Route of Infection That Induces a High Intensity of Gamma Interferon-Secreting T Cells in the Genital Tract Produces Optimal Protection against Chlamydia trachomatis Infection in Mice

The induction of local T helper type 1 (Th1)-mediated cellular immunity is crucial for resistance of mice to genital infection by the obligate intracellular bacterium Chlamydia trachomatis. We tested the hypothesis that the route of immunization that elicits relatively high numbers of chlamydia-specific, gamma interferon (IFN-γ)-secreting T lymphocytes (ISTLs) in the genital tract would induce optimal protective immunity against reinfection. Female BALB/c mice were infected intravaginally (i.v.), intranasally (i.n.), orally (p.o.), or subcutaneously (s.c.) with C. trachomatis. At days 7, 14, 21, and 28 postinfection, T cells isolated from the genital tract tissues were restimulated with chlamydial antigen in vitro, and the amounts of IFN-γ induced were measured by a sandwiched enzyme-linked immunosorbent assay method. At day 7 postinfection, i.n.- and i.v.-immunized mice had high levels of chlamydia-specific ISTLs in their genital tracts (203.58 ± 68.1 and 225.5 ± 12.1 pg/ml, respectively). However, there were no detectable ISTLs in the genital tracts of p.o.- or s.c.-infected mice. When preinfected mice were challenged i.v. 70 days later, animals preexposed by the i.n. route were highly resistant to reinfection, with greatly reduced chlamydial burden, and suffered an attenuated infection that resolved by day 6 postchallenge. Animals preexposed by the i.v. route were modestly protected, whereas p.o. and s.c. groups were indistinguishable in this regard from control mice. The resistance of i.n.-immunized mice (and to some extent the i.v.-exposed mice) to reinfection was associated with early appearance (within 24 h) of high levels of genital ISTLs compared with mice preinfected by other routes. Furthermore, although i.n. and i.v.-immunized mice had comparable levels of chlamydia-specific immunoglobulin A (IgA) antibodies in their vaginal washes, the levels of IgG2a were four- sixfold higher in i.n.-immunized mice than in any of the other groups. The results suggested that immunization routes that foster rapid induction of vigorous genital mucosal cell-mediated immune (CMI) effectors (e.g., IFN-γ), the CMI-associated humoral effector, IgG2a, and to some extent secretory IgA produce protective immunity against chlamydial genital infection. Therefore, i.n. immunization is a potential delivery route of choice in the development of a vaccine against Chlamydia.     

5-Lipoxygenase Negatively Regulates Th1 Response during Brucella abortus Infection in Mice

Brucella abortus is a Gram-negative bacterium that infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. A Th1-mediated immune response plays a critical role in host control of this pathogen. Recent findings indicate contrasting roles for lipid mediators in host responses against infections. 5-Lipoxygenase (5-LO) is an enzyme required for the production of the lipid mediators leukotrienes and lipoxins. To determine the involvement of 5-LO in host responses to B. abortus infection, we intraperitoneally infected wild-type and 5-LO-deficient mice and evaluated the progression of infection and concomitant expression of immune mediators. Here, we demonstrate that B. abortus induced the upregulation of 5-LO mRNA in wild-type mice. Moreover, this pathogen upregulated the production of the lipid mediators leukotriene B₄ and lipoxin A₄ in a 5-LO-dependent manner. 5-LO-deficient mice displayed lower bacterial burdens in the spleen and liver and less severe liver pathology, demonstrating an enhanced resistance to infection. Host resistance paralleled an increased expression of the proinflammatory mediators interleukin-12 (IL-12), gamma interferon (IFN-γ), and inducible nitric oxide synthase (iNOS) during the course of infection. Moreover, we demonstrated that 5-LO downregulated the expression of IL-12 in macrophages during B. abortus infection. Our results suggest that 5-LO has a major involvement in B. abortus infection, by functioning as a negative regulator of the protective Th1 immune responses against this pathogen.     

Crucial Role of Gamma Interferon-Producing CD4+ Th1 Cells but Dispensable Function of CD8+ T Cell,B Cell,Th2, and Th17 Responses in the Control of Brucella melitensis Infection in Mice

Brucella spp. are facultative intracellular bacterial pathogens responsible for brucellosis, a worldwide zoonosis that causes abortion in domestic animals and chronic febrile disease associated with serious complications in humans. There is currently no approved vaccine against human brucellosis, and antibiotic therapy is long and costly. Development of a safe protective vaccine requires a better understanding of the roles played by components of adaptive immunity in the control of Brucella infection. The importance of lymphocyte subsets in the control of Brucella growth has been investigated separately by various research groups and remains unclear or controversial. Here, we used a large panel of genetically deficient mice to compare the importance of B cells, transporter associated with antigen processing (TAP-1), and major histocompatibility complex class II-dependent pathways of antigen presentation as well as T helper 1 (Th1), Th2, and Th17-mediated responses on the immune control of Brucella melitensis 16 M infection. We clearly confirmed the key function played by gamma interferon (IFN-γ)-producing Th1 CD4⁺ T cells in the control of B. melitensis infection, whereas IFN-γ-producing CD8⁺ T cells or B cell-mediated humoral immunity plays only a modest role in the clearance of bacteria during primary infection. In the presence of a Th1 response, Th2 or Th17 responses do not really develop or play a positive or negative role during the course of B. melitensis infection. On the whole, these results could improve our ability to develop protective vaccines or therapeutic treatments against brucellosis.     

Clearance of Chlamydia trachomatis– induced polyserositis in SCID mice requires both CD4+ and CD8+ cells

To characterize the role of specific lymphocyte subsets in Chlamydia trachomatis infection, we established a murine model using the mouse pneumonitis agent (MoPn) of C. trachomatis and C.B-17 scid/scid (SCID) mice which lack functional B and T cells. After intraperitoneal inoculation with the bacteria, SCID mice developed polyserositis with pleuritis, pericarditis, and perihepatitis. Within 8 weeks post infection, SCID mice succumbed to the disease, whereas immunocompetent congenic C.B-17+/+ mice resolved the infection. Adoptive transfer of immune spleen cells into MoPn-infected SCID mice resulted in a complete elimination of the agent and prevention of polyserositis as measured by quantitative chlamydial culture, direct immunofluorescence and histopathological analysis. Selective reconstitution of MoPn-infected SCID mice with immune B lymphocytes, CD4⁺ T cells or CD8⁺ T cells alone did not influence the chlamydial load in the lung and liver of infected SCID animals, resulting in a polyserositis as observed in untreated MoPn-infected SCID mice. However, co-transfer of both CD4⁺ T cells and CD8⁺ T cells led to a significant reduction of chlamydiae in quantitative organ culture coupled with unremarkable histopathology. These data confirm that T cell-mediated immune responses are essential for immune protection in chlamydial infection, although total eradication of the agent could not be achieved. Further experiments are needed to stress the importance of a concerted action of B and T lymphocytes, as indicated by the complete protective efficacy of transferred splenocytes. Received: 9 April 1998     

Distinct Roles of CD28- and CD40 Ligand-Mediated Costimulation in the Development of Protective Immunity and Pathology during Chlamydia muridarum Urogenital Infection in Mice

Infection with Chlamydia muridarum in the mouse urogenital tract can induce both protective immunity and inflammatory pathologies, which has been used as a model for understanding the immune and pathogenic mechanisms of C. trachomatis infection. We compared the roles of CD28- and CD40 ligand (CD40L)-mediated costimulation in C. muridarum infection. Mice with CD28 or CD80/CD86 gene knockout (KO) displayed an infection course similar to that of wild-type mice during both primary and secondary infection, suggesting that CD28-mediated costimulation is not required for protection against C. muridarum infection. However, mice deficient in CD40L or CD40 displayed a prolonged infection course after primary or secondary infection, suggesting that CD40-CD40L costimulation plays an essential role in the development of anti-C. muridarum immunity. Interestingly, the CD28- or CD80/CD86-deficient mice displayed significantly lower levels of inflammatory pathologies in the upper genital tracts after primary infection, although the attenuation in inflammation was no longer significant during secondary infection. However, the CD40L or CD40 KO mice developed inflammatory pathologies as severe as those in wild-type mice following either primary or secondary infection despite the obvious deficits in adaptive immunity in these KO mice. The resistance of CD28 or CD80/CD86 KO mice to chlamydial infection correlated with production of gamma interferon, while the development of inflammatory pathologies in CD40L or CD40 KO mice correlated with the production of other proinflammatory cytokines in mouse urogenital tracts during the early stages of the infection. These observations together suggest that C. muridarum-induced protective immunity and inflammatory pathologies can be mediated by distinct costimulatory signals.Chlamydia trachomatis consists of multiple biovars with different tissue tropism. The three human biovars include the trachoma biovar that infects human ocular epithelium, causing preventable blindness (43); the genital biovar that invades human urogenital tract epithelial tissues, potentially leading to complications such as ectopic pregnancy and infertility in the affected women (40); and the LGV biovar (lymphogranuloma venereum) that can cause systemic infection (36). The mouse biovar known as mouse pneumonitis agent (MoPn) is now classified as a new species, C. muridarum. Although C. muridarum organisms cause no known human diseases, they have been extensively used to study the mechanisms of C. trachomatis pathogenesis and immunity (10, 24, 28, 30, 32). Using the C. muridarum urogenital tract infection mouse model in combination with strategies such as in vivo depletion and gene knockout (KO), CD4⁺ T-cell-dependent and gamma interferon (IFN-γ)-mediated immunity has been identified as a major protective mechanism for mice to control chlamydial infection (27). However, the same Th1 response may also contribute to the chlamydia-induced inflammatory pathologies. Since costimulation systems impact both T-cell activation and phenotypes, we compared the roles of CD28- and CD40 ligand (CD40L)-mediated costimulation in C. muridarum urogenital tract infection in the present study.T-cell activation requires both an antigen-specific signal generated by T-cell-receptor engagement with antigenic peptide-major histocompatibility complex on antigen-presenting cells, and a costimulatory signal that can be provided by CD28 or CD40L on T cells through ligation with their counterparts (CD80 [B7-1] and CD86 [B7-2] for CD28 or CD40 for CD40L) on antigen-presenting cells (18, 19, 23). The CD28-mediated costimulatory signal can result in an enhanced T-cell proliferation and cytokine production (23) and contribute to the development of various inflammatory diseases (6, 21, 31, 42, 44, 47). However, the role of CD28 costimulatory system in host defense against microbial infection has been less consistent (9, 13, 14, 26, 33, 46), and it is not known whether the CD28-mediated costimulation plays any role in C. muridarum infection. In contrast, the CD40L-CD40 costimulation system is known to play a critical role in host defense against intracellular pathogen infection by promoting Th1-dominant immunity (1, 12, 15, 16, 34, 41). Since Th1-dominant immunity is essential for controlling chlamydial infection, we hypothesize that the CD40L-CD40 costimulation is critical for developing antichlamydia immunity.In the present study, we found that CD28-mediated costimulation was not required for protection against C. muridarum infection but contributed to the development of inflammatory pathologies, whereas CD40L costimulation was necessary for developing anti-C. muridarum immunity but not required for inducing the inflammatory damages. These observations have clearly demonstrated that C. muridarum-induced protective immunity and inflammatory pathologies can be mediated via distinct costimulatory mechanisms.     

Interleukin-12 Is Essential for a Protective Th1 Response in Mice Infected with Cryptococcus neoformans

To analyze the roles of interleukin-12 (IL-12) and the IL-12-dependent Th1 response in resistance to Cryptococcus neoformans, we have established a chronic infection model in wild-type mice and in mice with targeted disruptions of the genes for the IL-12p35 and IL-12p40 subunits (IL-12p35^−/− and IL-12p40^−/− mice, respectively) as well as in mice with a targeted disruption of the IL-4 gene. Long-term application of exogenous IL-12 prevented death of infected wild-type mice for the entire period of the experiment (up to 180 days) but did not resolve the infection. Infected IL-12p35^−/− and IL-12p40^−/− mice died significantly earlier than infected wild-type mice, whereas infection of IL-4-deficient mice led to prolonged survival. Interestingly, infected IL-12p40^−/− mice died earlier and developed higher organ burdens than IL-12p35^−/− mice, which, for the first time in an infection model, suggests a protective role of the IL-12p40 subunit independent of the IL-12 heterodimer. The fungal organ burdens of IL-4-deficient mice and IL-12-treated wild-type mice were significantly reduced compared to those of untreated wild-type mice and IL-12-deficient mice. Histopathological analysis revealed reduction of the number of granulomatous lesions following treatment with IL-12. Susceptibility of both IL-12p35^−/− and IL-12p40^−/− mice was associated with marginal production of gamma interferon and elevated levels of IL-4 from CD4⁺ T cells, which indicates Th2 polarization in the absence of IL-12, whereas wild-type mice developed a Th1 response. Taken together, our data emphasize the essential role of IL-12 for protective Th1 responses against C. neoformans.     

Resolution of secondary Chlamydia trachomatis genital tract infection in immune mice with depletion of both CD4+ and CD8+ T cells

The essential role of T cells in the resolution of primary murine Chlamydia trachomatis genital tract infection is inarguable; however, much less is known about the mechanisms that confer resistance to reinfection. We previously established that CD4+ T cells and B cells contribute importantly to resistance to reinfection. In our current studies, we demonstrate that immune mice concurrently depleted of both CD4+ T cells and CD8+ T cells resisted reinfection as well as immunocompetent wild-type mice. The in vivo depletion of CD4+ and CD8+ T cells resulted in diminished chlamydia-specific delayed-type hypersensitivity responses, but antichlamydial antibody responses were unaffected. Our data indicate that immunity to chlamydial genital tract reinfection does not rely solely upon immune CD4+ or CD8+ T cells and further substantiate a predominant role for additional effector immune responses, such as B cells, in resistance to chlamydial genital tract reinfection.     

Vaccination with Plasmid DNA Encoding Mycobacterial Antigen 85A Stimulates a CD4+ and CD8+ T-Cell Epitopic Repertoire Broader than That Stimulated by Mycobacterium tuberculosis H37Rv Infection

Vaccination of mice with plasmid DNA carrying the gene for the major secreted mycobacterial antigen 85A (Ag85A) from Mycobacterium tuberculosis is a powerful technique for generating robust specific Th1 helper T-cell responses, CD8⁺-mediated cytotoxicity, and protection against M. tuberculosis challenge (K. Huygen et al., Nat. Med. 2:893–898, 1996). We have now analyzed in more detail the antigen-specific immune CD4⁺- and CD8⁺-T-cell responses induced in BALB/c mice vaccinated with Ag85A DNA and have compared these responses to those generated by intravenous infection with M. tuberculosis. T-cell-epitope mapping, as measured by interleukin-2 and gamma interferon secretion from splenic T cells restimulated in vitro with synthetic 20-mer peptides spanning the complete mature sequence of Ag85A, demonstrated that DNA vaccination stimulated a stronger and broader T-cell response than did M. tuberculosis infection. Moreover, elevated cytotoxic T lymphocyte (CTL) activity against Ag85A-transfected and peptide-pulsed P815 target cells could be generated exclusively by vaccination with plasmid DNA, not following M. tuberculosis infection. By using DNA vaccination, three Ag85A CTL epitopes with predicted major histocompatibility complex class I binding motifs were defined. One of them was previously reported as a dominant, promiscuously recognized T-cell epitope in healthy humans with primary infections. These data strengthen the potential of DNA vaccination with respect to inducing antituberculous immunity in humans.Tuberculosis remains a major health problem affecting millions of people worldwide (2). Combination chemotherapy is very effective in curing this disease, but unfortunately, the treatment is long and expensive and requires stringent compliancy to avoid the development of multidrug-resistant forms. The only tuberculosis vaccine currently available is an attenuated strain of Mycobacterium bovis, termed bacillus Calmette-Guérin (BCG). BCG continues to be widely administered to children in developing countries, yet its efficacy remains controversial, particularly against the pulmonary form of the disease in adults (3). Clearly, the development of a more effective vaccine could be an effective solution to the global threat of tuberculosis.Administration of plasmid DNA expression vectors has been shown to result in protein expression in vivo, the generation of humoral and cell-mediated immune responses, and protection in animal models of infectious diseases including influenza, human immunodeficiency virus infection, bovine herpes, rabies, malaria, leishmaniasis, herpes simplex, and cottontail papilloma (10, 32). Therefore, DNA vaccination seems to be a broadly applicable technique for generating protective immune responses against infectious pathogens without the need for live organisms, replicating vectors, or adjuvants.Recently, we and others have shown that vaccination with plasmid DNA containing Mycobacterium tuberculosis genes encoding hsp65 (31), the 38-kDa PstS-1 homolog (35), and the fibronectin-binding antigen 85 (Ag85) complex (18) is an effective means for inducing protective immunity in animal models. The three components of the Ag85 complex, a 30-32-kDa family of proteins (Ag85A, Ag85B, and Ag85C), constitute a major portion of the secreted proteins in M. tuberculosis and BCG culture filtrate (33). The bacteriostatic drug isoniazid enhances the expression of this Ag85 complex in M. tuberculosis culture filtrate (15) and has been described as possessing enzymatic trehalose mycolyltransferase activity (1a). The Ag85 complex induces strong T-cell proliferation, gamma interferon (IFN-γ) production, and cytotoxic T lymphocyte (CTL) activity in most healthy individuals infected with M. tuberculosis or Mycobacterium leprae and in BCG-vaccinated mice and humans (16, 22, 25, 28, 30), making it a promising candidate as a protective antigen. Genetic vaccination with plasmid DNA encoding the Ag85A component of the Ag85 complex (Ag85A DNA) was found to generate robust specific Th1-type helper T-cell responses and CD8-mediated cytotoxic T-cell responses and induced protection in a mouse model against aerosol or intravenous M. tuberculosis challenge (reference 18 and unpublished data). Vaccination with plasmid DNA encoding the Ag85B component but not the Ag85C component was also found effective for generating strong Th1- and CD8⁺-mediated immune responses (23).Here, we have analyzed in detail the immunogenicity of a DNA vaccine carrying the gene encoding Ag85A in comparison with the immunogenicity of intravenous M. tuberculosis infection. We show that in BALB/c mice, Ag85A DNA vaccination is more effective than live infection at inducing cellular immune responses, including Th1-type helper T-cell responses and CTL activity, against Ag85. Hence, DNA vaccination holds promise as a method of inducing immunity in humans.     

Autologous Hsp70 Induces Antigen Specific Th1 Immune Responses in a Murine T-Cell Lymphoma

Heat Shock protein-70 derived from tumor cells is highly immunogenic and induces specific anti-tumor immune response by directly activating cytotoxic CD8⁺ T cells. Additionally, Hsp70 is known to be a strong activator of antigen presenting cells and therefore, up regulates the production of pro-inflammatory cytokines and chemokines. In this study, we have shown the effect of tumor-derived Hsp70 on the induction of delayed type hypersensitivity reaction in a T cell lymphoma bearing mice. The autologous Hsp70 augments contact hypersensitivity and delayed type hypersensitivity responses in mice challenged with allergen in vehicle and antigens respectively. The adoptive transfer of splenocytes derived from Hsp70 immunized mice is able to enhance delayed type hypersensitivity response in antigen challenged normal and DL-bearing host. Furthermore, adoptive transfer of macrophages incubated with autologous Hsp70 also enhances DTH reactivity in mice. The pro-inflammatory cytokines and C-C chemokines are found to be elevated in the DTH footpad extract of antigen challenged normal and DL-bearing mice. Increased production of IFN-γ and MIP-1α± suggest that autologous Hsp70 augments the recruitment of antigen specific Th1 cells, which further secretes pro-inflammatory cytokines and C-C chemokines mediating the hypersensitivity reaction upon challenge with antigens.     

The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice.     

Expansion of a Novel Pulmonary CD3− CD4+ CD8+ Cell Population in Mice during Chlamydia pneumoniae Infection

A new pulmonary T-cell-like lymphocyte population with the phenotype CD3⁻ CD4⁺ CD8⁺ was discovered in mice. CD4⁺ CD8⁺ but CD3⁺ cells among murine intestinal intraepithelial lymphocytes have previously been described. We describe herein a dramatic expansion of the CD3⁻ CD4⁺ CD8⁺ cell population in response to experimental respiratory infection. After intranasal Chlamydia pneumoniae infection, CD4⁺ CD8⁺ cells became transiently the dominant lymphocyte type (maximum of 87% of all lymphocytes) in the lungs of NIH/S mice but remained virtually undetectable in spleen and blood. The enrichment of these cells was not a C. pneumoniae-specific event, since infection of NIH/S mice with influenza A virus also resulted in an increase in the number of CD4⁺ CD8⁺ cells (maximum of 42% of all lymphocytes). In addition to outbred NIH/S mice, two other mouse strains were studied: BALB/c (H-2^d) and C57BL/6 (H-2^b). C. pneumoniae-infected BALB/c mice responded with an intermediate increase in the number of CD4⁺ CD8⁺ cells in lungs, whereas C57BL/6 mice did not respond. The double-positive CD4⁺ CD8⁺ cells lacked a major part of the T-cell receptor complex, being both CD3⁻ and TCR αβ⁻. However, when they were stimulated in vitro with a T-cell mitogen, they responded by proliferation but did not secrete gamma interferon. The dramatic expansion of this cell population at the infection site suggests an active role for them in respiratory infection, but the specification of this requires further study.     

Differential regulation of IL-13 and IL-4 production by human CD8+ and CD4+ Th0, Th1 and Th2 T cell clones and EBV-transformed B cells

In the present study, the requirements and characteristics forthe production of IL-13 by human T cells, T cell clones andB cells were determined and compared with those of IL-4. IL-13was produced by human CD4⁺ and CD8⁺ T lymphocyte subsets isolatedfrom peripheral blood mononuclear cells and by CD4⁺ and CD8⁺T cell clones. CD4⁺ T cell clones belonging to T_h0, T_h1-likeand T_h2-like subsets produced IL-13 following antigen-specificor polyclonal activation. In addition, EBV-transformed B celllines expressed IL-13 mRNA and produced small amounts of IL-13protein. Expression of IL-13 mRNA and production of IL-13 proteinby peripheral blood T cells and T cell clones was induced rapidlyand was relatively long lasting, whereas IL-4 production bythese cells was transient In addition, IL-13 mRNA expressionwas induced by modes of activation that failed to induce IL-4mRNA expression. IL-13 shares many biological activities withIL-4 which Is compatible with the notion that the IL-13 andIL-4 receptors share a common component required for signaltransduction. However, IL-13 lacks the T cell-activating propertiesof IL-4. Here we have shown that this is related to the factthat T cells fall to bind radiolabeled IL-13 and do not expressthe IL-13-speclflc receptor component Taken together, theseresults indicate that the differences In expression and biologicalactivities of IL-4 and IL-13 on T cells may have consequencesfor the relative roles of these cytokines In the immune response.     

Oridonin promotes CD4+/CD25+ Treg differentiation, modulates Th1/Th2 balance and induces HO-1 in rat splenic lymphocytes

Objective and design: Oridonin is an ent-kaurene diterpenoid extracted from Isodon Serra, and we have previously demonstrated its immunosuppressive effect. Our goal was to study how Oridonin impacts CD4⁺/CD25⁺ regulatory T cells (Tregs) and Th1/Th2 balance, as well as its effect on the anti-inflammatory target HO-1. Material: Splenic lymphocytes were prepared from male 6–8-week-old SD rats. Treatment: Cells were cultured in four groups as Oridonin-L (Oridonin 12.5 μmol/l), Oridonin-H (Oridonin 25 μmol/l), Cobalt protoporphyrin (Copp 50 μmol/l) and control (DMSO) with stimulation of ConA (5 μg/ml) for 48 h or with no stimulation for 12 h. Method: We set up a model of Th1 polarization in vitro using ConA stimulation; ratios of CD4⁺/CD25⁺ Tregs (confirmed by the expression of Foxp3) were measured by flow cytometry, and levels of IL-2, IFN-γ, TGF-β and IL-10 were measured by ELISA. In addition, HO-1 expression was measured without stimulation with ConA by RT-PCR and Western blotting, and HO-1 level in vitro was then measured by enzyme activity assay. p<0.05 (t-test) was taken as the level of statistical significance. Result: Oridonin promoted differentiation towards CD4⁺/CD25⁺ Tregs, inhibited IL-2 and IFN-γ but induced TGF-β and IL-10, thus rectifed the Th1 polarization. Moreover, Oridonin induced the expression of HO-1 mRNA and protein, and HO-1 activity in vitro was enhanced accordingly. Conclusion: The results suggest that Oridonin has a distinct effect on promoting CD4⁺/CD25⁺ Treg differentiation and modulating Th1/Th2 balance, and this effect may be achieved via inducing the anti-inflammatory target HO-1. Received 16 October 2007; accepted by G. Wallace 26 November 2007     

Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases

CD4⁺ T cells play a key role in regulating immune system function. When these regulatory processes go awry, organ-specific autoimmune diseases may develop. Here, Roland Liblau, Steven Singer and Hugh McDevitt explore the thesis that a particular subset of CD4⁺ T cells, namely T helper 1 (Th1) cells, contributes to the pathogenesis of organ-specifc autoimmune diseases, while another subset, Th2 cells, prevents them.     

Differential CD28 and inducible costimulatory molecule signaling requirements for protective CD4+ T-cell-mediated immunity against genital tract Chlamydia trachomatis infection

Th1 cells and gamma interferon (IFN-gamma) production play critical roles in protective immunity against genital tract infections by Chlamydia trachomatis. Here we show that inducible costimulatory molecule (ICOS)(-/-) mice develop greatly augmented host resistance against chlamydial infection. Protection following a primary infection was characterized by strong Th1 immunity with enhanced CD4(+) T-cell-mediated IFN-gamma production in the genital tract and high expression of T-bet in the draining para-aortic lymph node. This Th1 dominance was associated with low expression of interleukin 10 (IL-10) mRNA in the uteruses of protected ICOS(-/-) mice. By contrast, CD28(-/-) mice were severely impaired in their adaptive immune response, demonstrating a lack of CD4(+) T cells and IFN-gamma in the genital tract, with a substantial delay in bacterial elimination compared to that seen in wild-type (WT) mice. Upon reinfection, WT mice exhibited a transient local infection with evidence of regulatory T-cell (Treg)/Foxp3 mRNA and a more balanced Th1 and Th2 response in the genital tract than ICOS(-/-) mice, whereas 90% of the latter mice developed sterile immunity, poor expression of local Treg/Foxp3 mRNA, and macroscopic signs of enhanced local immunopathology. Therefore, different requirements for CD28 signaling and ICOS signaling clearly apply to host protection against a genital tract infection by C. trachomatis. Whereas, CD28 signaling is critical, ICOS appears to be dispensable and can have a dampening effect on Th1 development by driving Th2 immunity and anti-inflammation through IL-10 production and promotion of the Foxp3(+) Treg populations in the genital tract. Both the CD28-deficient and the ICOS-deficient mice demonstrated poor specific antibody production, supporting the fact that antibodies are not needed for protection against genital tract chlamydial infections.     

Th1 cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T cells

The pathogenesis of pulmonary sarcoidosis has been related to an increased production of Th1-like cytokines. However, cytokine expression in sarcoidosis has not been systematically studied at a single-cell level. We therefore investigated the expression of IL-2, IL-4, IL-13, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) intracellularly in bronchoalveolar lavage (BAL) and peripheral blood CD3+ T lymphocytes from patients with pulmonary sarcoidosis (radiologic stage II-III, n = 8) and normal controls (n = 9) by flow cytometry. In contrast to IL-4 and IL-13, the percentage of T lymphocytes expressing intracellular IL-2 (49.3 +/- 21.3% versus 14.5 +/- 15.6%), IFN-gamma (75.5 +/- 14.9% versus 32.6 +/- 18.7%) and TNF-alpha (68.3 +/- 18.7% versus 36.8 +/- 20.8%) was significantly higher in patients with sarcoidosis than in normal controls (each P < 0.005). In contrast to BAL lymphocytes, expression of these cytokines in peripheral blood lymphocytes did not differ between patients with sarcoidosis and normal controls. Close correlations were observed between the percentages of BAL lymphocytes expressing intracellular IL-2, IFN-gamma and TNF-alpha, but not for IL-4 or IL-13. Analysis of the expression of these cytokines in T lymphocyte subsets revealed IL-2, IFN-gamma, and TNF-alpha in CD4+ as well as CD8+ T lymphocytes, suggesting a contribution of TC1 cells to the production of proinflammatory cytokines in sarcoidosis. We conclude that a Th1-like cytokine pattern can be observed in CD4+ as well as in CD8+ BAL T lymphocytes in patients with pulmonary sarcoidosis.     

Altered Th1/Th2 commitment in human CD4+ T cells with ageing

The human immune system undergoes continuous remodelling with the advancement of age. Since age-associated functional alterations in the immune system could be caused by a possible change in helper T cell regulation in elderly subjects, we comparatively studied the function of CD4+ T cells in peripheral blood obtained from both young and old healthy volunteers. Upon cell activation by phorbol myristate acetate and ionomycin, the proportion of CD4+ T cells containing interferon-gamma (IFN-gamma) was found to be greater in the old subjects. Utilizing a co-culture system, which activated CD4+ T cells via the TCR/CD3 complex and CD28, we found that CD4+ T cells from the old subjects secreted more IFN-gamma and IL-2, but less IL-4, than those from the young subjects. Upon cell activation by co-culture, CD4+ T cells from the old subjects expressed more CD26, CD40L, and LFA-1, but less CD30, than those from the young. These results together suggest that the microenvironment in which CD4+ T cells develop in older people may cause production of more cells committed to Th1 than that in younger subjects.