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Oral tolerance is defined as an inhibition of specific immune responsiveness to a previously ingested antigen. Paradoxically, we found an increased lymphocyte activity in tolerant mice alongside the specific inhibition. Orally-tolerant mice presented higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow; showed a greater variety of Ig classes being produced: IgM and IgA in the spleen and IgG and IgM in the bone marrow. ISC from immunized mice produced mainly IgG. Despite having the same number of regulatory and activated T cells in the spleen after immunization, these cells appeared earlier in tolerant mice, right after the primary immunization. Also, tolerant mice showed a prompt expression of regulatory cytokines (TGF-β and IL-10) and a transient expression of effector cytokines (IL-2 and IFN-γ). Thus, in addition to an inhibited specific responsiveness, orally-tolerant mice displayed an early and widespread mobilization of activated and regulatory lymphocytes.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.

B cell response to Trichinella spiralis (Ts) adult antigen (Ag) was studied in rats 1–20 days postinfection. B cell recoveries from the mesenteric lymph node (MLN), Peyer's patches (PP), thoracic duct lymph (TDL), and the spleen were determined by FACS analysis and Ag-specific antibody-producing cells (Ab-pc) in these tissues were enumerated using the immunoplaque assay. Total B cell numbers increased 2–70 times from day 3 postinfection in the MLN and TDL obtained from MLN-resected rats (MX) and such proliferation was not found in the PP or the spleen. Ab-pc of all isotypes increased from day 3 in the MLN and from day 2 in the MX-TDL. Among all isotypes, IgE- and IgG1-pc showed the strongest response. Immunofluorescence study revealed that these B cells were activated in the non-PP region of the small intestine. These results indicate an early isotype switch to IgG1 and IgE production in Ts-infected small intestine.

Anti-dinitrophenyl IgG1 and IgE antibody production were examined in vitro. Splenic B cells from mice immunized in vivo at various intervals with dinitrophenylated proteins were cultured in vitro with splenic T cells and splenic antigen-presenting cells from mice immunized previously (2 weeks) with keyhole limpet hemocyanin. Anti-dinitrophenyl IgG1 and IgE antibodies were determined in the supernatants by ELISA. Without T cells and antigen-presenting cells or without antigen, no anti-dinitrophenyl antibodies were detected. When the B cells were separated by panning with anti-murine IgG1 antibody, only the cells adhering to anti-IgG1 produced anti-dinitrophenyl IgG1 antibodies. Anti-dinitrophenyl IgE production was observed from cells adhering and from cells not adhering to the anti-IgG1-coated plates, as expected. However, anti-dinitrophenyl IgE was also produced from cells which adhered to the anti-IgG1-coated plates, but did not have surface IgE antibodies. Therefore, cells without surface IgE adhering to anti-IgG1 were capable of producing anti-dinitrophenyl IgE antibodies.

We compared the phenotypic characterization of antibody-secreting cells in the liver, the spleen, and Peyer's patch that had been subjected to various treatments in mice. The highest proportion of IgA-secreting cells was found in the liver mononuclear cells (MNC). They contained IgM-secreting cells in similar proportions to those in Peyer's patch but at lower proportions than in the spleen. In immunized mice, levels of IgG-secreting cells in the spleen and the liver were about 10 times higher than those in normal mice. The distribution pattern of immunoglobulin-secreting cells in Peyer's patch was virtually unchanged by any treatment. In the normal mice there were no significant differences among these organs in the distribution of the surface immunoglobulin phenotype in MNC. IgA-secreting cells in Peyer's patch secreted more antibodies per cell than those in the spleen or the liver.

In situ immunocytochemical-staining methods combined with computer-aided image analysis were employed to examine autoanti-idiotype antibody-forming cell expansion in vivo . Autoanti-idiotype antibody-forming cells were demonstrated in the spleens of C57BL/6J (B6) strain mice intranasally infected with the influenza virus A/Hong Kong/168/(H3N2)[R]X-31. Autoantiidiotype B cells were detected and elevated in spleen tissues after secondary influenza infections compared to normal B6 mice, and were specific for a dominant idiotype antibody called PY206 reactive with the influenza virus hemagglutinin. Influenza virus-infected BALB/c mice, which make little PY206 Id, did not have increased autoanti-Id against PY206 compared to normal mice. Results provide evidence for an idiotype network-mediated stimulation of autoanti-idiotype B cell production during influenza virus infection.

Mice infected with the protozoan parasite, Trypanosoma cruzi, are known to be immunosuppressed in responsiveness to heterologous antigens and parasite-specific antigens. This suppression is mediated by suppressor macrophages and is exemplified by deficient T cell activity and abnormal cytokine production. Neither the mechanism by which suppressor macrophages effect suppression nor the characteristics of these suppressor macrophages is known. In the present study, we analyzed the regulatory cell populations in splenocytes of infected mice (SCinf) and their interactions by limiting dilution-partition analysis, an approach which allows the functional separation of multiple regulatory cell subpopulations within cell mixtures. Our results demonstrate the presence of a complex immunoregulatory circuit in SCinf affecting the generation of anti-sheep erythrocyte antibody responses in vitro. Titration of SCinf (but not peritoneal exudate or lymph node cells) into Mishell-Dutton microcultures of normal spleen cells generated complex dose-response curves with two zones of suppressed responses following the addition of either low or high doses of SCinf to the cultures. Addition of intermediate doses of SCinf to the microcultures restored responsiveness. Both the low- and high-dose zones of suppression were shown to be mediated by macrophages, whereas T cells were responsible for the restored responsiveness at intermediate doses of SCinf. Examination of the development of this complex regulatory pattern during the course of the acute phase of infection indicated the sequential development of one suppressor macrophage population, followed by the development of the beneficial T cell population, and finally the expression of the second suppressive macrophage population.

We have found that a neoplastic Lyl + B cell clone (BCL 1 -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 β S and J chain mRNA levels. In the presence of both IL, increases in μ S and J china mRNA were additive and paralleled increases in IgM secretion. Using cells synchronized at the G 1 /S border with excess thymidine or in early G 1 using isoleicine-deficient media, IL-2 and IL-5 differed in their cell-cycle dependency for signal transmission. IL-5 appeared to act preferentially in late G 1 of the cell cycle. In contrast, IL-2 stimulated S and G 2 phase cells slightly more efficiently than cells in G 1 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 + BCL 1 -3B3 cells, they differ in their point of action during the cell cycle.

Primary anti-trinitrophenyl antibody production was investigated from spleen cells of mice immunized with trinitrophenylated-keyhole limpet hemocyanin, using the plaque-forming cell method and ELISA. Cells taken 5 days after antigen injection do not produce IgE, but do produce IgM and IgG1 anti-trinitrophenyl antibodies as demonstrated by plaque-forming cells. Substantial increase of IgM, IgG1, and IgE antibody production was seen from cells taken 7 days after immunization, followed by a rapid decline. By ELISA it was seen that cells taken 3 days after immunization already produce small amounts of anti-trinitrophenyl antibodies. Presence of antigen from the start of the cultures did not increase antibody production from cells taken 3 days after immunization, but potentiated antibody secretions from cells taken 5 days or later after immunization. This potentiation was interpreted as recruitment of antibody-forming cells from early memory B cells. The presence of IL-4 from the start of the cultures had no appreciable effect. Cell sorting with specific antibody-coated magnetic beads showed that plaque-forming cells from nonsorted cells, membrane IgE + or membrane IgE − cells secreted similar amounts of anti-trinitrophenyl IgG1 and IgE antibodies. No difference in anti-trinitrophenyl IgM, IgG1, or IgE production was found in controls; cells sorted negatively or positively for CD23. The data show that memory B cells can be demonstrated already on Day 5 after immunization, and their antigen-induced antibody secretion is IL-4 dependent.

The secondary immune response classically differs from the primary response in magnitude, avidity, and isotype of the antibodies produced. Cell transfer studies to assess the contribution of memory B and memory T cells to each of these parameters are described. Avidities of the anti-DNP plaque-forming cells (PFC) generated in lethally irradiated recipients of naive B cells and keyhole limpet hemocyanine (KLH)-primed T cells, followed by immunization with soluble DNPKLH, are medium to high, and do not differ significantly from the avidities of anti-DNP PFC in recipients of DNP-primed B cells and KLH-primed T cells. However, the number of indirect (I)-PFC and the ratio of I-PFC to direct (D)-PFC are significantly greater in the recipients of primed B and primed T cells. The results suggest that carrier primed T cells can selectively activate virgin B cells which are committed to produce medium- and high-avidity antibodies, and/or enhance the generation of somatic mutation which leads to antibodies of higher avidity. Priming of B cells is necessary for the increased magnitude of the I-PFC.

In vivo polyclonal activation of B cells in the lymph nodes and the spleens of mice injected with bacterial lipopolysaccharide (LPS) was compared. The peak of anti-trinitrophenylated sheep red blood cells plaque-forming cell (PFC) response in the lymph node was reached 6-8 days after the injection of LPS while that in the spleen was reached at 2 days. The maximal increase in the total number of Ig-producing cells in the lymph node also occurred at the later stage. These differences in time courses of polyclonal activation of B cells between the lymph node and the spleen were not due to the absence of B cells in the lymph node, migration of PFC from the spleen to the lymph node, or qualitative differences of B cells. This phenomenon was dependent on the environmental difference between the lymph node and the spleen, because B cells from the lymph node could respond to LPS rapidly in the spleen. Further, the polyclonal activation of B cells was accelerated in the lymph nodes of mice receiving prior injection of LPS. In in vitro cultures of lymph node cells of those mice, a significant amount of interleukin-1 could be detected by stimulation of LPS. It was possible that the delayed activation of B cells in the lymph node was due to the time lag necessary for construction of the environmental condition suitable for activation of B cells, whereas in the spleen this condition can be provided without delay.

On the basis of previous data that 1,25(OH)2D3 suppressed both helper and suppressor activities of CD4 and CD8 cells in the pokeweek mitogen-stimulated culture, we examined the further effect of 1,25(OH)2D3 on both cells to define how 1,25(OH)2D3 is involved in the deterioration of their functions. 1,25(OH)2D3 suppressed the pokeweed mitogen and phytohemagglutinin-induced DNA synthesis of CD4 and CD8 cells. The suppression by 1,25(OH)2D3 of DNA synthesis was caused by a time lag in reaching maximal response. 1,25(OH)2D3 also suppressed interleukin-2 production of CD4 and CD8 cells. 1,25(OH)2D3 did not, however, affect their interleukin-2 receptor expression detected within 24 hr after phytohemagglutinin stimulation. In addition, 1,25(OH)2D3 failed to suppress DNA synthesis of CD4 and CD8 cells when cultured with a large amount of interleukin-2. Suppression by 1,25(OH)2D3 of proliferation and interleukin-2 production in CD4 and CD8 cells would bring about the decrease of their helper or suppressor functions by inhibiting their expansion or maturation.

As shown previously, native or recombinant (r) human platelet factor 4 (PF4) alleviates the suppression induced by Con A or dimaprit, a histamine type 2 receptor (H2-R) agonist, in a murine system. The effect of rPF4 on human peripheral blood cells has now been studied, using as a model pokeweed mitogen (PWM)-induced, T-cell-mediated suppression of Ig-secreting cell (ISC) formation by Staphylococcus aureus and rIL-2 activated B cells. PWM, but not phytohemagglutinin (PHA), induced inhibitory activity in mitomycin-treated CD8+ T cells, but not unfractionated or CD4+ T cells, for both ISC formation and B cell proliferation. rPF4 and its C-terminal tridecapeptide alleviated the suppressive effect of PWM-activated CD8+ T cells on ISC production but not on proliferation. Heparin did not prevent this immunoregulatory activity of PF4. Neutralizing antibody to TGF-beta, but not to IFN-gamma or TNF-alpha, alleviated the suppression of ISC formation in some of the experiments. The H2-R appeared to play a part in inducing suppression, because the H2-R antagonist, cimetidine, prevented the PWM-induced suppression of ISC production. Furthermore, dimaprit induced suppression of ISC formation when added instead of PWM at the start of culture. Incubation of CD8+ T cells with dimaprit for only 3 hr prior to coculture with S. aureus + IL-2 activated B cells decreased the ISC response. This suppression was also alleviated by addition of rPF4 to the coculture. Similar to dimaprit, known cAMP upregulating agents, such as forskolin, dibutyryl cAMP, and cAMP analog, all induced this immunoregulatory activity in T cells. Moreover, the effect of dimaprit was prevented by the specific protein kinase A inhibitor, HA1004, suggesting strongly that upregulation of cAMP played a role in the H2-R-mediated effect. Cell contact appeared to be necessary, since supernatants from dimaprit or PWM activated T cells failed to suppress ISC production. We suggest that the known ability of PF4 to prevent TGF-beta-mediated effects on endothelial and other target cells may be involved in the alleviating effect of PF4 on the cell-contact-dependent CD8+ T-cell-mediated B cell suppression.

Previous in vivo studies have shown that murine T cells induced to express receptors specific for IgD (IgD-R) have humoral immunoaugmenting properties with respect to both primary and secondary antibody responses to various antigens. Such murine IgD-R+ T cells (T delta cells) belong to the CD4+ T cell population in contrast with human T delta cells which also include CD8+ cells. The purpose of these studies was to develop an in vitro assay system to examine the mechanism by which T delta cells facilitate enhanced antibody responses. Our studies demonstrate that B cell responses to both soluble and particulate antigens can, indeed, be enhanced in vitro by coculture with T cells previously induced to express IgD-R. This in vitro effect requires cognate interaction between T and B cells and is dependent on the presence of adherent cells or IL-1. T cell priming with antigen, while not a prerequisite, was found to result in more effective T delta-B cell interactions compared with naive T delta cells. Finally, evidence was obtained in support of an adhesion-mediated T delta-B cell interaction since the immunoaugmenting properties of T cells expressing IgD-R are completely blocked by the addition of very low doses of monomeric IgD to the cultures.

In this study, skin-infiltrating cells were prepared from guinea pig delayed-type hypersensitivity reaction sites, and their phenotypic properties and cytokine production were examined. Our cell preparations contained around 45% nonspecific esterase-positive cells and a few skin-derived cells. Skin-infiltrating cells produced TNF activity and macrophage chemotactic activity (MCA), whereas resident skin cells produced much lower activity. The highest TNF activity was produced by skin-infiltrating cells at 2 hr after antigen injection. In contrast, a larger amount of MCA was produced by skin-infiltrating cells at 24 and 48 hr than at 2 hr after antigen injection. These kinetics were in good agreement with our previous results regarding cytokine activity in skin extracts. Gel filtration analysis and neutralization assay with a monoclonal antibody against human macrophage chemotactic factor with a molecular weight of 1 kDa (MCF-1) indicated that a guinea pig homologue of human MCF-1 was partially responsible for the MCA.

Despite an extensive literature dealing with IL2-induced cytolytic activity, noncytotoxicity-related effects of IL2 on peripheral blood mononuclear cells (PBMC) or T cell function have received less attention. We have focused on the effects of irradiated, IL2-activated PBMC (PBMC*rIL2) on anti-CD3- and formalin-fixed heat-killed Staphylococcus aureus-induced polyclonal B cell differentiation in secondary cultures. PBMC*rIL2 act directly on B cells and cross major histocompatibility complex barriers to augment polyclonal B cell differentiation as measured by plaque-forming cell (PFC) generation. These effects are preferentially mediated by T (both CD4+ and CD8+) cells, and physical contact between effector PBMC*rIL2 and target B cells is not absolutely required for enhanced PFC generation. PBMC*rIL2 must be present for the initial 24 hr of the secondary cultures, indicating that some soluble B cell differentiation factor rapidly released by PBMC*rIL2 mediates the PFC-enhancing effect. Of IL2, IL4, IL5, IL6, IL10, IFN-gamma, and TNF-alpha, only IFN-gamma mRNA is appreciably and reproducibly increased in irradiated, IL2-activated T cells (T cells*rIL2). Nevertheless, exogenous rIFN-gamma cannot mimic and anti-IFN antibodies cannot block the PFC-enhancing effects of T cells*rIL2, indicating that some unidentified soluble factor(s) apart from or in addition to IFN-gamma is involved. IL2-induced effects on T cell noncytolytic function may help explain certain observed immune anomalies in IL2-treated patients, and a better understanding of the IL2-induced effects on T cell noncytolytic function may have ramifications for autoimmune diseases such as SLE.

In this study, we have analyzed the effects of the immunosuppressive agent rapamycin on the activation of highly purified normal human B lymphocytes. When the polyclonal activators Staphylococcus aureus (SA) and soluble CD40 ligand (CD40L) were used to stimulate B cells, rapamycin inhibited both interleukin 2 (IL2)-dependent and -independent proliferation, as well as IL2-dependent differentiation into antibody-secreting cells. Cell cycle analysis indicated that rapamycin inhibited the progression of SA+IL2-stimulated B cells past the mid-G1 phase of the cell cycle. To begin to identify rapamycin-sensitive signaling events essential for B cell activation, we examined the effects of rapamycin on p34cdc2 and p33cdk2 kinase activities. SA+IL2 stimulation induced the activation of both cyclin-dependent kinases. Of interest, rapamycin abrogated the activation of both p34cdc2 and p33cdk2. Our results indicate therefore that rapamycin inhibits a number of SA- and CD40L-inducible events that may be necessary for both entry into S phase and for permitting subsequent B cell differentiation. These studies emphasize the utility of this drug as a tool to begin to dissect the activation pathways utilized by human B cells, as well as to provide implications for the therapeutic use of rapamycin in vivo.

A minimal requirement in investigations of the behavior of the idiotypic network during immunization is the ability to quantitate both the idiotypic (Ab1) and anti-idiotypic (Ab2) responses. Quantitation of Ab2 in serum is complicated by the simultaneous presence of Ab1, so that Ab1-Ab2 immune complexes escape detection. In contrast, immune complexes should not complicate the enumeration of Ab2-producing lymphocytes in a hemolytic plaque assay. This study utilizes a procedure that allows detection of Ab2-producing cells in such an assay. The procedure relies upon the insertion of the appropriate antibody (Ab1) into the membrane of indicator SRBC through a covalently attached dipalmitoyl phosphatidylethanolamine (DPPE) tail. When the Ab2 response following murine immunization with DNP-Ficoll was analyzed using such an assay, peak plaque-forming cell (PFC) numbers were found to coincide with peak Ab1 PFC numbers in both the primary and secondary response. In addition, this Ab2 response was found to be T independent. The murine immune response to DNP-HGG demonstrated a peak Ab2 PFC response which followed the peak Ab1 PFC response after both primary and secondary immunization. This Ab2 response appeared to be T dependent. The secondary responses to both DNP-Ficoll and DNP-HGG showed increased levels of Ab2 PFC and decreased levels of Ab1 PFC in comparison to the primary responses to the same antigens, suggesting that immunoregulation may occur within these idiotypic networks.

Antibody response to phosphorylcholine, an immunodominant epitope of Streptococcus pneumoniae R36a (Pn), is characterized by a public idiotype, T15, that is expressed on a large proportion of antibody molecules produced by all mouse inbred strains. The ability of the immune system to produce an autologous antibody to T15 upon immunization with Pn vaccine was investigated using a modified ELISA plaque assay for detection of single antibody-forming cells (AFC). The limit of ELISA assay for detection of specific anti-T15 AFC is approximately 300 cells/spleen. However, our studies failed to detect any autologous anti-T15 AFC in the course of the primary antibody response to Pn vaccine in young/adult (2–4 months) BALB/c and C57BL/6 mice. Aged mice (20–22 months) also failed to develop any specific auto-anti-T15 AFC upon the primary Pn immunization, despite the fact that the anti-Pn response in these animals changes both quantitatively and qualitatively. In order to generate specific anti-T15 AFC, BALB/c mice had to be immunized repeatedly with Pn vaccine (four weekly injections) or immunized directly with T15 protein in CFA. Different results were obtained with D1 · LP mice that are low responders to Pn and express lower levels of Tl5 Id as compared to BALB/c. Young D1 · LP mice produced high numbers of auto-anti-T15 AFC of both IgM and IgG isotypes following a single immunization with Pn vaccine. The kinetics of auto-anti-T15 response in D1 · LP mice was similar to that of the antigen-specific response. These results demonstrate that the ability of the immune network to produce autologous antibody to a shared Id depends on the genetic makeup of the host, and that this response may be regulated by the level of Id expression.

Antigen-activated peripheral blood B cells were induced by parenteral immunization of healthy individuals with a polyvalent pneumococcal vaccine, or diphtheria toxoid. Seven to nine days after immunization, high frequencies of antigen-specific antibody-secreting cells, representing in vivo activated lymphoblastoid B cells, were detectable in peripheral blood or spleen. The B cellenriched fractions were stimulated for 7 days with different concentrations of rhIL-6. Both the frequency of antibody-secreting cells and the secreted amount of antibody to the immunizing antigen were increased by rhIL-6 in a dose-dependent fashion. Stimulation with rhIL-6 did not alter the isotype distribution of antibody-secreting cells. A polyclonal anti-IL-6 serum completely abrogated the stimulatory effect of rhIL-6 on the in vitro antibody secretion. Fluorescence-activated cell sorter analysis revealed that 25–29% of cells in the large B cell fraction which presumably contained the in vivo activated cells bore the IL-6 receptor. Thus, rhIL-6 enhances the terminal differentiation of in vivo activated B cells into antibody-secreting plasma cells.

After total splenectomy, a portion of the spleen was autotransplanted into subcutaneous ectopic sites of DBA/2 mice. These implanted fragments regenerated into splenic tissue that are microscopically indistinguishable from the structure of the original spleen. These implants are also immunologically functional as demonstrated by their ability to produce antibodies to sheep red blood cells (SRBC) and SRBC inhibition of splenic (implant) cell migration.

A new mitogenic system for in vitro immunoglobulin production induced by tetanus toxoid is presented and the role of T4 and T8 subsets in tetanus toxoid-induced in vitro immunoglobulin production is investigated. Purified T, T4, T8, and B cells from normal individuals previously immunized but not boosted with tetanus toxoid were cultured in helper and suppressor assays and the number of immunoglobulin-secreting cells were enumerated after culture using a hemolytic plaque assay. The regulatory roles of T4 and T8 cells in this tetanus toxoid system were compared with the role of these subsets after pokeweed mitogen stimulation. Although most of the immunoglobulin produced in the tetanus toxoid system was polyclonal, there were differences in the time course, the magnitude of the responses, the radiosensitivity of the subsets, and optimal T- to B-cell ratios for immunoglobulin production which distinguish the tetanus toxoid and pokeweed mitogen systems.

Evidence for a splenic role in regulating antibody production in other lymphoid tissue was obtained in a system in which cyclical fluctuations of splenic plaque-forming cells (PFC) occur following a single intravenous injection of aggregated human γ-globulin in rabbits. First, PFC arising simultaneously in the mesenteric nodes, peripheral blood, and spleen appear to be derived from the spleen since splenectomy prior to antigen injection abrogated these responses. Second, a noncyclical appearance of PFC in popliteal nodes of rabbits responding to subcutaneous injection of antigen was converted to a cyclical response by simultaneous intravenous injection of antigen, an effect which was abolished by splenectomy prior to antigen injection. It is suggested that, following an intravenous injection of antigen, both suppressor cells as well as antibody-forming precursors may be activated in the spleen and disseminated to other lymphoid tissue.

The role of genes linked to the H-2 locus in effecting an immune response to SRBC was examined using strains of mice which differ in the classes of antibodies produced following multiple injections with SRBC. While H-2-linked gene action appeared to be at the level of regulating the number of plaque-forming cells (PFC) present in the spleens of different strains following two injections with SRBC, non-H-2-linked immune response genes seemed to determine whether an IgM-IgG switch occurred as well as how much of each antibody class was produced by the number of PFC available as a result of H-2-linked gene intervention. Mapping studies showed that the H-2-linked genetic effects were due to either the requirement for two genes or the presence of genes located between I-B and H-2D.

Large lymphocytic cells of human tonsils concentrating in the lighter fractions of a BSA gradient contained both precursors of E rosette-forming cells and lymphocytes carrying μ and γ determinants. These two subpopulations of cells could be separated by retaining the Ig-carrying cells on an anti-Fab column. Cells which did not bind to the column matured within 3–5 days into E rosette-forming cells. The Ig-carrying cells were eluted by medium supplemented with immunoglobulins and continued to secrete γ determinants in vitro . Both types of cells were also distinguished by different profiles of spontaneous [ 3 H]thymidine incorporation, the persisting capacity for division of the E-rosette-maturing cells suggesting that they represent an earlier stage of lymphocyte maturation (precursor T cells).

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10 7 cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.

The relative affinity of specific antibody secreted by mouse spleen cells following primary immunization with SRBC was estimated by competitive inhibition assay of antibody secreted by PFC as well as by inhibition of observed PFC number. Inhibition of direct and of indirect anti-SRBC plaque assays by the addition of specific antigen (SRBC stromata) gave sigmoid inhibition profiles from which the concentration of antigen required to inhibit 50% of the plaques (PI 50 ) was determined, Alternatively, the sum of the cube of individual plaque diameters (Σd 3 ) provided a measure of total anti-SRBC antibody secreted by PFCs from which the concentration of antigen required to inhibit 50% of the antibody (Ab 50 ) was determined. Ab 50 , rather than PI 50 : (a) was a more sensitive measure of inhibition by antigen; (b) decreased following immunization indicating a progressive increase in mean antibody affinity; and (c) correlated with the results of hemolysin transfer experiments, an independent measure of mean affinity of circulating anti-SRBC antibody. From theoretical considerations, estimation of mean antibody affinity requires quantitative analysis of fractional antibody inhibition by antigen. Determination of Ab 50 , rather than PI 50 , provides an estimate of bound and of free antibody and therefore should provide a more valid estimate of the relative antibody affinity at the cellular level. Experimentally, utilizing Ab 50 analysis, the IgM and IgG responses of C3H mice to immunization with SRBC demonstrated a progressive increase in affinity during maturation of the immune response.

The natures of the IgM rheumatoid factor (RF)-, IgM-, and IgG-secreting cells in the human bone marrow as compared to the peripheral blood, have been investigated by (1) response to the polyclonal B-cell activator, the Epstein-Barr virus (EBV), (2) sensitivity to the S-phase specific antimetabolite hydroxyurea, (3) presence of the BA-1 and Ia antigens on the cell surface, and (4) cell size, as determined by counter flow elutriation. The EBV-inducible bone marrow IgM-RF precursors derived from medium to large B cells that were inhibited by hydroxyurea pretreatment. The marrow total IgM response derived from small to medium size cells, and was only partially inhibited by hydroxyurea. Hydroxyurea had no effect on IgM-RF or IgM synthesis by peripheral blood cells. These results indicate thet the marrow EBV-induced IgM-RF response is not representative of the response by peripheral blood cells, moreover; the marrow RF secreting response arises from a dividing cell pool that may represent newly generated autoreactive B cells.

Subcutaneous transplantation of the syngeneic P815 mastocytoma in DBA/2J mice induced an activation of splenic T cells which resulted in a hyperresponsiveness of the tumor-bearing animal to the unrelated antigens pneumococcal polysaccharide (Pn) and sheep red blood cells (SRBC). These tumor-activated T cells appeared to increase the plaque-forming cell (PFC) potential of suboptimal numbers of spleen cells, caused normal spleen cells to express increased numbers of PFC, and produced lymphokine(s) which also increased PFC responses of normal splenocytes. The tumor-activated T cells responsible for stimulating normal splenocytes in an in vitro antibody response were shown to be Ly1 + 2 − cells. The activity of the tumor-activated T-cell supernatants was not genetically restricted and required additional Ly1 T cells in order to induce rigorously clean B cells to produce antibody. The T cells capable of stimulating nonspecific antibody responses were also capable of slowing tumor growth when injected with tumor cells in normal recipient mice. These results suggest that T cells activated by tumor antigens release immunostimulatory lymphokines and, at the same time, are capable of leading to inhibition of tumor growth.

Cell viability and the production of direct PFC were studied in mouse spleen cell cultures after cortisol treatment in vivo or in vitro at various times relative to primary stimulation with SRBC in vitro . Cortisol treatment in vivo reduced spleen cell numbers by 88% after 48 hr, but cultures of the remaining cells produced as many PFC in vitro as did cultures of equal numbers of normal spleen cells. In normal spleen cell cultures incubated with cortisol for 4 hr prior to the addition of antigen, peak responses of PFC/culture and PFC/10 6 cells occurred 24 hr later than in controls and averaged, respectively, 27% and 141% of control values. Minimum viable cell numbers were observed in cortisol-treated cultures after 3 days; thereafter cell numbers gradually increased. These results were not significantly altered when cultures were treated simultaneously with cortisol and antigen. The response was not suppressed if the addition of antigen preceded that of cortisol by more than 4 hr. Suppression was also considerably reduced if fetal calf serum was used when preparing cells for culture.

It was generally believed until recently that the B-cell IgM response induced by type-II “thymusindependent” antigens, that are not mitogenic for B cells, was T cell independent. Neither has it been possible in the past to demonstrate that these antigens activate specific T cells. This has led to the general belief that type-II thymus-independent antigens have the anomalous property of not being able to activate such cells. Recent evidence has shown that the IgM response to hapten-Ficoll conjugates, the prototype of type-II thymus-independent antigens, is T cell dependent in at least some circumstances. Here evidence that these antigens activate T cells mediating hapten-specific delayed-type hypersensitivity (DTH) is presented. Furthermore, the conditions under which they do so are similar to those that allow thymus-dependent antigens to activate DTH-mediating T cells.

The ability of antigen to induce proliferation of memory B lymphocytes, thus perpetuating and expanding the memory cell pool, has been examined using an antibody-forming cell (AFC) assay. This method provided confirmation of previous studies in which serum antibody titer was used as a relative measure of pool size and demonstrated directly that the number of antibody-forming cells is increased. Memory cell subpopulations were prepared by lg velocity sedimentation from recently immunized donors (2 weeks) and tested for their ability to proliferate, thus expanding the memory cell pool. Both large and small immature DNP-specific memory cells displayed antigen-dependent and antigen-independent proliferation while mature cells (8 weeks postpriming) were capable only of antigen-dependent proliferation. Chicken γ-globulin (CGG) specific memory cells were also evaluated in this system and were found to differ from DNP-specific cells in several ways. (A) DNP-specific AFC were found to be concentrated in the spleen while CGG-specific AFC were found predominantly in the bone marrow early after transfer and in the spleen upon delayed challenge. (B) The rate of maturation of CGG-specific memory cells capable of antigen-driven proliferation and pool expansion was delayed in comparison to DNP-specific memory cells. The relationship of these functionally defined subsets to previously described memory cell subpopulations is discussed.

Tolerance to sheep erythrocytes (SRBC) has been induced by a combination of high doses of antigen and treatment with cyclophosphamide (CY). The influence of CY alone or in combination with SRBC has been investigated by using the treated animals as recipients for normal spleen cells. CY treatment appears to produce a mouse which is severely depleted of B cells. The injection of large doses of SRBC together with CY, in a schedule which induces tolerance, generates an environment which suppresses the production of antibody-forming cells by passively transferred normal spleen cells. However, transfer of cells from tolerant mice to irradiated mice failed to demonstrate the presence of suppressor cells.

The physical interaction between carrier-specific T hybridoma cells and long-term primed, TNP-specific memory B cells (TNP-MABC) exposed to TNP-OVA was compared to that of unprimed, TNP-specific virgin B cells (TNP-ABC). The direct conjugation of the T and B cells was visualized at the light microscopic level and the number of T/B conjugates was quantified directly. The results demonstrate that the TNP-MABC, as compared to TNP-ABC, formed T/B conjugates after a shorter exposure time to antigen and at a 10-fold lower concentration of antigen. Conjugate formation was inhibited almost completely by treating the TNP-MABC with concentrations of chloroquine that only partially inhibited the ability of the TNP-ABC to form conjugates. Exposure of the T hybridoma cells or the TNP-ABC to monoclonal antibodies directed against cell surface antigens prior to conjugation indicated that L3T4, Thy-1.2, and LFA-1 antigens on the T cells and LFA-1 and I-A antigens on the TNP-ABC are involved in conjugate formation. However, in contrast to the TNP-ABC where treatment of the B cells with anti-LFA-1 blocked T/B conjugate formation, pretreatment of the TNP-MABC with anti-LFA-1 had no effect.

Normal mouse thymocytes activated with concanavalin A (Con A) released soluble factors which selectively inhibited rosette formation between human peripheral blood lymphocytes (PBL) and ox erythrocytes (E O ) sensitized with rabbit IgM antibodies. The factors were removed by absorption with mouse IgM-coupled Sepharose, and were recovered from the beads by elution at acid pH. They neither bound to mouse IgG-Sepharose nor inhibited rosette formation of PBL with E o sensitized with rabbit IgG antibodies. Mouse IgM enhanced the formation of IgM-binding factors by Con A-activated thymocytes. Unstimulated normal mouse thymocytes also released IgM-binding factors upon incubation with mouse IgM. The molecular weights of IgM-binding factors were approximately 90–110 and 35–50 kDa as estimated by gel filtration. Each species of IgM-binding factors markedly suppressed the IgM-plaque-forming cell (PFC) response of sheep red blood cell-primed spleen cells and slightly suppressed the IgG PFC response.

The antifungal polyene amphotericin B (AmB) and its methyl ester derivative (AME) both show potent murine immunostimulant as well as B-cell activating effects. Under certain experimental conditions, AME is a much more potent polyclonal B-cell activator (PBA) than AmB. Notable features of the murine B-cell stimulation induced by AME include: (i) High concentrations of AME (50–100 μg/ml) are required and even at this level exhibit little or no spleen cell toxicity. (ii) Several lines of evidence suggest that the B-cell activating properties of AME are not involved in the cellular mechanism of adjuvant activity in vivo . (iii) There is a strong correlation between the magnitude of the in vitro PBA effects and the in vivo adjuvant effects of AME in a survey of different mouse strains. This evidence suggests that there is genetic control of the murine lymphoid cell-stimulatory effects of AME and that a small number of genes determines the responsive phenotype.

The influence of antigenic stimulation on the early development of the “spontaneously” occurring (“background”) IgM-, IgG-, and IgA-secreting cells has been studied in mice. To evaluate the effect of such exogenous stimulation by an evolving microbial microflora. the young of BALB/c mice that were kept under germ-free conditions and fed a low molecular weight chemically defined synthetic diet (GF-CD) were compared with the young of conventional BALB/c mice fed natural ingredients (CV-NI). The young were first suckling maternal milk and between Days 15 and 18 changed to the same diet as their parents. Background Ig-secreting cells in the spleen were enumerated in the protein A plaque assay. The specificity repertoire of the IgM-secreting cells was determined with plaque assays specific for sheep red blood cells (SRBC) that were haptenized with different concentrations of nitroiodophenyl (NIP). 4-hydroxy-3.5-dinitrophenyl (NNP), and 2,4,6-trinitrophenyl (TNP). The results show that during the first few weeks of life the numbers of background IgM-, IgG-, and IgA-secreting cells in the spleen develop faster in CV-NI mice than in GF-CD mice. At 4 weeks of age equal numbers of IgM-and IgG-secreting cells were found in both groups of mice, but the number of IgA-secreting cells remained reduced in GF-CD mice during the whole period of observation. The frequencies of IgM-secreting cells specific for the differently haptenized SRBC were the same in both groups of mice during the observation period of 10 weeks. This suggests that the ontogenetic appearance of IgM-, IgG-, and IgA-secreting cells in the spleen, and the specificity repertoire of the IgM-secreting cells, as far as was tested in our panel, is independent of exogenous antigenic and/or mitogenic stimulation. However, during neonatal development the rate of development of the background Ig synthesis is enhanced by environmental antigenic stimulation.

Human thymocytes from children less than 6 years of age were tested for their influence on differentiation of normal B cells. The addition of either thymocytes or a culture supernatant from thymocytes to normal peripheral blood lymphocytes (PBL) enhanced pokeweed mitogen-induced B-cell differentiation as tested in a plaque-forming assay for antibody to sheep red blood cells. The thymocytes, however, could not substitute for T lymphocytes in cultures of PBL which had been previously depleted of T lymphocytes. Further, prior treatment of thymocytes with concanavalin A did not result in generation of suppressor cells for either B-cell differentiation or for the responses of PBL to mitogens. Thus, although thymocytes were functionally immature by these assays as compared to mature T lymphocytes they exerted an influence on B-cell differentiation in cultures of normal peripheral blood lymphocytes.

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.

Lipopolysaccharide has been utilized as a carrier for the TNP hapten, producing an antigen which induces an in vivo thymus-independent antibody response to TNP as determined using athymic nude mice and their normal littermates. The immune response to TNP-LPS was investigated at both the antibody-forming cell and the serum antibody levels. The primary response to an optimal dose of TNP-LPS (1.0 μg) exhibited unusual kinetics reaching a sharp peak on day 3 of 58,000 anti-TNP PFC/spleen. Serum antibody to TNP was first detected on day 3 and reached a maximum log 2 titer of 17.5 on day 5, an uncommonly high level for hapten-carrier conjugates and most carriers. Both the anti-TNP serum antibody and PFCs were exclusively IgM. No IgG antibody was detected in the primary response through 28 days postimmunization, nor was any detected in any experiment described in this paper. The primary PFC response to 1.0 μg of TNP-LPS was specific for TNP, producing no evidence of polyclonal antibody synthesis. The relative affinities of PFC-secreted antibody were investigated using hapten inhibition. The hapten inhibition curves for TNP-LPS and TNP-SRBC were very similar, indicating that relatively high affinity antibody was elicited by TNP-LPS. The secondary response to this dose following priming with TNP-SRBC or TNP-LPS was similar to the primary response, though the peak was less sharp in both cases. The response to the homologous secondary challenge shifted somewhat, reaching a peak on days 3–4. The effect of various doses in priming or challenging for the secondary response to TNP-LPS was investigated. Using an increased PFC response as a criterion, no dose was optimal for priming or immunological memory to TNP-LPS. While the adoptive primary response to TNP-LPS reached a low level peak on day 7, the adoptive secondary attained a maximum on day 6. This shift in kinetics in intact mice and in adoptive hosts in comparing primary to secondary responses indicated that a state of B cell priming may be induced. However, its full expression may be suppressed by endogenous factors at the time of priming, such as the high level of circulating anti-TNP antibody or residual antigen. Adoptive transfer would remove the cells from these influences, allowing such B cell priming to manifest itself fully.

A method has been devised in which mouse spleen cells are cultured in such a way that individual “clones” of antibody-forming cells can be completely segregated. Assay of these clones allows the measurement of two parameters of an immune response, the number of initial events leading to antibody forming cells and the yield of antibody-forming cells from each event (immunological burst size). In this work, individual clones were segregated to allow the estimation of immunological burst sizes. The immune response is described as consisting of continual recruitment of new clones from a pool of precursors. The use of this culture method is discussed with reference to the biology of antibody formation.

Hybridization between plasmocytoma cells from Balb/C mice (X63-Ag8) and peritoneal cells from NZB mice, which secrete, in large amounts autoanibodies directed against bromelin-treated mouse red blood cells, have been achieved. Clones of hybridoma secreting permanently autoantibodies were isolated. The biochemistry and the immunochemistry of these monoclonal antibodies have been studied.

To investigate the B-cell repertoire for autoantibodies, the frequency of precursor cells capable of expressing anti-DNA antibodies in normal and autoimmune mice was evaluated by in vitro limiting dilution analysis. Spleen cell preparations from BALB/c, CBA/J, and MRL- lpr/lpr mice were stimulated at graded cell numbers in microcultures with LPS at 50 μg/ml in the presence of rat thymus filler cells. Culture supernatants were then assayed for IgM antibodies by an enzyme-linked immunosorbent assay; anti-DNA was measured as a model autoantibody response while the anti-TNP hapten response was determined as a control for an ordinary antibody response. The conditions for cell activation were found to be the same for the two responses and both demonstrated high precursor cell frequencies. Approximately 1 in 500 spleen cells was found committed to each response with similar frequencies in normal and autoimmune strains. These results indicate an abundant representation of some anti-DNA antibodies among normal B cells, without apparent augmentation by the autoimmune state. However, since not all anti-DNA antibodies may be pathogenic, it remains uncertain that the composition and disease potential of the B-cell repertoires of normal and autoimmune mice are comparable.

It is well known that Ir genes control the antibody production. To investigate whether they also influence B-memory-cell generation, CBA mice (nonresponders) were primed with dinitrophenyl-poly-(L-tyr,L-glu)-poly-(D,L-ala)-poly-(L-lys)-DNP-TGAL conjugate. At the same time animals were injected with ovalbumin (OVA) to activate OVA-specific T helpers. Two to four weeks later animals were challenged with DNP-OVA conjugate and the number of IgG-producing B cells was determined. The data presented indicate that carrier-specific MHC-restricted T helpers are not required for B-memory-cell generation. It is concluded that the defect of IgG response to DNP-TGAL in CBA mice is caused by a block in the maturation of memory cells to antibody-producing cells.

Efferent lymphatic cells from the popliteal lymph nodes of rabbits inoculated with SRBC into footpads indicated an outflow of only a small number of PFC until 3 days after antigen inoculation. If the efferent lymphatic cells in this period were added to normal spleen or lymph node cell culture, the primary response of these normal cells against SRBC, assayed by the plaque-forming method, was conspicuously enhanced. This effect of the primed efferent lymphatic cells was markedly reduced by the treatment of anti-rabbit thymus cell serum. Therefore it is suggested that the enhancement is largely due to the presence of T cells as the reacting cells in primed efferent lymphatic cells.

The relative migratory properties of [125I]iododeoxyuridine-labeled dividing mesenteric lymph node (MLN) and peripheral lymph node (PLN) cells were assessed in mice using the adoptive transfer system. MLN cells from virgin donors showed a greater tendency to localize in MLN, small intestine, and mammary glands (lactating recipients only) of virgin and lactating recipients. In addition, MLN cells were shown to selectively localize in the salivary (submandibular and sublingual) glands. The relative migratory properties of MLN and PLN cell populations were identical when donor cells were obtained from virgin or lactating animals. Selective depletion of IgA-bearing cells from the MLN transfer cell population abrogated the preferential localization in control mucosal tissues and salivary glands. These data show that the salivary glands can be included as an additional mucosal area populated by gut-derived IgA-bearing cells and provides direct evidence that the common mucosal immune sytstem extends to secretory tissue in the oral cavity.

Injection of immunodeficient, aged mice with synthetic thymosin-α1, a 28 amino acid residues peptide, before carrier priming enhances helper activity. Helper activity was titrated in vitro by adding graded numbers of HRBC-primed spleen cells from aging mice to cultures containing a constant number of spleen cells from 3-month-old normal mice and TNP-HRBC. The in vitro anti-TNP response is enhanced if the aging mice are injected with microgram quantities of α1 before carrier priming. As suggested by the shape of the titration curves α1 appears to increase T cell-T cell cooperation involved in the generation of helper cell activity. The in vitro proliferative response of lymph node cells from OVA-primed aging mice is also restored by injection of α1. Thus, T-cell functions which are markedly reduced in senescence can be efficiently repaired by injection of synthetic thymosin-α1.

Spleen cell cultures from mice of H-2 a, b, d, k, p, q, r, s haplotypes and BALB/c athymic nu/nu mice were stimulated by poly(TyrGluAlaGly), (TGAGly) n , as measured by an increase in the in vitro [ 3 H]thymidine incorporation. A kinetic study indicated that, as was the response to lipopolysaccharide (LPS), 2-day cultures gave the best response. (TGAGly) n and LPS when cultured together gave rise to synergistic responses during thymidine uptake. The synergism of LPS and (TGAGly) n occurred at both suboptimal and optimal concentrations. Of the strains tested for synergism between LPS and (TGAGly) n , mice of H-2 a, b, d, q, r, and s haplotypes showed positive reaction while H-2 k haplotype showed no synergism. Lipid A-associated protein component was found to be the active moiety in the synergistic response with (TGAGly) n . The synergism of LPS and (TGAGly) n was limited to the thymidine uptake only. There was no synergistic polyclonal response as assayed by PFC. The mitogenic responses to (TGAGly) n do not lead to polyclonal antibody responses. Two other related known-sequence polymers poly(TyrAlaGluGly), (TAGGly) n and poly(PheGluAlaGly), (PheGAGly) n , were also found to be mitogenic.

Thymus-independent primary antibody responses were studied in young and old (9 months) untreated and TLI-treated NZB/NZW and BALB/c mice. Untreated old NZB/NZW mice had a low primary response to Brucella abortus (BA) as compared to that of young NZB/NZW and BALB/c mice. However, TLI treatment resulted in a 130-fold increase in the IgG anti-BA primary antibody response at day 21 postimmunization, achieving similar levels to those of young NZB/NZW or nonautoimmune BALB/c mice. Anti-TNP responses to trinitrophenylated BA or Ficoll were masked by high background levels of anti-TNP antibodies. Despite the increase in the anti-BA response, spontaneous immunoglobulin secretion and autoantibody levels were markedly decreased after TLI in old NZB/NZW mice.

Stimulation of human tonsillar lymphocytes in culture with pokeweed mitogen (PWM) results in the production of a large number of antibody-forming cells which can be measured in the sheep erythrocyte (SE)-specific, direct haemolytic plaque-forming cell (PFC) assay. For optimal responsiveness in this system the human AB serum supplementing the culture medium must have been preabsorbed with SE. This effect is due, at least in part, to release during the absorption step of soluble immunogenic material from SE which acts synergistically with PWM to produce an enhanced response but which is unable to stimulate a response in the absence of the mitogen. In contrast to SE, absorption of AB serum with ox or horse erythrocytes failed to increase erythrocyte-specific responses. However, absorption with goat erythrocytes (GE) enhanced both SE- and GE-specific responses. An analysis of PFC specificities in cultures containing absorbed or unabsorbed serum revealed that enhanced responsiveness in the presence of absorbed serum was due to an increase in the proportion of PFC which cross-reacted with both SE and GE. The significance of this observation is discussed in terms of the antigenic requirements of interacting T- and B-cell subpopulations.