Human anti-inflammatory macrophages induce Foxp3+ GITR+ CD25+ regulatory T cells, which suppress via membrane-bound TGFbeta-1

CD4(+) T cell differentiation and function are critically dependent on the type of APC and the microenvironment in which Ag presentation occurs. Most studies have documented the effect of dendritic cells on effector and regulatory T cell differentiation; however, macrophages are the most abundant APCs in the periphery and can be found in virtually all organs and tissues. The effect of macrophages, and in particular their subsets, on T cell function has received little attention. Previously, we described distinct subsets of human macrophages (pro- and anti-inflammatory, m phi1 and m phi2, respectively) with highly divergent cell surface Ag expression and cytokine/chemokine production. We reported that human m phi1 promote, whereas m phi2 decrease, Th1 activation. Here, we demonstrate that m phi2, but not m phi1, induce regulatory T cells with a strong suppressive phenotype (T(m phi2)). Their mechanism of suppression is cell-cell contact dependent, mediated by membrane-bound TGFbeta-1 expressed on the regulatory T cell (Treg) population since inhibition of TGFbeta-1 signaling in target cells blocks the regulatory phenotype. T(m phi2), in addition to mediating cell-cell contact-dependent suppression, express typical Treg markers such as CD25, glucocorticoid-induced TNF receptor (GITR), and Foxp3 and are actively induced by m phi2 from CD25-depleted cells. These data identify m phi2 cells as a novel APC subset capable of inducing Tregs. The ability of anti-inflammatory macrophages to induce Tregs in the periphery has important implications for understanding Treg dynamics in pathological conditions where macrophages play a key role in inflammatory disease control and exacerbation.     

Interleukin-3 induces proliferation but not lymphokine activated killer activity from human and murine mononuclear cells

Recombinant IL-3 (rIL-3) is a potent colony stimulating factor capable of stimulating early hematopoietic pluripotential progenitor cells and of supporting the differentiation of multiple cells. IL-3 has also been shown to have effects on mature, differentiated circulating cells including eosinophils and T cells. We evaluated the role of exogenous rIL-3 in the generation of cells with LAK activity from murine splenocytes and human bone marrow, spleen, unseparated PBMC and purified null cell preparations. rIL-3 was unable to generate lytic activity from any of these populations by itself and appeared to decrease LAK activity in bone marrow cultures containing high dose IL-2, (bone marrow derived cells (n = 3) with LAK activity for fresh tumor, mean lytic units(LU) 94.6 +/- 63.5 vs 32.8 +/- 44.8 for IL-2 and IL-2 plus IL-3 cultures, respectively p2 less than 0.05). Unlike previous reports testing murine cells, IL-3 priming and subsequent culture in IL-2 of human unseparated bone marrow cells or human or murine splenocytes, failed to generate long-term cultures with lytic activity. IL-3 did, however, induce a dose dependent stimulation of bone marrow and null cell preparations (mean null cell stimulation (3H Thymidine incorporation) with IL-3, 436 +/- 168 cpm vs 9802 +/- 9799 cpm, for 0 vs 10(3) units of IL-3, respectively n = 4, p2 less than 0.05). Furthermore, in bone marrow, unseparated PBMC and null cell cultures, the addition of rIL-3 generated characteristic large blastic appearing cells with prominent basophilic granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

TLR-activated B cells suppress T cell-mediated autoimmunity

TLR sense microbial infections, and control activation of immune responses. Dendritic cells, macrophages, and B lymphocytes express TLR and the TLR-signaling adaptor protein MyD88. The impact of TLR-activated B cells on T cell-mediated inflammation is unknown. In this study, we have used mice carrying B cell-restricted deficiencies in MyD88 or in distinct TLR to examine the impact of TLR-activated B cells on a T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE). We demonstrate that TLR-signaling in B cells suppresses inflammatory T cell responses (both Th1 and Th17), and stimulates recovery from EAE. Only certain TLR are required on B cells for resolution of EAE, and these are dispensable for disease initiation, indicating that a category of TLR agonists preferentially triggers a suppressive function in B cells and thereby limits autoimmune disease. The TLR agonists controlling the regulatory function of B cells are provided by components of Mycobacterium tuberculosis present in the adjuvant. Thus, MyD88 signaling in B cells antagonizes MyD88 signaling in other cells, which drives differentiation of Th17 cells and is required for induction of EAE. Altogether, our data indicate that B cells link recognition of microbial products via TLR to suppression of a T cell-mediated autoimmune disease.     

Different antigen-presenting cells differ in their capacity to induce lymphokine production and proliferation of an apo-cytochrome c-specific T cell clone

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.     

Bystander central memory but not effector memory CD8+ T cells suppress allograft rejection

Memory T cells respond faster and more vigorously than their naive counterparts and are critical for adaptive immunity. However, it is unknown whether and how memory T cells react in the face of irrelevant Ags. It is generally accepted that bystander memory T cells are neutral in immune responsiveness. In this study, we present the first evidence that bystander central memory (TCM), but not effector memory (TEM), CD8+ T cells suppress allograft rejection as well as T cell proliferation in the draining lymph nodes (DLN) of recipient mice. Both bystander TCM and naive T cells, but fewer TEM cells, migrated to DLN, whereas TCM cells exhibited faster turnover than their naive counterparts, suggesting that bystander TCM cells have an advantage over their naive counterparts in suppression. However, bystander TEM cells migrated to inflammatory graft sites, but not DLN, and yet failed to exert their suppression. These findings indicate that bystander memory T cells need to migrate to lymph nodes to exert their suppression by inhibiting responder T cell activation or homeostatic proliferation. Moreover, the suppression mediated by bystander TCM cells was largely dependent on IL-15, as IL-15 was required for their homeostatic proliferation and TCM-mediated suppression of allograft rejection. This suppression also required the presence of TGFbeta1, as TCM cells expressed TGFbeta1 while neutralizing TGFbeta1 abolished their suppression. Thus, bystander TCM, but not TEM, CD8+ T cells are potent suppressors rather than bystanders. This new finding will have an impact on cellular immunology and may have clinic implications for tolerance induction.     

B7+ iris pigment epithelium induce CD8+ T regulatory cells; both suppress CTLA-4+ T cells

Ocular pigment epithelia contribute to immune privilege by suppressing T cell activation and converting T cells into regulatory T regulatory cells (Tregs) that inhibit bystander T cell activation. Iris pigment epithelium (IPE) does so through direct cell-cell contact with naive T cells, and this suppressive contact is via interactions between B7 expressed constitutively on IPE cells and CTLA-4 expressed on a subpopulation of CD8+ T cells. We have now examined whether TGFbeta is required in this process. We report that IPE produces both soluble and membrane-bound active TGFbeta, but that only the latter is actually delivered to CD8+ T cells. In turn, these T cells become IPE Tregs by up-regulating their own expression of B7-1/B7-2 and soluble and membrane-bound TGFbeta. IPE Tregs through their expression of B7 are able to engage CTLA-4+ bystander T cells, and thus precisely, target delivery of membrane-bound TGFbeta. We propose that this mechanism of suppression via TGFbeta ensures that soluble active TGFbeta is not released into the ocular microenvironment where it can have unregulated and deleterious effects, including elevation of intraocular pressure and development of glaucoma.     

Monocyte-derived human macrophages mediate anergy in allogeneic T cells and induce regulatory T cells

Activation of alloreactive T cells by APCs such as dendritic cells (DC) has been implicated as crucial step in transplant rejection. In contrast, it has been proposed that macrophages (Mphi) maintain tolerance toward alloantigens. It was therefore the aim of this study to further analyze the T cell-stimulatory capacity of mature DC and Mphi in vitro using the model of allogeneic MLR. There was a strong proliferative response in T cells cocultured with DC, which was further increased upon restimulation in a secondary MLR. In contrast, T cells did not proliferate in cocultures with Mphi despite costimulation with anti-CD28 and IL-2. Cytokine analysis revealed considerable levels of IL-10 in cocultures of T cells with Mphi, whereas high amounts of IL-2 and IFN-gamma were present in cocultures with DC. There was only minimal T cell proliferation in a secondary MLR when T cells were rescued from primary MLR with Mphi and restimulated with DC of the same donor, or DC of an unrelated donor (third party), whereas a strong primary proliferative response was observed in resting T cells, demonstrating induction of T cell anergy by Mphi. Functional analysis of T cells rescued from cocultures with Mphi demonstrated that anergy was at least partly mediated by IL-10-producing regulatory T cells induced by Mphi. These results demonstrate that Mphi drive the differentiation of regulatory T cells and mediate anergy in allogeneic T cells, supporting the concept that Mphi maintain peripheral tolerance in vivo.     

Characterization of a lymphokine produced by human T cells which inhibits collagen synthesis

Human lymphocytes, isolated from peripheral blood, were cultured for 48 hr in a defined medium containing 10 mg/ml bovine serum albumin and phytohemagglutinin. A lymphokine which inhibits collagen synthesis by cultured human dermal fibroblasts was purified from the lymphocyte incubation medium by successive steps of ammonium sulfate precipitation, gel filtration chromatography, and isoelectric focusing. Good recovery of this collagen synthesis inhibitory factor (CSIF) was obtained and a factor with an approximate molecular weight of 55,000 and a pI of 6.2 was isolated. The purification of the factor should permit further studies on its mechanism of action.     

Lung CD25 CD4 regulatory T cells suppress type 2 immune responses but not bronchial hyperreactivity

To study the effects of chronic Ag deposition in the airway mucosa on CD4(+) T cell priming and subsequent airway disease, transgenic mice were generated that expressed OVA under the control of the surfactant protein C promoter. CD4 T cells from these mice were tolerant to OVA but this was overcome among spleen CD4 T cells by crossing to OVA-specific DO11.10 TCR-transgenic mice. Lungs from the double-transgenic mice developed lymphocytic infiltrates and modest mucus cell hyperplasia. Infiltrating cells were unaffected by the absence of either Rag-1 or Stat6, although the latter deficiency led to the disappearance of mucus. In the lung of double-transgenic mice, a large number of Ag-specific CD4 T cells expressed CD25 and functioned as regulatory T cells. The CD25(+) CD4 T cells suppressed proliferation of CD25(-) CD4 T cells in vitro and inhibited type 2 immune responses induced by aerosolized Ags in vivo. Despite their ability to suppress allergic type 2 immunity in the airways, however, CD25(+) CD4 regulatory T cells had no effect on the development of bronchial hyperreactivity.     

IL-4, but not IL-5, can act synergistically with B cell activating factor (BCAF) to induce proliferation of resting B cells

B cell activating factor (BCAF) was initially identified in the supernatant of the murine T helper cell clone 52-3 (52-3 SN) because of its ability to promote activation and proliferation of resting B cells in the absence of any other costimulus. In this paper, we show that 52-3 T helper cells also secrete IL-4 and IL-5 and we have analyzed the influence of these two lymphokines on B cell proliferation induced by BCAF-containing 52-3 SN. Using the neutralizing anti-IL-4 monoclonal antibody 11B11, we observed partial inhibition of B cell proliferation. 52-3 SN free of IL-4 prepared using an immunoabsorbent column was still able to induce significant B cell proliferation. Although recombinant IL-4 alone does not induce B cell proliferation, it increased the proliferation induced by IL-4-free 52-3 SN. Kinetic studies showed that IL-4 is required at the start of B cell cultures in order to exert optimal synergistic effects. In contrast, anti-IL-5 monoclonal antibody NC17 did not affect the B cell proliferative activity of 52-3 SN whether or not IL-4 was present. When 52-3 SN was tested on dextran-sulfate-activated B cells, IL-5 and BCAF activities were detected but only the IL-5 activity was neutralized by monoclonal antibody NC17. These results demonstrate that (i) BCAF-containing SN can induce proliferation of resting B cells independently of IL-4 and IL-5, and (ii) IL-4, but not IL-5, can act synergistically with BCAF to induce B cell proliferation.     

Overexpression of DRG2 suppresses the growth of Jurkat T cells but does not induce apoptosis

Developmentally regulated GTP-binding protein (DRG) is a new subfamily within the superfamily of GTP-binding proteins. Its expression is regulated during embryonic development. To investigate the effect of the expression of DRG2 on cell growth, we constructed a human Jurkat-T-cell line that overexpresses DRG2. Overexpression of DRG2 suppressed the growth and the aggregation of Jurkat cells but did not induce apoptotic cell death. We used cDNA microarray analysis to examine the global changes in gene expression induced by an overexpression of DRG2. DNA array analyses identified genes that may suppress cell growth at a number of levels in multiple signaling cascades in Jurkat cells and also several prosurvival genes that may protect cells from apoptosis.     

Antigen presentation to Th1 but not Th2 cells by macrophages results in nitric oxide production and inhibition of T cell proliferation: interferon-gamma is essential but insufficient

The induction and role of nitric oxide (NO) during antigen presentation by macrophages to T helper (Th) cell subsets was examined. When cultured with Th1 clones, macrophage APC produced NO only in the presence of cognate Ag, which in turn suppressed T cell proliferation. IFN-gamma production by the activated Th1 cells was essential for the induction of NO. Th2 cells presented with the same cognate Ag did not induce NO production and proliferated uninhibited. Coactivation of Th1 and Th2 cells specific for the same Ag indicated that Th2 cells did not inhibit NO production, but were sensitive to NO induced by stimulated Th1 cells. Antigenic activation of Th2 cells in the presence of rIFN-gamma resulted in NO-mediated inhibition of proliferation. Th2 cells provided only a cell-associated cofactor, whereas Th1 cells secreted a soluble cofactor for IFN-gamma as well, i.e., TNF-alpha. Finally, a role for IFN-gamma and NO during immune responses was studied in spleen cells obtained from immunized IFN-gamma(-/-) mice. NO production and subsequent inhibition of Ag-specific proliferation ex vivo was observed only after the addition of rIFN-gamma. These studies suggest an IFN-gamma-dependent regulatory role for NO during Ag-specific Th cell activation involving macrophages, with obvious implications for Th subset-dependent immune responses in general.     

Bacteria induce lymphokine synthesis polyclonally in human B lymphocytes.

We have studied the ability of various bacteria to stimulate human lymphocytes to produce leukocyte migration inhibitory factor (LIF). Mononuclear cells from adult and cord blood as well as purified T and B lymphocytes were stimulated with killed bacteria. The culture supernatants were tested for the presence of LIF by the agarose migration method. All nine bacterial strains tested activated unseparated mononuclear cells and B lymphocytes but not T cells to produce LIF. LIF was also present in cord blood cell cultures suggesting that the stimulation of lymphocytes was polyclonal rather than antigenic. Therefore, we propose that one of the physiologic functions of B lymphocyte lymphokines might be to form part of the nonspecific defense mechanisms against microbial invasion.     

A replication-defective gammaherpesvirus efficiently establishes long-term latency in macrophages but not in B cells in vivo

Murine gammaherpesvirus 68 (γHV68 or MHV68) is genetically related to the human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), providing a useful system for in vivo studies of the virus-host relationship. To begin to address fundamental questions about the mechanisms of the establishment of gammaherpesvirus latency, we previously generated a replication-defective γHV68 lacking the expression of the single-stranded DNA binding protein encoded by orf6. In work presented here, we demonstrate that this mutant virus established a long-term infection in vivo that was molecularly identical to wild-type virus latency. Thus, despite the absence of an acute phase of lytic replication, the mutant virus established a chronic infection in which the viral genome (i) was maintained as an episome and (ii) expressed latency-associated, but not lytic replication-associated, genes. Macrophages purified from mice infected with the replication-defective virus harbored viral genome at a frequency that was nearly identical to that of wild-type γHV68; however, the frequency of B cells harboring viral genome was greatly reduced in the absence of lytic replication. Thus, this replication-defective gammaherpesvirus efficiently established in vivo infection in macrophages that was molecularly indistinguishable from wild-type virus latency. These data point to a critical role for lytic replication or reactivation in the establishment or maintenance of latent infection in B cells.     

Glutamine fuels proliferation but not migration of endothelial cells

Endothelial metabolism is a key regulator of angiogenesis. Glutamine metabolism in endothelial cells (ECs) has been poorly studied. We used genetic modifications and ¹³C tracing approaches to define glutamine metabolism in these cells. Glutamine supplies the majority of carbons in the tricyclic acid (TCA) cycle of ECs and contributes to lipid biosynthesis via reductive carboxylation. EC‐specific deletion in mice of glutaminase, the initial enzyme in glutamine catabolism, markedly blunts angiogenesis. In cell culture, glutamine deprivation or inhibition of glutaminase prevents EC proliferation, but does not prevent cell migration, which relies instead on aerobic glycolysis. Without glutamine catabolism, there is near complete loss of TCA intermediates, with no compensation from glucose‐derived anaplerosis. Mechanistically, addition of exogenous alpha‐ketoglutarate replenishes TCA intermediates and rescues cellular growth, but simultaneously unveils a requirement for Rac1‐dependent macropinocytosis to provide non‐essential amino acids, including asparagine. Together, these data outline the dependence of ECs on glutamine for cataplerotic processes; the need for glutamine as a nitrogen source for generation of biomass; and the distinct roles of glucose and glutamine in EC biology.     

Anti-Ia inhibits the activity of B cells but not a T cell-derived helper mediator

The effects of addition of anti-Ia sera to cultures of B cells responding to a number of different stimuli were studied. Anti-Ia sera inhibited the responses of B cells to thymus-dependent and thymus-independent antigens. The effects of antisera directed at different subregions within theI region were examined, using the same anti-Ia serum and mouse strains congenic atI. There was some indication that antisera directed at theI-C region might be more efficient at inhibiting responses to a thymus-independent antigen than to a thymus-dependent antigen. An antiserum to another B-cell surface component controlled by theH-2 complex, the D glycoprotein, had no effect on the response of B cells to a thymus-dependent antigen. By contrast, anti-Ia serum added to cultures had no effect on the activity of a T cell-derived, nonspecific, B-cell helper mediator (NSM). We concluded that the binding of anti-Ia sera to B-cell surfaces inhibited B-cell responses to antigen, either by competing directly with the binding of signal molecules, or by delivering an inhibitory signal to the B cell, such that it was subsequently refractory to stimulatory signals.     

Ultraviolet radiation-induced regulatory T cells not only inhibit the induction but can suppress the effector phase of contact hypersensitivity

Epicutaneous application of haptens to UV-exposed skin induces hapten-specific tolerance. This is mediated via regulatory T cells (Tr), as i.v. injection of T cells from UV-tolerized mice into naive animals renders the recipients unresponsive to the respective hapten. However, when UV-induced Tr are injected i.v. into sensitized mice, contact hypersensitivity (CHS) is not suppressed, suggesting that Tr inhibit the induction, but not the elicitation, of CHS and are inferior to T effector cells. As sensitization takes place in the lymph nodes, but elicitation occurs in the area of challenge, we postulated that Tr injected i.v. locate to the lymph nodes and not to the periphery and therefore only suppress the induction, not the elicitation, of CHS. Indeed, i.v. injection of Tr into sensitized mice did not inhibit CHS, although injection of Tr into the ears of sensitized mice suppressed the challenge. Inhibition was hapten specific, as injection of dinitrofluorobenzene (DNFB)-specific Tr into the ears of oxazolone (OXA)-sensitized mice did not affect challenge with OXA. However, when ears of OXA-sensitized mice were injected with DNFB-specific Tr and painted with DNFB before OXA challenge, CHS was suppressed. Inhibition correlated with the local expression of IL-10. Depletion studies and FACS analysis revealed that Tr express the lymph node-homing receptor L-selectin, but not the ligands for the skin-homing receptors E- and P-selectin, suggesting that UV-induced Tr, although able to inhibit T effector cells, do not suppress the elicitation of CHS upon i.v. injection, because they obviously do not migrate into the skin.     

Aluminum ions induce DNA synthesis but not cell proliferation in human fibroblasts in vitro

The effect in vitro of aluminum (Al) ions on DNA synthesis and human dermal fibroblast proliferation using [Al] concentrations from 1.85 to 74 μM and incubation periods of 1, 2, 3, 4, and 5 d was assessed. The lowest concentration of Al that exerted a slight positive, although not significant, effect on DNA synthesis was 1.85 μM, after d 3 or 5 of incubation. The stimulating action of Al was more evident and statistically significant from concentrations of 3.7 μM and 2 d exposure onward. This Al-induced effect on [³H] thymidine incorporation into DNA increased in a time-dependent manner as [Al] in the culture medium rose, provoking increments of up to 322% above the control at [Al] 74 μM and 5 d incubation. In contrast, Al salts moderately increased fibroblast division in a continuous manner only from 7.4 to 74 μM after 3 d of incubation. Although significant overall, the minimal and inconstant mitogenic activity of Al differs greatly from and is not parallel to DNA synthesis, which is not clearly related to exposure times or Al concentrations. Abnormalities in Al-induced cellular metabolic processes described herein and their influence on the cell cycle may constitute a toxicity mechanism for human tissues, leading to disease development. Further studies are required to determine whether these findings can be extrapolated to in vivo situations.     

B cells activated by lipopolysaccharide,but not by anti-Ig and anti-CD40 antibody,induce anergy in CD8+ T cells: role of TGF-beta 1

B cells recognize Ag through their surface IgRs and present it in the context of MHC class II molecules to CD4(+) T cells. Recent evidence indicates that B cells also present exogenous Ags in the context of MHC class I to CD8(+) T cells and thus may play an important role in the modulation of CTL responses. However, in this regard, conflicting reports are available. One group of studies suggests that the interaction between B cells and CD8(+) T cells leads to the activation of the T cells, whereas other studies propose that it induces T cell tolerance. For discerning this dichotomy, we used B cells that were activated with either LPS or anti-Ig plus anti-CD40 Ab, which mimic the T-independent and T-dependent modes of B cell activation, respectively, to provide accessory signals to resting CD8(+) T cells. Our results show that, in comparison with anti-Ig plus anti-CD40 Ab-activated B cells, the LPS-activated B cells (LPS-B) failed to induce significant levels of proliferation, cytokine secretion, and cytotoxic ability of CD8(+) T cells. This hyporesponsiveness of CD8(+) T cells activated with LPS-B was significantly rescued by anti-TGF-beta1 Ab. Moreover, it was found that such hyporesponsive CD8(+) T cells activated with LPS-B had entered a state of anergy. Furthermore, LPS-B expresses a significantly higher level of TGF-beta1 on the surface, which caused the observed hyporesponsiveness of CD8(+) T cells. Therefore, this study, for the first time, provides a novel mechanism of B cell surface TGF-beta1-mediated hyporesponsiveness leading to anergy of CD8(+) T cells.