Immunoglobulin secretion by human splenic lymphocytes in vitro: the effects of antibodies to IgM and IgD.

The effects of F(ab')2 fragments of affinity-purified rabbit anti-human mu chain antibody (RaHmu) and rabbit anti-human delta chain antibody (RaHdelta) on spontaneous and mitogen-stimulated immunoglobulin (Ig) secretion by normal human spleen cells were studied. IgM and IgG secretion by human spleen cells cultured in vitro was measured by incubating the cells with 3H-amino acids precipitating the secreted labeled Ig with anti-Ig, and analyzing the precipitates by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Both RaHmu and RaHdelta suppressed spontaneous and LPS-induced IgM and IgG secretion as well as PWM-stimulated IgG secretion. In different experiments, RaHmu and RaHdelta either suppressed or augmented PWM-induced IgM secretion. The anti-Ig induced augmentation of PWM-triggered IgM secretion was most apparent when spleen cells were cultured at lower cell densities or when lower concentrations of anti-Ig were employed. These date indicate that perturbation of B cell surface immunoglobulin receptors with specific anti-Ig antibody can alter markedly the ability of these cells to differentiate into antibody-secreting cells.     

The isolation and characterization of the human helper inducer T cell subset

Monoclonal antibody anti-4B4 was produced by fusing NS1 myeloma with spleen cells of a mouse immunized with Saguinus oedipus lymphocyte. This anti-4B4 antibody defines a 135-KD cell surface protein that is widely distributed throughout the hematopoietic system. More importantly, anti-4B4 is reactive with functionally unique human T cell subsets. Anti-4B4 antibody was reactive with approximately 41% of unfractionated T cells, 41% of T4+ inducer cells, and approximately 43% of T8+ cytotoxic/suppressor population. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+4B4+ subset proliferates relatively poorly upon stimulation with Con A and autologous cell antigens (AMLR) but well on exposure to soluble antigens, and it provides a good helper signal for PWM-induced Ig synthesis. The T4+4B4- subset, in contrast, proliferates well to Con A stimulation and autologous cell antigen (AMLR) but relatively poorly to soluble antigen stimulation, and provides little help to B cells for PWM-induced Ig synthesis. The T4+4B4- subset is largely 2H4+ and functions as the inducer of the T8+ suppressor cells. Thus, the present results suggest that one can divide the human T4 population into two major subsets that are phenotypically and functionally distinct, the human helper inducer subset (T4+4B4+/H.I.) and its reciprocal population defined by anti-2H4, the suppressor inducer subset (T4+2H4+/S.I.).     

Changes of the blood lymphocyte subpopulations and their functions following 131I treatment for nodular goitre and 32P treatment for polycythemia vera

The blood lymphocyte population was examined in 34 patients who were treated with 131I for toxic or atoxic nodular goitre. One to three doses of 300-550 MBq of 131I were administered at 1-week intervals. Lymphocyte counts were found to be significantly reduced at both 1 and 6 weeks after treatment. This decrease was accompanied by a changed composition of the lymphocyte subpopulations. The frequency of lymphocytes expressing membrane receptors for C'3 (EAC-rosette forming cells) was significantly reduced at 1 and 6 weeks following 131I administration. At 6 weeks there was a small but statistically significant increase of the frequency of T cells as identified by Leu 1 monoclonal antibodies. This was essentially due to an increased proportion of helper/inducer T cells as identified by Leu 3 monoclonals. 131I treatment also decreased the capacity of lymphocytes to secrete immunoglobulins (Ig) when stimulated with pokeweed mitogen (PWM). The greatest effect was observed for IgM. Secretion of IgG and IgA were less reduced. Mitogenic stimulations of lymphocytes with phytohemagglutinin (PHA) and concanavalin A were not significantly changed. It is concluded that these findings, with the exception of mitogen reactivity, are largely similar to those occurring following external radiation therapy for cancer. It is suggested that blood lymphocytes passing through the continuously irradiated gland are damaged mainly by beta-rays. The effect of 32P treatment on the blood lymphocyte population was examined in 16 patients with polycythemia vera. Before treatment the lymphocyte counts were within the normal range but the expression of certain membrane structures, as identified by monoclonal antibodies against total T cells (Leu 1 and 4), helper/inducer (Leu 3) and suppressor/cytotoxic T cells (Leu 2), were slightly decreased. Moreover, mitogenic responses of the lymphocytes to PHA and PWM-induced Ig secretion were impaired. Following a single oral dose of 32P (150-305 MBq), which normalized the production of erythrocytes and/or platelets, the blood lymphocyte counts were reduced by approximately 40 per cent 12 weeks after treatment. Examination of subsets demonstrated that the proportion of B-cells, as identified by B1 monoclonal antibodies, was decreased by the highest relative extent. On the other hand, lymphocytes expressing the above-mentioned T cell markers were somewhat increased. 32P treatment markedly increased PHA reactivity but it further reduced PWM-induced Ig secretion. The latter observation was in agreement with the finding that serum concentrations of Ig were reduced after treatment.     

Human B lymphocytes capable of spontaneous Ig production in short-term cultures: characterization in the circulation and lymphoid tissues

In the present work we have studied the occurrence and the characteristics of human B lymphocytes which are capable of spontaneous immunoglobulin (Ig) production in short-term cultures. It was found that this type of B cells secreted predominantly IgG and were present in the circulation as well as in the lymphoid tissue (tonsil and lymph node) of normal subjects. Tissular and circulating cell subsets exhibited many similarities: These cells produced Ig during a 3-day culture period without apparent need for T cells or monocytes. Protein and DNA synthesis were required for Ig secretion to occur. In Percoll fractionation experiments these cells showed low density, as they were mainly collected in the 42.5-45% Percoll fractions. These subsets consisted of cells considerably larger in size than the majority of B lymphocytes, as determined by Ig sedimentation. They were commonly defined as SmIg- Ia+ B cells by panning fractionation techniques. All these common characteristics suggest that these B cells have reached an advanced stage of maturation in vivo in both circulation and lymphoid tissue. Further surface marker analysis demonstrated that tissular but not circulating B-cell subset reacted with peanut agglutinin and the BA-2 monoclonal antibody which probably indicates their germinal center origin.     

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.     

PPD-induced immunoglobulin production in human peripheral blood lymphocytes. I. Necessary conditions for inducing the response.

The ability of PPD to induce Ig production in human PBL was investigated. PPD proved to be a good B cell activator for inducing polyclonal Ig production in PBL from healthy Japanese. Comparative studies of this response with PWM-induced Ig production showed that the cellular mechanisms involved in the two responses were different. First, PBL from an atypical individual with a deficient IgM production to PWM responded normally to PPD with IgM production as well as IgG production. Secondly, in IgG production, the effects of the two mitogens (PPDand PWM) were additive. An analysis of the cellular requirements in PPD-induced Ig production clearly demonstrated that T cells played a role in this response as well as in the PWM-induced response. However, the head-to-head comparative study on the titration curves of helper T cells in the two responses showed that PWM-induced helper activity was 2 to 5 times more effective than PPD-induced helper activity. Moreover, PPD-induced helper activity was shown to be more sensitive to ionizing radiation than was PWM-induced helper activity. Thus, this system of PPD-induced Ig production may provide a useful tool for understanding the human antibody production system as well as the PWM-induced response.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.     

Activation of human B lymphocytes: XVI. Cellular requirements,interactions, and immunoregulation of pokeweed mitogen-induced total-immunoglobulin producing plaque-forming cells in peripheral blood

The optimal culture and assay conditions for the detection of spontaneously occurring and pokeweed mitogen (PWM)-induced polyvalent Ig (IgG + IgM + IgA) and individual Ig class-specific plaque-forming cells (PFC) in human peripheral blood have been described in detail. Culture conditions are critical, particularly with regard to cell density and batches of supplemental serum. Fetal calf serum is a much more supportive serum supplement for PWM-induced PFC than is human serum. The assay system is a modified reverse hemolytic PFC assay using staphylococcal protein A coupled to sheep red blood cells by the chromic chloride method. PFC are developed by rabbit anti-human polyvalent Ig or anti-human individual Ig class antisera. Human peripheral blood contains 468 (±78) spontaneously occurring Ig secreting PFC per 10⁶ lymphocytes at Day 0 and 20,500(± 1971) PWM-induced Ig secreting PFC after 6 days in culture. The response is T-cell dependent; however, T cells can be replaced by a soluble T-cell factor prepared from a 48-hr allogeneic mixed lymphocyte reaction supernatant. The relative dependence on monocytes is a reflection of the culture conditions employed. Under the conditions of round-bottom tubes which promote cell-to-cell contact, depletion of monocytes to 0 to 2% does not result in a diminution of PFC responses. In fact, under such conditions, in certain individuals monocytes are markedly suppressive such that removal of monocytes results in a substantial enhancement of PFC responses. This system is simple and reproducible and should prove extremely useful in the delineation of the mechanisms of B-cell triggering and immunoregulation in normals and in disease states.     

The isolation and characterization of the human suppressor inducer T cell subset

Immunization of mice with lower primate lymphoid cells has provided a useful strategy for raising monoclonal antibodies against functionally important surface determinants on human T lymphocytes. We have developed a monoclonal antibody, anti-2H4, which defines functionally unique human T cell subsets. This anti-2H4 antibody was reactive with approximately 42% of unfractionated T cells, 41% of T4+ inducer cells, and was reactive with approximately 54% of T8+ cytotoxic/suppressor population. Anti-2H4 was not reactive with human thymocytes, but reacted with subsets of peripheral blood B cells and null cells. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+2H4+ subset proliferate well to concanavalin A (Con A) stimulation, but poorly to soluble antigen stimulation, and provides poor help to B cells for PWM-induced Ig synthesis. The T4+2H4- subset, in contrast, proliferates poorly upon stimulation with Con A, but well on exposure to soluble antigen, and provides a good helper signal for PWM-induced Ig synthesis. What is, perhaps, most important, the T4+2H4+ subset functions as the inducer of the T8+ suppressor cells. Previous attempts to define the latter subset of cells has relied heavily on the use of specific autoantibodies present in the sera of patients with juvenile rheumatoid arthritis (JRA) and systemic lupus erythematosus (SLE). The present results suggest that anti-2H4 antibody defines the human suppressor induced subset of lymphocyte previously described as T4+JRA+. Last, the results reemphasize the previously documented remarkable structural conservation of certain T cell-specific determinants on lymphocytes of phylogenetically distant primates.     

Analysis of T cell and B cell function in Peyer's patch and lamina propria of New Zealand Black and DBA/2 mice

We have analyzed gastrointestinal immune function in both DBA/2 and spontaneously autoimmune New Zealand Black (NZB) mice. We have studied both in vitro proliferation and differentiation of Peyer's patch cells and have measured immunoglobulin (Ig) secretion by cultured jejunal segments. Peyer's patch B cells and T cells from both DBA/2 and NZB mice showed similar proliferative responses to Con A and lipopolysaccharide (LPS), respectively. Unlike NZB splenic B cells, isolated Peyer's patch B cells from NZB mice did not spontaneously secrete Ig of any isotype. Seven-day cultures of equal numbers of Peyer's patch T cells and B cells resulted in similar patterns of secretion of IgA, IgG, and IgM in both strains. The addition of Con A to cultures of DBA/2 Peyer's patch cells consistently resulted in a onefold to threefold increase in IgA secretion after 7 days. Con A stimulation of NZB Peyer's patch cells did not produce any increment in IgA secretion. LPS stimulation of Peyer's patch cells from either strain resulted in a similar increase in IgG secretion with little effect on IgA secretion. The in vivo correlate of this finding was seen in the IgA to IgG ratio of Ig secreted by cultured jejunal fragments. In DBA/2 mice the rates of IgA/IgG varied from 2.36 to 4.85, whereas in NZB mice the ratio never exceeded 0.5. These experiments show that defects on the T cell compartment of NZB mice encompass gut-associated lymphoid tissue. The possible relationship of these findings and previously observed defects in oral tolerance is discussed.     

T cell regulation of polyclonally induced immunoglobulin secretion in humans

We measured the pokeweed mitogen (PWM)-induced secretion of IgG by the unfractionated mononuclear cells (MNC) of young adult donors, and correlated the results with the functional activity of cell suspensions enriched for T helper (T4+) and T suppressor/cytotoxic (T8+) cells. The distribution of IgG levels secreted by MNC differs from a Gaussian curve, implying that the group is composed of distinct heterogeneous populations. When donors were compared who were judged to be very low responders or very high responders on the basis of IgG secretion levels by MNC (less than 700 ng/ml or greater than 2500 ng/ml), no differences were found in the capacity of T4+-enriched cells to support PWM-driven IgG secretion by a common B cell pool. In contrast, the addition of 0.2 X 10(5) T8+ cells from these low responders to PWM-stimulated cultures of 0.5 X 10(5) T4+ cells plus 0.5 X 10(5) B cells resulted in significantly less IgG secretion (389 +/- 121 ng/ml) than did the addition of the same number of T8+ cells from the high responders (2241 +/- 548 ng/ml, p less than 0.01). Normalized percent suppression by T8+ cells was higher in low responders than in high responders (77.0 +/- 9.9% vs 33.0 +/- 8.5%, p less than 0.01). Both high and low responders markedly suppressed IgG secretion when 0.5 X 10(5) T8+ cells were added. No correlation was found either between proportion of T3+, T8+, T4+, or M1+ cells within the MNC population and levels of IgG secretion by MNC or between T8+ numbers and levels of suppression induced by a constant number of T8+-enriched cells. Our data indicate that differences in the functional activity of T8+ cells, rather than quantitative differences, account for the wide range of PWM-induced IgG secretion by MNC.     

IL-4-dependent IgE switch in membrane IgA-positive human B cells

IgE responses by human B cells, separated according to membrane Ig classes, were analyzed in a clonal assay using EL-4 thymoma cells as helper cells, T cell supernatant, and rIL-4. In cultures seeded by means of the autoclone apparatus of the FACS, IgE responses were generated frequently by either IgM (mu+/gamma-alpha-) or IgA (alpha +/mu-)-positive B cells (16 and 14% of the Ig producing wells, respectively), but rarely by IgG (gamma +/mu-)-positive B cells (1.3% of Ig producing wells). The total amounts of Ig secreted by IgM-, IgG-, or IgA-positive cells and the total proportions of responding autoclone wells (23-27%) were comparable. All IgE secretion was IL-4 dependent. When the Ig secretion patterns from alpha +/mu- vs alpha +/mu-epsilon- B cells were compared, most autoclone wells from both types of cells produced IgA only, and similar proportions of IgA producing wells (6.2 and 6.0%) also secreted IgE. In addition, IgE restricted responses occurred 6 times more frequently with alpha +/mu- than with alpha +/mu-epsilon- cells, which suggests that membrane IgA+E double-positive, IgE committed B cells occur in vivo. The isotype pattern generated by alpha +/mu-epsilon- B cells cannot be explained by a chance assortment of separate IgA and IgE precursors or by cytophilic antibody. Thus, IL-4 dependent switch to IgE occurred frequently in IgM- or IgA-positive, but rarely among total IgG-positive, B cells. This could be relevant to IgE production in mucosal tissues rich in IgA expressing B cells.     

Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies. Identification of a functional polymorphism to human IgG2 anti-CD3.

T cell activation induced by mouse anti-CD3 mAb has shown to be dependent on the Ig isotype of these antibodies. A study of isotype dependency of human antibodies, however, seems more relevant to human effector systems, especially in view of the availability of humanized antibodies for clinical applications. We constructed a panel of mouse and mouse/human chimeric anti-CD3 mAb, which differ only in their CH region and hence have identical binding sites and affinity. By using these antibodies, we now studied their ability to induce T cell proliferation in human PBMC and analyzed the classes of IgG FcR involved in these responses. The human (h)IgG1, hIgG3, and hIgG4, as well as mouse (m)IgG2a and mIgG3 anti-CD3 mAb induced an Fc gamma RI (CD64)-dependent T cell proliferation in all donors. Activation with hIgG2 and mIgG1 anti-CD3 mAb was observed to be mediated via the low affinity Fc gamma RII (CD32). It was found that leukocytes in a normal donor population display a functional polymorphism with respect to hIgG2 anti-CD3 responsiveness. This polymorphism was found to be inversely related to the previously defined Fc gamma RII-polymorphism to mIgG1 anti-CD3 mAb. Monocytes expressing the Fc gamma RII mIgG1 low responder (LR) allele support hIgG2 anti-CD3 induced T cell proliferation efficiently, whereas cells homozygous for the Fc gamma RII mIgG1 high responder (HR) allele do not. This observation could be confirmed in T cell activation studies using hFc gamma RIIa-transfected mouse fibroblasts, expressing either the mIgG1 anti-CD3 HR or LR Fc gamma RII-encoding cDNA.     

Induction of immunoglobulin-secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man

The existence of functionally distinct T lymphocyte subsets in man, initially demonstrated with heteroantisera, has been confirmed with monoclonal reagents. Two major subsets have been defined: Leu-2 cells, which effect cell mediated lymphocytotoxicity (CML), and Leu-3 cells, which amplify CML and other T cell functions. This study is an effort to determine the effects of these subsets on the immunoglobulin secretion induced in the human mixed leukocyte reaction (MLR). T cells were separated from non-T cells by rosetting with sheep erythrocytes and were fractionated into Leu-2 and Leu-3 subsets by solid phase immunoabsorption with monoclonal antibodies. T cell subsets were cultured with autologous non-T cells and irradiated allogeneic stimulator non-T cells. Secretion of IgM and IgG was measured by a reverse hemolytic plaque assay. MLR-induced antibody secretion was specifically dependent on the Leu-3 T lymphocyte subset. The Leu-2 subset was incapable of generating large numbers of IgM- or IgG-secreting cells, and, in fact, suppressed the Leu-3-induced response. Exposure of Leu-3 cells to a dose of irradiation sufficient to prevent their proliferation in MLR did not reduce induced immunoglobulin secretion. Leu-2-mediated suppression, however, was sensitive to low dose irradiation. Thus Ig secretion in human MLR is regulated by a balance of helper activity from the Leu-3 subset and suppressor activity from the Leu-2 subset.     

Induction of IgG2a secretion from mIgG2a+ B-lymphoid tumor cells by BCDF present in several antigen-specific T-helper clones

In order to study the soluble factor(s) that play an important role for the differentiation of IgG2-secreting B cells, we examined whether membrane IgG2a (mIgG2a)-bearing BALB/c B-lymphoid tumor cells, A20, could be induced to secrete IgG2a after treatment with soluble factors. We detected a potent B-cell differentiation activity inducing the Ig secretion of A20 tumor cells (BCDF-A20) in supernatants of several soluble antigens as well as alloantigen-specific T-cell clones of various genetic backgrounds. Thus, this BCDF-A20 activity was working in an antigen-nonspecific and MHC-nonspecific manner and abundant in many T-cell clones. It was shown that neither interleukin 1, interleukin 2, interferon, T-cell replacing factor, B-cell maturation factor, nor B-cell stimulatory factor-1 alone had any significant effect on the induction of Ig secretion of A20 tumor cells. Using isotype-specific rabbit anti-mouse Ig developers, we showed that mIgG2a+ A20 tumor cells secreted IgG2a after the treatment with soluble factors. The peak of the response of A20 tumor cells to BCDF-A20 was obtained 3 days after the treatment with culture supernatants of T-cell clones. In this study, we have clearly shown that mIgG2a+ A20 tumor cells were able to secrete IgG2a after treatment with T-cell soluble factors.     

Heterogeneity of B cell clones: immunoglobulin-secreting subsets

Staphylococcus aureus Cowan I bacteria (SpA CoI) is known to be a polyclonal B-cell activator of human lymphocytes. In this study, we investigated which of the B-cell subsets SpA CoI could stimulate and induce immunoglobulin (Ig) production. B-Cell subsets were separated from peripheral blood and tonsil lymphocytes by rosette formation with E, EA_IgG, EAC, anti-Ig-conjugated ox erythrocytes (OE-anti-Ig), and protein A-conjugated OE (OE-Pro A), or on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E^?, C3 receptor-positive (C3R⁺), Fc receptor-negative (FcR^?), and surface Ig-positive (SIg⁺) B-cell subsets. These B-cell populations responded well to SpA CoI and produced significant amounts of IgG, IgM, and a lesser amount of IgA. Among SIg⁺ B cells, IgG, IgA, and IgM⁺ B-cell subsets responded to SpA CoI and produced large amounts of Ig belonging to each corresponding Ig class. IgD⁺ B cells failed to produce Ig of any class, except for minimal amounts of IgG and IgM. While both the protein A receptor-positive (Pro A · R⁺) and negative (Pro A · R^?) cells responded well to SpA CoI, Pro A · R⁺ B cells produced IgG mainly and Pro A · R^? B cells produced IgM. Fractionation of B cells on a BSA gradient revealed that comparatively small-sized and denser B-cell subsets responded well to SpA CoI and produced every class of Ig.     

alpha-Interferon increases immunoglobulin production in cultured human mononuclear leukocytes

Interferons (IFN) are known to modulate immune responses in either an inhibitory or a stimulating manner. The present study was initiated to investigate the mechanisms by which alpha-IFN modulates Ig production of human peripheral blood mononuclear cells (PBMC). IgG and IgM production was measured in pokeweed mitogen- (PWM) stimulated 7-day cultures of PBMC. Significant enhancement of IgM and IgG production was observed when alpha-IFN was added. Overnight preincubation followed by washing also produced significant enhancement. The effect of alpha-IFN was not obtained in the absence of PWM or T cells. The effect of alpha-IFN on cultures of B and T cells was not altered by irradiation of T cells (2000 rad). alpha-IFN was not shown to enhance the production of helper factor but did increase the responsiveness of B cells to helper factor if the B cells were preincubated with alpha-IFN. Finally, alpha-IFN did not increase the Ig production of PBMC induced by Epstein Barr virus (EBV), and the outgrowth of EBV-infected PBMC was not affected. Overall, these results show for the first time that the effect of alpha-IFN on PBMC is due to an enhanced responsiveness of B cells to helper factors produced by radioresistant T cells.     

Synthesis and secretion of IgG in synchronized mouse myeloma cells

A mutant of the MPC-11 mouse myeloma cell line which grows as a monolayer has been used to study the synthesis and secretion of IgG in relation to the cell cycle. The mitotic detachment method has been used to obtain a pure population of mitotic cells which were then allowed to progress through the G1, S, and G2 phases of the cell cycle. The synthesis and the rate of secretion of IgG have been studied in each phase of the cycle by incubation of cells with ¹⁴C-amino acids, followed by immunoprecipitation and quantitation of synthesized and secreted IgG_2b. The data are consistent with the idea that synthesis and secretion of Ig are not a cell cycle dependent event in myeloma cells.     

Rapid loss of IgM expression by normal murine B cells undergoing IgG1 and IgE class switching after in vivo immunization

Injection of mice with polyclonal goat anti-mouse IgD antibody (G alpha M delta) stimulates a potent T cell-dependent immune response characterized by large increases in serum IgG1 and IgE concentrations and by the generation of substantial numbers of membrane (m)IgG1+ B cells. The onset of this response occurs 6 days after G alpha M delta injection and peaks by day 7 to 8. Utilizing two color fluorescence analysis and cell sorting we demonstrate that most mIgG1-expressing B cells lack mIgM during the period of onset of Ig isotype switching (day 6). Both IgG1 and IgE are produced predominantly by mIgM- cells. On day 6, IgG1 and IgE are secreted predominantly by cells expressing mIgG1 and mIgE, respectively. By day 8, a majority of the IgG1 secretion occurs among the mIgG1- cells but virtually all IgE secretion continues to come from the mIgE+ population. B cells that strongly express mIgG1 secrete little IgM or IgE. Freshly harvested B cells expressing mIgG1, 6 days after G alpha M delta injection, have undergone substantial deletion of CH mu-specific DNA in contrast to their mIgG1- counterparts. Hence, the great majority of B cells that switch to the IgG1 or IgE isotypes in vivo rapidly lose their expression of IgM.