The type-specific capsular carbohydrate of Hemophilus influenzae B is a potent mitogen for murine B lymphocytes

In this study we investigated the in vitro mitogenic properties of the capsular carbohydrate of Hemophilus influenzae b, polyribosylribitolphosphate (PRP). PRP was found to be a potent polyclonal activator of murine B lymphocytes. PRP induced normal B cells to undergo blastogenesis, DNA synthesis, and differentiation to IgM and IgG secretion. IgG3 accounted for the majority of the IgG. No PRP-specific antibody was detectable, indicating the polyclonal origin of the secreted immunoglobulin (Ig). T lymphocytes were neither activated by PRP nor required for B cell proliferation or Ig secretion. In addition, T cell-depleted spleen cells also depleted of accessory (A) cells by passage through Sephadex G-10 retained responsiveness to PRP. Trace lipopolysaccharide (LPS) contamination was not responsible for the mitogenic effect, as shown by the ability of C3H/HeJ spleen cells to proliferate in response to PRP and by the failure of polymyxin B to inhibit PRP-induced DNA synthesis. The B cell responses induced by PRP and LPS were similar with respect to T cell and A cell independence, to the magnitude of DNA synthesis, and to Ig secretion and the Ig isotypes expressed. These data, taken with the finding that the combination of optimal doses of PRP and LPS did not give an additive DNA synthetic response, indicate that PRP and LPS were activating similar B cell populations. However, in contrast to LPS, PRP was capable of inducing significant DNA synthesis in cultures containing as few as 1,000 B cells, suggesting that PRP-driven proliferation was less dependent on cellular interactions than the response to LPS. The differential ability of PRP and LPS to stimulate C3H/HeJ B cells and to stimulate B cell proliferation at low density indicates basic differences between these two mitogens in their mechanisms of B cell activation.     

Human lymphocyte differentiation antigens HB-10 and HB-11. I. Ontogeny of antigen expression

T, B, and NK cells appear to represent separate lymphocyte lineages, but indirect evidence suggests that they may be related via a common lymphoid precursor cell. We have produced two monoclonal antibodies, HB-10 (IgM) and HB-11 (IgG1), by fusing spleen cells from mice immunized with the human B cell line SB, and have shown that both antibodies react with lymphocyte-specific cell surface antigens present on T, B, and NK cells, but not on other types of blood cells. The antibodies were reactive with most cell lines and malignancies of B cell origin and with some of T and NK cell lineage. Although the populations of cells expressing these two antigens were virtually identical, the HB-10 and HB-11 antibodies identified separate protease-sensitive determinants on the cell surface. The HB-11 antigenic determinant was also sensitive to neuraminidase and periodate treatments, but the HB-10 determinant was not. Antigen expression by lymphocytes from fetal, newborn, and adult tissues was examined. Within the B cell lineage, these antigens were expressed by most pre-B cells in bone marrow (88% +/- 5) and almost all B cells, but were not expressed by mature plasma cells. Virtually all of the granular lymphocytes in blood marked by the Leu-7 and Leu-11 (anti-Fc receptor) antibodies were HB-10+ and 11+. Among T lineage cells, the HB-10 and 11 antigens were expressed by a subset of relatively mature T3+ thymocytes and by greater than 90% of the T cells in newborn blood. In adults, however, only 65% of blood T cells and 24 to 30% of splenic or tonsillar T cells expressed the HB-10 and HB-11 antigens. The postnatal emergence of T cells which, like plasma cells, do not express these antigens suggests that post-thymic T lymphocyte maturation occurs and may be an activation-dependent process.     

Detection of HLA-DRw (Ia-like) antigens on human T lymphocytes grown in tissue culture.

Human T lymphocytes that proliferate in the presence of conditioned medium from PHA-stimulated allogeneic peripheral blood cells were shown to express IPA antigens after the 8th culture day. Ia antigens as detected by xenogeneic antisera were present on 80 to 90% of the cultured cells which were also strongly reactive with xenogeneic antisera defining a human T cell antigen and formed E rosettes. Control cultures with PHA or no conditioned medium expressed T cell but not Ia antigens. These cultured T cells also express the same HLA-DRw determinants as the B cells of the donor they were derived from. Absorption of xenogeneic Ia, and HLA-DRw alloantisera with cultured T cells completely removed the reactivity of these sera for enriched peripheral blood B lymphocytes from normal donors.     

Representation of heavy but not light chain Ig idiotypes on T cell receptors for alloantigens.

We have analyzed idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants by the use of different anti-idiotypic antibodies. Such antisera were produced in (Lewis X DA) F1 rats against Lewis anti-DA alloantibodies (= B cell product) and Lewis T lymphocyte receptors with the same specificity. We found that B lymphocytes bear unique idiotypic determinants which are not present on the corresponding T lymphocytes. T cell unique (not shared by B lymphocytes) idiotypes were so far not detected. T cells idiotypic determinants which are present on heavy but not light chains of the corresponding alloantibodies.     

Effects of anti-beta-2 microglobulin antibodies on human lymphocytes.

Anti-β2 microglobulin antisera prepared in rabbits immunized with β2m purified from the urine of a single patient were cytotoxic for human T and B lymphocytes of all donors tested; lymphocytotoxicity could be fully inhibited by all human sera tested, not by serum from other animal species. Anti-β2 microglobulin antibodies and their F(ab′)2 fragments had little effect on E and EAC rosette formation, suggesting that β2m is not closely associated with receptors for sheep erythrocytes on T lymphocytes or receptors for C3 on B cells. Anti-β2m IgG and F(ab′)2 fragments inhibited EA rosette formation though the latter did not impair lysis of antibody-coated xenogeneic erythrocytes by lymphocytes bearing receptors for the Fc portion of IgG. Some of the antisera had a mild mitogenic effect, all of them inhibited mitogen and antigen-induced lymphocyte proliferation at high concentrations whereas they potentiated these responses at low concentrations. In mixed lymphocyte cultures pretreatment of responding cells markedly depressed the response whereas coating of stimulating cells with β2m antibodies had little or no effect.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

The decline in murine splenic PHA and LPS responsiveness with age is primarily due to an intrinsic mechanism

Changes in splenic B and T lymphocyte number and mitogenic activity with age were quantitated in (A X C57BL/6)F1 (AB6F1) hybrid mice. Although both the B and T lymphocyte proliferative reactivity to their respective mitogens, lipopolysaccharide (LPS) and phytohemagglutinin (PHA), declined significantly with age, an earlier and more marked reduction was recorded for the T cell response. The decline in B and T lymphocyte mitogenic activity with age could not be correlated with a corresponding reduction in the percentage of splenic B or T lymphocytes. The main focus of this study was to determine if the reduction in T and B lymphocyte mitogenic activity with age results primarily from a mechanism intrinsic to the lymphoid lineage itself or from adverse extracellular factors that increase with age. Bone marrow cells (BMC) derived from individual young and old donor AB6F1 mice were transplanted into the neutral environment of young, lethally irradiated syngeneic recipients. Number and mitogenic activity of splenic T and B lymphocytes were recorded for the original BMC donors as well as for the recipients of the young and old BMC lines 9 mo after the BMC transplants. A predominance of the donor (male) rather than recipient (female) karyotype within the mitogen-responding populations of recipient mice confirmed a donor BMC take. The PHA and LPS response levels exhibited by the old donors were 30% and 70% of those of the young donors, respectively. These differences in PHA and LPS reactivity recorded between young and old donors were maintained between recipients of young and old donor BMC lines. Thus, even under the influence of a young recipient environment, old BMC were incapable of giving rise to mitogen responding cells with a functional competence equivalent to that of their younger counterparts. This finding would lend further support to the theory that an intrinsic mechanism is responsible for the decline in murine mitogenic activity with age.     

Rabbit antisera to human B cell alloantigens: effects on the mixed lymphocyte response.

Antisera were produced in rabbits to two glycoproteins (31,000 MW and 23,000 MW by SDS-gel electrophoresis) isolated from papain digests of membranes from a human B lymphoblastoid cell line (LCL). After minimal absorption with a T LCL the antisera reacted with two glycoproteins (35,000 MW and 27,000 MW) present in detergent-solubilized membranes from human B LCLs. The immunizing molecules are proposed to have arisen by proteolysis of the intact molecules in the detergent-solubilized membranes. The glycoproteins were detectable on human B LCLs and macrophages, but absent from T LCLs and fibroblasts. The molecules were identified as B cell alloantigens by their reactivity with alloantisera specific for human B cells. The rabbit antisera reacted with peripheral blood B lymphocytes, but not with T lymphocytes, platelets or erythrocytes.Pretreatment of human peripheral blood mononuclear cells with the rabbit antisera in a uni- or bidirectional mixed lymphocyte response (MLR) rendered them unable to stimulate, but they were able to respond. Addition of the antisera at various intervals during the MLR to block continuous stimulation indicated that cells were activated at different times. The presence of an F(ab′)₂ or Fab′ preparation of the antisera throughout the MLR did not inhibit the response at the concentrations tested. Further experiments suggest that, while a responding lymphocyte can replicate several times without restimulation, there is a delay between commitment to and commencement of division.     

Dissociation of autologous and allogeneic mixed lymphocyte reactivity by using a monoclonal antibody specific for human T helper cells

A monoclonal antibody defining a population of human T helper cells was developed and shown to specifically block the autologous mixed lymphocyte reaction (AMLR). This antibody, termed KT69-7 (IgG1), recognized 62% of peripheral blood E rosette-positive (E+) cells while demonstrating negligible reactivity with E- cells, monocytes, granulocytes, EBV-transformed B cell lines, and mouse splenocytes. Separation of E+ cells into KT69-7+ and KT69-7- populations revealed that KT69-7+ T cells provided helper function in PWM-driven B cell differentiation, whereas KT69-7- T cells provided no help and may suppress this response. Modulation of membrane moieties by using KT69-7 or OKT4 plus goat anti-mouse IgG removed reactivity to both these antibodies, suggesting an association between these molecules recognized by these antibodies. In functional studies, KT69-7 selectively blocked the AMLR while demonstrating minimal or no effect on the allogeneic MLR (allo-MLR). Blocking of the autoreactivity occurred when either autologous B lymphocytes or macrophages were used as stimulators. The failure of KT69-7 to block the allo-MLR was not attributable to excessive allogeneic stimulus; KT69-7 failed to block even under conditions of limiting numbers of stimulator cells. KT69-7 thus appears to recognize a molecule on the surface of T helper cells required for recognition of autologous class II antigens.     

Lymphokine-like activity of 8-mercaptoguanosine: induction of T and B cell differentiation

Lymphocyte proliferation and differentiation result from ordered cellular interactions governed by soluble products (lymphokines). Dissecting the individual steps in these processes has been difficult, due to a paucity of pure lymphokines. Recently, it was reported that the derivatized ribonucleoside 8-mercaptoguanosine (8MGuo) has both mitogenic and differentiative effects on murine B cells. In the present studies, we tested 8MGuo for its ability to stimulate both B and T cell responses. In contrast to the murine studies, 8MGuo does not stimulate rat B cells to proliferate and, when tested for B cell growth factor-like activity, no stimulation was observed. The addition of 8MGuo (0.5 to 1 mM final concentration) to mitogen-stimulated B cells led to a marked increase in IgM and a modest increase in IgG secretion. When mixed with conditioned medium, 8MGuo acted synergistically in stimulating secretion of both isotypes, arguing that 8MGuo has both B cell-differentiating factor-mu (BCDF-mu) and BCDF-gamma activity. 8MGuo had no IL 2-like activity when tested on a mouse IL 2-dependent cell line, and no IL 1-like activity on addition to mouse thymocytes with or without submitogenic doses of lectin. However, when added to cultures of murine allogeneic cells in which the stimulating cell populations had been heat-inactivated, 8MGuo induced the generation of specific allogeneic cytotoxic T lymphocytes. Together, these results suggest that a simple derivatized nucleoside can induce both T and B cell differentiation without concomitant proliferation, and thus represent a unique probe for studying events in lymphocyte differentiation.     

T cell reactivity with allergoids: influence of the type of APC

The use of allergoids for allergen-specific immunotherapy has been established for many years. The characteristic features of these chemically modified allergens are their strongly reduced IgE binding activity compared with the native form and the retained immunogenicity. T cell reactivity of chemically modified allergens is documented in animals, but in humans indirect evidence of reactivity has been concluded from the induction of allergen-specific IgG during immunotherapy. Direct evidence of T cell reactivity was obtained recently using isolated human T cells. To obtain further insight into the mechanism of action of allergoids, we compared the Ag-presenting capacity of different APC types, including DC and macrophages, generated from CD14+ precursor cells from the blood of grass pollen allergic subjects, autologous PBMC, and B cells. These APC were used in experiments together with Phl p 5-specific T cell clones under stimulation with grass pollen allergen extract, rPhl p 5b, and the respective allergoids. Using DC and macrophages, allergoids exhibited a pronounced and reproducible T cell-stimulating capacity. Responses were superior to those with PBMC, and isolated B cells failed to present allergoids. Considerable IL-12 production was observed only when using the DC for Ag presentation of both allergens and allergoids. The amount of IL-10 in supernatants was dependent on the phenotype of the respective T cell clone. High IL-10 production was associated with suppressed IL-12 production from the DC in most cases. In conclusion, the reactivity of Th cells with allergoids is dependent on the type of the APC.     

DRw antisera react with activated T cells.

Nylon wool-purified T cells appear to be nonreactive in a lymphocytotoxicity assay with HLA-DRw antisera and complement before cell activation. However, after activation in mixed lymphocyte culture, responder cells express determinants that are strongly reactive with DRw alloantisera after 6 days and gradually disappear by 16 to 18 days. Restimulation of the primed cells resulted in re-expression of the blast determinants. Mitogenic stimulation with Con A or purified PHA (HA-17) also resulted in temporary expression of these determinants; reactivity usually conformed to DRw genetic restriction; however, occasional extra reactions occurred that were variable depending on the method of activation (i.e., MLC, Con A, or HA-17). The results suggest the presence of additional allospecificities within some of the DRw antisera that react with "Ia-like" antigens on activated cells from unique subsets of T cells. Whether these DRw antisera contain antibodies against T cells or agains activation or differentiation T cell antigens is not as yet clear.     

Specific antigen-binding and antibody-secreting lymphocytes associated with the erythrocyte autoantibody responses of NZB and genetically unrelated mice.

The receptor characteristics as well as incidence of antigen-binding lymphocytes (ABL) or B and T cell classes with membrane receptors specific for the exposed (X) and cryptic (HB) murine erythrocyte autoantigens were examined in NZB and nine control strains of mice. Whereas only NZB and NZB hybrid mice synthesize anti-X autoantibody pathogenetically implicated in the genetically determined autoimmune hemolytic anemia, the NZB as well as control strains synthesize the ubiquitous anti-HB anti-erythrocyte autoantibody. By utilizing immunocytoadherence assays, maximum numbers of specific ABL of both B and T lymphocyte classes were optimally demonstrated at erythrocyte:lymphocyte ratios of 20:1 and after lymphocyte fixation at 56 degrees C for 20 min. Surface membrane receptor specificity was established by inhibition with semi-purified soluble X or HB autoantigen. Inhibition of immunocyto-adherence with class specific antisera to mouse immuno-globulins demonstrated that the receptors on both B and T cells were of IgM class. Specific receptors regenerated in vitro after trypsinization which excluded the role of cytophilic antibody in the immunocytoadherence reactions. B lymphocyte ABL reactive with the X autoantigen were demonstrable in NZB, NZB hybrid, and control mice. Only in NZB and NZB hybrid mice, strains that uniformly synthesize anti-X autoantibody, were X ABL of T lymphocyte class demonstrated. The presence and incidence of T lymphocyte X ABL is compatible with the expression of a single dominant gene carried by the NAB strain. The incidence of B lymphocyte X ABL increased with age, suggesting proliferation of this cell population. HB ABL of both B and T lymphocyte classes were observed in all strains, concordant with the ubiquitous presence of humoral anti-HB autoantibodies. Differentiation of precursor B cells are evaluated by PFC assay of cells secreting specific autoantibodies. Anti-X PFC were observed only in NZB and NZB hybrid mice; and the observed frequency suggested that less than 3.5% of the specific ABL were differentiated for the secretion of anti-X autoantibody. Anti-HB PFC were observed in all strains and represented as high as 11.8% of specific ABL. Genetic determination of the anti-X anti-erythrocyte autoantibody response does not prescribe the presence of precursors of the antibody-forming cell, but rather appears to influence regulation of the differentiation of these cells. These data suggest that circumvention of immunologic tolerance to this specific erythrocyte autoantigen may occur at the level of the T lymphocyte; or alternatively, that T lymphocytes as well as B lymphocytes, are induced to proliferate and differentiate in the NZB strain.     

Amplification of lymph node cell tube leukocyte adherence inhibition (LAI) reactivity by leukocyte adherence inhibition factor (LAIF)

This investigation examines the immunologic basis for specific antigen-induced tube leukocyte adherence inhibition (LAI) reactivity of draining lymph node cells (LNC) from dogs with canine transmissible venereal sarcoma (CTVS). CTVS regressor LNC, macrophage-depleted LNC, and enriched T lymphocyte fractions, but not enriched B lymphocyte fractions, were specifically reactive to CTVS antigen extract in direct tube LAI. In addition, regressor LNC amplified tube LAI responses by generating supernatants with leukocyte adherence inhibition factor (LAIF) activity for normal dog indicator LNC and enriched peripheral blood mononuclear cells (PBMC) in an indirect tube LAI assay. However, macrophage-depleted LNC and enriched T lymphocyte fractions failed to generate supernatants with LAIF activity, suggesting that macrophage accessory cells play a central role in the amplification of tube LAI. Interestingly, CTVS regressor peripheral blood leukocytes (PBL) and PBMC, which were specifically reactive in direct tube LAI, also failed to generate supernatants with LAIF activity. These findings demonstrate a distinction between LAIF-mediated amplification and direct tube LAI reactivity, and suggest that leukocyte populations with differing cellular proportions and from different immunologic compartments may participate in tube LAI via different mechanisms.     

Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro.

Autologous cell mixtures containing T cells, B cells, and adherent accessory cells from individuals primed to the malaria parasite Plasmodium falciparum by repeated natural infections were investigated for induction of Ig and antibody secretion in vitro. In vitro activation of cell cultures with two synthetic peptides corresponding to immunodominant T cell epitopes of the merozoite Ag ring-infected erythrocyte surface Ag (Mr 155,000) (Pf155/RESA), one from its carboxyl-terminal repeat and one from its nonrepeated amino-terminal region, gave rise to significant IgG secretion. Supernatants from lymphocyte cultures activated with either one of these peptides contained antibodies reacting with P. falciparum Ag in immunofluorescence assays and with Pf155/RESA peptides in a slot blot assay. No anti-P. falciparum antibodies were induced in the medium controls by lymphocyte stimulation with either tetanus toxoid or PWM. Induction in vitro of anti-Pf155/RESA antibodies was correlated with the presence of such antibodies in the sera of the lymphocyte donors, suggesting that the induction of antibody secretion reflected a secondary response in vitro of in vivo primed cells. Inspection of antibody profiles in individual donors revealed that the peptide corresponding to a sequence in the 3' repeat region induced anti-Pf155/RESA peptide antibodies reacting with identical or related and cross-reacting sequences in the 3' or 5' repeat region of the molecule. In contrast, the peptide corresponding to a nonrepeated T cell epitope in the amino terminus of the molecule only induced antibodies to an immunodominant amino-terminal B cell epitope partly overlapping with the T cell reactive sequence. Similar findings were made in the lymphocyte donors' plasma, frequently displaying significant correlations between antibody reactivities to the repeat peptides but not between these reactivities and those to the amino-terminal peptide. The marked specificity of this antibody formation in vitro suggests an underlying process of cognate recognition involving Ag-specific T and B cells reacting with different segments of the inducer peptide. The present experimental system should be well suited for identification of Th epitopes capable of inducing the production of antibodies of defined specificity in the human system.     

Cell types involved in cytotoxicity induced by heated cells and inhibition of this cytotoxicity by anti-human B cell sera.

We have studied the induction of cytotoxic activity in human peripheral blood lymphocytes by heated allogeneic cells. By separating T and B cells from the responder and stimulator cell populations we found that cytotoxic cells are generated in responder T cell populations by both T and the B stimulator cells. Rabbit antisera to a membrane glycoprotein complex (33,000 and 27,000 m.w. by SDS-gel electrophoresis) isolated from a human B cell line were utilized to explore further the nature of the effector cells in this type of cytotoxicity. This antiserum, present during the 6-day-culture period, blocked generation of cytotoxic effector cells. Depletion of cells bearing the B cell antigen from the responder cell population by anti-B cell serum and complement (C) eliminated cytotoxicity. Furthermore, heated cell-induced cytotoxicity was blocked by simply pretreating the responder or the stimulator cell populations with anti-B cell serum in the absence of C. Apparently the human lymphocyte that functions as the effector cell in heated cell-induced cytotoxicity bears the Ia-like antigen that might be important in triggering this type of cytotoxicity.     

Functional recognition of bacterial mitogens by B lymphoma cells: reactivity of WEHI 279.1 to lipopolysaccharide and selection of nonreactive variants

To analyze functional mitogen recognition by reactive B lymphocytes, we studied the effects of bacterial lipopolysaccharide (LPS) on the growth of the WEHI 279.1 B lymphoma line (W279). We found that LPS inhibits, in a dose-dependent manner, the growth of W279 cells in culture and that it reduces the frequency of cells growing as clones under limiting dilution conditions. Furthermore, we show that differential reactivity of "wild-type" cells to increasing LPS concentrations reflects the heterogeneity in the lymphoma cell population and the frequencies of "resistant" variants to each mitogenic concentration. This allowed us to derive variant tumor cell lines and clones, no longer LPS sensitive, either from mass cultures or, in a single-step selection, under limiting dilution conditions in the presence of low and high concentrations of LPS. Although mitogen reactivity is progressively lost upon prolonged culture, resistance to LPS was found to be a stable trait in selected variants, suggesting that it results from loss of functional mitogen recognition by the reactive cells. The specificity of mitogen reactivity or resistance was shown by the fact that some of the variant clones are still reactive to T helper cell-derived factors and others are not. Thus reactivity to LPS and to T cell factors can be separated, suggesting that the cell lines described here provide new tools for the biochemical analysis of B cell activation.     

Lectin extracts of champedak seeds demonstrate selective stimulation of T lymphocyte proliferation.

The effect of extracts of champedak (Artocarpus integer) seed lectin on the proliferation of normal human lymphocyte was investigated. The IgA1 binding lectin was demonstrated to stimulate the proliferation of human peripheral blood mononuclear cells. Action of the lectin on enriched T and B cell populations demonstrated T lymphocyte specificity. The lectin was not mitogenic to B lymphocytes. Optimal stimulation of proliferative response was achieved when cells were subjected to 5 days exposure to the crude lectin at 20 micrograms/ml.     

Polyclonal B cell stimulation and interleukin 1 induction by the mucoid exopolysaccharide of Pseudomonas aeruginosa associated with cystic fibrosis

Mucoid exopolysaccharide isolated from Pseudomonas aeruginosa obtained from colonized cystic fibrosis patients was found to be a potent mitogen for mouse lymphocytes. The responding lymphocyte was a B cell, and we found no evidence that T cell could proliferate or synergize with B cells in response to the mucoid exopolysaccharide. Proliferation was not inhibitable by polymyxin B, which blocks lipopolysaccharide (LPS)-induced proliferation, indicating that a minor LPS contaminant in the purified exopolysaccharide was not the mitogenic component. Mucoid exopolysaccharide induced secretion of IgG, suggesting that it is polyclonal mitogen. It also induced splenic adherent cells (macrophages) to produce interleukin 1. We propose that mucoid exopolysaccharide produced by P. aeruginosa present in the lungs of cystic fibrosis patients may have potent in vivo consequences resulting in aberrant immunoregulation and inhibition of effective immune elimination of P. aeruginosa.     

Distinctive functional properties of human blood L lymphocytes: a comparison with T lymphocytes, B lymphocytes, and monocytes.

Human blood lymphocytes with high affinity Fc receptors have been operationally named L lymphocytes because of membrane-labile IgG markers. L lymphocytes lack membrane-incorporated immunoglobulin and do not form rosettes with sheep red blood cells coated with IgM antibody and mouse complement. These lymphocytes are capable of binding IgG in normal human serum at 4 degrees C and will form rosettes with human lymphocytes coated with Ripley IgG. In this study, functional in vitro properties of isolated L lymphocytes were compared with T lymphocytes, B lymphocytes, and monocytes. To obtain these mononuclear populations, first, plastic adherent monocytes were harvested. T lymphocytes were then isolated by centrifugation of E rosette-forming cells, and other rosetting techniques were employed to isolate L and B lymphocytes by negative selection. The functional properties of L lumphocytes were completely unlike those of T cells, B cells, or monocytes. L lymphocytes did not proliferate in response to mitogens, soluble antigens, or cell surface antigens. Moreover, this population could not replace monocytes in helping T lymphocytes respond to concanavalin A and pokeweed mitogen. Once T cells were supplemented with monocytes, however, the addition of L lymphocytes to the culture greatly enhanced the T lymphocytes proliferative response to phytohemagglutinin, concanavalinA, purified protein derivative (PPD), and streptokinase/streptodornase. L lymphocytes were not a subset of B cells. They did not spontaneously develop surface Ig in culture, and pokeweek mitogen could not induce them to transform and generate cytoplasmic Ig detectable by immunofluorescence. Mixtures of B cells and T cells responded to pokeweed mitogen better than do T cells alone. In contrast, enhanced reactivity with L and T cell combinations was not observed. Another sharp difference between these two populations was the stimulator capacity of each in mixed lymphocyte culture. When B and L lymphocytes were carefully monocyte-depleted, only B cells were effective stimulators of autologous and allogeneic lymphocytes. In comparison with T cells, B cells, and monocytes, L lymphocytes were the only effective killers of human blood lymphocytes sensitized with IgG. L lymphocytes, then, have cytotoxic potential, but cannot proliferate in response to various stimulants or become antibody-producing cells. These findings suggest that L lymphocytes comprise a third lymphocyte population.