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International Congress of Immunology (ICI), Melbourne, AUSTRALIA, AUG 21-26, 2016; International audience; no abstract

International audience; no abstract

The relative contribution of IL-4 and IL-13 to the regulation of IgE synthesis has remained relatively poorly characterized, partially because of lack of suitable animal models. We have studied the roles of IL-4 and IL-13 in human IgE synthesis induced by supernatants derived from activated CD4+ or CD8+ T cell clones. Neutralizing anti-IL-4 and anti-IL-13 monoclonal antibodies (mAbs) inhibited IgE synthesis induced by anti-CD40 mAbs and supernatants from CD4+ T cells by an average 61% and 42%, respectively (n = 25). Recombinant IL-13 had additive effects on IL-4-induced IgE synthesis, but only when IL-4 was present at low concentrations. Accordingly, IL-4 was the dominant IgE synthesis-inducing cytokine derived from highly polarized T helper (TH)2 cells. However, anti-IL-13 mAbs also significantly inhibited IgE synthesis induced by two of three supernatants derived from allergen-specific T(H2)-like cell lines generated from the skin of patients with atopic dermatitis. Furthermore, anti-IL-13 mAbs almost completely inhibited IgE synthesis induced by supernatants from T(H1) cells or CD8+ T cell clones. Taken together, these data indicate that IL-13, in addition to IL-4, contributes to IgE synthesis induced by all T helper cell subsets, including allergen-specific T(H2) cells. Moreover, IL-13 appears to be the major IgE synthesis-inducing cytokine derived from T(H1) cells or CD8+ T cells.

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 3-13 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.

Infection and transformation by human T cell leukemia virus type I (HTLV-I) up-regulates expression of several inducible genes including those coding for cytokines involved in the proliferation of normal and leukemic T cells. We demonstrate that HTLV-I can also shut off expression of the CD3-gamma, delta, epsilon, and zeta genes that code for the constant elements of the TCR for Ag. In addition, the T cell-specific CD3-epsilon enhancer was found to be inactive in a HTLV-I-infected T cell clone. This HTLV-I-infected T cell clone (827-p19-II) that could be cultured in the absence of IL-2 lacked the CD3 proteins but did express the TCR-alpha and -beta proteins intracellularly. In the absence of the CD3-gamma, delta, epsilon, and zeta polypeptide chains the disulfide bridged TCR-alpha/beta heterodimer was not formed and the Ag receptor did not appear at the cell surface. These results allowed two major conclusions: first, HTLV-I infection has an effect on the T cell specific regulatory elements that coordinately regulate CD3-gamma, delta, epsilon, and zeta expression and second, the CD3-gamma, delta, epsilon, and zeta proteins are necessary for formation and routing the variable TCR-alpha/beta (or -gamma/delta) heterodimer to the human T cell surface.

In the present study, we investigated the lymphokine production patterns in a series of CD4+ and CD8+ host-reactive T cell clones isolated from PBL of a SCID patient, who was immunologically reconstituted by two allogeneic fetal liver and thymus transplantations 13 years ago. We demonstrate that these donor-derived T cell clones, specifically reacting with the MHC Ag expressed on the recipient cells, do not produce IL-4 and do not express IL-4 mRNA upon Ag or polyclonal stimulations. In contrast, CD4+ tetanus toxin-specific T cell clones isolated from the same patient and having the same HLA phenotype produced normal amounts of IL-4 upon activation. These data suggest that the failure to produce IL-4 is a specific characteristic of these host-reactive clones and is not due to a genetic defect of the transplanted cells. Furthermore, different modes of activation resulted in simultaneous production of IL-5, IL-2, IFN-gamma, granulocyte/macrophage-CSF, and transcription of the TNF-beta gene by the host-reactive clones, indicating that the lack of IL-4 production is not related to the mode of activation. The finding that some of these clones produce significant levels of IL-5 but no IL-4 indicates that the IL-4 and IL-5 genes are not always coexpressed in activated human T cells.

Differential expression of cytokine receptors accounts for an important regulatory mechanism in differentiation of Th1/Th2 subsets. Here, we report that human Th0 and Th2 clones constitutively express transcripts for the IFN-gamma R beta-chain, whereas mRNA for this signaling component of the IFN-gamma receptor is absent in Th1 clones. Activation of T cell clones, however, resulted in a transient induction or enhancement of IFN-gamma R beta-chain mRNA expression in Th1 clones and Th0/Th2 clones, respectively. IL-12-mediated Th1 cell differentiation of naive CD4(+), CD45RA(+) cord blood T cells, which constitutively express IFN-gamma R beta-chain mRNA, resulted in a loss of expression of this cytokine receptor chain after 6 to 12 days of culture. In contrast, Th2 populations, differentiated from CD4(+), CD45RA(+) cord blood T cells in the presence of IL-4, continued to express high levels of IFN-gamma R beta-chain transcripts. The loss of IFN-gamma R beta-chain expression in Th1 populations was accompanied by a failure of IFN-gamma to induce the expression of the IFN-gamma-inducible gene, IFN response factor-1, whereas IFN-gamma was effective in inducing IFN response factor-1 mRNA expression in Th0 and Th2 cells. These results indicate that down-regulation of the IFN-gamma R beta-chain correlates with impaired IFN-gamma-induced signaling in Th1 cells. Finally, Th2 populations, generated in the presence of both IL-4 and IFN-gamma, expressed levels of IFN-gamma R beta-chain transcripts similar to those produced by cells differentiated in the presence of IL-4 only, demonstrating that IFN-gamma does not modulate the expression of its receptor. Together, these data indicate that human Th0/Th2 and Th1 subsets, respectively, can be distinguished based on the expression of the IFN-gamma R beta-chain.

Despite the short history of interleukin-10, accumulated evidence indicates that this interleukin plays a major role in suppressing immune and inflammatory responses. Yet interleukin-10 also maintains cell viability and acts as a cofactor to promote the growth of lymphoid and myeloid cells in vitro. Here we review the present knowledge on the structure and function of interleukin-10.

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma-delta+, CD4+ and TcR alpha-beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha-beta/CD3 or TcR gamma-delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha-beta+, CD8+, and TcR gamma-delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha-beta+ or TcR gamma-delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha-beta+, CD4+, TcR gamma-delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA- producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha-beta+, CD4+ and TcR gamma-delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco) protein that is induced by activation of both TcR alpha-beta and TcR gamma-delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoyl-phorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.

NK cell clones obtained from three different donors were tested for their ability to present soluble proteins to Ag-specific T cell clones. All NK clones were CD2+CD3-CD56+, whereas the expression of CD16 varied from clone to clone. The NK cell clones were able to process and present tetanus toxoid (TT) to TT-specific T cell clones in a class II HLA restricted manner. The capacity of NK cell clones to function as APC was also observed using the house dust mite allergen Der p I and the Der p I-derived peptide Val89-Cys117. As with EBV-transformed B cell line, NK cell clones could present the peptide 313 derived from the 65-kDa heat shock protein of Mycobacterium leprae, but they were unable to present the whole M. leprae Ag. Freshly isolated NK cells, IL-2-activated NK cells, and NK cell lines expanded in vitro could also process and present TT. The ability of the different NK populations to act as accessory cells correlated with their levels of class II HLA expression. These data demonstrate that NK cell clones can efficiently function as APC, however they may be restricted in the types of Ag that they can process.

In the present study, it is demonstrated that cloned surface IgM- positive human B cells can be induced to proliferate and to switch with high frequencies to IgG4 and IgE production after a contact-mediated signal provided by T cell clones and interleukin 4 (IL-4). This T cell signal is antigen nonspecific and is provided by activated CD4+ cells, whereas activated CD8+ or resting CD4+ T cell clones are ineffective. 15-35% of the B cell clones cultured with cloned CD4+ T cells and IL-4 produced antibodies; 35-45% of those wells in which antibodies were produced contained IgE and IgG4. In addition to B cell clones that produced IgG4 or IgE only, B cell clones producing multiple isotypes were observed. Simultaneous production of IgG4 and IgE, IgM, IgE, and IgM, or IgG4 and IgE was detected, suggesting that during clonal expansion switching might occur in successive steps from IgM to IgG4 and IgE. In addition, production of only IgM, IgG4, and IgE during clonal expansion indicates that this isotype switching is directed by the way a B cell is stimulated and that it is not a stochastic process.

Interleukin 10 (IL-10) and viral IL-10 (v-IL-10) strongly reduced antigen-specific proliferation of human T cells and CD4+ T cells clones when monocytes were used as antigen-presenting cells. In contrast, IL-10 and v-IL-10 did not affect the proliferative responses to antigens presented by autologous Epstein-Barr virus-lymphoblastoid cell line (EBV-LCL). Inhibition of antigen-specific T cell responses was associated with downregulation of constitutive, as well as interferon gamma-or IL-4-induced, class II MHC expression on monocytes by IL-10 and v-IL-10, resulting in the reduction in antigen-presenting capacity of these cells. In contrast, IL-10 and v-IL-10 had no effect on class II major histocompatibility complex (MHC) expression on EBV-LCL. The reduced antigen-presenting capacity of monocytes correlated with a decreased capacity to mobilize intracellular Ca2+ in the responder T cell clones. The diminished antigen-presenting capacities of monocytes were not due to inhibitory effects of IL-10 and v-IL-10 on antigen processing, since the proliferative T cell responses to antigenic peptides, which did not require processing, were equally well inhibited. Futhermore, the inhibitory effects of IL-10 and v-IL-10 on antigen-specific proliferative T cell responses could not be neutralized by exogenous IL-2 or IL-4. Although IL-10 and v-IL-10 suppressed IL-1-alpha, IL-1-beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 production by monocytes, it was excluded that these cytokines played a role in antigen-specific T cell proliferation, since normal antigen-specific responses were observed in the presence of neutralizing anti-IL-1, -IL-6, and -TNF-alpha mAbs. Furthermore, addition of saturating concentrations of IL-1-alpha, IL-1-beta, IL-6, and TNF-alpha to the cultures had no effect on the reduced proliferative T cell responses in the presence of IL-10, or v-IL-10. Collectively, our data indicate that IL-10 and v-IL-10 can completely prevent antigen-specific T cell proliferation by inhibition of the antigen-presenting capacity of monocytes through downregulation of class II MHC antigens on monocytes.

Recent studies in the mouse have established that IL-4 and IL-12 direct the development of Ag-stimulated naive CD4 T cells into, respectively, type 2 and type 1 Th cells. We report that prolonged exposure of immunologically naive and unstimulated human neonatal CD4 T cells to IL-4 or to IL-4 plus either IL-2 or IL-12 markedly affects their cytokine production on primary stimulation with PMA and ionomycin. IL-4 induces long-term proliferation of neonatal T cells and after 3 wk of culture these are capable of producing high levels of Th1 (IL-2, IFN-gamma) but no Th2 (IL-4, IL-5) cytokines; IL-4-primed cells are homogenously CD45RO-/RA+ and CD31+. After culture in the presence of IL-4 + IL-2 or IL-4 + IL-12, neonatal T cells are enriched in CD45RO+ and CD31- cells and they can produce Th2 as well as Th1 cytokines. In response to primary stimulation with PMA and ionomycin, cells primed with IL-4 + IL-2 produce IL-4, IL-5, and IL-10 and the same levels of IL-2 and IFN-gamma as IL-4-primed cells. Cells primed with IL-4 + IL-12 produce very high levels of both IL-4 and IFN-gamma but no IL-5. Endogenous IFN-gamma that is detected in primary cultures containing IL-4 + IL-12 does not inhibit, but rather enhances, the ability of the cells to produce IL-4. Further analysis of IL-4 + IL-12-primed cells reveals that IL-4 is mainly produced by CD31- cells and that these cells can trigger B cells to synthesize Ig including IgE. Finally, positively selected CD31+ cells remain CD31+ and are poor IL-4 producers after 3 wk of culture with IL-4 + IL-12, suggesting that these two cytokines promote the selective expansion of the small number of CD31- cells that are present in freshly isolated neonatal CD4 T cells.

In the present study it is demonstrated that human B cells can be induced to switch to IgE production following a contact-mediated signal provided by activated T cell receptor (TcR) gamma delta+, CD4+ and TcR alpha beta+, CD4+ T cell clones and interleukin (IL)-4. The signal provided by these T cell clones was antigen nonspecific, indicating that the TcR alpha beta/CD3 or TcR gamma delta/CD3 complexes were not involved in these T-B cell interactions. Activated TcR alpha beta+, CD8+, and TcR gamma delta+, CD4-CD8-, or resting CD4+ T cell clones were ineffective. Intact TcR alpha beta+ or TcR gamma delta+, CD4+ T cell clones could be replaced by plasma membrane-enriched fractions isolated from these activated CD4+ T cell clones. In contrast, membranes isolated from resting TcR alpha beta+, CD4+, TcR gamma delta+, CD4+ T cell clones or an Epstein-Barr virus (EBV)-transformed B cell line (EBV-LCL) failed to provide the costimulatory signal that, in addition to IL-4, is required for induction of IgE synthesis. As described for intact CD4+ T cells, CD4+ T cell membranes induced purified surface IgM+ B cells to switch to IgG4- and IgE- but not to IgA-producing cells, excluding the possibility of a preferential outgrowth of IgG4- and IgE-committed B cells. The membrane activity was inhibited by protease or heat treatment. Induction of IgE synthesis by B cells co-cultured with both TcR alpha beta+, CD4+ and TcR gamma delta+, CD4+ T cell clones and membrane preparations of these cells was blocked by anti-class II major histocompatibility complex (MHC) monoclonal antibodies (mAb), whereas various anti-CD4 mAb had differential blocking effects. Murine L cells, or EBV-LCL transfected with CD4 could not replace CD4+ T cell clones. These results indicate that, although CD4 and class II MHC antigens are required for productive CD4+ T cell clone-B cell interactions, an additional signal, provided by a membrane associated (glyco)protein that is induced by activation of both TcR alpha beta and TcR gamma delta, CD4+ T cells, is needed for induction of IgE production in the presence of IL-4

A new monoclonal antibody (mAb) of the IgG1 subclass, mAb 104, has been obtained after immunization of mice with the Burkitt lymphoma cell line Jijoye. It only weakly binds to a small proportion of non-activated normal B cells and binds to a larger proportion of in vitro-activated normal B cells. All tested Epstein-Barr virus (EBV)-transformed B-cell lines, Burkitt lymphoma cell lines and freshly isolated follicular B-lymphoma cell preparations strongly bound mAb 104. mAb 104 did not bind to peripheral monocytes or tested myelomonocytic cell lines, or to resting and activated normal T cells, T-cell lines and T-cell clones. However, the recognized antigen is expressed on HTLV-1-infected T-cell lines and HTLV-1-transformed T-cell clones. mAb 104 immunoprecipitates, from Jijoyce cell lysates, a single polypeptide with an apparent MW of 45,000-60,000 and an isoelectric point of 5.6. Competition studies with the anti-B7 antibody (Freedman et al., 1987) demonstrated that mAb 104 and the anti-B7 block each others' binding. Furthermore, mAb 104 binds to transfected COS cells (Freedman et al., 1989) expressing the B7 antigen. Thus mAb 104 and and anti-B7 define the same antigen. The restricted distribution of the 104/B7 antigen to activated B cells and HTLV-1-transformed T cells may make it a useful marker for the study of pathological states linked to lymphocyte activation and for the functional study of B-cell subpopulations.