IFN‐α‐mediated increase in cytolytic activity of maturing NK cell upon exposure to HSV‐infected myelomonocytes

Impaired control of chronic pathogen replication may be associated to alterations of NK‐cell function. Whether mechanisms underlying this dysfunction involve perturbations of differentiating NK cells is still unknown. We studied an “in vitro” model of differentiation from CD34⁺Lin^? precursors growing only myelomonocytes and maturing NK cells and where myelomonocytes could be suitably infected with HSV, HIV, or vaccinia. Cultures were evaluated by cytofluorometry and cytotoxicity assays for perturbations in differentiating NK cells. Increased expression of natural cytotoxicity receptors on maturing NK cells with increased cytolytic activity was observed with HSV‐1 infection, and with vaccinia while no modulation of NK‐cell phenotype nor cytotoxic activity were evident with an ssRNA lentivirus (HIV‐1). In the presence of constant IL‐12 and IL‐15 concentrations, the observed effect did not require cell contact, involved IFN‐αand was not reproduced by the addition of TLR9 agonist, nor blocked by TLR9 antagonists. Virus replication at sites of NK‐cell precursor development may have different outcomes depending on the interaction between invading viruses and maturing NK cells. Thus, NK‐cell precursors may be involved in the immune response to dsDNA viruses and possibly contribute to efficient control of virus infection.     

CEACAM1 on activated NK cells inhibits NKG2D‐mediated cytolytic function and signaling

Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is expressed on activated natural killer (NK) cells wherein it inhibits lysis of CEACAM1‐bearing tumor cell lines. The mechanism for this is unknown. Here, we show that interleukin‐2‐induced expression of CEACAM1 on both mouse and primary human NK cells impairs the ability of NK gene complex group 2 member D (NKG2D) to stimulate cytolysis of CEACAM1‐bearing cells. This process requires the expression of CEACAM1 on the NK cells and on the tumor cells, which is consistent with the involvement of trans‐homophilic interactions between CEACAM1. Mechanistically, co‐engagement of NKG2D and CEACAM1 results in a biochemical association between these two surface receptors and the recruitment of Src homology phosphatase 1 by CEACAM1 that leads to dephosphorylation of the guanine nucleotide exchange factor Vav1 and blockade of downstream signaling that is associated with the initiation of cytolysis. Thus, CEACAM1 on activated NK cells functions as an inhibitory receptor for NKG2D‐mediated cytolysis, which has important implications for understanding the means by which CEACAM1 expression adversely affects tumor immunity.     

Submandibular gland morphogenesis: Stage‐specific expression of TGF‐α/EGF,IGF, TGF‐β, TNF,and IL‐6 signal transduction in normal embryonic mice and the phenotypic effects of TGF‐β2, TGF‐β3, and EGF‐r null mutations

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell‐cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF‐α/EGF/EGF‐R; IGF‐II/IGF‐IR/IGF‐IIR; TGF‐βs and cognate receptors; TNF, IL‐6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF‐β2, TGF‐β₃, EGF‐R). Among many observations, the following seem of particular importance: (1) TGF‐α and EGF‐R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF‐R1 signalling; (3) TGF‐β2 and TGF‐β3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF‐R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies. Anat Rec 256:252–268, 1999. © 1999 Wiley‐Liss, Inc.     

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.     

Attenuated TGF‐β1 responsiveness of dendritic cells and their precursors in atopic dermatitis

The responsiveness of DCs and their precursors to transforming growth factor beta1 (TGF‐β1) affects the nature of differentiating DC subsets, which are essential for the severity of atopic dermatitis (AD). To evaluate TGF‐β signaling in monocytes and monocyte‐derived DCs of AD patients compared with that of controls, in vitro generated Langerhans cell (LC) like DCs, expression of TGF‐β receptors, phospho‐Smad2/3 and Smad7 were evaluated. Furthermore, TNF‐α expression and synergistic effects of TNF‐α upon TGF‐β signaling and DC generation were evaluated. We found LC‐like DC differentiation of monocytes from AD patients in response to TGF‐β1 was remarkably reduced and TGF‐β1 receptor expression was significantly lower compared with that of healthy controls. Attenuated TGF‐β1 responsiveness mirrored by lower phospho‐Smad2/3 expression after TGF‐β1 stimulation and higher expression of inhibitory Smad7 was observed in monocytes from AD patients. During DC generation, mRNA expression of Smad7 was relatively higher in LC‐like DCs of AD patients. Lower TNF‐α expression of monocytes from AD patients might further contribute to attenuated TGF‐β signaling in the disease since TNF‐α had synergistic effects on TGF‐β1 signaling and LC generation through mediating the degradation of Smad7. Our results demonstrate alleviated TGF‐β1 signaling together with the amount of soluble co‐factors might direct the nature of differentiating DCs.     

TGF‐β‐producing regulatory B cells induce regulatory T cells and promote transplantation tolerance

Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti‐T cell immunoglobulin domain and mucin domain‐1 and anti‐CD45RB antibody treatment results in tolerance to full MHC‐mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen‐specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4⁺CD25^? T cells than with naive B cells. We also show that Breg cells express the TGF‐β associated latency‐associated peptide and that Breg‐cell mediated graft prolongation post‐adoptive transfer is abrogated by neutralization of TGF‐β activity. Breg cells, like Treg cells, demonstrate preferential expression of both C‐C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody‐induced transplantation tolerance, Breg cells promote graft survival by promoting Treg‐cell development, possibly via TGF‐β production.     

TGF‐β regulation of encephalitogenic and regulatory T cells in multiple sclerosis

Transforming growth factor beta (TGF‐β) is a pleiotropic cytokine that has been shown to influence the differentiation and function of T cells. The role that TGF‐β plays in immune‐mediated disease, such as multiple sclerosis (MS), has become a major area of investigation since CD4⁺ T cells appear to be a major mediator of autoimmunity. This review provides an analysis of the literature on the role that TGF‐β plays in the generation and regulation of encephalitogenic and regulatory T cells (Treg) in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as well as in T cells of MS patients. Since TGF‐β plays a major role in the development and function of both CD4⁺ effector and Treg, which are defective in MS patients, recent studies have found potential mechanisms to explain the basis for these T‐cell defects to establish a foundation for potentially modulating TGF‐β signaling to restore normal T‐cell function in MS patients.     

TGF‐β indirectly favors the development of human Th17 cells by inhibiting Th1 cells

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti‐proliferative effect of TGF‐β than Th1 and Th2 clones or circulating Th1‐oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl‐2 expression and reduced apoptosis in the presence of TGF‐β, in comparison with Th1 cells. Umbilical cord blood naïve CD161⁺CD4⁺ T cells, which contain the precursors of human Th17 cells, differentiated into IL‐17A‐producing cells only in response to IL‐1β plus IL‐23, even in serum‐free cultures. TGF‐β had no effect on constitutive RORγt expression by umbilical cord blood CD161⁺ T cells but it increased the relative proportions of CD161⁺ T cells differentiating into Th17 cells in response to IL‐1β plus IL‐23, whereas under the same conditions it inhibited both T‐bet expression and Th1 development. These data suggest that TGF‐β is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.     

IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

Exosomes with membrane‐associated TGF‐β1 from gene‐modified dendritic cells inhibit murine EAE independently of MHC restriction

We have previously demonstrated that exosomes from dendritic cells (DCs) secreting TGF‐β1 (sTGF‐β1‐EXOs) delay the development of murine inflammatory bowel disease (IBD). In this study, we isolated exosomes from DCs expressing membrane‐associated TGF‐β1 (mTGF‐β1‐EXOs) and found mTGF‐β1‐EXOs had more potent immunosuppressive activity than sTGF‐β1‐EXOs in vitro. Treatment of mice with mTGF‐β1‐EXOs inhibited the development and progression of myelin oligodendrocyte glycoprotein (MOG) peptide‐induced EAE even after disease onset. Treatment of mice with mTGF‐β1‐EXOs also impaired Ag‐specific Th1 and IL‐17 responses, but promoted IL‐10 responses ex vivo. Treatment with mTGF‐β1‐EXOs decreased the frequency of Th17 cells in EAE mice, which might be associated with the down‐regulation of the p38, ERK, Stat3, and NF‐κB activation and IL‐6 expression in DCs. Treatment with mTGF‐β1‐EXOs maintained the regulatory capacity of Treg cells, and adoptive transfer of CD4⁺Foxp3⁺ Treg cells from mTGF‐β1‐EXO‐treated EAE mice dramatically prevented the development of EAE in the recipients. Moreover, treatment with mTGF‐β1‐EXOs from C57BL/6 mice effectively prevented and inhibited proteolipid protein (PLP) peptide‐induced EAE in BALB/c mice. These results indicate that mTGF‐β1‐EXOs possess powerful immunosuppressive ability and can effectively inhibit the development and progression of EAE in different strains of mice.     

TGF‐β downregulates KLRG1 expression in mouse and human CD8+ T cells

The inhibitory receptor killer cell lectin‐like receptor G1 (KLRG1) and the integrin α_E (CD103) are expressed by CD8⁺ T cells and both are specific for E‐cadherin. However, KLRG1 ligation by E‐cadherin inhibits effector T‐cell function, whereas binding of CD103 to E‐cadherin enhances cell–cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8⁺ T cells from untreated and virus‐infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8⁺ T cells‐infiltrating hepatocellular carcinomas. As TGF‐β is known to induce CD103 expression in CD8⁺ T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF‐β signaling in mouse as well as in human CD8⁺ T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8⁺ T‐cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8⁺ T cells if lymphocytes from tissues expressing high levels of TGF‐β are analyzed.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

Membrane protein GARP is a receptor for latent TGF‐β on the surface of activated human Treg

Human Treg and Th clones secrete the latent form of TGF‐β, in which the mature TGF‐β protein is bound to the latency‐associated peptide (LAP), and is thereby prevented from binding to the TGF‐β receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF‐β and bear LAP on their surface. Here, we show that latent TGF‐β, i.e. both LAP and mature TGF‐β, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF‐β mediated by binding to GARP may be necessary for the ability of Treg to activate TGF‐β upon TCR stimulation. However, it is not sufficient as lentiviral‐mediated expression of GARP in human Th cells induces binding of latent TGF‐β to the cell surface, but does not result in the production of active TGF‐β upon stimulation of these Th cells.