Ecto-5'-nucleotidase expression during human B cell development. An explanation for the heterogeneity in B lymphocyte ecto-5'-nucleotidase activity in patients with hypogammaglobulinemia

Ecto-5'-nucleotidase (ecto-5'-NT) activity was measured in human B cells at different stages of development. Ecto-5'-NT activity of B cell preparations from fetal spleen and cord blood was 5.08 and 5.59 +/- 2.8 nmol/hr/10(6) cells, respectively; that of B cell preparations from adult peripheral blood, spleen, or lymph node was fivefold to sixfold higher (27.9 +/- 12, 29.2 and 33.8 nmol/hr/10(6) cells, respectively). The increased enzyme activity in B cell preparations from adult peripheral blood as compared with cord blood paralleled increased percentages of 5'-NT+ cells (69 +/- 12% vs 32 +/- 17%) and an average of twice as much enzyme activity per positive cell. Small, resting B cells that cannot synthesize Ig in vitro in response to pokeweed mitogen (PWM) were isolated from adult peripheral blood by mouse erythrocyte rosetting. Total ecto-5'-NT activity and the percentage of 5'-NT+ cells were equivalent in total B cells and the mouse erythrocyte rosette-positive subpopulation. Thus, ecto-5'-NT activity is acquired before B cells gain the ability to differentiate into Ig-secreting plasma cells in response to PWM. Ecto-5'-NT activity was also measured in B cell preparations from eight patients with common variable immunodeficiency. Six had reduced ecto-5'-NT activity (2.83 to 15.4 nmol/hr/10(6) cells), and two had normal activity (34.7 and 58.2 nmol/hr/10(6) cells). B cells from all six patients with low ecto-5'-NT activity failed to synthesize Ig when cultured with PWM and normal irradiated T cells. Of the two patients with normal B cell ecto-5'-NT activity, one also had B cells unresponsive to PWM, but B cells from the other patient appeared to more normal, in that they synthesized IgM and IgG when cultured with PWM plus irradiated allogeneic T cells. Thus, measurement of B cell ecto-5'-NT activity allows the subclassification of patients who have a common inability to synthesize immunoglobulin in vitro response to PWM. B cells with low ecto-5'-NT activity are presumably blocked at an earlier stage in development than B cells with normal ecto-5'-NT activity. Evaluation of ecto-5'-NT activity along with the expression of other B cell surface antigens should aid in the definition of discrete stages of B cell development.     

Pathophysiologic analysis of peripheral blood lymphocytes from patients with primary immunodeficiency. I. Ig synthesis by peripheral blood lymphocytes stimulated with either pokeweed mitogen or Epstein-Barr virus in vitro

In the present study 5 patients with common variable hypogammaglobulinemia (CVH) and 4 patients with selective IgA deficiency (IgA-D) were analyzed for the cellular defects responsible for impaired Ig synthesis with use of peripheral blood lymphocytes stimulated with either PWM or EBV in vitro. By the use of co-culture with PWM, all the patients examined had intrinsic B cell defects restricted to the synthesis of Ig class corresponding to the low or absent Ig class(es) in the sera. Two types of excessive suppressor T activity were found, which were abrogated by irradiation. One was isotype-nonspecific and the other was IgA-specific. Moreover, failure of IgA-specific helper T activity was demonstrated. The use of EBV as an agent that polyclonally activates B cells independently of T cells and monocytes should allow a clearer delineation of the level of the B cell defects. When co-cultured with EBV, B cells from 3 patients with CVH produced normal to subnormal quantities of IgM although they could produce no IgM upon co-culturing with normal T cells and PWM. B cells from 2 patients with CVH could produce IgM normally by stimulation with either PWM or EBV; however, there was no restoration to produce IgG or IgA in these patients. In addition, B cells from 2 patients with IgA-D produced not only IgG and IgM but also IgA almost normally at 4 days after in vitro stimulation with EBV.     

Immune deficiency in the X-linked lymphoproliferative syndrome. II. Immunoregulatory T cell defects

Surface phenotypic markers and the function of lymphocytes in patients affected with the X-linked lymphoproliferative syndrome (XLP) were studied. This syndrome is characterized by a defective response to infection with Epstein Barr virus (EBV). Normal numbers of B and T cells were detected with anti-Ig and monoclonal OKT3 antisera, respectively. T cell subset values, however, were persistently altered: cells reacting with OKT8 were significantly elevated in five of nine patients, accompanied by a slight decrease in the percentage of OKT4-positive cells, leading to abnormally low OKT4 to OKT8 ratios. One patient had a high OKT4 to OKT8 ratio due to low number of OKT8-positive cells. Lymphocytes from patients showed normal proliferation after stimulation with T and B cell mitogens. In contrast, Ig synthesis by lymphocytes after stimulation with B cell mitogens was markedly deficient: low or undetectable levels of one or all classes of Ig were detected, whereas cell lines established from EBV-infected B lymphocytes from patients produced normal quantities of Ig. These studies imply immune regulatory impairments in the patient with XLP.     

Immunoglobulin synthesis after HLA-identical marrow grafting. V. The role of peripheral blood monocytes in the regulation of in vitro immunoglobulin secretion stimulated by pokeweed mitogen

The ability of purified monocytes to regulate in vitro immunoglobulin (Ig) production was examined in 12 patients after HLA-identical marrow grafting. Five patients were studied less than 3 mo after grafting and seven more than 1 yr after grafting. One of the former had acute graft-vs-host disease and five of the latter had chronic graft-vs-host disease. Ficoll-Hypaque-separated peripheral blood mononuclear cells from patients, normal marrow donors, or healthy unrelated individuals were separated into T and non-T cells by sheep erythrocyte rosetting. Highly enriched monocyte and B cell subpopulations were obtained by placing the non-T cells over discontinuous Percoll gradients. Co-cultures of patient or normal monocyte populations with either normal or patient T and B cells with pokeweed mitogen were performed. A hemolytic plaque assay was used to assess Ig secretion after 6 days of culture. Co-culture of T and non-T cells from 10 of 12 patients failed to produce Ig. Monocyte-enriched fractions from all patients provided normal accessory cell functions when co-cultured with normal T and B cells. Two of five patients with chronic graft-vs-host disease had monocytes that suppressed Ig synthesis at high ratios of monocytes to normal T and B cells. Normal monocyte-enriched fractions did not restore Ig production to T and B cells of patients whose T and non-T cells failed to produce Ig. These data indicate that the observed defects in pokeweed mitogen-driven Ig secretion after marrow grafting are due primarily to defective T and B cell functions and that the monocyte accessory function is intact in most patients studied.     

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of the expression of CD5 by human B cells and correlation with functional activity.

B cells expressing the CD5 marker in the mouse have been suggested to be a separate lineage and a major source of autoantibody production. In man, this relationship is less clear. Studies were therefore undertaken to determine whether human CD5+ B cells represent a distinct lineage of cells that differ in patterns of antibody production from CD5- B cells. In normal B cell populations, CD5 was expressed by a mean of 24.0 +/- 2.8% (n = 10) of CD20+ B cells. Of note, an increased frequency of CD5+ B cells was not found in patients with systemic lupus erythematosus (mean of 17.9 +/- 2.8%, n = 16). Analyzing CD5+ B cells for cell membrane Ig isotype expression demonstrated similar frequencies of IgG and IgA expressing cells as were found on the CD5- B cell population, although the frequency of IgM+ cells was slightly increased. Incubation of CD20+ B cells with phorbol myristate acetate (PMA) for 72 hr increased the frequency of CD5 expressing B cells by more than threefold. CD5 expression was also increased by coculture with anti-CD3-activated T cells and most markedly by simultaneous stimulation with both PMA- and anti-CD3-activated T cells (greater than 50% positive). Analysis of CD5- B cells clearly indicated that stimulation with PMA or anti-CD3-activated T cells induced the majority to become CD5+ transiently. Functional analysis of Ig production by CD5+ and CD5- B cells stimulated with anti-CD3-activated T cells indicated that both populations in normals produced IgM and a variety of autoantibodies in comparable amounts, whereas the CD5- B cells produced greater quantities of IgG. B cells were activated with anti-CD3-stimulated T cells followed by separation into CD5+ and CD5- populations. The largest amount of Ig was produced by CD5- B cells that were induced to express CD5, although all populations produced some Ig. These data suggest that CD5 behaves as an activation marker on human B cells rather than as a marker for a distinct lineage of cells. Moreover, CD5 expression does not appear to identify a population of resting B cells with a greater capacity to produce antibodies to DNA or other autoantibodies.     

Multiple myeloma: an immunologic profile. IV. The EA rosette-forming cell is a Leu-1 positive immunoregulatory B cell

Patients with myeloma have a depressed capacity to respond to antigenic challenge. Studies in this laboratory have previously described an unclassified lymphoid cell which binds human erythrocytes coated with human immunoglobulin G (IgG) anti-D antibody (EA) as important in the inhibition of Ig synthesis in myeloma patients. Using monoclonal antibodies, two-color fluorescence studies, and flow cytometry, we characterized this EA cell as a Leu-1+ (cluster designation (CD) 5), Leu-12+ (CD 19), Leu-16+ (CD 20), B2+ (CD 21), Leu-14+ (CD 22), and HLA-DR+ B cell. The cell was negative for antibodies to Leu-2 (CD 8), Leu-3 (CD 4), Leu-4 (CD 3), Leu-5 (CD 2), Leu-7, Leu-8, Leu-11 (CD 16), Leu-M1 (CD 15), Leu-M3, and CALLA (CD 10). This profile is consistent with a Leu-1+ B cell and excludes a T cell, natural killer cell, and monocyte. Comparison of the relative role of these cells to the role of monocytes in the suppression of pokeweed mitogen-stimulated Ig synthesis was determined in serial studies on 19 myeloma patients. The mean (+/- SEM) percentage of inhibition of Ig synthesis by monocytes from stage I myeloma patients was 14 +/- 2.2%, from stage II patients was 37 +/- 3.5%, and from stage III patients was 51 +/- 4.7%. Inhibition of Ig synthesis by Leu-1+ EA cells was 46 +/- 1.5%, 48 +/- 1.6%, and 43 +/- 3.7% in stage I, II, and III patients, respectively. Immunosuppressive B cells are an important component of inhibition of Ig synthesis in the immunodeficiency of myeloma.     

Lymphocyte cholesterol synthesis in chronic lymphocytic leukemia

Studies in B and T lymphocytes from two patients with B cell chronic lymphocytic leukemia (CLL) showed that the rate of cholesterol synthesis and hydroxymethylglutaryl (HMG)-CoA reductase activity in the malignant cell--the B lymphocyte--did not differ from that in normals, before or after the treatment. Cholesterol synthesis and HMG-CoA reductase activity in T cells, on the other hand, from one patient (H.E.) before therapy, were only one-fifth of that in normals and in the other patient (J.R.) receiving treatment. Following chemotherapy and treatment with prednisone, T lymphocytes from H.E. exhibited normal cholesterol metabolism.     

Activation and function of an autoreactive T cell clone with dual immunoregulatory activity

Previously it was demonstrated that the human autoreactive CD4+ T cell clone MTC-4 is bifunctional, having the capacity to augment differentiation of autologous B cells into Ig-secreting cells in the absence of PWM and the capacity to suppress such differentiation in the presence of PWM. In the present study it was shown that these two functions of MTC-4 are mediated by distinctly different mechanisms. In the presence of autologous class II MHC Ag, MTC-4 releases one or more non-MHC-restricted soluble factors which stimulate B cell differentiation. The helper factors are different from IL-2, and act on both resting (small) and activated (large) B cells. The suppressor function of MTC-4 cells is elicited when MTC-4 cells are co-cultured with autologous non-T cells preincubated with PWM for 4 h, but not with non-T cells preincubated with PWM for 24 h; thus, activated autologous non-T cells have a transient capacity to induce MTC-4 suppressor function. Induction of MTC-4 suppressor activity is not associated with increased proliferation of MTC-4 and is mediated by low numbers of these cells. Unlike helper function, MTC-4 suppression of Ig synthesis can occur late in B cell cultures, and MTC-4 suppresses Ig production by autologous B cells, but not by allogeneic B cells. Finally, in co-cultures with activated autologous non-T cells and allogeneic B cells, MTC-4 can simultaneously produce helper factors that augment Ig synthesis by allogeneic B cells and suppress Ig synthesis by autologous B cells. In summary, exposure of MTC-4 to autologous non-T cells causes release of non-MHC-restricted factors which augment Ig production by both resting and activated autologous B cells, whereas exposure of MTC-4 to recently activated B cells causes MTC-4 to express the additional function of directly suppressing Ig production by differentiated autologous B cells. Thus autoreactive T cells may be uniquely suited to regulate Ig production.     

Inhibition of N-linked oligosaccharide trimming mannosidases blocks human B cell development.

Deoxymannojirimycin (dMM) or swainsonine (SW), which block conversion of high-mannose to complex-type N-linked glycans, strongly inhibited the production of immunoglobulin (Ig) when added to cultures of human lymphocytes together with the polyclonal B cell activators pokeweed mitogen (PWM) and Staphylococcus aureus (SAC). To obtain the inhibitory effect, inhibitor had to be present during the first 36 h of culture. Addition at later timepoints was less effective and showed that neither inhibitor interfered with rate of production or secretion of Ig as such. Viability and proliferation of the lymphocytes, as defined by cell number and rate of DNA synthesis, were not influenced by the presence of dMM or SW, and no changes in the relative number of helper (T4+) or suppressor (T8+) cells were observed. Thus, for normal differentiation of human B lymphocytes into Ig secreting (plasma) cells in response to PWM and SAC, conversion of high-mannose to complex N-linked glycans is essential.     

The effect of 1,25-dihydroxyvitamin D3 on in vitro immunoglobulin production in human B cells

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently suppressed immunoglobulin (Ig) production of human B cells, as evaluated by IgG-plaque-forming cells (IgG-PFC) in the culture of pokeweed mitogen (PWM)-activated B cells. Similar suppressive effect of 1,25(OH)2D3 on Ig production of B cells was observed in the Staphylococcus aureus Cowan I(SAC)-induced Ig-producing system. The mean percentage of inhibitions at a concentration of 10(-9) M were 60.0 +/- 8.2% (mean +/- SE, n = 6) and 65.1 +/- 4.7% (n = 10) in PWM- and SAC-stimulated cultures, respectively. The suppression was strongly exhibited only when 1,25(OH)2D3 was added at the start of the 6-day culture, accompanied by a decrease in DNA synthesis of B cells in both culture systems. On the other hand, the addition of 1,25(OH)2D3 on day 4, when DNA synthesis reached at plateau and IgG-PFC began to be detectable, had no noticeable affect on either the number of PFC or DNA synthesis of B cells. Furthermore, 1,25(OH)2D3 suppressed Ig production even when B cells were exposed to the agent for 4 hr after the activation with PWM or SAC, but not before the activation. These results indicate that 1,25(OH)2D3 inhibits B cell proliferation before differentiation to Ig-secreting cells, consequently reducing Ig production; and that its action appears to be mediated by the cytosol receptors expressed on activated B cells. Thus, the agent may serve as an immunoregulating hormone in vivo, as well as in vitro.     

Phospholipid synthesis by activated human B lymphocytes

Pokeweed mitogen- (PWM) stimulated DNA and Ig synthesis in human B cells is dependent on the presence of T cells and adherent cells, but the influence of these regulatory cells on earlier activation events is unknown. We have studied the T cell and monocyte influence on the incorporation of [methyl-14C]choline chloride into B cell phospholipids (PL) after varying periods of in vitro culture with or without pokeweed mitogen (PWM). By separating B and T cells after choline pulsing, a peak in PWM-induced PL synthesis of B cells at days 1 to 2 was revealed, whereas the T cell response was later (days 2 to 3). In the first 4 hr of culture, the purified B cell plus monocyte fraction incorporated choline four to six times faster than the T cell fraction, but PWM did not increase choline incorporation, whether these fractions were cultured separately or together. When cultures were pulsed with choline between 16 and 20 hr with or without PWM, monocytes incorporated choline six to nine times faster than T cells, and B cells were intermediate. Also at 16 to 20 hr of culture, a significant PWM-induced increase in choline incorporation by B cells was evident and was dependent on the presence of T cells and monocytes. The monocytes showed no increased choline incorporation due to PWM. Thus, the influence of regulatory cells on the PWM response in B cells is evident within the first 24 hr.     

Cells and mediators which participate in immunoglobulin synthesis by human mononuclear cells. I. The obligatory requirement of null lymphocytes in the synthesis of immunoglobulins in vitro

The circulating mononuclear cells of normal adult human volunteers were fractionated into T and non-T lymphocytes. The T cells were then fractionated into TM and non-TM cells and the non-T cells were further fractionated, by both positive and negative isolation, into B cells and null cells. Culture of non-T cells, TM cells, and PWM for 7 to 8 days resulted in cytoplasmic Ig (cIg) synthesis by all B cells, as detected by immunofluorescence. However, culture of either purified B or null cells with TM cells and PWM resulted in a marked reduction (75%) of cIg-synthesizing cells. The addition of monocytes did not enhance cIg synthesis. Culture of reconstituted B and null cells with TM cells and PWM restored the capacity to synthesize cIg to the cells. It is concluded that null cells are required, as well as TM cells and PWM, for cIg synthesis by B cells.     

Pokeweed mitogen-induced immunoglobulin secretion by peripheral blood lymphocytes from patients with primary intracranial tumors. Characterization of T helper and B cell function

We have previously demonstrated that patients with primary malignant brain tumors have impaired in vivo and in vitro cell-mediated immunity. The purpose of the present research was to employ pokeweed mitogen (PWM)-induced secretion of immunoglobulin (Ig) by peripheral blood lymphocytes (PBL) to further investigate impaired lymphocyte function in these patients. The PWM response of PBL from normal individuals averaged 8384 plaque-forming cells (PFC) per 10(6) cells, whereas the response of PBL from patients averaged 1590 PFC/10(6). The decreased PWM response of PBL patients could not be improved by varying the number of PBL placed in culture or employing different concentrations of PWM. Co-culture experiments to detect the presence of suppressor cells in PBL and purified T cell preparations from patients demonstrated that enhanced suppressor cell activity was not evident. Next, experiments were performed to assess the T-helper cell activity present in purified T cell preparations obtained from patients. The results demonstrated that T cells from patients lacked the ability to provide adequate helper activity in the PWM response. Moreover, studies with monoclonal antibodies directed against T cell subsets revealed that PBL from patients have a reduced percentage of T-helper cells (40%) as compared with normal values (55%). In concert with T-helper cell anomalies, B cell function in these patients also is diminished. Thus, these observations indicate that a combined T-helper and B cell defect may contribute to the broad impairment of host immunocompetence observed in patients with primary gliomas.     

Two phenotypically distinct suppressor T cell subpopulations inhibit the induction of B cell differentiation by phytohemagglutinin

Human B lymphocytes can be induced to differentiate into antibody-secreting plasma cells by Leu-3+ T lymphocytes stimulated with pokeweed mitogen (PWM), a polyclonal T cell activator. In contrast, other polyclonal T cell mitogens, such as phytohemagglutinin (PHA), also activate Leu-3+ T cells but are relatively ineffective inducers of B cell differentiation. We have performed a series of experiments to investigate the mechanism underlying this apparent paradox. When human B cells were cultured with unfractionated T cells and PWM or PHA, only PWM was able to induce plasma cell formation and immunoglobulin (Ig) secretion. However, when the T cells were treated with mitomycin C (MMC) before culture, both PWM and PHA were able to induce significant B cell differentiation. These data indicated that both mitogens were able to activate the helper T cells required for B lymphocyte differentiation and suggested that MMC-sensitive suppressor T cells were responsible for inhibiting the induction of antibody-secreting cells by MMC-untreated T cells stimulated with PHA. Phenotypic analysis of the T cells capable of suppressing PHA-induced B cell differentiation revealed that small numbers of either Leu-2+ or Leu-3+ T cells could profoundly suppress the B cell differentiation induced by PHA. In contrast, significant suppression of PWM-stimulated B cell differentiation was observed only with relatively large numbers of Leu-2+ T cells. These data confirm previous reports that OKT4+/Leu-3+ T cells can suppress human B cell differentiation and indicate that the difference in B cell differentiation induced by PWM and PHA with MMC-untreated T cells is largely a reflection of the relative potency of these mitogens to activate these phenotypically distinct suppressor T cell subpopulations.     

Immunodeficiency with thymoma: failure to induce Ig production in immunodeficient lymphocytes cocultured with normal T cells.

Blood mononuclear cells of two individuals having immunodeficiency with thymoma (ID-THY) were cocultured with normal mononuclear cells or treated mononuclear cell fractions in an attempt to correct an imbalance of regulatory cells postulated to be responsible for the failure of pokeweed mitogen-induced Ig synthesis in vitro. Treatment included abrogation of suppressor cell activity by irradiation or incubation with prednisilone in vitro. T cell help was provided by cocultivating lymphocytes of related and unrelated persons, and in some cases autologous treated cells. Ig secretion failed to be induced by any experimental maneuver suggesting that the primary problem in the above ID-THY cells was related to defective or deficient B cells rather than an imbalance of T regulatory cells. Prednisilone treatment in vitro decreased suppressor cell activity in allogeneic cocultures of two ID-THY persons (S1 and S2) but not of an individual (S3) with variable immunodeficiency suggesting heterogeneity of suppressor cells.     

Role of interleukin 2, interleukin 4 and interleukin 5 in the T helper cell-driven B cell polyclonal differentiation in the elderly.

There is evidence for an impaired T cell-mediated B cell response during senescence. In thirty aged donors, pokeweed mitogen (PWM)-driven immunoglobulin (Ig) synthesis by B cells co-cultured with autologous enriched CD4+ lymphocytes and low amounts of monocytes, was evaluated. Under such experimental conditions, elderly cultures displayed a reduced IgG and/or IgM production when compared with the younger counterpart. Moreover, interleukin (IL)-2 and/or IL-5 addition to cultures led to an enhancement of Ig release. In contrast, IL-4 supplementation failed to positively modulate B cell differentiation. At the same time, aged cells cultured in the presence of IL-2 + IL-5 exhibited an increased Ig synthesis, while the addition of IL-2 + IL-4 or IL-4 + IL-5 mixtures did not induce any significant effect in comparison with homologous untreated samples. The results suggest a critical role for IL-2, IL-4 and IL-5 in the modulation of T helper cell-driven B cell polyclonal responsiveness in the elderly.     

Regulation of immunoglobulin production in human peripheral bood leukocytes: cellular interactions.

The intercellular influences regulating immunoglobulin (Ig) synthesis by normal human peripheral blood leukocytes (PBL) were investigated in cells stimulated by pokeweed mitogen (PWM). This system was shown to be totally T lymphocyte dependent as purified B lymphocytes (less than or equal to 1% T lymphocytes) failed to make significant amounts of Ig. No evidence was obtained for an Ig class switch as all classes of Ig (IgM, IgG, IgA) were shown to be produced in increasing amounts over a 6-day time period. T lymphocytes demonstrated maximum helper effect when mixed with equal numbers of B cells. This helper effect was mediated through the dual mechanisms of increasing the number of B lymphocytes containing cytoplasmic Ig and by increasing the maturity of these B lymphocytes as demonstrated by an increasing Ig production per B lymphocyte. When present in higher numbers, T lymphocytes were also capable of suppressing Ig production. This T-mediated suppression was first evident as a decrease in the Ig produced per B lymphocyte (decreased maturity). With maximum T suppression Ig-containing B lymphocyte numbers were also diminished. T lymphocyte help was relatively independent of macrophages (phagocytic cells) and did not require DNA synthesis for expression. Both T help and suppression were shown to cross allogeneic barriers. Immature T lymphocytes (thymocytes) were incapable of mediating either activity. Normal human PBL contain T lymphocytes campable of mediating both T help and suppression and the Ig produced by PBL was shown to be the balance of these activities. This balance probably represent the participation of distinct T lymphocyte subpopulations analogous to the T helper (Ly 1+) and T suppressor (Ly 2+, 3+) populations in the mouse.     

Human peripheral blood B lymphocyte subpopulations: functional and phenotypic analysis of surface IgD positive and negative subsets

Highly purified human peripheral blood B cells stimulated with Cowan I Staphylococcus aureus (SA) and mitogen-activated T cell supernatants (T supt) generated large numbers of immunoglobulin (Ig)-secreting cells (ISC), whereas fewer ISC developed in cultures containing T supt in the absence of SA. To determine whether surface Ig isotype expression defined responsive B cell subsets, IgD+ and IgD- B cells were prepared with the fluorescence-activated cell sorter. Whereas both the IgD+ and IgD- B cells responded to SA + T supt, only the IgD- subset generated substantial numbers of ISC in response to T supt alone. Analysis of secreted Ig revealed that IgG and IgA were the predominant isotypes secreted by IgD- B cells in response to T supt or SA + T supt. By contrast, the IgD+ cells secreted predominantly IgM in response to SA + T supt but not to T supt alone. When responsiveness to pokeweed mitogen (PWM) was examined in the presence of supplemental T cells, the IgD- subset was found to be greatly enriched for responsive cells, and again, IgG and IgA were the predominant isotypes secreted, although these cells were also capable of secreting some IgM. The magnitude of the response induced by PWM from IgD- B cells was usually greater than that induced by SA + T supt. Although IgD+ B cells responded poorly to PWM, the differentiation of a small number of IgM-secreting cells was routinely stimulated by this polyclonal activator in the presence of T cells. The magnitude of the PWM response by IgD+ B cells was always greatly diminished compared with that stimulated by SA + T supt. Cell cycle analysis after acridine orange staining, cell volume measurement, and staining for expression of activation antigens (transferrin receptor and 4F2) indicated that the IgD- B cells were largely resting, but did contain a population of activated cells. Removal of activated 4F2+ cells from the IgD- subset diminished but did not abolish their capacity to generate ISC in response to SA + T supt or PWM in the presence of T cells. These results suggest that the IgD- population contains both an activated 4F2+ and a resting 4F2- subset. The data emphasize that multiple subpopulations of peripheral blood B cells contain precursors of ISC. Moreover, the responsiveness of the subsets to various stimuli and the Ig isotype subsequently secreted appear to be intrinsic features of each subset.     

NK cell function in severe combined immunodeficiency (SCID): evidence of a common T and NK cell defect in some but not all SCID patients

The immunologic work-up of eight infants with the clinical diagnosis of severe combined immunodeficiency (SCID) was performed with special emphasis on natural killer (NK) cell function and ontogeny. Contrary to previous reports, our study shows that not all SCID patients lack NK activity; some may even express very high NK- and antibody-dependent cellular cytotoxicity (ADCC). The present group of eight SCID infants was homogeneous with respect to normal levels of the purine metabolism enzymes adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). They all had low serum Ig levels and were defective for specific antibody formation against BSA and diphtheria toxin (DiT). None of the infants' peripheral blood mononuclear cells (PBMC) proliferated significantly upon in vitro stimulation with PHA, concanavalin A (Con A), pokeweed mitogen (PWM), and irradiated allogeneic lymphocytes. Seven of eight patients, however, responded significantly to mitogenic factors present in a lectin-free interleukin 2 (IL 2) preparation, and two exhibited a positive costimulation as well with simultaneous exposure to IL 2 + Con A. The lymphocyte marker analysis revealed high percentages of OKT10+ cells in seven of eight infants, whereas peripheral T cells (OKT3+) with suppressor/killer (OKT8+) or helper/inducer (OKT4+) phenotypes were abnormally low in all infants with one exception. The PBMC of two patients formed low to normal percentages of E rosettes but expressed no B cell markers (B-/SCID). The six other infants had high percentages of B cells (B+/SCID) but lacked E rosette-forming cells. High NK and ADCC activity was found in the two B-/SCID patients. The B+/SCID infants either totally lacked NK and ADCC function (four of six) or expressed low to normal NK activity together with some T cell markers as revealed by monoclonal antibody staining but not by E rosette formation (two of six). From the data presented, an ontogenic model is proposed that assumes the status of an independent cell lineage in between T cells and monocytes for human NK cells, or that places these cells in close proximity to early differentiation steps of the T cell lineage. In any case, NK cell function clearly constitutes an additional parameter of heterogeneity in the immunologic analysis of SCID.