Th1 Cell Reactivity Research Articles

Low CD46 expression on activated CD4+ T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors.

Previous research showed that the intracellular complement system, with CD46 as its central molecule, regulates the Th1 response associated with IFN-γ production and transition to a type 1 regulatory response (Tr1) characterized by IL-10 production. This transition can be influenced by a vitamin D (calcitriol), favouring a shift towards Tr1 cells and increased IL-10 production, as described in some autoimmune diseases. It is unknown whether calcitriol modulates CD46-induced Th1 response towards regulatory type 1 T cells (Tr1) in allergic eosinophilic asthma and its value in relation to reducing inflammatory response. CD4+ T cells from 58 patients with allergic eosinophilic asthma (AEA) and 49 healthy donors (HDs) were stimulated with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol in vitro for 60 h and analyzed by flow cytometry. IFN-γ and IL-10 levels in cell culture supernatants were measured using ELISA. CD4+ T cells from patients with AEA demonstrated elevated CD46 expression in both the non-activated state and under stimulation conditions with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol. Moreover, CD46 expression in AEA patients fluctuated with the pollen season, showing a significant increase during period of low pollen exposure. Calcitriol further induced CD4+Tr1 cells from in vitro generated CD4+Th1 cells in both HDs and AEA patients. However, in both cohorts were individuals (HDs: 35/49, AEA: 40/58) who responded to calcitriol with a more pronounced regulatory response. The calcitriol-induced regulatory effect manifested by a stronger surface decrease of CD46 on activated CD4+ T cells (by 40% in HDs and by 26% in AEA), accompanied by a significant inhibition of IFN-γ and increased IL-10 production (by 31% in HDs and by 85% in AEA). These individuals were termed as the CD46D group. Contrary to this, calcitriol induced an increase in CD46 expression at the CD4+ T cell surface in a minor group of HDs (14/49), and AEA patients (18/58), who were termed as the CD46I group. In CD46I group, CD4+ T cells produced less IFN-γ in comparison with CD46D group (by 33% in HDs and by 43% in AEA) and were unable to upregulate IL-10 production following stimulation with αCD3/αCD46/IL-2/Calcitriol. Our results suggest the potential existence of a key for stratifying individuals suitable for calcitriol treatment in the context of low serum vitamin D levels. After validation in clinical studies, this key could be used as an adjunctive therapy not only for patients with allergic eosinophilic asthma, but also for other diseases.

OP0092 Differential systemic and synovial TH1 cell reactivity towards escherichia coli antigens in patients with ankylosing spondylitis and rheumatoid arthritis

BackgroundAnkylosing spondylitis (AS) is associated with clinical and subclinical mucosal inflammation suggesting that commensal bacteria contribute to the pathogenesis of the disease [1,2].ObjectivesWe determined the frequency of Th1 cells reacting...

Differential synovial Th1 cell reactivity towards Escherichia coli antigens in patients with ankylosing spondylitis and rheumatoid arthritis

ObjectiveAnkylosing spondylitis (AS) is associated with clinical and subclinical mucosal inflammation, suggesting that commensal bacteria contribute to the pathogenesis of the disease.MethodsThe frequency of Th1 cells reacting towards conserved Escherichia...

Kinetics of Local Tissue and Regional Lymph Node IL‐2 and IFN‐γ Responses in Experimental Oral Mucosa and Skin Contact Sensitivity in Mice

Using ELISA, we have quantified the levels of IL-2 and IFN-gamma in the oral mucosa, ear skin and regional and distant lymph nodes in an experimental murine model of contact sensitivity (CS), induced by the hapten oxazolone (OXA). Compared to normal conditions, the levels of IL-2 peaked early (4-6 h) after hapten exposure in the hapten-exposed tissues analysed both during the first hapten exposure (sensitization) and the second (elicitation) phase, thereafter quickly to subside. The oral mucosa displayed maximal 24-fold increase in IL-2 levels after sensitization and 39-fold increase after elicitation. Respective figures for ear skin were x27 and x35 and for regional lymph nodes x8 and x9, respectively. The distant lymph nodes displayed only minor cytokine increases at any time. IFN-gamma-levels did not increase after sensitization with OXA. An increase in IFN-gamma was seen after the second exposure, peaking at 8-24 h, thereafter quickly subsiding. The oral mucosa IFN-gamma increased x14 after elicitation, the ear skin x8 and regional lymph nodes x37. The weight of the four regional lymph nodes increased from 10 to 38 mg, and the total number of cells in these lymph nodes was increased x11, peaking 48 h after the elicitation. We conclude that in CS reactions, tissue levels of IL-2 increased in buccal mucosa, ear skin and in regional lymph nodes after hapten exposure and re-exposure, IFN-gamma appeared only after re-exposure to the hapten. The increased weight of the regional lymph nodes was mainly attributed to cell proliferation. The common ectodermal origin and the similarity of the CS reactions on skin and in buccal mucosa indicate that these tissues share common immunological patterns of Th1 cell reactivity, at least in dealing with haptens like OXA.

Th1 Cell Reactivity and HLA‐DR Binding Prediction for Promiscuous Recognition of MPT63 (Rv1926c), a Major Secreted Protein of Mycobacterium tuberculosis

MPT63 (Rv1926c), a major secreted protein of Mycobacterium tuberculosis, is immunoreactive in antibody assays in humans and animals and provides protection as a combined DNA vaccine in mice. This study was undertaken to determine the reactivity of MPT63 in T helper 1 (Th1) cell assays, i.e. antigen-induced proliferation and interferon-gamma secretion, using peripheral blood mononuclear cells (PBMCs) obtained from 72 Mycobacterium bovis Bacille Calmette-Guérin vaccinated healthy subjects. PBMCs were tested with complex mycobacterial antigens and pools of synthetic peptides corresponding to MPT63, MPB70, MT24, PPE68, CFP10 and ESAT-6. The results showed that MPT63 induced moderate Th1 cell reactivity which was equivalent to the reactivity induced by other secreted antigens of M. tuberculosis, i.e. MT24 and MPB70. Furthermore, human leucocyte antigen (HLA) heterogeneity of the responding donors suggested that MPT63 was presented to Th1 cells promiscuously. Analysis of the MPT63 sequence and its peptides for binding to 51 alleles of 9 serologically defined HLA-DR molecules, using a virtual matrix-based prediction program (ProPred) showed that MPT63 sequence could bind to all the 51 alleles, whereas 9 of the 10 peptides of MPT63 were also predicted to bind promiscuously. When tested with PBMCs of HLA-DR heterogeneous donors that responded to MPT63 in interferon-gamma assays, at least 9 of the 10 peptides of MPT63 were recognized by PBMCs from HLA heterogeneous donors. These results suggested that promiscuous Th1 cell reactive epitopes are scattered throughout the sequence of MPT63, and further support the inclusion of this protein in an antigen cocktail to develop a new anti-tuberculosis vaccine.

HLA-Promiscuous Th1-Cell Reactivity of MPT64 (Rv1980c), a Major Secreted Antigen of Mycobacterium tuberculosis, in Healthy Subjects

Objective: To determine HLA-promiscuous Th1-cell reactivity of MPT64 (Rv1980c) in healthy humans. Materials and Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from healthy subjects (n = 61) and HLA typed genomically. PBMCs were tested for Th1-cell reactivity (antigen-induced proliferation and interferon-γ secretion) with complex antigens (whole cells, culture filtrate and cell walls) and single secreted antigens (Ag85B, MPT64 and MPB70) of Mycobacterium tuberculosis. In addition, culture-filtrate-induced T-cell lines were established from PBMCs of 14 donors and tested with the above antigens in Th1-cell assays. Furthermore, 3 T-cell lines were tested for cytotoxic activity against MPT64-pulsed monocytes/macrophages, and a T-cell line was analysed for HLA restriction in antigen presentation using anti-HLA class I and class II monoclonal antibodies and HLA-DR-typed antigen-presenting cells. Results: PBMCs showed strong Th1-cell reactivity with all of the complex mycobacterial antigens, whereas MPT64 induced moderate Th1-cell reactivity, which was comparable to the reactivity induced by other previously characterized secreted antigens of M. tuberculosis, i.e. Ag85B and MBP70. Furthermore, HLA heterogeneity of the responding donors suggested that MPT64 was presented to Th1 cells promiscuously. Testing of the T-cell lines confirmed that Th1 cells contributed to the promiscuous antigen-specific reactivity observed with PBMCs and exhibited cytotoxic activity against MPT64-pulsed monocytes/macrophages. In addition, a T-cell line investigated for HLA restriction analysis showed that MPT64 was presented to T cells in association with HLA-DR molecules in a promiscuous manner. Conclusion: MPT64 is promiscuously recognized by human Th1 cells with cytotoxic activity and therefore deserves consideration as a candidate vaccine against tuberculosis in humans.

155. Oral Delivery of Recombinant Vaccinia Viruses Expressing Adjuvanted Islet Autoantigens Protects NOD Mice from Autoimmune Diabetes

Oral administration of autoantigens can delay or suppress clinical disease in experimental autoimmune disorders. However, repeated feeding of large tolerogen amounts is required over long periods and is only partially effective in animals systemically sensitized to the ingested antigen. The cholera toxin B subunit adjuvant (CTB) when linked to autoantigen epitopes acts as a strong adjuvant to enhance suppression of inflammatory Th1 cell reactivity in both naive and immune animals. Enhanced suppression of inflammatory Th1 cell reactivity was demonstrated in both naive and immune NOD mice following oral inoculation with islet autoantigens coupled to CTB. To stimulate adjuvant-autoantigen fusion protein biosynthesis in the gut mucosae, we evaluated oral inoculation of juvenile NOD mice with recombinant vaccinia virus (rVV) expressing fusion genes encoding CTB linked to the pancreatic islet autoantigens proinsulin (INS) and a 55 kDa peptide from glutamate decarboxylase (GAD55). Oral inoculation with rVV significantly reduced pancreatic insulitis. Hyperglycemia in NOD mice inoculated with CTB::GAD fusion protein was reduced by 40% in comparison with rVV-GAD inoculated mice. Measurement of antibody isotype titers in rVV infected mice suggested activation of autoantigen specific Th2 lymphocytes. These experiments demonstrate the feasibility of vaccinia virus mediated delivery of adjuvanted autoantigens to the mucosae of prediabetic mice for suppression of autoimmune diabetes.

Escherichia coli heat-labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+ T cells.

The receptor-binding B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a highly stable, nontoxic protein that is capable of modulating immune responses. This study was conducted to determine whether mucosal administration of EtxB can block collagen-induced arthritis (CIA) and to investigate the mechanisms involved. Clinical arthritis in DBA/1 mice was monitored following mucosal administration of EtxB on 4 occasions. The dependence of disease prevention on receptor binding by EtxB and the associated alterations to the immune response to type II collagen (CII) were assessed. Adoptive transfer experiments and lymph node cell cocultures were used to investigate the underlying mechanisms. Both intranasal and intragastric delivery of EtxB were effective in preventing CIA; a 1-microg dose of EtxB was protective after intranasal administration. A non-receptor-binding mutant of EtxB failed to prevent disease. Intranasal EtxB lowered both the incidence and severity of arthritis when given either at the time of disease induction or 25 days later. EtxB markedly reduced levels of anti-CII IgG2a antibodies and interferon-gamma (IFNgamma) production while not affecting levels of IgG1, interleukin-4 (IL-4), or IL-10. Disease protection could be transferred by CD4+ T cells from treated mice, an effect that was abrogated upon depletion of the CD25+ population. In addition, CD4+CD25+ T cells from treated mice were able to suppress anti-CII IFNgamma production by CII-primed lymph node cells. Mucosal administration of EtxB can be used to prevent or treat CIA. Modulation of the anti-CII immune response by EtxB is associated with a reduction in Th1 cell reactivity without a concomitant shift toward Th2. Instead, EtxB mediates its effects through enhancing the activity of a population of CD4+ regulatory T cells.

Antigen-Specific Mediated Suppression of β Cell Autoimmunity by Plasmid DNA Vaccination

In this study, we have investigated the use of plasmid DNA (pDNA) vaccination to elicit Th2 effector cell function in an Ag-specific manner and in turn prevent insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. pDNA recombinants were engineered encoding a secreted fusion protein consisting of a fragment of glutamic acid decarboxylase 65 (GAD65) linked to IgGFc, and IL-4. Intramuscular injection of pDNA encoding GAD65-IgGFc and IL-4 effectively prevented diabetes in NOD mice treated at early or late preclinical stages of IDDM. This protection was GAD65-specific since NOD mice immunized with pDNA encoding hen egg lysozyme-IgGFc and IL-4 continued to develop diabetes. Furthermore, disease prevention correlated with suppression of insulitis and induction of GAD65-specific regulatory Th2 cells. Importantly, GAD65-specific immune deviation was dependent on pDNA-encoded IL-4. In fact, GAD65-specific Th1 cell reactivity was significantly enhanced in animals immunized with pDNA encoding only GAD65-IgGFc. Finally, NOD.IL4(null) mice treated with pDNA encoding GAD65-IgGFc and IL-4 continued to develop diabetes, indicating that endogenous IL-4 was also required for disease prevention. These results demonstrate that pDNA vaccination is an effective strategy to elicit beta cell-specific Th2 regulatory cell function for the purpose of preventing IDDM even at a late stage of disease development.

Interleukin‐4 and ‐10 exacerbate candidiasis in mice

Neutralization of endogenous interleukin (IL)-4 or IL-10 in mice with Candida albicans infection initiates or accelerates development of a T helper (Th)1-associated protective response. Here, we report the effect of IL-4 and IL-10 administration on the course of systemic or gastrointestinal (GI) candidiasis and on the development of Th immunity using yeast/host combinations that result either in Th1-associated self-limiting infection (healer mice) or in Th2-associated progressive disease (nonhealer mice). Treatment with IL-4 or IL-10 greatly exacerbated the course of systemic infection in nonhealer mice and rendered healer mice, inoculated with attenuated yeast cells, susceptible to infection. Under the latter conditions of yeast challenge and IL-4/IL-10 administration, the development of a fatal disease was associated with inhibition of IL-12 production and detection of progressive Th2 cell dominance. In contrast, in healer mice allowed to resolve their infections and to develop long-lived anti-candidal resistance, the expression of this acquired resistance was not impaired by IL-4 and/or IL-10, as shown by the outcome of reinfection with virulent yeast cells. In the GI model of infection, both IL-4 and IL-10 were found to exacerbate the course of infection and to induce the appearance of CD4+ T cells producing high levels of IL-4 and IL-10 in Peyer's patches. These findings demonstrate that exogenous IL-4 and IL-10 may greatly affect the development of Th responses to C. albicans in vivo, but do not modify the expression of established and predominant Th1 cell reactivity.

Comparison of Th1- and Th2-associated immune reactivities stimulated by single versus multiple vaccination of mice with irradiated Schistosoma mansoni cercariae.

Mice immunized against Schistosoma mansoni by a single percutaneous exposure to radiation-attenuated parasite larvae demonstrate partial resistance to challenge infection that has been shown to correlate with development of cell-mediated immunity, whereas mice hyperimmunized by multiple exposure to attenuated larvae produce antibodies capable of transferring partial protection to naive recipients. Measurement of Ag-specific lymphokine responses in these animals suggested that the difference in resistance mechanisms may be due to the differential induction of Th subset response by the two immunization protocols. Thus, upon Ag stimulation, singly immunized mice predominantly demonstrated responses associated with Th1 reactivity, including IL-2 and IFN-gamma production, whereas multiply immunized animals showed increased IL-5, IL-4, and IgG1 antibody production associated with enhanced Th2 response. These responses demonstrated some degree of organ compartmentalization, with splenocytes demonstrating higher Th1-related lymphokine production and cells from draining lymph nodes showing stronger proliferation and Th2 type reactivity. However, hyperimmunized mice also continued to demonstrate substantial Th1-associated immune reactivity. Moreover, in vivo Ag challenge elicited activated larvacidal macrophages in hyperimmunized animals. These observations indicate that protective cell-mediated mechanisms associated with induction of CD4+ Th1 cell reactivity predominate in singly vaccinated mice. Further vaccination stimulates Th2 responses, such as enhanced IgG1 production, that may also contribute to protective immunity.