In vitro 2D and 3D cancer models to evaluate compounds that modulate macrophage polarization.

In vitro cancer models that can identify novel immunomodulating compounds are essential. Using a 3D multicellular tumor spheroid (MCTS) model comprising cancer cells, fibroblasts, and macrophages, we tested tumor-associated macrophage (TAM)-inhibiting compounds (CCL2 Ab, CSF1R inhibitor, CSF1R Ab) and TAM-reprograming compounds (poly I:C, CD40 Ab, CD40 ligand) for their effects on monocyte infiltration and polarization in tumor spheroids. For characterization of macrophage polarization, we measured the expression of CD206, CD163, CD86, MHC II, CD40, and CD14 and measured 43 soluble factors in the 3D MCTS cultures. 2D macrophage models were evaluated for comparison. A CSF1R inhibitor prevented infiltration of monocytes into pancreatic cancer spheroids, and macrophages treated with the inhibitor showed decreased expression of M2 markers. Treatment with a CD40 ligand and poly I:C induced M1 macrophage polarization in our models. We propose that these models can be used to improve the drug screening process of anti-cancer immunotherapies targeting macrophages.

Development of a flow cytometry-based potency assay for prediction of cytokine storms induced by biosimilar monoclonal antibodies.

Cytokine release syndrome (CRS) is an undesired immune reaction that may cause dangerous side effects after the administration of novel biological therapies. In vitro cytokine release assays (CRA) are used for preclinical safety assessment prior to first-in-man dose administration of therapeutic monoclonal antibodies (mAbs). A variety of CRA platforms has been developed where the analysis of secreted cytokines is performed. Analysis of T cell activation markers is not performed routinely in CRA platforms and few studies have described intracellular cytokine levels after stimulation with therapeutic mAbs. In the present study, we performed a CRA using intracellular cytokine staining and assessment of extracellular T cell activation markers by flow cytometry. We used commercially available reference mAbs for the stimulation of peripheral blood mononuclear cells (PBMCs). We found that stimulation using solid phase (SP) dry coating with two different CD28 antibodies and muromonab-CD3 increased the percentage of IFN-É£ + CD4+ and CD8+ T cells as well as of CD3-CD56+ NK cells compared to stimulation with antibodies in aqueous phase (AP). Expression of the T cell activation markers CD25 and CD69 on CD4+ and CD8+ T cells was also increased upon SP muromonab-CD3 stimulation. Using multiplex cytokine assessment, we showed that stimulation in AP using ANC28.1, CD28.2 and muromonab-CD3 led to an increase of IFN-É£, GM-CSF, TNF-α, and IL-2 secretion. Stimulation of PBMCs preincubated at high-density culture led to an increase in IFN-É£ production but not in the expression of activation markers compared to low-density culture. Our findings demonstrated that flow cytometry analyses for assessing relevant T cell and NK cell markers may be used as a supplement to multiplex cytokine analysis in CRAs. The approach may be a valuable addition that enables a more precise description of the mechanisms leading to CRS.

Monocyte Infiltration and Differentiation in 3D Multicellular Spheroid Cancer Models.

Tumor-associated macrophages often correlate with tumor progression, and therapies targeting immune cells in tumors have emerged as promising treatments. To select effective therapies, we established an in vitro 3D multicellular spheroid model including cancer cells, fibroblasts, and monocytes. We analyzed monocyte infiltration and differentiation in spheroids generated from fibroblasts and either of the cancer cell lines MCF-7, HT-29, PANC-1, or MIA PaCa-2. Monocytes rapidly infiltrated spheroids and differentiated into mature macrophages with diverse phenotypes in a cancer cell line-dependent manner. MIA PaCa-2 spheroids polarized infiltrating monocytes to M2-like macrophages with high CD206 and CD14 expression, whereas monocytes polarized by MCF-7 spheroids displayed an M1-like phenotype. Monocytes in HT-29 and PANC-1 primarily obtained an M2-like phenotype but also showed upregulation of M1 markers. Analysis of the secretion of 43 soluble factors demonstrated that the cytokine profile between spheroid cultures differed considerably depending on the cancer cell line. Secretion of most of the cytokines increased upon the addition of monocytes resulting in a more inflammatory and pro-tumorigenic environment. These multicellular spheroids can be used to recapitulate the tumor microenvironment and the phenotype of tumor-associated macrophages in vitro and provide more realistic 3D cancer models allowing the in vitro screening of immunotherapeutic compounds.

Cladribine inhibits secretion of pro-inflammatory cytokines and phagocytosis in human monocyte-derived M1 macrophages in-vitro.

Cladribine (Cd) is a purine nucleoside analogue which in an oral formulation is approved for treatment of patients with multiple sclerosis (MS). It is known to mediate the effect through a short-term selective reduction of lymphocytes with minimal effect on the innate immune system. However, a few studies have emerged, that also demonstrate a beneficial immunomodulatory effect of cladribine on monocyte-derived cells. As cladribine crosses the blood-brain barrier this effect could have clinical meaningful impact in the treatment of MS, where recruitment of innate cells such as M1 macrophages play a role in plaque development. Here, we investigated the in-vitro effect on monocyte differentiation into M1 and M2 macrophages and dendritic cells as well as the effect on activation of M1 macrophages. In our experiments, cladribine in therapeutic relevant in-vitro concentrations, did not lead to apoptosis in differentiated M1, M2 macrophages or DCs and did not interfere with the phenotype of these differentiated cells. In M1 macrophages, cladribine reduced the secretion of IL-6 and TNF-α observed after activation with LPS. Similar, cladribine reduced the phagocytic capacity of LPS activated M1 macrophages but did not affect unactivated cells. We conclude, that such reduction of inflammatory potential as well as reduced M1 phagocytic activity, e.g. within an MS plaque, could be an additional clinical meaningful effect of cladribine in the treatment of MS while at the same time it would leave M1 macrophages intact for the protection against infections.

Development of an In Vitro Assay to Assess Pharmacological Compounds and Reversion of Tumor-Derived Immunosuppression of Dendritic Cells.

BACKGROUND: Cancer immunotherapies have achieved much success and have become the forefront treatment of cancers previously associated with poor prognosis. However, a major challenge in cancer immunotherapies remains the heterogeneity of the immunoregulatory capacities of cancers, and not all patients of a given cancer responds to current therapeutic strategies. To address this issue and to facilitate the development of new pharmacological compounds, we here describe an in vitro model of dendritic cell suppression by cancer cells. METHODS: We treated monocyte-derived dendritic cells with conditioned medium from cancer cell lines and assessed their maturation using ELISA and flow cytometry. In addition, we assessed their ability to induce T cell activation and differentiation. RESULTS: We found that both the phenotypic and functional maturation of dendritic cells was suppressed by the conditioned medium. The expression of IL-12p70, TNF-α, CD80, CD83, and CD86 was significantly reduced by conditioned medium from the 786-O and HeLa cell lines, and CD4+ T cells had a weaker TH1 phenotype with significantly decreased expression of IFN-γ and T-bet following co-culturing. Furthermore, we use our model to characterize the differential immunoregulatory capacities of primary cancers by using conditioned medium of cultured primary cancer cells. CONCLUSION: This model can be used to screen pharmacological compounds seeking to alleviate the immunosuppression of the tumor microenvironment and can furthermore be used to investigate the immunoregulatory capacities of primary cancer cells, which could be a helpful prognostic tool following tumor resection.

Adipose Tissue-Derived Stromal Cells Induce a Highly Trophic Environment While Reducing Maturation of Monocyte-Derived Dendritic Cells.

Allogeneic cell-based therapies using adipose tissue-derived stromal cells (ASCs) offer an off-the-shelf alternative to autologous therapy. An underlying assumption is that ASC can modulate the immune response of the recipient. However, in vitro models are required to explore and identify cell interactions and mechanisms of action, to ensure sufficient and sustained effects, and to document these. In this study, we shed light on the effect of ASC manufactured for clinical use on monocyte-derived dendritic cells and an inflammatory microenvironment. ASCs were isolated from healthy voluntary donors, expanded using a human platelet lysate in bioreactors, and cryopreserved as per clinical use. Monocyte-derived dendritic cells were generated by isolation of monocytes and differentiation with GM-CSF and IL-4. Dendritic cells were cocultured with different ratios of ASC and matured with LPS and IFN-γ. Dexamethasone was included as an immunosuppressive control. Dendritic cells were analyzed by flow cytometry for CD11c, CD40, CD80, CD83, CD86, PD-L1, and HLA-DR, and supernatants were analyzed for FGF2, HGF, IL-10, IL-12p70, LIF, MIF, PDGF, PlGF, and IDO. Reduced expression of maturation markers was observed on ASC-treated dendritic cells, while high levels of PD-L1 were maintained. Interestingly, the expression of CD83 was elevated. Escalating ratios of ASC did not affect the concentration of IL-10 considerably, whereas the presence of IL-12 was reduced in a dose-dependent manner. Besides offsetting the IL-12/IL-10 balance, the concentrations of IDO and MIF were elevated in cocultures. Concentrations of FGF2, HGF, LIF, and PIGF were high in ASC cocultures, whereas PDGF was depleted. In a robust coculture model, the addition of ASC to dendritic cells inhibited the dendritic maturation substantially, while inducing a less inflammatory and more tolerogenic milieu. Despite the exposure to dendritic cells and inflammatory stimuli, ASC resulted in supernatants with trophic factors relevant for regeneration. Thus, ASC can perform immunomodulation while providing a regenerative environment.

Sym021, a promising anti-PD1 clinical candidate antibody derived from a new chicken antibody discovery platform.

Discovery of therapeutic antibodies is a field of intense development, where immunization of rodents remains a major source of antibody candidates. However, high orthologue protein sequence homology between human and rodent species disfavors generation of antibodies against functionally conserved binding epitopes. Chickens are phylogenetically distant from mammals. Since chickens generate antibodies from a restricted set of germline genes, the possibility of adapting the Symplex antibody discovery platform to chicken immunoglobulin genes and combining it with high-throughput humanization of antibody frameworks by "mass complementarity-determining region grafting" was explored. Hence, wild type chickens were immunized with an immune checkpoint inhibitor programmed cell death 1 (PD1) antigen, and a repertoire of 144 antibodies was generated. The PD1 antibody repertoire was successfully humanized, and we found that most humanized antibodies retained affinity largely similar to that of the parental chicken antibodies. The lead antibody Sym021 blocked PD-L1 and PD-L2 ligand binding, resulting in elevated T-cell cytokine production in vitro. Detailed epitope mapping showed that the epitope recognized by Sym021 was unique compared to the clinically approved PD1 antibodies pembrolizumab and nivolumab. Moreover, Sym021 bound human PD1 with a stronger affinity (30 pM) compared to nivolumab and pembrolizumab, while also cross-reacting with cynomolgus and mouse PD1. This enabled direct testing of Sym021 in the syngeneic mouse in vivo cancer models and evaluation of preclinical toxicology in cynomolgus monkeys. Preclinical in vivo evaluation in various murine and human tumor models demonstrated a pronounced anti-tumor effect of Sym021, supporting its current evaluation in a Phase 1 clinical trial. Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; CD, cluster of differentiation; CDC, complement-dependent cytotoxicity; CDR, complementarity determining region; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence activated cell sorting; FR, framework region; GM-CSF, granulocyte-macrophage colony-stimulating factor; HRP, horseradish peroxidase; IgG, immunoglobulin G; IL, interleukin; IFN, interferon; mAb, monoclonal antibody; MLR, mixed lymphocyte reaction; NK, natural killer; PBMC, peripheral blood mono-nuclear cell; PD1, programmed cell death 1; PDL1, programmed cell death ligand 1; RT-PCR, reverse transcription polymerase chain reaction; SEB, Staphylococcus Enterotoxin B; SPR, surface Plasmon Resonance; VL, variable part of light chain; VH, variable part of heavy chain.

Reconstitution of Th17, Tc17 and Treg cells after paediatric haematopoietic stem cell transplantation: Impact of interleukin-7.

Successful reconstitution of T lymphocytes after allogeneic haematopoietic stem cell transplantation (HSCT) is needed to establish the graft-versus-leukaemia effect and an effective anti-microbial defense, but the ratio between functionally different T-cell subsets needs to be balanced to avoid graft-versus-host disease (GVHD). IL-7 is essential for T-cell generation in the thymus and peripheral T-cell homeostasis. High IL-7 levels have been associated with impaired T-cell reconstitution, increased risk of acute GVHD and treatment-related mortality, but the underlying cellular mechanisms behind these associations have not been investigated previously. We hypothesized that increased levels of IL-7 post-transplant alters the balance between immune-regulatory T cell subsets during the post-transplant lymphocyte recovery towards a more pro-inflammatory profile. We quantified Th17 cells, Tc17 cells and Tregs in 29 children following HSCT. Th17 cell and Treg counts rose significantly from day +90 to +180 post-HSCT, and prior acute GVHD was associated with significant changes in the concentration of Tregs (9.4×106/L vs. 1.3×106/L, P=0.0052) and the Th17/Treg ratio (1.5 vs. 4.2, P=0.025). The plasma level of IL-7 at day +90 correlated inversely with Th17 cell counts (rs=-0.65, P=0.0002) and the proportion of Tc17 cells (rs=0.64, P=0.0005) at day +90, but not with Tregs. Furthermore, high IL-7 levels at day +7 were predictive of a less naïve T-cell phenotype at day +90. These findings add further evidence that IL-7 is a key regulatory factor that may tune the balance between functionally different T-cell subsets following HSCT.

Human adipose-derived stromal cells in a clinically applicable injectable alginate hydrogel: Phenotypic and immunomodulatory evaluation.

BACKGROUND AIMS: Clinical trials have documented beneficial effects of mesenchymal stromal cells from bone marrow and adipose tissue (ASCs) as treatment in patients with ischemic heart disease. However, retention of transplanted cells is poor. One potential way to increase cell retention is to inject the cells in an in situ cross-linked alginate hydrogel. METHODS: ASCs from abdominal human tissue were embedded in alginate hydrogel and alginate hydrogel modified with Arg-Gly-Asp motifs (RGD-alginate) and cultured for 1 week. Cell viability, phenotype, immunogenicity and paracrine activity were determined by confocal microscopy, dendritic cell co-culture, flow cytometry, reverse transcriptase quantitative polymerase chain reaction, Luminex multiplex, and lymphocyte proliferation experiments. RESULTS: ASCs performed equally well in alginate and RGD-alginate. After 1 week of alginate culture, cell viability was >93%. Mesenchymal markers CD90 and CD29 were reduced compared with International Society for Cellular Therapy criteria. Cells sedimented from the alginates during cultivation regained the typical level of these markers, and trilineage differentiation was performed by standard protocols. Hepatocyte growth factor mRNA was increased in ASCs cultivated in alginates compared with monolayer controls. Alginates and alginates containing ASCs did not induce dendritic cell maturation. ASCs in alginate responded like controls to interferon-gamma stimulation (licensing), and alginate culture increased the ability of ASCs to inhibit lymphocyte proliferation. DISCUSSION: ASCs remain viable in alginates; they transiently change phenotype in alginate hydrogel but regain the phenotype of monolayer controls upon release. Cells maintain their paracrine potential while in alginates; the combination of ASCs and alginate is non-immunogenic and, in fact, immunosuppressive.

Monocyte targeting and activation by cationic liposomes formulated with a TLR7 agonist.

OBJECTIVES: Monocytes are one of the major phagocytic cells that patrol for invading pathogens, and upon activation, differentiate into macrophages or antigen-presenting dendritic cells (DCs) capable of migrating to lymph nodes eliciting an adaptive immune response. The key role in regulating adaptive immune responses has drawn attention to modulate monocyte responses therapeutically within cancer, inflammation and infectious diseases. We present a technology for targeting of monocytes and delivery of a toll-like receptor (TLR) agonist in fresh blood using liposomes with a positively charged surface chemistry. METHODS: Liposomes were extruded at 100 nm, incubated with fresh blood, followed by leukocyte analyses by FACS. Liposomes with and without the TLR7 agonist TMX-202 were incubated with fresh blood, and monocyte activation measured by cytokine secretion by ELISA and CD14 and DC-SIGN expression. RESULTS: The liposomes target monocytes specifically over lymphocytes and granulocytes in human whole blood, and show association with 75 - 95% of the monocytes after 1 h incubation. Formulations of TMX-202 in cationic liposomes were potent in targeting and activation of monocytes, with strong induction of IL-6 and IL-12p40, and differentiation into CD14(+) and DC-SIGN+ DCs. CONCLUSION: Our present liposomes selectively target monocytes in fresh blood, enabling delivery of TLR7 agonists to the intracellular TLR7 receptor, with subsequent monocyte activation and boost in secretion of proinflammatory cytokines. We envision this technology as a promising tool in future cancer immunotherapy.

Development of assay platforms for in vitro screening of Treg modulating potential of pharmacological compounds.

CD4 + CD25+ regulatory T cells (Tregs) are believed to be pivotal in controlling chronic inflammation as well as in opposing the effect of cancer immunotherapy. Therefore, identification of novel drug compounds that interfere with Treg function is of high priority together with research that investigates Treg modulation by current drugs. For such research as well as for novel cell based therapies based on Treg infusions, rapid in vitro assays as well as functional assays based on inhibitory capacity of Tregs are required. Here, we report on such assays using highly pure fluorescence-activated cell sorting (FACS) sorted CD4 + CD25(high)CD127(dim/-)CD45RA+ naïve Treg cells followed by in vitro expansion. We report on the use of these cells in a short-term assay based on Treg mediated inhibition of the early effector T cell activation markers CD69 and CD154. Additionally, we investigate the use of highly pure Tregs in a functional assay based on Treg mediated inhibition of effector T cell proliferation. We report highly reproducible Treg function in assays that test the effect of well-known model compounds such as CpG-A, anti-IL-6R (tocilizumab), anti-TNF-α (adalimumab) or a combination of IL-6 and TNF-α. In conclusion, these assays have the potential for use in pharmacological screening and discovery in relation to drug development in immunology.

Regulation of the IL-10/IL-12 axis in human dendritic cells with probiotic bacteria.

In this study, we have used monocyte-derived dendritic cells (DCs) to design a screening model for the selection of microorganisms with the ability to suppress DC-secreted IL-12p70, a critical cytokine for the induction of T-helper cell type 1 immune responses under inflammatory conditions. By the treatment of DCs with cocktails containing TLR agonists and proinflammatory cytokines, the cells increased the secretion of the Th1-promoting cytokine IL-12p70. Clinically used probiotics were tested for their IL-10- and IL-12p70-stimulating properties in immature DCs, and showed a dose-dependent change in the IL-10/IL-12p70 balance. Lactobacillus acidophilus NCFM(™) and the probiotic mixture VSL#3 showed a strong induction of IL-12p70, whereas Lactobacillus salivarius Ls-33 and Bifidobacterium infantis 35624 preferentially induced IL-10. Escherichia coli Nissle 1917 induced both IL-10 and IL-12p70, whereas the probiotic yeast Saccharomyces boulardii induced low levels of cytokines. When combining these microorganisms with the Th1-promoting cocktails, E. coli Nissle 1917 and B. infantis 35624 were potent suppressors of IL-12p70 secretion in an IL-10-independent manner, indicating a suppressive effect on Th1-inducing antigen-presenting cells. The present model, using cocktail-stimulated DCs with potent IL-12p70-stimulating capacity, may be used as an efficient tool to assess the anti-inflammatory properties of microorganisms for potential clinical use.

Differential induction of inflammatory cytokines by dendritic cells treated with novel TLR-agonist and cytokine based cocktails: targeting dendritic cells in autoimmunity.

BACKGROUND: Dendritic cells (DC) are main gate-keepers of the immune system, bridging the innate and adaptive immune system. DCs are able to mature into inflammatory DCs at sites of inflammation in both autoimmune and allergic disease, thereby sustaining a continuous activation of the adaptive immune system at sites of inflammation. This function of DCs makes them attractive target cells for therapeutic intervention in inflammatory diseases. We have designed a DC-based screening model by which drug candidates can be evaluated for their ability to suppress DC maturation into an inflammatory and disease promoting phenotype. METHODS: Human monocyte derived DCs were differentiated using IL-4 and GM-CSF to immature DCs (imDCs). The imDCs were treated with various combinations of TLR-agonists and pro-inflammatory cytokines to identify cocktails with ability to mature imDCs into inflammatory DCs. The effect of the cocktails on DC maturation was evaluated using ELISA and cytokine arrays to measure secreted cytokines and chemokines. FACS analysis was used to assess expression of maturation markers, and functional studies were carried out using naïve allogeneic T-cells to assay for a Th1-promoting DC phenotype. RESULTS: Nine cocktails were designed with potent ability to induce secretion of the Th1-promoting cytokines IL-12p70 and TNFalpha from imDCs, and three were able to induce the Th17-promoting cytokine IL-23. The cocktails were further characterized using cytokine arrays, showing induction of inflammation related cytokines and chemokines like CXCL10, CCL2, CCL4, CCL8, CCL15, CCL20 and IL-8, of which some are present in a range of autoimmune pathologies. Prostaglandin E2 secretion was identified from DCs treated with TLR agonists poly I:C and peptidoglycan, but not LPS. The cocktails were able to induce DC maturation markers like HLA-DR, CD40, CD80, CD83 and CD86, except the TLR7/8 agonist R848. Functional end-points made by co-culture of allogeneic CD4+ T cells with the cocktail treated DCs, showed that five cocktails in particular could induce a classical Th1-phenotype with ability to secrete high amounts of the hall-mark cytokine IFNgamma. The model was validated using dexamethasone and two COX-inhibitors, which were able to suppress the cocktail driven pro-inflammatory DC maturation. CONCLUSIONS: The identification of novel Th1-promoting cocktails allows screening of anti-inflammatory drug candidates by assessing the ability to suppress the activation and differentiation of imDCs into inflammatory DCs with a specific Th1-promoting phenotype. The model thus provides a screening tool, which can identify potential anti-inflammatory effects on the natural regulator of the immune response, the dendritic cell.

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.

Dexamethasone/1alpha-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.

Genome-wide expression profiling during protection from colitis by regulatory T cells.

BACKGROUND: In the adoptive transfer model of colitis it has been shown that regulatory T cells (Treg) can hinder disease development and cure already existing mild colitis. The mechanisms underlying this regulatory effect of CD4(+)CD25(+) Tregs are not well understood. METHODS: To identify pathways of importance for immune regulation in protected mice we studied the genome-wide expression profile in the inflamed rectum of SCID mice with CD4(+) T cell transfer colitis and in the uninflamed rectum of mice protected from colitis by Treg cells. We used DNA microarray technology (Affymetrix GeneChip Mouse Genome 430 2.0 Array), which enabled an analysis of a complete set of RNA transcript levels in each sample. Array results were confirmed by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Data were analyzed using combined projections to latent structures and functional annotation analysis. The colitic samples were clearly distinguishable from samples from normal mice by a vast number of inflammation- and growth factor-related transcripts. In contrast, the Treg-protected animals could not be distinguished from either the normal BALB/c mice or the normal SCID mice. mRNA expression profiles of cytokine, chemokine, and growth factor genes were significantly altered in colitic as opposed to noncolitic mice. In particular, the transcription factors STAT3, GATA2, and NFkappaB, the cytokine IL1beta, and the chemokine receptors CXCR3 and CCR1 as well as their ligands all seemingly play central roles in the inflammatory processes. CONCLUSIONS: We suggest that these molecules alone or in combination could be future therapeutic targets.

Chemokines involved in protection from colitis by CD4+CD25+ regulatory T cells.

Chemokines are small proteins involved in the direction of migration of immune cells both during normal homeostasis and inflammation. Chemokines have been implicated in the pathology of many different inflammatory disorders and are therefore appealing therapeutic targets. Using a chemokine/chemokine receptor-specific gene expression profiling system of 67 genes, the authors have determined the expression profile of chemokine and chemokine receptor genes in the rectum of colitic mice and in mice that have been protected fromcolitis by CD4CD25 regulatory T cells. In mice protected from colitis, the authors found down regulation of the mRNA expression of the inflammatory chemokine receptors CCR1 and CXCR3 and their ligands CXCL9, CXCL10, CCL5, and CCL7. Also the transcripts for CCR9, CCL25, CCL17, and CXCL1 are found down regulated in protected compared with colitic animals. In addition, the authors' results suggest that CCL20 is used by CCR6 regulatory T cells in the complex process of controlling colitis because transcripts for this chemokine were expressed to a higher level in protected animals. The chemokine pathways identified in the present study may be of importance for the development of new targets for anti-inflammatory treatment strategies in human inflammatory bowel disease.

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

BACKGROUND: CD4CD25 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 CXCR3CD4CD25T 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 CD4CXCR3CD25 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 CD4CD25 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 CD4CD25 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.

Reactivity of naive CD4+CD25- T cells against gut microflora in healthy mice.

We have previously shown that conventional as well as germ-free CD4+ T cells depleted of CD25+ cells from the gut-associated lymphoid tissue and the periphery proliferate specifically in response to enterobacterial antigen exposure whereas unfractionated CD4+ T cells are not reactive under these conditions. Here we show that the majority of the enteroantigen-specific CD4+ CD25- T cells are naive cells expressing a CD45RB high, CD62L high and CD44 low phenotype. These cells are also present in the thymus and data from adult thymectomized mice show that they represent late (>6 weeks) thymic emigrants. Upon enteroantigen activation, the CD4+ CD25- T cells secrete IL-4, IL-5, IL-10, granulocyte macrophage colony-stimulating factor, tumor necrosis factor-alpha and IFN-gamma. Clonotype mapping of the TCRBV regions 1-18 of enteroantigen-reactive CD4+ CD25- T cells by TCR clonotype mapping revealed the polyclonal nature of this subset. In conclusion, we have for the first time demonstrated the presence of an evolutionary, functionally conserved subset of CD4+ T cells, which are reactive against enterobacterial antigens. This subset resides both in the thymus and the periphery; it is not dependent on previous antigen experience and represents late thymic emigrants, which by enteroantigen-induced activation express a mixed Th 1-Th 2 phenotype. At homeostatic conditions, CD25+ T cells maintain peripheral tolerance in this CD4+ T cell subset.

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 2x10 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-gamma 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.