Differential activation of Brucella-reactive CD4+ T cells by Brucella infection or immunization with antigenic extracts.

In order to induce acquired cellular resistance to facultative bacterial pathogens, infection with live organisms is required. We have previously demonstrated that spleen cells from Brucella-infected mice produced gamma interferon (IFN-gamma) and interleukin-2 (IL-2) in response to Brucella antigens in vitro, while spleen cells from mice immunized with soluble Brucella proteins (SBP) produced substantial amounts of IL-2 but no detectable amount of IFN-gamma. In this study, we further analyzed the response of T cells from Brucella-infected mice and SBP-immunized mice and demonstrated that CD4(+)-enriched cells from SBP-immunized mice also produced significant amounts of IL-4, which was not detected in bulk cultures of spleen cells from infected mice. Limiting dilution analysis showed that infection resulted in a higher precursor frequency of IFN-gamma-producing CD4+ T cells and a lower precursor frequency of IL-4-producing CD4+ T cells, while immunization with SBP resulted in a higher precursor frequency of IL-4-producing cells and a very low frequency of IFN-gamma-producing cells. The precursor frequencies of IL-2-producing cells for the two groups were similar. Furthermore, IFN-gamma-producing CD4+ T cells from infected donor mice were capable of mediating resistance against challenge infection in recipient mice, but IL-4-producing CD4+ T cells from immunized mice failed to do so. These results indicate that the form of antigen has a profound influence on the outcome of the immune response. The results are discussed in light of the supposed dichotomy between Th1 and Th2 cytokine responses.     

The influence of dietary protein antigen on Th1/Th2 balance and cellular immunity.

Dietary administration of ovalbumin (OVA) antigen (Ag) into OVA-specific T cell receptor alphabeta-transgenic (TCR-Tg) mice resulted in the induction of activated CD4+ Th cells expressing CD69 early activation Ag. However, the number of CD4+ Th cells rather decreased by dietary administration of OVA antigen. The production of Th1-cytokines such as IFN-gamma and IL-2 markedly reduced in spleen of OVA-fed mice compared to mice fed with normal diet. In sharp contrast, the production of Th2-cytokine, IL-4 greatly increased in spleen of OVA-fed mice though the number of CD4+ T cells decreased to less than 10% of control mouse spleen. The decrease of IFN-gamma production and the increase of IL-4 production by CD4+ T cells was demonstrated at a single cell level by intracellular cytokine staining analysis. Moreover, such a polarized cytokine production pattern was also demonstrated using highly purified CD4+ T cells obtained from mice fed with OVA. In addition to the decrease of Th1-cytokine production, TCR-Tg mice fed with OVA-containing diet showed greatly reduced in vivo generation of NK cells, LAK cells and CTL. These results suggested that dietary protein antigen caused the polarization of Th1/Th2 balance into Th2-dominant immunity and inhibited cellular immunity.     

Islet-cell antigen-reactive T cells show different expansion rates and Th1/Th2 differentiation in type 1 diabetic patients and healthy controls

The low frequency of islet-cell antigen-reactive T cells in type 1 diabetes makes their direct measurement difficult. Commonly used in vitro expansion could alter in vivo frequencies and Th1/Th2 differentiation states. Using IFN-gamma/IL-4 double color ELISPOT, we tested longitudinally the reactivity of PBMC from HLA-matched diabetic patients and healthy controls to GAD65, IA-2, and proinsulin peptides ex vivo and after in vitro culture. The peptide-reactive T cells showed IFN-gamma bias in the patients' PBMC in the primary assay. During in vitro culture, both IFN-gamma- and IL-4-producing cells were induced in controls, suggesting that the precursor cells were uncommitted naive T cells in vivo. In contrast, in diabetic patients, the ex vivo IFN-gamma response was conserved during culture, suggesting their Th1 commitment. Using CFSE-dye-dilution, we demonstrate that naive T cells expand in vitro at a faster rate than memory cells, which might account for the differences in expansion rates between diabetic patients and controls.     

Helper CD4+ T cells for IgE response to a dietary antigen develop in the liver

BACKGROUND: Although T-cell responses to food antigens are normally inhibited either by deletion, active suppression, or both of antigen-specific T cells, T helper cells for IgE response to a food antigen still develop by unknown mechanisms in a genetically susceptible host. OBJECTIVE: We determined the site at which those IgE helper T cells develop. METHODS: We administered ovalbumin (OVA) orally to DO11.10 mice and studied CD4+ T cells in Peyer's patches, the spleen, and the liver. Helper activity for IgE response was assessed by adoptively transferring those CD4+ T cells to naive BALB/c mice, followed by systemic immunization with OVA. RESULTS: OVA-specific CD4+ T cells were deleted by cell death in the liver and Peyer's patches of DO11.10 mice fed OVA. OVA-specific CD4+ T cells that survived apoptosis in the liver expressed Fas ligand and secreted IL-4, IL-10, and transforming growth factor beta(1). CD4+ T cells producing IFN-gamma were deleted in the liver by repeated feeding of OVA. On transfer of CD4+ T cells to naive mice and systemic immunization with OVA, a marked increase in OVA-specific IgE response developed only in the mice that received hepatic CD4+ T cells from OVA-fed mice, the effect of which was not observed in the recipients of hepatic CD4+ T cells deficient in IL-4. In addition, significant suppression of delayed-type hypersensitivity and IgG(1)/IgG(2a) responses to OVA was observed in the recipients of hepatic CD4+ T cells, and this suppression required Fas/Fas ligand interaction. CONCLUSION: Together, these results suggested that a food antigen might negatively select helper T cells for IgE response to the antigen by preferential deletion of T(H)1 cells in the liver.     

Failure of rIL-12 administration to inhibit established IgE responses in vivo is associated with enhanced IL-4 synthesis by non-B/non-T cells

Administration of rIL-12 offers a widely successful tactic for preferential induction of type 1 immune responses in vivo. Its use to modulate ongoing cytokine or effector responses has proven to be substantially more difficult. Immediate hypersensitivity is the most common human immunologic disease. Here, rIL-12 was administered to C57Bl/6 and outbred CD1 mice with ongoing ovalbumin (OVA)-specific IgE responses in an attempt to redirect established type 2 cytokine and antibody production. Despite use of a broad range of treatment protocols for >4 months following initial immunization, recall IgE responses were consistently unaffected. rIL-12-treated mice exhibited strong in vivo and in vitro IFN-gamma responses, increased approximately 40-fold relative to controls, but also markedly enhanced (15- to 20-fold) OVA-specific IL-4 production. CD4 T cell function was successfully transformed from a type 2- to a type 1-dominated pattern following long-term IL-12 administration in vivo, as measured by strongly reduced IL-4 and IL-10 responses in antigen-stimulated primary culture, and 5-fold reductions in the frequencies of IL-4- and IL-10-producing OVA-specific CD4 T cells. However, chronically rIL-12-treated mice exhibited increased numbers of non-B/non-T cells that when re-stimulated with specific allergen, produce IL-4 at levels 20-fold higher than did CD4 T cells while IL-13 responses are unaffected. Collectively, the data indicate that even effectively shifting CD4 T cell activation from a type 2- to a type 1-dominated response does not in itself lead to altered effector (IgE) responses upon antigen re-exposure.     

Leishmania donovani-reactive Th1- and Th2-like T-cell clones from individuals who have recovered from visceral leishmaniasis.

Infections in humans by Leishmania donovani parasites can result in a fatal disease, visceral leishmaniasis (VL), or in a self-limiting asymptomatic infection. In murine models of the infection employing Leishmania major, the course of the disease can be directed into a VL-like syndrome by interleukin-4 (IL-4)-producing Th2 cells, or cure may result by Th1 cells secreting gamma interferon (IFN-gamma). The present study examined the potential of human T cells to generate Th1 or Th2 responses to L. donovani. The profiles of IFN-gamma, IL-4, and lymphotoxin secretion after antigen stimulation were analyzed in a panel of L. donovani-reactive CD4+ human T-cell clones generated from individuals who had recovered from VL after antimonial treatment. Two of the T-cell clones produced large amounts of IL-4 without production of IFN-gamma, seven clones produced both IFN-gamma and IL-4, and eight produced only IFN-gamma. This is the first report of a Th1- and Th2-type response in human leishmaniasis. These results suggest that in analogy with murine models, there is a dichotomy in the human T-cell response to L. donovani infections. Preferential activation of IL-4-producing Th2-like cells may be involved in the exacerbation of human VL, whereas activation of IFN-gamma-producing Th1 cells may protect the host from severe disease. Identification of leishmanial antigens activating one or the other type of T cells will be important in the development of vaccines against leishmaniasis.     

Modulation of naive CD4+ T-cell responses to an airway antigen during pulmonary mycobacterial infection

During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.     

IFN-gamma-producing CD45RA+CD8+ and IL-10-producing CD45RA-CD4+ T cells generated in response to LACK in naive subjects never exposed to Leishmania

Leishmania guyanensis (L.g.)-specific CD8+ T cells can be isolated from PBMC of subjects who have never been previously exposed to Leishmania. Cells that produce IFN-gamma in response to live L.g. are generated from naive CD45RA+CD8+ T cells. The generation of L.g.-specific CD8+ T cells requires the presence of whole L.g. or UV-irradiated parasite but not the soluble antigens from L.g. promastigotes. The IFN-gamma-producing T cells recognize a specific antigen, the Leishmania homologue of receptors of activated C kinases (LACK) and this antigen but not live L.g. can produce a strong IL-10 response in CD45RA-CD4+ memory T cells from naive subjects. A single epitope (amino acid 156-173) is found to induce the IL-10 synthesis. While the IFN-gamma-producing cells are present among CD45RA+CD8+ T cells that are CD62L-CDR7- and CLA-, the LACK-reactive IL-10-producing cells are CD4+ T cells that are CD62L+CCR7- and CLA-.     

Oral tolerance in T cells is accompanied by induction of effector function in lymphoid organs after systemic immunization

The physiological ramifications of oral tolerance remain poorly understood. We report here that mice fed ovalbumin (OVA) exhibit oral tolerance to subsequent systemic immunization with OVA in adjuvant, and yet they clear systemic infection with a recombinant OVA-expressing strain of Salmonella enterica serovar Typhimurium better than unfed mice do. Mice fed a sonicated extract of S. enterica serovar Typhimurium are also protected against systemic bacterial challenge, and the protection is Th1 mediated, as feeding enhances clearance in interleukin-4-null (IL-4(-/-)) and IL-10(-/-) mice but not in gamma interferon-null (IFN-gamma(-/-)) mice. When T-cell priming in vivo is tracked temporally in T-cell receptor-transgenic mice fed a single low dose of OVA, CD4 T-cell activation and expansion are restricted largely to mucosal lymphoid organs. However, T cells from spleens and peripheral lymph nodes of fed mice proliferate and secrete IFN-gamma when restimulated with OVA in vitro, indicating the presence of primed T cells in systemic tissues following oral exposure to antigen. Nonetheless, oral tolerance can be observed in the fed mice as reduced recall responses following subsequent systemic immunization with OVA in adjuvant. Soluble OVA administered systemically has similar effects in vivo, and the "tolerance" seen in both cases can be partially reversed if the initial priming is made more immunogenic. Together, the results indicate that antigen exposure under poor adjuvantic conditions, whether oral or systemic, may lead to T-cell commitment to effector rather than proliferative capabilities, necessitating a reassessment of therapeutic modalities for induction of oral tolerance in allergic or autoimmune states.     

Th1 and Th2 cell involvement in immune response to Salmonella typhimurium porins.

In understanding the regulation of the specific immune response to Salmonella typhimurium, the role of a surface major component (porins) was studied. In this study we demonstrate that purified porins are able to induce a different response to that induced by the porins present on the S. typhimurium cell surface. Porin-treated or orally infected mice show anti-porin antibodies with bactericidal activity. The complete adoptive transfer of resistance to S. typhimurium is achieved only using splenic T cells from survivor mice after experimental infection. After stimulation with specific antigen in vitro CD4+ cells from porin-immunized mice released large amounts of interleukin-4 (IL-4), at a time when CD4+ cells from S. typhimurium-infected mice predominantly secreted interferon-gamma (IFN-gamma). Limiting dilution analysis showed that infection resulted in a higher precursor frequency of IFN-gamma-producing CD4+ T cells and a lower precursor frequency of IL-4-producing CD4+ T cells, while immunization with porins resulted in a higher precursor frequency of IL-4-producing cells and a low frequency of IFN-gamma-producing cells. Analysis of polymerase chain reaction-amplified cDNA from the spleens of infected mice revealed that IFN-gamma, IL-2 and IL-12 p40 mRNA were found 5 days after in vitro challenge and increased after 15 days; IL-10 expression was barely present after both 5 and 15 days, while IL-4 mRNA expression was not detected. In immunized mice, the IL-4 mRNA expression increased after 15 days, IFN-gamma mRNA expression disappeared entirely after 15 days, while IL-2, IL-10 and IL-12 mRNA remained relatively unchanged.     

Relaxin favors the development of activated human T cells into Th1-like effectors.

Differentiation of naive CD4+ helper T (Th) cells into Th1 or Th2 effectors, as characterized by their opposite pattern of cytokine production, can be influenced by several factors, including hormones. In this study, we demonstrate that porcine relaxin, at concentrations ranging from 10(-10) to 10(-6) M, favors the in vitro development of human antigen-specific T cells into Th1-like effectors and enhances both IFN-gamma mRNA expression and IFN-gamma production by established human T cell clones. The promoting effect of relaxin on the development of IFN-gamma-producing cells was not due to a relaxin-induced release of IL-12 and/or IFN-alpha by antigen-presenting cells. These results suggest that relaxin may contribute to the regulation of the immune homeostasis during pregnancy and may also play some role in counteracting Th2-dominated disorders.     

Synovial fluid T cells from patients with rheumatoid arthritis are refractory to the T helper type 2 differentiation-inducing effects of interleukin-4

The balance between T helper type 1 (Th1) and Th2 cytokines is thought to be important in the initiation and outcome of autoimmune diseases. The goal of the present study was to compare the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by synovial fluid (SF) and peripheral blood (PB) CD4+ and CD8+ cells from patients with rheumatoid arthritis (RA) using three-colour immunofluorescence staining and flow cytometry, and to investigate the capacity of IL-4, IL-10 and IL-12 to modify the cytokine production profile of SF T cells. The frequency of IFN-gamma-producing CD4+ and CD8+ cells was significantly increased in SF when compared with PB. In contrast to IFN-gamma, the expression of IL-4 in SF and PB T cells was comparable. The majority of IL-4-producing cells in SF belonged to Th0/T cytotoxic (Tc) type 0 phenotype, whereas there were significantly more Th2/Tc2 cells in PB than in SF. Interestingly, IL-4 was unable to induce differentiation of non-adherent SF mononuclear cells (SFMC) into Th2 cells, whereas PB mononuclear cells (PBMC) under similar culture conditions differentiated into cells producing high levels of IL-4, IL-10 and IL-13. In contrast, there were no major differences in the effects of IL-10 and IL-12 on the cytokine production profile of SFMC when compared with PBMC. Taken together, the present results suggest that SF T cells from patients with RA are terminally differentiated into Th1/Tc1-like phenotype, and Th2/Tc2 differentiation-inducing agents, such as IL-4, may not be able to reverse the inflammatory process occurring in the joints.     

Instruction of naive CD4+ T-cell fate to T-bet expression and T helper 1 development: roles of T-cell receptor-mediated signals

Using T-cell receptor (TCR) transgenic mice, we demonstrate that TCR stimulation of naive CD4(+) T cells induces transient T-bet expression, interleukin (IL)-12 receptor beta2 up-regulation, and GATA-3 down-regulation, which leads to T helper (Th)1 differentiation even when the cells are stimulated with peptide-loaded I-A(b)-transfected Chinese hamster ovary cells in the absence of interferon-gamma (IFN-gamma) and IL-12. Sustained IFN-gamma and IL-12 stimulation augments naive T-cell differentiation into Th1 cells. Intriguingly, a significant Th1 response is observed even when T-bet(-/-) naive CD4(+) T cells are stimulated through TCR in the absence of IFN-gamma or IL-12. Stimulation of naive CD4(+) T cells in the absence of IFN-gamma or IL-12 with altered peptide ligand, whose avidity to the TCR is lower than that of original peptide, fails to up-regulate transient T-bet expression, sustains GATA-3 expression, and induces differentiation into Th2 cells. These results support the notion that direct interaction between TCR and peptide-loaded antigen-presenting cells, even in the absence of T-bet expression and costimulatory signals, primarily determine the fate of naive CD4(+) T cells to Th1 cells.     

Potentiation of antigen-specific, Th1 immune responses by multiple DNA vaccination with an ovalbumin/interferon-gamma hybrid construct.

The preferential differentiation of T helper (Th) cells to Th1 or Th2 subsets is important with respect to susceptibility or resistance to particular infections, or to autoimmune diseases and allergic diseases. To more effectively drive immune responses toward antigen-specific Th1 responses, we constructed a mammalian expression vector (pOVA/IFN-gamma) carrying a hybrid gene in which the ovalbumin (OVA) (a model antigen) cDNA was covalently linked to murine interferon-gamma (IFN-gamma) cDNA. Intramuscular injection of BALB/c mice with the pOVA/IFN-gamma DNA increased both the production of OVA-specific IFN-gamma by CD4+ T cells and the ratio of anti-OVA immunoglobulin G (IgG) 2a to IgG1 isotypes, while the injection with the pOVA alone, or with the mixture of the pOVA and pIFN-gamma, caused no or little increase. Furthermore, the OVA-specific, Th1 immune responses were dramatically augmented by multiple injections with the pOVA/IFN-gamma DNA. These studies indicate that the direct linkage of an OVA gene to an IFN-gamma gene in the expression plasmid is required for efficiently confining the Th1 effects of IFN-gamma to the OVA-specific cells, and the linkage effect of the OVA/IFN-gamma DNA can be potentiated by multiple vaccination.     

Multiparameter grouping delineates heterogeneous populations of human IL-17 and/or IL-22 T-cell producers that share antigen specificities with other T-cell subsets

The ontogenic relationship between pro-inflammatory populations of interleukin-17 (IL-17A)- and/or IL-22-producing T cells and other T-cell subsets is currently unclear in humans. To appreciate T helper cell-lineage commitment, we combined cytokine production profiles of in vitro expanded T-cell clones with T-cell receptor (TCR) clonotypic signatures. Moreover, ex vivo cytokine production profiles at the single-cell level were analyzed using an original approach based on the hierarchical cluster analysis of multiparametric flow cytometry data. These combined approaches enabled the delineation of distinct functional T-cell subsets, including Th1, Th2, Tr1, Th17 cells and a highly polyfunctional IL-22-producing T-cell population. Cluster analysis highlighted that the IL-22-producing T-cell population should be considered independently from the Th17 and Th1 subsets, although it was more closely related to the former. In parallel, we observed extensive TCRαβ sharing across all five subsets defined. The strategy described here allows the objective definition of cellular subsets and an unbiased insight into their similarities. Together, our results underscore the ontogenic plasticity of CD4(+) T-cell progenitors, which can adopt a differentiation profile irrespective of antigen specificity.     

Induction of IFN-gamma-producing CD4+ natural killer T cells by Mycobacterium bovis bacillus Calmette Guérin

The CD4+ natural killer (NK)T cells in the liver are potent IL-4 producers and hence may promote Th2 cell development. Following Mycobacterium bovis bacillus Calmette Guérin (BCG) infection, IL-4-producing CD4+ NKT cells become undetectable in liver mononuclear cells of normal density (interface between 40 and 70% Percoll) by flow cytometry. The present study shows that M. bovis BCG infection changes the density of liver CD4+ NKT cells and shifts cytokine production from IL-4 to IFN-gamma. The number of CD4+ NK1+ TCR alpha/beta(intermediate) cells increased in the low-density fraction (<40% Percoll density gradient) in parallel to the reduction of this cell population in the fraction of normal density. The number of IL-4-producing cells, however, was small and high frequencies of IFN-gamma-secreting cells were identified in the low-density fraction after TCR/CD3 ligation. Accordingly, selected low-density CD4+ NKT cells encompassed high numbers of IFN-gamma producers and minute numbers of IL-4-secreting cells. Induction of low-density CD4+ NKT cells by M. bovis BCG was abrogated by endogenous IL-12 neutralization which also caused increased bacterial growth in the liver. We assume that M. bovis BCG infection changes cytokine secretion by the CD4+ NKT cell population from IL-4 to IFN-gamma through IL-12 induction. Thus, CD4+ NKT cells may contribute to host resistance against intracellular bacteria prior to conventional IFN-gamma-producing Th1 cells.     

Th2 cells shape the differentiation of developing T cell responses during interactions with dendritic cells in vivo

During priming, naive CD4(+) Th cells differentiate into cells that produce either IFN-gamma or IL-4. Even though the cascade of pathways that induces IL-4-producing Th2 cells has been determined in vitro, the signals promoting Th2 differentiation under physiological conditions remain enigmatic, especially the natural role of the single most important Th2-inducing signal,IL-4. Using Th2 and naive Th cells, each expressing a distinct transgenic TCR, here we show that Th2 cells migrate with the same dynamics as naive Th cells in draining lymph nodes and bind to the same DC, when driven by antigen in complete Freund's adjuvant (CFA). Th2-cell-derived IL-4 deviates CFA-induced Th1 development toward a Th2 phenotype, if both cell populations co-localize in the same T cell area, and are activated simultaneously. Thus, intranodal Th2 cells directly influence Th cell differentiation in vivo, but only under restricted conditions. These findings have implications for the design of cytokine-based therapies and explain the spreading of Th2 responses to multiple aeroallergens in allergic asthma, where naive Th and Th2 cells co-localize in lung-draining lymph nodes.     

The enhanced antigen-specific production of cytokines induced by pertussis toxin is due to clonal expansion of T cells and not to altered effector functions of long-term memory cells

Pertussis toxin (PT) has been shown to act as an adjuvant that enhances the production of both Th1 and Th2 cytokines to coinjected protein antigens. It has remained unresolved, however, how PT affects the clonal sizes, long-term effector functions, and Th1/Th2/Th0 differentiation of the T cell responses induced. We have studied the effects of PT on the development of the CD4(+) T cell response to a prototypic antigen, hen eggwhite lysozyme (HEL). HEL injection with incomplete Freund's adjuvant (IFA) resulted in an IFN-gamma(-)/IL-5(+) Th2 recall response. In comparison, co-administration of PT with HEL:IFA enhanced the frequencies of IL-5-producing T cells up to eightfold, and induced the differentiation of high frequencies of IFN-gamma-producing CD4(+) T cells. The results showed that the IFN-gamma and IL-5 produced, originated from clonally expanded Th1 and Th2, but not Th0 cells, and that the effector functions of long-term memory cells were unaffected. Adoptive transfer experiments suggested that PT mediated these effects via activation of APC, not by acting on the T cells directly. The effects of PT on the developing T cell response required the presence of the holotoxin (A- and B-subunit); the individual subunits did not show adjuvant effects. The data suggest that PT enhanced cytokine production by promoting differentiation and vigorous clonal expansion of Th1 and Th2 cells via activation of APC.