IL-6 Inhibits Upregulation of Membrane-Bound TGF-ß 1 on CD4+ T Cells and Blocking IL-6 Enhances Oral Tolerance.

Oral administration of Ag induces regulatory T cells that express latent membrane-bound TGF-ß (latency-associated peptide [LAP]) and have been shown to play an important role in the induction of oral tolerance. We developed an in vitro model to study modulation of LAP+ on CD4+ T cells. The combination of anti-CD3 mAb, anti-CD28 mAb, and recombinant IL-2 induced expression of LAP on naive CD4+ T cells, independent of Foxp3 or exogenous TGF-ß. In vitro generated CD4+LAP+Foxp3- T cells were suppressive in vitro, inhibiting proliferation of naive CD4+ T cells and IL-17A secretion by Th17 cells. Assessing the impact of different cytokines and neutralizing Abs against cytokines, we found that LAP induction was decreased in the presence of IL-6 and IL-21, and to a lesser extent by IL-4 and TNF-α. IL-6 abrogated the in vitro induction of CD4+LAP+ T cells by STAT3-dependent inhibition of Lrrc32 (glycoprotein A repetitions predominant [GARP]), the adapter protein that tethers TGF-ß to the membrane. Oral tolerance induction was enhanced in mice lacking expression of IL-6R by CD4+ T cells and by treatment of wild-type mice with neutralizing anti-IL-6 mAb. These results suggest that proinflammatory cytokines interfere with oral tolerance induction and that blocking the IL-6 pathway is a potential strategy for enhancing oral tolerance in the setting of autoimmune and inflammatory diseases.

Mucosal administration of CD3-specific monoclonal antibody inhibits diabetes in NOD mice and in a preclinical mouse model transgenic for the CD3 epsilon chain.

CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-ß and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans.

The Host Shapes the Gut Microbiota via Fecal MicroRNA.

The host gut microbiota varies across species and individuals but is relatively stable over time within an individual. How the host selectively shapes the microbiota is largely unclear. Here, we show that fecal microRNA (miRNA)-mediated inter-species gene regulation facilitates host control of the gut microbiota. miRNAs are abundant in mouse and human fecal samples and present within extracellular vesicles. Cell-specific loss of the miRNA-processing enzyme, Dicer, identified intestinal epithelial cells (IEC) and Hopx-positive cells as predominant fecal miRNA sources. These miRNAs can enter bacteria, such as F. nucleatum and E. coli, specifically regulate bacterial gene transcripts, and affect bacterial growth. IEC-miRNA-deficient (Dicer1(ΔIEC)) mice exhibit uncontrolled gut microbiota and exacerbated colitis, and WT fecal miRNA transplantation restores fecal microbes and ameliorates colitis. These findings identify both a physiologic role by which fecal miRNA shapes the gut microbiota and a potential strategy for manipulating the microbiome.

MicroRNA-21 promotes Th17 differentiation and mediates experimental autoimmune encephalomyelitis.

Accumulation of IL-17-producing Th17 cells is associated with the development of multiple autoimmune diseases; however, the contribution of microRNA (miRNA) pathways to the intrinsic control of Th17 development remains unclear. Here, we demonstrated that miR-21 expression is elevated in Th17 cells and that mice lacking miR-21 have a defect in Th17 differentiation and are resistant to experimental autoimmune encephalomyelitis (EAE). Furthermore, we determined that miR-21 promotes Th17 differentiation by targeting and depleting SMAD-7, a negative regulator of TGF-ß signaling. Moreover, the decreases in Th17 differentiation in miR-21-deficient T cells were associated with defects in SMAD-2/3 activation and IL-2 suppression. Finally, we found that treatment of WT mice with an anti-miR-21 oligonucleotide reduced the clinical severity of EAE, which was associated with a decrease in Th17 cells. Thus, we have characterized a T cell-intrinsic miRNA pathway that enhances TGF-ß signaling, limits the autocrine inhibitory effects of IL-2, and thereby promotes Th17 differentiation and autoimmunity.

The short isoform of the CEACAM1 receptor in intestinal T cells regulates mucosal immunity and homeostasis via Tfh cell induction.

Carcinoembryonic antigen cell adhesion molecule like I (CEACAM1) is expressed on activated T cells and signals through either a long (L) cytoplasmic tail containing immune receptor tyrosine based inhibitory motifs, which provide inhibitory function, or a short (S) cytoplasmic tail with an unknown role. Previous studies on peripheral T cells show that CEACAM1-L isoforms predominate with little to no detectable CEACAM1-S isoforms in mouse and human. We show here that this was not the case in tissue resident T cells of intestines and gut associated lymphoid tissues, which demonstrated predominant expression of CEACAM1-S isoforms relative to CEACAM1-L isoforms in human and mouse. This tissue resident predominance of CEACAM1-S expression was determined by the intestinal environment where it served a stimulatory function leading to the regulation of T cell subsets associated with the generation of secretory IgA immunity, the regulation of mucosal commensalism, and defense of the barrier against enteropathogens.

Induction of immunological tolerance by oral anti-CD3.

In recent years, our knowledge about immunoregulation and autoimmunity has significantly advanced, but nontoxic and more effective treatments for different inflammatory and autoimmune diseases are still lacking. Oral tolerance is of unique immunologic importance because it is a continuous natural immunologic event driven by exogenous antigen and is an attractive approach for treatment of these conditions. Parenteral administration of anti-CD3 monoclonal antibody is an approved therapy for transplantation in humans and is effective in autoimmune diabetes. Orally administered anti-CD3 monoclonal antibody is biologically active in the gut and suppresses experimental models of autoimmune diseases. Orally delivered antibody does not have side effects including cytokine release syndromes, thus oral anti-CD3 antibody is clinically applicable for chronic therapy. Here we review findings that identify a novel and powerful immunologic approach that is widely applicable for the treatment of human autoimmune conditions.

In vivo induction of Tr1 cells via mucosal dendritic cells and AHR signaling.

BACKGROUND: Type 1 regulatory T (Tr1) cells, characterized by the secretion of high levels of the anti-inflammatory cytokine interleukin-10 (IL-10), play an important role in the regulation of autoimmune diseases and transplantation. However, effective strategies that specifically induce Tr1 cells in vivo are limited. Furthermore, the pathways controlling the induction of these cells in vivo are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that nasal administration of anti-CD3 antibody induces suppressive Tr1 cells in mice. The in vivo induction of Tr1 cells by nasal anti-CD3 is dependent on IL-27 produced by upper airway resident dendritic cells (DCs), and is controlled by the transcription factors aryl hydrocarbon receptor (AHR) and c-Maf. Subsequently, IL-21 acts in an autocrine fashion to expand and maintain the Tr1 cells induced in vivo by nasally administered anti-CD3. CONCLUSIONS/SIGNIFICANCE: Our findings identify a unique approach to generate Tr1 cells in vivo and provide insights into the mechanisms by which these cells are induced.

Oral tolerance.

The gut-associated lymphoid tissue is the largest immune organ in the body and is the primary route by which we are exposed to antigens. Tolerance induction is the default immune pathway in the gut, and the type of tolerance induced relates to the dose of antigen fed: anergy/deletion (high dose) or regulatory T-cell (Treg) induction (low dose). Conditioning of gut dendritic cells (DCs) by gut epithelial cells and the gut flora, which itself has a major influence on gut immunity, induces CD103(+) retinoic acid-dependent DC that induces Tregs. A number of Tregs are induced at mucosal surfaces. Th3 type Tregs are transforming growth factor-ß dependent and express latency-associated peptide (LAP) on their surface and were discovered in the context of oral tolerance. Tr1 type Tregs (interleukin-10 dependent) are induced by nasal antigen and forkhead box protein 3(+) iTregs are induced by oral antigen and by oral administration of aryl hydrocarbon receptor ligands. Oral or nasal antigen ameliorates autoimmune and inflammatory diseases in animal models by inducing Tregs. Furthermore, anti-CD3 monoclonal antibody is active at mucosal surfaces and oral or nasal anti-CD3 monoclonal antibody induces LAP(+) Tregs that suppresses animal models (experimental autoimmune encephalitis, type 1 and type 2 diabetes, lupus, arthritis, atherosclerosis) and is being tested in humans. Although there is a large literature on treatment of animal models by mucosal tolerance and some positive results in humans, this approach has yet to be translated to the clinic. The successful translation will require defining responsive patient populations, validating biomarkers to measure immunologic effects, and using combination therapy and immune adjuvants to enhance Treg induction. A major avenue being investigated for the treatment of autoimmunity is the induction of Tregs and mucosal tolerance represents a non-toxic, physiologic approach to reach this goal.

Study of oral tolerance and its indirect effects in adoptive cell transfer experiments.

Parenteral exposure to antigens to which oral tolerance had been previously induced results in the inhibition of immune responses to other unrelated antigens. Herein we tested whether indirect effects of oral tolerance could be adoptively transferred. Anti-Ova- and antihemoglobin-specific responsiveness as well as oral tolernace to Ova were transferred to irradiated, but not to normal, nonirradiated recepients. Irradiation, thus, facilitated adoptive transfer of oral tolerance. However, the inhibitory (indirect) effects upon the unrelated immunogen were not adoptively transferred, even to irradiated recepients. In addition, we studied adoptively transferred CFSE-labeled spleen cells by flow cytometry in recipient spleen, inguinal lymph nodes, and bone marrow, both in irradiated and nonirradiated recipients, 1, 3, or 5 days after cell transfer. Comparing the percent and absolute number of CFSE-labeled cells in each organ displayed significant differences in the dynamics of decay of adoptively transferred cells from tolerant or immune donors.