Interferon and the cellular immune response: separation of interferon-producing cells from DNA-synthetic cells

Spleen cells from mice were separated by albumin discontinuous density gradient centrifugation into six fractions. Cells from each fraction were cultured with a variety of general mitogens and a specific antigen PPD. The cultures then were assayed for mitogenesis and interferon production. The fraction of cells producing interferon were found to belong to a different population of cells from those undergoing a DNA synthetic response. Treatment of the interferon-producing fraction of cells with anti-theta serum and complement eliminated the interferon-producing capabilities of the remaining cells after exposure to PHA, Con A, and PPD but not after exposure to PWM. The results suggest at least a two-cell requirement for the production of interferon in response to PHA, Con A or PPD stimulation. They also suggest that the interferon-producing cells do not belong to the thymus-dependent lymphocyte subpopulation.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

Expression of lectin receptors on the surface of granulocyte-macrophage progenitor cells (CFU-gm)

It has been emphasized that specific bindings between membrane glycoproteins and membrane lectin-like substances are important in cell-to-cell interactions. We explored the surface of granulocyte-macrophage precursor cells (CFU-gm) by the differential agglutination technique. Enrichment of CFU-gm in the agglutinated fraction, containing the cells which have lectin receptors, from marrow treated with soybean agglutinin (SBA), peanut agglutinin (PNA) and concanavalin A (Con A), suggests the presence of reactive galactosyl and mannosyl residues on the surface of CFU-gm. On the other hand, wheat germ agglutinin (WGA), phytohemagglutinin (PHA) and ulex europaeus agglutinin (UEA), which bind to reactive N-acetylglucosamine, N-acetylgalactosamine and fucose, respectively, did not specifically agglutinate CFU-gm. Thus, reactive groups containing galactosyl and mannosyl structures on the surface of CFU-gm may possibly play a role in the process of cell-to-cell interactions between CFU-gm and marrow stromal cells.     

T11: a new protein marker on activated murine T lymphocytes

Murine lymphocytes were activated in vitro in mixed lymphocyte cultures (MLC) or by the addition of the mitogens concanavalin A (Con A), phytohemagglutinin (PHA), or E. coli lipopolysaccharide (LPS). Activated lymphocytes were internally labeled with 35S-methionine and then disrupted by hypotonic lysis. A plasma membrane-enriched fraction was isolated from each cell population, and the 35S-labeled proteins in this fraction were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). An intensely labeled band, the position of which indicated an apparent m.w. of 11,000, was observed when plasma membrane-enriched fractions from MLC- and Con A-activated cells were subjected to SDS-PAGE. In contrast, plasma membrane-enriched fractions from normal spleen cells, LPS-activated cells, PHA-activated cells, and EL4, RDM4, and P815 tumor cells possessed little or none of this protein, which we have designated T11. T11 was not found in the soluble cytoplasmic protein from MLC-activated cells. Hence the presence of T11 in the plasma membrane-enriched fraction from these cells cannot be attributed to contamination by cytoplasmic protein. Removal of T cells from populations of MLC-activated cells by treatment with monoclonal anti-Thy 1 and complement removed T11. These results suggest that T11 may represent a new protein marker on a subclass of activated T lymphocytes.     

Effect of T- and B-lymphocyte mitogens on interactions between lymphocytes and macrophages.

The mitogenic response of murine T cells ² to Con A, S-Con A and PHA was found to be macrophage-dependent. Optimal mitogenic responses were obtained when macrophage-depleted T-cell populations were reconstituted with 5% normal peritoneal macro-phages. Studies were carried out to investigate the effect of T- and B-cell mitogens on in vitro physical interactions between murine lymphocytes and macrophages. This was done by determining the number of T- or B cells binding to macrophages in the absence and in the presence of T- and B cell mitogens, and comparing the results of these experiments with the induction of lymphocyte proliferation. Con A increased the binding of T cells to macrophages when used in mitogenic doses (1–5 μg/ml). Dose response experiments showed that the same dose of Con A which produced maximal mitogenic stimulation also induced the greatest number of T cells to bind to macrophages. Nonmitogenic doses of Con A (20–50 μg/ml) did not enhance the binding of T cells, while identical doses of S-Con A both induced T cell mitogenesis and increased the number of T cells bound to macrophages. Similar results were obtained with PHA. None of the B-cell mitogens tested (LPS, EPO 127 and LAgl) increased the binding of either T or B cells to macrophages. PWM, which is mitogenic for both T and B cells, increased the binding of T cells to macrophages, but not that of B cells. In brief, the four T-cell mitogens tested (Con A, S-Con A, PHA, and PWM) induced specific physical interactions between T cells and macrophages, while none of the B-cell mitogens had any effect on the physical interactions between either B or T cells and macrophages when used in mitogenic doses.     

Responses of spleen cells from mice with X-linked B-cell defect to polyclonal B-cell activators, purified protein derivative of tuberculin, and dextran sulfate

Polyclonal responses to LPS, PPD, and DxS of spleen cells from mice expressing a X-linked B-cell defect were examined. Spleen cells from young (CBA/N × BALB/c)F₁ male mice responded slightly lower to LPS, significantly lower to PPD than the cells from age-matched F₁ female mice, and showed no response to DxS stimulation. This hypo- or unresponsiveness of F₁ male cells to PPD or DxS could not be explained by a shift in the dose-response or time kinetics of the responding cells, and also could not be due to the defect in the function of T cells or macrophages. Suppressor T cells to polyclonal response to PPD or DxS could not be shown in F₁ male spleen cells. The response of F₁ male cells to PPD was dramatically improved with age but not to DxS. These results suggest that B cells responsive to DxS may belong to a distinct subpopulation from the cells responsive to LPS or PPD.     

Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response

Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process. The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified. Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes. Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines. PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay. In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells. The same results were obtained with PNA+ and PNA- cells from LAF1 mice. Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS. B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS. Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less. These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation. IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells. IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation. In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5. The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population.     

Separation of polypeptides which induce a mitogen response of thymocytes from the culture supernatant of a thymic epithelial cell line

To examine thymic hormonal factors, four polypeptide fractions (estimated molecular weight: I, 10 K; II, 7 K; III, 3 K; IV, 2.5 K) were separated from the culture supernatant of a rat thymic epithelial cell line by high-pressure liquid chromatography (HPLC) with a gel-filtration column. The effects of the fractions on response to mitogens of three small-lymphocyte subsets were studied. All fractions enhanced response to concanavalin A (Con A) of the lighter subset containing mainly immature thymocytes, but only fractions II and IV increased response to phytohemagglutinin (PHA) of the heavier subset containing relatively mature thymocytes. When fraction IV was subfractionated by reversed-phase HPLC, the polypeptides that enhanced response to Con A and PHA were separated into hydrophobic and hydrophilic subfractions, respectively. Fraction I was subfractionated by a similar method, and the inducing activity of Con A response was found in a relatively hydrophobic subfraction. These data suggested that the cell line secretes several kinds of bioactive polypeptides that affect the thymocytes at different stage of maturation.     

Modulation of lymphocyte functions by group A streptococcal membrane

Protoplast membranes isolated from group A streptococci suppress functions of mouse B cells in vivo and in vitro. Intraperitoneal injection 24 or 72 hr (but not 12 hr) before collection of lymphoid cells results in a selective decrease in the mitogenic response of bone marrow cells to dextran sulfate (DS). The response of bone marrow cells to lipopolysaccharide (LPS), and spleen cells to both DS and LPS, is unaltered. In vitro exposure of lymphocytes to membranes concomitantly with mitogen reduces the response to both DS and LPS, however, the DS response is more susceptible to low doses of membrane. Suppression of the response to DS in vitro is not mediated by cells bearing Thy 1.2 antigen. Neither the phytohemagglutinin (PHA)-responsive cells nor the adherent cells participate in suppression of the LPS response in vitro. In contrast to the suppression of B-cell functions neither the PHA nor concanavalin A (Con A) response of mouse bone marrow, spleen, or thymus cells is altered by streptococcal protoplast membranes injected 24 hr before collection of cells. In vitro exposure of spleen cells to a limited range of concentrations of membrane results in an enhanced proliferative response of spleen cells stimulated by suboptimal doses of PHA. This synergism is not mediated by the adherent cells. Addition of membranes to spleen cell cultures in vitro has no effect upon the response of spleen cells to suboptimal doses of Con A or to optimal doses of either Con A or PHA. Higher concentrations of membranes reduce the proliferative response of both control and mitogen-stimulated cells. This nonselective suppression by high doses of membranes is not due to toxicity. Delayed hypersensitivity to sheep erythrocytes is potentiated by injection of membranes. These studies suggest that streptococcal membranes preferentially suppress the immature B cells and enhance certain T-cell functions.     

The lack of generalized immunosuppression in C57BL/6 mice during progressive growth of syngenic T lymphoma EL-4 and Lewis lung carcinoma 3LL

The immune competence of C57Bl/6 mice implanted with EL-4 lymphoma of Lewis Lung carcinoma 3LL was investigated during 3 weeks after implantation. Splenic lymphocyte responses to mitogens (Con A, PHA, LPS, PWM) cytotoxic T lymphocytes (CTL) and interleukin-2 (IL-2) production were assessed. A dramatic reduction in mitogenic responses to Con A and PHA was observed during tumour progression. LPS and PWM responses were less depressed. Con A-induced IL-2 production correlated with Con A and PHA responses. Allospecific CTL response to mastocytoma P 815 was not decreased in syngeneic tumour-bearing mice.     

Inhibition of the mitogenic response to lipopolysaccharide (LPS) in mouse spleen cells by polymyxin B.

The addition of low doses of the cationic polypeptide antibiotic, polymyxin B (PB), to cultures of mouse spleen cells inhibits lipopolysaccharide-(LPS) induced DNA synthesis but not that stimulated by PPD, PHA, or Con A. Inhibition is stoichiometric; the mitogenic response is suppressed by 50% at a weight ratio of PB:LPS of 0.055 to 1. Furthermore, PB-LPS complexes have a much reduced mitogenic capacity. These complexes inhibit the mitogenic response of spleen cells to unmodified LPS but not to PPD, Con A, or PHA. The inhibitory activity of PB is less effective when added after LPS is mixed with responding cells, achieving 50% inhibition when addition is made at 4 to 6 hr. Time course experiments indicate that partial inhibition is a reflection of a lower rate of DNA synthesis. Thus, PB inhibition of LPS mitogenesis apparently occurs as a result of formation of PB-LPS complexes with reduced mitogenic capacity. Specific inhibition by the complexes of mitogenesis induced by native LPS suggests that the inactive complex may bind to B cells but is unable to trigger them.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Plant Lectins Induce the Production of a Phytoalexin in Pisum sativum

The effects of several plant lectins on the production of apea phytoalexin, pisatin, were examined. Con A, PHA, PNA andPSA each induced the production of pisatin in pea epicotyl tissues,demonstrating that plant lectins can act as elicitors. The productionof pisatin in response to PHA, PNA or PSA was not affected bythe simultaneous presence of the respective hapten sugars, whereashaptens specific for Con A, such as -D-mannose and methyl--D-mannoside,abolished the induction of pisatin by Con A. These results indicatethat the elicitor effect of Con A is attributable to its abilityto bind to specific carbohydrates in pea cells. Induction ofthe production of pisatin by Con A was markedly inhibited bythe suppressor derived from a pea pathogen, Mycosphaerella pinodes,and by several inhibitors related to signal-transduction pathways.It is suggested, therefore, that the Con A-induced productionof pisatin in pea tissues might be associated with activationof a signal-transduction pathway. An additive effect on theaccumulation of pisatin was observed when Con A was presentwith a polysaccharide elicitor from M. pinodes, suggesting thatexogenous Con A does not compete with the recognition site(s)for the fungal elicitor in pea cells. The present data alsoindicate that Con A may be useful for characterization of thesignal-transduction system that leads to the synthesis of phytoalexinin pea epicotyl tissues. (Received November 16, 1994; Accepted April 20, 1995)     

Functional diversity of polypeptides in primary culture supernatant of thymus epithelial cells

Polypeptide fractions A-C (M.W., 7 kd, 4.7 kd, and 3 kd) were obtained from the primary culture supernatant of thymus epithelial cells from Wistar rats by high-pressure liquid chromatography with a gel-filtration column. Changes in the mitogen responses of rat thymocytes and their subpopulations with addition of a fraction were studied. One subpopulation was rich in non-rosette-forming cells (non-RFCs), and the other was cortisone resistant thymocytes (CRTs). These subpopulations were incubated with a fraction for 24 hrs. before mitogen stimulation. Fractions A and C increased the response of the non-RFCs to concanavalin A (Con A) and that of total thymocytes and CRTs to phytohemagglutinin (PHA). Fraction B inhibited Con A and PHA response of total thymocytes and CRTs. Fraction B was cytotoxic toward total thymocytes and CRTs when viability was evaluated by [3H]uridine prelabelling. This cytotoxicity was suppressed by treatment with trypsin. Subfractions B3 and B4 obtained by reversed-phase column chromatography were cytotoxic toward CRTs. The effects of the fractions on thymocyte maturation were different, showing their functional diversity.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

In vitro proliferation of blood,spleen and thymus leukocytes from the turtle Mauremys caspica

Cell-mediated immune responses is commonly evaluated by cell proliferation assays. Mitogens are known to induce a vigorous proliferative response in lymphoid cells from mammals but relatively fewer studies have investigated mitogen-mediated lymphoproliferation in non-mammalian animals. In the present work, we incubated spleen, thymus and blood leukocytes with phytohaemagglutinin (PHA), concanavalin A (Con A), lipopolysaccharide (LPS) and pokeweed mitogen (PWM), by different times of incubation (96 and 120 h) and at different concentrations. Our results show that the optimal mitogen concentrations inducing proliferation on leukocytes from Mauremys caspica were 20 microg/ml PHA, 1 microg/ml Con A, 12.5 microg/ml LPS and 1/150 dilution PWM. The optimal time of incubation was dependent on the type of leukocytes (peripheral blood leukocytes, splenic leukocytes or thymic cells) and the mitogen utilized.     

Characteristics of complement receptor-bearing cells in the spleens of tumor-bearing mice.

In the course of mammary tumor development, a population of nylon nonadherent cells with CR appears in the spleens of tumor-bearing mice although none are ever detected in normal mice. These cells apparently arise in response to immunologic stimulation. In a series of studies we have further characterized subsets of T cells (CR+ and CR-) with regard to their responses to mitogens in the lymphocyte transformation assay. Nylon column nonadherent cells from the spleens of tumor-bearing mice were rosetted in a complement receptor assay using EAC rosetting, and CR+ cells were separated from CR- by centrifugation in a discontinuous Ficoll gradient. CR+ T cells responded strongly to PHA and Con A and in addition responded to LPS, an activity not usually associated with conventional T cells. In contrast, CR- T cells from tumor-burdened mice responded to PHA but failed to respond to Con A or LPS.     

Trypanosoma rhodesiense Bloodstream Trypomastigote and Culture Procyclic Cell Surface Carbohydrates1, 2, 3

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A < PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.     

Responses of B cells with or without C3 receptors to polyclonal B cell activators

Responses of B cells with or without receptors for C3 (CR) to polyclonal B cell activators (PBA) were studied. Mouse spleen cells were incubated with sheep red blood cells (SRBC) coated with antibody and complement to form rosettes, and they were separated by Ficoll-Hypaque density sedimentation into populations depleted of and enriched with lymphocytes bearing CR (CRL). These 2 populations were cultured with lipopolysaccharide (LPS), purified protein derivative of tuberculin (PPD), or dextran sulfate (DxS) and assayed for anti-TNP PFC. The CRL-depleted population responded well to LPS, poorly to PPD, and it showed practically no response to DxS, whereas the CRL-enriched population seemed to respond poorly to LPS but well to both PPD and DxS. The low responsiveness of the cRL-depleted population to PPD and DxS could not be explained by a shift of time-kinetics, by the dose-response profile of the responding cells, or by the depletion of adherent cells. Suppressor T cells did not take part in the reduced responses, since the treatment of the population with anti-Thy 1.2 plus complement could not restore the responses. These results indicate that B cells with CR [CR(+) B cells] respond well both to PPD and DxS, whereas the cells without CR [CR(-) B cells] respond poorly to PPD and DxS. It was difficult to evaluate the low responsiveness of CR(+) B cells to LPS because of the high background PFC of the cRL-enriched population.