Foxp3, a winged-helix family transcription factor, serves as the master switch for CD4+ regulatory T cells (Treg). We identified a unique and evolutionarily conserved CpG-rich island of the Foxp3 nonintronic upstream enhancer and discovered that a specific site within it was unmethylated in natural Treg (nTreg) but heavily methylated in naive CD4+ T cells, activated CD4+ T cells, and peripheral TGFβ-induced Treg in which it was bound by DNMT1, DNMT3b, MeCP2, and MBD2. Demethylation of this CpG site using the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (Aza) induced acetylation of histone 3, interaction with TIEG1 and Sp1, and resulted in strong and stable induction of Foxp3. Conversely, IL-6 resulted in methylation of this site and repression of Foxp3 expression. Aza plus TGFβ-induced Treg resembled nTreg, expressing similar receptors, cytokines, and stable suppressive activity. Strong Foxp3 expression and suppressor activity could be induced in a variety of T cells, including human CD4+CD25− T cells. Epigenetic regulation of Foxp3 can be predictably controlled with DNMT inhibitors to generate functional, stable, and specific Treg.

Blockade of C3aR/C5aR signaling in nT reg cells augments in vitro and in vivo suppression, abrogates autoimmune colitis, and prolongs allogeneic skin graft survival.

SUMMARY Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8+ T cell immunity and promoted CD4+Foxp3+ Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN+ suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

No abstract

The leukocyte immunoglobulin-like receptor (LILR) family comprises a set of paired immunomodulatory receptors expressed among human myeloid and lymphocyte cell populations. While six members of LILR subfamily A (LILRA) associate with membrane adaptors to signal via immunoreceptor tyrosine-based activating motifs (ITAM), LILR subfamily B (LILRB) members signal via multiple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIM). Ligand specificity of some LILR family members has been studied in detail, but new perspective into the immunoregulatory aspects of this receptor family in human myeloid cells has been limited. LILRB receptors and the murine ortholog, paired immunoglobulin-like receptor B (PIRB), have been shown to negatively regulate maturation pathways in myeloid cells including mast cells, neutrophils, dendritic cells, as well as B cells. Our laboratory further demonstrated in mouse models that PIRB regulated functional development of myeloid-derived suppressor cell and the formation of a tumor-permissive microenvironment. Based on observations from the literature and our own studies, our laboratory is focusing on how LILRs modulate immune homeostasis of human myeloid cells and how these pathways may be targeted in disease states. Integrity of this pathway in tumor microenvironments, for example, permits a myeloid phenotype that suppresses antitumor adaptive immunity. This review presents the evidence supporting a role of LILRs as myeloid cell regulators and ongoing efforts to understand the functional immunology surrounding this family.

CD4+Foxp3+ regulatory T cells (Treg) are critical regulators of immune homeostasis and self-tolerance. Whereas thymic-derived or natural Treg (nTreg) stably express Foxp3, adaptive or induced Treg (iTreg) generated from peripheral CD4 T cells are susceptible to inflammation-induced reversion to pathogenic effector T cells (Teff). Building upon our previous observations that T cell-expressed receptors for C3a (C3aR) and C5a (C5aR) drive Th1 maturation, we tested the impact of C3aR/C5aR signaling on induction and stability of alloreactive iTreg. We observed that genetic deficiency or pharmacological blockade of C3aR/C5aR signaling augments murine and human iTreg generation, stabilizes Foxp3 expression, resists iTreg conversion to IFNγ/TNFα-producing Teff, and as a consequence, limits the clinical expression of graft-versus-host disease. Together the findings highlight the expansive role of complement as a crucial modulator of T cell alloimmunity and demonstrate proof-of-concept that targeting C3a/C3aR and C5a/C5aR interactions could facilitate iTreg-mediated tolerance to alloantigens in humans.

Results of studies published since 2002 reveal that T cells and antigen-presenting cells (APCs) produce complement proteins. The immune cell-derived, alternative pathway complement components activate spontaneously, yielding local, but not systemic, production of C3a and C5a. These anaphylatoxins to bind their respective G-protein coupled receptors, C3aR and C5aR, expressed on both partners. The resultant complement-induced T cell and APC activation drives T cell differentiation, expansion and survival. Complement deficiency or blockade attenuates T cell mediated autoimmunity and delays allograft rejection in mice. Increasing complement activation, achieved by genetic removal of the complement regulatory protein decay accelerating factor (DAF), enhances murine T cell immunity and accelerates allograft rejection. The findings support the need for design and testing of complement inhibitors in humans.

Summary Complement proteins are generated both by the liver (systemic compartment) and by peripheral tissue-resident cells and migratory immune cells (local compartment). The immune cell–derived, alternative pathway complement components activate spontaneously, yielding local, but not systemic, production of C3a and C5a. These anaphylatoxins bind to their respective G-protein–coupled receptors, the C3a receptor and the C5a receptor, expressed on T cells and antigen-presenting cells, leading to their reciprocal activation and driving T-cell differentiation, expansion, and survival. Complement deficiency or blockade attenuates T-cell–mediated autoimmunity and delays allograft rejection in mice. Increasing complement activation, achieved by genetic removal of the complement regulatory protein decay accelerating factor, enhances murine T-cell immunity and accelerates allograft rejection. Signaling through the C3a receptor and the C5a receptor reduces suppressive activity of natural regulatory T cells and the generation and stability of induced regulatory T cells. The concepts, initially generated in mice, recently were confirmed in human immune cells, supporting the need for testing of complement targeting therapies in organ transplants patients.