APC activation by IFN-alpha decreases regulatory T cell and enhances Th cell functions.

Type I IFNs are central to a vast array of immunological functions. Their early induction in innate immune responses provides one of the most important priming mechanisms for the subsequent establishment of adaptive immunity. The outcome is either promotion or inhibition of these responses, but the conditions under which one or the other prevails remain to be defined. The main objective of the current study was to determine the involvement of IFN-alpha on murine CD4(+)CD25(-) Th cell activation, as well as to define the role played by this cytokine on CD4(+)CD25(+) regulatory T (Treg) cell proliferation and function. Although IFN-alpha promotes CD4(+)CD25(-) Th cells coincubated with APCs to produce large amounts of IL-2, the ability of these cells to respond to IL-2 proliferative effects is prevented. Moreover, in medium supplemented with IFN-alpha, IL-2-induced CD4(+)CD25(+) Treg cell proliferation is inhibited. Notably, IFN-alpha also leads to a decrease of the CD4(+)CD25(+) Treg cell suppressive activity. Altogether, these findings indicate that through a direct effect on APC activation and by affecting CD4(+)CD25(+) Treg cell-mediated suppression, IFN-alpha sustains and drives CD4(+)CD25(-) Th cell activation.

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression.

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.

Cytotoxic T-lymphocyte antigen-4 inhibits GATA-3 but not T-bet mRNA expression during T helper cell differentiation.

Naive CD4+ T-cell differentiation to T helper 1 (Th1) and Th2 cells is dependent on T-bet and GATA-3 factors, respectively. T-bet and GATA-3, indeed, through chromatin remodelling allow transcriptional activation of Ifngamma and Th2 cytokine (Il4, Il5, Il13) genes, respectively. We investigated the effects of the negative costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) on GATA-3 and T-bet mRNA expression and Th cell differentiation in mouse naive CD4+ T cells. Our results show that CTLA-4 inhibits GATA-3 mRNA expression and Th2 cell differentiation. At variance, CTLA-4 does not affect T-bet mRNA expression and Th1 cell differentiation. GATA-3 mRNA expression is inhibited when CD4+ cells are stimulated under both neutral (i.e. absence of cytokines) and Th2-polarizing (i.e. presence of interleukin (IL)-4) conditions, the effect being larger under the latter condition. Hence CTLA-4 might affect the IL-4/signal transducer and activator of transcription-6 (STAT6) pathway leading to GATA-3 mRNA up-regulation. We found, indeed, that CTLA-4 engagement inhibits STAT6 activation leaving unaffected the STAT6 protein level. Moreover, CTLA-4 engagement drastically inhibits IL-4Ralpha mRNA and protein up-regulation under Th2-polarizing conditions. Thus, CTLA-4 exerts a tight control on Th2 cell differentiation by negatively regulating both the CD3/CD28 and the IL-4/STAT6 pathways.

IL-4 modulation of CD4+CD25+ T regulatory cell-mediated suppression.

Murine CD4(+)CD25(+) T regulatory (Treg) cells were cocultured with CD4(+)CD25(-) Th cells and APCs or purified B cells and stimulated by anti-CD3 mAb. Replacement of APCs by B cells did not significantly affect the suppression of CD4(+)CD25(-) Th cells. When IL-4 was added to separate cell populations, this cytokine promoted CD4(+)CD25(-) Th and CD4(+)CD25(+) Treg cell proliferation, whereas the suppressive competence of CD4(+)CD25(+) Treg cells was preserved. Conversely, IL-4 added to coculture of APCs, CD4(+)CD25(-) Th cells, and CD4(+)CD25(+) Treg cells inhibited the suppression of CD4(+)CD25(-) Th cells by favoring their survival through the induction of Bcl-2 expression. At variance, suppression was not affected by addition of IL-13, although this cytokine shares with IL-4 a receptor chain. When naive CD4(+)CD25(-) Th cells were replaced by Th1 and Th2 cells, cell proliferation of both subsets was equally suppressed, but suppression was less pronounced compared with that of CD4(+)CD25(-) Th cells. IL-4 production by Th2 cells was also inhibited. These results indicate that although CD4(+)CD25(+) Treg cells inhibit IL-4 production, the addition of IL-4 counteracts CD4(+)CD25(+) Treg cell-mediated suppression by promoting CD4(+)CD25(-) Th cell survival and proliferation.

Role of immune responsiveness and DNA repair capacity genes in ageing.

The genetic factors that determine immune responsiveness and DNA repair capacity are reviewed as major elements influencing the life span. Within this framework two sets of new data obtained in mice and humans are reported and discussed. As to the first set, the role of immune response genes was investigated in Biozzi mice genetically selected for high (H) or low (L) antibody response. After 15-20 generations of assortative mating, H and L mice exhibited almost complete intraline homozygosity and interline polymorphism including distinct H-2 haplotypes, such as q in H and s in L mice. In order to analyze the impact of quantitative trait loci (QTL) on the antibody response as well as on the DNA repair capacity in cells of the immune system independently of the selected H-2 haplotype, congenic Hq and Hs as well as Lq and Ls mice were produced and analysed. Both the antibody response and DNA repair capacity were found to be independent of the H-2 haplotype and determined by QTL. As to the second set of data, DNA repair was also studied in irradiated peripheral blood mononuclear cells (PBMC) from ageing humans. The levels of ku 70, ku 80, DNA-PKcs, phosphorylated ku 80 as well as the DNA-binding activity of the ku70/ku 80 heterodimer were determined in the cytoplasmic and nuclear extracts obtained, before and after irradiation, from young and elderly subjects. The results of this study suggest that the decreased DNA repair capacity in PBMC from elderly subjects may be related to impaired migration of the phosphorylated ku 80 from the cytoplasm to the nucleus. This finding helps to elucidate questions related to the impairment of DNA repair during ageing.

CTLA-4 engagement inhibits Th2 but not Th1 cell polarisation.

CTLA-4 deficient mice show severe lymphoproliferative disorders with T helper sub-population skewed toward the Th2 phenotype. In the present work, we investigated the role of CTLA-4 in T helper cell subset differentiation. Naïve CD4+ cells were stimulated with anti-CD3 and anti-CD28 mAbs in the presence of either IL-12 or IL-4 to induce polarisation to Th1 or Th2 cells, respectively. Under these two polarising conditions cells express comparable levels of CTLA-4. CTLA4 was stimulated by plastic-bound mAb. The frequency of IFN-gamma- and IL-4-producing cells were estimated by FACS analysis. In parallel cultures, polarised Th1 and Th2 cells were re-stimulated with anti-CD3 and anti-CD28 mAbs for 48 h and their culture supernatants analysed by ELISA. Results show that CTLA-4 engagement during differentiation inhibits polarisation of naive CD4+ cells to the Th2 but not the Th1 cell subset. At variance, once cells are polarised, CTLA-4 engagement inhibits cytokine production in both effector Th2 and Th1 cells. Altogether these data indicate that CTLA-4 may interfere not only in the signalling involved in acute transcriptional activation of both Th1 and Th2 cells but also in the development of one of the Th cell subsets.

DNA damage recognition and repair capacities in human naïve and memory T cells from peripheral blood of young and elderly subjects.

T cells accumulate genetic damage over time but nai;ve cells display higher genomic stability and longer lifespan as compared to memory cells. We found in nai;ve and memory T cells from young and elderly subjects that DNA damage in unirradiated cells is higher in memory than in nai;ve T cells, and is increased by radiation in both cell types. Repair of the radiation-induced DNA damage was much higher in nai;ve than in memory T cells from young subjects but null in both cell types from elderly subjects. Molecular mechanisms involved in DNA damage recognition and repair were analyzed in both cell subsets from young subjects. The intracellular distribution and amount of the DNA-dependent protein kinase (DNA-PK) complex components (ku 70, ku 80, DNA-PKcs), which are involved in the recognition and repair of DNA breaks caused by ionizing radiations, V(D)J recombination and isotype switching, was assessed in nai;ve and memory T cells from young subjects. While the expression of ku 70 and ku 80 was at comparable levels in both T cell subsets, DNA-PKcs, phosphorylated ku 80, and DNA-binding of ku 70/80 were mostly evident in nai;ve but negligible or absent in memory T cells. These findings may account for the higher genomic stability and longer lifespan of nai;ve as compared to memory human T cells from young subjects.

Cytotoxic T lymphocyte-associated antigen-4 inhibits integrin-mediated stimulation.

The negative role exerted by cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in the regulation of T-cell activity, as induced by T-cell receptor (TCR)/CD3 and CD28 costimulation, has been widely described. In the present work we investigated the role of CTLA-4 in the control of cell activation, as induced by costimulation of the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) in murine CD4+ T cells. Results show that CTLA-4 engagement inhibits interleukin-2 (IL-2) production, not only when induced by CD3/CD28 costimulation, but also when CD4+ T cells are costimulated by anti-CD3 and anti-LFA-1 monoclonal antibodies (mAbs). LFA-1 has been described to induce Ca2+ mobilization also in the absence of TCR engagement. Moreover, we found that CTLA-4 engagement negatively affects Ca2+ mobilization and NF-AT activation, as induced by LFA-1 engagement alone. PLCgamma1 phosphorylation was also dampened within minutes after CTLA-4 engagement. Altogether these data indicate that through the control of signals induced by different receptors, CTLA-4 could be a global attenuator of T-cell activation.

The DNA repair protein ku is involved in gp130-mediated signal transduction events in PBMC from young but not from elderly subjects.

Ku, composed of 70kDa (ku 70) and 86kDa (ku 80) proteins, is the DNA-targeting subunit of the DNA-dependent serine/threonine kinase (DNA-PK), which plays a crucial role in DNA double strand break recognition and repair in mammalian cells. We have investigated the effects of an IL-6-type cytokine (K-7/D-6), known to trigger gp130, on the expression and function of the ku protein in cytoplasmic and nuclear extracts of freshly isolated human peripheral blood mononuclear cells (PBMC) from subjects of different ages. DNA-binding of nuclear ku was found to be increased by cytokine treatment of cells from young donors but only to a negligible extent from elderly subjects. This cytokine effect was correlated with a greater amount of phosphorylated ku 80, rather than increased expression of ku 70 and ku 80 proteins. DNA-binding activity of cytoplasmic ku was hardly discernible, as compared to nuclear ku, in both young and elderly subjects and was unaffected by the cytokine treatment regardless of age. Regarding the mechanisms whereby ku and gp130 signaling are coupled in PBMC, results from co-immunoprecipitation experiments have shown that ku in the cytoplasm of PBMC from young, but not from elderly subjects, is associated with Tyk-2, a kinase involved in signal transduction events after gp130 triggering by IL-6-type cytokines. This association was independent of PHA stimulation. Moreover, the present results indicate that after gp130 signaling both Tyk-2 and ku 80 are phosphorylated, suggesting their activation by K-7/D-6.