Functional differentiation of B lymphocytes in congenital agammaglobulinemia. I. Generation of hemolytic plaque-forming cells.

Despite the absence of B lymphocytes, peripheral blood lymphocytes (PBL) from four of five patients with congenital agammaglobulinemia (cAgamma) generated a specific hemolytic plaque-forming cell (HcPFC) response in vitro to sheep red blood cells and ovalbumin. The kinetics, antigenic, and cellular requirements were similar to normals, but significantly less HcPFC were found in patient cultures. Normal but not patient HcPFC-precursor cells were inactivated by treatment with anti-mu antisera whereas generated HcPFC in both controls and patients were sensitive to treatment with anti-mu. Pokeweed mitogen (PWM) and dextran sulfate (DXS) enhanced the HcPFC-response of normal PBL; cAgamma-cells were unresponsive to DxS and, in the presence of PWM, the development of HcPFC was inhibited. These findings indicate the presence of B lymphocyte precursors in the majority of patients with cAgamma investigated.     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. VII. The effect of immunodeficiency disease.

Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and antibody-dependent cellular cytotoxicity (ADCC) was assessed in 13 patients with immunodeficiency diseases—immunodeficiency-thymoma syndrome (1), Bruton type agammaglobulinemia (3), and common variable hypogammaglobulinemia (9). SLMC and ADCC function were intact (and possibly enhanced) in the patient with immunodeficiency thymoma. Both ADCC and SLMC were detectable in the three patients with X-linked agammaglobulinemia, one of whom had lower than expected SLMC. In all of the immunodeficient patients, the relative inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC, although in three of the CVH patients this was lower than normal. In every case, removal of Fc receptor-bearing cells from the patients' lymphocyte preparations severely depleted SLMC (and ADCC when tested), but cytotoxicity was either unchanged or enhanced by depletion of E rosette forming T cells. The effects of Fc receptor-positive cell depletion, T-cell depletion, culture serum variation, or the addition of antibody-coated erythrocytes to the assay were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and whether patients' or normal lymphocytes were tested. The possible significance of the results with respect to surveillance against cancer is discussed.     

Ecto-5'-nucleotidase activity in T and B lymphocytes from normal subjects and patients with congenital X-linked agammaglobulinemia.

Ecto-5'-nucleotidase activity was measured in lymphocyte subpopulations isolated from normal subjects and patients with congenital X-linked agammaglobulinemia. B lymphocytes from normal subjects have at least three times more ecto-5'-nucleotidase activity than T lymphocytes. Patients with X-linked agammaglobulinemia have 56% of normal activity in their T cells, and lack a lymphocyte subpopulation high in nucleotidase activity. High activity of ecto-5'-nucleotidase may be a biochemical marker for mature surface immunoglobulin-bearing B cells.     

B lymphocyte precursors in human bone marrow: an analysis of normal individuals and patients with antibody-deficiency states.

Lymphoid cells containing cytoplasmic IgM but lacking stable surface IgM are believed to be the direct precursors of B lymphocytes. We have characterized these pre-B cells in the bone marrow of normal individuals and patients with a variety of immunoglobulin deficiencies or hematologic disorders by using immunofluorescence and autoradiography. Pre-B cells comprised 5.8 +/- 5.7% of lymphoid cells in normal bone marrow. Eleven patients with infantile X-linked agammaglobulinemia (X-LA) lacked B lymphocytes but had a normal frequency (3.8 +/- 3.6%) of bone marrow pre-B cells. A smaller proportion of marrow pre-B cells from patients with X-LA were engaged in spontaneous DNA synthesis than was found for normal controls. In individuals other than the group with X-LA, the number of circulating B cells was positively correlated with the frequency of marrow pre-B cells. These results indicate that patients with X-LA have a defect in maturation of pre-B cells, and suggest that some patients with acquired B lymphocyte deficiency may have lost the capacity to generate pre-B cells from stem cells.     

Role of t lymphocytes in the humoral immune response. II. T cell-mediated regulation of antibody avidity.

The effect of activated T lymphocytes (ATC) on the avidity distribution of PFC in the secondary response was studied in normal mice. The total PFC response was not significantly changed for either direct or indirect PFC by administration of ATC before secondary antigen challenge. However, marked suppression occurred of indirect PFC that secreted high avidity antibody; no suppression was seen of high avidity direct PFC. At the same time, significant stimulation was seen of relative and absolute frequencies of indirect PFC that secreted middle and low avidity antibody. These effects were dependent on Thy 1-bearing, nylon nonadherent cells which demonstrated carrier specificity. In further characterization of these effects, it was found that increasing the number of ATC transferred produced progressive loss of high avidity PFC and compensatory increase in lower avidity PFC. Moreover, in these experiments, suppression of the high avidity response was inducible with the administration of ATC 5 weeks before to 3 days after the secondary immunization. Thus, it is likely that the avidity-modifying effects are dependent on T lymphocytes which influence the late stages of B lymphocyte maturation.     

The nature of the suppressive effect of interferon and interferon inducers on the in vitro immune response

Viral-induced interferon inhibition of the primary in vitro plaque-formong cell (PFC) response in the mouse (C57B1/6) involves a dynamic relationship between the nature of the antigen, the concentration of interferon added to antigen-stimulated cultures, and the time of addition of interferon relative to antigen addition. The PFC response to a thymus-dependent antigen (sheep red blood cells) was more easily suppressed by viral-induced interferon than was that to a thymus-independent antigen (E. coli 0127 LPS), both in terms of inhibitory concentrations of interferon and the time at which the interferon could be added to cultures after antigen and still inhibit the PFC response. These differential effects of interferon could be related to the difference in cellular requirements (B and T lymphocytes) of the two antigens. Interferon was effective in inhibiting the in vitro PFC response of antigen-primed spleen cells, indicating that it can block the response of memory lymphocytes. By using interferon inducers as inhibitors of the in vitro PFC response, it was possible to show that at least two antigenically distinct interferons may be involved in suppressing the immune response. It is known that one type of interferon is induced by virus and synthetic double-stranded polyribonucleotides. The other type is stimulated by antigen and T cell mitogens. A model is proposed to explain the nature of these interferon inhibitory effects in terms of mediation of immune suppressor cell effects.     

Human lymphocyte-mouse myeloma somatic cell hybrids: selective hybrid formation.

Fusion of unfractionated human lymphocytes with mouse myeloma cells resulted in proliferating hybrid colonies, almost all producting human Ig. We examined whether this high frequency of Ig production was the result of selective formation of human B lymphocyte-mouse myeloma hybrids, rather than induction of Ig genes in T lymphocytes. Unfractionated peripheral lymphocytes and B lymphocytes from patients with the common variable form of agammaglobulinemia formed proliferating somatic cell hybrid colonies. In contrast, peripheral lymphocytes from a patient with agammaglobulinema who lacked B lymphocytes, as well as albumin gradient fractions of peripheral blood which do not contain B lymphocytes, failed to produce somatic cell hybrids with three different myeloma parent cell lines. B, T, and precursor lymphocytes all had Sendai virus receptors, as witnessed by viral agglutination. We conclude that fusion of human lymphocytes with mouse myeloma cells results in selective hybrid formation, rather than activation of Ig genes in disparate cell types. Only B lymphocyte-mouse myeloma heterokaryons form hybrid cells.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Activation of human B lymphocytes. IX. Modulation of antibody production by products of activated macrophages.

Human monocytes, after in vitro activation by mixed lymphocyte culture (MLC) supernatants produce a monokine (MK) that enhances the plaque-forming cell (PFC) response of pokeweed mitogen (PWM)-stimulated human B lymphocytes. Technical conditions and kinetics of MK production were established. Irradiation of monocytes (5000 rads) does not abolish MK production but heat-killed cells are unable to release the factor. Highly T cell-depleted monocyte populations still produced the PFC-enhancing factor. The same MK has an inconsistent enhancing effect on the PFC responses of lipopolysaccharide (LPS) and nocardia water-soluble mitogen (NWSM)-stimulated B cells. Other macrophage activators such as LPS, phytohemagglutinin (PHA), and latex particles failed to induce consistently the liberation of the PFC-enhancing MK. The target cell for the MK activity on PWM-stimulated B cells appears to be the B lymphocyte itself. These studies demonstrate that soluble monocyte products can have substantial modulatory effects on human B cell function.     

Functional differentiation of B lymphocytes in congenital agammaglobulinemia. II. Immunochemical analysis of the in vitro primary immune response.

Cultures of peripheral blood lymphocytes (PBL) in which specific hemolytic plaque-forming cells (HcPFC) had been induced were labeled with 14C-amino acids. Antigen-specific products in the culture supernatants were characterized by using indirect immune precipitation in conjunction with specific immunoabsorbents and/or gel filtration followed by SDS-polyacrylamide gel electrophoresis. After 5 days of culture with antigen (sheep red blood cells or ovalbumin) newly synthesized IgM and specific IgM antibody were demonstrated in culture supernatants from normal donors and from four out of five patients with congenital agammaglobulinemia (cAgamma). Secreted products bound specifically to antigen and pretreatment of labeled supernatants with anti-mu and anti-L chain antisera, but not with anti-gamma antiserum, prevented binding. Typical mu- and L chains constituted only a proportion of the anigen-binding peptides recognized by the anti-mu reagents. Induction of IgM antibody synthesis was dependent on the presence of antigen and was correlated with the generation of HcPFC. No major differences between the antigen-induced products of cAgamma and normal PBL were observed. These findings suggest that in the absence of terminal B cell differentiation in vivo, certain patients with cAgamma possess precursor cells that can respond to antigen in vitro with the synthesis of specific humoral products, including IgM antibody.     

Effects of estrogen on the lymphoid regeneration and immune response in irradiated and marrow-reconstituted mice.

Various doses of estriol (E3) were given to mice intraperitoneally, immediately after lethal irradiation and marrow reconstitution. The assessment of the plaque-forming cell (PFC) response to sheep erythrocytes in the spleen and the histological assessment of lymphoid tissues were carried out 30 days later. The effects appeared to be dose-dependent and resulted in a marked suppression of the PFC response. The depletion of lymphocytes was dramatic and dose-dependent in the thymus, and in the thymus-dependent and in the thymus independent areas of the peripheral lymphoid tissues. These results suggest that E3 acts on the differentiation of stem or precursor cells toweard both the populations of T and B lymphocytes. Although E3, given on day 7 after irradiation and marrow reconstitution, suppressed the lymphoid regeneration and PFC response markedly, E3 given on day 14 had no effect. On day 7 the majority of regenerating lymphoid tissues were large pyroninophilic cells and on day 14, small lymphocytes. These results suggest that the precursor or immature lymphocytes are sensitive to E3, while mature lymphocytes are resistant. Lymphoid regeneration and PFC response were retarded in mice irradiated and reconstituted with bone marrow cells from donors pretreated with E3. These results suggest that E3 acts on the stem or precursor cells capable to differentiate in the direction of lymphoid populations and reduce their number in the bone marrow.     

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.     

Immune competence of splenic lymphocytes following graft-vs-host disease in mouse allogeneic radiation chimeras.

The abnormal immune response of long-term mouse allogeneic chimeras is reflected by qualitative deficiencies in either T or B lymphocytes. The present study was undertaken to determine if a relationship existed between the severity of graft-vs-host disease (GVHD) that these animals had experienced and a functional defect in either the T or B cell population. The in vitro PFC response of chimera spleen cells to sheep red blood cells (SRBC) was evaluated in the presence of normal T or B lymphocytes 4 to 8 months after marrow transplantation and well beyond the GVHD period. In an analysis of several different allogeneic radiation chimeras, our results showed no relationship between the severity of GVHD experienced and the immunologic capacity of either T or B cells. Thus, different chimera combinations showing similar degrees of GVHD were functionally deficient in one or the other of these two cells types or both with no apparent predilection for abnormality in either population. In examining the quantitative in vitro PFC response to sheep RBC by spleen cells from individual chimeras, we found that the number of PFC formed was related to the severity of GVHD experienced by that animal. A general relationship between severity of GVHD and PFC capacity may also exist between chimeras of different genetic combinations. However, this relationship is not precise since gross exceptions occur. Our results, although documenting further the qualitative abnormalities in T and/or B lymphocytes of radiation chimeras, do not reveal the factor or mechanisms by which these cells are made unresponsive. It is suggested that the tolerance-inducing mechanism of these animals, whether it be humoral blocking factors or suppressor cells, is in some way interfering with the collaboration of T and B cells for antibody production.     

Pregnancy-associated growth factor. II. A T-dependent polyclonal activator of human adult peripheral blood lymphocytes (PBL)

This study examined the ability of pregnancy-associated growth factor (PAGF), a substance found in crude human chorionic gonadotropin (hCG), to induce plaque-forming cells (PFC) in cultured human peripheral blood lymphocytes (PBL). PAGF, 0.25 to 1 mg/ml, induced maximal PFC at 6 to 7 days as measured by the staphylococcal protein A-coupled SRBC reverse hemolytic plaque assay with a rabbit anti-human Ig antiserum. PAGF-induced PFC/culture ranged from 1800 to 39,000 with a mean of 11,524 in unfractionated PBL (N = 24), as compared to 540 to 77,840 with a mean of 17,303 for pokeweed (PWM) (N = 22). Comparison of PAGF- and PWM-induced PFC showed that both induced specific IgG, IgA, and IgM PFC. In most individuals, PAGF induced more IgM and PWM more IgG PFC. The kappa: lambda ratio was 1.5 for unstimulated PBL, and approximately 3.5 for PAGF and PWM. To see if PAGF was a T-dependent polyclonal activator of B cells, T and non-T populations were obtained by SRBC rosettes and negatively selected T4 and T8 cells by complement-mediated lysis of SRBC+(T) cells. Only the recombined subsets which included T4 cells and non-T cells supported PAGF- and PWM-induced PFC. These data indicate that PAGF, a substance derived from commercial extracts of pregnancy urine, is a T4-dependent polyclonal activator of normal human B cells.     

Activation of human B lymphocytes. V. Kinetics and mechanisms of suppression of plaque-forming cell responses by concanavalin A-generated suppressor cells.

The kinetics and mechanisms of suppression of the PWM-induced PFC response of human PB lymphocytes by Con A-activated suppressor cells were investigated. It was necessary that Con A suppressor cells be present early in the process of activation of human B cells toward antibody syntheses, but maximal suppression of the PFC response occurred later in the culture period. In addition, Con A-activated cells, although suppressing the PFC response to PWM greater that 90% of control, did not significantly suppress the blastogenic response to PWM after 3 or 5 days in culture. On the contrary, after 3 days in culture, background tritiated thymidine incorporation as well as tritiated thymidine incorporation to PWM stimulation was increased when Con A suppressor cells are added to fresh autologous peripheral blood lymphocytes. This increased blastogenic response after three days most likely represented an autologous mixed lymphocyte reaction (MLR) or Con A suppressor cells against fresh autologous non-T cells. The induction of autoreactive cells may be one of several modes of suppression of PFC responses by Con A activated suppressor cells.     

Modulation of human B cell immunoglobulin secretion by the C3b component of complement

The human C3b component of complement was found to inhibit the differentiation of human B lymphocytes into immunoglobulin-secreting cells in vitro. Pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses were inhibited by C3-coated zymosan particles and by purified human C3b. C3b inhibited the PWM-driven responses in a dose-dependent fashion, and it was necessary for C3b to be present in the early phases of the cultures. C3b acted directly on B cells rather than on helper T cells because it inhibited the PFC responses of MNC depleted of T cells and subsequently stimulated with a T cell-independent Epstein Barr virus mitogen. Furthermore, C3b failed to stimulate the generation of suppressor lymphocytes and/or monocytes that might have been responsible for the inhibition of B cell responses. Our results indicate that C3b or its fragments exert negative modulatory effects on human B lymphocyte responses.     

The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.     

T cell activation of primed rabbit blood lymphocytes by antigen.

Activation of primed rabbit PBL by homologous antigen in the early proliferative phase (on days 3--5) mainly involves lymphocytes which neither secrete specific antibody nor contain immunoglobulin in their cell membrane. This stimulation is antigen-specific, and evidence is given that the major proportion of these cells are T lymphocytes. The B cells forming Av-CHO-specific PFC were studied by autoradiography on days 6 and 12 of culture. Since incorporation of radioactive thymidine was found in the majority of PFC, these cells are also in proliferation.     

Application of carrier testing to genetic counseling for X-linked agammaglobulinemia.

Bruton X-linked agammaglobulinemia (XLA) is a phenotypically recessive genetic disorder of B lymphocyte development. Female carriers of XLA, although asymptomatic, have a characteristic B cell lineage-specific skewing of the pattern of X inactivation. Skewing apparently results from defective growth and maturation of B cell precursors bearing a mutant active X chromosome. In this study, carrier status was tested in 58 women from 22 families referred with a history of agammaglobulinemia. Primary carrier analysis to examine patterns of X inactivation in CD19+ peripheral blood cells (B lymphocytes) was conducted using quantitative PCR at the androgen-receptor locus. Obligate carriers of XLA demonstrated > 95% skewing of X inactivation in peripheral blood CD19+ cells but not in CD19- cells. Carrier status for mothers of isolated affected males could be assessed in 10 of 11 families: 7 women showed skewing, and 3 did not. Five carriers were found in six families in which there were no living affected males. Among all those tested, one individual's carrier status was considered to be indeterminate and five women were noninformative for the carrier test. Results obtained by the carrier test were congruent with linkage analysis (where applicable) using the RFLPs DXS178 and DXS94 and two newly developed polymorphic microsatellite markers, DXS178CA and DXS101AAT. Refinements in techniques for primary carrier testing and genetic mapping of XLA now make possible an ordered approach to diagnosis, prenatal diagnosis, and genetic counseling.     

The plaque-forming-cell (PFC) response of human blood lymphocytes. II. PFC response by mixtures of allogeneic lymphocytes cultured with formalin-treated staphylococci

Formalin-treated Staphylococci (FSA) induce anti-SRBC PFC in cultures of human lymphocytes. Regulation of the PFC response induced by FSA in cultures containing lymphocytes from two allogeneic donors was studied. The PFC response observed in such cocultures could not be predicted from the responses of lymphocytes from the two donors cultured individually. The PFC response of approximately one half the cocultures was less than expected. The remaining cocultures generated more PFC than expected. The depression or augmentation in the PFC response which was observed in cocultures was reproducible when lymphocytes from the same pair of donors were cocultured. Cocultures containing lymphocytes from identical twins generated the expected PFC response. The data suggest that suppressor or helper activity may be generated during a “two-way” allogeneic mixed lymphocyte reaction (MLR). Much less deviation from the expected PFC response was observed during a “one-way” MLR. Anti-Ia antiserum treatment of either donor's lymphocyte population tended to eliminate the deviation from the expected PFC response in coculture. The data suggest that a feedback loop, involving cells from both donors, may be operating in the “two-way” MLR, which leads to the generation of suppressor or helper activity.