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Background: The mechanisms by which human dendritic cells (DCs) activate a TH1-polarizing or TH2-polarizing program are still partially unclear. Objective: Study of the mechanisms responsible for the TH1/TH2-polarizing activity of human circulating myeloid DCs before and after ligation of their Toll-like receptors (TLRs). Methods: IL-4 and IFN-γ production by CD4+ T cells was assessed in cocultures with myeloid DCs before or after TLR triggering. Expression of Jagged-1 and Delta-4 Notch ligands and of GATA-3 and T-box expressed in T cells transcription factors was evaluated by real-time quantitative PCR. Signal transducer and activator of transcription 4 and 6 phosphorylation was assessed by flow cytometry. Knockdown of Jagged-1 or Delta-4 was performed by transfection of DCs with appropriate silencing mRNAs. Results: Myeloid immature DCs constitutively expressed Jagged-1, which induces in CD4+ T cells a TH2 polarization, as shown by Jagged-1 gene silencing. The TH2 polarization associated with high GATA-3/T-box expressed in T cells ratio and was at least partially dependent on the early induction of IL-4. Maturation of DCs by TLR ligation resulted in the reduction of Jagged-1 and upregulation of Delta-4, which was at least in part responsible for the polarization of CD4+ T cells to the TH1 phenotype. Conclusion: CD4+ T-cell responses are usually characterized by a prevalent TH2 phenotype unless TLRs are triggered on DCs by microbial components. [Copyright &y& Elsevier]

Background: CD4+CD25+Foxp3+ T-regulatory (Treg) cells play a fundamental role in the control of autoimmunity. Whether human CD4+CD25+Foxp3+ Treg cells that recognize foreign antigens also exist is less clear. Objective: To investigate the existence in humans of circulating Treg cells able to recognize exogenous antigens, including allergens. Methods: CD4+CD25highFoxp3+ and CD4+CD25-Foxp3- cells were purified from human peripheral blood and cultured for 15 days with autologous dendritic cells (DCs), unloaded, or loaded with Der p 1 allergen or the bacterial antigen streptokinase (SK). Results: CD4+CD25highFoxp3+ circulating T cells obtained from healthy nonatopic subjects and cultured with Der p 1–loaded DCs, but not those cultured with either unloaded or SK-loaded DCs, suppressed the proliferative response to Der p 1 of autologous Der p 1–specific T cells generated from the CD4+CD25-Foxp3- subset. The antigen specificity of either Der p 1–CD4+CD25highFoxp3+ or SK–CD4+CD25highFoxp3+ T cells was confirmed even at clonal level. Finally, under the same experimental conditions, functionally active Der p 1–specific Treg cells were obtained from the pool of circulating CD4+CD25highFoxp3+ T cells of Der p 1–sensitive, atopic individuals. Conclusion: These data provide undoubted demonstration of the existence of human CD4+CD25highFoxp3+ circulating Treg cells specific for exogenous antigens, including the Der p 1 allergen, and indicate that CD4+CD25highFoxp3+ Treg cells specific for Der p 1 are present and functionally active in both nonatopic and Der p 1–sensitive, atopic individuals. Clinical implications: Caution should be advised in interpreting allergic disorders as simply resulting from defective Treg cell activity. [Copyright &y& Elsevier]

Background: The identification of mycobacteria represents the gold standard in the diagnosis of tuberculosis (TB), but it is not applicable in all patients, and immunological tests, such as the tuberculin skin test (TST), are not specific and sensitive enough. Methods: By flow cytometry, we measured the CD4 T-cell response to purified protein derivative (PPD) and early secretory antigenic target-6 (ESAT-6) protein using the intracellular cytokine staining technique on whole blood samples obtained from active TB (n = 16), latent TB (n = 17), Bacille Calmette-Guérin (BCG)-vaccinated (n = 11) and healthy (n = 10) donors. All the patients were also tested with conventional TST. Results: The identification by flow cytometry of PPD-specific T lymphocytes upon antigen stimulation of whole blood enables the discrimination of active TB, latent TB and BCG-vaccinated subjects from healthy individuals, whereas the ESAT-6 response discriminated active TB from healthy and BCG-vaccinated individuals. Moreover, this test enables identification of active TB patients who were negative on TST and to distinguish between TB and non-typical mycobacteria TB infections. Conclusions: The identification by flow cytometry of antigen-specific T lymphocytes upon antigen stimulation of whole blood has a better positive predictive value than TST, and could represent a further tool in the diagnosis of TB infection. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Two variants of the CXCR3 receptor exist, one (CXCR3-A) reactive with CXCL9, CXCL10, and CXCL11 and the other (CXCR3-B) also reactive with CXCL4. Both variants are contemporarily expressed by human T cells. Objective: We sought to investigate the in vitro effects of CXCL10 and CXCL4 on the production of TH1 or TH2 cytokines. Methods: The cytokine profile of antigen-specific human CD4+ T-cell lines obtained in the absence or presence of CXCL10 or CXCL4 was evaluated by means of quantitative RT-PCR, flow cytometry, and ELISA. Results: CXCL10 upregulated IFN-γ and downregulated IL-4, IL-5, and IL-13 production, whereas CXCL4 downregulated IFN-γ and upregulated TH2 cytokines. Similar effects were also observed on polyclonally activated pure naive CD4+ T cells. The opposite effects of CXCL10 and CXCL4 on TH1 and TH2 cytokine production were inhibited by an anti-CXCR3 antibody able to neutralize both CXCR3-A and CXCR3-B and were apparently related to the activation of distinct signal transduction pathways. Moreover, CXCL10 upregulated mRNA levels of T-box expressed in T cells and downregulated GATA-3 expression, whereas CXCL4 downregulated T-box expressed in T cells and upregulated GATA-3. Finally, CXCL4, but not CXCL10, induced direct activation of IL-5 and IL-13 promoters. Conclusion: CXCL10 and CXCL4 exert opposite effects on the production of human TH1 and TH2 cytokines, likely through their respective interaction with CXCR3-A or CXCR3-B and the consequent activation of different signal transduction pathways. This might represent an internal regulatory pathway of TH cell responses and might contribute to the modulation of chronic inflammatory reactions, including allergy. [Copyright &y& Elsevier]

Background: IL-17A has been suggested to play a pathogenic role in bronchial asthma and other allergic disorders. Objective: Study of the relationship between human IL-17A–producing CD4+ TH cells (TH17) and IL-4–producing CD4+ TH (TH2) cells. Methods: T-cell clones generated from the CCR6+CD161+ fraction of human circulating CD4+ T cells, which contains virtually all TH17 cells, as well as circulating CD4+ T cells from both healthy subjects and patients with asthma, were assessed by flow cytometry for their cytokine production profile. Results: A small proportion of CCR6+CD161+CD4+ T-cell clones showed the ability to produce both IL-17A and IL-4 (TH17/TH2). TH17/TH2 clones also produced IL-5, IL-8, IL-9, IL-13, IL-21, and IL-22 and displayed the ability to induce the in vitro secretion of IgE. A very few TH17/TH2 cells were found among circulating CD4+ T cells from normal subjects, but their proportions were significantly increased in the circulation of patients with chronic asthma. TH17/TH2 cells could not be derived from naive umbilical cord blood CD4+ T cells under any experimental condition. However, when circulating memory CCR6+CD161+CD4+ T cells were cloned under appropriate polarizing conditions, TH17/TH2 clones originated in the presence of IL-4, suggesting that an IL-4–rich microenvironment may induce the shifting of memory TH17 cells into TH17/TH2 cells. Conclusion: Because of its peculiar functional properties and the increased numbers in the circulation of patients with bronchial asthma, this previously unknown population of TH17/TH2 cells may play some role in the pathogenesis of this disease. [Copyright &y& Elsevier]

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) are multipotent, nonhemopoietic progenitors that also possess regulatory activity on immune effector cells through different mechanisms. We demonstrate that human BM-derived MSCs expressed high levels of Toll-like receptors (TLRs) 3 and 4, which are both functional, as shown by the ability of their ligands to induce nuclear factor kappaB (NF-kappaB) activity, as well as the production of interleukin (IL)-6, IL-8, and CXCL10. Of note, ligation of TLR3 and TLR4 on MSCs also inhibited the ability of these cells to suppress the proliferation of T cells, without influencing their immunophenotype or differentiation potential. The TLR triggering effects appeared to be related to the impairment of MSC signaling to Notch receptors in T cells. Indeed, MSCs expressed the Notch ligand Jagged-1, and TLR3 or TLR4 ligation resulted in its strong downregulation. Moreover, anti-Jagged-1 neutralizing antibody and N[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch signaling, hampered the suppressive activity of MSCs on T-cell proliferation. These data suggest that TLR3 and TLR4 expression on MSCs may provide an effective mechanism to block the immunosuppressive activity of MSCs and therefore to restore an efficient T-cell response in the course of dangerous infections, such as those sustained by double-stranded RNA viruses or Gram-negative bacteria, respectively.

Mesenchymal stem cells (MSCs) inhibit the proliferation of HLA-unrelated T lymphocytes to allogeneic stimulation, but the mechanisms responsible for this activity are not fully understood. We show here that MSCs suppress the proliferation of both CD4+ and CD8+ T lymphocytes, as well as of natural killer (NK) cells, whereas they do not have an effect on the proliferation of B lymphocytes. The antiproliferative effect of MSCs was not associated with any effect on the expression of cell-activation markers, induction of cell apoptosis, or mimicry/enhancement of T regulatory cell activity. The suppressive activity of MSCs was not contact-dependent and required the presence of interferon (IFN)-gamma produced by activated T cells and NK cells. Accordingly, even activated B cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added IFN-gamma. The suppressive effect of IFN-gamma was related to its ability to stimulate the production by MSCs of indoleamine 2,3-dioxygenase activity, which in turn inhibited the proliferation of activated T or NK cells. These findings suggest that the beneficial effect on graft-versus-host disease induced by in vivo coinfusion with the graft of MSCs may be due to the activation of the immunomodulatory properties of MSCs by T cell- derived IFN-gamma.