Regulation and functions of the IL-10 family of cytokines in inflammation and disease.

The IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and the more distantly related IL-28A, IL-28B, and IL-29. Evolutionarily, IL-10 family cytokines emerged before the adaptive immune response. These cytokines elicit diverse host defense mechanisms, especially from epithelial cells, during various infections. IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers. Members of this family can promote innate immune responses from tissue epithelia to limit the damage caused by viral and bacterial infections. These cytokines can also facilitate the tissue-healing process in injuries caused by infection or inflammation. Finally, IL-10 itself can repress proinflammatory responses and limit unnecessary tissue disruptions caused by inflammation. Thus, IL-10 family cytokines have indispensable functions in many infectious and inflammatory diseases.

IL-22 bridges the lymphotoxin pathway with the maintenance of colonic lymphoid structures during infection with Citrobacter rodentium.

Colonic patches (CLPs) and isolated lymphoid follicles (ILFs) are two main lymphoid structures in the colon. Lymphoid tissue-inducer cells (LTi cells) are indispensable for the development of ILFs. LTi cells also produce interleukin 17 (IL-17) and IL-22, signature cytokines secreted by IL-17-producing helper T cells. Here we report that IL-22 acted downstream of the lymphotoxin pathway and regulated the organization and maintenance of mature CLPs and ILFs in the colon during infection with Citrobacter rodentium. Lymphotoxin (LTα(1)ß(2)) regulated the production of IL-22 during infection with C. rodentium, but the lymphotoxin-like protein LIGHT did not. IL-22 signaling was sufficient to restore the organization of CLPs and ILFs and host defense against infection with C. rodentium in mice lacking lymphotoxin signals, which suggests that IL-22 connects the lymphotoxin pathway to mucosal epithelial defense mechanisms.

Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161.

Innate lymphoid cells (ILCs) are emerging as a family of effectors and regulators of innate immunity and tissue remodeling. Interleukin 22 (IL-22)- and IL-17-producing ILCs, which depend on the transcription factor RORγt, express CD127 (IL-7 receptor α-chain) and the natural killer cell marker CD161. Here we describe another lineage-negative CD127(+)CD161(+) ILC population found in humans that expressed the chemoattractant receptor CRTH2. These cells responded in vitro to IL-2 plus IL-25 and IL-33 by producing IL-13. CRTH2(+) ILCs were present in fetal and adult lung and gut. In fetal gut, these cells expressed IL-13 but not IL-17 or IL-22. There was enrichment for CRTH2(+) ILCs in nasal polyps of chronic rhinosinusitis, a typical type 2 inflammatory disease. Our data identify a unique type of human ILC that provides an innate source of T helper type 2 (T(H)2) cytokines.

Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells.

Interleukin 22 (IL-22) is a member of the IL-10 cytokine family that is involved in inflammatory and wound healing processes. Originally considered a T helper type 1 (T(H)1)-associated cytokine, IL-22 has since been shown to be produced mainly by IL-17-producing helper T cells (T(H)-17 cells). Here we describe a previously uncharacterized IL-22-producing human helper T cell population that coexpressed the chemokine receptor CCR6 and the skin-homing receptors CCR4 and CCR10. These cells were distinct from both T(H)-17 cells and T(H)1 cells. Downregulation of either the aryl hydrocarbon receptor (AHR) or the transcription factor RORC by RNA-mediated interference affected IL-22 production, whereas IL-17 production was affected only by downregulation of RORC by RNA-mediated interference. AHR agonists substantially altered the balance of IL-22- versus IL-17-producing cells. This subset of IL-22-producing cells may be important in skin homeostasis and pathology.

Human fetal lymphoid tissue-inducer cells are interleukin 17-producing precursors to RORC+ CD127+ natural killer-like cells.

The human body contains over 500 individual lymph nodes, yet the biology of their formation is poorly understood. Here we identify human lymphoid tissue-inducer cells (LTi cells) as lineage-negative RORC+ CD127+ cells with the functional ability to interact with mesenchymal cells through lymphotoxin and tumor necrosis factor. Human LTi cells were committed natural killer (NK) cell precursors that produced interleukin 17 (IL-17) and IL-22. In vitro, LTi cells gave rise to RORC+ CD127+ NK cells that retained the ability to produce IL-17 and IL-22. Postnatally, similar populations of LTi cell-like cells and RORC+ CD127+ NK cells were present in tonsils, and both secreted IL-17 and IL-22 but no interferon-gamma. Our data indicate that lymph node organogenesis is controlled by an NK cell precursor population with adaptive immune features and demonstrate a previously unappreciated link between the innate and adaptive immune systems.

Regulation of cytokine secretion in human CD127(+) LTi-like innate lymphoid cells by Toll-like receptor 2.

Lymphoid tissue inducer cells are members of an emerging family of innate lymphoid cells (ILC). Although these cells were originally reported to produce cytokines such as interleukin-17 (IL-17) and IL-22, we demonstrate here that human CD127(+)RORC(+) and CD56(+)CD127(+) LTi-like ILC also express IL-2, IL-5, and IL-13 after activation with physiologic stimuli such as common γ-chain cytokines, Toll-like receptor (TLR) 2 ligands, or IL-23. Whereas TLR2 signaling induced IL-5, IL-13, and IL-22 expression in a nuclear factor κB (NF-κB)-dependent manner, IL-23 costimulation induced only IL-22 production. CD127(+) LTi-like ILC displayed clonal heterogeneity for IL-13 and IL-5 production, suggesting in vivo polarization. Finally, we identified a role for autocrine IL-2 signaling in mediating the effects of TLR2 stimulation on CD56(+)CD127(+) and CD127(+) LTi-like ILC. These results indicate that human LTi-like ILC can directly sense bacterial components and unravel a previously unrecognized functional heterogeneity among this important population of innate lymphoid cells.

Generation of potent and stable human CD4+ T regulatory cells by activation-independent expression of FOXP3.

Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naïve and memory CD4(+) T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4(+) Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications.

A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism.

Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Tregs). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Tregs. We report the generation of a fully human anti-IL-2 antibody, F5111.2, that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalomyelitis and protected mice against xenogeneic graft-versus-host disease. These results suggest that IL-2-F5111.2 may provide an immunotherapy to treat autoimmune diseases and graft-versus-host disease.

Flow cytometry-based methods for studying signaling in human CD4+CD25+FOXP3+ T regulatory cells.

T regulatory (Treg) cells have a fundamental role in the establishment and maintenance of peripheral tolerance. It is well established that Treg cells have a phenotype and function that is distinct from conventional T effector cells, although how these two T cell subsets differ in terms of molecular signaling cascades remains largely unknown. Analysis of signaling events in Treg cells using classical biochemistry has been hampered due to difficulties in isolating homogeneous populations and limited cell numbers. In order to overcome these challenges, we defined the optimal conditions for culture, in vitro expansion, and stimulation of human CD4(+)CD25(+) Treg and T effector cells to study intracellular signaling events by flow cytometry. In order to avoid the pitfalls associated with cell isolation based on CD25 expression, we developed methodology to analyze subpopulations of FOXP3 positive and negative cells from ex vivo CD4(+) T cells. In addition to examination of ex vivo cells, we optimized expansion conditions for analysis of signaling in Treg and T effector cell lines. Using these methods, we found that human FOXP3(+) Treg cells displayed a greater capacity to phosphorylate the extracellular regulated kinase (ERK) compared to T effector cells, upon TCR-mediated activation. In contrast, FOXP3(+) Treg cells showed a significantly diminished capacity to phosphorylate AKT. This methodology provides a foundation for future investigation into the molecular events that regulate the phenotype and function of Treg cells, and may ultimately lead to the identification of Treg-cell specific therapeutic targets.

The IL-17 pathway as a major therapeutic target in autoimmune diseases.

Th17 cells are a subset of T helper cells that have been recently found to play important functions in host defense and the pathogenesis of various human autoimmune and inflammatory diseases. Th17 cells produce IL-17A, IL-17F, IL-22, and IL-21, of which IL-17A and IL-17F mediate many of the downstream pathologic functions of these cells. IL-17A and IL-17F signal through IL-17RA and IL-17RC heterodimeric receptors that are mainly expressed on tissue epithelial cells and fibroblasts. While IL-17A and IL-17F are important for host defense against many extracellular pathogens, they can also cause excessive tissue damage and exacerbate proinflammatory responses during autoimmunity. The IL-17 pathway, therefore, is a primary therapeutic target downstream of Th17 cells.

Human NKp44+IL-22+ cells and LTi-like cells constitute a stable RORC+ lineage distinct from conventional natural killer cells.

Lymphoid tissue inducer (LTi) cells are required for lymph node formation during fetal development, and recent evidence implies a role in mucosal immunity in the adult. LTi cells share some phenotypic features of conventional natural killer (NK; cNK) cells; however, little is known to date about the relationship between these two cell types. We show that lineage(-) (Lin(-)) CD127(+)RORC(+) LTi-like cells in human tonsil are precursors to CD56(+)CD127(+)RORC(+)NKp46(+) cells, which together comprise a stable RORC(+) lineage. We find that LTi-like cells and their CD56(+) progeny can be expanded and cloned ex vivo without loss of function and without conversion into cNK cells. Clonal analysis reveals heterogeneity of cytokine production within the CD127(+) LTi-like population. Furthermore, we identify within the tonsil a cNK precursor population that is characterized as Lin(-)CD117(+)CD161(+)CD127(-) cells. Overall, we propose that CD127(+)RORC(+) cells, although they share some characteristics with cNK cells, represent a functionally and developmentally distinct lineage.

Altered activation of AKT is required for the suppressive function of human CD4+CD25+ T regulatory cells.

Suppression by T regulatory cells (Treg cells) is a major mechanism by which the immune system controls responses to self and nonharmful foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cell-surface IL-2Ralpha (CD25). We investigated whether altered T-cell-receptor (TCR)-mediated signaling in pure populations of ex vivo human CD4+CD25+ Treg cells might underlie their unique phenotype, including hyporesponsiveness to TCR-mediated activation and lack of cytokine production. CD4+CD25+ Treg cells displayed a consistent defect in phosphorylation of AKT at serine 473 and reduced phosphorylation of the AKT substrates FOXO and S6. Restoration of AKT activity via lentiviral-mediated expression of an inducibly active form of the kinase revealed that reduced activity of this pathway was necessary for the suppressive function of CD4+CD25+ Treg cells. These data represent the first demonstration of a causal association between altered signaling and the function of CD4+CD25+ Treg cells. Moreover, we have created the first system allowing inducible abrogation of suppression through manipulation of the suppressor cells. This system will be a powerful tool to further study the mechanism(s) of suppression by CD4+CD25+ Treg cells.

Activation-induced FOXP3 in human T effector cells does not suppress proliferation or cytokine production.

Forkhead box P3 (FOXP3) is currently thought to be the most specific marker for naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs). In mice, expression of FoxP3 is strictly correlated with regulatory activity, whereas increasing evidence suggests that in humans, activated T effector cells (Teffs) may also express FOXP3. In order to better define the role of FOXP3 in human Teff cells, we investigated the intensity and kinetics of expression in ex vivo Teff cells, suppressed Teff cells and Teff cell lines. We found that all dividing Teff cells expressed FOXP3, but only transiently, and at levels that were significantly lower than those in suppressive nTregs. This temporary expression in Teff cells was insufficient to suppress expression of reported targets of FOXP3 repressor activity, including CD127, IL-2 and IFN-gamma, and was not correlated with induction of a nTreg phenotype. Thus expression of FOXP3 is a normal consequence of CD4(+) T cell activation and, in humans, it can no longer be used as an exclusive marker of nTregs. These data indicate that our current understanding of how FOXP3 contributes to immune tolerance in humans needs to be re-evaluated.

Human CD4+ T cells express TLR5 and its ligand flagellin enhances the suppressive capacity and expression of FOXP3 in CD4+CD25+ T regulatory cells.

Germline encoded pattern recognition receptors, such as TLRs, provide a critical link between the innate and adaptive immune systems. There is also evidence to suggest that pathogen-associated molecular patterns may have the capacity to modulate immune responses via direct effects on CD4+ T cells. Given the key role of both CD4+CD25+ T regulatory (Treg) cells and the TLR5 ligand flagellin in regulating mucosal immune responses, we investigated whether TLR5 may directly influence T cell function. We found that both human CD4+CD25+ Treg and CD4+CD25- T cells express TLR5 at levels comparable to those on monocytes and dendritic cells. Costimulation of effector T cells with anti-CD3 and flagellin resulted in enhanced proliferation and production of IL-2, at levels equivalent to those achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+CD25+ Treg cells, but rather, potently increased their suppressive capacity and enhanced expression of FOXP3. These observations suggest that, in addition to their APC-mediated indirect effects, TLR ligands have the capacity to directly regulate T cell responses and modulate the suppressive activity of Treg cells.