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Hypomorphic mutations in the zinc finger domain of NF-kappaB essential modulator (NEMO) cause X-linked hyper-IgM syndrome with ectodermal dysplasia (XHM-ED). Here we report that patient B cells are characterized by an absence of Ig somatic hypermutation (SHM) and defective class switch recombination (CSR) despite normal induction of activation-induced cytidine deaminase (AID) and Iepsilon-Cepsilon transcripts. This indicates that AID expression alone is insufficient to support neutralizing antibody responses. Furthermore, we show that patient B cells stimulated with CD40 ligand are impaired in both p65 and c-Rel activation, and whereas addition of IL-4 can enhance p65 activity, c-Rel activity remains deficient. This suggests that these NF-kappaB components have different activation requirements and that IL-4 can augment some but not all NEMO-dependent NF-kappaB signaling. Finally, using microarray analysis of patient B cells we identified downstream effects of impaired NF-kappaB activation and candidate factors that may be necessary for CSR and SHM in B cells.

Hyper-IgM syndrome (HIGM) is a heterogeneous condition characterized by impaired Ig class-switch recombination (CSR). The molecular defects that have so far been associated with this syndrome - which affect the CD40 ligand in HIGM type 1 (HIGM1), CD40 in HIGM3, and activation-induced cytidine deaminase (AID) in HIGM2 - do not account for all cases. We investigated the clinical and immunological characteristics of 15 patients with an unidentified form of HIGM. Although the clinical manifestations were similar to those observed in HIGM2, these patients exhibited a slightly milder HIGM syndrome with residual IgG production. We found that B cell CSR was intrinsically impaired. However, the generation of somatic hypermutations was observed in the variable region of the Ig heavy chain gene, as in control B lymphocytes. In vitro studies showed that the molecular defect responsible for this new HIGM entity (HIGM4) occurs downstream of the AID activity, as the AID gene was induced normally and AID-induced DNA double-strand breaks in the switch micro region of the Ig heavy chain locus were detected during CSR as normal. Thus, HIGM4 is probably the consequence of a selective defect either in a CSR-specific factor of the DNA repair machinery or in survival signals delivered to switched B cells.

X-linked hyper-IgM syndrome (XHIM) results from mutations in the gene encoding for CD40 ligand (CD154). Patients with the syndrome suffer from infections with opportunistic pathogens such as Cryptosporidium and Pneumocystis carinii. In this study, we demonstrate that activated T cells from patients with XHIM produce markedly reduced levels of IFN-gamma, fail to induce antigen-presenting cells to synthesize IL-12, and induce greatly reduced levels of TNF-alpha. In addition, we show that the patients' circulating T lymphocytes of both the CD4(+) and CD8(+) subsets contain a markedly reduced antigen-primed population, as determined by CD45RO expression. Finally, we demonstrate that the defects in antigen priming are likely due to the lack of CD154 expression and insufficient costimulation of T cells by CD80/CD86 interactions. Taken together, this study offers a basis for the increased susceptibility of patients with XHIM to certain opportunistic infections.

The present study analyzed peripheral blood B cell populations separated by IgD and CD27 expression in six males with X-linked hyper-IgM syndrome (XHIM). Costimulation of mononuclear cells from most of the patients induced no to low levels of class switching from IgM to IgG and IgA with Staphylococcus aureus Cowan strain (SAC) plus IL-2 or anti-CD40 mAb (anti-CD40) plus IL-10. Measurable levels of IgE were secreted in some of the patients after stimulation with anti-CD40 plus IL-4. Costimulation with SAC plus IL-2 plus anti-CD40 plus IL-10 yielded secretion of significant levels of IgG in addition to IgM, but not IgA. The most striking finding was that peripheral blood B cells from all of the six patients were composed of only IgD+ CD27(-) and IgD+ CD27(+) B cells; IgD- CD27(+) memory B cells were greatly decreased. IgD+ CD27(+) B cells from an XHIM patient produced IgM predominantly. Our data indicate that the low response of IgG production in XHIM patients is due to reduced numbers of IgD- CD27(+) memory B cells. However, the IgG production can be induced by stimulation of immunoglobulin receptors and CD40 in cooperation with such cytokines as IL-2 and IL-10 in vitro.

Recent studies show that most patients with X-linked hyper IgM syndrome have defects in the gene for CD40 ligand. We evaluated 17 unrelated males suspected of having X-linked hyper IgM syndrome. Activated T cells from 13 of the 17 patients failed to bind a soluble CD40 construct. In these patients, the sequence of CD40 ligand demonstrated mutations. By contrast, T cells from the remaining four patients exhibited normal binding to the CD40 construct. Sequencing of the cDNA for CD40 ligand from these patients did not show mutations. The possibility that hyper IgM syndrome in these four patients was due to abnormalities in the B cell response to CD40-mediated signals was examined. Peripheral blood lymphocytes were stimulated with anti-CD40 alone, IL4 alone or anti-CD40 plus IL4. In comparison with B cells from controls or patients with hyper IgM syndrome and mutant CD40 ligand, B cells from the patients with hyper IgM syndrome and normal CD40 ligand were defective in their ability to secrete IgE (P < 0.02) or express activation markers, CD25 and CD23 (P < 0.02) in response to stimulation with anti-CD40. The failure of these B cells to respond to CD40-mediated activation could not be attributed to a generalized deficiency in B cell activation because IL4 induced normal up-regulation of CD23 and CD25 expression. These findings indicate that hyper IgM syndrome may result from defects in expression of CD40 ligand by activated T cells or defects in CD40-mediated signal transduction in B cells.

In the current study, we investigated whether Staphylococcus aureus grown from affected skin of atopic dermatitis (AD) patients secreted identifiable toxins that could act as allergens to induce IgE-mediated basophil histamine release. The secreted toxins of S. aureus grown from AD patients were identified by ELISA using antibodies specific for staphylococcal enterotoxin (SE) exfoliative toxin (ET), or toxic shock syndrome toxin (TSST-1). S. aureus isolates from 24 of 42 AD patients secreted identifiable toxins with SEA, SEB, and TSST accounting for 92% of the isolates. 32 of 56 AD sera (57%) tested contained significant levels of IgE primarily to SEA, SEB, and/or TSST. In contrast, although SEA, SEB, or TSST secreting S. aureus could be recovered from the skin of psoriasis patients, their sera did not contain IgE antitoxins. Freshly isolated basophils from 10 AD patients released 5-59% of total histamine in response to SEA, SEB, or TSST-1 but only with toxins to which patients had specific IgE. Basophils from eight other AD patients and six normal controls who had no IgE antitoxin failed to demonstrate toxin-induced basophil histamine release. Stripped basophils sensitized with three AD sera containing IgE to toxin released 15-41% of total basophil histamine only when exposed to the relevant toxin, but not to other toxins. Sensitization of basophils with AD sera lacking IgE antitoxin did not result in release of histamine to any of the toxins tested. These data indicate that a subset of patients with AD mount an IgE response to SEs that can be grown from their skin. These toxins may exacerbate AD by activating mast cells, basophils, and/or other Fc epsilon-receptor bearing cells armed with the relevant IgE antitoxin.

The hyper-IgE (HIE) syndrome is characterized by high IgE serum levels, chronic dermatitis, and recurrent infections. The mechanisms responsible for hyperproduction of IgE in HIE patients are presently unknown. We investigated whether spontaneous in vitro IgE synthesis by PBMC from seven HIE patients was sensitive to signals (cell adhesion, T/B cell cognate interaction and lymphokines: IL-4, IL-6, and IFN-gamma) known to regulate IgE induction in normals. Our results show that, unlike IL-4 dependent IgE synthesis induced in normals, spontaneous IgE production by PBMC from HIE patients was not blocked by monoclonal antibodies to CD2, CD4, CD3, and MHC class II antigens. Furthermore, antibodies to IL-4 and IL-6 did not significantly suppress IgE production. IFN-gamma had no significant effects on spontaneous in vitro IgE synthesis. To test whether an imbalance in lymphokine production might underlie hyperproduction of IgE in HIE patients, mitogen-induced secretion of IL-4 and IFN-gamma by PBMC was assessed. No significant difference was detected between HIE patients and normal controls. Thus, ongoing IgE synthesis in the HIE syndrome is largely independent of cell-cell interactions and endogenous lymphokines, and is due to a terminally differentiated B cell population, no longer sensitive to regulatory signals.

The chemotactic responsiveness of the neutrophils of 10 patients with the hyperimmunoglobulin E-recurrent infection syndrome (HIE) were compared with neutrophils from normal volunteers over a 10-mo period. HIE neutrophils as a group displayed significantly less chemotactic motility than control neutrophils. The data from individual patients were variable, being normal or abnormal on different days. Mononuclear cells from HIE patients, when cultured for 24 h in the absence of serum or a mitogen, produced a factor that inhibited normal neutrophil and monocyte chemotaxis. Mononuclear cells from normal volunteers with and without atopy or from patients with parasites or bacterial infections did not produce such an inhibitory factor. The production of this chemotactic inhibitory factor in vitro was variable over time, but it correlated with the presence of an in vitro neutrophil chemotactic defect. The chemotactic inhibitory factor was partially purified and was found to contain protein, to be stable at 56 degrees C, and to have a molecular weight of approximately 61,000. Irreversible inhibitors of serine esterases do not inactivate the factor. The factor is produced by esterase-negative mononuclear cells and is not toxic to neutrophils. This chemotactic inhibitory factor may be the basis of the variable chemotactic defect in HIE neutrophils.

To gain insight into a possible role for antibody-dependent cell-mediated cytotoxicity in vivo, we examined the ability of leukocytes from 28 patients with primary immunodeficiency and from 20 normal controls to lyse three different types of antibody-coated targets in vitro. Mean cytotoxic indices +/-1 SD elicited by unfractionated mononuclear cells from normal controls were 28.74+/-13.26 for human HLA antibody-coated lymphocyte targets, 42.79+/-8.27 for rabbit IgG antibody-coated chicken erythrocyte targets, and 47.58+/-10.34 for human anti-CD (Ripley)-coated O+ erythrocyte targets. Significantly (P=<0.05) lower than normal mean cytotoxic indices against lymphocyte targets were seen with effector cells from 10 patients with X-linked agammaglobulinemia (3.7+/-4.33), in 10 with common variable agammaglobulinemia (16.05+/-7.74), in 3 with immunodeficiency with hyper IgM (18.41+/-4.88), and in 2 with severe combined immunodeficiency (3.94+/-0.3). Antibody-dependent cytotoxicity against chicken erythrocytes was significantly (P=<0.05) lower than normal only in the common variable agammaglobulinemic group (33.33+/-12.3) and against human erythrocytes only in the common variable (34.36+/-9.59) and hyper IgM (27.54+/-0.66) groups. Rosette and anti-F(ab')(2) depletion studies with normal leukocytes indicated that a nonadherent, nonphagocytic, non-Ig-bearing, non-C receptor-bearing, Fc receptor-bearing lymphocyte was the only effector capable of lysing HLA antiboyd-coated lymphocyte targets. Patients with infantile X-linked agammaglobulinemia and severe combined immunodeficiency appear to have a marked deficiency in this type of effector cell function.

Concentrations of IgD and IgE were measured in sera from 165 patients with well-defined immunodeficiency in an effort to find information possibly relevant to the roles of antibodies of these classes in host defense. Values for both immunoglobulins were generally quite low in patients who had marked deficiencies of all three major immunoglobulins, although occasional normal or high normal values for IgD were seen in hypogammaglobulinemic patients. Group mean IgD concentrations were also depressed in patients with Wiskott-Aldrich syndrome and in those with selective IgA deficiency; IgE concentrations were depressed in patients with X-linked immunodeficiency with hyper-IgM and in those with ataxia telangiectasia. IgD and IgE were both significantly elevated in patients with extreme hyperimmunoglobulinemia E and undue susceptibility to infection and in a patient with the Nezelof syndrome; none of these patients had histories suggestive of atopy. In addition, the mean IgE concentration was significantly elevated in patients with selective IgA deficiency, many of whom were atopic, and in those with the Wiskott-Aldrich syndrome. The highest IgD concentration (163 mg/100 ml) was found in serum from a boy with variable immunodeficiency who had a lifelong history of severe recurrent pharyngeal infections, primarily streptococcal in etiology. Recurrent staphylococcal infection was a feature common to many but not all patients with elevated serum IgE concentration. These data may prove useful in the future delineation of biologic roles for antibodies in these two immunoglobulin classes.

The metabolism of human IgE was studied in normals, severe atopics, and patients with the hyperimmunoglobulin E-recurrent infection (HIE; Job's) syndrome to determine whether IgE metabolism is altered in patients with marked elevation of serum IgE. Purified polyclonal 125I-IgE was administered intravenously and serial plasma and urine samples were obtained. After analysis, the metabolic data support previously published evidence that IgE (at concentrations found in normal individuals) is catabolized at a higher fractional rate than other immunoglobulins and is catabolized by both an intravascular and an extravascular pathway. In addition, the data show that the fractional catabolic rate for IgE is significantly less for the atopic patients (mean +/- SEM = 0.20 +/- 0.01) and for the HIE patients (0.15 +/- 0.02) than for the normal volunteers (0.52 +/- 0.06; P less than 0.01) and is inversely related (r = -0.851; P less than 0.001) to the serum IgE concentration. These findings have specific importance in showing that decreased fractional catabolic rate contributes substantially to elevation of IgE in atopic and HIE patients. In addition, the findings have general significance in that they lead to a unifying hypothesis of immunoglobulin catabolism.

Affinity-purified IgE-binding factors from the plasma of patients with the hyper IgE syndrome (HIE) were assessed for their capacity to enhance IgE synthesis by B cells derived from patients with allergic rhinitis or normal nonatopic donors. IgE-binding factors from three of four HIE patients enhanced IgE synthesis by B cells from patients with perennial allergic rhinitis, or with seasonal allergic rhinitis (SAR) and recent pollen exposure, but did not enhance IgE synthesis by B cells from nonatopic donors or from SAR patients with no recent pollen exposure. IgG synthesis was not affected by HIE IgE binding factors. In contrast, IgE binding factors from three of three nonatopic donors failed to enhance IgE or IgG synthesis. Plasma IgE-binding factors from the fourth patient with HIE contained a mixture of IgE-potentiating activity and IgE-suppressive activity. These two activities could be separated on concanavalin A Sepharose or peanut agglutinin agarose columns. Human IgE potentiating factor, but not IgE suppressive factor, had affinity for concanavalin A but not peanut agglutinin and fractionated into two peaks on gel filtration over Sephadex G-75: one peak with a molecular size of approximately 15,000 D and the other with a molecular size of approximately 60,000 D. The isolation of functional IgE binding factors which potentiate IgE synthesis from the plasma of patients with HIE suggests that IgE-binding factors play an important role in the in vivo regulation of IgE synthesis in man.

Immunological evaluations (lymphocyte markers, B cell differentiation, T cell function) were performed on peripheral blood mononuclear cells from four individuals with hyper IgM immunodeficiency. Number, proportion, and proliferation of T lymphocytes and T lymphocyte subpopulations were relatively normal in affected individuals. The percentage and number of B cells expressing surface IgM and IgD were either normal or elevated in both blood and lymph nodes. However, surface IgG- and IgA-bearing B lymphocytes were completely absent. In vitro stimulation of blood lymphocytes with both T cell-dependent and T-cell independent polyclonal B cell activators resulted in normal numbers of IgM plasma cells and IgM secretion in cultures, but failed to induce any IgG- or IgA-producing cells. This failure of isotype switching was intrinsic to the B cell population and did not involve aberrant T cell help or suppression. Therefore, individuals with this disorder possess an intrinsic B cell dysfunction that is not related to abnormal T cell regulation.

Patients with the hyperimmunoglobulin E and recurrent infection syndrome (HIE) characteristically have frequent skin and respiratory infections caused by Staphylococcus aureus. We have developed a set of enzyme-linked immunosorbent assays that use whole S. aureus (Wood's strain) immobilized on 0.22-micrometers filters and highly specific, affinity-purified enzyme conjugates of goat anti-human IgE, anti-human IgD, anti-human IgG, anti-human IgA, and anti-human IgM. These reagents were used to determine S. aureus-specific immunoglobulin (Ig) levels. As previously published, 10 patients with HIE had markedly higher levels of anti-S. aureus IgE than did 5 patients with eczema and recurrent superficial S. aureus infections (P less than 0.001). The HIE patients were also found to have a deficit of anti-S. aureus serum IgA as compared with 12 normal subjects, 12 patients with chronic granulomatous disease, 5 patients with chronic eczema and recurrent superficial S. aureus infections, and 3 patients with the Chediak-Higashi syndrome (P less than 0.01 for each comparison). In addition the HIE patients had an excess of anti-S. aureus IgM as compared with normal subjects (P less than 0.01). An expected excess of anti-S. aureus IgG was absent. These abnormalities cannot be explained by variations of total serum Ig levels or by a general inability to produce antigen-specific IgA because levels of naturally occurring IgA antibody against Escherichia coli lipopolysaccharide and the antigens of the pneumococcal vaccine are normal. Parotid saliva from patients with HIE contained less salivary IgA per milligram of protein (P less than 0.01) and less salivary anti-S. aureus IgA per milligram of protein (P less than 0.05) than did normal controls. The incidence of infection at mucosal surfaces and adjacent lymph nodes correlated inversely with serum anti-S. aureus IgA (r = -0.647, P = 0.034), serum anti-S. aureus IgE (r = -0.731, P = 0.016), total serum IgE (r = -0.714, P = 0.020), and total serum IgD (r = -0.597, P = 0.049). These findings are evidence of a previously undescribed immunoregulatory defect in patients with HIE, which may contribute to the increased susceptibility to infection in this syndrome.

The status of suppressor T cells (Ts) was assessed in seven children with the hyper IgE syndrome (recurrent staphylococcal infections, eczematous skin rash, and elevated serum IgE) to determine whether a deficiency in Ts is associated with increased IgE synthesis. When circulating T cells and their subsets were enumerated with the aid of monoclonal antibodies that identify T cells (T3), helper/inducer T cells (T4), and suppressor/cytotoxic T cells (T8), there was a selective deficiency of T3+ cells (51.7+/-11.2% vs. 66+/-5% for normal controls) and of T8+ cells (7.5+/-4.4% vs. 22+/-4% for normal controls) but not of T4+ cells (36.5+/-7.5% vs. 37+/-3% for normal controls). Suppressor T cell function was assessed by examining the ability of mononuclear cells incubated for 48 h with concanavalin A to suppress the proliferation of fresh autologous mononuclear cells in response to the mitogens phytohemagglutinin and pokeweed mitogen. All seven patients were severely deficient in concanavalin A-inducible suppressor cells. In vitro de novo synthesis of IgE in 6-d cultures of peripheral blood lymphocytes was measured in four patients by a solid-phase radioimmunoassay. Mononuclear cells from all four patients synthesized spontaneously increased quantities of IgE in vitro (4,950+/-3,760 pg/10(6) cells vs. 250+/-215 pg/10(6) cells for eight normal controls). IgE synthesis was suppressed by the addition of parental T cells to the culture. Elimination of the T8+ subset, but not of the T4+ subset, by complement-dependent lysis resulted in the loss of the capacity of parental T cells to suppress IgE synthesis. These results suggest that a deficiency of Ts underlies the elevated IgE levels observed in the hyper IgE syndrome.

The neoplastic T cells of a series of seven patients with chronic T-cell neoplasia were tested for helper activity on pokeweed mitogen (PWM)-induced and interleukin 2 (IL-2)-induced Ig synthesis. The neoplastic T cells of all patients had a T3+4+8-11+I1- phenotype but differed in expression of the 3A1 antigen. The neoplastic T cells of three patients had helper activity on both PWM- and IL-2-driven Ig synthesis, and in addition produced IL-2 in response to PWM stimulation. Two of these patients had hypergammaglobulinemia. In contrast, the neoplastic T cells in the remaining four patients did not produce IL-2 and did not support PWM-driven Ig synthesis. The T4+ cells of these four patients, however, provided excellent helper activity on IL-2-driven Ig synthesis. These findings emphasize the role of IL-2 in T cell-dependent Ig synthesis and clearly show that IL-2 production is required for helper activity in the PWM-driven system. It is concluded that the combined use of PWM- and IL-2-driven Ig synthesis systems allows separate analysis of IL-2 production and T-helper activity in health and disease.