Antigen-nonspecific suppression of the immune response in mice as affected by pertussis vaccine

A study was made of the suppressorgenic action of killed whole-cell pertussis vaccine prepared from B. pertussis strains 475 and 305. Thymic and splenic lymphocytes of CBA mice 3-7 days following intraperitoneal or intravenous inoculation of pertussis vaccine were shown to inhibit in an antigen-nonspecific manner the plaque-forming cell (PFC) production in the adoptive transfer experiments. Suppression of graft-versus-host reaction was also observed, estimated by the survival of irradiated (CBA X C57BL/6) Fl mice, or by measuring the endogenous colony formation. Suppression-mediating cells were found to be susceptible to complement-dependent lysis by the anti-I-Jk alloantiserum against the specific marker of suppressor T cells, antigen I-J. Furthermore, thymocytes of pertussis vaccine-treated mice were shown to inhibit the endogenous colony formation in syngeneic mice irradiated in sublethal dose. Thus, B. pertussis vaccination of CBA mice resulted in appearance of suppressor T cells that exerted various inhibitory activities in several experimental test-systems.     

Specific mitogenic activity of 8-Br-guanosine 3',5'-monophosphate (Br-cyclic GMP) on B lymphocytes.

8-Br-cyclic GMP has been found to be a specific B cell mitogen; it triggers athymic nude mice spleen cells and "B mice" spleen cells, nylon adherent, anti-theta and complement-treated cells to proliferate. It does not stimulate thymocytes or purified T cells. The kinetics of the response to Br-cyclic GMP and LPS are almost identical. The mitogenic effect of LPS and Br-cyclic GMP is additive when the two mitogens are given together to cells. Spleen cells (C3H/HeJ strain) that did not respond to LPS were triggered by Br-cyclic GMP to make DNA. In order to achieve maximal stimulation by Br-cyclic GMP, the drug had to be in contact with the cells for more than 24 hr. Br-cyclic GMP was found to be mitogenic for spleen cells from five different mouse strains, but not for human leukocytes. DB-cyclic AMP was found to inhibit the DNA synthesis of T lymphocytes after they interacted with Con A; DB-cyclic AMP had no effect on the ability of the B lymphocytes to be transformed by LPS. The differential effects of cyclic nucleotides on B vs. T lymphocytes are discussed.     

T and B lymphocytes of irradiated, tumor-bearing mice treated with an immunostimulator, pyran.

The T and B splenic lymphocyte populations of BALB/c mice were determined in Madison lung 109 carcinoma-bearing animals. Concurrently, some groups of tumored mice were exposed to 500 rads of whole body irradiation and treated with one dose of a nonspecific immunostimulating agent, pyran. By indirect immunofluorescence, it was found that the percentage of splenic T lymphocytes were significantly depressed in the tumored irradiated mice. Mitogenic studies revealed that the PHA-sensitive T lymphocytes were more depressed in the tumored irradiated mice than in the corresponding Con A-sensitive T lymphocytes. Pyran was relatively effective in reconstituting the T cell compartment of these splenic T lymphocytes.The B cell compartment of the splenic lymphocytes of the tumored irradiated mice was found to be extremely radiosensitive. Utilizing a specific anti-F(ab')₂ serum, no B lymphocytes were detected throughout the duration of testing. Blastogenic studies using LPS as the mitogenic probe revealed that the incorporation of [³H]TdR of the tumored irradiated mice was just slightly higher than background values. Pyran proved to be relatively ineffective in reconstituting the splenic B cells of the tumored irradiated mice.     

Induction of phenotypic lymphocyte differentiation in LPS unresponsive mice by an LPS-induced serum factor and by lipid A-associated protein.

Spleen cells from C3H/HeJ mice are known to be unresponsive to mitogenic stimulation by LPS. We show here that T and B cell precursors of C3H/HeJ mice are unresponsive to induction of differentiation by LPS. Phenotypic differentiation of C3H/HeJ lymphocytes can be induced with DB-cAMP, Lipid A-associated protein, and with a serum factor induced by LPS in LPS responder strains.     

T and B lymphocyte populations of the spleen and thymus in normal and immunosuppressed BALB/c mice.

The antituinor agent 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) has been studied in order to determine its effect on thymic and splenic T and B lymphocytes in normal and immunosuppressed BALB/c mice. Utilizing indirect immunofluorescence and lymphocyte proliferation studies we detected an initial reduction of splenic T and B cells as a result of the administration of an optimal dose, 30 mg/kg, of BCNU. The population dynamics of the thymic lymphocytes are totally different in their mitogenic reactivity than that of the splenic lymphocytes. An initial decline in the PHA and LPS-sensitive splenic lymphocytes of BCNU-treated mice was temporary. However, there was no return to normal levels detected for the Con A-sensitive splenic lymphocytes. On the other hand, the PHA-sensitive thymic lymphocytes of BCNU-treated mice not only failed to repopulate but were totally depleted by the tenth day.     

The use of cytotoxic T cells in the regulation of tumour growth in syngeneic mice

Summary Normal C57BL/6 (B6) spleen cells were cultured with syngeneic EL4 tumour cells, expanded in IL2-containing medium, and tested for anti-tumour activity in vitro and in vivo. The activated cells were highly cytotoxic for EL4 and to a lesser degree killed syngeneic B6 blasts and allogeneic (D2) P815 tumour cells. B6 or BDF1 mice that received these cultured cells by IP injection cleared ¹²⁵IUdR-labelled EL4 cells faster than untreated mice. However, this enhanced clearance was evident only 7–12 days after injection. Since the injected cells had a short half-life (<10% remaining after 48 h) the effect of these cells in vivo was most probably due to the activation of the host's immune system. Mice that received cultured cells survived significantly longer than untreated mice following a lethal dose of EL4 cells. Cultured cells were much more effective in prolonging survival when used in conjunction with cyclophosphamide (CY). In animals receiving either cultured cells with or without CY or CY alone tumour clearance was markedly enhanced 7–12 days after injection.When challenged with a small dose of EL4 tumour cells (1×10⁴ SC per mouse) three of ten B6 mice treated with B6 anti-EL4 cultured cells were able to survive indefinitely. The frequency of CTL precursors to EL4 from the spleen cells of these surviving animals was about five-fold higher than that of normal spleen cells. Furthermore, CTL derived from primed spleen cells were more specific for EL4 than those derived from normal spleen cells.Abbreviations B6 C57BL/6J - BDF1 (C57BL/6J×DBA/2J) F1 - ConA SN concanavalin A supernatant - CTL cytotoxic T lymphocytes - CTL-P cytotoxic T-lymphocyte precursors - CY cyclophosphamide - E/T effector-to-target ratio - IL2 interleukin 2 - IP intraperitoneal - IUdR iododeoxyuridine - IV intravenous - LPS lipopolysaccharide - MST mean survival time     

Mitogenic action of the liquid phase of a microbial suspension of Bordetella pertussis

The suspension of B. pertussis cells in 0.15 M NaC1 solution, used for the preparation of corpuscular pertussis vaccine contains components loosely bound to microbial cells and producing pronounced mitogenic effect on mouse splenocytes at a concentration of 10 micrograms/ml. The mitogenic activity of B. pertussis is due to complex substances (lipopolysaccharide, protein, nucleic acids) with a wide range of molecular weights (70,000 to greater than 400,000). The mitogenic factor showing no leukocyte-stimulating and protective activity has been isolated by sedimentation with ammonium sulfate and gel filtration on Sephadex G-200. The mitogenic activity of B. pertussis lipopolysaccharide in the blast transformation test has been confirmed.     

Regulation of inflammatory responses to Bordetella pertussis by N(G)-monomethyl-L-arginine in mice intranasally infected

To investigate effect of MMLA, an inhibitor of nitric oxide (NO) production, on regulation of inflammatory responses to Bordetella pertussis infection, mice were infected intranasally, and treated with various concentrations of MMLA. Ten days after infection, mice treated with MMLA at dosage of 100 mg/kg, given intraperitoneally in a single dose or for 5 consecutive days, showed at histopathologic examination, a significant decrease of intensity of inflammation (scores, 0.6 +/- 0.2 and 0.9 +/- 0.5 respectively). A decrease of cellular accumulation of neutrophils and lymphocytes in the bronchoalveolar lavage (BAL) fluid was observed in infected mice treated with MMLA, especially at dosage of 10 mg/kg, given in a single dose intraperitoneally. In addition, BP-infected mice treated with MMLA (100 mg/kg, intraperitoneally) for 5 consecutive days showed higher mortality rate than untreated mice infected with B. pertussis, and the number of B. pertussis in lungs of mice treated with MMLA was significantly increased. However, MMLA treatment of infected mice had some effect on levels of IFN-gamma and nitrite/nitrate (end-stable products of NO) in the BAL fluid. This study indicates that NO may play a role either as microbiocidal agent or as a modulator of immune regulation, inasmuch as it may upregulate tissue inflammatory response to B. pertussis.     

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.     

The significance of chorionic gonadotropin in regulating the antigen-independent differentiation of immunocompetent splenic cells]

The functional activity of fractionated splenocytes were investigated in the syngeneic transfer system. Chorionic gonadotrophin (CG) was injected to ovariectomized and noncastrated lymphocyte donors, the T or B lymphocytes being isolated on a column with a nylon fiber and by means of anti-BaO-serum accordingly. In some series CG was introduced only into recipients before their being irradiated and before syngeneic cells transferring. CG has been stated to be capable of influencing either independently or by means of ovarian hormones splenic B lymphocytes without exhibiting a significant effect on T helper precursors as well as radioresistant cells of the splenic stroma. The character of immunoregulating effect was completely dependent on the presence of ovaries in mice and on the hormone dose.     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

Differential effect of vanadate on DNA synthesis induced by mitogens in T and B lymphocytes

Summary The effect of sodium orthovanadate on enhancement of DNA synthesis by T and B cell mitogenic agents was studied using murine thymocytes and splenocytes. Addition of vanadate to thymocyte cultures inhibited the mitogenic response induced by concanavalin A in a dose dependent manner (50% inhibition at 10 M). On the other hand, DNA synthesis induced in thymocytes by pokeweed lectin and periodate treatment essentially was not inhibited at the lower vanadate concentrations that were markedly effective for concanavalin A induced synthesis. In addition, no significant inhibition of mitogenesis of splenic B cells in response to lipopolysaccharide and dextran was detectable at lower vanadate concentrations. In the absence of added mitogens, vanadate was found to be mitogenic for a subpopulation of thymus cells but not for splenocytes or T cell enriched splenocyte populations. These results suggest that vanadate affects the mitogenic responses in lymphocytes and that the interaction of vanadate with T and B cells is different.     

Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and β2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2(-/-) mice responded to anti-IgM worse than those of the wild-type or α7(-/-) mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not β2(-/-) cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7(-/-) B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a "reserve" receptor, which partly compensates the absence of α7 nAChR in α7(-/-) cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B 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.     

Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin

Together with macrophages and dendritic cells, mast cells have recently been shown to interact with certain pathogenic bacteria and present microbial antigens to the immune system. We show here that Bordetella pertussis can adhere to and be phagocytosed by mast cells. In addition, mast cells are able to process and present B. pertussis antigens to T lymphocytes. Furthermore, exposure of mast cells to B. pertussis induced the release of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). The release of IL-6 was strongly reduced by pertussis toxin expressed by B. pertussis . The production of IL-10, but not that of IL-4, by mast cells was also inhibited by pertussis toxin. Depletion of mast cells in vivo resulted in significant reduction of early TNF-α production in bronchoalveolar lavage (BAL) fluids of B. pertussis -infected mice. These data suggest that mast cells may play a role in the induction of immune responses against B. pertussis through the release of cytokines, especially TNF-α.     

IL-2 production, IL-2 receptor expression, and IL-2 responsiveness of spleen and mesenteric lymph node cells from inbred mice infected with Trichinella spiralis

The in vitro production of IL-2 and IL-2R expression by lymphoid cells of inbred mice of strong (NFS), intermediate (C3H), or weak (B10.BR) in phenotype of Trichinella spiralis (TS) rejection was measured during a primary infection. Maximum production of IL-2 by spleen and mesenteric lymph node (MLN) cells occurred at 5 days postinfection. Cell depletion experiments demonstrated that Lyt-1.2+ T cells were predominantly responsible for in vitro IL-2 production. Cells from strong-responder NFS mice produced more IL-2 than cells from intermediate-responder C3H or weak-responder B10.BR mice. Similarly, after TS infection, NFS mice had significantly more IL-2R expressing MLN cells than B10.BR or C3H MLN cells. All mouse strains displayed a dose-dependent increase in in vitro IL-2 production after infection with 100 to 800 TS. This effect was most pronounced in NFS mice. Limiting dilution analysis of day 5 infected MLN cells demonstrated that the frequency of TS-reactive CD4+ cells was threefold higher in NFS mice than B10.BR and fourfold higher than in C3H mice. Finally, MLN cells taken from infected NFS mice responded to an exogenous source of IL-2, whereas MLN cells from infected C3H or B10.BR mice were unable to do so. We conclude that strong responsiveness in parasite rejection may be related to the amount of IL-2 produced as well as to the capacity of the lymphocytes of each mouse strain to respond to IL-2. Although these differences help explain the strong rejection phenotype of NFS mice, they fail to separate C3H and B10.BR mice where TS-responsive CD4+ precursors, IL-2 production, and dose responsiveness are all lower for the intermediate phenotype (worm rejection) C3H than the weak phenotype B10.BR mice.     

Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-gamma responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.     

The effects of sodium metaperiodate on T and B lymphocytes.

The differential mitogenic response of T and B lymphocytes to sodium metaperiodate has been investigated. It was found that periodate treatment leads to lymphocyte stimulation in spleen cells from Balb/c mice but not in spleen cells from the congenitally athymic nu/nu mice. In addition, treatment of Balb/c spleen cells with anti-θ serum plus complement lowers the mitogenic response to periodate and to concanavalin A without affecting the response to lipopolysaccharide. These results suggest a requirement for the presence of T lymphocytes in the initiation of a response to periodate. Spleen cells from nude mice also react with periodate, and their ability to respond to B cell mitogens is impaired after treatment with the chemical reagent.     

Differentiation of B lymphocytes in C3H/HeJ mice: the induction of Ia antigens by lipopolysaccharide.

The lipid A moiety of bacterial lipopolysaccharide (LPS) elicits several types of responses in murine B lymphocytes. First, lipid A induces the nonproliferative expression of cell surface antigens in more immature cell types. Second, lipid A induces a mitogenic response in more mature B cell types. Lipid A induces the expression of Ia antigens on bone marrow cells from C3H/DiSn but not C3H/HeJ mice. The Ia-inducible cells possess surface immunoglobulin. Agents that elevate intracellular levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) induce the appearance of Ia antigens on B lymphocytes from both C3H/HeJ and C3H/DiSn mice, suggesting that lipid A exerts its inductive effects by increasing cyclic AMP levels in cells. In contrast to what is observed by using other strains of mice, mature B lymphocytes from C3H/HeJ mice do not support a mitogenic response to lipid A. The subpopulation of B lymphocytes in C3H/HeJ mice that normally respond mitogenically to LPS not only appear to lack an LPS-response mechanism utilized in the mitogenic pathway, but they lack the LPS-response pathway of the immature B cell types. A lipid A-bound protein (LAP) induces both the expression of Ia and a mitogenic response in the different subpopulations of B lymphocytes from C3H/HeJ and C3H/DiSn mice. The genetic defect in C3H/HeJ mice that limits responses to lipid A may be associated with a receptor that is normally expressed on many different cell types.     

T and B lymphocyte populations of tumor-bearing mice treated with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) or pyran

Summary The immunostimulator pyran copolymer and the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) have been studied to determine their effect on T and B lymphocytes from BALB/c mice bearing a syngeneic tumor, Madison lung 109 carcinoma. Indirect immunofluorescent and mitogenic assays revealed that the tumor-bearing controls usually had T and B cell populations lower than those observed for the normal controls. The tumor-bearing group treated with BCNU generally had T and B cell levels lower than the tumor controls. The administration of pyran resulted in an increase of T cells and a temporary increase in the B cell population. Although this increase in the B cell population of tumor-bearing mice treated with pyran is higher than that seen for the untreated tumor controls it was never higher than in the normal control mice. The experimental results indicate that pyran appears to reconstitute the depressed T cell population and has a transitory effect on the B cell population resulting from tumor burden.