Human T-helper clones induce IgG production in a subclass-specific fashion.

The mechanisms governing the induction of IgG subclasses by T-helper cells in humans were investigated. As preliminary bulk-culture experiments had indicated that a direct B cell contact with viable T cells was an essential requirement for optimal IgG subclass production, 256 CD4+ human T cell clones were preactivated with PHA and cultured in direct contact with autologous B cells. These clones induced IgG production in a strikingly subclass-specific fashion. Moreover, the distribution of subclass-specific helper clones was very similar to the IgG subclass profile observed in serum and peripheral lymphoid tissue plasma cells (IgG1 approximately 60%, IgG2 approximately 30%, IgG3 approximately 5-10%, IgG4 less than or equal to 5%) and unlike that observed in resting B cells (which is IgG1 approximately 40% and IgG2 approximately 50%). It would, therefore, seem that a predominance of T cells capable of delivering IgG1-specific, as opposed to IgG2-specific, help is an essential factor for the preferential induction of IgG1 antibodies during B cell proliferation and differentiation. There was no relationship between IL2, IL4, IL6, and IFN-gamma secreted by the T-helper clones and their IgG subclass induction patterns. In addition, only a few supernatants were able to reproduce the helper effects of the clones themselves. Therefore, direct contact of B cells with helper clones is crucial for IgG-subclass production in humans.     

Analysis of human IgG and IgA subclass antibody-secreting cells from localized chronic inflammatory tissue

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.     

The IgE and IgG subclass responses of mice to four helminth parasites

To investigate whether the formation of IgE is linked in vivo to an IgG subclass, mice were infected with four helminth parasites, Nippostrongylus brasiliensis (Nbr), Mesocestoides corti, Taenia crassiceps and Trichinella spiralis, and the changes in the serum levels of the different Ig isotypes as well as the antibody response to M. corti and T. crassiceps antigen extracts were determined by radioimmunoassays. All four parasites induced a concomitant increase of the IgE and IgG1 serum levels and usually a decrease of the IgG2a level. They also induced an increase of the IgM level but had little effect on the IgG2b, IgG3, and IgA serum levels. The specific antibodies to an M. corti antigen extract were mainly of the IgG1 subclass, whereas it was of both IgG1 and IgG2a subclasses to T. crassiceps. Injections of dead M. corti induced an increase of all IgG subclasses and similar levels of IgG1 and IgG2a anti-parasite antibodies. Subcutaneous instead of intraperitoneal infection with T. crassiceps induced higher IgG2a than IgG1 levels and 10-fold lower IgE levels than the natural ip infection; however, despite the greater IgG2a polyclonal response, anti-parasite antibodies were predominantly of the IgG1 subclass. The data demonstrate that natural infection with four different helminth parasites induces a concomitant polyclonal IgG1 and IgE response. These in vivo observations corroborate the recent in vitro findings demonstrating that interleukin-4 induces lipopolysaccharide-activated murine B cells to secrete both IgG1 and IgE, suggesting that the regulation of these two isotypes is linked.     

Mitogen-induced human IgG subclass expression

To investigate human isotype expression among lymphocyte populations, we have studied IgG subclass production by splenocytes, tonsil cells, and peripheral blood lymphocytes (PBL) after stimulation with a panel of nine mitogens. Response magnitudes varied with tissues: all mitogens produced the strongest responses with splenocytes and the smallest with PBL. In addition, the IgG subclass maximally stimulated by a particular mitogen also depended on the tissue studied. For example, LPS mainly stimulated IgG2 in PBL and IgG1 in spleen. Interestingly, the response patterns seen in splenocytes suggest a large and coordinate expression of IgG1 and IgG3 subclasses. Implications of these findings are discussed with respect to immunoglobulin gene organization and human disease states.     

In vitro regulation of IgA subclass synthesis. II. The source of IgA2 plasma cells

In vitro regulation of IgA subclass synthesis was investigated in pokeweed mitogen (PWM)-stimulated cultures of peripheral blood lymphocytes. In past experiments we have demonstrated that 50% of the IgA plasma cells derived from PWM-stimulated cultures are positive for IgA1 and 50% are positive for IgA2. This observation is surprising because approximately 80% of the IgA B cells in the peripheral circulation bear IgA1 and 20% bear IgA2. To determine if the shift toward IgA2 predominance in PWM-stimulated cultures might be due to an enriched source for IgA2 plasma cells from a precursor pool of immature B cells, we used panning techniques to separate immature precursors that express surface IgM (sIgM+) from mature precursors that no longer express IgM (sIgM-). These separated B cells were cultured with equal numbers of T cells and PWM for 7 days. In all 10 experiments there was an enrichment for IgA2 in the sIgM+ cultures; 55 +/- 9.6% of the total IgA plasma cells were positive for IgA2 in the sIgM+ cultures vs 38 +/- 6.3% in the sIgM- cultures (p less than 0.001). These results indicate that both sIgM+ and sIgM- cells can give rise to IgA plasma cells in PWM-stimulated cultures and that there is an enrichment for IgA2 precursors in the sIgM+ population. Other possible regulatory mechanisms were also investigated. To determine if there was isotype switching from IgA1 to IgA2, monoclonal anti-IgA1 antibodies were added to PWM cultures. These antibodies resulted in a mean suppression of IgA1 plasma cell production of 82% with a concomitant 45% suppression of total IgA but only 4.6% suppression of IgA2. These results make it unlikely that IgA2 plasma cells in PWM-stimulated cultures are derived from cells that initially produced IgA1. To investigate the possibility that one IgA subclass might be more T cell dependent than the other, T and B cells were separated and B cells were reconstituted with T cells in ratios that varied from 1:10 to 10:1 or with irradiated T cells. These procedures did not alter the proportion of IgA plasma cells positive for IgA1 or IgA2, indicating that the two subclasses do not differ in their response to T cell signals in PWM-stimulated cultures.     

The subclass distribution of human IgG rheumatoid factor

The subclass distribution of IgG rheumatoid factor (RF) was determined by a sensitive ELISA assay in sera from patients with rheumatoid arthritis and from normal controls. In both instances, the most important subclasses were IgG1 and IgG4. The IgG4 RF was directed against the Fc region of IgG, and recognized human as well as rabbit IgG. Although human IgG4 myeloma proteins bound to rabbit IgG better than did myelomas of other IgG subclasses, the IgG4 RF activity in rheumatoid sera showed an additional specificity, because the fraction of IgG4 RF/total IgG4 for rheumatoid arthritis sera was far greater than for myelomas. This inference was supported by the observation that there was persistent, albeit diminished, IgG RF activity in pepsin-digested, RF-containing sera (but not myeloma proteins), indicating that a critical component of IgG4 RF activity was contained within the Fab region of the IgG4 molecule. The finding of large quantities of IgG4 RF was not due to a bias of the assay, because the preponderance of IgG4 did not extend to the subclass distribution of antibodies directed against other antigens. The demonstration of an important role for IgG4 as a RF is of special interest because of the relative inability of this subclass to fix complement or to bind to Fc receptors, and because of its potential role as a mediator of increased vascular permeability.     

Differential effect of vasoactive intestinal peptide, somatostatin, and substance P on human IgE and IgG subclass production.

We studied the effect of vasoactive intestinal peptide (VIP), somatostatin (SOM), and substance P (SP) on IL-4-stimulated human IgE and IgG subclass production. VIP and SOM, but not SP, inhibited IgE production without affecting IgM or IgA production by mononuclear cells (MNC) from nonatopic donors from 10 pM to 10 nM. These neuropeptides also differentially modulated IgG subclass production. While IgG1 production was not affected by VIP, SOM, or SP, all of the neuropeptides enhanced IgG2 production. By contrast, SOM and SP, but not VIP, inhibited IgG3 production, whereas VIP and SP, but not SOM, enhanced IgG4 production. The effect by neuropeptides was specific since each peptide effect was specifically blocked by each antagonist. To achieve this effect, neuropeptides must be added at the start of the culture and be present throughout the entire culture period. The inhibition of IgE production was not mediated by known inhibitors of IgE production, IFN-gamma or PGE2, because the addition of anti-IFN-gamma mAb (10 micrograms/ml) or indomethacin (0.1 microM) did not overcome the inhibition of IgE production. In contrast to MNC, neuropeptides did not affect IgG subclass production in purified B cells. IgE production was not induced by IL-4 in purified B cells. Neuropeptides also failed to modulate IgG subclass production in cultures of B cells with either T cells or monocytes. However, they modulated IgE production and IgG subclass production in B cells in the presence of T cells and monocytes. In purified B cells, IL-4 plus anti-CD40 mAb induced IgE production which was not inhibited by VIP or SOM. However, VIP or SOM, but not SP, inhibited IgE production in B cells cultured with both T cells and monocytes. Finally, the mechanism of modulation of IgE and IgG4 production was dependent on IL-4-induced switching, since neuropeptides modulated IgG4 and IgE production in surface IgG4-negative (sIgG4-) and sIgE- B cells, respectively. In contrast, modulation of IgG2 and IgG3 production was not due to switching, since neuropeptides did not affect either IgG2 or IgG3 production in sIgG2- or sIgG3- B cells, respectively.     

Differential regulation by Ly-5 and Lyb-2 of IgG production induced by lipopolysaccharide and B cell stimulatory factor-1 (IL-4)

We have previously shown that mAb Ly-5 which on B cells recognizes a 220,000-Da (B220) molecule, inhibits LPS-induced IgG responses without affecting IgM or proliferative responses, whereas mAb Lyb-2 which modulates B cell activation processes induced by B cell stimulatory factor-1 (BSF-1) or IL-4, has no effect on LPS-induced B cell responses. In this report we further examined the cellular mechanisms of Ly-5 antibody action and the effect of Lyb-2 antibody in IgG responses induced by LPS and BSF-1. The results presented demonstrated that the inhibitory effect of Ly-5 antibody seems to be restricted to the IgG class and is observed in all IgG subclasses induced by LPS. Limiting dilution analysis showed that the Ly-5 antibody reduces primarily the precursor frequency of IgG-secreting cells and that the effect on the clone size is partial. Lyb-2 antibody, on the other hand, greatly inhibited IgG1 induction initiated by LPS and BSF-1 by the action on processes triggered by BSF-1, although it could not reverse the reduced IgG2b or IgG3 responses. Limiting dilution analysis revealed that Lyb-2 antibody reduces the precursor frequency but not the clone size of BSF-1-induced IgG1-producing cells, supporting our previous proposition that Lyb-2 plays a critical role in the B cell differentiation mediated by BSF-1. Taken together, these results indicate that both Ly-5 and Lyb-2 are important molecules in IgG subclass regulation, each acting on a distinct activation step.     

Strain-dependent IgG subclass response patterns

Previous studies have shown that antigens preferentially stimulate IgG subclasses. However, the immunologic processes responsible for the patterns of IgG subclasses stimulated by antigens are probably complex and are certainly unclear. To define some of the genetic controls of IgG subclass expression in mice, we have studied the patterns of IgG subclasses elicited by antigens in BALB/cAn, C57BL/6N, derived recombinant inbred strains, and derived Ig congenic strains. This study shows that both thymus-independent antigens and thymus-dependent antigens stimulate different patterns of IgG subclasses in BALB/cAn and C57BL/6N. Furthermore, analysis using recombinant inbred strains and Ig congenic strains shows that the patterns of IgG subclasses stimulated by all antigens are linked to Ig allotype. In contrast, only the IgG subclass patterns stimulated by thymus-dependent antigens are linked to major histocompatibility complex haplotype. This study also shows that the Ig allotype-linked controls of IgG subclass response patterns are located telomeric to a BAB14 intra-heavy chain variable region recombinant site. Therefore, this region of mouse chromosome 12 may contribute to the control of IgG subclass selection in the B cell.     

Differentiation of B lineage cells from liver of neonatal mice: generation of immunoglobulin isotype diversity in vitro

The development and differentiation of B cells expressing different immunoglobulin (Ig) isotypes was studied in cultures of murine neonatal liver cells. Before culture, 5 to 15% of the liver cells were mu + pre-B cells; 1 to 3% had surface IgM and less than 0.1% had slgG. During 4 days in culture the number of pre-B cells declined, whereas the number of IgM B cells increased greater than 20-fold; IgG B cells also increased in number. Of the four subclasses, IgG3+ and IgG2b+ cells predominated, each representing 3 to 10% of the total B cells at day 4. IgG1+ and IgG2a+ cells were present in lower numbers, representing 1 to 5% and 0.3 to 2.5% of B cells, respectively. Most IgG+ cells also expressed sIgM. Only a minority (less than 10%) of the sIgM+ cells were sIgD+, and most sIgG+ cells were sIgD-. Few T cells were present in these cultures (less than 0.5% in newborn liver), and sIgG+ cells were generated in normal frequencies in cultures of cells from nude mice. The numbers of B cells expressing each IgG subclass were similar in cultures from athymic nu/nu mice, nu/+ heterozygous littermates, and normal BALB/c mice. Plasmablasts and plasma cells appeared over a 14-day culture interval, and these expressed cytoplasmic IgM, IgG3, IgG1, IgG2b, IgG2a, and IgA. Measurable amounts of the first four isotypes were detected in the culture supernatants by radioimmunoassay. These results indicate that neonatal B cells can undergo isotype switching in the absence of T cell help, and that the expression of sIgD may not be a prerequisite for cells to switch Ig isotypes.     

Cytokine regulation of localized inflammation. Induction of activated B cells and IL-6-mediated polyclonal IgG and IgA synthesis in inflamed human gingiva

It is well established that increased numbers of plasma cells occur in the localized tissues of chronic inflammatory diseases such as adult periodontitis, and enzymatic isolation has shown that most B lineage cells produce IgG-subclass with some IgA-subclass responses. It would be of importance to determine if excess production of cytokines in the localized lesion account for these responses and in the present study we have assessed gingival mononuclear cell (GMC) supernatants for cytokines that activate B cells including IL-6R expression and for levels of IL-6 present. Inasmuch as limited numbers (approximately 1 to 3 x 10(6) cells) of GMC were obtained from surgically removed tissues (approximately 400 mg), we have focused on the analysis of IL-6 production by GMC in this study. Further, initial evidence of additional cytokines that are produced by GMC and induce expression of IL-6R on resting B cells has been obtained. The GMC and PBMC from individual patients were cultured in the presence (or absence) of Con A. Higher levels of IL-6 were produced spontaneously by GMC when compared with Con A-stimulated PBMC. When PBMC cultures were supplemented with GMC supernatants obtained from the same patient, high numbers of spot-forming cells (SFC), mainly of IgG followed by IgA isotype, were seen. The induction of SFC by GMC supernatants was inhibited by incubation with a goat anti-human IL-6 antibody. When the effect of GMC supernatants on subclasses of PBMC SFC was determined, the response was IgG1 greater than IgG2 greater than IgG3 = IgG4 and IgA1 greater than IgA2, a pattern remarkably similar to the distribution of plasma cells in the GMC itself. To assess for cytokines in GMC supernatants that mediated B cell activation, supernatants containing anti-IL-6 were cultured with PBMC or purified B cells for 72 h. This treatment induced small proliferative B cell responses and elevated expression of IL-6R on B cells, but did not induce SFC responses. Further, incubation of B cells with GMC supernatants induced resting B cells (G0/G1) to enter the cell cycle (S and G2/M). Addition of human rIL-6 to these cultures on day 3 restored IgG- and IgA-subclass SFC responses by day 7. Cytokine-induced IL-6R expression also occurred in vivo because freshly isolated GMC expressed high levels of this receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative evaluation of serum IgG subclass levels by an immunoenzyme method

We developed a enzyme linked immunosorbent assay (ELISA) for measuring IgG subclasses concentration in serum. For this we used monoclonal antibodies. The specificity of these antibodies was evaluated with a panel of myeloma proteins belonging to the 4 IgG subclasses. The ELISA was sensitive (allowing the detection of subclasses at ng level) and accurate (inter-assay coefficient of variation of 14%). Using the WHO serum 67/97 as reference, we determined the concentration of IgG subclasses in a pool of sera. In addition concentrations were measured in 69 healthy adults to study the distribution of each IgG subclass. A good correlation (r = 0.78) was obtained between the sum of the subclasses measured by ELISA and total IgG measured by immunonephelometry.     

J-chain expression in human cells producing IgG subclasses

Previous studies have demonstrated that J chain is expressed not only in cells that produce polymeric immunoglobulins, but also in those engaged in synthesis of monomers including IgG and IgD. The presence of J chain in these cells suggested that its role may not be restricted to the formation of polymers. For the present study, fluorochrome-labeled polyclonal anti-J-chain and monoclonal antibodies to IgG subclasses were used to determine the distribution of J chain in IgG plasma cells from normal human tissues and from pokeweed mitogen (PWM)-stimulated human peripheral blood lymphocytes. The results indicate that J chain is not equally distributed among cells producing different IgG subclasses. The percentages of PWM-stimulated cells containing J chain were: 22 +/- 5 (SE) for IgG1, 49 +/- 6 for IgG2, 17 +/- 7 for IgG3, and 64 +/- 11 for IgG4. Examination of sections of various human lymphoid tissues revealed that the frequency of IgG cells that coexpressed J chain was lower than that observed in the PWM system and displayed variable distribution among IgG subclasses. The frequency of J-chain expression in IgG-producing cells may be related to the degree of cellular maturation and may differ according to the origin of cells.     

Serum immunoglobulin IgG subclass distribution of antibody responses to Yop proteins and lipopolysaccharide of Yersinia enterocolitica in patients with yersiniosis.

To assess the humoral immunological responses at the IgG subclass level in yersiniosis specific antibody responses against lipopolysaccharide of Yersinia enterocolitica 03 (LPS) and Yersinia Yop proteins were analyzed by ELISA. Thirty five patients with arthritis and forty nine patients with uncomplicated yersiniosis were included in the study. Analysis of the IgG subclass responses to the LPS revealed that the subclass distribution for both groups of patients was IgG2>IgG1>IgG3. The concentration of IgG4 was below detection level. The predominant antibody responses to Yop proteins were IgG1>IgG3>IgG2>IgG4 but the frequency of detection of particular IgG subclass antibodies were dependent on the age of patients. Generally, the frequency of occurrence of IgG2 antibodies for Yop proteins of Yersinia together increased with age reaching its peak among individuals aged above 40 years. On the other hand, IgG1 for Yop proteins and IgG3 for Y. enterocolitica LPS were diagnosed more often in serum samples obtained from children than from adults. We also found significantly higher frequency of IgG4 to Yop proteins of Y. enterocolitica in men than in women.     

Regulation of IgG1 and IgG2 subclass expression by adjuvant-activated splenic suppressor T cells

Spleen cells from mice immunized with different adjuvants are able to suppress secondary in vitro IgG plaque-forming cell (PFC) responses. The suppressive effect is mediated by Lyt-2-positive T cells. IgG subclasses are affected differentially depending on the number of T suppressor (Ts) cells added in the assays. At low Ts cell concentration IgG2a and IgG2b PFC responses are selectively inhibited. At higher Ts cell concentration IgG1 responses could also be completely inhibited, but IgA and IgM responses are not affected. Suppressor cells responsible for IgG1, IgG2a, and IgG2b suppression are never found in the draining lymph nodes of adjuvant-immunized animals.     

Effects of cocaine and morphine on IgG production by human peripheral blood lymphocytes in vitro

Elevated serum levels of IgG are amongst the immunological abnormalities exhibited by intravenous drug addicts. We therefore addressed the hypothesis that cocaine and morphine (the major metabolite of heroin) exert a direct effect on human B cell function in vitro. Human peripheral blood mononuclear cells from normal individuals were incubated for 7 days with the T cell-dependent B cell activator pokeweed mitogen (PWM) and serial dilutions of either cocaine or morphine. At the end of this time total IgG was measured by use of a sandwich ELISA incorporating a biotin-labelled affinity-purified anti-IgG and streptavidin peroxidase. At concentrations relevant to those found in plasma, morphine and cocaine did not affect PWM-stimulated IgG synthesis in vitro. We suggest that these drugs of abuse do not directly influence human B cells, but in vivo exert immune modulatory effects via indirect mechanisms.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

Phospholipid synthesis by activated human B lymphocytes

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