It is becoming increasingly apparent that probiotics are important to the health of the host. The absence of probiotic bacteria in the gut can have adverse effects not only locally in the gut, but has also been shown to affect central HPA and monoaminergic activity, features that have been implicated in the aetiology of depression. To evaluate the potential antidepressant properties of probiotics, we tested rats chronically treated with Bifidobacteria infantis in the forced swim test, and also assessed the effects on immune, neuroendocrine and central monoaminergic activity. Sprague-Dawley rats were treated for 14 days with B. infantis. Probiotic administration in naive rats had no effect on swim behaviours on day 3 or day 14 following the commencement of treatment. However, there was a significant attenuation of IFN-gamma, TNF-alpha and IL-6 cytokines following mitogen stimulation (p<0.05) in probiotic-treated rats relative to controls. Furthermore, there was a marked increase in plasma concentrations of tryptophan (p<0.005) and kynurenic acid (p<0.05) in the bifidobacteria-treated rats when compared to controls. Bifidobacteria treatment also resulted in a reduced 5-HIAA concentration in the frontal cortex and a decrease in DOPAC in the amygdaloid cortex. The attenuation of pro-inflammatory immune responses, and the elevation of the serotonergic precursor, tryptophan by bifidobacteria treatment, provides encouraging evidence in support of the proposition that this probiotic may possess antidepressant properties. However, these findings are preliminary and further investigation into the precise mechanisms involved, is warranted.

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-gamma of inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 in mouse macrophages. Here, we show that these analogues are also extremely potent inducers of the phase 2 response [e.g., elevation of NAD(P)H-quinone oxidoreductase and heme oxygenase 1], which is a major protector of cells against oxidative and electrophile stress. Moreover, like previously identified phase 2 inducers, the TP analogues use the antioxidant response element-Nrf2-Keap1 signaling pathway. Thus, induction of the phase 2 response and suppression of the iNOS induction was abrogated in nrf2(-/-) and keap1(-/-) mouse embryonic fibroblasts. The high potency of TP analogues in inducing the phase 2 response and blocking inflammation depends on the presence of activated Michael reaction (enone) functions at critical positions in rings A and C. The most potent TP doubles NAD(P)H-quinone oxidoreductase in murine hepatoma cells at 0.28 nM and has an IC(50) for suppression of iNOS induction in primary mouse macrophages of 0.0035 nM. The direct interaction of this TP with thiol groups of the Keap1 sensor for inducers is demonstrated spectroscopically. The antiinflammatory and phase 2 inducer potencies of 18 TP are closely linearly correlated (r(2) = 0.91) over 6 orders of magnitude of concentration. Thus, in addition to blocking inflammation and promoting differentiation, these TP exhibit another very important protective property: the induction of the phase 2 response.

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is a disease controlled by the major histocompatibility complex (MHC) which results from T-cell-mediated destruction of pancreatic beta-cells. The incomplete concordance in identical twins and the presence of autoreactive T cells and autoantibodies in individuals who do not develop diabetes suggest that other abnormalities must occur in the immune system for disease to result. We therefore investigated a series of at-risk non-progressors and type 1 diabetic patients (including five identical twin/triplet sets discordant for disease). The diabetic siblings had lower frequencies of CD4-CD8- Valpha24JalphaQ+ T cells compared with their non-diabetic sibling. All 56 Valpha24JalphaQ+ clones isolated from the diabetic twins/triplets secreted only interferon (IFN)-gamma upon stimulation; in contrast, 76 of 79 clones from the at-risk non-progressors and normals secreted both interleukin (IL)-4 and IFN-gamma. Half of the at-risk non-progressors had high serum levels of IL-4 and IFN-gamma. These results support a model for IDDM in which Thl-cell-mediated tissue damage is initially regulated by Valpha24JalphaQ+ T cells producing both cytokines; the loss of their capacity to secrete IL-4 is correlated with IDDM.

OBJECTIVE

Glioblastoma (GBM) is the most common form of malignant glioma in adults. Although protected by both the blood-brain and blood-tumor barriers, GBMs are actively infiltrated by T cells. Previous work has shown that IDO, CTLA-4, and PD-L1 are dominant molecular participants in the suppression of GBM immunity. This includes IDO-mediated regulatory T-cell (Treg; CD4(+)CD25(+)FoxP3(+)) accumulation, the interaction of T-cell-expressed, CTLA-4, with dendritic cell-expressed, CD80, as well as the interaction of tumor- and/or macrophage-expressed, PD-L1, with T-cell-expressed, PD-1. The individual inhibition of each pathway has been shown to increase survival in the context of experimental GBM. However, the impact of simultaneously targeting all three pathways in brain tumors has been left unanswered.

RESULTS

In this report, we demonstrate that, when dually challenged, IDO-deficient tumors provide a selectively competitive survival advantage against IDO-competent tumors. Next, we provide novel observations regarding tryptophan catabolic enzyme expression, before showing that the therapeutic inhibition of IDO, CTLA-4, and PD-L1 in a mouse model of well-established glioma maximally decreases tumor-infiltrating Tregs, coincident with a significant increase in T-cell-mediated long-term survival. In fact, 100% of mice bearing intracranial tumors were long-term survivors following triple combination therapy. The expression and/or frequency of T cell expressed CD44, CTLA-4, PD-1, and IFN-γ depended on timing after immunotherapeutic administration.

CONCLUSIONS

Collectively, these data provide strong preclinical evidence that combinatorially targeting immunosuppression in malignant glioma is a strategy that has high potential value for future clinical trials in patients with GBM.

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.

Exposure to dextran sulfate sodium (DSS) induces acute colitis, which is normally resolved after DSS removal. To study chronicity, mice are typically subjected to three to five cycles of weekly DSS exposures, each followed by a 1- to 2-wk rest period. Here, we describe a novel and convenient way of inducing chronic, progressive colitis by a single exposure to DSS. C57BL/6 mice exposed to DSS for 5 days developed acute colitis that progressed to severe chronic inflammation. The plasma haptoglobin levels remained high during the chronic phase, showing that the inflammation was active. Surprisingly, the mice regained their original weight along with the progression of colitis, and the only apparent symptom was loose feces. Histopathological changes 4 wk after DSS removal were dense infiltrates of mononuclear cells, irregular epithelial structure, and persistent deposits of collagen. A progressive production of the cytokines IL-1beta, IL-12 p70, and IL-17 correlated with the extensive cellular infiltration, whereas high IFN-gamma production was mainly found late in the chronic phase. Similar to C57BL/6 mice, BALB/c mice exposed to 5 days of DSS developed acute colitis as previously described. The acute colitis was accompanied by elevated plasma levels of haptoglobin and increased colonic levels of IL-1alpha/beta, IL-6, IL-18, and granulocyte colony-stimulating factor. However, soon after DSS removal, BALB/c mice recovered and were symptom free within 2 wk and completely recovered 4 wk after DSS removal in terms of histopathology, haptoglobin levels, and local cytokine production. In summary, these data stress the effect of genetic background on the outcome of DSS provocation. We believe that the present protocol to induce chronic colitis in C57BL/6 mice offers a robust model for validating future therapies for treatment of inflammatory bowel disease.

Immune interferon gamma (IFN-γ) is essential for mammalian host defense against intracellular pathogens. IFN-γ induces nearly 2000 host genes, yet few have any assigned function. Here, we examined a complete mouse 65-kilodalton (kD) guanylate-binding protein (Gbp) gene family as part of a 43-member IFN-γ-inducible guanosine triphosphatase (GTPase) superfamily in mouse and human genomes. Family-wide loss-of-function analysis found that at least four Gbps--Gbp1, Gbp6, Gbp7, and Gbp10--conferred cell-autonomous immunity to listerial or mycobacterial infection within macrophages and gene-deficient animals. These Gbps solicited host defense proteins, including the phagocyte oxidase, antimicrobial peptides, and autophagy effectors, to kill intracellular bacteria. Thus, specific 65-kD Gbps coordinate a potent oxidative and vesicular trafficking program to protect the host from infection.

B-lymphocyte stimulator (BLyS) is a recently identified novel member of the tumor necrosis factor ligand superfamily shown to exist in a membrane-bound and soluble form. BLyS was found to be specifically expressed on cells of myeloid lineage and to selectively stimulate B-lymphocyte proliferation and immunoglobulin production. The expression of a cytokine involved in potentiation of humoral immune responses, such as BLyS, is expected to be strictly controlled. The goal of the present study was to examine regulation of BLyS levels in monocytic cells in response to cytokines and during their differentiation to macrophages and dendritic cells. The presence of BLyS on the cell surface and in the culture medium of both normal blood monocytes and on tumor cells of myelomonocytic origin was demonstrated. BLyS gene expression and levels of membrane-associated and soluble BLyS were found to be regulated by cytokines, in particular interferon (IFN)-gamma and to a lesser extent interleukin-10 (IL-10). The expression of BLyS on monocyte membranes was retained following differentiation into macrophages, but detection on the surface of monocyte-derived dendritic cells required stimulation with IFN-gamma. Both IFN-gamma and IL-10 enhanced the release of soluble BLyS that was active in B-cell proliferation assays. Cells transfected with BLyS complementary DNA mutated in a predicted cleavage site failed to release BLyS into the culture medium, thereby suggesting that soluble BLyS was derived from the membrane form. These results provide further support for an important role for BLyS expressed in myeloid cells in B-cell expansion and antibody responses.

Innate immune cells may regulate adaptive immunity by balancing different lineages of T cells and providing negative costimulation. In addition, CD11b(+)Gr-1(+) myeloid-derived suppressor cells have been described in tumor, parasite infection, and severe trauma models. In this study, we observe that splenic CD11b(+) cells markedly increase after experimental autoimmune encephalomyelitis (EAE) immunization, and they suppress T cell proliferation in vitro. Although >80% of CD11b(+) cells express varying levels of Gr-1, only a small population of CD11b(+)Ly-6C(high) inflammatory monocytes (IMC) can efficiently suppress T cell proliferation and induce T cell apoptosis through the production of NO. IFN-gamma produced by activated T cells is essential to induce IMC suppressive function. EAE immunization increases the frequencies of IMC in the bone marrow, spleen, and blood, but not in the lymph nodes. At the peak of EAE, IMC represent approximately 30% of inflammatory cells in the CNS. IMC express F4/80 and CD93 but not CD31, suggesting that they are immature monocytes. Furthermore, IMC have the plasticity to up-regulate NO synthase 2 or arginase 1 expression upon different cytokine treatments. These findings indicate that CD11b(+)Ly-6C(high) IMC induced during EAE priming are powerful suppressors of activated T cells. Further understanding of suppressive monocytes in autoimmune disease models may have important clinical implications for human autoimmune diseases.

Interferons (IFNs) are essential for host defense. Although the antiviral effects of the type 1 IFNs IFN-alpha and IFN-beta (IFN-alpha/beta) have been established, their immunoregulatory functions, especially their ability to regulate IFN-gamma production, are poorly understood. Here we show that IFN-alpha/beta activate STAT4 directly (STAT, signal transducers and activators of transcription) and that this is required for IFN-gamma production during viral infections of mice, in concert with T cell receptor-derived signals. In contrast, STAT1 appears to negatively regulate IFN-alpha/beta induction of IFN-gamma. Thus, type 1 IFNs, in addition to interleukin-12, provide pathways for innate regulation of adaptive immunity, and their immunoregulatory functions are controlled by modulating the activity of individual STATs.

Foxo transcription factors integrate extrinsic signals to regulate cell division, differentiation and survival, and specific functions of lymphoid and myeloid cells. Here, we showed the absence of Foxo1 severely curtailed the development of Foxp3(+) regulatory T (Treg) cells and those that developed were nonfunctional in vivo. The loss of function included diminished CTLA-4 receptor expression as the Ctla4 gene was a direct target of Foxo1. T cell-specific loss of Foxo1 resulted in exocrine pancreatitis, hind limb paralysis, multiorgan lymphocyte infiltration, anti-nuclear antibodies and expanded germinal centers. Foxo-mediated control over Treg cell specification was further revealed by the inability of TGF-β cytokine to suppress T-bet transcription factor in the absence of Foxo1, resulting in IFN-γ secretion. In addition, the absence of Foxo3 exacerbated the effects of the loss of Foxo1. Thus, Foxo transcription factors guide the contingencies of T cell differentiation and the specific functions of effector cell populations.

The intestinal mucosal CD8 T cell response to infection with Listeria monocytogenes was measured using MHC class I tetramers and was compared with the response in peripheral blood, secondary lymphoid tissue, and liver. To assess the vaccination potential of Listeria and to analyze responses in C57BL/6 mouse strains, a recombinant Listeria expressing OVA (rLM-ova) was generated. The response peaked at 9 days postinfection with a much larger fraction of the intestinal mucosa and liver CD8 T cell pool OVA specific, as compared with the spleen. However, these differences were not linked to bacterial titers in each site. The higher responses in lamina propria and liver resulted in a larger CD8 memory population in these tissues. Furthermore, the level of memory induced was dependent on infectious dose and inversely correlated with the magnitude of the recall response after oral challenge. Recall responses in the tissues were most robust in the lamina propria and liver, and reactivated Ag-specific T cells produced IFN-gamma. Infection of CD40- or MHC class II-deficient mice induced poor CD8 T cell responses in the intestinal mucosa, but only partially reduced responses in the spleen and liver. Overall, the results point to novel pathways of tissue-specific regulation of primary and memory antimicrobial CD8 T cell responses.

Dendritic cells (DCs) are known to induce the growth and function of natural killer (NK) cells. Here, we address the capacity of DCs to interact with NK cells in human lymphoid organs and identify the role of specific DC-derived cytokines. We demonstrate that DCs colocalize with NK cells in the T cell areas of lymph nodes. In culture, DCs from either blood or spleen primarily stimulate the CD56(bright)CD16- NK cell subset, which is enriched in secondary lymphoid tissues. Blocking of IL-12 abolished DC-induced IFN-gamma secretion by NK cells, whereas membrane-bound IL-15 on DCs was essential for NK cell proliferation and survival. Maturation by CD40 ligation promoted the highest IL-15 surface presentation on DCs and led to the strongest NK cell proliferation induced by DCs. These results identify secondary lymphoid organs as a potential DC/NK cell interaction site and identify the distinct roles for DC-derived IL-12 and IL-15 in NK cell activation.

An in vitro cell culture system was used to study the effect of interferon gamma (IFN-gamma) on Chlamydia trachomatis growth and differentiation. The effect of IFN-gamma on chlamydiae was dose-dependent. IFN-gamma at 2 ng/ml completely inhibited chlamydial growth and differentiation; however, persistent infection was established when chlamydiae were cultured with IFN-gamma at 0.2 ng/ml. Persistent infection was characterized by the development of noninfectious atypical chlamydial forms from which infectious progeny could be recovered only when IFN-gamma was removed from the culture system. Analysis of persistently infected cells by immunofluorescent microscopy and immunoblotting with specific antibodies revealed that the atypical chlamydial forms had near-normal levels of the 60-kDa heat shock protein, an immunopathologic antigen, and a paucity of the major outer membrane protein, a protective antigen. Furthermore, steady-state levels of other outer membrane constituents, such as the 60-kDa cysteine-rich outer membrane protein and lipopolysaccharide, were greatly reduced. If IFN-gamma causes similar events to occur in vivo, then persistently infected cells could augment the pathogenesis of the chronic inflammatory sequelae that follow chlamydial infection by serving as depots of antigen capable of stimulating a sustained inflammatory response.

Activation with lipopolysaccharide induces macrophages to produce the enzymes arginase and nitric oxide (NO) synthase. Both enzymes use as a substrate the amino acid L-arginine, which can be either hydrolyzed by arginase to urea and ornithine or oxidized by NO synthase to NO and citrulline. NO is important in the bactericidal and cytotoxic activities of macrophages. An equivalent functional role of arginase and its products is not known. We tested the induction of arginase in bone marrow-derived macrophages by endogenous mediators that are known to induce NO synthase, such as interferon-gamma (IFN-gamma), or suppress the induction of this enzyme, such as interleukin (IL)-4, IL-10, and prostaglandin E2 (PGE2). We find that PGE2 and the TH2 cytokines IL-4 and IL-10 are potent inducers of arginase. In contrast, the TH1 cytokine IFN-gamma does not induce arginase. Simultaneous application of both types of mediators leads to reduced induction of both arginase and NO synthase. Exposure of macrophage cultures to inducers of NO synthase exhausts their ability to respond subsequently to inducers of arginase. Conversely, exposure of the cells to inducers of arginase exhausts their ability to respond subsequently to inducers of NO synthase. The results are consistent with a competition of both enzymes for their substrate, L-arginine, with a reciprocal inhibition in the induction of both enzymes, or a combination of both phenomena. The enzymes NO synthase and arginase appear to define two alternate functional states of macrophages, induced by TH1 and TH2 cytokines, respectively.

Mendelian susceptibility to mycobacterial diseases confers predisposition to clinical disease caused by weakly virulent mycobacterial species in otherwise healthy individuals. Since 1996, disease-causing mutations have been found in five autosomal genes (IFNGR1, IFNGR2, STAT1, IL12B, IL12BR1) and one X-linked gene (NEMO). These genes display a high degree of allelic heterogeneity, defining at least 13 disorders. Although genetically different, these conditions are immunologically related, as all result in impaired IL-12/23-IFN-gamma-mediated immunity. These disorders were initially thought to be rare, but have now been diagnosed in over 220 patients from over 43 countries worldwide. We review here the molecular, cellular, and clinical features of patients with inborn errors of the IL-12/23-IFN-gamma circuit.

Although CD8 T cell-mediated immunosuppression has been a well-known phenomenon during the last three decades, the nature of primary CD8 T suppressor cells and the mechanism underlying their generation remain enigmatic. We demonstrated that naive CD8 T cells primed with allogeneic CD40 ligand-activated plasmacytoid dendritic cells (DC)2 differentiated into CD8 T cells that displayed poor secondary proliferative and cytolytic responses. By contrast, naive CD8 T cells primed with allogeneic CD40 ligand-activated monocyte-derived DCs (DC1) differentiated into CD8 T cells, which proliferated to secondary stimulation and killed allogeneic target cells. Unlike DC1-primed CD8 T cells that produced large amounts of interferon (IFN)-gamma upon restimulation, DC2-primed CD8 T cells produced significant amounts of interleukin (IL)-10, low IFN-gamma, and no IL-4, IL-5, nor transforming growth factor (TGF)-beta. The addition of anti-IL-10-neutralizing monoclonal antibodies during DC2 and CD8 T cell coculture, completely blocked the generation of IL-10-producing anergic CD8 T cells. IL-10-producing CD8 T cells strongly inhibit the allospecific proliferation of naive CD8 T cells to monocytes, and mature and immature DCs. This inhibition was mediated by IL-10, but not by TGF-beta. IL-10-producing CD8 T cells could inhibit the bystander proliferation of naive CD8 T cells, provided that they were restimulated nearby to produce IL-10. IL-10-producing CD8 T cells could not inhibit the proliferation of DC1-preactivated effector T cells. This study demonstrates that IL-10-producing CD8 T cells are regulatory T cells, which provides a cellular basis for the phenomenon of CD8 T cell-mediated immunosuppression and suggests a role for plasmacytoid DC2 in immunological tolerance.

Formation of the atherosclerotic intima must involve altered metabolism of the elastin-rich arterial extracellular matrix. Proteases potentially involved in these processes remain unclear. This study examined the expression of the potent elastases cathepsins S and K in human atheroma. Normal arteries contained little or no cathepsin K or S. In contrast, macrophages in atheroma contained abundant immunoreactive cathepsins K and S. Intimal smooth muscle cells (SMC), especially cells appearing to traverse the internal elastic laminae, also contained these enzymes. Extracts of atheromatous tissues had approximately twofold greater elastase-specific activity than extracts of uninvolved arteries, mostly due to cysteine proteases. Cultured human SMC displayed no immunoreactive cathepsins K and S and exhibited little or no elastolytic activity when incubated with insoluble elastin. SMC stimulated with the atheroma-associated cytokines IL-1beta or IFN-gamma secreted active cathepsin S and degraded substantial insoluble elastin (15-20 microg/10(6) cells/24 h). A selective inhibitor of cathepsin S blocked>> 80% of this elastolytic activity. The presence of cathepsins K and S at sites of vascular matrix remodeling and the ability of SMC and macrophages to use these enzymes to degrade elastin supports a role for elastolytic cathepsins in vessel wall remodeling and identifies novel therapeutic targets in regulating plaque stability.

The purpose of this study was to explore whether there is a linkage between the infiltration of CD8(+) T cells and the risk of death from esophageal cancer. Cases of 70 consecutive patients in whom esophageal squamous cell carcinomas ( n = 33) or adenocarcinomas (n = 37) were R0-resected between 1993 and 1999 were reviewed. The presence of activated CD8(+) T cells was evaluated by quantitative real-time PCR and immunohistochemistry and compared to clinical and pathological stages. The primary end point analyzed was overall survival, and a multivariate analysis was performed to distinguish any factors conferring an improved survivorship. Intratumoral (i.t.) CD8(+) T cells accumulating within the epithelial complexes were detected in 11 of all (16%) cases: in 9 of 25 (36%) patients with Union Internationale Contrele Cancer stage I or II versus 2 of 45 (4%) patients with Union Internationale Contrele Cancer stage III or IV (P = 0.001). Intratumoral CD8(+) T cell infiltrations showed proliferative activity and also IFN-gamma secretion. The presence of i.t. CD8(+) T cell infiltration more than peritumoral infiltration was associated with a good prognosis in both squamous cell and adenocarcinomas. Multivariate analysis showed that i.t. CD8(+) T cell infiltration was an independent prognostic factor (hazard ratio, 0.5; P = 0.0004) indicating favorable outcome. In conclusion, the presence of CD8(+) T cell infiltration in esophageal carcinomas is a favorable prognostic factor that should have diagnostic and therapeutic implications.

Stimulated human monocytes/macrophages are a source of mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and prostaglandin E2 (PGE2), which can modulate inflammatory and immune reactions. Therefore, the ability to control the production of such mediators by monocytes/macrophages may have therapeutic benefits, and it has been proposed that glucocorticoids may act in this way. Purified human monocytes, when stimulated in vitro with lipopolysaccharide (LPS) or with LPS and gamma interferon (IFN-gamma), produce TNF-alpha, IL-1, and PGE2. Cotreatment of stimulated cells with the purified human lymphokine, interleukin 4 (IL-4 greater than or equal to 0.1-0.5 unit/ml; 12-60 pM) dramatically blocked the increased levels of these three mediators; for TNF-alpha and IL-1, the inhibition was manifest at the level of mRNA. Thus, IL-4 can suppress some parameters of monocyte activation and, as for B cells, have opposite effects to IFN-gamma. The effects of IL-4 on human monocytes are similar to those obtained with the glucocorticoid dexamethasone (0.1 microM).

Experimental autoimmune encephalomyelitis (EAE) and other organ-specific autoimmune diseases are induced by autoantigen-specific Th1 cells. In contrast, transfer of autoantigen-reactive Th2 cells that produce IL-4 and IL-10 can prevent and/or reverse EAE. The relative roles of these two Th2 cytokines in the regulation of EAE has not been evaluated. Utilizing IL-4 and IL-10 knockout mice deficient for these cytokines and IL-10 and IL-4 transgenic mice overexpressing these cytokines, we demonstrate that IL-10-deficient mice (IL-10(-/-)) are more susceptible and develop a more severe EAE when compared with IL-4-deficient mice (IL-4(-/-)) or wild-type mice. T cells from IL-10(-/-) mice exhibit a stronger Ag-specific proliferation, produce more proinflammatory cytokines (IFN-gamma and TNF-alpha) when stimulated with an encephalitogenic peptide, and induce very severe EAE upon transfer into wild-type mice. In contrast, while IL-4 transgenic mice develop similar disease compared with their nontransgenic littermates, mice transgenic for IL-10 are completely resistant to the development of EAE. Taken together, our data suggest that IL-10 plays a more critical role in the regulation of EAE by regulating autopathogenic Th1 responses.

Historically, cancer-directed immune-based therapies have focused on eliciting a cytotoxic T cell (CTL) response, primarily due to the fact that CTL can directly kill tumors. In addition, many putative tumor antigens are intracellular proteins, and CTL respond to peptides presented in the context of MHC class I which are most often derived from intracellular proteins. Recently, increasing importance is being given to the stimulation of a CD4+ T helper cell (Th) response in cancer immunotherapy. Th cells are central to the development of an immune response by activating antigen-specific effector cells and recruiting cells of the innate immune system such as macrophages and mast cells. Two predominant Th cell subtypes exist, Th1 and Th2. Th1 cells, characterized by secretion of IFN-gamma and TNF-alpha, are primarily responsible for activating and regulating the development and persistence of CTL. In addition, Th1 cells activate antigen-presenting cells (APC) and induce limited production of the type of antibodies that can enhance the uptake of infected cells or tumor cells into APC. Th2 cells favor a predominantly humoral response. Particularly important during Th differentiation is the cytokine environment at the site of antigen deposition or in the local lymph node. Th1 commitment relies on the local production of IL-12, and Th2 development is promoted by IL-4 in the absence of IL-12. Specifically modulating the Th1 cell response against a tumor antigen may lead to effective immune-based therapies. Th1 cells are already widely implicated in the tissue-specific destruction that occurs during the pathogenesis of autoimmune diseases, such as diabetes mellitus and multiple sclerosis. Th1 cells directly kill tumor cells via release of cytokines that activate death receptors on the tumor cell surface. We now know that cross-priming of the tumor-specific response by potent APC is a major mechanism of the developing endogenous immune response; therefore, even intracellular proteins can be presented in the context of MHC class II. Indeed, recent studies demonstrate the importance of cross-priming in eliciting CTL. Many vaccine strategies aim to stimulate the Th response specific for a tumor antigen. Early clinical trials have shown that focus on the Th effector arm of the immune system can result in significant levels of both antigen-specific Th cells and CTL, the generation of long lasting immunity, and a Th1 phenotype resulting in the development of epitope spreading.

HIV-1- and cytomegalovirus (CMV)-specific CD4 T-cell-mediated antiviral immunity was evaluated by assessing the frequency of interleukin 2 (IL-2)- and interferon gamma (IFN-gamma)-secreting cells following antigen-specific stimulation in blood and lymph node. HIV-1-infected subjects with progressive disease at early stage of infection with no previous history of antiretroviral therapy (ART), subjects with nonprogressive disease, and HIV-negative subjects were studied. On the basis of the ability to secrete IL-2 and IFN-gamma, 3 functionally distinct populations of CD4 T cells were identified: (1) IL-2-secreting cells; (2) IL-2/IFN-gamma-secreting cells; and (3) IFN-gamma-secreting cells. CMV-specific CD4 T cells were almost equally distributed within the 3 functionally distinct cell populations in the 3 study groups as well as HIV-1-specific CD4 T cells in subjects with nonprogressive disease. However, a skewing toward IFN-gamma-secreting cells (70% of HIV-1-specific CD4 T cells) was observed in subjects with progressive disease, and IL-2- and IL-2/IFN-gamma-secreting cells were almost absent. The frequencies of IL-2- and of IL-2/IFN-gamma-secreting HIV-1-specific CD4 T cells were negatively correlated with the levels of viremia. Interestingly, prolonged ART was able to correct the skewed representation of different populations of HIV-1-specific CD4 T cells but was associated with only a partial recovery of IL-2-secreting cells. These results indicate that the composition of the pool of functionally distinct virus-specific CD4 T cells is important for virus control.

Whole cell pertussis vaccines (Pw) induce Th1 responses and protect against Bordetella pertussis infection, whereas pertussis acellular vaccines (Pa) induce Ab and Th2-biased responses and also protect against severe disease. In this study, we show that Pw failed to generate protective immunity in TLR4-defective C3H/HeJ mice. In contrast, protection induced with Pa was compromised, but not completely abrogated, in C3H/HeJ mice. Immunization with Pw, but not Pa, induced a population of IL-17-producing T cells (Th-17), as well as Th1 cells. Ag-specific IL-17 and IFN-gamma production was significantly lower in Pw-immunized TLR4-defective mice. Furthermore, treatment with neutralizing anti-IL-17 Ab immediately before and after B. pertussis challenge significantly reduced the protective efficacy of Pw. Stimulation of dendritic cells (DC) with Pw promoted IL-23, IL-12, IL-1beta, and TNF-alpha production, which was impaired in DC from TLR4-defective mice. B. pertussis LPS, which is present in high concentrations in Pw, induced IL-23 production by DC, which enhanced IL-17 secretion by T cells, but the induction of Th-17 cells was also dependent on IL-1. In addition, we identified a new effector function for IL-17, activating macrophage killing of B. pertussis, and this bactericidal activity was less efficient in macrophages from TLR4-defective mice. These data provide the first definitive evidence of a role for TLRs in protective immunity induced by a human vaccine. Our findings also demonstrate that activation of innate immune cells through TLR4 helps to direct the induction of Th1 and Th-17 cells, which mediate protective cellular immunity to B. pertussis.