Role of macrophages in elevated IgA and IL-6 production by Peyer's patch cultures following acute oral vomitoxin exposure.

Oral vomitoxin (VT) exposure in mice results in elevated cytokine gene expression, increased production of IgA, and IgA nephropathy. To determine the potential role of macrophages (Mphi) in these effects, an ex vivo model was devised whereby Peyer's patch (PP) and spleen cells were prepared from mice 2 h after oral exposure to 0 or 25 mg/kg body wt VT, cultured, and then evaluated for IgA and cytokine IL-6 production. Both PP and, to a lesser extent, spleen cells from treatment mice produced more IgA over a 7-day period than did corresponding control cells when cultured without a costimulus or in the presence of either phorbol myristate acetate plus ionomycin (PMA + ION) or lipopolysaccharide (LPS); IgA elevation was most marked in LPS-treated cultures. The VT effect was completely ablated in PP cultures that were depleted of Mphi but not in Mphi-depleted spleen cultures. VT exposure similarly increased production of IL-6, an important helper factor for IgA secretion, in LPS-stimulated PP and spleen cell cultures. IL-6 production was also ablated by Mphi depletion. A potential costimulatory role for Mphi was further suggested because both IgA and IL-6 production increased when Mphi-depleted PP cells from VT-treated animals were cocultured with peritoneal Mphi from VT-treated animals. Similar effects were observed when an analogous ex vivo approach was used with purified PP B cells and peritoneal Mphi. PP B cells from control animals also secreted elevated levels of IgA when cocultured with splenic CD4(+) cells from VT-treated animals, thus confirming previous studies showing that T cell help also contributes to increased IgA production. Potential roles for soluble mediators and cell contact in this process were suggested when IgA production was measured in cultures of PP cells separated from VT-treated Mphi by a semipermeable membrane. Taken together, these and previous results suggest that Mphi may play a key mechanistic role in elevated IgA production and IgA nephropathy in VT-exposed mice.

Potential role for IL-5 and IL-6 in enhanced IgA secretion by Peyer's patch cells isolated from mice acutely exposed to vomitoxin.

Dietary exposure to vomitoxin (VT) results in hyperelevated serum IgA and IgA nephropathy in mice. To assess the possible role of cytokines in this IgA dysregulation, the effects of a single oral exposure in B6C3F1 male mice to 0, 5 or 25 mg/kg BW VT on production of IgA and cytokines in Peyer's patch (PP) and spleen cell cultures were evaluated. IgA levels were increased significantly in PP cell cultures prepared from mice at 2 or 24 h after oral exposure to VT and subsequently stimulated with phorbol myristate acetate (PMA) and ionomycin (ION) or with lipopolysaccharide (LPS). Significant effects on IgA production were not observed in spleen cell cultures. Since cytokines such as IL-2, IL-4, IL-5 and IL-6 have been shown to promote IgA production, the effect of the same VT exposure regimen on secretion of these mediators was determined in PP and spleen cultures. Supernatant IL-2 and IL-4 levels were unaffected by the prior treatment of animals with VT. In contrast, IL-5 levels were increased significantly in 7-day PP cell cultures obtained 2 h after VT exposure both with and without PMA + ION exposure but not in other cultures. IL-6 levels were increased significantly in LPS-treated cultures prepared from PP at 2 and 24 h following exposure to VT. IL-6 levels were also elevated significantly in both PMA + ION or LPS treated cultures from spleen isolated at 2 h but not 24 h post VT exposure. To determine whether IL-5 or IL-6 play a role in IgA hyperelevation in vitro, PP and spleen cells from mice obtained 2 h after exposure to 25 mg/kg VT were cultured in the presence of neutralizing cytokine antibodies (Abs) and IgA production was monitored. Consistent with IL-5's previously documented role in IgA production, anti-IL-5 decreased IgA levels to background in cultures of both control and VT-exposed PP or spleen cells in the presence of either PMA + ION or LPS. Similar results were seen with addition of anti-IL-6. IgA levels were decreased to a lesser extent in PP cells cultured with LPS and in spleen cells cultured with PMA + ION from VT-exposed mice to which anti-IL-2 Ab was added. Thus, the potential for enhanced IgA production exists in lymphocytes as early as 2 h and as late as 24 h after a single oral exposure to VT and this may be related to the increased capacity to secrete helper cytokines of T cell and macrophage origin. Taken together, the results suggest that the superinduction of cytokine expression may, in part, be responsible for upregulation of IgA secretion in mice exposed orally to VT.

Elevated gene expression and production of interleukins 2, 4, 5, and 6 during exposure to vomitoxin (deoxynivalenol) and cycloheximide in the EL-4 thymoma.

Aberrant elevation of serum IgA and induction of murine IgA nephropathy following dietary exposure to the naturally occurring trichothecene vomitoxin (VT or deoxynivalenol) may involve dysregulation of cytokine production at the T cell level. EL4.IL-2 (EL-4), a cloned thymoma that produces interleukins (IL)-2, 4, 5, and 6, was used as a T cell model to investigate the in vitro effects of VT on interleukin production and gene expression. When supernatants of cells stimulated with phorbol 12-myristate 13-acetate (PMA) were assessed by enzyme-linked immunosorbent assay, IL-2, 4, and 5 were increased in the presence of 50 and/or 100 ng/ml VT for 2 and/or 8 days of culture. IL-2, 5, and 6 were also significantly elevated in the presence of 10-100 ng/ml of cycloheximide (CHX), another protein synthesis inhibitor, after 8 days of culture. As demonstrated by Northern analysis, VT at the levels between 50 and 100 ng/ml superinduced IL-2, 4, 5, and 6 mRNAs in PMA-stimulated EL-4 cells during a 24 hr culture period. Similar effects in PMA-treated samples were observed for CHX at 50, 100, 250, 1000, and 10000 ng/ml. mRNA levels for both IL-4 and IL-5, but not IL-2 and IL-6, were increased in unstimulated EL-4 cultures exposed to 50 and 100 ng/ml VT for 48 hr when analyzed by reverse transcriptase-polymerase chain reaction. Using [3H]leucine incorporation as a measurement of protein synthesis, IC50s for VT and CHX were estimated to be 280 and 55 ng/ml, respectively. This study indicates that VT as well as CHX could increase production of several interleukins in the EL-4 model even when present at concentrations that partially inhibited protein synthesis, whereas IL mRNA superinduction occurred across a broader range of concentrations that included maximal protein synthesis inhibition.

B-1 cells in systemic autoimmune responses: IgM+, Fc epsilon Rdull B cells are lost during chronic graft-versus-host disease but not in murine AIDS or collagen-induced arthritis.

The potential role of B-1 cells (i.e. the CD5+ B cell and "sister" B cell subsets) in autoimmunity is controversial. CD5+ B cells have been shown to secrete antibodies of similar specificity as those found in many systemic autoimmune diseases; in addition, increases in CD5+ B cell frequency have been reported in patients suffering from rheumatoid arthritis, Sjögren's syndrome, myasthenia gravis, insulin-dependent diabetes mellitus and Hashimoto's thyroiditis. Whether these increases are due to expansion of B-1 lineage cells in the human or due to activation-induced expression of CD5 by conventional B cells is unclear. In the present study, we used three murine models of systemic autoimmunity: murine acquired immunodeficiency syndrome (MAIDS), chronic graft-versus-host disease (cGvHD), and collagen-induced arthritis (CIA) to determine whether increases in B-1 cell frequency are universally seen in models of autoimmunity which are mechanistically distinct. In contrast to the aforementioned human systemic autoimmune diseases which exhibit an increase in CD5+ B cell frequency, the percentage of CD5+ B cells declined in all three murine models of systemic autoimmune disease. Even though there was a decrease in the frequency of CD5+ B cells there was no change in the actual number of CD5+ B cells. Thus, the apparent decline in CD5+ B cell frequency was due to increases in either T cells, conventional Fc epsilon R+ B cells, or both. The only actual decline in a B cell subset was the loss of IgM+, Fc epsilon Rdull cells in both the spleen and peritoneal cavity of mice undergoing a chronic graft-versus-host reaction. Therefore, our data suggests that expansion of the B-1 subset does not occur as a general feature of murine systemic autoimmune disease. These observations, consistent with previous studies of Ig gene usage in autoreactive antibodies, support the view that expansion and differentiation of the CD5+ B cell subset is not a central event leading to autoantibody production.

Polyspecific and autoreactive IgA secreted by hybridomas derived from Peyer's patches of vomitoxin-fed mice: characterization and possible pathogenic role in IgA nephropathy.

A total of 122 immunoglobulin (Ig)A-producing hybridoma clones were isolated from the Peyer's patches of vomitoxin-fed BALB/c mice and the resultant antibodies were characterized for their antigenic specificity and pathogenic potential. When reactivity was tested against a panel consisting of DNA, sphingomyelin, thyroglobulin, collagen, casein, cardiolipin and bovine serum albumin conjugates of phosphorylcholine, inulin and trinitrophenol that were representative of self and non-self antigens, approximately 95% of the monoclonal IgAs bound to at least one of the panel antigens and 80% bound to more than one antigen. The polyspecificity of some of the monoclonal IgAs was further suggested by demonstrating the capacity of one antigen to inhibit binding of monoclonal IgA to another antigen. Protein staining and Western blotting of gradient native polyacrylamide gels, indicated that trimeric IgA predominated in the isolated monoclonal IgAs. Repeated injections of mice with representative monoclonal IgAs induced microhaematuria in three of four of the clones tested but not IgA deposition in the kidney glomerulus. In addition, three of the four monoclonal IgAs caused IgG and C3 deposition in the kidney mesangium. These and previous results suggest that dietary vomitoxin promotes the polyclonal activation and expansion of IgA-secreting B cells at the Peyer's patch level and that resultant polyspecific, autoreactive IgA may contribute to kidney pathogenesis.

IL-2 and IL-5 both induce mu S and J chain mRNA in a clonal B cell line, but differ in their cell-cycle dependency for optimal signaling.

We have found that a neoplastic Lyl+ B cell clone (BCL1-3B3) can be stimulated to secrete IgM by a Th1-derived cytokine, IL-2, and/or by a Th2-derived cytokine, IL-5. At suboptimal concentrations these interleukins acted synergistically to enhance IgM secretion. Both IL-2 and IL-5 induced increases in microseconds and J chain mRNA levels. In the presence of both ILs, increases in microseconds and J chain mRNA were additive and paralleled increases in IgM secretion. Using cells synchronized at the G1/S border with excess thymidine or in early G1 using isoleucine-deficient media, IL-2 and IL-5 differed in their cell-cycle dependency for signal transmission. IL-5 appeared to act preferentially in late G1 of the cell cycle. In contrast, IL-2 stimulated S and G2 phase cells slightly more efficiently than cells in G1 of the cell cycle. Furthermore, a twofold increase in high-affinity IL-2R was observed as the cells entered S phase. The results suggest that although IL-2 and IL-5 can independently and additively induce differentiation of the Lyl+ BCL1-3B3 cells, they differ in their point of action during the cell cycle.

Elevated membrane IgA+ and CD4+ (T helper) populations in murine Peyer's patch and splenic lymphocytes during dietary administration of the trichothecene vomitoxin (deoxynivalenol).

Recent investigations indicate that dietary exposure to the trichothecene vomitoxin increases total and antigen-specific serum immunoglobulin A (IgA) and glomerular IgA accumulation in mice. In this study, the effects of 25 ppm dietary vomitoxin on the histological and lymphocytic profile of component immune organs in the mucosal lymphocyte migratory pathway were evaluated in the B6C3F1 mouse. Vomitoxin administration resulted in marked stimulation of the size and frequency of germinal centres in Peyer's patches, mesenteric lymph nodes and the spleen. A slight increase in the percentage of B cells in the Peyer's patch was observed, although vomitoxin treatment had no effect on the percentage of B cells in the spleen. The percentage of IgA+ cells in Peyer's patches and spleen were approximately twice that of controls at 4, 8 and 12 wk of vomitoxin exposure whereas the percentage of IgG+ cells decreased in these two organs. Exposure to vomitoxin increased the percentage of T cells in Peyer's patches and the spleen. The percentage of CD4+ cells (T helper subset) increased slightly in Peyer's patches and more markedly (30-50%) in the spleen following vomitoxin treatment. Contrastingly, there was only a slight increase in the percentage of CD8+ cells (T cytotoxic/suppressor subset) in the spleens of vomitoxin-treated mice in comparison with controls, and no effect in Peyer's patches. The relative effects of vomitoxin on these two T cells populations was also reflected in increased CD4+: CD8+ ratios in Peyer's patches and spleen. These results are consistent with the hypothesis that dietary vomitoxin modulates normal regulation of the IgA response at the Peyer's patch level and that this is manifested in an altered lymphocyte distribution pattern in both the mucosal and systemic compartment. Notably increased levels of IgA+ and CD4+ cells are indicative of IgA-producing progenitors and T helper subsets, respectively, that in tandem could favour IgA hyperproduction and elevated IgA in serum.

Characterization of a neoplastic B cell clone that secretes IgM in response to Th2-derived lymphokines.

Recent advances of T cell cloning have allowed the classification of T helper cells in terms of the lymphokines they secrete. The functional significance of segregating lymphokine production to unique T cell subsets is still being evaluated, but undoubtedly plays a key role in the regulatory mechanisms of the immune system. Initial studies have indicated that the Th1 cells which secrete IL-2 and IFN-gamma may be primarily responsible for augmenting cell-mediated responses, whereas Th2 cells, which release IL-4, IL-5, and IL-6, provide help for humoral responses. However, it is also known that B cells can respond to both IL-2 and IFN-gamma. This raises the question of the homogeneity of B lymphocyte activation requirements. Are all B cells responsive to all of the lymphokines with the end-result of stimulation depending largely on the relative concentrations of the various lymphokines, or are there B cell subsets which only respond to Th1-derived lymphokines and others which respond to Th2-derived lymphokines? Such differential activation requirements might be present to allow these subsets to play unique roles in immunological responses. Since B cell cloning techniques have not yet been developed to obtain a homogenous B cell population for studies of activation requirements, regulation of lymphokine receptors, and regulation of gene expression, we must utilize lymphokine-responsive neoplastic B cells. The vast majority of spontaneous B cell lymphomas appear to belong to a minor B cell subset which expresses the Ly1 marker. This subset is clearly not representative of the majority of splenic B cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropin (ACTH) functions as a late-acting B cell growth factor and synergizes with interleukin 5.

In recent years there has been considerable discussion of possible cross-regulatory mechanisms involving the immune system and the neuroendocrine system. Certainly, evidence of hormonal communication between these two systems would provide at least a partial explanation for the many anecdotal observations of physical and mental stress affecting disease course. In previous studies of a neoplastic lymphokine-responsive B cell clone, BCL1-3B3, we noted that these cells produced a lymphokine which could affect normal B cell growth and viability. Physical characterization of this lymphokine indicated that its molecular weight was identical to that of the neuroendocrine hormone adrenocorticotropin (ACTH). Since Blalock and colleagues had reported the production of ACTH by virally-infected B cells, we have investigated whether ACTH can functionally mimic the BCL1-3B3-derived lymphokine. The neuroendocrine hormone adrenocorticotropin (ACTH) can increase in vitro murine B lymphocyte proliferation when added at physiologically relevant concentrations between 10(-9) to 10(-11) M. ACTH does not mimic the action of any lymphokine known to be required for B cell proliferation such as IL-2, IL-4, or IL-5. ACTH requires the presence of one or more of these known B cell stimulatory factors for its action and the most marked increase in B cell proliferation were noted in assays for IL-5 activity where 10(-10) M ACTH increased thymidine incorporation up to five-fold. Using two-stage assays, we determined that ACTH acts during the latter stages of B cell activation (i.e., 3-4 days after initial stimulation with either the combination of IL-4, GAMIg-Sepharose, and IL-1 or the combination of DxS and IL-5). These data indicate a direct role for a stress-induced neuroendocrine hormone in modulating the course of a humoral immune response.

Activation of murine B cells with Salmonella typhimurium mitogen (STM), lipopolysaccharide (LPS), and dextran sulfate (DxS). I. Cell-cycle analysis and induction of cytoplasmic immunoglobulin.

There have been two recent reports concerning a B cell-specific mitogen that induces proliferation, but not differentiation of rat and human B cells. This mitogen, which is derived from Salmonella typhimurium (STM), appears to be providing the signals required for anti-immunoglobulin-treated (anti-Ig) B cells to enter cycle and divide, but may not be inducing responsiveness to B cell differentiation factors (BCDF). In this report, we have compared STM to the other known murine B cell polyclonal activators: lipopolysaccharide (LPS), dextran sulfate (DxS), and the combination of LPS/DxS. STM was the most potent stimulus of B cell proliferation as determined by uptake of 3H-thymidine, viable cell numbers and cell cycle analysis utilizing acridine orange (AO). STM did not induce significant proliferation of murine T lymphocytes. In addition, the proliferative effect of STM on B cells shows minimal, if any, macrophage dependence. However, in contrast to its effect on human and rat B cells, STM induces differentiation of murine B cells. The levels of cytoplasmic Ig induced by STM are equivalent or greater to those induced by LPS/DxS. Thus, in the murine system, STM will be useful as a polyclonal activator which induces proliferation and differentiation of the vast majority of the B cell population without stringent accessory cell requirements.

Activation of murine B cells from different tissues with different mitogens. II. Isotype distribution of secreted immunoglobulins in the presence and absence of IL-4-containing T cell supernatants.

In the accompanying report, we have compared a B cell-specific protein mitogen, Salmonella thyphimurium mitogen (STM), to lipopolysaccharide (LPS), dextran sulfate (DxS), and the combination of LPS/DxS with regard to their ability to induce B cell proliferation and differentiation. The results of these studies demonstrated that STM, LPS, and LPS/DxS induce significant expression of cytoplasmic immunoglobulin (Ig). In this report, we have analyzed the distribution of isotypes induced by each of these mitogens in the presence or absence of interleukin-4 (IL-4) containing T cell supernatants (SN), since IL-4 increases the secretion of IgG1 and IgE and decreases the secretion of IgG3 and IgG2b in LPS-stimulated splenic B cells. These experiments were designed to determine whether LPS was unique in its ability to "program" B cells to respond to IL-4 by secreting IgG1, as has been suggested in our previous studies. The current experiments also addressed the issue of whether splenic B cells were unique in their response to IL-4 by using B cells from other tissues. The efficiency of induction of cytoplasmic Ig measured in the preceding report was STM greater than LPS/DxS greater than LPS. DxS did not induce a significant level of cell proliferation, cytoplasmic Ig, or secreted IgM and inhibited the LPS-induced IgM response by approximately 50%. In contrast, STM induced an extremely high level of IgM secretion in splenic B cells. In this report, we demonstrate that the addition of IL-4-containing T cell SN to splenic B cells stimulated with LPS, LPS/DxS or STM increased the level of IgG1 and reduced the level of IgM, IgG3, and IgG2b secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant IL 2 but not recombinant interferon-gamma stimulates both proliferation and IgM secretion in a Ly-1+ clone of neoplastic murine B cells (BCL1).

We have used a lymphokine-responsive clone (3B3) of B leukemia cells (BCL1) to examine the effects of several recombinant and purified lymphokines. Cells from BCL1-3B3 were induced to secrete IgM in the presence of recombinant interleukin 2 (rIL 2) (10 to 50 U/ml); a concomitant increase in proliferation was observed. Recombinant interferon-gamma (rIFN-gamma) was a potent inhibitor of proliferation. In addition, rIFN-gamma did not induce an increase in IgM secretion and, when added to rIL 2-stimulated BCL1-3B3 cells, completely blocked IgM secretion at a concentration of 10 U/ml. Purified and recombinant IL 1 (rIL 1) had no significant effect on differentiation either alone or in combination with rIL 2 and/or rIFN-gamma. However, rIL 1 was able to synergize with rIL 2 in enhancing the proliferation of BCL1-3B3. The ability of cells to respond to rIL 2 was limited to the in vitro (Ly-1+) clones of BCL1 cells since the in vivo derived (Ly-1-) BCL1 cells did not differentiate in response to IL 2. Consistent with their functional response to rIL 2, cells from the in vitro clone (3B3) are IL 2-receptor-positive (IL-2R+) and the in vivo derived BCL1 cells are IL-2R-. A second set of neoplastic B cell clones derived from the AKR 225 lymphoma did not respond to rIL 2 even though they expressed receptors for IL 2 and could be induced by T cell supernatant to secrete IgM, thus indicating that expression of IL 2R is not the sole requirement for IL 2 responsiveness. The monoclonal anti-IL 2R antibody (7D4) mimicked IL 2 in its ability to stimulate differentiation of BCL1-3B3 cells. These data suggest that rIL 2 and the monoclonal anti-IL-2R antibody are capable of inducing a differentiative response in the Ly-1+ BCL1-3B3 cells that is functionally equivalent to the response evoked by the previously described lymphokine B cell differentiation factor for IgM (BCDF mu). Thus, two distinct lymphokines appear to be providing a similar signal to a clonal neoplastic B cell population. Furthermore, rIL 2 is capable of providing both a proliferative and a differentiative signal.

Cell cycle-related expression of the receptor for a B cell differentiation factor.

Cloned, neoplastic B cells (BCL1) have been used to evaluate the expression of the receptor for the B cell differentiation factor, BCDF mu. These cells do not secrete IgM before stimulation with BCDF mu-containing T cell supernatants (SN). By inducing cell cycle synchrony in this homogeneous population, the expression of the BCDF mu receptor could be evaluated as a function of the cell cycle. Responsiveness to BCDF mu-containing SN is maximal when the cells are in S and G2 phases of the cell cycle, and a 2-hr exposure of cells to BCDF mu-containing SN during S/G2 results in optimal IgM secretion 5 days later. Cells in S/G2 are also maximally effective in absorbing BCDF mu activity from SN. These data support the hypothesis that B cells do not respond to differentiative signals until after they are committed to at least one round of cell division.

A B cell growth factor-like activity is secreted by cloned, neoplastic B cells.

Supernatants from synchronized clones of neoplastic B cells (BCL1) were found to contain a B cell growth factor (BCGFI)-like activity. The BCGF activity in the BCL1 supernatants (SN) could synergize with EL-4 SN in the late phases of an IL 1-dependent BCGF I assay (days 5 through 8). These SN did not contain any detectable BCGF II, IL, 1, or IL 2 activity. In contrast to EL-4-derived BCGF I, which has a m.w. of approximately 18,000, the BCL1-BCGF activity has a m.w. of approximately 4500. Because the BCGF activity in BCL1 SN fluctuates with cell cycle in synchronized cultures, this BCL1-BCGF may play an auto-stimulatory role in B cell proliferation.

The correlation between the activation state of B cells and their capacity for in vitro propagation of immunologic memory.

The B-cell population responsible for in vitro antigen-mediated proliferation and expansion of the memory B-cell population is a large activated blast. Such cells predominate early after antigen priming and can be regenerated by adjuvant (Bordetella pertussis) stimulation in vivo. Although these cells are proliferating in vivo, additional stimuli are needed for expansion of the memory population in vitro. These triggering requirements include specific antigen (DNP-OVA) and the assistance of adherent accessory cells. Although T cells are present in the culture, their role in the propagation of memory is not completely clear. Using the unrelated antigen, sheep erythrocytes, we have shown that "bystander" T-cell help can mediate differentiation of these memory B-cell blasts to AFC, but it cannot induce expansion of the memory-cell population. However, the fact that the TI-2 antigen DNP-Ficoll is a relatively ineffective inducer of memory-cell propagation (inducing an expanded response which is less than 10% of that induced by the T-cell-dependent antigen, DNP-OVA) suggests that T cells may be involved, possibly via production of B-cell growth factor. Thus, the minimal requirements for triggering the propagation of B-cell memory include (i) a blastogenic signal which can be mediated by adjuvant, (ii) specific antigen, and (iii) adherent accessory-cell help.

In vitro antigen-mediated clonal expansion of memory B lymphocytes.

An in vitro model for the propagation and expansion of the memory B lymphocyte population is described. DNP-BGG immune cells were mixed with OVA immune cells and challenged immediately with DNP-OVA. After the 1st response had begun to wane, the cells were rechallenged with DNP-OVA (day 11 of culture). An average of 13-fold more PFC were observed after delayed challenge (day 11). This expansion in the PFC response was an antigen-dependent process and did not involve recruitment of new memory cells from the virgin lymphocyte pool. The level of expansion of the memory cell pool was also calculated using limiting dilution analysis and was found to fall in a range of 16- to 67-fold increase in precursor frequency. In addition to the expansion of the memory B cell population, we also observed the development of 2 immunoregulatory cycles previously observed only in vivo. First, in the presence of persistent antigen, a cyclical PFC response was seen. Second, after day 10 of culture, optimal PFC numbers were observed only when DNP-lysine was added to the plaque assay. Such hapten-augmentable PFC responses have been reported by other investigators as indicative of anti-idiotypic regulation. This possibility is examined more extensively in the following communication.