Soluble CD30 and lymphocyte activation gene-3 (CD223), as potential serological markers of T helper-type cytokine response induced by acellular pertussis vaccine.

T cell responses are involved in vaccine-induced immunity to pertussis but no easy-to-monitor, serological markers are available to assess these responses. The lymphocyte activation gene-3 (CD223) molecule is present on, and released by, activated T helper (Th) 1 cells, whereas CD30 molecules have been associated with Th2 immune responses. Starting from the recent knowledge of the cytokine profile induced by pertussis vaccination, we examined the levels of soluble (s)CD223 and sCD30 proteins in child recipients of acellular pertussis (aP) and diphtheria-tetanus (DT) vaccines and in children receiving DT vaccine only, as control. The correlation of the two proteins with specific antibody and T cell responses was assessed. The main findings are: i) sCD223 and sCD30 levels are inversely related, suggesting that the two markers are the expression of different and counter-regulated T-cell responses; ii) sCD30 level correlated with induction of T cell proliferation to pertussis vaccine antigens and antibody response to pertussis toxin. Overall, sCD30 and sCD223 levels seem to be promising candidate markers to assess the induction of Th-type responses in vaccine recipients.

Signaling through CD38 induces NK cell activation.

Human CD38 is a signal transduction molecule, and, concurrently, an ectoenzyme catalyzing the synthesis and degradation of cyclic ADP-ribose (cADPR), a potent Ca2+ mobilizer. One facet of CD38 that has not yet been addressed is its role in NK cells. To this end, the events triggered by CD38 ligation with agonistic mAb were analyzed on freshly purified human NK cells. Ligation was followed by (i) a significant rise in the intracellular level of Ca2+, (ii) increased expression of HLA class II and CD25, and (iii) tyrosine phosphorylation of discrete cytoplasmic substrates. The phosphorylation cascade involved CD3-zeta and FcepsilonRIgamma chains, zeta-associated protein (ZAP)-70 and the proto-oncogene product c-Cbl. NK effector functions were then analyzed: CD38 signaling was able (iv) to induce release of IFN-gamma and, more prominently, of granulocyte macrophage colony stimulating factor, as assessed by measuring both mRNA and protein products; and, lastly, (v) to induce cytolytic effector functions on target cells after IL-2 activation, as shown both by cytotoxicity assays and ultrastructural changes. The tyrosine-phosphorylated substrates and all the effects mediated by CD38 were similar to those observed following triggering via CD16 (FcgammaRIIIA); moreover, Ca2+ mobilization via CD38 no longer operated in NK-derived cell lines lacking CD16. These results suggest that the activation signals transduced by CD38 in NK cells elicit relevant cellular events. The effects are similar to those elicited via CD16 and possibly rely on common signaling pathways.

Human CD38 and its ligand CD31 define a unique lamina propria T lymphocyte signaling pathway.

CD38, a nonlineage-restricted surface glycoprotein, is an ecto-enzyme (ADP ribosyl cyclase/cADPR hydrolase/EC 3.2.2.6) that regulates cytoplasmic Ca2+ and cell-cell interactions. The molecule also delivers trans-membrane signals, despite a structural ineptitude to the scope. To reconcile these issues in a unitarian model, we compared the effects of CD38 signaling in circulating and residential T lymphocytes, the latter represented by those colonizing the intestinal lamina propria. Results are as follows: 1) LP T cells express an enzymatically active form of CD38, characterized by a modified ratio between cyclase and hydrolase functions; 2) LP T cells do not mobilize Ca2+ upon CD38 ligation, as seen in PB T cells (this condition is due to a lack in activation of PLC- g, constantly observed in PB T lymphocytes); 3) The early steps of CD38 signaling involve activation of lck, syk, and LAT; 4) Late events include synthesis and release of IL-2, IL-4, IL-5, IL-10, IFN-g and GM-CSF; 5) The uniqueness of the CD38 pathway in LP T cells is not caused by impaired interactions with the CD31 ligand. The differences observed concern the signaling machinery that CD38 exploits for its own use and not the interplay with its ligand.

Cell-mediated immunity and antibody responses to Bordetella pertussis antigens in children with a history of pertussis infection and in recipients of an acellular pertussis vaccine.

Cell-mediated immunity (CMI) and antibody responses to Bordetella pertussis antigens were assessed 4-6 years after primary infant immunization with diphtheria-tetanus tricomponent acellular pertussis (DTaP) or diphtheria-tetanus (DT) vaccine in a country with high endemicity of B. pertussis infection. CMI to the B. pertussis antigens (especially to the pertussis toxin [PT]) was more frequent and/or intense in DTaP than in DT recipients. No lymphoproliferation differences were found between those with and without a history of pertussis although the DT recipients produced very little interferon-gamma after antigen (particularly PT and filamentous hemagglutinin [FHA]) stimulation. In contrast, seropositivity to PT, but not to pertactin or FHA, was more frequent in DT recipients with history of pertussis than in all other subjects. Thus, years after disease or vaccination, CMI response to PT or circulating PT antibodies appears to be the main distinctive feature of pertussis-protected DTaP recipients or pertussis-affected DT recipients.

Functional topography of discrete domains of human CD38.

In addition to its catalytic activities as ecto-NAD+ glycohydrolase (NADase), CD38 displays the ability to transduce signals of biological relevance. Indeed, ligation of CD38 on peripheral blood mononuclear cells (PBMC) by agonistic monoclonal antibodies (mAbs) is followed by the transcription and secretion of a vast array of regulatory cytokines. The present work addresses the issue of whether the signals leading to calcium (Ca2+) mobilization, lymphocyte proliferation and release of cytokines is dependent on the epitopes recognized by the individual mAbs. Competition binding analysis identifies two families of mAbs, namely IB4, IB6 and AT2 on one side and OKT10, SUN-4B7 and AT1 on the other. Each mAb family binds epitopes that are completely or partially common. However, the functional activities of the CD38 molecule can not be simply attributed to the epitopes engaged: for instance, IB4 and OKT10 mAbs, which bind different epitopes, perform as agonistic mAbs in inducing PBMC proliferation and interferon (IFN)-gamma secretion. SUN-4B7 yields intermediate effects, whereas IB6, AT1 and AT2 mAbs are totally ineffective. The effects mediated by IB4 and OKT10 mAbs are apparent in 80% of the healthy individuals studied, whereas the effects of SUN-4B7 mAb operate only in 25% of the donors. Interleukin (IL)-6 secretion was observed in all individuals analyzed, irrespective of the epitopes triggered and of mAbs used to ligate the CD38 molecule. In addition, IB4 is the only mAb able to induce significant intracellular Ca2+ fluxes.

Decreased DTH response to recall antigens in mice injected with human immunodeficiency virus type 1-infected U937 cells and infected with Candida albicans.

In previous studies, we have reported that the intraperitoneal (i.p.) injection of HIV-1 infected human U937 cells into normal mice resulted in long-term persistence of anti-HIV antibodies and in a small percentage (10-20%) of HIV-1 infected animals at 6-12 months after the injection. The study reported here was undertaken to detect T immune defects in U937-HIV-1-injected mice. Eight months after the initial injection, a marked decrease in DTH response against U937 cells was detected in HIV injected animals. In addition, a consistent decrease in DTH response against a soluble mannoprotein antigen of Candida albicans cell wall (MP-F2) was also observed in U937-HIV-1-injected mice, chronically infected with low-virulent strain of the fungus. No decreases in DTH response was observed in control-injected animals. These data indicate that U937-HIV-1-injected mice become unable to mount a normal antigen-specific immune response. Although the mechanisms involved in the generation of these T cell defects remain unclear, these events appear to be somehow related to the HIV-1 infection and should be considered in the current studies of HIV-1 infection with transgenic mice.

A 70-kilodalton recombinant heat shock protein of Candida albicans is highly immunogenic and enhances systemic murine candidiasis.

The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-gamma), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-gamma was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-gamma upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.

Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member.

Human CD38 is a cell surface molecule involved in the regulation of lymphocyte adhesion to endothelial cells. This suggests that HUVEC bear a ligand(s) for CD38 on the cell surface. By means of the mAb Moon-1, which specifically inhibits CD38-mediated cell adhesion, we have identified a trans-membrane 130-kDa molecule acting as a ligand for CD38. Here, we report that the molecule recognized by the Moon-1 mAb is CD31, a member of the Ig superfamily. This conclusion is based on 1) cross-inhibition assays between Moon-1 and reference anti-CD31 mAbs; 2) sequential immunoprecipitation experiments using Moon-1 and known anti-CD31 mAbs, and 3) reactivity of the Moon-1 mAb with CD31 transfectants. Further, CD31 and CD38 cognate interactions were found to modulate heterotypic adhesion as well as to implement cytoplasmic calcium fluxes identical to those obtained by means of agonistic anti-CD38 mAbs. Other effects tested included the synthesis of messages for a panel of cytokines, markedly increased upon receptor-ligand interactions. These results suggest that the interplay between CD38 and its ligand CD31 is an important step in the regulation of cell life and of the migration of leukocytes (and CD38+ cancer cells) through the endothelial cell wall.

Responsiveness of human polymorphonuclear cells (PMNL) to stimulation by a mannoprotein fraction (MP-F2) of Candida albicans; enhanced production of IL-6 and tumour necrosis factor-alpha (TNF-alpha) by MP-F2-stimulated PMNL from HIV-infected subjects.

IL-1beta, IL-6, IL-8 and TNF-alpha production by PMNL from 21 HIV-infected (HIV+), including 11 full-blown AIDS, and 20 HIV-uninfected (HIV-) subjects (matched for age and sex to HIV+ ones) was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA. PMNL from both categories of subjects were strongly stimulated in their actual cytokine production by a mannoprotein fraction (MP-F2) of Candida albicans, as well as by the bacterial lipopolysaccharide (LPS). These stimulatory effects were apparently due to increased cytokine gene expression and were substantially reversed by the physiological inhibitor IL-10. However, PMNL from HIV+ subjects showed increased IL-6 and TNF-alpha gene expression and produced more IL-6 and TNF-alpha than PMNL from HIV- controls, under similar stimulation conditions. This difference could not be attributed to a given stage of HIV infection, any associated medication, or to a generalized increase of gene expression, as quantitatively similar beta-actin and IL-1beta transcripts were detected. Moreover, no significant difference in IL-8 production by the PMNL from HIV+ and HIV- subjects was observed. Our studies suggest that PMNL from HIV+ subjects might add to other cellular sources of IL-6 and TNF-alpha (e.g. monocytes-macrophages) in contributing to the cytokine-dysregulated pattern typical of the HIV+ patient.

Role of the human CD38 molecule in B cell activation and proliferation.

Human CD38 is a surface molecule which has been attributed the function of a signaling channel leading to cellular activation and proliferation, an ectoenzyme with multiple function as well as an inducer of Ca2+ mobilization from cytoplasmic stores. The effect mediated by CD38 have been studied in different cell populations: the results obtained in human B cells are apparently contradictory, with CD38 simultaneously leading to apoptosis in early B cells while increasing survival in cells derived from lymph node germinal center. Other effects recently reported concern a different potential in terms of signaling in early B cells and derived cell lines or in more detailed disease models of human leukemia, namely B chronic lymphocytic leukemia cells. To complete the picture of the effects mediated by CD38 in the B cell compartment, we have studied the signals elicited by ligation of the human molecule in mature B cells from circulating pool and also from spleen of normal individuals. The information obtained completes the picture of CD38 and mature B cells, where we also studied the contribution of relevant cytokines involved in maintenance and differentiation of these normal cells, namely IL-1 alpha, IL-2, IL-4 and IL-6. Our results indicate that human CD38 plays a key role as a co-receptor in mature B cells from normal individuals.

Acellular vaccines induce cell-mediated immunity to Bordetella pertussis antigens in infants undergoing primary vaccination against pertussis.

The induction of cell-mediated immunity (CMI) to Bordetella pertussis antigens (whole, heat-inactivated bacterial cells [BPC], pertussis toxin [PT], filamentous haemagglutinin [FHA], pertactin [PRN]) was assessed by a lymphoproliferation assay in vitro in a cohort of children enrolled in a randomized clinical trial of pertussis vaccines efficacy in Italy. Four vaccination groups were compared: children receiving acellular pertussis (aP) vaccines from SmithKline Beecham (SB) or Chiron Biocine (CB) or whole-cell vaccine (wP) from Connaught, each combined with diphtheria and tetanus toxoids (DT), or a DT vaccine only. When the purified antigens were used, statistically significant differences in CMI responses were observed between pre- and post-vaccination samples. In particular, CMI responses to FHA and PRN were detected in the majority of both aP vaccines recipients, whereas DTwP-recipients were CMI-positive in a much lower proportion. Clear-cut differences in PT responses were detected between DTwP and DTaP vaccine recipients, in favour of the latter. These differences were maintained up to 24 months after completion of the primary vaccination schedule. Thus, CMI responses could be a useful adjunct to serology in studying the immune responses to pertussis vaccines.

Secretion of IFN-gamma, IL-6, granulocyte-macrophage colony-stimulating factor and IL-10 cytokines after activation of human purified T lymphocytes upon CD38 ligation.

Human CD38, a surface glycoprotein expressed by different immunocompetent cells, is associated with distinct transmembrane signaling molecules and plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. This study reports that CD38 ligation by specific monoclonal antibodies (mAb) in purified peripheral blood T cells is followed by secretion of discrete cytokines. IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-gamma, and IL-10 mRNA expression were constant findings. Low levels of IL-2 mRNA were also detected in CD38-activated T lymphocyte cultures of all subjects studied. Low levels of IL-4 and IL-5 mRNA were detected in the majority of CD38-activated T cultures. Moreover, CD38 mediated cytokine induction does not require T cell proliferation or the addition of antigen presenting cells. In conclusion, human CD38 runs an activation pathway in purified T cells which operates through the induction of a cytokine profile shared by Th1 or Th2 cells.

Late acquisition of hyporesponsiveness to lipopolysaccharide by Mycobacterium avium-infected human macrophages in producing tumor necrosis factor-alpha but not interleukin-1 beta and -6.

To investigate whether infection with Mycobacterium avium modifies the cytokine response of human macrophages (Mphi) to lipopolysaccharide (LPS), the release of interleukin (IL)-1 beta, IL-6, and tumor necrosis factor (TNF)-alpha was determined in infected and uninfected Mphi, unstimulated or stimulated with LPS. In unstimulated Mphi, the release of IL-1 beta and IL-6 increased with the progress of infection while that of TNF-alpha progressively decreased. When Mphi were stimulated with LPS, IL-1 beta and IL-6 levels were always higher in infected than in uninfected cells, but levels of TNF-alpha significantly decreased in infected Mphi. A similar trend was obtained for TNF-alpha mRNA expression. Altogether, these results indicate that infected Mphi react to LPS stimulus with enhanced levels of IL-1 beta and IL-6 but are unable to restore the production of TNF-alpha impaired by the growth of the intracellular mycobacteria.

CD38 ligation induces discrete cytokine mRNA expression in human cultured lymphocytes.

Human CD38 is a surface glycoprotein expressed by different immuno-competent cells such as immature and activated lymphocytes, plasma cells and natural killer cells. It has recently been reported that the CD38 molecule exerts adenosine diphosphate ribosyl cyclase activity and is associated with distinct transmembrane signaling molecules. This study reports that ligation of CD38 by specific monoclonal antibodies (mAb) induces multiple cytokine mRNA expression in cultured peripheral blood mononuclear cells (PBMC). The mRNA for tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-12 were always detected, whereas interferon-gamma and IL-10 mRNA expression were seen in most, but not all PBMC cultures. Low levels of IL-2, IL-4 and IL-5 mRNA were also found. The key observation of this work is that CD38 ligation in PBMC induces a large spectrum of cytokines, many of which overlap with those induced via CD3 activation. The main differences between CD38 and CD3 activation are the low to undetectable levels of IL-2 mRNA, and the sustained IL-1 beta and IL-6 mRNA accumulation found in PBMC cultures following treatment with anti-CD38 mAb. Furthermore, PBMC proliferation was not found to be a prerequisite for CD38-mediated cytokine induction. Together, these results suggest that human CD38 activates a signaling pathway which leads to the induction of a discrete array of cytokines, and that this pathway only partially overlaps with that controlled by T cell receptor CD3.

Defective expression of interferon-gamma, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and interleukin-6 in activated peripheral blood lymphocytes from glioma patients.

The ability of a mannoprotein antigen from Candida albicans (MP) or interleukin-2 (IL-2) to induce cytokines in cultures of peripheral blood mononuclear cells (PBMC) of glioma patients and healthy controls was evaluated by mRNA expression and by protein secretion. The subjects studied were all responsive to both MP and IL-2, as assayed by lymphoproliferation of PBMC cultures. In control subjects, MP and IL-2 were strong inducers of IFN-gamma, IL-1 beta, TNF-alpha, and GM-CSF mRNA expression, but only MP was able to induce considerable levels of IL-6 and IL-2 mRNA expression. In MP-activated PBMC from glioma subjects, a highly defective IFN-gamma, together with a significant reduction in TNF-alpha and GM-CSF mRNA expression, was observed. This impairment was paralleled by a decreased accumulation of IL-6 and IL-2 mRNA. The pattern of cytokine mRNAs in IL-2-activated PBMC of glioma patients confirmed the impairment of IFN-gamma mRNA expression paralleled by a reduction in IL-6, TNF-alpha and GM-CSF mRNA, compared with healthy subjects. Coherently, in PBMC cultures from glioma patients, there was a clear-cut decrease in the secretion of IL-6 and TNF-alpha and especially of IFN-gamma compared with healthy controls. No or very low levels of IL-4, IL-10, and TGF-beta 2 mRNA expression were detected in PBMC cultures of both glioma and control populations, irrespective of the activation conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

CD8+ T lymphocytes provide helper activity for IgE synthesis in human immunodeficiency virus-infected patients with hyper-IgE.

Increased levels of serum IgE and eosinophilia have been described in human immunodeficiency virus (HIV) infection, almost exclusively in patients with CD4+ cell count < 200 cells/microliters. IgE production is regulated by CD4+ T helper type 2 (Th-2) lymphocytes, producing interleukin 4 (IL-4) and expressing a ligand for the B cell-specific CD40 molecule (CD40 ligand [L]). A shift to a Th-2-like pattern of cytokine secretion has been postulated to be associated with progression toward acquired immunodeficiency syndrome (AIDS). We studied three AIDS patients with very high levels of IgE and almost complete depletion of CD4+ lymphocytes, suggesting that IgE synthesis could not be driven by CD4+ cells. IgE in vitro synthesis by cells from such patients was, however, inhibited by anti-IL-4. We show that both CD8+ T cell lines and the majority of CD8+ T cells clones derived from these patients produce IL-4, IL-5, and IL-6 in half of the cases together with interferon gamma (IFN-gamma). 44% of CD8+ T cell clones expressed a CD40L, and the supernatants of the clones were capable of inducing IgE synthesis by normal B cells costimulated with anti-CD40. CD8+ T cells in these patients therefore functionally mimic Th-2 type cells and may account for hyper-IgE and eosinophilia in the absence of CD4+ cells. The presence of such CD8+ cells may also provide a source of IL-4 directing the development of predominant Th-2 responses in HIV infection.

Cytokine gene expression in human peripheral blood mononuclear cells stimulated by mannoprotein constituents from Candida albicans.

The expression of cytokine genes in cultures of human peripheral blood mononuclear cells (PBMC) stimulated with mannoprotein constituents (MP) of Candida albicans has been studied by means of S1 nuclease mapping analysis, polymerase chain reaction, and enzyme-linked immunosorbent assay. MP induced early, consistent, and long-lasting production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, and IL-6 mRNAs. Similar results were obtained when the same PBMC cultures were stimulated with the purified protein derivative (PPD) from Mycobacterium tuberculosis or with IL-2, although lower levels of IL-6 mRNA were detected in IL-2-stimulated cells than in MP- or PPD-stimulated cells. MP, PPD, and IL-2 induced appreciable levels of granulocyte-macrophage colony-stimulating factor and gamma interferon, but only MP and PPD were able to induce IL-2 mRNA. MP were unable to stimulate a consistent expression of the genes encoding for IL-4, IL-5, and IL-10, while low, sometimes barely detectable levels of these cytokine mRNAs were observed in PPD- or IL-2-stimulated PBMC cultures. When protein synthesis of MP-stimulated PBMC was inhibited by cycloheximide, a superinduction of mRNAs for IL-4 and IL-10 and, more markedly, gamma interferon was observed. Overall, these results highlight the powerful, selective induction of cytokine gene expression by MP constituents of C. albicans in human PBMC cultures, thus providing some functional clues to explain the efficient state of the anticandidal response in normal human subjects.

Identification of a 65-kDa mannoprotein as a main target of human cell-mediated immune response to Candida albicans.

To identify molecular targets of anticandidal cell-mediated immunity (CMI) in humans, a highly immunogenic mannoprotein fraction (MP-F2) of Candida albicans was studied. SDS-PAGE and gel-permeation chromatography separated MP-F2 into polydisperse mannoproteins of > 200-31.5 kDa. However, only a 65-kDa constituent specifically induced proliferation of human peripheral blood mononuclear cells (PBMC). Lymphoproliferation was accompanied by production of interleukin (IL)-1 beta, interferon-gamma, and IL-6 but not IL-4. MP-F2- and MP-65-induced PBMC proliferation was inhibited by an antagonist anti-T cell receptor antibody. Neither the purified protein derivative of Mycobacterium tuberculosis nor MP-65 activated naive lymphocytes from umbilical cord blood, although these cells proliferated extensively in response to both phytohemagglutinin and IL-2. These data strongly suggest that MP-65 is an immunodominant mannoprotein antigen that is ordinarily expressed as a target of anti-Candida CMI in healthy humans.