Jonathan S. Bromberg

Gr-1+CD115+ Immature Myeloid Suppressor Cells Mediate the Development of Tumor-Induced T Regulatory Cells and T-Cell Anergy in Tumor-Bearing Host

The accumulation of myeloid suppressor cells (MSCs) is associated with immune suppression in tumor-bearing mice and in cancer patients. The suppressive activity of MSC correlates with the expression of the myeloid markers Gr-1, CD115 (macrophage colony-stimulating factor receptor), and F4/80. Gr-1(+)CD115(+) MSCs, in addition to being able to suppress T-cell proliferation in vitro, can induce the development of Foxp3(+) T regulatory cells (Treg) in vivo, which are anergic and suppressive. Furthermore, the secretion of interleukin (IL)-10 and transforming growth factor-beta by Gr-1(+)CD115(+) MSCs was induced and enhanced, respectively, on IFN-gamma stimulation. The development of Treg requires antigen-associated activation of tumor-specific T cells, depends on the presence of IFN-gamma and IL-10, and is independent of the nitric oxide-mediated suppressive mechanism by MSC. Our data provide evidence that Gr-1(+)CD115(+) MSC can mediate the development of Treg in tumor-bearing mice and show a novel immune suppressive mechanism by which MSCs can suppress antitumor responses.

Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts.

The induction of alloantigen-specific unresponsiveness remains an elusive goal in organ transplantation. Here we identify plasmacytoid dendritic cells (pDCs) as phagocytic antigen-presenting cells essential for tolerance to vascularized cardiac allografts. Tolerizing pDCs acquired alloantigen in the allograft and then moved through the blood to home to peripheral lymph nodes. In the lymph node, alloantigen-presenting pDCs induced the generation of CCR4+CD4+CD25+Foxp3+ regulatory T cells (Treg cells). Depletion of pDCs or prevention of pDC lymph node homing inhibited peripheral Treg cell development and tolerance induction, whereas adoptive transfer of tolerized pDCs induced Treg cell development and prolonged graft survival. Thus, alloantigen-presenting pDCs home to the lymph nodes in tolerogenic conditions, where they mediate alloantigen-specific Treg cell development and allograft tolerance.

The origin and development of nonlymphoid tissue CD103+ DCs

CD103+ dendritic cells (DCs) in nonlymphoid tissues are specialized in the cross-presentation of cell-associated antigens. However, little is known about the mechanisms that regulate the development of these cells. We show that two populations of CD11c+MHCII+ cells separated on the basis of CD103 and CD11b expression coexist in most nonlymphoid tissues with the exception of the lamina propria. CD103+ DCs are related to lymphoid organ CD8+ DCs in that they are derived exclusively from pre-DCs under the control of fms-like tyrosine kinase 3 (Flt3) ligand, inhibitor of DNA protein 2 (Id2), and IFN regulatory protein 8 (IRF8). In contrast, lamina propria CD103+ DCs express CD11b and develop independently of Id2 and IRF8. The other population of CD11c+MHCII+ cells in tissues, which is CD103−CD11b+, is heterogenous and depends on both Flt3 and MCSF-R. Our results reveal that nonlymphoid tissue CD103+ DCs and lymphoid organ CD8+ DCs derive from the same precursor and follow a related differentiation program.

Epstein-Barr virus-induced posttransplant lymphoproliferative disorders

Epstein-Barr virus-induced posttransplant lymphoproliferative disease (EBV-PTLD) continues to be a major complication after solid organ transplantation in high-risk patients. Despite the identification of risk factors that predispose patients to develop EBV-PTLD, limitations in our knowledge of its pathogenesis, variable criteria for establishing the diagnosis, and lack of randomized studies addressing the prevention and treatment of EBV-PTLD hamper the optimal management of this transplant complication. This review summarizes the current knowledge of EBV-PTLD and, as a result of two separate international meetings on this topic, and provides recommendations for future areas of study.

TGF-β induces Foxp3 + T-regulatory cells from CD4 + CD25 - precursors

CD4 + CD25 + regulatory T cells (Tregs) are potent suppressors, playing important roles in autoimmunity and transplantation tolerance. Understanding the signals necessary for the generation and expansion of Tregs is important for clinical cellular therapy, but only limited progress has been made. Recent reports suggest a role for TGF‐β in the generation of Tregs from CD4 + CD25 − precursors, but the mechanism remains unknown. Here, we demonstrate that TGF‐β2 triggers Foxp3 expression in CD4 + CD25 − precursors, and these Foxp3 + cells act like conventional Tregs. The generation of Foxp3 + Tregs requires stimulation of the T‐cell receptor, the IL‐2R and the TGF‐β receptor. More importantly, strong costimulation through CD28 prevents Foxp3 expression and suppressive function in an IL‐4‐dependent manner. Furthermore, TGF‐β‐driven Tregs inhibit innate inflammatory responses to syngeneic transplanted pancreatic islets and enhance islet transplant survival. Thus, TGF‐β is a key regulator of the signaling pathways that initiate and maintain Foxp3 expression and suppressive function in CD4 + CD25 − precursors. TGF‐β and signaling through TGF‐β receptor, CD28 costimulation and IL‐4 may be key components for the manipulation of Treg. The de novo generation of Foxp3 + cells from CD4 + cells has the potential to be used for treatment of autoimmune diseases and induction of transplant tolerance.

Epigenetic Regulation of Foxp3 Expression in Regulatory T Cells by DNA Methylation

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.

Perioperative glucocorticoid coverage. A reassessment 42 years after emergence of a problem.

ObjectiveThe authors review the historical basis for the provision of perioperative glucocorticoid coverage, and detail the evolution in the understanding of the role of the hypothalamic-pituitary-adrenal cortical (HPA) axis in response to physical stressors. New recommendations are proposed for glucocorticoid-dependent patients who require anesthesia and surgery. Summary Background DataIn 1952, a patient developed surgery-associated adrenal insufficiency as a result of preoperative withdrawal from glucocorticoid therapy. That case report, and one other in the ensuing 12 months, prompted the publication of recommendations for perioperative glucocorticoid coverage, which became the standard of care. The understanding of the role of the HPA axis in the stress response has been subsequently refined; however, recommendations for perioperative glucocorticoid coverage have not been altered in parallel. MethodsStudies were identified beginning with the first reports of the physiologic actions of the adrenal glands (1855) and the description and clinical use of cortisone (1930–1993). Studies were selected for review if they were related to or evaluated the provision of stress-related glucocorticoid administration. All clinical studies were evaluated to determine the basis for the provision of perioperative glucocorticoid coverage and the validity of the data used to justify these conclusions. ConclusionsClinical and experimental evidence support the concept that the current amount of perioperative glucocorticoid coverage is excessive and has been based on anecdotal information. New recommendations are proposed which suggest that the amount and duration of glucocorticoid coverage should be determined by: a) the preoperative dose of glucocorticoid taken by the

Role of CCR8 and Other Chemokine Pathways in the Migration of Monocyte-derived Dendritic Cells to Lymph Nodes

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.

Epigenetic mechanisms of regulation of Foxp3 expression

Regulatory T cells play important roles in the control of autoimmunity and maintenance of transplantation tolerance. Foxp3, a member of the forkhead/winged-helix family of transcription factors, acts as the master regulator for regulatory T-cell (Treg) development and function. Mutation of the Foxp3 gene causes the scurfy phenotype in mouse and IPEX syndrome (immune dysfunction, polyendocrinopathy, enteropathy, X-linked syndrome) in humans. Epigenetics is defined by regulation of gene expression without altering nucleotide sequence in the genome. Several epigenetic markers, such as histone acetylation and methylation, and cytosine residue methylation in CpG dinucleotides, have been reported at the Foxp3 locus. In particular, CpG dinucleotides at the Foxp3 locus are methylated in naive CD4+CD25- T cells, activated CD4+ T cells, and TGF-beta-induced adaptive Tregs, whereas they are completely demethylated in natural Tregs. The DNA methyltransferases DNMT1 and DNMT3b are associated with the Foxp3 locus in CD4+ T cells. Methylation of CpG residues represses Foxp3 expression, whereas complete demethylation is required for stable Foxp3 expression. In this review, we discuss how different cis-regulatory elements at the Foxp3 locus are subjected to epigenetic modification in different subsets of CD4+ T cells and regulate Foxp3 expression, and how these mechanisms can be exploited to generate efficiently large numbers of suppressive Tregs for therapeutic purposes.

Regulatory T cells sequentially migrate from inflamed tissues to draining lymph nodes to suppress the alloimmune response.

To determine the site and mechanism of suppression by regulatory T (Treg) cells, we investigated their migration and function in an islet allograft model. Treg cells first migrated from blood to the inflamed allograft where they were essential for the suppression of alloimmunity. This process was dependent on the chemokine receptors CCR2, CCR4, and CCR5 and P- and E-selectin ligands. In the allograft, Treg cells were activated and subsequently migrated to the draining lymph nodes (dLNs) in a CCR2, CCR5, and CCR7 fashion; this movement was essential for optimal suppression. Treg cells inhibited dendritic cell migration in a TGF-beta and IL-10 dependent fashion and suppressed antigen-specific T effector cell migration, accumulation, and proliferation in dLNs and allografts. These results showed that sequential migration from blood to the target tissue and to dLNs is required for Treg cells to differentiate and execute fully their suppressive function.