We have previously reported that T cells bearing T cell receptors (TCRs) of gamma/delta type appear at a relatively early stage of primary infection with Listeria monocytogenes in mice. To characterize the early-appearing gamma/delta T cells during listeriosis, we analyzed the specificity and cytokine production of the gamma/delta T cells in the peritoneal cavity in mice inoculated intraperitoneally with a sublethal dose of L. monocytogenes. The early-appearing gamma/delta T cells, most of which were of CD4-CD8- phenotype, proliferated and secreted IFN-gamma and macrophage chemotactic factor in response to purified protein derivative from Mycobacterium tuberculosis, or recombinant 65-kD heat-shock protein derived from M. bovis but not to heat-killed Listeria. To further elucidate the potential role of the gamma/delta T cells in the host-defense mechanism against primary infection with Listeria, we examined the effects of in vivo administration of monoclonal antibodies (mAbs) against TCR-gamma/delta or TCR-alpha/beta on the bacterial eradication in mice infected with Listeria. Most of alpha/beta T cells or gamma/delta T cells were depleted in the peripheral lymphoid organs at least for 12 d after an intraperitoneal injection of 200 micrograms TCR-alpha/beta mAb or 200 micrograms TCR-gamma/delta mAb, respectively. An exaggerated bacterial multiplication was evident at the early stage of listerial infection in the gamma/delta T cells-depleted mice, whereas the alpha/beta T cell-depleted mice exhibited much the same resistance level as the control mice at this stage although the resistance was severely impaired at the late stage after listerial infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloantigen-stimulated CD8+ mouse spleen cells, either spontaneously or in the presence of IL-12 or IFN gamma plus anti-IL-4, differentiate into CD8+ T cells secreting a Th1-like cytokine pattern (IL-2 and IFN gamma). IL-4 induced differentiation into CD8+ T cells secreting Th2 cytokines (IL-4, IL-5, IL-6, and IL-10), whereas anti-IFN gamma suppressed the development of CD8+ cells secreting IFN gamma. Clones of IL-4- or IFN gamma-producing CD8+ T cells were relatively stable, as IL-4 or IFN gamma did not cause interconversion of committed CD8+ T cells. Both CD8+ subsets were cytotoxic, failed to provide cognate help for B cell antibody production, and remained CD4-, CD8 alpha+ CD8 beta+. We propose the names TC1 and TC2 for cytotoxic CD8+ T cells secreting Th1-like and Th2-like cytokines, respectively.

OBJECTIVE

To understand the current status of knowledge in the basic field of polarized specific immune responses mediated by CD4+ T helper (Th) lymphocytes, based on their profile of cytokine production (type 1 or Th1 and type 2 or Th2).

METHODS

Relevant articles and publications from the medical literature, especially review articles dealing with properties, mechanisms of polarization, transcription regulatory factors, and role in different human pathophysiological conditions of Th1 and Th2 cells.

CONCLUSIONS

Th1 cells, which produce interferon (IFN)-gamma, interleukin (IL)-2 and tumor necrosis factor (TNF)-beta, evoke cell-mediated immunity and phagocyte-dependent inflammation. Th2 cells, which produce IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13, evoke strong antibody responses (including those of the IgE class) and eosinophil accumulation, but inhibit several functions of phagocytic cells (phagocyte-independent inflammation). Both environmental and genetic factors act in concert to determine the Th1 or Th2 polarization. Further, Th1-dominated responses are involved in the pathogenesis of organ-specific autoimmune disorders, Crohn's disease, sarcoidosis, acute kidney allograft rejection, and some unexplained recurrent abortions. In contrast, allergen-specific Th2 responses are responsible for atopic disorders in genetically susceptible individuals. Further, Th2-dominated responses play a pathogenic role in both progressive systemic sclerosis and cryptogenic fibrosing alveolitis, and favor a more rapid evolution of HIV infection towards the full-blown disease. Finally, the Th1/Th2 paradigm can provide the basis for the development of new types of vaccines against infectious agents and of novel strategies for the therapy of allergic and autoimmune disorders.

The direct effects of IL-10 on the proliferation and lymphokine production of human peripheral blood T cells and CD4+ T cell clones representing the Th0, Th1-like, and Th2-like Th cell subsets were investigated in the absence of professional APC. IL-10 partially inhibited the proliferative responses of CD4+ human T cell clones induced by anti-CD2 or anti-CD3 mAb cross-linked on CD32 (Fc gamma RII)-transfected mouse L cells. Transfection of ICAM-1 or LFA-3 in CD32+ L cells resulted in enhanced proliferative responses of CD4+ T cell clones after activation by anti-CD3 mAb, whereas transfection of B7 in CD32+ L cells enhanced proliferative responses of CD4+ T cell clones after activation by anti-CD2 mAb. In addition, B7 expression on CD32+ L cells was required for activation of small resting T cells by anti-CD3 or anti-CD2 mAb. IL-10 inhibited the proliferation of T cell clones induced by anti-CD2 or anti-CD3 mAb on CD32+ L cells expressing these accessory molecules, indicating that interactions of LFA-3, ICAM-1, and B7 with their ligands on T cells did not overcome the inhibitory effects of IL-10. Inhibition of proliferation of T cell clones by IL-10 was in all instances completely neutralized by relatively low concentrations of IL-2, whereas IL-4 was ineffective. IL-10 did not affect the expression of the TCR/CD3 complex, CD2, LFA-1, CD28, or IL-2R alpha- or beta-chains, nor did it inhibit the induction of the latter two molecules on T cells after activation. Inhibition of proliferation was found to be the result of specific inhibition of IL-2 production by the responding T cell subsets, which occurred at the mRNA level. The production and mRNA levels of IL-4, IL-5, IFN-gamma, and granulocyte/macrophage-CSF were not affected by IL-10. Taken together, these results indicate that IL-10/IL-10R interaction on CD4+ T cell clones and peripheral blood T cells results in signaling pathways that specifically interfere with activation processes leading to IL-2 production. These direct inhibitory effects on IL-2 production by activated T cells may contribute to the general immunosuppressive activities of IL-10.

Regulatory CD25(+)CD4(+) T cells are considered as important players in T cell homeostasis and self-tolerance. Here we report that the integrin alpha(E)beta(7), which recognizes epithelial cadherin, identifies the most potent subpopulation of regulatory CD25(+) T cells. Strikingly, CD25-negative alpha(E)+CD4(+) T cells displayed regulatory activity. Both alpha(E)+ subsets, CD25(+) and CD25(-), express CTLA-4, suppress T cell proliferation in vitro, and protect mice from colitis in the severe combined immunodeficient model (SCID) in vivo. Whereas alpha(E)+CD25(+) T cells produce almost no cytokines, alpha(E)+CD25(-) T cells represent a unique subset in which high IL-2, IFN-gamma and T helper 2-cytokine production is linked with suppressive function. Thus, the integrin alpha(E)beta(7) can be regarded as a novel marker for subsets of highly potent, functionally distinct regulatory T cells specialized for crosstalk with epithelial environments.

The cytokine interferon (IFN)-gamma regulates immune clearance of parasitic, bacterial, and viral infections; however, the underlying mechanisms are poorly understood. Recently, a family of IFN-gamma-induced genes has been identified that encode 48-kD GTP-binding proteins that localize to the endoplasmic reticulum of cells. The prototype of this family, IGTP, has been shown to be required for host defense against acute infections with the protozoan parasite Toxoplasma gondii, but not for normal clearance of the bacterium Listeria monocytogenes and murine cytomegalovirus (MCMV). To determine whether other members of the gene family also play important roles in immune defense, we generated mice that lacked expression of the genes LRG-47 and IRG-47, and examined their responses to representative pathogens. After infection with T. gondii, LRG-47-deficient mice succumbed uniformly and rapidly during the acute phase of the infection; in contrast, IRG-47-deficient mice displayed only partially decreased resistance that was not manifested until the chronic phase. After infection with L. monocytogenes, LRG-47-deficient mice exhibited a profound loss of resistance, whereas IRG-47-deficient mice exhibited completely normal resistance. In addition, both strains displayed normal clearance of MCMV. Thus, LRG-47 and IRG-47 have vital, but distinct roles in immune defense against protozoan and bacterial infections.

Human mesenchymal stem cells (MSCs) were evaluated for their ability to activate allogeneic T cells in cell mixing experiments. Phenotypic characterization of MSCs by flow cytometry showed expression of MHC Class I alloantigens, but minimal expression of Class II alloantigens and costimulatory molecules, including CD80 (B7-1), CD86 (B7-2), and CD40. T cells purified from peripheral blood mononuclear cells (PBMCs) did not proliferate to allogeneic MSCs. Lack of response was not due to a deficiency of costimulation, since retroviral transduction of MSCs with either B7-1 or B7-2 costimulatory molecules did not result in lymphoproliferation. Although these results suggested that MSCs were immunologically inert or potentially tolerogenic, T cells cultured with MSCs produced IFN-gamma and displayed secondary kinetics to restimulation with PBMCs, indicating alloantigen priming rather than tolerance induction by the MSCs. To determine whether MSCs suppressed alloreactive T cells, MSCs were added to primary mixed lymphocyte reaction (MLR) cultures. MSCs suppressed cell proliferation when added at the initiation of culture or when added to an ongoing MLR culture. Suppression was dose-dependent, genetically unrestricted, and occurred whether or not MSCs were pretreated with IFN-gamma. MSCs in transwell chambers suppressed primary MLR cultures, indicating that suppression was mediated by soluble molecules. Analysis of cytokines in suppressed MLR cultures demonstrated up-regulation of IFN-gamma and IL-10, and down-regulation of TNF-alpha production relative to control cultures. We conclude that MSCs can initiate activation of alloreactive T cells, but do not elicit T cell proliferative responses due to active suppressive mechanisms.

It has been shown that all three classes of interferons enhance the expression of the major histocompatibility class I antigens (HLA-A,B,C;H-2) on a wide variety of cell types (1-10). However, their effect on the expression of the class II antigens (HLA-DR, Ia), which play a major part in cellular interactions that initiate an immune response, is more controversial. The predominate findings have been that the interferons specifically increase the synthesis and expression of only the class I antigens (3, 4, 6, 8, 10, 11). We report here that recombinant interferon-gamma (IFN-gamma) increases the synthesis and expression of the HLA-DR (la-like) antigens as well as beta 2-microglobulin (beta 2-m), a low m.w. subunit of HLA, on human melanoma cells. No increase in HLA-DR was detected on these melanoma cells with leukocyte interferon (IFN-alpha) at doses 400 times higher than the maximum dose of IFN-gamma. These findings were extended to show that pure IFN-gamma also increases the expression of the HLA-DR antigens on normal peripheral blood monocytes, whereas recombinant IFN-alpha at a similar dose had little effect on the expression of this surface antigen. These findings suggest a specialized role for IFN-gamma in immune regulation in comparison with IFN-alpha.

Functional and phenotypic characterization of virus-specific CD8 T cells against cytomegalovirus, Epstein-Barr virus, influenza (flu), and HIV-1 were performed on the basis of the ability of CD8 T cells to secrete IFN-gamma and IL-2, to proliferate, and to express CD45RA and CCR7. Two functional distinct populations of CD8 T cells were identified: (i) dual IFN-gamma/IL-2-secreting cells and (ii) single IFN-gamma-secreting cells. Virus-specific IFN-gamma/IL-2-secreting CD8 T cells were CD45RA-CCR7-, whereas single IFN-gamma CD8 T cells were either CD45RA-CCR7- or CD45RA+CCR7-. The proportion of virus-specific IFN-gamma/IL-2-secreting CD8 T cells correlated with that of proliferating CD8 T cells, and the loss of HIV-1-specific IL-2-secreting CD8 T cells was associated with that of HIV-1-specific CD8 T cell proliferation. Substantial proliferation of virus-specific CD8 T cells (including HIV-1-specific CD8 T cells) was also observed in CD4 T cell-depleted populations or after stimulation with MHC class I tetramer-peptide complexes. IL-2 was the factor responsible for the CD4-independent CD8 T cell proliferation. These results indicate that IFN-gamma/IL-2-secreting CD8 T cells may promote antigen-specific proliferation of CD8 T cells even in the absence of helper CD4 T cells.

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

We have found that an important Th2 cytokine, IL-10, is produced by tissues from patients acutely infected with Leishmania donovani. In all individuals tested, IL-10 mRNA production was increased in lymph nodes taken during acute disease over that observed in postacute samples. In contrast, both pre- and posttreatment lymph nodes had readily detected mRNA for IFN-gamma and IL-2. A down-regulating effect of IL-10 on leishmania-induced proliferative responses was demonstrated when Hu rIL-10 was added to cultures of PBMC from clinically cured individuals. PBMC from individuals with acute visceral leishmaniasis responded to stimulation with leishmania lysate by producing IL-10 mRNA. Simultaneously cultured PBMC collected from the same patients after successful chemotherapy produced no detectable IL-10 mRNA after leishmania antigen stimulation. Neutralizing anti-IL-10 mAb added to PBMC from patients with acute visceral leishmaniasis markedly increased the proliferative response to leishmania lysate. Finally, we observed mRNA for IL-10 and IFN-gamma concurrently in a lesion from a patient with post-kala-azar dermal leishmaniasis (PKDL). These results indicate the production of IL-10 during L. donovani infection, and suggest a role for this cytokine in the regulation of immune responsiveness during visceral leishmaniasis.

OBJECTIVE

Maintenance of the mucosal barrier is a critical function of intestinal epithelia. Myosin regulatory light chain (MLC) phosphorylation is a common intermediate in the pathophysiologic regulation of this barrier. The aim of this study was to determine whether a membrane permeant inhibitor of MLC kinase (PIK) could inhibit intracellular MLC kinase and regulate paracellular permeability.

METHODS

Recombinant MLC and Caco-2 MLC kinase were used for kinase assays. T84 and Caco-2 monolayers were treated with enteropathogenic Escherichia coli (EPEC) or tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma to induce barrier dysfunction.

RESULTS

PIK inhibited MLC kinase in vitro and was able to cross cell membranes and concentrate at the perijunctional actomyosin ring. Consistent with these properties, apical addition of PIK reduced intracellular MLC phosphorylation by 22% +/- 2%, increased transepithelial resistance (TER) by 50% +/- 1%, and decreased paracellular mannitol flux rates by 5.2 +/- 0.2-fold. EPEC infection induced TER decreases of 37% +/- 6% that were limited to 16% +/- 5% by PIK. TNF-alpha and IFN-gamma induced TER decreases of 22% +/- 3% that were associated with a 172% +/- 1% increase in MLC phosphorylation. Subsequent PIK addition caused MLC phosphorylation to decrease by 25% +/- 4% while TER increased to 97% +/- 6% of control.

CONCLUSIONS

PIK can prevent TER defects induced by EPEC and reverse MLC phosphorylation increases and TER decreases induced by TNF-alpha and IFN-gamma. The data also suggest that TNF-alpha and IFN-gamma regulate TER, at least in part, via the perijunctional cytoskeleton. Thus, PIK may be the prototype for a new class of targeted therapeutic agents that can restore barrier function in intestinal disease states.

Due to the dependency on aromatic precursors, the growth of Salmonella typhimurium aroA- is limited in immunocompetent mice. Here we show that H-21-A beta-/- mice (lacking MHC class II molecules and thus devoid of mature CD4+ TCR-alpha beta cells), TCR-beta-/- mice (devoid of TCR-alpha beta cells), and IFN-gamma R-/- mice (unresponsive to IFN-gamma) are highly susceptible to S. typhimurium aroA- infection compared with heterozygous controls. In contrast, beta 2m-deficient mice (lacking surface MHC class I and thus devoid of conventional CD8+ T cells) or TCR-delta-/- mice (devoid of TCR-gamma delta cells) were equally as resistant to S. typhimurium aroA- infection as their heterozygous littermates. These findings emphasize the vital role of CD4+ TCR-alpha beta cells and IFN-gamma in resistance against S. typhimurium aroA-. Sublethal inocula of S. typhimurium aroA- led to permanent infection in H-21-A beta-/- mice, suggesting that bacterial starvation is insufficient for sterile clearance in immunocompetent mice and that MHC class II-dependent immune mechanisms are required for pathogen eradication. The TCR-beta-/- mice suffered from salmonellosis more severely than the MHC class II-deficient mutants, suggesting an auxiliary function of CD8+ T cells. Recombinant S. typhimurium aroA-, secreting listeriolysin (Hly) of Listeria monocytogenes, are capable of escaping from the phagosome into the cytosol of the host cell. However, the course of infection of these recombinant S. typhimurium SL7207 Hlys and control strains did not differ in beta 2m-/- mutants. This finding argues against direct correlation of cytosolic location of S. typhimurium SL7207 Hlys with CD8+ T cell dependency of protection.

T follicular helper (Tfh) cells help development of antibody responses via interleukin-21 (IL-21). Here we show that activated human dendritic cells (DCs) induced naive CD4(+) T cells to become IL-21-producing Tfh-like cells through IL-12. CD4(+) T cells primed with IL-12 induced B cells to produce immunoglobulins in a fashion dependent on IL-21 and inducible costimulator (ICOS), thus sharing fundamental characteristics with Tfh cells. The induction of Tfh-like cells by activated DCs was inhibited by neutralizing IL-12. IL-12 induced two different IL-21-producers: IL-21(+)IFN-gamma(+)T-bet(+) Th1 cells and IL-21(+)IFN-gamma(-)T-bet(-) non-Th1 cells, in a manner dependent on signal transducer and activator of transcription 4 (STAT4). IL-12 also regulated IL-21 secretion by memory CD4(+) T cells. Thus, IL-12 produced by activated DCs regulates antibody responses via developing IL-21-producing Tfh-like cells and inducing IL-21 secretion from memory CD4(+) T cells. These data suggest that the developmental pathway of Tfh cells differs between mice and humans, which have considerable implications for vaccine development.

BACKGROUND

In previous animal studies, interleukin 12 (IL 12) was shown to inhibit the growth of a wide spectrum of tumors in vivo but to have no direct effect on tumor cells in vitro. Also, contrary to the expectation of a T-cell-mediated effect, the antitumor activity of IL 12 was not completely abrogated in tests of T-cell-deficient mice. These observations suggest that IL 12 may possess antiangiogenic properties that account for its tumor-inhibitory effects in vivo.

OBJECTIVE

Our goal was to investigate the hypothesis that IL 12 has antiangiogenic properties.

METHODS

A model of basic fibroblast growth factor-induced corneal neovascularization in mice was used to evaluate the effects of IL 12 and interferon gamma (IFN gamma) on angiogenesis in vivo. Different strains of male mice, e.g., immunocompetent C57BL/6 mice, severe combined immune-deficient (SCID) mice, natural killer cell-deficient beige mice, and T-cell-deficient nude mice, were treated with IL 12 (1 microgram/day) intraperitoneally for 5 consecutive days. The extent of neovascularization in response to a basic fibroblast growth factor pellet and the inhibition of neovascularization by IL 12 or IFN gamma were assessed by measuring the maximal vessel length and the corneal circumference involved in new blood vessel formation. The antitumor activities of IL 12 and of the angiogenesis inhibitor AGM-1470 were evaluated in Lewis lung carcinoma-bearing mice. In vitro proliferation studies were performed on bovine capillary endothelial cells, mouse pancreatic islet endothelial cells, and mouse hemangioendothelioma cells.

RESULTS

IL 12 treatment almost completely inhibited corneal neovascularization in C57BL/6, SCID, and beige mice. This potent suppression of angiogenesis was prevented by the administration of IFN gamma-neutralizing antibodies, suggesting that the suppression was mediated through IFN gamma. In addition, the administration of IFN gamma reproduced the antiangiogenic effects observed during treatment with IL 12. Treatment with IL 12 and AGM-1470 combined did not increase toxicity and showed a trend toward enhanced antitumor efficacy in Lewis lung carcinoma-bearing mice.

CONCLUSIONS

IL 12 strongly inhibits neovascularization. This effect is not mediated by a specific cell type of the immune system. Instead, IL 12 has been shown to induce IFN gamma, which, in turn, appears to play a critical role as a mediator of the antiangiogenic effects of IL 12.

CONCLUSIONS

Recognition of the mechanisms of the antiangiogenic properties of IL 12 may be crucial in planning its clinical applications, including a possibility of coadministration with other inhibitors of neovascularization.

Activated mouse peritoneal macrophages produce nitric oxide (NO) via a nitric oxide synthase that is inducible by interferon gamma (IFN-gamma): iNOS. We have studied the mechanisms by which transforming growth factor beta 1 (TGF-beta) suppresses IFN-gamma-stimulated NO production. TGF-beta treatment reduced iNOS specific activity and iNOS protein in both cytosolic and particulate fractions as assessed by Western blot with monospecific anti-iNOS immunoglobulin G. TGF-beta reduced iNOS mRNA without affecting the transcription of iNOS by decreasing iNOS mRNA stability. Even after iNOS was already expressed, TGF-beta reduced the amount of iNOS protein. This was due to reduction of iNOS mRNA translation and increased degradation of iNOS protein. The potency of TGF-beta as a deactivator of NO production (50% inhibitory concentration, 5.6 +/- 2 pM) may reflect its ability to suppress iNOS expression by three distinct mechanisms: decreased stability and translation of iNOS mRNA, and increased degradation of iNOS protein. This is the first evidence that iNOS is subject to other than transcriptional regulation.

Emerging evidence suggests that increases in activated T cell populations in adipose tissue may contribute toward obesity-associated metabolic syndrome. The present study investigates three unanswered questions: 1) Do adipose-resident T cells (ARTs) from lean and obese mice have altered cytokine production in response to TCR ligation?; 2) Do the extralymphoid ARTs possess a unique TCR repertoire compared with lymphoid-resident T cells and whether obesity alters the TCR diversity in specific adipose depots?; and 3) Does short-term elimination of T cells in epididymal fat pad without disturbing the systemic T cell homeostasis regulate inflammation and insulin-action during obesity? We found that obesity reduced the frequency of naive ART cells in s.c. fat and increased the effector-memory populations in visceral fat. The ARTs from diet-induced obese (DIO) mice had a higher frequency of IFN-gamma(+), granzyme B(+) cells, and upon TCR ligation, the ARTs from DIO mice produced increased levels of proinflammatory mediators. Importantly, compared with splenic T cells, ARTs exhibited markedly restricted TCR diversity, which was further compromised by obesity. Acute depletion of T cells from epididymal fat pads improved insulin action in young DIO mice but did not reverse obesity-associated feed forward cascade of chronic systemic inflammation and insulin resistance in middle-aged DIO mice. Collectively, these data establish that ARTs have a restricted TCR-Vbeta repertoire, and T cells contribute toward the complex proinflammatory microenvironment of adipose tissue in obesity. Development of future long-term T cell depletion protocols specific to visceral fat may represent an additional strategy to manage obesity-associated comorbidities.

CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25- or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-gamma and TNF-alpha secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-beta and IL-10. In addition, the expression of ERK, NF-kappaB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling.

NF-κB is an integral component of the immune response to Toxoplasma gondii. Although evidence exists that T. gondii can directly modulate the NF-κB pathway, the parasite-derived effectors involved are unknown. We determined that type II strains of T. gondii activate more NF-κB than type I or type III strains, and using forward genetics we found that this difference is a result of the polymorphic protein GRA15, a novel dense granule protein which T. gondii secretes into the host cell upon invasion. A GRA15-deficient type II strain has a severe defect in both NF-κB nuclear translocation and NF-κB-mediated transcription. Furthermore, human cells expressing type II GRA15 also activate NF-κB, demonstrating that GRA15 alone is sufficient for NF-κB activation. Along with the rhoptry protein ROP16, GRA15 is responsible for a large part of the strain differences in the induction of IL-12 secretion by infected mouse macrophages. In vivo bioluminescent imaging showed that a GRA15-deficient type II strain grows faster compared with wild-type, most likely through its reduced induction of IFN-γ. These results show for the first time that a dense granule protein can modulate host signaling pathways, and dense granule proteins can therefore join rhoptry proteins in T. gondii's host cell-modifying arsenal.

A molecular understanding of the regulation of IgG class switching to IL-4-independent isotypes, particularly to IgG2a, remains largely unknown. The T-box transcription factor T-bet directly regulates Th1 lineage commitment by CD4 T cells, but its role in B lymphocytes has been largely unexplored. We show here a role for T-bet in the regulation of IgG class switching, especially to IgG2a. T-bet-deficient B lymphocytes demonstrate impaired production of IgG2a, IgG2b, and IgG3 and, most strikingly, are unable to generate germ-line or postswitch IgG2a transcripts in response to IFN-gamma. Conversely, enforced expression of T-bet initiates IgG2a switching in cell lines and primary cells. This function contributes critically to the pathogenesis of murine lupus, where the absence of T-bet strikingly reduces B cell-dependent manifestations, including autoantibody production, hypergammaglobulinemia, and immune-complex renal disease and, in particular, abrogates IFN-gamma-mediated IgG2a production. Classical T cell manifestations persisted, including lymphadenopathy and cellular infiltrates of skin and liver. These results identify T-bet as a selective transducer of IFN-gamma-mediated IgG2a class switching in B cells and emphasize the importance of this regulation in the pathogenesis of humorally mediated autoimmunity.

Periodontal diseases (PD) are chronic infectious inflammatory diseases characterized by the destruction of tooth-supporting structures, being the presence of periodontopathogens required, but not sufficient, for disease development. As a general rule, host inflammatory mediators have been associated with tissue destruction, while anti-inflammatory mediators counteract and attenuate disease progression. With the discovery of several T-cell subsets bearing distinct immunoregulatory properties, this pro- vs. anti-inflammatory scenario became more complex, and a series of studies has hypothesized protective or destructive roles for Th1, Th2, Th17, and Treg subpopulations of polarized lymphocytes. Interestingly, the "protective vs. destructive" archetype is usually considered in a framework related to tissue destruction and disease progression. However, it is important to remember that periodontal diseases are infectious inflammatory conditions, and recent studies have demonstrated that cytokines (TNF-α and IFN-γ) considered harmful in the context of tissue destruction play important roles in the control of periodontal infection. Therefore, in this review, the state-of-the-art knowledge concerning the protective and destructive roles of host inflammatory immune response will be critically evaluated and discussed from the tissue destruction and control-of-infection viewpoints.

Naive CD4+ T cells activated through TCR/CD28 under Th1 or Th2 conditions expressed canonical cytokine patterns irrespective of cell division. Only cells that had divided fewer than four times were capable of reexpressing alternative cytokines when restimulated under opposing conditions. Although T cells transcribed both IFN-gamma and IL-4 within hours in a Stat4-/Stat6-independent manner, neither T-bet nor GATA-3 was induced optimally without Stat signals, and polarized cytokine expression was not sustained. Cytokine genes were positioned apart from heterochromatin in resting T cell nuclei, consistent with rapid expression. After polarization, the majority of silenced cytokine alleles were repositioned to heterochromatin. Naive T cells transit through sequential stages of cytokine activation, commitment, silencing, and physical stabilization during polarization into differentiated effector subsets.

Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4+ T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-α Mtb-specific CD4+ T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry-based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion of single-positive TNF-α Mtb-specific CD4+ T cells is a new tool for the rapid diagnosis of active tuberculosis disease.

The cell surface Fas antigen is a membrane-associated polypeptide which can mediate apoptosis. cDNA clones encoding the Fas antigen were isolated from a cDNA library constructed with mRNA from the mouse macrophage cell line BAM3. The nucleotide sequence and the deduced amino acid sequence of the mouse Fas antigen were 58.5 and 49.3% identical, respectively, to the corresponding sequences of human Fas antigen cDNA. The mouse Fas antigen consists of 306 amino acids with a calculated Mr of 34,971 and contains a single transmembrane domain which divides the molecule into extracellular and cytoplasmic domains. A 2.1-kb mRNA coding for the Fas antigen was detected in the mouse thymus, heart, liver, and ovary but not in brain and spleen. The expression of the Fas antigen gene in mouse fibroblast L929 and macrophage BAM3 cell lines was significantly induced by treatment with IFN-gamma but not by IFN-alpha/beta. Interspecific backcross analysis indicated that the gene coding for the Fas antigen is in the distal region of mouse chromosome 19.