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Normal human peripheral blood mononuclear cells, depleted of most monocytes and virtually all CD8-positive cells, were stimulated in vitro with pokeweed mitogen plus Staphylococcus aureus Cowan I in the presence or absence of various neutralizing anti-cytokine antibodies. Numbers of CD5+ and CD5- immunoglobulin-secreting cells were determined using the protein A haemolytic plaque assay after labelling B1a cells with anti-CD5-coated beads. Antibodies against IL-2, IL-5 and IL-10 had little or no effect on plaque-forming cell (PFC) induction; anti-IL-6, -TNF alpha and -TGF beta enhanced PFC induction; anti-IL-1 alpha, -IL-1 beta, -IL-4, -IFN gamma and -IL-13 suppressed PFC induction. B1a and B2 cells were equally affected by cytokine deprivation using these 11 neutralizing antibodies. In contrast, neutralizing anti-IL-12 suppressed induction of CD5+ but not CD5- PFC. Furthermore, recombinant IL-12, if added during the first 48 h of culture, enhanced CD5+ PFC induction while marginally suppressing (IgG-) or not affecting (IgA-, IgM-) induction of CD5- PFC. IL-12 did not preferentially increase survival in culture of B1a cells nor induce expression of CD5 on B2-cells. Further studies are required to determine whether manipulation of B1a and B2 subsets in vivo using IL-12 could be achieved in clinical situations where imbalances in the two populations have been observed.

The relative contributions of CD5+ and CD5- B-cells in production of rheumatoid factors (RF) was evaluated in polyclonally activated B-cells from patients with IgA nephropathy (IgAN), rheumatoid arthritis (RA) and Graves' disease (GD). In IgAN and RA, diseases in which RFs are believed to be involved in pathogenesis, there were 10- and 4-fold decreases respectively in CD5+ IgG-RF-secreting B-cells compared with controls. Furthermore, the number of CD5- IgG-RF- and IgA-RF-secreting B-cells were increased 12- and 14-fold in IgAN and 9- and 4-fold in RA. Such abnormalities were not apparent in GD, in which RFs have not been implicated in pathogenesis. These findings are compatible with the concept of CD5+ RF-secreting B-cells normally acting to prevent production of potentially pathogenic RFs by CD5- B-cells. When IgAN or RA patients' B-cells were activated in the presence of control instead of autologous CD4+ cells, numbers of RF-secreting CD5- B-cells were reduced to the levels seen with control B-cells plus control T-helper cells. Presumably lymphokine secretion profiles of T-helper cells would be important in determining whether CD5+ or CD5- B-cells are activated to secrete RFs, and perhaps therapeutic manipulation of these profiles could restore normal activity of CD5+ B-cells in IgAN and RA.

We report six Chinese boys with hypogammaglobulinaemia. All but one had very low or undetectable circulating B-lymphocytes, two had reversed CD4/CD8 ratios (in one of whom this latter became normal), one had reduced lymphocyte proliferative responses to concanavalin A and pokeweed mitogen and three had deficient responses to OKT3. Generation of antibody-secreting cells in response to pokeweed mitogen was markedly defective in all patients. Co-cultures of purified lymphocyte subsets from the patients with those of normal donors revealed that in addition to B-cell deficiency seen in all patients, two had T-helper cell deficiency and two had T-suppressor cell hyperactivity. One of the latter patients was treated with cimetidine in an attempt to ablate histamine type 2 receptor-bearing suppressor cells: the absolute number of such cells was temporarily reduced but there was no concurrent correction of the functional hyperactivity. These studies point to the variable nature of T-regulatory cell deficiencies in hypogammaglobulinaemia.

We report two Chinese boys with Wiskott-Aldrich syndrome presenting with gastro-intestinal bleeding, eczema and recurrent infection. They had thrombocytopenia and the mean platelet volume was small. Serum IgG and IgA were elevated and lymphocyte proliferation in response to phytohaemagglutinin, concanavalin A and pokeweed mitogen was defective. Despite documented herpes simplex virus type 1 and cytomegalovirus infection in one patient, he did not mount any humoral response. The generation of antibody-secreting cells in response to pokeweed mitogen was markedly defective in a plaque-forming cell assay. Both patients' regulatory T-cell and B-cell functions were defective in this assay. The genetic defect in Wiskott-Aldrich syndrome therefore affects T-cells, B-cells and platelets.

Pokeweed mitogen (PWM)-stimulated cultures were established with varying numbers of CD4+ or CD8+ cells and CD5+ immunoglobulin-secreting cells were subsequently identified using a combination of haemolytic plaque formation and rosetting with immunobeads coated with anti-Leu-1. It was found that CD5+ and CD5- B-cells required similar numbers of CD4+ cells for half-maximum plaque-forming cell (PFC) induction and that 50% suppression of both CD5+ and CD5- PFC occurred with similar numbers of CD8+ cells. Suppression of both CD5+ and CD5- PFC could occur when T-helper signals were supplied by a PWM-stimulated culture supernatant, indicating that both subsets could be directly suppressed by CD8+ cells rather than by indirect suppression of T-helper cells. It is concluded that CD5+ B-cells are as responsive to T-regulatory influences as conventional CD5- B-cells.

This paper describes a new method, with high specificity and sensitivity, for evaluating cell surface markes such as differentiation antigens and cytokine receptors on immunoglobulin-secreting cells. Mononuclear cells, freshly derived from peripheral blood or following stimulation in vitro with pokeweed mitogen or Staphylococcus aureus Cowan I, are partially depleted of T cells and monocytes using immunomagnetic beads (Dynabeads) coated with anti-CD2. The cells are incubated with Dynabeads coated with monoclonal antibody against the cell marker under investigation and then used in a protein A haemolytic plaque assay. Plaque-forming cells (PFC) with (marker-positive) and without (marker-negative) attached beads can be readily enumerated. Values are given for percentages of IgG-, IgA- and IgM-PFC bearing CD19, CD38, CD25 and CD5.

Peripheral blood mononuclear cells (PBMN) that were depleted of OKT8+ cells and stimulated with pokeweed mitogen (PWM) produced higher cell yields and higher numbers of plaque-forming cells than unfractionated PBMN. Conversely, OKT8-enriched PBMN, prepared by mixing unfractionated and OKT8+ cells in a ratio of 3:1, gave reduced cell growth and B-cell activation. In OKT8-depleted cultures, B cells, OKT4+ cells, OKT8+ cells, and OKM1+ cells increased in number between days 4 and 7 of culture by factors of 9.8, 5.9, 20.1, and 5.6 respectively, whereas growth rates for these subsets were 2.4, 1.0, 2.0, and 1.3 in unfractionated cultures and 0.9, 1.0, 1.2, and 0.6 in cultures enriched for OKT8+ cells. On day 7 of culture, 73 +/- 10% of B cells secreted immunoglobulin in unfractionated cultures, whereas only 21 +/- 10% of B cells were activated in OKT8-enriched cultures. Surprisingly, PWM stimulation of OKT8-depleted PBMN produced only 40 +/- 12% activated B cells.

Using the protein A haemolytic plaque assay (PrA-HPA) and standardized, optimal conditions, pokeweed mitogen (PWM) stimulated peripheral blood lymphocytes (PBL) from normal donors produced 5,500-48,000 (mean 16,955) IgG plaque-forming cells (PFC) per 10(6) lymphocytes, 3,375-38,000 (mean 14,179) IgA-PFC/10(6) and 5,500-69,750 (mean 18,684) IgM-PFC/10(6). Addition of 4 micrograms/ml concanavalin A (Con A) at the start of PWM stimulation of PBL from normal donors gave 59-98% (mean 84%) suppression of IgG-PFC, 46-99% (mean 79%) suppression of IgA-PFC and 31-100% (mean 82%) suppression of IgM-PFC. When PFC/10(6) values in PWM-stimulated co-cultures of unfractionated PBL from pairs of unrelated normal donors were compared with PFC/10(6) obtained in the individual PWM-stimulated cultures, observed/expected ratios were between 0.81 and 1.24 (mean 0.99) for IgG-PFC, 0.85 and 1.23 (mean 0.98) for IgA-PFC and 0.86 and 1.25 (mean 1.03) for IgM-PFC. Cord blood lymphocytes (CBL) produced few PWM-stimulated PFC and co-culture of CBL with normal PBL gave markedly reduced observed/expected ratios, indicating hyperactivity of T-suppressor cells in cord blood. PBL from a patient with partial DiGeorge syndrome also responded poorly in the PWM-driven PrA-HPA, but co-culture of this patient's PBL with normal PBL and PWM gave rise to higher than expected values, indicating reduced T-helper cell function. These simple methods could be employed in clinical evaluation of the cellular defect in humoral immunodeficiency and autoimmune disease states.

OKT4+ (T helper/inducer) and OKT8+ (T cytotoxic/suppressor) subsets were depleted from peripheral blood lymphocytes (PBL) by complement-mediated lysis and residual cells examined for responsiveness to pokeweed mitogen (PWM) using a protein A haemolytic plaque assay for immunoglobulin secreting B cells. It was shown that: (1) three cycles of cell killing were required to totally abolish T helper function; (2) OKT4- PBL did not respond to PWM, but in a co-culture system, an equal number of unfractionated normal PBL could entirely reconstitute responsiveness of the residual B cells; (3) OKT8- PBL gave enhanced numbers of PWM-induced plaque forming cells (PFC); (4) addition of 4 micrograms/ml concanavalin A (con A) to PWM stimulated OKT8- PBL failed to suppress PFC generation, but suppression was induced by 12.5 and 25 micrograms/ml con A and (5) kinetics of PWM-induced PFC development were similar in the presence or absence of OKT8+ cells.

Double-marker assays were developed for enumerating CD5+ B-cells and CD25+ activated B-cells. To measure CD5+ B-cells, CD19+ (pan B-specific) cells were first isolated from peripheral blood mononuclear cells using anti-Leu12-coated immunomagnetic beads (Dynabeads). Positively selected cells were stained for surface immunoglobulin (sIg) and then incubated with anti-Leu1-coated Dynabeads. Fluorescent cells which also bound Dynabeads could be readily enumerated. The proportion of CD5+ B-cells was found to be significantly elevated in 9 of 20 patients with definite rheumatoid arthritis. B-cells bearing the interleukin-2 receptor (IL-2-R, CD25) were identified in pokeweed mitogen-stimulated PBM by sIg fluorescence and rosetting with anti-Tac-labelled Dynabeads. The kinetics of CD25 expression following activation were found to be different for B- and non-B-cells.

We have compared T and B cell analyses using whole blood, separated lymphocytes, and separated, frozen, then thawed lymphocytes to see how long blood can be kept before separation and analysis. We also examined the effect of various anticoagulants and the effect of diluting blood in culture media on T and B cell analysis over time. We found that the whole blood method is a very reliable method for T and B cell analysis, even 4 days after the blood is drawn, provided that heparin or ACD is the anticoagulant used. Separated lymphocytes and cryopreserved lymphocytes from blood that was separated within 24 h of collection was satisfactory; however, results were less consistent if separation was delayed more than 24 h. For lymphocyte separation, blood collected in heparin or ACD held up better over time than did blood collected in EDTA, and dilution with either RPMI 1640 or McCoy's medium gave better lymphocyte separation in older blood.

Methods are described for distinguishing between intrinsic B-cell defects, T-helper-cell defects, and suppressor cell hyperactivity in patients who fail to secrete immunoglobulin when peripheral blood mononuclear cells are stimulated with pokeweed mitogen. Control cells which respond to pokeweed mitogen are made unresponsive by depleting B cells or OKT4+ cells, and the missing subset, purified from the patient's peripheral blood mononuclear cells, is added back to examine its functional activity. Alternatively, hyperactivity of OKT8+ putative suppressor T cells or suppressor monocytes is evaluated by depleting these populations from the patient's peripheral blood mononuclear cells. Four patients who produced few plaque-forming cells in response to pokeweed mitogen were investigated: two had intrinsic B-cell deficiencies, one had T-helper-cell deficiency, and one had T-suppressor-cell hyperactivity.

Pokeweed mitogen (PWM)-induced B-cell activation was entirely abrogated by pre-treating peripheral mononuclear cells (PBM) with T4 (anti-T-helper-inducer subset) or T11 (anti-sheep erythrocyte receptor, ie. anti-pan-T-cell) monoclonal antibody plus complement. Immunoglobulin secretion was restored in T11- but not T4- cultures by adding the culture supernatant from PWM-stimulated PBM (T-replacing factor, TRF). Since T8+ (T-suppressor-cytotoxic subset) cells were present in high concentrations in T4- but absent from T11- cultures, it appeared that these cells could inhibit B-cell activation by PWM plus TRF and that suppression could occur in the absence of T4+ cells. Incubation of TRF with purified, unstimulated T8+ cells prior to addition to T11- cultures abrogated subsequent PWM-induced B-cell activation, whereas T8- PBM had no effect on TRF. T8+ cells did not secrete suppressor factors during incubation with TRF. Absorption of one or more of the factors required for B-cell growth and differentiation by T8+ cells appears to be an important suppressor mechanism operating in the PWM system.