Up-regulation of IL-12 in monocytes: a fundamental defect in common variable immunodeficiency.

We show that LPS-stimulated circulating CD14-positive monocytes from patients with common variable immunodeficiency (CVID) express a higher proportion of intracellular IL-12-positive cells than monocytes from patients with X-linked agammaglobulinemia or normal subjects. We used four-color flow cytometry and measured IL-12 with an Ab to the p40 subunit following stimulation with LPS. The raised IL-12 is associated with an increased frequency of IFN-gamma-positive T cells, but not of IFN-gamma-positive CD56+ NK cells. These increases in frequency of cytokine-positive cells are due to a decrease in the absolute numbers of circulating monocytes and T cells that are negative for IL-12 and IFN-gamma, respectively. The increased frequency of IL-12-positive monocytes appears to be selective because TNF-alpha was not increased, and is thus unlikely to reflect a general activation. Chronic infection is also unlikely to explain our data since cells from X-linked agammaglobulinemia patients with a similar Ig deficiency do not show these changes. Our data suggest a fundamental abnormality in the IL-12/IFN-gamma circuit in CVID, with up-regulation of IL-12 being the "primary" factor. This imbalance is likely to skew the immune response away from Ab production and also explains the failure of CVID T cells to make Ag-specific memory cells and the chronic inflammatory and granulomatous complications that are a feature of CVID. This disease appears to be a rare example of a polarized Th1-type response and may in part be due to a genetic defect in the control of IL-12 production.

In vivo modulation of cytokine synthesis by intravenous immunoglobulin.

We examined the effects of intravenous immunoglobulin (IVIG) on cytokine regulation in vivo using samples taken before and after replacement-dose (200-400 mg/kg) IVIG in a group of patients with common variable immunodeficiency (CVID) and X-linked agammaglobulinaemia (XLA). The intracellular cytokine content of CD4+ and CD8+ lymphocytes, and their CD28+/- subsets, were measured following in vitro activation with phorbol myristate acetate (PMA) and ionomycin. The cytokines IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and the early activation marker CD69, were assessed by four-colour flow cytometry of whole blood cultures taken before and after IVIG infusion. There was a significant increase in IL-2 expression in CD4+ (and CD4+28-) cells and an increase in TNF-alpha expression in CD8+28- cells following IVIG in CVID, but not in XLA patients. IFN-gamma and CD69 expression were not affected by IVIG infusion. This increase in TNF-alpha and IL-2, combined with unchanged IFN-gamma expression, is evidence against the putative 'anti-inflammatory' role of IVIG, and may explain the failure of resolution of granulomata in CVID patients treated with IVIG alone.

Primary defect in CD8+ lymphocytes in the antibody deficiency disease (common variable immunodeficiency): abnormalities in intracellular production of interferon-gamma (IFN-gamma) in CD28+ ('cytotoxic') and CD28- ('suppressor') CD8+ subsets.

We have measured by flow cytometry the ability of subsets of CD8+CD3+ lymphocytes within mononuclear cell preparations to make intracellular cytokines (IL-2, tumour necrosis factor-alpha (TNF-alpha) and IFN-gamma) on stimulation in vitro with phorbol myristate acetate (PMA) and ionomycin for 16 h. These CD8+ subsets were defined by the presence or absence of CD28 or HLA-DR. Subsets of normal CD8+ cells were compared with cells from the antibody deficiency disease common variable immunodeficiency (CVID). In CVID there was a significant increase in the production of IFN-gamma in the CD8+CD28+ subset ('cytotoxic'). This reflects a shift in this disease towards an excessive Th1 response away from B cell help. Paradoxically, some CVID patients also showed a reduction in IFN-gamma production in the CD8+CD28- subset ('suppressor') which was associated with a failure of these cells to maintain a state of activation after a stimulus in vitro. The B cell problem in this disease is known to be related to a failure of T cell help shown by an inability to produce the antigen-specific CD4+ memory T cells needed for successful B cell maturation. The two pathological CD28 subsets of CD8+ cells we have found in CVID may both be detrimental to a normal CD4-dependent immune response. The CD28- suppressor subset expands and is unable to maintain activation and cytokine secretion, and the CD28+ cytotoxic subset is over-producing the Th1 cytokine IFN-gamma.

Determination of intracellular cytokines by flow-cytometry following whole-blood culture.

Various methods have been reported for measuring intracellular cytokines in peripheral blood mononuclear cells isolated by density-gradient centrifugation. In this report, we describe a whole-blood method for the determination of intracellular cytokines (IFN-gamma, TNF-alpha and IL-2) that uses small-volume (500 microl) blood samples. Directly conjugated anti-cytokine antibodies and commercial cell membrane fixation and permeabilisation reagents were used. Blood was cultured in a 1:3 dilution with a combination of PMA and ionomycin to reveal the cytokine synthetic potential of each cell, together with monensin to increase the sensitivity by retaining cytokines within the cell to detectable levels. The optimum concentrations of PMA (10 ng/ml (16.2 nmol/l)), ionomycin (2 micromol/l) and monensin (3 micromol/l) were determined. Kinetic studies showed maximal cytokine expression after 2 h of culture for TNF-alpha and IFN-gamma and 4 h for IL-2. Assessment of TNF-alpha and IFN-gamma production within the CD4 and CD8 lymphocytes from 10 normal volunteers showed that considerably more CD8 + than CD4 + cells produced IFN-gamma. This technique could be used by routine immunology laboratories and will be of use in studies to determine whether cytokine assays are of value in the investigation of immune disorders.

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry.

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) induced in human CD4+ and CD8+ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20 h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4+ cells can make IL-2 than the other two cytokines, and that there are more TNF-alpha-positive CD4+ cells than cells with IFN-gamma. For normal CD8+ cells the highest production of cytokine is of IFN-gamma (up to 50% of the cells) especially at longer times (10-20 h) of stimulation. For CD8+ cells, IL-2-positive cells exceed those with TNF-alpha. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20 h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4+ or CD8+ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-alpha but significantly raised levels of production of IFN-gamma in both CD4+ and CD8+ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.

Co-stimulation with anti-CD28 (Kolt-2) enhances DNA synthesis by defective T cells in common variable immunodeficiency.

In normal T cells, an anti-CD28 MoAb (Kolt-2) will synergize with the mitogenic stimuli phytohaemagglutinin (PHA), anti-CD3 (OKT3) or a combination of anti-CD2 antibodies (OKT11 and GT2) in the induction of DNA synthesis. A subgroup of patients with common variable immunodeficiency (CVID) show a defect in DNA synthesis by T cells stimulated in vitro with the above mitogens. We have now investigated whether anti-CD28 will correct the defect. This strategy partially restored DNA synthesis, providing evidence that the CD28 co-stimulatory pathway in CVID T cells is normal. Ligation of CD28 acts through co-stimulating IL-2 secretion. The natural ligand (B7) for CD28 on antigen-presenting cells from CVID patients is expressed normally. We conclude that the defect in CVID T cells lies in pathways that lead to transcription of the IL-2 gene other than that induced by ligation of CD28 with Kolt-2.

LFA-1-dependent OKT3-driven T cell clusters in common variable immunodeficiency.

The triggering of the TCR/CD3 complex by anti-CD3 (OKT3) antibody leads to the formation of T cell clusters. In cultures of T lymphocytes from most normal individuals, the peak of cluster formation occurs at 24 h, but with cells from patients with common variable immunodeficiency (CVI) it was seen earlier at 4-9 h; in addition, the clusters were larger than normal, particularly at 9 h. Cluster formation by CVI and normal cells was dependent on temperature and divalent cations, but did not require Fc receptors. Since OKT3 clustering is known to be dependent on the LFA-1/ICAM-1 adhesion system, the effect of monoclonal antibodies directed against these molecules was tested. A potent inhibitor was the antibody against the common beta chain of the integrin family (CD18), but of four MoAbs against the alpha chains (CD11), three inhibited and one stimulated T cell aggregate formation. Increased expression of LFA-1 or ICAM-1 on CVI patients' T cells could not be demonstrated. The accelerated clustering was therefore probably due to an increase in the proportion of cells carrying the activated form of LFA-1. The formation of large numbers of homotypic lymphocyte clusters might reduce the effective interaction between B and T cells, thus contributing to the depression of immunoglobulin synthesis observed in this disease.

The writing road to reading: From theory to practice.

Orton-based programs include essential elements that insure success for teaching language to regular and special education children. This paper traces the theoretical foundations of The Writing Road to Readingby Romalda B. Spalding (1990) from the beginning concepts taught Mrs. Spalding by Dr. Samuel T. Orton through its validation in current cognitive science and learning theory. Pilot project locations and success statistics with regular and special education children in Arizona, Louisiana, Maine, and Texas are presented. It explains how direct, multisensory instruction in seven processes necessary for skilled reading and principles of skill learning and instruction are incorporated in the Spalding Method.

Raised serum levels of CD8, CD25 and beta 2-microglobulin in common variable immunodeficiency.

Soluble CD8, soluble CD4, soluble CD25 (IL-2 receptor), beta 2-microglobulin and the cytokine tumour necrosis factor-alpha (TNF-alpha) were measured in sera from patients with common variable immunodeficiency (CVI). Levels of soluble CD8, soluble CD25 and beta 2-microglobulin but not of soluble CD4 and TNF-alpha were raised significantly above levels in normal sera. Sera from patients with X-linked agammaglobulinaemia, who are also antibody deficient, did not show this marked elevation. The raised levels of soluble CD8, soluble CD25 and beta 2-microglobulin in CVI, correlated with the extent of the defects in the B lymphocytes assessed in vitro, as well as with the clinical severity of the disease. The selective release of these molecules into sera may indicate that abnormal cellular activation occurs in most CVI patients. It is also possible that the raised levels of these soluble molecules play a part in the immunodeficiency.

Defects in proliferative responses of T cells from patients with common variable immunodeficiency on direct activation of protein kinase C.

DNA synthesis in response to mitogens has been studied in T cells from nine patients with common variable immunodeficiency (CVI) and seven normal individuals. Five out of the nine patients had cells with subnormal responses to the mitogen phytohaemagglutinin (PHA). As PHA-induced responses are largely mediated through activation of Ca(2+)-dependent protein kinase C, we studied whether the defective response was still present on direct activation of protein kinase C. This was done using combinations of concentrations of phorbol 12,13,-dibutyrate and the calcium ionophore ionomycin which induced proliferation in normal T cells. We found that in CVI patients with T cells which had normal responses to PHA, responses to phorbol ester and ionomycin were at the same level as in normal T cells. However, with this treatment, in which the linkage between the membrane receptor and protein kinase C is bypassed, the level of DNA synthesis was still depressed in the patient group whose T cells had subnormal responses to PHA. IL-2 failed to restore the DNA synthesis to normal levels when added with the phorbol ester and ionomycin to T cells from one patient in this group. These data suggest that in a group of CVI patients there are defects in T cell activation pathways at or down-stream of protein kinase C.

Role of interleukin-2 and interleukin-6 in the mitogen responsiveness of T cells from patients with 'common-variable' hypogammaglobulinaemia.

We have assessed the ability of interleukin-2 (IL-2) and interleukin-6 (IL-6) to augment the proliferative response of T lymphocytes from 'common-variable' hypogammaglobulinaemia (CVH) patients and from normal controls, to the mitogens phytohaemagglutinin (PHA) and OKT3. We show that with cells from the control group and from those patients whose T cells respond to PHA within the control range, both IL-2 and IL-6 will significantly augment the response to OKT3. However, in those patients with a T cell defect in which the PHA response is below the control range, neither IL-2 nor IL-6 could restore the PHA or OKT3 response to normal. Responses to IL-2 or IL-6 alone were always in or above the control range.

Defective DNA synthesis by T cells in acquired 'common-variable' hypogammaglobulinaemia on stimulation with mitogens.

We have studied T cell defects in acquired 'common-variable' hypogammaglobulinaemia (CVH) by measuring the synthesis of DNA, RNA and protein in vitro in response to mitogens and to interleukin 2 (IL-2). We have confirmed that some patients have defective DNA synthesis in response to PHA and shown that this extends to responses to cell-derived B cell growth factor (c-BCGF) which is also mitogenic to T cells. DNA synthesis induced by IL-2 was not defective in these patients suggesting IL2-receptor induction is normal. The mitogen-related defect in DNA synthesis was not accompanied by any reduction in synthesis of RNA or of protein. Levels of the rate limiting enzyme (thymidylate synthetase EC 2.1.1.45) responsible for de novo DNA synthesis in the absence of endogenous thymidine were measured following PHA stimulation and found to be in the normal range. In the CVH patients (but not in normal individuals) the relationship between the levels of thymidylate synthetase and DNA synthesis in response to PHA approached significance, suggesting that this pathway becomes more important in CVH patients than in normal individuals perhaps because of defects in the thymidine 'salvage' pathway.

T-cell depletion of allogeneic bone marrow prevents acceleration of graft-versus-host disease induced by exogenous interleukin 2.

Highly purified human recombinant interleukin 2 (IL-2) markedly accelerated lethal GVHD in the H-2-identical B10.BR----CBA combination, but had no effect when the donor cells were depleted of mature (Thy-1.2-positive) T lymphocytes, indicating a strong immunopotentiating effect of IL-2 on mature T cells causing GVHD. In the same donor-host combination, IL-2 did not influence the recovery from the post-transplantation bone marrow aplasia. The results suggest that IL-2 could be considered for adjuvant hormonal therapy to enhance immune recovery in recipients of T-cell-depleted allogeneic marrow.

The role of B cell differentiation factors and specific T cell help in the pathogenesis of primary hypogammaglobulinemia.

We have examined the function of T and B cells from patients with late onset primary acquired hypogammaglobulinemia (PHG). T cells from these patients give effective help to normal B cells for antigen-dependent antibody synthesis. PHG mononuclear cells also synthesize normal quantities of B cell differentiation factors, which enhance IgG, IgM and antigen-dependent antibody synthesis by normal lymphocytes. While patient T cells appear to behave appropriately, the responsiveness of patient B cells is abnormal. Although they respond to differentiation factors with increased synthesis of IgM, overall levels are 10-50-fold lower than normal B cells, and they produce little or no IgG. This pattern of response is not altered if normal T cells are the source of help. The poor response of the B cell appears to represent immaturity rather than an inherent defect, as IgG-secreting clones can be obtained after Epstein-Barr virus transformation of lymphocytes from certain patients, and some of these clones respond to differentiation factors with increased IgG production. The lack of any functional defect in the T population, and the apparent immaturity rather than abnormality of the B cells, may implicate accessory cells in the pathogenesis of the disease.

Non-specific factor enhancement of human in vitro antigen-dependent antibody synthesis: role of B cell activation and T cell help.

Lectin-free supernatants obtained from PWM-stimulated lymphocytes, enable B cells to proliferate and secrete immunoglobulin. Both functions are augmented by the addition of irradiated T cells. In the presence of antigen, these supernatants also enhance specific anti-tetanus toxoid antibody production. The components of the supernatant responsible for these activities have a molecular weight between 30,000 and 60,000, and have the characteristics of non-specific factors: they are genetically unrestricted, and do not bind to either antigen or anti-DR affinity columns. There is no evidence that the partial T dependency of these factors is an indication that their target is a T cell. Instead, T cells appear necessary to move the B cell into a state of activation in which it becomes responsive to the factor. Alternative activation signals such as Staph. A. Cowan can substitute for T cell help in the proliferative response, but not for immunoglobulin or antibody synthesis. The implications of these results for the approaches used to detect and classify B cell growth factors are discussed.

Production of antibody by human B cells under serum-free conditions.

A method is described for growing human B cells in 20 microliter hanging drops in Terasaki plates under serum-free conditions. B cell proliferation and differentiation has a critical dependence for added soybean lipid, while T cell proliferation does not. In this medium, pokeweed mitogen stimulation of separated human B cells induces high levels of immunoglobulin in a T dependent manner. Cells from donors vaccinated with tetanus toxoid and shown to be responders by a conventional culture system, produce high levels of IgG anti-tetanus antibody after antigen stimulation in these serum-free microcultures. The serum free culture system has the novel features of high sensitivity to dose of mitogen or antigen, low background responses and high antibody production with low cell numbers.

The interaction of specific T-cell help and non-specific B-cell growth factors in the production of anti-tetanus antibody by human B cells grown in serum-free microcultures.

Apart from a brief period after in-vivo immunization, only a minority of human donors provide peripheral lymphocytes that synthesize specific antibody on stimulation with tetanus toxoid in vitro. A 20 microliters hanging drop microculture technique using serum-free medium has been adapted to analyse the conditions under which B cells mature into antibody-secreting cells. Multiple permutations of antigen dose, cell concentration and T:B cell ratios have been examined. The results indicate that in-vitro failure of antigen response by the majority of donors is not due simply to an inappropriate choice of culture conditions. The addition to antigen-stimulated cultures of a lectin-free conditioned medium derived from pokeweed mitogen-stimulated peripheral lymphocytes, enables B cells from the majority of donors to produce high titres of specific antibody, in a T-dependent manner, for up to 24 months after immunization. The observed failure of prolonged antigen responsiveness in vitro thus appears to represent a failure to expand a population of antigen-specific B cells, rather than indicating an absence of such clones.

Human B cell growth factors overcome T cell-mediated inhibition of specific antibody production: a possible mechanism for the exacerbation of autoimmune disease.

Under normal circumstances, antigen-dependent antibody production in man requires autologous T cells, B cells and macrophages. If allogeneic T cells are substituted, then antibodies are not synthesized, due to the development of inhibitory interactions. Addition of B cell growth and differentiation factors changes this pattern of response, and allows antibodies to be produced even when allogeneic T cells are the source of help. There is evidence that such B cell growth factors are released during most normal immune responses: we suggest that their ability to allow B cells to escape from inhibitory interactions and secrete antibodies, may underlie the observed exacerbation of certain autoimmune diseases by intercurrent infection.