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 naive 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

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

Molecular phenotype of human CD4+CD25+ T regulatory cells

Suppression by T regulatory cells (Tregs) is a major mechanism by which the immune system controls responses to self and innocuous foreign proteins. Although there are many different types of Treg cells, the best characterized are those that constitutively express cellsurface IL-2 R (CD25). The aim of this research was to; increase understanding of the molecular phenotype of human CD4[sup +]CD25[sup +] Tregs, and was addressed using 3 different approaches. I) Certain pathogens are known to be capable of modulating the function of CD4[sup +]CD25[sup +] Tregs, but it was unclear i f this was via a direct or indirect mechanism. The expression of TLR5 on human CD4[sup +]T cells was studied, and it was observed that both CD4[sup +]CD25[sup +] effectors and CD4[sup +]CD25[sup +] Tregs express TLR5 at functionally significant levels. Further, the TLR5 ligand flagellin is a co-stimulatory molecule for CD4[sup +]CD25[sup +] effector cells, and flagellin can increase the suppressive capacity of CD4[sup +]CD25[sup +] Tregs in the absence o f antigen presenting cells. II) In an effort to understand the mechanism underlying the unique functional phenotype of CD4[sup +]CD25[sup +] Tregs, including hypo-responsiveness to T cell receptor (TCR) mediated activation and lack o f cytokine production, TCR-mediated signaling in pure populations of ex vivo human CD4[sup +]CD25[sup +] Tregs was investigated. A consistent defect in AKT activation in CD4[sup +]CD25[sup +] Tregs was observed, which when reversed, abrogated CD4[sup +]CD25[sup +] Treg suppressive capacity. Ill) Clinical applications and experimental manipulations have been hampered by the lack of a unique cell surface marker for CD4[sup +]CD25[sup +] Tregs that is not also an activation marker for CD4[sup +]CD25[sup +] T effector cells. Microarray analysis on single cell-derived Treg and T effector clones was performed as an initial screen, followed by quantitative R T - PCR validation on polyclonal populations of human CD4[sup +]CD25[sup +] Tregs. It was observed that human CD4[sup +]CD25[sup +] Tregs specificallyexpress the membrane channel protein Aquaporin 9. In summary, the research described within is of considerable import to the study of human CD4[sup +]CD25[sup +] Tregs and their control of immune homeostasis, and represents a significant contribution to the field of immunology.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

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 patholog

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 gamma-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 kappa B (NF-kappa B)-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 cell