Expression of the tumor necrosis factor locus is not necessary for the cytolytic activity of T lymphocytes

In order to test whether tumor necrosis factors alpha (TNF-alpha) or beta (TNF-beta, also known as lymphotoxin) are involved in the lysis of target cells by cytolytic T lymphocytes, we probed for the presence of the TNF mRNAs in several quiescent and activated CTL clones. No TNF mRNA could be found in constitutively cytolytic Lyt-2+ clones, and only two out of three clones tested accumulated TNF mRNA after stimulation with phorbol myristate acetate and ionomycin. Of two L3T4+ clones that can be induced to become cytolytic by a combination of antigen and IL-1, only one accumulated TNF-beta mRNA in the process. The PC60 rat X mouse T cell hybrid, which becomes cytolytic in response to a combination of IL-1 and IL-2, also failed to accumulate TNF mRNA after stimulation with these agents. Our results strongly suggest that TNF-alpha or -beta are not necessary agents of the cytolytic activity exhibited by antigen-specific T lymphocytes.     

TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells

TNF-related activation-induced cytokine (TRANCE) is a member of the TNF family recently identified in activated T cells. We report here that TRANCE mRNA is constitutively expressed in memory, but not naive, T cells and in single-positive thymocytes. Upon TCR/CD3 stimulation, TRANCE mRNA and surface protein expression are rapidly up-regulated in CD4+ and CD8+ T cells, which can be further enhanced on CD4+ T cells by CD28-mediated costimulation. However, TRANCE induction is significantly suppressed when cells are stimulated in the presence of IL-4, but is not modified in the presence of IFN-alpha, IFN-gamma, TGF-beta, TNF-alpha, or IL-2. High levels of TRANCE receptor expression are found on mature dendritic cells (DCs). In this study we show that activated T and B cells also express TRANCE receptor, but only at low levels. TRANCE, however, does not exert any significant effect on the proliferation, activation, or survival of those cells. In DCs, TRANCE induces the expression of proinflammatory cytokines (IL-6, IL-1) and T cell growth and differentiation factors (IL-12, IL-15) in addition to enhancing DC survival. Moreover, TRANCE cooperates with CD40 ligand or TNF-alpha to further increase the viability of DCs, suggesting that several TNF-related molecules on activated T cells may cooperatively regulate the function and survival of DCs to enhance T cell-mediated immune responses.     

Stimulating lymphotoxin beta receptor on the dendritic cells is critical for their homeostasis and expansion

The increased number of dendritic cells (DCs) inside lymphoid tissue may contribute to the enhanced priming of lymphocytes. The homeostasis of splenic DCs has mostly been attributed to their migration to the spleen via the chemokine microenvironment induced by lymphotoxin beta receptor (LTbetaR) signaling on splenic stromal cells. In this study we show that the lack of direct LTbetaR signaling on DCs is associated with the reduction of the number of DCs in the spleen independently of chemokine gradients. LTbetaR-/- mice have reduced DCs and reduced BrdU incorporation on DCs, and fewer DCs from LTbetaR-/- mice are detected in the spleen. Furthermore, increased expression of LIGHT (homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpes virus entry mediator on T cells) on T cells, a member of the TNF family (TNFSF14) and a ligand for LTbetaR, could dramatically increase the number of T cells and DCs, which leads to severe autoimmune diseases in a LTbetaR-dependent fashion. In vitro, LIGHT could directly promote accumulation of bone marrow-derived DCs. Furthermore, intratumor expression of LIGHT can dramatically expand DCs in situ, and inoculation of DCs into tumor tissues enhanced tumor immunity. Therefore, LTbetaR signaling on DCs is required for their homeostasis during physiology and pathological conditions, and increased LIGHT-LTbetaR interaction could stimulate DC expansion for T cell-mediated immunity.     

Differential role for p38 mitogen-activated protein kinase in regulating CD40-induced gene expression in dendritic cells and B cells

We investigated whether human monocyte-derived dendritic cells (DCs) differed from tonsillar B cells in the set of cell fate genes they express constitutively and in the way these genes are affected after CD40 ligation. In particular, Bcl-2, TNF receptor-associated factor-2 (TRAF2), and TRAF4 were clearly inducible via CD40 in B cells but not in DCs. DCs, unlike B cells, were induced to increase expression of IL-1beta, IL-1Ra, IL-8, IL-12 p40, RANTES, macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1 after CD40 ligation. We next tested whether CD40-induced signaling pathways were different in DCs vs B cells. In DCs, as in B cells, CD40 ligation activated p38 mitogen-activated protein kinase (MAPK), its downstream target, MAPKAPK-2, and the c-Jun N-terminal kinase. The p38 MAPK-specific inhibitor, SB203580, blocked CD40-induced MAPKAPK-2 activation, but did not affect activation of c-Jun N-terminal kinase. Furthermore, unlike in B cells, extracellular signal-regulated kinase-1 and -2 were activated after CD40 ligation in DCs. SB203580 strongly blocked CD40-induced IL-12 p40 production in DCs at both mRNA and protein levels, while having minimal effect on CD40-induced expression of the chemokine RANTES. In contrast, no detectable IL-12 p40 protein was secreted in CD40-stimulated B cells. Furthermore, CD40-induced mRNA expression of cellular inhibitor of apoptosis protein-2 was also dependent on the p38 MAPK pathway in DCs and differed compared with that in B cells. In conclusion, CD40 induces distinct programs in DCs and B cells, and the set of p38 MAPK-dependent genes in DCs (IL-12 p40 and cellular inhibitor of apoptosis protein-2) is different from that in B cells (IL-10 and IL-1beta).     

LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response

LIGHT is a recently identified member of the TNF superfamily and its receptors, herpesvirus entry mediator and lymphotoxin beta receptor, are found in T cells and stromal cells. In this study, we demonstrate that LIGHT is selectively expressed on immature dendritic cells (DCs) generated from human PBMCs. In contrast, LIGHT is not detectable in DCs either freshly isolated from PBMCs or rendered mature in vitro by LPS treatment. Blockade of LIGHT by its soluble receptors, lymphotoxin beta receptor-Ig or HVEM-Ig, inhibits the induction of DC-mediated primary allogeneic T cell response. Furthermore, engagement of LIGHT costimulates human T cell proliferation, amplifies the NF-kappaB signaling pathway, and preferentially induces the production of IFN-gamma, but not IL-4, in the presence of an antigenic signal. Our results suggest that LIGHT is a costimulatory molecule involved in DC-mediated cellular immune responses.     

IL-23 is increased in dendritic cells in multiple sclerosis and down-regulation of IL-23 by antisense oligos increases dendritic cell IL-10 production

IL-23 is a heterodimeric cytokine comprising a p19 subunit associated with the IL-12/23p40 subunit. Like IL-12, IL-23 is expressed predominantly by activated dendritic cells (DCs) and phagocytic cells, and both cytokines induce IFN-gamma secretion by T cells. The induction of experimental autoimmune encephalitis, the animal model of multiple sclerosis (MS), occurs in mice lacking IL-12, but not in mice with targeted disruption of IL-23 or both IL-12 and IL-23. Thus, IL-23 expression in DCs may play an important role in the pathogenesis of human autoimmune diseases such as MS. We quantified the expression of IL-23 in monocyte-derived DCs in MS patients and healthy donors and found that DCs from MS patients secrete elevated amounts of IL-23 and express increased levels of IL-23p19 mRNA. Consistent with this abnormality, we found increased IL-17 production by T cells from MS patients. We then transfected monocyte-derived DCs from healthy donors with antisense oligonucleotides specific for the IL-23p19 and IL-12p35 genes and found potent suppression of gene expression and blockade of bioactive IL-23 and IL-12 production without affecting cellular viability or DCs maturation. Inhibition of IL-23 and IL-12 was associated with increased IL-10 and decreased TNF-alpha production. Furthermore, transfected DCs were poor allostimulators in the MLR. Our results demonstrate that an abnormal Th1 bias in DCs from MS patients related to IL-23 exists, and that antisense oligonucleotides specific to IL-23 can be used for immune modulation by targeting DC gene expression.     

Negative regulation of NK cell activities by inhibitory receptor CD94/NKG2A leads to altered NK cell-induced modulation of dendritic cell functions in chronic hepatitis C virus infection

NK cells are potent activators of dendritic cells (DCs), but it remains obscure how third-party cells affect the ability of NK cells to modulate DC functions. We show here that NK cells derived from healthy donors (N-NK), when cocultured with human liver epithelial cells, induced maturation as well as activation of DCs, such as increased migratory capacity as well as T cell stimulatory activity. In contrast, NK cells from chronic hepatitis C virus-infected donors (HCV-NK) were not capable of activating DCs under the same conditions. In comparison to N-NK, HCV-NK showed higher expression of CD94/NKG2A and produced IL-10 and TGFbeta when cultured with hepatic cells, most of which express HLA-E, a ligand for CD94/NKG2A. Blockade of NKG2A restored the ability of HCV-NK to activate DCs, which appeared to result from the reduced NK cell production of IL-10 and TGFbeta. The blockade also endowed HCV-NK with an ability to drive DCs to generate Th1-polarized CD4+ T cells. These findings show that NK cell modulation of DCs is regulated by third-party cells through NK receptor and its ligand interaction. Aberrant expression of NK receptors may have an impact on the magnitude and direction of DC activation of T cells under pathological conditions, such as chronic viral infection.     

Transformation of T lymphocytes by the v-fos oncogene

Activation of T lymphocytes through the T cell antigen receptor has been shown to stimulate a rapid and transient accumulation of c-fos mRNA and protein. Transfection of a normal murine T lymphocyte clone with the FBJ-v-fos oncogene resulted in generation of a cell line that was morphologically transformed, had lost the requirement for IL-2 for proliferation, and was tumorigenic in adult syngeneic mice; however, the transformed cells retained the ability to proliferate in response to IL-2. The transformed cells did not show constitutive expression of IL-2 or c-fos mRNA, although the promoter regions of both IL-2 and c-fos genes contain AP-1 sites that are expected to be targets for binding of Fos/Jun complexes. In contrast, the transformed T cells showed increased constitutive expression of IL-2R alpha and c-myc mRNA; these genes may represent cellular targets for transformation by v-fos and physiologic activation by c-fos. We discuss the possibility that these transformed cells behave as cells partially activated through the TCR, and that transformation occurs through a mechanism independent of IL-2.     

Opposite effects of IL-10 on the ability of dendritic cells and macrophages to replicate primary CXCR4-dependent HIV-1 strains

We investigated the effect of IL-10 on replication of primary CXCR4-dependent (X4) HIV-1 strains by monocyte-derived dendritic cells (DCs) and macrophages (M Phis). M Phis efficiently replicated CXCR4-dependent HIV-1 (X4 HIV-1) strains NDK and VN44, whereas low levels of p24 were detected in supernatants of infected DCs. IL-10 significantly increased X4 HIV-1 replication by DCs but blocked viral production by M Phis as determined by p24 levels and semiquantitative nested PCR. IL-10 up-regulated CXCR4 mRNA and protein expression on DCs and M Phis, suggesting that IL-10 enhances virus entry in DCs but blocks an entry and/or postentry step in M Phis. The effect of IL-10 on the ability of DCs and M Phis to transmit virus to autologous CD4(+) T lymphocytes was investigated in coculture experiments. DCs exhibited a greater ability than did M Phis to transmit a vigorous infection to CD4(+) T cells despite their very low replication capacity. IL-10 had no effect on HIV-1 replication in DC:T cell cocultures but markedly decreased viral production in M Phi:T cell cocultures. These results demonstrate that IL-10 has opposite effects on the replication of primary X4 HIV-1 strains by DCs and M Phis. IL-10 increases X4-HIV-1 replication in DCs but does not alter their capacity to transmit virus to CD4(+) T lymphocytes. These findings suggest that increased levels of IL-10 observed in HIV-1-infected patients with disease progression may favor the replication of X4 HIV-1 strains in vivo.     

Plexin-B2 and Plexin-D1 in Dendritic Cells: Expression and IL-12/IL-23p40 Production

Plexins are a family of genes (A,B,C, and D) that are expressed in many organ systems. Plexins expressed in the immune system have been implicated in cell movement and cell-cell interaction during the course of an immune response. In this study, the expression pattern of Plexin-B2 and Plexin-D1 in dendritic cells (DCs), which are central in immune activation, was investigated. Plexin-B2 and Plexin-D1 are reciprocally expressed in myeloid and plasmacytoid DC populations. Plasmacytoid DCs have high Plexin-B2 but low Plexin-D1, while the opposite is true of myeloid DCs. Expression of Plexin-B2 and Plexin-D1 is modulated upon activation of DCs by TLR ligands, TNFα, and anti-CD40, again in a reciprocal fashion. Semaphorin3E, a ligand for Plexin-D1 and Plexin-B2, is expressed by T cells, and interestingly, is dramatically higher on Th2 cells and on DCs. The expression of Plexins and their ligands on DCs and T cells suggest functional relevance. To explore this, we utilized chimeric mice lacking Plxnb2 or Plxnd1. Absence of Plexin-B2 and Plexin-D1 on DCs did not affect the ability of these cells to upregulate costimulatory molecules or the ability of these cells to activate antigen specific T cells. Additionally, Plexin-B2 and Plexin-D1 were dispensable for chemokine-directed in-vitro migration of DCs towards key DC chemokines, CXCL12 and CCL19. However, the absence of either Plexin-B2 or Plexin-D1 on DCs leads to constitutive expression of IL-12/IL-23p40. This is the first report to show an association between Plexin-B2 and Plexin-D1 with the negative regulation of IL-12/IL-23p40 in DCs. This work also shows the presence of Plexin-B2 and Plexin-D1 on mouse DC subpopulations, and indicates that these two proteins play a role in IL-12/IL-23p40 production that is likely to impact the immune response.     

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.     

Expression of surface lymphotoxin and tumor necrosis factor on activated T, B, and natural killer cells.

The expression of membrane-associated forms of lymphotoxin (LT) and TNF were examined on cell lines of T, B, and myeloid origin, IL-2 dependent T cell clones, and peripheral blood lymphocytes. Inducible and constitutive patterns of surface LT expression were found on T cells as exemplified by the II-23.D7, a CD4+T cell hybridoma, and HUT-78, a T cell lymphoma. Phorbol ester induced surface LT expression on Ramos, an EBV transformed B cell line, but at a slower rate of appearance when compared to the II-23.D7. Secretion of LT was rapidly inducible by phorbol ester in II-23.D7 and also in HUT-78 but with slower kinetics; surface LT expression continued in both lines after secretion had ceased. Low levels of membrane TNF were transiently induced on II-23.D7 and HUT-78, but none was observed on Ramos. Peripheral blood monocytes and some myeloid tumor lines did not express surface LT. Several T cell clones expressed surface LT after Ag-specific stimulation, and expression persisted several days. Stimulation through the TCR or by IL-2 rapidly induced surface LT on resting peripheral T cells and CD56+ NK cells; pokeweed mitogen activation induced expression on CD20+ B cells. Consistent with previous results, immunoprecipitation with anti-LT mAb showed that LT was complexed with a distinct 33 kDa glycoprotein (p33) on cells that expressed surface LT, whereas secreted LT was not associated with p33. Surface and secreted modes of LT expression by activated T, B, and NK cells suggests that LT can be utilized as either a localized or diffusible mediator in immune responses.     

Tumor necrosis factor combines with IL-4 or IFN-gamma to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-1-dependent and -independent binding mechanisms.

The adhesion of lymphocytes to vascular endothelium is the first step in their passage from the blood into inflammatory tissues. By modulating endothelial cell (EC) adhesiveness for lymphocytes, cytokines may regulate lymphocyte accumulation and hence the nature and progression of inflammatory responses. IL-1, TNF, IFN-gamma, and IL-4 each increase EC adhesiveness for T cells when used alone in adhesion assays in vitro. As cytokines are more likely to act in combination at sites of inflammation in vivo, we have studied the stimulating effect of different combinations of cytokines on EC adhesiveness for T cells and polymorphonuclear leukocytes (PMN). Acting alone IL-1, TNF, IFN-gamma, and IL-4 each significantly enhanced EC adhesiveness for T cells (p less than 0.005), whereas only IL-1 (p less than 0.005) and TNF (p less than 0.005) but not IFN-gamma or IL-4 significantly enhanced adhesiveness for PMN. When EC were stimulated with optimal concentrations of TNF in combination with IL-4 or IFN-gamma, there was a significant further increase in adhesiveness for T cells (p less than 0.003), but not PMN, over that seen with TNF alone. The additive effect of TNF and IL-4 was more marked than that of TNF and IFN-gamma. Although approximately equal proportions of T cells and PMN bound to TNF-stimulated EC, nearly double the proportion of T cells compared with PMN bound EC preincubated with TNF and IL-4 together. A similar interaction with IL-4 or IFN-gamma was exhibited by lymphotoxin. mAb-inhibition studies indicated that the extra increase in binding caused by stimulating EC with TNF and IL-4 in combination was mediated by VCAM-1 whereas that caused by stimulating with TNF and IFN-gamma in combination was substantially mediated through leukocyte function-associated Ag-1- and VCAM-1-independent mechanisms. These observations suggest that whereas IL-1 and TNF alone are unselective in terms of leukocyte adhesion to EC, the combination of TNF (or LT) with IL-4 or IFN-gamma may be of key importance in determining the recruitment of a lymphocyte-predominant infiltrate in immune mediated inflammation, and in initiating the transition from acute to chronic inflammation.