Association between IL-6 and CD40 signaling. IL-6 induces phosphorylation of CD40 receptors.

CD40 mAb at subsaturating doses inhibit the growth of transformants of the M12 murine cell line expressing intact full length CD40 molecules (M12/CD40+ cells) but do not inhibit the growth of two M12 transformants expressing either a mutant CD40 cDNA missing most of the cytoplasmic tail (CD40/tailless) or a mutant cDNA with a substitution at residue 234 (CD40/234A, Ala for Thr). Using these transformants, we tested a panel of cytokines for the ability to mimic CD40 mAb. rIL-6 behaved like CD40 mAb and inhibited the growth of M12/CD40+ cells but not of CD40/tailless or CD40/234A mutants. The effect of IL-6 on M12/CD40+ cells not only required intact CD40 including threonine 234 but also was specific because IL-6 mAb blocked the inhibitory activity. The M12/CD40+ cells responsive to IL-6 expressed greater than 300,000 CD40 molecules/cells but, like M12/CD40-controls, expressed only small numbers (less than 50/cell) of high affinity IL-6R, indicating that CD40 is not a receptor for IL-6. Nevertheless, IL-6 utilizes intact CD40 efficiently when it signals these cells: treatment of M12/CD40+ cells with IL-6 induced increased phosphorylation of CD40. Conversely, triggering CD40 on M12/CD40+ cells leads to IL-6 production. Similar effects were evident in human CD40+ B cells: IL-6 increased the phosphorylation of CD40 in the IL-6-responsive cell line, CESS, and CD40 mAb induced IL-6 production in activated human B cells. Thus, CD40 may function to receive and regulate IL-6-dependent signals in B cells.     

CD40-40L signaling in vascular inflammation

Ligation of CD40 in circulating cells or in the vessel wall may promote mononuclear cell recruitment, participate in the weakening of the plaque, and contribute to thrombosis. This process appears to be redox-sensitive, but the precise signaling mechanism by which the interaction between CD40L and its receptor CD40 mediates inflammatory secretion is unclear. Our previous studies have shown that the CD40-CD40L interaction modulates release of reactive oxygen species (ROS) and the current findings demonstrate that in endothelial cells CD40L dose dependently induces intracellular CD40L and MCP1 release in a redox sensitive manner. Pharmacological inhibition of phosphatidylinositol 3-kinase and p38 MAPK as well as adenovirus-mediated inactivation of Akt and p38 MAPK inhibited CD40L effects on endothelial cells. Akt, in particular, appeared to mediate CD40L-induced CD40L synthesis and MCP1 release by endothelial cells in a redox sensitive manner via NFkappaB activation. In addition, using confocal microscopy, exogenous addition of recombinant CD40L or adenoviral mediated CD40L overexpression was found to stimulate nuclear translocation of NFkappaB, which was further augmented by Akt overexpression and inhibited by Akt inactivation. These data support a mechanism whereby redox-sensitive CD40-CD40L interactions induce activation of Akt and p38 MAPK, leading to stimulation of NFkappaB and enhanced synthesis of CD40L and MCP1. Increased CD40L and MCP1 may contribute to the adherence of CD40-positive cells, such as platelets and monocytes, to the vessel wall modulating atherothrombosis.     

IL-10-independent STAT3 activation by Toxoplasma gondii mediates suppression of IL-12 and TNF-alpha in host macrophages

Infection of mouse macrophages by Toxoplasma gondii renders the cells resistant to proinflammatory effects of LPS triggering. In this study, we show that cell invasion is accompanied by rapid and sustained activation of host STAT3. Activation of STAT3 did not occur with soluble T. gondii extracts or heat-killed tachyzoites, demonstrating a requirement for live parasites. Parasite-induced STAT3 phosphorylation and suppression of LPS-triggered TNF-alpha and IL-12 was intact in IL-10-deficient macrophages, ruling out a role for this anti-inflammatory cytokine in the suppressive effects of T. gondii. Most importantly, Toxoplasma could not effectively suppress LPS-triggered TNF-alpha and IL-12 synthesis in STAT3-deficient macrophages. These results demonstrate that T. gondii exploits host STAT3 to prevent LPS-triggered IL-12 and TNF-alpha production, revealing for the first time a molecular mechanism underlying the parasite's suppressive effect on macrophage proinflammatory cytokine production.     

OX40-mediated differentiation to effector function requires IL-2 receptor signaling but not CD28, CD40, IL-12Rbeta2, or T-bet

Ag-specific CD4 T cells transferred into unirradiated Ag-bearing recipients proliferate, but survival and accumulation of proliferating cells is not extensive and the donor cells do not acquire effector functions. We previously showed that a single costimulatory signal delivered by an agonist Ab to OX40 (CD134) promotes accumulation of proliferating cells and promotes differentiation to effector CD4 T cells capable of secreting IFN-gamma. In this study, we determined whether OX40 costimulation requires supporting costimulatory or differentiation signals to drive acquisition of effector T cell function. We report that OX40 engagement drives effector T cell differentiation in the absence of CD28 and CD40 signals. Two important regulators of Th1 differentiation, IL-12R and T-bet, also are not required for acquisition of effector function in CD4 T cells responsive to OX40 stimulation. Finally, we show that CD25-deficient CD4 T cells produce little IFN-gamma in the presence of OX40 costimulation compared with wild type, suggesting that IL-2R signaling is required for efficient OX40-mediated differentiation to IFN-gamma secretion.     

Modulation of CLA, IL-12R, CD40L, and IL-2Ralpha expression and inhibition of IL-12- and IL-23-induced cytokine secretion by CNTO 1275

Cytokines interleukin (IL)-12 and IL-23 are implicated in the pathogenesis of psoriasis. IL-12 causes differentiation of CD4+ T cells to interferon-gamma (IFN-gamma)-producing T helper 1 (Th1) cells, while IL-23 induces differentiation to IL-17-producing pathogenic Th17 cells. The effects of the monoclonal antibody to IL-12/23 p40 subunit (CNTO 1275) on IL-12 receptor (IL-12R) expression, markers associated with skin homing, activation, and cytokine secretion were investigated in vitro using human peripheral blood mononuclear cells (PBMCs) from healthy donors. PBMCs were activated in the presence or absence of recombinant human (rh) IL-12 or rhIL-23, with or without CNTO 1275. CNTO 1275 inhibited upregulation of CLA, IL-12R, IL-2Ralpha and CD40L expression and also inhibited IL-12- and IL-23-induced IFN-gamma, IL-17A, tumor necrosis factor (TNF)-alpha, IL-2, and IL-10 secretion. Thus, the therapeutic effect of CNTO 1275 may be attributed to the IL-12/23 neutralization, resulting in decreased expression of skin homing and activation markers, and IL-12- and IL-23-induced cytokine secretion.     

Production of IL-10 and IL-12 in CD40 and interleukin 4-activated mononuclear cells from patients with Graves' disease

We investigated the effect of T cell-dependent B cell activation on the production of IL-10 and IL-12 by peripheral blood mononuclear cells (PBMCs) obtained from patients with Graves' disease vs Hashimoto's thyroiditis, type 1 diabetes or normal controls. Incubation of PBMCs, from each of the subject groups, with a combination of anti-CD40 monoclonal antibodies and interleukin 4 (IL-4)-activated B cells, as shown by an increased level of soluble CD23. There was also a notable increase in the number of CD23(+)cells in PBMCs from patients with Graves' disease as compared to the other subject groups. This combination of B cell stimulants increased production of IL-10 in PBMCs obtained from patients with Graves' disease relative to those patients with Hashimoto's thyroiditis, type 1 diabetes, or the control subjects. The production of IL-12 showed wide variation that depended on the basal IL-12 level. In subjects with a low basal IL-12 level there was a positive correlation between the production of IL-12 and that of IL-10 from PBMCs stimulated with anti-CD40 antibodies plus IL-4. On the contrary, in the patients with a high basal IL-12 level, no change or a decrease of IL-12 production was observed after the stimulation. Thus, T cell-dependent B cell activation via a CD40 pathway triggers the overproduction of IL-10 and overcome the effect of IL-12 to shift the Th(1)/Th(2)balance to Th(2)dominance in patients with Graves' disease but not in Hashimoto's thyroiditis or type 1 diabetes.     

Evidence for interleukin-1-independent stimulation of interleukin-12 and down-regulation by interleukin-10 in Helicobacter pylori-infected murine dendritic cells deficient in the interleukin-1 receptor

Helicobacter pylori infection is characterized by infiltration of cells of the immune system, including dendritic cells, into the gastric mucosa. During chronic inflammation with Helicobacter pylori infection, a variety of cytokines are secreted into the mucosa, including interleukin-1beta (IL-1beta). The role of IL-1 in H. pylori infection was investigated using bone-marrow-derived dendritic cells from wild-type and IL-1 receptor-deficient (IL-1R-/-) mice. Dendritic cells were incubated with H. pylori at a multiplicity of infection of 10 and 100, and cytokine production evaluated. Helicobacter pylori SS1, H. pylori SD4, and an isogenic cagE mutant of SD4 stimulated IL-12, IL-6, IL-1beta, IL-10, and tumor necrosis factor-alpha at comparable levels in dendritic cells from both wild-type and IL-1R-/- mice. IL-10 production required the higher inoculum, while IL-12 was decreased at this bacterial load. Pretreatment of dendritic cells with an antibody to IL-10 resulted in an increased production of IL-12, confirming the down-regulation of IL-12 by IL-10. cagE was required for maximum stimulation of IL-12 by H. pylori. We speculate that the down-regulation of IL-12 by IL-10 at the higher multiplicity of infection represents the modulation of the host inflammatory response in vivo by H. pylori when the bacterial load is high, allowing for persistent colonization of the gastric mucosa.     

IL-12p70 production by Leishmania major-harboring human dendritic cells is a CD40/CD40 ligand-dependent process

Leishmaniasis, a vector-borne parasitic disease, is transmitted during a sandfly blood meal as the parasite is delivered into the dermis. The parasite displays a unique immune evasion mechanism: prevention of IL-12 production within its host cell, the macrophage (i.e., where it differentiates and multiplies). Given the close proximity of skin dendritic cells (DC) to the site of parasite delivery, their critical role in initiating immune responses and the self-healing nature of Leishmania major (Lm) infection, we examined the interaction between myeloid-derived human DC and Lm metacyclic promastigotes (infectious-stage parasites) to model the early "natural" events of infection. We found that DC can take up Lm and, after this internalization, undergo changes in surface phenotype suggesting "maturation". Despite the intracellular location of the parasite and resultant up-regulation of costimulatory and class II molecules, there was no detectable cytokine release by these Lm-harboring DC. However, using intracellular staining and flow cytometry to analyze cytokine production at the single-cell level, we found that Lm-harboring DC, but not monocytes, produce large amounts of IL-12p70 in a CD40 ligand (CD40L)-dependent manner. Finally, DC generated from mononuclear cells from patients with cutaneous leishmaniasis (Lm), once loaded with live metacyclic promastigotes, were found to reactivate autologous primed T lymphocytes and induce a CD40L-dependent IFN-gamma response. Our results link the required CD40/CD40L interactions for healing with DC-derived IL-12p70 production and provide a mechanism to explain the genesis of a protective T cell-mediated response in the face of local immune evasion within the macrophage at the site of Leishmania delivery.     

Novel regulatory mechanisms of CD40-induced prostanoid synthesis by IL-4 and IL-10 in human monocytes

Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE(2), a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE(2) via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE(2) production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-kappaB. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-kappaB in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-kappaB pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.     

Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40hiCD5+ regulatory B cells in vitro and in vivo

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40^hiCD5⁺ B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40^hi is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10^−/−CD5⁺CD19⁺ B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40^hiCD5⁺ Breg cells in mice. However, the population of CD40^hiCD5⁺ B cells was minimal in IL-10^−/− mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40^hiCD5⁺ B cells and the autocrine effect of IL-10 is critical to the formation of CD40^hiCD5⁺ Breg cells. [BMB Reports 2015; 48(1): 54-59]     

Sustained linked stimulation via CD3 and CD4 is required for the IL-4-independent development of IL-4 synthesizing CD4+ T cells

Previous work has shown that CD4 engagement can promote the development of interleukin-4-producing cells from naive CD4+ T cells activated with anti-CD3 antibody and interleukin-2 in the absence of other exogenous signals, including interleukin-4 itself. When CD44low CD4+ T cells were activated with immobilized anti-CD3 antibody and interleukin-2, they proliferated and produced interferon-gamma but not interleukin-4. Co-immobilization of antibodies to CD3 and CD4 enhanced cell recoveries and induced interleukin-4 as well as interferon-gamma synthesis. Here we show that these effects of CD4 ligation were not observed when anti-CD4 antibody was replaced with another CD4 ligand, interleukin-16, or when the anti-CD3 and anti-CD4 antibodies were spatially separated by immobilization on different beads. Removal of the anti-CD4 antibodies within the first three days of stimulation also prevented the development of detectable interleukin-4-producing cells. The data suggest that interleukin-4-independent priming of interleukin-4-producing cells in this system requires sustained stimulation via both the T cell receptor and CD4 with close physical association between the ligands for these two receptors.     

An IL-12-independent role for CD40-CD154 in mediating effector responses: studies in cell-mediated glomerulonephritis and dermal delayed-type hypersensitivity

Crescentic glomerulonephritis (GN) results from IL-12-driven Th1-directed cell-mediated responses (akin to delayed-type hypersensitivity (DTH)) directed against glomerular Ags. CD40-CD154 interactions are critical for IL-12 production and Th1 polarization of immune responses. Crescentic anti-glomerular basement membrane GN was induced in C57BL/6 (wild-type (WT)) mice (sensitized to sheep globulin) by planting this Ag (as sheep anti-mouse glomerular basement membrane globulin) in their glomeruli. Crescentic GN did not develop in CD40(-/-) mice due to significantly reduced nephritogenic Th1 responses. IL-12 was administered to CD40(-/-) mice with GN to dissect interactions between IL-12 and CD40 in inducing nephritogenic immunity and injury. Administration of IL-12 to CD40(-/-) mice restored Th cell IFN-gamma production, and up-regulated intrarenal chemokines and glomerular T cell and macrophage accumulation compared with WT control mice. Despite this, renal macrophages were not activated and renal injury and dermal DTH were not restored. Thus, CD40-directed IL-12 drives Th1 generation and effector cell recruitment but CD40 is required for activation. To test this hypothesis, activated OT-II OVA-specific CD4(+) cells and OVA(323-339)-loaded nonresponsive APCs were transferred into footpads of WT, CD40(-/-), and macrophage-depleted WT mice. WT mice developed significant DTH compared with CD40(-/-) and macrophage-depleted WT mice. This study demonstrated that CD40-induced IL-12 is required for generation of systemic Th1 immunity to nephritogenic Ags, and that IL-12 enhances Th1 effector cell recruitment to peripheral sites of Ag presentation via generation of local chemokines. Effector cell activation, renal DTH-like injury, and dermal DTH require direct Th1 CD154/macrophage CD40 interactions.     

Following direct CD40 activation, human primary microglial cells produce IL-12 p40 but not bioactive IL-12 p70

There is accumulating evidence that interleukin 12 (IL-12) is involved in the pathogenesis of multiple sclerosis. In the periphery, this cytokine is produced by antigen-presenting cells (APCs) following interaction with activated T cells. CD40 ligation plays a crucial role in this production. Microglial cells are thought to play a major role in antigen presentation in the central nervous system. In this work, we examined IL-12 production by human primary microglial cells after CD40 ligation. These cells expressed CD40 and MHC class II following interferon-gamma activation. IL-12 p40 mRNA and protein, but not bioactive IL-12 p70, were detected in response to direct CD40 activation. Microglial cells co-cultured with activated allogenic CD4+ T lymphocytes also produced IL-12 p40 but not IL-12 p70. This IL-12 p40 production was inhibited by anti-CD40 ligand. Altogether, these results suggest that CD40-CD40-ligand interaction provides a signal that triggers IL-12 p40 expression. However, other interaction(s) may be required during antigen presentation for bioactive heterodimeric IL-12 p70 to be produced by microglial cells.     

Synergy between CD40 ligation and IL-4 on fibroblast proliferation involves IL-4 receptor signaling.

Fibrosis can be an undesired consequence of activated cellular immune responses. The purpose of this work was to determine whether CD40 ligation and the pro-fibrotic cytokine IL-4 interact in regulating fibroblast proliferation and collagen production, and, if so, the mechanisms used. This study found that the combination of IL-4 and ligation of CD40 on the fibroblast cell surface had synergistic effects in stimulating fibroblast proliferation. In contrast, CD40 ligation negated the inhibitory effects of IFN-gamma on fibroblast proliferation. Western blotting analyses of fibroblast crude lysates revealed that a potential mechanism of the synergy between CD40 ligation and IL-4 was the phosphorylation of proteins at 130 kDa and, to a lesser degree, at 95, 85, and 75 kDa. Immunoprecipitation-Western blotting experiments showed that phosphorylation levels of IL-4Ralpha, Janus kinase 1, insulin receptor substrate 1, and insulin receptor substrate 2, factors with molecular mass close to the observed 130 kDa major phosphorylation band, increased in response to the combined CD40 ligation and IL-4 action. In contrast, there was no evidence that synergy was mediated by an increased expression of IL-4Ralpha chain, CD40, or the autocrine profibrotic cytokines IL-6 and TGF-beta. These findings suggest that CD40-CD40 ligand contacts between fibroblasts and cells secreting IL-4 may promote the profibrotic effects of IL-4 by affecting signal transduction and reducing the anti-fibrotic effects of IFN-gamma.     

Context-dependent IL-6 potentiation of interferon- gamma-induced IL-12 secretion and CD40 expression in murine microglia

Interleukin-6 (IL-6) is produced by neurons, astrocytes, and microglia, and elevated levels of IL-6 within the CNS have been documented in multiple neurological disorders including Alzheimer's disease, stroke, epilepsy, attention deficit disorder, cerebral palsy, and multiple sclerosis. Here, we sought to understand how IL-6 regulates microglial signal transduction and their immune properties. Using highly enriched cultures of neonatal murine microglia we show that IL-6 alone has direct effects on microglia as it activates STAT3 and extracellular signal-regulated kinase pathways in a time- and dose-dependent fashion and it enhances interferon-gamma (IFNγ)-stimulated IL-12 secretion. However, other immune properties were only weakly modulated by IL-6 when administered without the soluble IL-6 receptor (sIL-6R). For instance, IFNγ-induced expression of the co-stimulatory molecule, CD40 was dependent on sIL-6R administration. IL-6 with or without sIL-6R did not affect major histocompatability complex class II expression. In granulocyte–macrophage colony-stimulating factor (GMCSF)-induced dendritic cell-like microglia, IL-6/sIL-6R and IFNγ stimulated an even greater increase in CD40 expression compared with primary microglia. Altogether, our results demonstrate that microglial responses to IL-6 are not simple in that the effects of IL-6 are context-dependent. In particular, the presence or absence of sIL-6R, IFNγ or GMCSF will alter the type and amplitude of their response.     

双歧杆菌对过敏性哮喘儿童树突状细胞分泌IL-10、IL-12等细胞因子的影响

目的探讨双歧杆菌对过敏性哮喘儿童外周血单核细胞（PBMC）来源的树突状细胞（DC）分泌IL-1β、IL-6、IL-10、IL-12、IL-23和IFN-γ的影响。方法从15例过敏性哮喘儿童和15例非哮喘儿童的外周血单个核细胞诱导生成未成熟DC,加入双歧杆菌后继续培养DC2d,用ELISA方法检测培养上清中IL-1β、IL-6、IL-10、IL-12、IL-23和IFN-γ的水平。结果双歧杆菌能明显刺激哮喘儿童DC分泌IL-12、IFN-γ,IL-1β及IL-6和非哮喘儿童DC分泌IL-12、IL-10、IL-1β及IL-23水平增高。结论双歧杆菌能够刺激过敏性哮喘儿童DC分泌IL-12和IFN-γ,可能改变Th2优势分化,纠正Th1／Th2失衡。同时双歧杆菌还能刺激哮喘儿童DC分泌IL-1p及IL-6增高,达到促进,Th17细胞分化的作用。     

Signaling through CD40 ligand decreases CD80 expression on murine Langerhans cells and enhances IL-12 p40 production

We previously showed that murine Langerhans cells (LC) express CD40 ligand (CD40L). In this study, we further investigated the function of CD40L on LC using agonistic antibodies and CD40L knockout (KO) mice. Signaling through CD40L decreased CD80 expression on LC 48 h after stimulation and the decrease was more remarkable in the presence of interferon-gamma (IFN-gamma). Signaling through CD40 enhanced the production of IL-12 p40 from LC, and simultaneous signaling through CD40L slightly augmented this effect. Addition of IFN-gamma further enhanced IL-12 p40 production. LC from CD40L KO mice expressed similar levels of surface molecules such as CD40, CD80, CD86, and MHC class II, compared with those from wild-type mice. However, they produced less amount of IL-12 p40 during 48 h after purification. These results suggest that signaling through CD40L on LC is important in regulating IL-12 production, which is critical for Th1 type immune responses.     

Reprogramming of IL-10 activity and signaling by IFN-gamma

One important mechanism of cross-regulation by opposing cytokines is inhibition of signal transduction, including inhibition of Janus kinase-STAT signaling by suppressors of cytokine signaling. We investigated whether IFN-gamma, a major activator of macrophages, inhibited the activity of IL-10, an important deactivator. Preactivation of macrophages with IFN-gamma inhibited two key anti-inflammatory functions of IL-10, the suppression of cytokine production and of MHC class II expression. Gene expression profiling showed that IFN-gamma broadly suppressed the ability of IL-10 to induce or repress gene expression. Although IFN-gamma induced expression of suppressor of cytokine signaling proteins, IL-10 signal transduction was not suppressed and IL-10 activation of Janus kinases and Stat3 was preserved. Instead, IFN-gamma switched the balance of IL-10 STAT activation from Stat3 to Stat1, with concomitant activation of inflammatory gene expression. IL-10 activation of Stat1 required the simultaneous presence of IFN-gamma. These results demonstrate that IFN-gamma operates a switch that rapidly regulates STAT activation by IL-10 and alters macrophage responses to IL-10. Dynamic regulation of the activation of different STATs by the same cytokine provides a mechanism by which cells can integrate and balance signals delivered by opposing cytokines, and extends our understanding of cross-regulation by opposing cytokines to include reprogramming of signaling and alteration of function.     

IL-12-independent LIGHT signaling enhances MHC class II disparate CD4+ T cell alloproliferation, IFN-gamma responses, and intestinal graft-versus-host disease

Inhibition of LIGHT (a cellular ligand for herpes virus entry mediator and lymphotoxin receptor)/herpes simplex virus entry mediator (HVEM) and LIGHT/lymphotoxin beta receptor (LT beta R) interactions decreases mortality in MHC class I and II disparate graft-vs-host disease (GVHD). The present studies assessed the effects of these interactions on the generation of CD4+ T cell alloresponses in MHC class II-disparate MLC and GVHD. An inhibitor protein of LIGHT and LT alpha beta2 (LT beta R-Ig) and an inhibitor protein of LIGHT (HVEM-Ig) caused similar decreases in alloresponses of control B6 or B6.129S1-IL12rb2(tm1Jm) (B6.IL12R-/-) spleen cells (SpC) in a MHC class II-disparate MLC. GVHD-induced wasting disease in MHC class II-disparate recipients of B6 CD4+ SpC who received either the LT beta R-Ig-encoding adenovirus (LT beta R-Ig Adv; 13.1 +/- 10.9%; n = 10; p = 0.0004) or the HVEM-Ig-encoding adenovirus (HVEM-Ig Adv; 16.4 +/- 9.9%; n = 13; p = 0.0008) was significantly reduced compared with that in recipients of a control adenovirus (30.4 +/- 8.8%; n = 13). Furthermore, gut GVHD histologic scores of recipients of B6 CD4+ SpC who received the LT beta R-Ig Adv (0.8 +/- 0.8; n = 5; p = 0.0007) or the HVEM-Ig Adv (1.4 +/- 0.5; n = 5; p = 0.008) were reduced compared with scores of recipients of a control adenovirus (2.5 +/- 0.75; n = 11). In the intestine, both LT beta R-Ig Adv and HVEM-Ig Adv decreased CD4+ T cells (0.35 +/- 0.4 x 10(6) (n = 6) vs 0.36 +/- 0.02 x 10(6) (n = 9); p = 0.03 and p = 0.007) compared with control adenovirus (0.86 +/- 0.42 x 10(6); n = 9). LIGHT is critical for optimal CD4+ T cell alloresponses in MHC class II-disparate MLC and GVHD.