anti-SRBC Plaque-forming Cell Response Research Articles

IgA responses in xid mice: oral antigen primes Peyer's patch cells for in vitro immune responses and secretory antibody production.

Because the gut-associated lymphoreticular tissue (GALT), e.g., Peyer's patches (PP), of X-linked immunodeficient (xid) mice possesses a subpopulation of mature B cells, we have characterized the ability of xid mice to respond to the thymic-dependent antigen sheep erythrocytes (SRBC) given by the oral route. Gastric intubation of SRBC to xid (CBA/N X DBA/2) F1 male or CBA/N mice, followed by the in vitro culture of dissociated PP cells with SRBC, resulted in IgM, IgG1, IgG2, and high IgA anti-SRBC plaque-forming cell (PFC) responses. The addition of unprimed PP but not splenic T cells to splenic xid B cell cultures resulted in IgM anti-SRBC PFC responses, suggesting the importance of GALT T cells for support of the immune responses to SRBC by splenic B cells from xid mice. Furthermore, purified PP T cells from SRBC orally primed xid mice supported in vitro IgA anti-SRBC PFC responses in B cell cultures from either the PP or the spleens of nonprimed xid mice. Higher IgA responses, however, occurred in PP, when compared with splenic B cell cultures. Additional evidence that the GALT of xid mice contains functional IgA precursor cells was provided by the finding that cloned H-2k PP T helper cells (PP Th A) supported IgA responses in PP B cell cultures derived from (CBA/N X C3H/HeN) F1 male (xid) mice. On the other hand, splenic B cells from these xid mice, in the presence of PP Th A cells, did not support in vitro responses. These results suggest that unique subpopulations of T cells occur in the GALT of xid and normal mice; one T cell subpopulation may induce immature B cells to become precursor IgA cells in the PP. A separate GALT T cell subpopulation, e.g., isotype-specific helper T cells, effectively collaborates with mature IgA B cells for the induction of IgA responses to orally administered antigen. When xid mice were gastric intubated with SRBC, followed by i.p. injection of SRBC, good splenic IgA anti-SRBC PFC responses were seen. Salivary and serum IgA antibodies were also detected in these xid mice. Nevertheless, the magnitude of the anti-SRBC response in xid mice was lower than that seen in similarly treated normal mice. These studies indicate that the GALT of both xid and normal mice possess unique populations of T cells that support in vitro responses in xid B cell cultures from either the spleen or the PP, which direct the mature B cell populations present toward IgA isotype-specific responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: III. Augmentation of the Antibody Response to Subcutaneously Injected Sheep Red Blood Cells by the Adjuvant Polysaccharide

The adjuvant action of the O3 antigen of Klebsiella (KO3) on the antibody response to sheep red blood cells (SRBC) was elucidated by injecting both KO3 and SRBC subcutaneously at the right inguinal region of SMA mice. We demonstrated that KO3 exhibits a novel ability to augment anti-SRBC plaque-forming cell responses in both the local lymph node and the spleen at a relatively late stage of immunization. Escherichia coli lipopolysaccharide, dextran sulfate and concanavalin A showed such an action only minimally. In parallel with the development of the adjuvant action, KO3 definitely activated B cells in the local lymph node polyclonally for either IgM or IgG synthesis, suggesting that the mechanism of the adjuvant action includes direct stimulation of B cells by KO3 at the late stage. Neither increase in trapping of lymphocytes in the local lymph node nor change in tissue distribution of antigen was shown to be primarily involved in the mechanism of the adjuvant action.

In vivo immune response to a T-cell-dependent antigen by cultures of disassociated murine Peyer's patch.

The first line of defense against pathogens that enter the host by the oral route appears to involve the gut-associated lymphoreticular tissue-e.g., Peyer's patches (PP). Although animals can readily be immunized by orally administered antigen that mobilizes the secretory immune system, there is a total lack of local antibody synthesis in the PP and the cellular basis for this deficiency remains a mystery. A lymphoreticular cell population, obtained when murine PP were treated with a neutral protease (Dispase), consisted of accessory cells [macrophages (MPhi)] and T and B lymphocytes. In vitro cultures of these PP cell preparations with the thymic-dependent antigen sheep erythrocytes (SRBC) resulted in good anti-SRBC plaque-forming cell (PFC) responses. The time courses of these responses were identical to those seen with spleen cell cultures. Submitogenic concentrations of concanavalin A (Con A) and optimal doses of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) and lipopolysaccharide (LPS) enhanced in vitro responses of PP cell cultures to SRBC. PP possess fully functional antigen-presenting MPhi because incubation of optimal proportions of splenic T and B cells with purified populations of PP MPhi supported good in vitro immune responses. Murine PP possess all of the necessary elements for an IgA immune response because PP cell cultures derived from mice orally primed with SRBC and immunized with SRBC in vitro gave high IgA anti-SRBC PFC responses. All of the adjuvants tested (LPS, MDP, and Con A) enhanced IgA responses in PP cell cultures from orally primed mice; however, Con A induced the greatest enhancement. These results demonstrate that murine PP possess MPhi capable of accessory cell functions for in vitro immune responses and that oral priming with antigen induces the precursor T- and B-cell populations necessary for IgA responses, that are potentiated by adjuvants, in PP cell cultures. Thus, murine PP possess the lymphoreticular cells required for antibody responses; however, the tissue architecture likely prevents local responses in vivo. The finding that enzymatically dissociated PP contain all of the necessary cellular components for antibody synthesis, whereas the in vivo tissue architecture prevents the complex interactions necessary for this response, suggests that the initial inductive events take place in situ, and additional cell interactions are required for final differentiation of IgA-synthesizing plasma cells to occur at distant mucosal sites.

Immunopharmacological profile of TEI-3096: a new immunomodulator.

TEI-3096 [6-p-chlorobenzyl-5H-2,3,6,7-terahydro-5,7-dioxothiazolo (3,2-a) pyrimidine], a novel thiazolopyrimidine compound has been shown to suppress adjuvant arthritis in rats without any effect on conventional inflammation. We examined the immuno-pharmacological profile of TEI-3096 in murine lymphocytes. The blastformation induced by Con A or LPS was inhibited by addition of 50-500 microM concentrations of TEI-3096. This compound suppressed elevated plaque-forming cell (PFC) response against T cell-dependent antigen. However, TEI-3096 had no effect either on normal PFC response or on antibody formation against T cell independent antigen. Although orally administered TEI-3096 had no obvious effect on anti-SRBC PFC response in normal mice, it normalized the colchicine-induced enhancement of antibody formation against TNP-HGG. TEI-3096 also enhanced the delayed type hypersensitivity in mice and rats. These results suggests that TEI-3096 restores the abnormal immune response. Therefore, it may be useful for the treatment of auto-immune disease such as rheumatoid arthritis.

Restoration by cyclic guanosine monophosphate and extracellular calcium of butylated hydroxyanisole-suppressed primary murine thymus-dependent antibody response.

The effect of dibutyryl cyclic guanosine monophosphate (dbc-GMP) on butylated hydroxyanisole (BHA)-induced suppression of the primary in vitro thymus-dependent antibody response of BDF1 mouse spleen cultures was studied. When added at 0 hr relative to antigen addition, 1 mg of dbc-GMP (8 mM) restored by greater than 70% the BHA-inhibited primary immunoglobulin (Ig)M plaque-forming cell (PFC) response to sheep erythrocytes (SRBC). The suppression was not reversed by the addition of 50 microgram of dibutryl cyclic adenosine monophosphate (dbc-AMP), which is known to reverse suppressor T-cell activity. The addition of 10 mM extracellular calcium (Ca2+) at the same time as antigen to BHA-inhibited cultures resulted in more than 80% restoration of the anti-SRBC PFC response. Quantitation of c-GMP by radioimmunoassay demonstrated that BHA lowered by 58% the c-GMP content of splenic lymphocytes and abrogated the ability of lipopolysaccharide of E. coli (LPS) to elevate c-GMP levels in splenic lymphocytes. The data suggest that BHA exerts its immunosuppressive effect on the primary in vitro antibody response by inhibiting guanylate cyclase activity and effectively lowering c-GMP levels; exogenous dbc-GMP and Ca2+ can freely reverse the immunosuppressive effect of BHA.

Activation of human B lymphocytes: XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

We have previously described a system for the in vitro induction of anti-sheep red blood cell (SRBC) plaque-forming cells (PFC) in human peripheral blood (PB) following polyclonal triggering with pokeweed mitogen (PWM). A soluble antigen derived from hemolyzed SRBC was added to cultures of PB mononuclear cells. Addition of this antigen to PWM-stimulated cultures led to a two- to three-fold increase in the anti-SRBC PFC response seen with PWM alone. The synergistic response was specific for soluble SRBC antigen (SRBC-SA). Depletion of adherent cells eliminated the synergy between PWM and SRBC-SA. The plaques produced were macroscopic, inhibited by the addition of puromycin, and were not seen in the absence of complement. The synergistic response was dependent on the presence of human A sera absorbed with SRBC. Thus, these studies demonstrate clear-cut synergy between SRBC-SA and PWM in the SRBC-specific response of human PB B cells indicating the potential role of nonspecific signals in the antigen-specific antibody response of human B cells.

Antibody feedback regulation in vitro: T helper cell activation and T-B cell cooperation are not impaired by anti-carrier antibody.

Effects of antibody feedback regulation on T helper (Th) cell functions required in plaque-forming cell (PFC) responses were studied in vitro. Using a sensitive functional assay, it was found that serum from immunized mice did not inhibit sheep erythrocyte (SRBC)-, or keyhole limpet hemocyanin-specific Th cell activation. SRBC-"pulsed" splenic adherent cells activated Th cells more efficiently when pulsed in the presence of anti-SRBC antiserum. PFC responses against, 2,4,6-trinitrophenyl (TNP) coupled to soluble carriers were studied under conditions in which linkage between hapten and the priming carrier was either required or not. Under both conditions, anti-carrier antiserum had an enhancing or no effect; only anti-hapten antiserum or monoclonal anti-hapten antibody were inhibitory. Thus, these results did show that antibody feedback was essentially hapten-specific and did not interfere with Th cell functions, except, possibly, that anti-hapten antibody interfered with linked cooperation at the level of hapten-B cell interaction, in addition to its inhibitory effect detectable under conditions of unlinked cooperation. Since SRBC-pulsed, adherent cells were very inefficient in stimulating anti-SRBC PFC responses, the results further suggested that phagocytosed antigen could only be recognized by Th cells but not by B cells. Finally, by attempting to further study hapten-B cell interactions, no PFC responses against TNP coupled to soluble carriers could be generated by substituting for Th cells with different types of secondary mixed leukocyte culture supernatant in cultures with pure surface Ig-positive cells. Only anti-SRBC PFC responses were generated in such cultures.

Mechanism of Epstein-Barr virus-induced human B-lymphocyte activation

The mechanism of Epstein-Barr virus (EBV) activation of human B lymphocytes toward Ig synthesis was investigated in a direct anti-sheep red blood cell (SRBC) antibody plaque-forming cell (PFC) system. Exposure of human peripheral blood lymphocytes to EBV in vitro resulted in an anti-SRBC PFC response in 12 of 16 normal donors. The EBV-induced anti-SRBC PFC response did not require the presence of autologous helper T lymphocytes, but was inhibited by the presence of autologous concanavalin A-generated suppressor T cells. Live virus was required for B-cell activation since the EBV-induced PFC response was inhibited by exposure of EBV to ultraviolet light. Using fluorescent techniques which detected simultaneous intracytoplasmic (ICP) Ig production and the presence of EB nuclear antigen, we found that most, if not all, EBV-activated ICP Ig-positive cells were virally infected. Thus, these studies suggest that viral infection of Ig-producing B lymphocytes is required for EBV-induced polyclonal B-lymphocyte activation. Although the participation of T lymphocytes is not required for the induction of EBV-triggered B-lymphocyte Ig production, activated T lymphocytes can serve as modulators of this response.

The possible role of prostaglandins in mediating immune suppression by nonspecific T suppressor cells

Nonadherent splenic lymphoid cells from tumor-bearing animals exhibit a poor primary in vitro plaque-forming cell (PFC) response to SRBC. This is attributable to the presence, in this population, of a T suppressor cell. This nonadherent population suppressed the in vitro anti-SRBC PFC response of normal spleen cells (NSC). This nonspecific suppressive effect was eliminated by adding indomethacin, a prostaglandin synthetase inhibitor, to the in vitro cultures. These effects were seen at indomethacin concentrations which have no direct effect on the NSC response. When exogenous prostaglandins were added to NSC cultures plus SRBC, prostaglandins PGE 1 and PGE 2 were shown to markedly inhibit the PFC response at a wide range of concentrations, whereas PGA 1, PGF 1α, and PGF 2α were inhibitory only at the highest concentrations tested.

Separation of a serum-derived tumoricidal factor from a helper factor for plaque-forming cells.

C3H/HeN mice administered BCG followed by lipopolysaccharide 14 days later released into their serum a cytotoxic factor for tumor cells and a factor that restored the anti-SRBC plaque-forming cell response of nude mouse spleen cells (helper activity). Gel filtration of serum containing the cytotoxic and the helper activities indicated that both factors exhibited an apparent m.w. of 125,000 to 150,000. The helper activity was also found at lower m.w. (60,000 and 13,000) suggesting the possibility that this factor existed in aggregated forms. Gel filtration of ammonium sulfate (40 to 60% saturation) precipitated serum in a high ionic strength buffer (1.6 M NaCl) resulted in shifts in the apparent m.w. of both factors. The cytotoxic factor now exhibited a m.w. of 55,000. The helper activity eluted with an apparent m.w. of 13,000, and thus was clearly separated from the cytotoxic factor. The helper activity was further shown to co-elute with macrophage-derived lymphocyte activating factor (LAF). This as well as other data represent the first demonstration of in vivo produced LAF.

Feedback suppression of the immune response in vitro. II. IgVH-restricted antibody-dependent suppression.

Feedback suppression of the primary humoral immune response to sheep erythrocytes (SRBC) in vitro was induced with cell-free supernate material derived from antigen-(SRBC) activated B (sIg+) cells. This soluble products bears Ig determinants and binds to the eliciting antigen (SRBC). The activity of this antibody in suppressing anti-SRBC plaque-forming cell responses is restricted to spleen cell cultures containing B cells sharing VH genes with the B cells producing the suppressive antibody. The anti-hapten (trinitrophenyl) response to derivatized SRBC is not affected by antigen-primed B cells or their products. These data are compatible with suppression being mediated by anti-antigen antibody, either (a) via blockade of different SRBC epitopes recognized by a limited set of B cell clones in each mouse strain, (b) via triggering of an anti-idiotypic response, either antibody or suppressor T cell in nature, restricted to activity in cultures containing B cells sharing VH structures with the original antibody, or (c) via interference by preformed antibody with T cell help directed at idiotype bearing B cells.

In vitro idiotypic suppression of chronic lymphocytic leukemia lymphocytes secreting monoclonal immunoglobulin M anti-sheep erythrocyte antibody.

A patient with chronic lymphocytic leukemia was found to have B cells with surface immunoglobulin (Ig)M manifesting anti-sheep erythrocyte (SRBC) specificity together with a high titer serum monoclonal anti-SRBC IgM antibody. By immunizing a sheep with the monoclonal IgM antibody, followed by multiple absorptions against normal human IgM, an anti-idiotype (Id) antibody was obtained. The serum IgM anti-SRBC antibody was then demonstrated to share the same idiotypic determinants with the surface IgM with anti-SRBC specificity on the patient's B cells. The anti-Id antibody suppressed the spontaneous secretion of anti-SRBC antibody as well as the pokeweed mitogen-induced anti-SRBC antibody production as measured by a hemolysis-in-gel plaque-forming cell assay. In contrast, pokeweed mitogen-induced anti-SRBC plaque-forming cell responses of normal individuals were not suppressed by the anti-Id antibody. Thus, this study demonstrates in vitro suppression of human B-cell function by anti-Id antibody.

Conditions for induction of specific and polyclonal antibody production by Cowan 1 bacteria and by pokeweed mitogen.

Cowan 1 bacteria and pokeweed mitogen (PWM) were used to induce the formation of direct plaque-forming cells (PFC) against sheep erythrocytes (SRBC) by human peripheral blood lymphocytes in vitro. It was necessary to absorb the serum supplement with SRBC before culture to obtain anti-SRBC PFC. Alternatively, sheep serum could be added to the cultures. The PFC response was specific, and the response was equally high in cultures with a mixture of absorbed and non-absorbed serum as in cultures with absorbed serum only. Cowan 1 and PWM could also induce synthesis and secretion of both IgM and IgG polyclonal antibodies. Absorption with SRBC or addition of sheep serum had no effect on this synthesis. Thus it seems likely that the induction of anti-SRBC PFC by Cowan 1 or PWM needs the presence of SRBC antigen and is the result of a synergism between mitogen and antigen. Consequently, the anti-SRBC PFC response obtained after stimulation with Cowan 1 or PWM in SRBC-absorbed serum does not reflect a true polyclonal antibody response.

Emergence of Suppressor Cells of Immunoglobulin Synthesis During Acute Epstein-Barr Virus-Induced Infectious Mononucleosis

Abstract In vitro polyclonal immunoglobulin (Ig) production and immunoregulation were studied in peripheral blood lymphocytes (PBL) from 17 acute (<2 weeks after onset of symptoms) and 14 convalescent (2 to 5 weeks after onset of symptoms) Epstein-Barr virus (EBV)-induced infectious mononucleosis (IM) patients. PBL from both groups developed fewer anti-sheep red blood cell (SRBC) plaque-forming cells (PFC) (p < 0.001) and fewer intracytoplasmic (ICP) Ig-positive cells (p < 0.01) than normal PBL after in vitro stimulation with pokeweed mitogen (PWM). However, there was a significant trend toward increased PWM-induced anti-SRBC PFC with increasing time after onset of IM symptoms (R = 0.389, p < 0.025). PBL from acute but not convalescent IM patients significantly suppressed the PWM-induced anti-SRBC PFC response (15 ± 8% of expected, p < 0.001) and the ICP Ig response (21 ± 8% of expected, p < 0.001) of normal PBL in allogeneic co-cultures. The suppressor cell activity in PBL of acute IM patients was removed by treatment with anti-T cell antiserum and complement as well as by the addition of 10-5 M hydrocortisone to the cell cultures. In addition, acute IM cells in the presence of normal T cell help were able to respond normally in vitro to PWM to generate both anti-SRBC PFC and ICP Ig. Thus, PBL from both acute and convalescent IM patients manifest decreased Ig production after in vitro polyclonal stimulation. These decreased responses were associated with the emergence of corticosteroid sensitive suppressor T cells that were demonstrable in PBL of acute IM patients, and were no longer demonstrable in PBL of convalescent IM patients. The transient presence of peripheral blood suppressor T cells in these acute IM patients was associated with an uneventful clinical course and may represent in vivo immunoregulatory cell activation in response to EBV-induced polyclonal B cell activation.

The macrophage, target cell of the synthetic adjuvant muramyl dipeptide

The mechanism of adjuvant activity of the synthetic glycopeptide N-acetylmuramul-L-alanyl-D-isoglutamine or muramyl dipeptide (MDP) was studied using in vitro plaque-forming cell (PFC) response to sheep erythrocytes (SRBC). Addition of MDP to DBA/2 mouse spleen cell cultures resulted regularly in a 2 to 3-fold increase of PFC numbers/10(6) recovered cells (p less than 0.01). Supernates (SPN) from MDP-stimulated cultures added to standard spleen cell + SRBC cultures brought about even more important increases of PFC numbers (p less than 0.01 to p less than 0.001). SPN from cultures supplemented with MDP alone (without SRBC) were more active than those of cell + MDP + SRBC cultures, and SPN removed on day 3 of culture were more active than those of day 5. This activity of SPN was maintained accross an H-2 histocompatibility barrier. Although pretreatment of spleen cells with anti-theta antigen serum entirely suppressed the anti-SRBC PFC response in spite of the presence of MDP, SPN from these cultures were as active as SPN from normal spleen cell MDP-stimulated cultures. In contrast, pretreatment of spleen cells with specific rabbit anti-mouse macrophage serum entirely suppressed both anti-SRBC response and SPN activity. It was concluded that the target cell for MDP is the macrophage which releases factors ultimately acting on B cells through T cell mediation.

Suppression of in vitro antibody responses by Listeria primed spleen cells

Spleen cells from mice primed with virulent Listeria monocytogenes do not develop an anti-SRBC plaque forming cell response to SRBC in culture. Furthermore, when Listeria primed spleen cells are co-cultured with normal spleen cells and SRBC, the anti-SRBC response of the normal cells is suppressed. Listeria primed spleen cells from T cell depleted donors are equally effective at immunosuppression. The immunosuppressive effect does not appear to be due to the presence of the bacterium or its products per se in the cultures. Furthermore, the effect cannot be transferred across a 0.45 μm pore membrane. Kinetic studies show that the immunosuppressive effect develops by 2 days post- Listeria inoculation and peaks by Day 6. Low doses of Listeria are not immunosuppressive and produce some enhancement effect. From these results, it is suggested that a population of non-T cell dependent cells develop in Listeria primed hosts that nonspecifically suppress the response of B cells to an unrelated antigen in culture.

Nonspecific elicitation of antibody-forming cells in the mouse spleen by bacterial lipopolysaccharide.

Mechanisms of nonspecific elicitation of anti-sheep erythrocyte (SRBC) hemolytic antibody plaqueforming cells (PFC) in mouse spleens with an injection of bacterial endotoxin (lipopolysaccharide (LPS)) were studied in comparison with the genesis of naturally occurring "background" PFC in normal mouse spleens and of rapidly arising PFC in mouse spleens after immunization with SRBC. The cytokinetic pattern of anti-SRBC PFC response after an injection of LPS was quite different from that of the response elicited after immunization with SRBC. In addition, even though LPS nonspecifically elicited anti-SRBC PFC response in mice, LPS could not confer any immunological memory on mouse immunocytess for a "secondary-type" anti-SRBC PFC response to restimulation with LPS or SRBC. The administration of rabbit anti-mouse thymocyte immunoglobulin or anti-SRBC antiserum in mice markedly suppressed the PFC response after immunization with SRBC, but did not do so after stimulation with LPS. Neonatally thymectomized mice could still respond to stimulation with LPS, producing anti-SRBC PFC in their spleens. Injections of actinomycin D or cyclophosphamide into mice resulted in obvious reductions of the PFC responses elicited by either LPS or SRBC. However, injections of these immunosuppressive antisera or drugs did not affect the number of anti-SRBC PFC in normal mouse spleens. These results suggest that the genesis of anti-SRBC PFC developed under different conditions, i.e., background PFC, LPS-stimulated PFC, and antigen-stimulated PFC, are quite different from each other, and that the nonspecific elicitation of anti-SRBC PFC by LPS does not require the helper function of T lymphocytes. No obvious difference, however, was observed in the time of ontogenic maturation among these three different anti-SRBC PFC in the mouse spleens judging from when they were first manifested after birth.

Antibody formation in mouse bone marrow: II. Evidence for a memory-dependent phenomenon

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity in a primary response to sheep red blood cells (SRBC), while PFC activity in the secondary response to SRBC can be clearly demonstrated. This phenomenon was studied by means of cell transfer experiments. T cells, which are involved in an anti-SRBC PFC response, were shown to be very scarce in normal mouse bone marrow. This is considered to be the cause of the low PFC activity in the marrow during the primary response to SRBC. In normal mouse bone marrow precursors of IgM-PFC but not of IgG- and IgA-PFC could be found. Priming with SRBC induced the appearance of IgM-, IgG-, IgA- and T-memory cells in the marrow. These B- and T-memory cells were shown to be specific for the antigen which induced their appearance. It is thought that after a second injection of SRBC the IgM-, IgG- and IgA-memory cells can differentiate with the help of the T-memory cells within the bone marrow into IgM-, IgG- and IgA-PFC respectively. The sequence of appearance of the B-memory cells in the bone marrow was shown to be IgM-IgG-IgA. Six months after the intravenous injection of SRBC, the presence of B-memory cells could be demonstrated not only in spleen and bone marrow, but also in peripheral lymph nodes, mesenteric lymph node, Peyer's patches, thymus and blood. The increase in amount of B-memory cells was most prominent in the spleen.