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A combination of immunomagnetic cell sorting and ELISPOT techniques has been evaluated to permit enrichment and characterization of antibody-secreting cells (ASC). Cell suspensions containing putative ASC were first incubated with magnetic microbeads coated with antibodies specific for a given cell surface marker. After separation of bead-cell clusters and free cells, the resulting cell populations were examined for the presence of ASC by an ELISPOT assay. As a model system, the expression of selected cell differentiation markers by human circulating ASC has been evaluated after parenteral tetanus vaccination and during the course of a Leishmania infection. Prior treatment of blood MNC with beads coated with antibodies to CD38, HLA-DR or CD19 permitted the isolation of virtually all blood ASC. Further, prior immunomagnetic removal of T (CD2+) cells from blood MNC, followed by isolation of CD38+ cells facilitated the detection of Leishmania major-specific ASC in all six patients examined, whereas parasite-specific ASC among unfractionated blood mononuclear cells could only be detected in 3 out of these six patients. Simple and rapid, this approach provides not only accurate estimates of the frequency of ASC within a given B cell population or subpopulation, but can also efficiently enrich functional ASC from complex cell suspensions and thus should be particularly useful in situations where ASC are present at low frequencies.

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or beta-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7- cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.

The first product of the humoral response to antigen is low-affinity antibody, produced by extrafollicular foci of antibody-forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6-parameter flow cytometry to isolate and examine the characteristics of (4-hydroxy-5-nitrophenyl)acetyl-specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T-dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme-linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick-end labeling.

Problem: Isolation of viable cervical lymphocyte populations and characterization of their function in healthy tissue is necessary to understand immunity in the genital tract., Methods: Normal, cervical tissue was digested using a multi-enzymatic digestion procedure. Lymphocytes were characterized using FACS analysis and ELISPOT analysis for immunoglobulin secreting cells., Results: Following the digestion procedure, 0.16 x 10(6) +/- 0.8 cells/g of tissue with a viability of 90-98% were isolated from normal cervical tissue. FACS analysis determined that B lymphocytes were the predominant cell type in normal cervical tissue representing a significantly higher percentage than that found in peripheral blood (P = 0.015). T lymphocytes and NK cells represented a significantly lower percentage than that found in peripheral blood (P = 0.0001 and 0.026, respectively). The largest percentage of immunoglobulin secreting cells isolated were secreting IgG followed by IgA. A limited number of IgM secreting cells were detected. IgA2 secreting cells represented 34.46 +/- 4.6% of the total number of IgA plasma cells., Conclusion: These studies represent the first analysis of viable mononuclear cells isolated from normal cervical tissue. The results form a baseline from which it will now be possible to compare changes that occur at the cervical squamocolumnar junction in response to infection or neoplasia.

We have encountered two types of variability in the numbers of cells making antibodies in vitro to sheep erythrocytes (PFC) in pokeweed-stimulated human peripheral blood mononuclear lymphocytes (PBM). One is a variation in number of PFC generated by the PBM of one individual compared -o that of another, which is probably an in vitro counterpart to the well known individual variation in in vivo antibody responsiveness and presumed to be T cell controlled. The other variability is in numbers of PFC found in replicate tubes from a given individual. This variation is not T cell controlled, but rather is releated to the relative number of monocytes in the system. Enhancement by monocytes of the numbers of PFC generated is also demonstrated.

Mice were immunized against fluorescein isothiocyanate (FITC)-labelled human gamma globulin (HGG) in the absence or presence of different adjuvants. The immune response was assayed every other day with regard to both total Ig-secreting cells and FITC-specific plaque-forming cells (PFC). The adjuvants influenced the type of immune response induced to the same antigenic determinant. Thus, addition of Freund's complete (FCA) or incomplete (FIA) adjuvant preferentially led to the secretion of IgG1 PFC of an average high affinity. Most newly appearing IgG-secreting cells were also detected as FITC-specific PFC. The use of lipopolysaccharide (LPS) as an adjuvant resulted in the induction of both IgM and IgG, particularly of the IgG3 and IgG2b subclasses. However, these antibodies had relatively low affinity, and a large number of total IgG-secreting cells induced by LPS had no detectable FITC specificity. The FCA/FIA- and LPS-induced responses to FITC-HGG were additive when injected together, indicating that they act on distinct subpopulations of B lymphoid cells. The adjuvant response to LPS, but not the response to FCA/FIA, was totally absent in mice of the C3H/Hej strain, which are non-responders to the polyclonal activating properties of LPS. Finally, the response induced by FCA or FIA was T-cell-dependent and the LPS response T-cell-independent as assayed in nude mice.

Primary and secondary anti-Thy-1 responses were elicited in C57BL/6Kh and (C57BL/6Kh X BALB/cKh)F1 mice by injection of either intact or sonicated Thy-1 disparate thymocytes as the source of the two forms of Thy-1 antigen--cell bound and cell free, respectively. The primary response was characterized by a high number of anti-Thy-1 plaque-forming cells in the spleen and a high titer of serum antibodies that belonged predominantly to the IgM class. The secondary response consisted of a moderate number of plaque-forming cells and a high titer of serum antibodies that belonged to the IgG and IgM classes. Only secondary responses ensued in mice primed and boosted with the same form of antigen, whereas primary and secondary responses developed concomitantly in animals primed with one form and boosted with both forms simultaneously. These data suggested that the two forms of Thy-1 antigen activate in a given responder two discrete subsets of T cells.

A 67-year-old man with stable chronic neutropenia, lymphocytosis, multiple auto-antibodies, and recurring infections was studied in order to characterize the abnormal lymphocytes and the mechanism of neutropenia. One-half of the circulating mononuclear leukocytes were large granular lymphocytes that did not rosette with sheep erythrocytes and did not have surface immunoglobulin or receptors for C3 or IgG Fc. Virtually all of the mononuclear leukocytes, however, reacted with three monoclonal antibodies specific for mature T lymphocytes--OKT3, 3A1, and 12.1--the latter with a bimodal pattern on FACS analysis. The nonadherent, non-E-rosetting lymphocytes consisted of a homogeneous population of T cells with a novel phenotype: 3A1+, OKT3+, OKT8+, Ia+, 12.1+, OKT4-, 9.6-, 10.2-, Fc gamma receptor-. The abnormal phenotype of these cells suggested that they may have developed clonally. In spite of their OKT8+ phenotype, these lymphocytes were functionally abnormal, since they did not suppress B-cell immunoglobulin production in vitro. A humoral immune mechanism of neutropenia was indicated by increased levels of neutrophil-reactive IgG and in vivo kinetic studies with autologous neutrophils demonstrating shortened intravascular survival. The granulocyte turnover was normal, however, suggesting a blunted marrow response to the neutropenia. Corroborating the kinetic data, in vitro cultures of the patient's blood and marrow cells showed reduced numbers of granulocytic progenitors (CFU-C). Neither blood lymphocytes nor serum from the patient suppressed growth of allogeneic CFU-C nor did removal of OKT8+ cells from the marrow increase CFU-C expression. Thus, the disorder in this patient is characterized by proliferation of abnormal OKT8+ lymphocytes with impaired suppressor function. Our findings suggest that the neutropenia was due to anti-neutrophil autoantibodies that may have resulted from abnormal T-cell immunoregulation.

Peripheral blood mononuclear cells (PBM) from four patients with IgG myeloma and four patients with benign monoclonal gammopathy (BMG) were stimulated with pokeweed mitogen (PWM), and the in vitro immunoglobulin production over 7 days was measured with an enzyme-linked immunosorbent assay. All myeloma patients were sufficiently treated with cytostatic drugs. Their PBM did not produce monoclonal Ig in vitro, as opposed to PBM from two patients with BMG. Unseparated PBM from myeloma patients produced smaller amounts of polyclonal Ig than unseparated cells from normal donors. However, macrophage-depleted PBM from myeloma patients produced amounts of Ig comparable to those of normal donors when autologous or allogeneic adherent cells were returned in defined numbers. T cells from three of the four myeloma patients could provide help for the Ig production by B cells from healthy donors. These results indicate that functionally normal polyclonal B cells circulate in the blood of myeloma patients. The circulating T-cell population also has no obvious defect. In contrast, blood macrophages seemed to be altered with respect to their regulating function for polyclonal Ig production. The results obtained by using cell populations from patients with BMG did not differ from those of healthy people.

In vivo immunization of normal volunteers with tetanus toxoid induces the formation of a circulating B cell subset that has the capacity to secrete specific antibody in vitro without the need for T cell help or mitogen stimulation. From earlier studies it was not clear whether the spontaneous antibody-secreting lymphoblastoid (LB) B cell was at a terminal stage of differentiation or if it had the capacity to give rise to additional B cell subsets, such as memory cells or more fully mature antibody-producing cells. In this study we have shown that at least two distinct waves of spontaneous antibody secretion can occur in vitro when cultures are initiated with the lymphocytes from individuals immunized 6 days earlier. The first production of antibody was completed by 3 days of in vitro culture and the second production of antibody did not initiate until day 7 or 8 of culture and was completed by day 12. The B cells responsible for the second stage of antibody production appeared derived from a portion of the antibody-secreting cells present on day 3 in that 1) treatment of the cultures on day 0 with BuDr and light equally inhibited the first and second rounds of antibody synthesis; 2) when isolated from the blood, both B cell subsets were in the large cell fraction after 1 X G sedimentation; and 3) under conditions of limiting numbers of cells, the cells responsible for the second wave of antibody production were almost exclusively found in cultures positive for a B cell that had produced antibody on days 1 to 3. Although only a portion (10 to 30%) of the LB B cells present on day 0 had the capacity to again produce antibody on days 8 to 12, the two cells were capable of producing similar quantities of antibody on a per cell basis. These results indicate that the mature circulating LB cell induced in vivo by immunization is not terminally differentiated, but under appropriate conditions has the capacity to give rise to additional antibody-secreting cells.

Staphylococcus aureus Cowan I strain (SAC) activates human peripheral blood B lymphocytes to proliferate but does not induce secretion of immunoglobulin (Ig) unless pokeweed mitogen (PWM)-activated T cells are added. Using a Percoll gradient separation method, we show that a B-cell subpopulation isolated at 50/60% Percoll interface is the target for SAC stimulation. After SAC activation, small, dense B cells become larger, less-dense B cells which proliferate. These activated B blasts differentiate into Ig-secreting cells when cultured with PWM-activated T cells. The culture supernatant of PWM-activated T cells is capable of supporting proliferation and Ig secretion of the activated B blasts but not of resting small B cells. This result suggests that the culture supernatant contains B-cell growth factors.

We identified the presence in the Aleutian skate, Bathyraja aleutica, of two classes of immunoglobulins (Ig), a high molecular weight Ig analogous to mammalian IgM and a low molecular weight Ig, similarly to the spiny rasp skate, Raja kenojei, (Kobayashi, K. et al., Mol. Immunol. 1984. 21: 397), using an immunological cross-reaction with the specific antisera to the spiny rasp skate Ig components. The antigenic similarity of the heavy chains of the Ig of the Aleutian skate and those of the spiny rasp skate was less than that between their light chains. Two types of Ig-producing cells, one producing the high molecular weight and the other forming the low molecular weight Ig, were present in the spleen of embryos and adults in the Aleutian skate at a ratio of 5-6:4-5. The number of these Ig-producing cells increased with advancing development of the embryos but was 1/20 to 1/50 of those of adults. Cells, each of which were capable of forming both classes of Ig, were found in the spleen of embryos but not in that of adults. These results suggested that the spleen of the Aleutian skate is the primary lymphoid organ for B lymphocyte differentiation and proliferation, possibly equivalent to the bursa of birds.

In past experiments, using limited dilution analysis, we have demonstrated that a high percentage of immunoglobulin-secreting clones derived from Epstein-Barr virus- (EBV) stimulated lymphocytes secrete IgA. To further characterize the IgA produced by these clones, the IgA subclass of supernatants from clones stimulated 4 to 6 wk previously with EBV was determined by radioimmunoassay. All of 17 IgA-producing clones secreted IgA1; none secreted IgA2. Because we have shown that surface IgM+ (sIgM+) B cells are an enriched source of IgA2 plasma cell precursors, panning techniques were used to purify sIgM+ B cells from tonsils. Of 103 clones derived from these sIgM+ B cells, 102 secreted IgA1 and only one secreted IgA2. The relative absence of IgA2-producing clones could not be attributed to an absence of EBV receptors on IgA2 cells. A mean of 84 +/- 4% of freshly isolated IgA2 B cells and 78 +/- 6% of IgA1 B cells could be stained with a monoclonal antibody binding the EBV receptor; and there was no failure of EBV to infect IgA2 plasma cells precursors. Of IgA2 plasma cells derived from peripheral blood lymphocytes stimulated 7 days previously with EBV, 54 +/- 7% were positive for the EBV nuclear antigen, compared with 54 +/- 18% of IgA1 plasma cells from the same cultures. Seven days after EBV stimulation, a mean of 25% of the total IgA plasma cells were positive for cytoplasmic IgA2, whereas by 21 days after stimulation only 7% were positive for IgA2. This shift in the proportions of IgA1 and IgA2 plasma cells could be attributed to a failure of the IgA2 plasma cell number to increase after 10 days in culture. There was no evidence for selective suppression of IgA2 production by T cells or selective lysis of IgA2 plasma cells by infectious EBV particles. These results demonstrate that although precursors for both IgA1- and IgA2-producing cells can be stimulated to differentiate in response to EBV, there is preferential transformation of IgA1-producing cells.

The immunoglobulin M (IgM) and the IgG1, IgG2ab, and IgG3 subclasses of plaque-forming cells (PFC) specific for lipopolysaccharide (LPS) were measured after immunization of mice with LPS alone and compared with the responses to LPS in combination with nonbacterial proteins and with bacterial membrane phospholipid vesicles or two major outer membrane proteins from Proteus mirabilis. The relative numbers of IgG PFC belonging to the IgG1, IgG2, or IgG3 subclasses induced by immunization with LPS alone depended upon the type of LPS administered. Phospholipids and the proteins effected characteristic alterations in not only the strength but also the subclass of the IgG responses to LPS. The results suggest that the hydrophobic-hydrophilic nature or state of aggregation of the preparations plays a role in the induction of IgG1 and IgG2 subclasses of PFC specific for LPS. Complex formation with LPS and adjuvant was apparently necessary to obtain these effects.

Germinal center cells from the rabbit appendix were fractionated by velocity sedimentation and isopycnic gradient centrifugation. Subsets were analysed with respect to cell size and surface markers, and were functionally characterized by testing the capacities for primary antibody synthesis, memory cell production, and formation of new germinal centers in an autologous transfer system. The migratory behaviour of the germinal center cell subsets within the spleen of homologous recipients was also studied using autoradiography. Both cell fractionation methods yielded a separation of large and small cells. Surface immunoglobulin and C3 receptors were equally expressed on germinal center cells differing in size and density. The different subsets were also equally capable in giving rise to IgM-antibody-forming cells and memory cells upon antigenic stimulation. Furthermore, large germinal centers were newly formed in the spleen of the recipients, irrespective of the cell subset injected. It was concluded that the results do not support the hypothesis that, inside germinal centers, the differentiation of large lymphoid cells (centro-blasts) into small centrocytes also implies a maturation process. Subsets of germinal center cells, however, showed a different and characteristic migratory behaviour; while small cells migrated preferentially to the corona of lymphocytes in spleen follicles, large, light cells showed an affinity for the germinal center area. We postulate that, upon stimulation, immature B cells develop an affinity for the germinal center microenvironment, to participate in a germinal center reaction.

This report shows that, in 8- to 10-month-old BALB/c mice immunized intraperitoneally with dextran B1355, approximately 75% of IgG3 anti-alpha (1----3) polyglucan (anti-dex) plaque-forming cells (PFC) detected in the spleen were identified as double-Ig class producers secreting simultaneously IgG3 and IgM antibodies with the same specificity for the dex epitope. Under the same conditions of immunization, however, IgA anti-dex PFC were mostly single-class secretors. IgA PFC developed in the spleen in highest numbers (equal to IgM), but in Peyer's patches IgA PFC were sevenfold more numerous than IgM. Furthermore, spleen IgG3 anti-dex PFC responses were low compared with spleen IgA and IgM anti-dex PFC responses and appeared only late in ontogeny. The possibility is discussed whether a TH dependence of the IgA anti-dex response and a TH-independent generation of the IgG3 response are responsible for the different pattern of isotype expression.

The kinetics of appearance of immunoglobulin- and antibody-containing cells in rat lymph nodes was studied by immunoenzymatic techniques between days 12 and 90 after a single injection of horse-radish peroxidase. Three distinct phases appeared during the primary repsonse. Between days 8 and 17, essentially no antibody-containing cells were detectable, while non-specific immunoglobulin-containing cells amounted to up to 9% of the total cell population. The peak of antibody-forming cells was observed between days 18 and 24, and their number decreased slowly during the last phase. Days 18-20 were marked by a sharp peak of a distinct category of cells containing antibodies in a limited area of their cytoplasm. One day before this event, the percentage of immunoglobulin-forming cells decreased drastically, and remained very low throughout the response. These results are discussed on the basis of a previously published model, which proposes that non-specific immunoglobulin-containing cells are precursors to antibody-containing cells, and that the transformation between these two categories of cells involves a stage where antibody synthesis is restricted to a few endoplasmic reticulum vesicles.

Since patients with rheumatoid arthritis (RA) exhibit serum hypergammaglobulinemia and autoantibody (rheumatoid factor) production, we compared elaboration and control of in vitro RA mononuclear cell (MNC), Ig assayed by enzyme-linked immunoassays or by hemolytic plaque formation, in 37 RA patients and 17 normal subjects. We found (1) RA spontaneous plaque-forming cells were significantly reduced (RA 344 vs normal 627 PFC/10(6) MNC, P less than 0.002); (2) RA spontaneous IgG and IgM (but not IgA) elaboration was significantly diminished (IgG RA 339, normal 776; IgM RA 255, normal 869 ng/ml, P less than 0.001; IgA RA 87, normal 124); (3) RA stimulated IgG and IgM production (but not IgA) was also decreased (IgG RA 2434, normal 3862, P less than 0.06; IgM RA, 1676, normal 3323, P less than 0.005; IgA RA 1859, normal 2315); (4) reduced RA Ig elaboration was not clearly due to altered numbers of T or non-T cells, age, medications, clinical features of disease, or response kinetics; (5) relative improvement of RA in vitro IgG, but not usually IgM, secretion followed removal of adherent cells, addition of indomethacin or addition of mitomycin C-treated T cells; (6) MNC from synovial fluids, but not bone marrows, exhibited spontaneous Ig production in excess of stimulated synovial fluid cellular or peripheral blood Ig elaboration. These observations indicate selective impairment of peripheral blood MNC IgG and, particularly, IgM secretion in RA. This defect appears to reflect accessory cell influences which differ from normal as well as the sequestration of primed or activated cells in the synovial fluid.

Studies of an EBV-transformed and TNP-specific human B cell line revealed that, unlike myeloma or hybridoma cell lines that consist mainly of fully differentiated cells, most of the cloned EBV-transformed cells were not fully differentiated, as judged by inability to bind TNP-SRBC and to secrete anti-TNP antibody. The minority of more differentiated cells were selected by TNP-SRBC rosetting. They were found to proliferate to a lesser extent than nonrosetting cells and to contain increased numbers of antibody-secreting cells. This inverse relationship between proliferation and differentiation was also shown to be cell cycle related in that the TNP-SRBC rosetting cells resided, to a greater extent than the nonrosetting cells, in the G1 phase of the cell cycle. The finding that the G1 phase of the cell cycle was associated with differentiation into anti-TNP secreting cells was confirmed by demonstrating that treatment with hydroxyurea, which arrests the cells in G1, resulted in decreased proliferation and an increased proportion of antibody-secreting cells. Similarly, addition of phorbol ester resulted in increased antibody secretion and decreased proliferation, suggesting a role for protein kinase C in this differentiation pathway. The strategy of increasing the number of antibody-producing cells in this human EBV line, by promoting differentiation of the cells in G1, may be relevant to the large scale production of specific human mAb for the treatment and diagnosis of human diseases.

The chronic inflammatory diseases in humans have been intensively investigated, however the immune mechanisms underlying diseases such as rheumatoid arthritis (RA), inflammatory bowel disease, and periodontal disease (PD) remain elusive. In this study, we have analyzed the distribution of IgM, IgG, and IgA secreting cells with emphasis on the IgG and IgA subclasses among mononuclear cell populations isolated from gingiva at different stages of PD. Surgically removed tissues were treated with Dispase to gently dissociate cells and the Ficoll-Hypaque gradient centrifugation was used to enrich for viable mononuclear cells rich in lymphocytes, macrophages, and plasma cells. The total numbers of plasma cells increased with the severity of disease. Immunofluorescence analysis showed that most Ig-containing cells were of the IgG isotype; however, significant numbers of IgA-positive cells but few IgM-positive cells were seen. This isolation procedure allowed analysis, at the single cell level, of the distribution of IgG and IgA subclasses of antibody-secreting cells with monoclonal antibodies to human IgG and IgA subclasses. For this, we selected four monoclonal anti-IgG subclass (anti-gamma 1, -gamma 2, -gamma 3, and -gamma 4) antibodies with no subclass cross reactivity for use in the enzyme-linked immunospot assay. Analysis of slight, moderate, and advanced stages of PD showed a progressive increase in spotforming cells (SFC) numbers, and the major isotype of SFC was IgG followed by IgA. The major IgG subclass SFC seen was IgG1 followed by IgG2 whereas similar numbers of IgG3 and IgG4 SFC were observed, a pattern also seen with cells from synovium of RA patients and in mitogen-triggered spleen and PBMC. In terms of the IgA subclass distribution, IgA1 predominated in moderate stages, whereas a selective increase in IgA2 SFC were seen in the more advanced stage of PD. These results show that significant numbers of viable plasma cells/Ig-secreting cells can be isolated from inflamed gingival tissues. Further, careful analysis has shown that IgG subclass responses in gingiva are similar to those found in synovia of RA subjects, and in stimulated PBMC and spleen. However, it should be noted that the number of IgG4- and IgA2-secreting cells increased in the advanced stage of PD.

Suppression of an antigen-specific plaque-forming cell response of human blood lymphocytes can be effected by T mu+ cells that have been primed previously by antigen in vitro for 6 days. While lacking the capacity to suppress the plaque-forming response directly, these primed T mu+ suppressor-inducer cells stimulate a subpopulation of unprimed T mu gamma- cells to differentiate to T gamma + suppressor-effector cells. The T mu+, T gamma+ and T mu gamma- subsets have been shown to be heterogeneous populations of cells. Therefore, the functionally defined T suppressor-inducer, -precursor and -effector cells were characterized by OKT monoclonal antibodies and by the capacity to form rosettes with autologous erythrocytes (ar+). Evidence will be presented that in vitro a T4+mu+ar- cell induces a T8+mu gamma-ar+ precursor cell to differentiate to a T8+gamma+ar- suppressor-effector cell. A similar T suppressor-effector cell can also be isolated directly from peripheral blood of normal donors.

As a marked local immunoglobulin G (IgG) response has previously been found to be the most prominent immunopathological feature of both ulcerative colitis and Crohn's disease, the subclass distribution of colonic IgG-producing immunocytes was examined. This study included tissue specimens from 10 patients with ulcerative colitis and 8 with Crohn's colitis. Paired immunofluorescence staining was performed with subclass-specific murine monoclonal antibodies combined with a rabbit antibody reagent of IgG; the proportion of cells belonging to each subclass could thereby be determined in relation to the total number of mucosal IgG immunocytes. A significantly higher median proportion of IgG1 immunocytes was found in ulcerative colitis (81.3%) than in Crohn's colitis (66.5%). Conversely, the median proportion of IgG2 immunocytes was significantly higher in Crohn's colitis (24.9%) than in ulcerative colitis (9.4%). This disparity in the local IgG subclass response might reflect dissimilar mucosal exposure to mitogenetic or antigenic stimuli or genetically determined immunoregulatory differences in the two categories of patients.

Sheep red blood cells conjugated with protein A from Staphylococcus aureus were frozen in liquid nitrogen. After thawing these cells were used as indicator cells in a plaque-forming cell assay visualizing human B lymphocytes secreting IgG, IgM or IgA. The thawed erythrocytes performed as well in the assay as erythrocytes freshly conjugated with protein A. Recovery of conjugated erythrocytes after freezing and thawing averaged 66%. By the method described indicator cells for a number of PFC assays may be prepared in bulk, frozen in suitable portions and thawed when needed. This prevents possible batch-to-batch variations and saves time.

Plasma cells synthesizing rheumatoid factors (RF) were identified by fluorescent staining of sections of synovium and macrophage-depleted cells from dispersed synovial tissue. The latter avoided problems related to sampling errors in studying tissue sections and in the uncertainty raised by the staining of macrophages with intracellular complexes. Plasma cells producing IgG predominated, and seropositive patients had a higher proportion of IgM producers than seronegative subjects. None the less, in both groups of patients more than 90% of the IgM plasma cells were synthesizing RF, whereas the corresponding figure for IgG was between 50% and 60%. Only around 10% of IgA plasma cells were positive for RF. The high percentage of IgM plasma cells making RF would tend to argue for an IgG-specific response and against direct polyclonal activation as the stimulus. The percentage of IgG-producing cells positive for RF is also consistent with a dominant response to IgG. Accepting the difference in the relative proportion of total IgM- to IgG-producing plasma cells in seropositive as against seronegative patients, the close similarity between the two groups in the fraction of cells making RF favours the view that the two groups have a comparable underlying immunopathology dependent on IgG autosensitization. From the technical standpoint, the dispersed cell method gives results in line with those obtained with sections but which are easier to read, whereas the fluorescent techniques described give clear and reproducible results for the detection of RF of different heavy-chain isotype.

The total number of spontaneously occurring ("background") IgM-, IgG-, and IgA-secreting cells and the frequency of antigen-specific IgM-, IgG-, and IgA-secreting cells were determined in germ-free BALB/c mice fed a chemically defined ultrafiltered diet (GF-CD), in specific pathogen-free BALB/c mice fed an autoclaved natural ingredient diet (SPF-NI), and in conventional BALB/c mice fed nonautoclaved natural ingredients (CV-NI). This was done by means of the ELISA-plaque assay. The results did not show differences among the various groups of mice with regard to the total numbers of IgM-secreting cells in the various lymphoid organs. Also the frequencies of IgM-secreting cells specific for DNP27-BSA and the anti-idiotypic monoclonal antibodies Ac38 and Ac146 did not differ significantly among GF-CD, SPF-NI, and CV-NI mice. GF-CD mice, however, did show substantially decreased numbers of IgG- and IgA-secreting cells in their lymphoid organs. Furthermore, there were striking differences in the frequencies of antigen-specific IgG- and IgA-secreting cells between GF-CD mice and the two other groups of mice. These results indicate that exogenous antigenic stimulation has a great effect on both the total numbers and the specificity repertoires of background IgG- and IgA-secreting cells. Such an influence could not be detected with regard to the background IgM-secreting cells. This suggests two distinct compartments of background Ig-secreting cells: a very stable, endogenously regulated compartment consisting mainly of IgM-secreting cells, and another compartment, consisting mainly of IgG- and IgA-secreting cells, whose numbers and specificity repertoire appeared to be influenced by exogenous antigenic stimulation.

We have investigated in vitro the induction of antibody responses to phosphorylcholine (PC) by cloned T helper (Th) cell lines. The cloned Th cells are antigen specific, in this case ovalbumin (OVA), self-Ia recognizing, and induce antibody secretion only if the hapten, PC, is physically linked to the carrier (OVA) molecule. The plaque-forming cell (PFC) response generated in the presence of cloned Th cells is idiotypically diverse with 5-40% of the secreting B cells bearing the TEPC-15 (T15) idiotype. The interaction of the cloned Th cells and unprimed B cells requires recognition of B cell surface Ia glycoproteins for all B cells activated to secrete anti-PC antibody, whether they be T15-bearing or not. More importantly, however, effective interaction between a cloned Th cell and a B cell is determined by the quantity of B cell surface Ia glycoproteins. Our results indicate that quantitative differences in B cell surface Ia antigens are directly related to B cell activation by the cloned Th cell. The high Ia density B cells are most easily activated by cloned Th cells, and these appear to be mainly non-T15-bearing. These data suggest that the failure of cloned Th cells to effectively activate T15-bearing B cells in vitro may be due to the lower relative Ia density of these B cells and therefore to their inability to interact effectively with cloned Ia-recognizing Th cells. These results imply that monoclonal T cells may distinguish between T15-bearing and non-T15-bearing B cells based on their Ia density.

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. micron+ and micron- cells were rosetted with anti-gamma-ox red blood cells and separated by density centrifugation into fractions enriched or depleted of gamma + cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the micron- fraction was by gamma + cells. In the microm+ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the micron+ gamma- and micron+ gamma+ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that gamma was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody production in vitro. Studies performed to determine the culture requirements of micron- and micron+ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some micron- cells could produce IgG in the presence of T cells alone.

A helper cell population with phenotypic characteristics of both B and T cells is described. This helper population, called BH, is present in normal unprimed C57BL/6 mice and preferentially helps the expression of NPb idiotype-bearing plaque-forming B cells in the absence of T helper cells. Its surface phenotype is Lyt-1.2+, Ig+, Lyb-3+, Thy-1.2-, Lyt-2.2-. The helper activity of the BH population is IgH restricted and BH cells selectively bind NPb idiotypic determinants. Collectively the data demonstrate that this unique subpopulation can regulate the response of antibody-secreting B cells through specific recognition of idiotypic determinants.

The polyclonal B cell activators (PBA) dextran sulphate (DxS) and lipopolysaccharide (LPS) induce a marked synergistic response in mouse splenic B cells. Such activated B cell blasts could be maintained in culture for a total period of 15 to 20 days, provided the cells were subcultured repeatedly in fresh medium containing the PBA. After this time period activation could no longer be achieved. The B cell blasts responded at all times better to LPS plus DxS than to either PBA added alone. Furthermore, LPS-induced B cell blasts also responded better to LPS plus DxS than to LPS alone. Activated cells secreted IgM and switched to IgG3 and IgG2b secretion. At the end of the culture period, approximately one cell out of four secreted IgG. The kinetics of IgG3 and IgG2b production were similar, and the fraction of cells secreting these subclasses increased at similar rates throughout the culture period. We conclude that high-rate secreting and proliferating B cells have a limited life span. Furthermore, most activated B cells do not seem to go through a continuous subclass switch but keep on secreting the subclass they originally switched to.

A highly reproducible paired immunofluorescence staining method was used to map the relative distribution of IgA1- and IgA2-producing cells in peripheral lymphoid organs and various secretory tissues. Spleen, peripheral lymph nodes, and tonsils all contained a marked predominance (91 to 95%) of IgA1 immunocytes. However, striking variations were demonstrated among the secretory tissues with regard to the median proportion of IgA1-producing cells: nasal mucosa, 96%; lacrimal glands, 81%; major salivary glands, 66%; mammary glands, 63%; gastric and proximal small intestinal mucosa, 84 to 77%; ileum, 55%; and large bowel, 41%. Thus, IgA2 production is relatively enhanced mainly in the distal gut and in mammary and salivary glands, in that order.

The nature of the IgA B cell precursors that receive preferential help from selected clones of T helper cells from mouse Peyer's patches (PP Th A) were studied. Activation of the PP Th A clones required the presence of antigen, sheep erythrocytes (SRBC), in a culture system supporting development of antibody-secreting plasma cells. Two types of PP Th A cells were used. Both gave vigorous IgA responses; the first also supported low IgM, and the second low IgM and IgG subclass antibody responses. Removal of sIgA+ B cells from either splenic or PP B cell cultures selectively depleted precursors of IgA antibody producers. Cultures of purified sIgA+ B cells, cloned PP Th A cells and SRBC, selectively yielded IgA antibody producers. Finally, PP Th A cells did not support IgA responses in B cell cultures derived from spleens of young mice (days 1-25), and full IgA responses were not seen until the donor mice were 6-7 wk of age. These results suggest that cloned T helper cells can recognize and collaborate with mature, IgA committed B cells.

The role of IgD (delta) in the induction of tolerance in murine B lymphocytes was explored in the in vivo adoptive antibody response to bovine serum albumin (BSA). Delta+ or delta- B cells purified on the fluorescence-activated cell sorter were injected, with or without purified T cells, into lethally irradiated intermediate hosts, which were rendered tolerant by an injection of deaggregated BSA one day after the cell transfer. Spleen cells from the intermediate hosts were treated with anti-Thy-1.2 antiserum and complement mixed with normal T cells and were transferred to final hosts. The ability of the B cells of the final hosts to respond to the tolerogen BSA and to dinitrophenylated human gamma globulin antigen was examined. This approach enabled examination of the function of the B cell subpopulations in an environment free of tolerogen and of induced T suppressor cells. The results revealed that in the presence of T cells the delta+ subpopulation was highly resistant to tolerance induction, whereas the delta- subpopulation was highly susceptible to tolerance induction. However, there was no difference in susceptibility to tolerance induction between the two subpopulations when tolerance was induced in the absence of T cells.