helper cell proliferation were measured by flow cytometry. Human gammadelta T cells showed poor proliferation upon CD3/CD28 stimulation and suppressed T helper cell growth stronger than CD4(+) CD25(+) T cells, although gammadelta T cells were FOXP3 negative. They secreted little IL-2 but high concentrations of IFN-gamma, IL-10 and TGF-beta. When looking at LAP expression the Vdelta1 subset was found to be the main TGF-beta producer compared to Vdelta2 T cells. Taken together, peripheral gammadelta T cells have in vitro a more potent regulatory potential than CD4(+) CD25(+) cells regarding T helper cell suppression. This is most likely the result of strong TGF-beta secretion, particularly by the Vdelta1 subset.

Aggravation of different types of experimental colitis by depletion or adhesion blockade of neutrophils.

BACKGROUND & AIMS: Neutrophils are generally thought to play an important proinflammatory role in the pathogenesis of inflammatory bowel disease. The objective of this study was to evaluate whether blocking the invasion of neutrophils by anti-L-selectin monoclonal antibodies modulates chemically induced colitis and how this modulation is accomplished. METHODS: Trinitrobenzene sulfonic acid/dinitrobenzene sulfonic acid (TNBS/DNBS)-induced colitis was studied in rats on treatment with anti-L-selectin monoclonal antibodies (mAb) or antineutrophil antiserum. Different anti-L-selectin mAb, either blocking or nonblocking, as well as F(ab)(2) fragments were evaluated. Additionally, leukocyte migration was examined using intravital microscopy. Furthermore, the effect of neutrophil depletion in rat TNBS-induced colitis was studied either prior to or after colitis induction as well as murine CD4(+)CD45RB(high) transfer colitis. Finally, bacterial translocation during DNBS-induced colitis was studied in neutrophil-depleted and control rats. RESULTS: Anti-L-selectin mAb treatment resulted in increased mortality and bowel inflammation as well as hemorrhagic eye secretion. No clear difference was found between blocking and nonblocking mAb or F(ab)(2) fragments. For all investigated antibodies/fragments, either complete blockade of leukocyte invasion or marked neutrophil depletion was found. Accordingly, neutrophil depletion by antiserum resulted in aggravation of rat DNBS-induced colitis as well as murine transfer colitis. CONCLUSIONS: Adhesion blockade or neutrophil depletion aggravates rat TNBS/DNBS-induced colitis together with extraintestinal manifestations of the eyes. Therefore, neutrophils appear to have an important role in mucosal repair processes. Importantly, adhesion blockade as a therapeutic concept can be detrimental in inflammatory bowel disease.

Aggravation of intestinal inflammation by depletion/deficiency of gammadelta T cells in different types of IBD animal models.

The role of gammadelta T cells in inflammatory bowel disease (IBD) is still controversial. Although gammadelta T cells induce IBD in immunodeficient animals, others suggest a protective role of gammadelta T cells. Therefore, this study was conducted in order to elucidate the effect of gammadelta T cell depletion/deficiency on different IBD animal models. Mice depleted of or deficient in gammadelta T cells were exposed to dextran sodium sulfate (DSS) in order to induce colitis. In addition, gammadelta T cells were depleted in mice with terminal ileitis (TNFDeltaARE) or colitis due to interleukin 2 deficiency (IL-2 ko). Finally, DSS-induced colitis was studied in mice deficient in interferon gamma (IFN-gamma ko) upon gammadelta T cell depletion. Depletion of gammadelta T cells aggravated DSS-induced colitis and terminal ileitis of TNFDeltaARE mice. Exacerbated DSS-induced colitis was also found in gammadelta T cell-deficient mice. IL-2 ko mice showed increased mortality upon early (starting at 4 wk of age) but not late depletion (starting at 8 wk of age). Early gammadelta T cell depletion or deficiency resulted in increased IFN-gamma production by both lamina propria lymphocytes and splenocytes in every model investigated herein. In IFN-gamma ko mice, gammadelta T cell depletion did not affect the development and course of DSS-induced colitis. The protective effect of gammadelta T cells in IBD was confirmed in various IBD animal models. Particularly, during the early phase of intestinal inflammation, gammadelta T cells appear to be important. The mechanism seems to involve the control of IFN-gamma production and epithelial regeneration.

CD2 deficiency partially prevents small bowel inflammation and improves parasite control in murine Toxoplasma gondii infection.

AIM: To investigate whether bowel inflammation and/or parasite control is altered in the absence of the T cell adhesion molecule CD2. METHODS: Wildtype (WT) and CD2 deficient (CD2(-/-)) mice were infected with 100 cysts of Toxoplasma gondii (T. gondii) (ME49) by gavage. On d 7 after infection mice were killed. Necrosis and the number of parasites/cm ileum were determined. Cytokine levels of stimulated cells as well as sera were evaluated. Secondly, survival of WT vs CD2(-/-) mice was analysed using Kaplan-Meier analysis. RESULTS: CD2(-/-) mice survived longer than WT mice (mean: 23.5 vs 7.1 d, P = 0.001). Further, CD2(-/-) mice showed less weight loss and less ileal inflammation than WT mice at d 7 post infection. In addition, the number of parasites in the ileum was significantly lower in CD2(-/-) mice than in WT mice (88 +/- 12 vs 349 +/- 58 cm, P < 0.01). This was paralleled by lower production of IFN-gamma and IL-6 from TLA-stimulated mLN cells and increased IFN-gamma production by splenocytes. CONCLUSION: CD2 deficient mice are more resistant to T. gondii infection than WT mice. In contrast to most current immunosuppressive or biological therapies CD2 deficiency reduces intestinal inflammation and at the same time helps to control infection.

Gammadelta T lymphocytes: a new type of regulatory T cells suppressing murine 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis.

BACKGROUND: The intestinal immune system is continuously challenged by antigen without becoming dysregulated. However, injury of the mucosa by, i.e. dextran sulphate sodium causes severe inflammation in gammadelta T-cell-deficient mice. We therefore asked whether gammadelta T cells have regulatory functions. MATERIALS AND METHODS: gammadelta T cells were isolated from spleens and mesenteric lymph nodes of C57BL/6 wild-type (wt) mice. Proliferation and cytokine secretion of gammadelta T cells were quantified by [(3)H] thymidine incorporation and ELISA. Additionally, proliferation of carboxyfluorescein diacetate succinimidylester-labelled CD4(+) T cells cocultured with gammadelta T cells was analysed by flow cytometry. Finally, gammadelta T cells from wt or interleukin-10 transgenic (IL-10tg) mice were transferred into congenic mice with 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. RESULTS: gammadelta T cells were hyporesponsive to CD3/CD28 stimulation and suppressed CD4(+) T-cell proliferation (up to 66+/-7% suppression) in vitro. Further, the preventive transfer of wt or IL-10tg gammadelta T cells ameliorated TNBS-induced colitis resulting in prolonged survival and reduced histological damage (1.5+/-0.4 and 1.3+/-0.2, respectively vs. 3.8+/-0.3 in untransferred mice, p<0.05). This was accompanied by reduced TNF-alpha and increased IL-10 and TGF-beta secretion from intestinal and splenic lymphocytes. CONCLUSIONS: Murine gammadelta T cells are a new type of regulatory T cells in vitro and act protective on mouse TNBS-induced colitis in vivo. Future studies have to define the underlying mechanism and to investigate whether gammadelta T cells can be used for immunotherapy of human inflammatory bowel disease.

Lamina propria T cell activation: role of the costimulatory molecule CD2 and its cytoplasmic tail for the regulation of proliferation and apoptosis.

BACKGROUND/AIMS: Accumulation of T lymphocytes in the gut is a hallmark of inflammatory bowel disease probably caused by insufficient T cell apoptosis. Activated peripheral T cells, or "resting" lamina propria T lymphocytes (LPLs), are highly susceptible to apoptosis induction, e.g., using the mitogenic anti-CD2 monoclonal antibody (mAb) pair T11(2+3). It is, however, unknown how CD2-mediated LPL apoptosis is related to proliferation and whether the whole CD2 molecule is required for apoptosis induction. MATERIALS AND METHODS: Mapping of anti-CD2 mAb was performed using erythrocyte rosetting assays and cross-blocking enzyme-linked immunosorbent assay (ELISA). Lamina propria mononuclear cells (LPMNCs) or phytohemagglutinin (PHA) blasts were stimulated with a panel of 18 anti-CD2 mAbs followed by apoptosis analysis [Annexin V expression on propidium iodide (PI)-negative cells, 4c6-diamidino-2-phenylindole x 2HCl (DAPI) staining]. Proliferation was measured by [(3)H]-thymidine incorporation. For structural analysis, EL4 cells were used which were transfected with human CD2 (wild type (WT), cytoplasmic-deficient, cytoplasmic CD28). Sorting was performed employing standard techniques RESULTS: All three mitogenic anti-CD2 mAb pairs induced apoptosis of LPMNC and PHA blasts. Two out of four submitogenic anti-CD2 mAb, AICD2.M3, and ICRFCD2.3 lead to LPMNC proliferation but no apoptosis. Importantly, apoptosis was also detected in cytoplasmic-deficient CD2 tg or CD2/CD2/CD28 tg EL4 cells. Sorted CD45(high) huCD2 WT EL4 had higher apoptosis rates compared to WT huCD2tg EL4 cells CONCLUSION: LPMNC apoptosis induction via CD2 is always associated with proliferation, although proliferation is not necessarily associated with apoptosis. The cytoplasmic tail of CD2 is not required, and CD45 appears to transmit apoptotic signals entering the T cell via CD2.

Alpha CD 2 mAb treatment safely attenuates adoptive transfer colitis.

Increased proliferation, defective apoptosis, and cytokine dysregulation of T lymphocytes are thought to be important for the pathogenesis of inflammatory bowel disease. Since these phenomena can be corrected by alpha CD 2 mAb, we asked whether CD2 directed immunotherapy safely prevents and/or ameliorates adoptive transfer colitis. Colitis was induced by transfer of CD4(+) T cell blasts to syngenic RAG 1(-/-) mice or CD 45 RB(high) CD4(+) T cells to SCID mice. The alpha CD 2 mAb 12-15 or rat IgG was given, starting either initially or upon first signs of colitis. Disease activity was assessed by clinical monitoring, microscopic scoring, hemoccult, endoscopy, and blood count analysis. Cytokine production of stimulated LPL was measured by ELISA and cell proliferation by [(3)H]-thymidine incorporation. Parasite control was analyzed in a murine model of infection with Toxoplasma gondii. The alpha CD 2 mAb significantly increased mean survival time when starting at transfer of blasts (survival >35 days: alpha CD 2 69% vs 0% of controls, P<0.001). In the SCID colitis model hematochezia and macroscopic colitis were delayed. When used in established T-cell blast colitis, the benefit was less pronounced, even in combination with dexamethasone (mean survival+/-s.e.m.: alpha CD 2+dexa: 13.5+/-2.9 vs dexa+IgG: 6.3+/-1.0, P<0.05). In the preventive experiment the alpha CD 2 mAb markedly reduced IL-2 secretion and T-cell proliferation. The immune response towards Toxoplasma gondii was not impaired. These studies show for the first time that CD2 directed immunotherapy can attenuate or delay adoptive transfer colitis and ameliorate established colitis. Most likely inhibition of IL-2 secretion and T-cell proliferation are responsible for these effects. Still, immune defence towards T. gondii is maintained.

Animal models of inflammatory bowel disease: an overview.

Inflammatory bowel disease (IBD) research has been performed in human in vitro studies and in in vivo studies using appropriate animal models. Such animal models allow both the examination of inflammatory processes (both early and late events) as well as the evaluation of new therapeutic modalities. Since the first description of the immune complex colitis in rabbits in 1961, overall 63 models have been described, most of which within the last decade. These IBD animal models can be divided into 5 different categories: (1) antigen-induced colitis and colitis induced by microbials; (2) other inducible forms of colitis (chemical, immunological, and physical); (3) genetic colitis models (transgenic and knock-out models); (4) adoptive transfer models, and (5) spontaneous colitis models. In spite of the high overall number of models, none of them is the 'perfect' model and therefore numerous aspects need to be considered when choosing one model for a particular study. Importantly, most clinical aspects (e.g. extraintestinal manifestations or fistula) have recently been described in one or the other model allowing further studies with relevance for almost all aspects of IBD. So far, IBD animal models have taught us important lessons, e.g. the requirement of T-helper cells in most models, the need of a particular genetic background, and the role of the flora in the initiation of IBD. It is expected that our understanding of IBD will further increase in a number of additional areas using animal models, e.g. exploring the role of the innate immune system in IBD.