CD4 ؉ CD25 ؉ Foxp3 ؉ regulatory T cells (Tregs) are potent suppressors of the adaptive immune system, but their effects on innate immune cells are less well known. Here we demonstrate a previously uncharacterized function of Tregs, namely their ability to steer monocyte differentiation toward alternatively activated macrophages (AAM). AAM are cells with strong antiinflammatory potential involved in immune regulation, tissue remodeling, parasite killing, and tumor promotion. We show that, after coculture with Tregs, monocytes/ macrophages display typical features of AAM, including up-regulated expression of CD206 (macrophage mannose receptor) and CD163 (hemoglobin scavenger receptor), an increased production of CCL18, and an enhanced phagocytic capacity. In addition, the monocytes/ macrophages have reduced expression of HLA-DR and a strongly reduced capacity to respond to LPS in terms of proinflammatory mediator production (IL-1␤, IL-6, IL-8, MIP-1␣, TNF-␣), NFB activation, and tyrosine phosphorylation. Mechanistic studies reveal that CD4 ؉ CD25 ؉ CD127 low Foxp3 ؉ Tregs produce IL-10, IL-4, and IL-13 and that these cytokines are the critical factors involved in the suppression of the proinflammatory cytokine response. In contrast, the Tregmediated induction of CD206 is entirely cytokine-independent, whereas the up-regulation of CD163, CCL18, and phagocytosis are (partly) dependent on IL-10 but not on IL-4/IL-13. Together these data demonstrate a previously unrecognized function of CD4 ؉ CD25 ؉ Foxp3 ؉ Tregs, namely their ability to induce alternative activation of monocytes/macrophages. Moreover, the data suggest that the Tregmediated induction of AAM partly involves a novel, cytokineindependent pathway. alternatively activated macrophages ͉ mannose receptor ͉ phagocytosis ͉ proinflammatory response ͉ interleukin-10

Objective. In mice, CD4؉CD25؉ regulatory T cells play a pivotal role in preventing autoimmunity. Regulatory T cells are also present and functional in healthy humans. We investigated the presence, phenotype, and function of CD4؉CD25؉ regulatory T cells in peripheral blood (PB) and synovial fluid (SF) from patients with rheumatoid arthritis (RA).Methods. The presence and phenotype of CD4؉CD25؉ regulatory T cells were determined by flow cytometry. Anergy and suppressive activity were assessed by culturing CD4؉CD25؊ and CD4؉CD25؉ T cells with anti-CD3 monoclonal antibodies and antigen-presenting cells, followed by proliferation and cytokine detection.Results. The percentage of CD4؉CD25؉ T cells in RA SF was significantly increased compared with that in RA PB, and both of these percentages were higher than that in PB from controls. The cells in RA PB were similar in phenotype and function to CD4؉CD25؉ regulatory T cells from controls. In SF, however, ϳ40-50% of CD4؉CD25؉ T cells expressed an activated phenotype, i.e., CD69؉, class II MHC؉, OX-40؉, with high levels of CTLA-4 and glucocorticoid-induced tumor necrosis factor receptor. These synovial CD4؉CD25؉ T cells displayed an increased suppressive capacity compared with blood CD4؉CD25؉ T cells. However, this enhanced suppressive activity was counterbalanced, because activated responder T cells from SF were less susceptible to CD4؉CD25؉ T cell-mediated suppression than were responder cells from PB.Conclusion. We demonstrate that CD4؉CD25؉ regulatory T cells are present and functional in patients with RA, with higher numbers of regulatory T cells with increased suppressive activity found in SF compared with PB. These findings suggest a negative feedback system that is active at the site of inflammation. The balance between activated responder and regulatory T cells appears to influence the extent of immunoregulation in RA.

Anergic/suppressive CD4+CD25+ T cells exist in animal models but their presence has not yet been demonstrated in humans. We have identified and characterized a human CD4+CD25+ T cell subset, which constitutes 7–10 % of CD4+ T cells in peripheral blood and tonsil. These cells are a CD45RO+CD45RBlow highly differentiated primedT cell population that is anergic to stimulation. Depletion of this small subset from CD4+ T cells significantly enhances proliferation by threefold in the remaining CD4+CD25– T cells, while the addition of isolated CD4+CD25+ T cells to CD4+CD25– T cells significantly inhibits proliferative activity. Blocking experiments suggest that suppression is not mediated via IL‐4, IL‐10 or TGF‐β and is cell‐contact dependent. Isolated CD4+CD25+ T cells are susceptible to apoptosis that is associated with low Bcl‐2 expression, but this death can be prevented by IL‐2 or fibroblast‐secreted IFN‐β. However, the anergic/suppressive state of these cells is maintained after cytokine rescue. These human regulatory cells are therefore a naturally occurring, highly suppressive, apoptosis‐prone population which are at a late stage of differentiation. Further studies into their role in normal and pathological situations in humans are clearly essential.

Acetaminophen‐induced acute liver failure (AALF) is associated with innate immunity activation, which contributes to the severity of hepatic injury and clinical outcome. A marked increase in hepatic macrophages (h‐mϕ) is observed in experimental models of AALF, but controversy exists regarding their role, implicating h‐mϕ in both aggravation and resolution of liver injury. The role of h‐mϕ in human AALF is virtually unexplored. We sought to investigate the role of chemokine (C‐C motif) ligand 2 (CCL2) in the recruitment of circulating monocytes to the inflamed liver and to determine how the h‐mϕ infiltrate and liver microenvironment may contribute to tissue repair versus inflammation in AALF. We evaluated circulating monocytes, their chemokine (C‐C motif) receptor 2 (CCR2) expression, and serum CCL2 levels in patients with AALF. Cell subsets and numbers of circulation‐derived (MAC387+) or resident proliferating (CD68/Ki67+) h‐mϕ in hepatic immune infiltrates were determined by immunohistochemistry. Inflammatory cytokine levels were determined in whole and laser microdissected liver tissue by proteome array. In AALF, circulating monocytes were depleted, with the lowest levels observed in patients with adverse outcomes. CCL2 levels were high in AALF serum and hepatic tissue, and circulating monocyte subsets expressed CCR2, suggesting CCL2‐dependent hepatic monocyte recruitment. Significant numbers of both MAC387+ and CD68+ h‐mϕ were found in AALF compared with control liver tissue with a high proportion expressing the proliferation marker Ki67. Levels of CCL2, CCL3, interleukin (IL)‐6, IL‐10, and transforming growth factor‐β1 were significantly elevated in AALF liver tissue relative to chronic liver disease controls. Conclusion: In AALF, the h‐mϕ population is expanded in areas of necrosis, both through proliferation of resident cells and CCL2‐dependent recruitment of circulating monocytes. The presence of h‐mϕ within an anti‐inflammatory/regenerative microenvironment indicates that they are implicated in resolution of inflammation/tissue repair processes during AALF. (HEPATOLOGY 2012)

No abstract

While memory T cells are maintained by continuous turnover, it is not clear how human regulatory CD4 + CD45RO + CD25 hi Foxp3 + T lymphocyte populations persist throughout life. We therefore used deuterium labeling of cycling cells in vivo to determine whether these cells could be replenished by proliferation. We found that CD4 + CD45RO + Foxp3 + CD25 hi T lymphocytes were highly proliferative, with a doubling time of 8 days, compared with memory CD4 + CD45RO + Foxp3 -CD25 -(24 days) or naive CD4 + CD45RA + Foxp3 -CD25 -populations (199 days). However, the regulatory population was susceptible to apoptosis and had critically short telomeres and low telomerase activity. It was therefore unlikely to be self regenerating. These data are consistent with continuous production from another population source. We found extremely close TCR clonal homology between regulatory and memory CD4 + T cells. Furthermore, antigen-related expansions within certain TCR Vb families were associated with parallel numerical increases of CD4 + CD45RO + CD25 hi Foxp3 + Tregs with the same Vb usage. It is therefore unlikely that all human CD4 + CD25 + Foxp3 + Tregs are generated as a separate functional lineage in the thymus. Instead, our data suggest that a proportion of this regulatory population is generated from rapidly dividing, highly differentiated memory CD4 + T cells; this has considerable implications for the therapeutic manipulation of these cells in vivo. IntroductionBoth memory and regulatory populations of T cells must be maintained in tandem in order to generate controlled immunity for the lifetime of the organism. Since the thymus involutes early in life, memory T cells have to largely be maintained by lifelong turnover of preexisting populations of specific T cells in adults (1, 2). The corollary of this is that thymic involution during aging will also severely restrict the production of Tregs by this organ. The source of these cells in adult humans and the relative contributions of long-term survival and ongoing turnover to the maintenance of CD4 + CD25 hi Foxp3 + Treg populations remain unknown.The naturally occurring CD4 + CD25 hi Treg subset that expresses the lineage marker Foxp3 represents an important population of suppressive T cells that can prevent reactivity to both self and nonself antigens (3-5). These cells also downregulate immune responses as pathogen is cleared (3)(4)(5). Early studies demonstrated that in mice, CD4 + CD25 hi Tregs are generated as a distinct population in the thymus (6). Indeed, in mice, there is substantial overlap of TCR repertoires between thymic and peripheral CD4 + Foxp3 + Tregs, suggesting that the thymic regulatory pool makes a significant contribution to the peripheral regulatory cells (7). However, murine CD4 + CD25 hi Tregs, which are phenotypically and functionally identical to the thymus-derived population can also be

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vβ repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RBlow subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen‐specific stimulation in vivo. This suggests that anergic/suppressiveCD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non‐professional antigen‐presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.

The suppressive effects of CD4+CD25+ regulatory T cells (Tregs) on T cells have been well documented. Here we investigated whether human CD4+CD25+ Tregs can inhibit the proinflammatory properties of monocytes/macrophages. Monocytes and T cells were isolated from peripheral blood of healthy volunteers by magnetic cell separation and cocultured for 40 h. Monocytes were analyzed directly for cytokine production and phenotypic changes or repurified and used in T-cell stimulation and lipopolysaccharide challenge assays. Coculture with CD4+CD25+ Tregs induced minimal cytokine production in monocytes, whereas coculture with CD4+CD25- T cells resulted in large amounts of proinflammatory (tumor necrosis factor-alpha, interferon-gamma, interleukin-6) and regulatory (interleukin-10) cytokines. Importantly, when these CD4+CD25+ Treg-treated monocytes were repurified after coculture and challenged with lipopolysaccharide, they were severely inhibited in their capacity to produce tumor necrosis factor-alpha and interleukin-6 compared with control-treated monocytes. In addition, monocytes that were precultured with CD4+CD25+ Tregs displayed limited upregulation of human leukocyte antigen class II, CD40 and CD80, and downregulation of CD86 compared with control-treated monocytes. This altered phenotype had functional consequences, as shown by the reduction in T cell-stimulatory capacity of Treg-treated monocytes. Together, these data demonstrate that CD4+CD25+ Tregs can exert direct suppressive effects on monocytes/macrophages, thereby affecting subsequent innate and adaptive immune responses.