A comparative study on the humoral immune responses in germ-free and conventional mice.

The plaque-forming cell (PFC) responses to sheep red blood cell (SRBC) dinitrophenyl-lysine-Ficoll (DNP-lys-Ficoll), and dinitrophenylated bovine serum albumin (DNP-BSA) have been studied in both germ-free and conventionally reared ICR mice. In germ-free mice, the IgG response to SRBC and the IgM and IgG responses to DNP-BSA were lower than in conventional mice, but no difference was observed in the IgM response to SRBC or the IgM and IgG responses to DNP-lys-Ficoll. Further, the number of 0-bearing cells in the spleen was smaller, and the mitogenic response of spleen cells to PHA was lower in germ-free mice than in conventional mice. These observations suggest that T cells of germ-free mice remain functionally immature.     

Antibody formation by human tonsil cells in vitro.

The ability of human tonsil lymphocytes to give an anti-SRBC response in conventional cultures and in microcultures was studied. It was found that about two-thirds of tonsils responded with a significant number of plaque-forming cells (PFC), and that in some instances the response could be augmented if allogeneic tonsil cell (irradiated or intact) were added. Moreover, tonsil lymphocytes which failed to give a response on their own often responded upon addition of an appropriate number of allogeneic tonsil cells. The response was remarkably improved if allogeneic tonsil supernatant or conditioned medium were added. An anti-SRBC response was also obtained if SRBC was omitted from the cultures. The frequency of anti-SRBC specific B cells was estimated as 1/60000 (f = 1-7 X 10(-5)).     

Immunomodulation by Corynebacterium parvum. 1. Variable effects on anti-sheep erythrocyte antibody responses.

Corynebacterium parvum injected i.p. 1--16 days prior to i.p. antigen inoculation virtually abolished both IgM and IgG primary responses to 1 X 10(8) SRBC. The suppression was significantly marked at antigen doses ranging from 1 X 10(6)--1 X 10(9) SRBC but not at 5 X 10(9) SRBC. As little as 56 microgram C. parvum caused a marked suppression of the response to 1 X 10(8) SRBC. In secondary responses C. parvum given either one day before priming with 1 X 10(8) SRBC or one day before secondary challenge caused a dramatic suppression of both IgM and IgG PFC responses. In contrast with i.p. injected C. parvum, i.v. injection of the vaccine enhanced immune responses to i.p. or i.v. injected SRBC. Similarly C. parvum injected i.p. prior to i.v. immunization resulted in an augmented anti-SRBC response. An enhancement of anti-SRBC response was also noted when C. parvum was injected i.p. on the day of i.p. immunization. The suppressed responses in C. parvum injected animals could be explained partly by the reduced splenic localization of the antigen.     

Antigen-specific B-cell function in human autoimmune thyroiditis

T-B cells from the peripheral circulation of patients with autoimmune thyroiditis were cocultured with sheep red blood cells (SRBC) or soluble human thyroglobulin (Tg), a self-antigen. The B-cell mitogen, Staphylococcus aureus, combined with macrophage-derived B-cell differentiating factor, induced in vitro lymphoid activation and proliferation in the presence or absence of Tg or SRBC, which was monitored after 6 days by specific anti-Tg and anti-SRBC plaque-forming cell (PFC) responses (expressed as PFC per 10(6) T-B cells). In the presence of SRBC, significantly more anti-SRBC PFC (322 +/- 113, SE) were generated in normals (N = 5) compared with autoimmune thyroiditis patients (58 +/- 36) (N = 8) (P less than or equal to 0.001), data consistent with an antigen-specific T-cell defect. Anti-Tg PFC, not detectable in normal controls, were observed in patients in the absence (111 +/- 41) and presence (171 +/- 64) of Tg (10-1000 ng/ml). However, variable responses were noted after such coculture experiments with Tg. Three patients demonstrated amplification of anti-Tg PFC, while three showed antigen-related inhibition of anti-Tg PFC. These data indicated heterogeneity of responses to Tg antigen in patients with autoimmune thyroid disease compounded by significantly depressed antigen-specific induction mechanisms.     

The effect of maternal antigenic stimulation upon the active immune responsiveness of their offspring.

Pregnant mice were stimulated by sheep erythrocytes (SRBC) and the active immune responses of their offspring were investigated. The offspring whose mothers were stimulated with SRBC did not develop either IgM or IgG plaque-forming cell (PFC) target cells. From the dose response of pregnant mice for inducing suppression, enough doses (10(8)-10(10) cells of SRBC) for inducing primary anti-SRBC PFC could establish suppression in the young. Both intravenous and intraperitoneal administration of SRBC induced almost complete suppression of the specific PFC response (98.6-95.2%), but only partial suppression (57.8%) was induced by subcutaneous injection. For suppression to take place, female mice had to be injected with SRBC from 2 days before fertilization to day 16 of gestation. Suppression of the PFC response was not obtained when SRBC were given 3 days before fertilization or just 24 hr before delivery. This suppressive effect on the PFC response persisted until the 15th week after birth. In these newborn mice, detectable amounts of specific anti-SRBC antibodies were found. After exchanging mothers and newborns, the stimulated newborns fostered by normal mothers were still unresponsive following antigenic stimulation, even though a specific antibody from the mother was not detected. However, normal newborns nursed by stimulated mothers could respond to SRBC injection, no matter how the specific antibodies were transferred. Possible mechanisms of immunosuppression of the PFC response by antibody transmitted by the mother to her offspring are discussed.     

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.     

Effect of Mechlorethamine on SRBC-Induced Secondary Antibody Response in Mice

Abstract

The effect of low-dose mechlorethamine (5μg/kg) on secondary humoral response to sheep red blood cells (SRBC), depending on time of exposure to the drug in relation to priming and challenge was studied in Balb/c mice. It was found that mechlorethamine in a dose of 5 μg/kg stimulated primary humoral response to SRBC resulting in the increased number of the plaque forming cells (PFC) and hemagglutinin titre (19S + 7S). However, this effect waned 10 days after immunization. On the other hand, the same mechlorethamine dose potentiated secondary humoral response to SRBC and increased the number of PFC and anti-SRBC hemagglutinin titres (notably 7S), which was due to the challenging antigenic stimulus. In each immunization, mechlorethamine administration prolonged the potentiating effect of the drug on anti-SRBC hemagglutinin titre. When mechlorethamine was administered to the mice only after priming, the number of PFC increased, but anti-SRBC hemagglutinin titre (7S) remained unchanged. This was likely due to the fact that mechlorethamine administered after priming increases the number of long-lived lymphocytes B, which in turn affect secondary humoral response.     

Effect of Mechlorethamine on SRBC-Induced Secondary Antibody Response in Mice

The effect of low-dose mechlorethamine (5μg/kg) on secondary humoral response to sheep red blood cells (SRBC), depending on time of exposure to the drug in relation to priming and challenge was studied in Balb/c mice. It was found that mechlorethamine in a dose of 5 μg/kg stimulated primary humoral response to SRBC resulting in the increased number of the plaque forming cells (PFC) and hemagglutinin titre (19S + 7S). However, this effect waned 10 days after immunization. On the other hand, the same mechlorethamine dose potentiated secondary humoral response to SRBC and increased the number of PFC and anti-SRBC hemagglutinin titres (notably 7S), which was due to the challenging antigenic stimulus. In each immunization, mechlorethamine administration prolonged the potentiating effect of the drug on anti-SRBC hemagglutinin titre. When mechlorethamine was administered to the mice only after priming, the number of PFC increased, but anti-SRBC hemagglutinin titre (7S) remained unchanged. This was likely due to the fact that mechlorethamine administered after priming increases the number of long-lived lymphocytes B, which in turn affect secondary humoral response.     

Major histocompatibility complex restriction of maternally induced suppression in young adult mice.

This study focused on the mode in which maternal T cells induce suppression of plaque-forming cell (PFC) response in offspring. The maternal T cells of C57BL/6J pregnant mice, which had been intraperitoneally injected with 2 x 10(8) of sheep red blood cells (SRBC) on day 12 of gestation, were transferred, 5 days after immunization, into (C3H/HeJ x C57BL/6J)F1 normal pregnant mice on day 12 of gestation. The (C3H/HeJ x C57BL/6J)F1 x C3H/HeJ offspring of (C3H/HeJ x C57BL/6J)F1 recipient pregnant mice were reared to more than 6 weeks of age, and their anti-SRBC PFC responses were examined. Suppression of anti-SRBC PFC response was observed in H-2bxk but not H-2k offspring. Thus, maternal T cells of SRBC-immunized pregnant mice induce suppression of anti-SRBC PFC in offspring with restriction to major histocompatibility complex (MHC) haplotype utilized in maternal T-cell responses during pregnancy. Maternal CD4+ T cells are responsible for the MHC-restricted induction of PFC suppression in offspring. Furthermore we demonstrated, in this report, using adoptive transfer of maternal T cells from SRBC-immunized pregnant mice and in vitro secondary PFC assay in the offspring, that maternal T-cell-mediated suppression results from the development of CD4+ suppressor T cells in offspring. Moreover, the activation of suppressor T cells in offspring depends on the recognition of SRBC antigens presented in association with the same MHC haplotype as that utilized in the maternal T-cell response during pregnancy. Thus, the maternal T cells of SRBC-immunized pregnant mice generate a repertoire of suppressor T cells in their offspring.     

In Vitro Studies on the Effect of Anti-Lymphocyte Serum on the Humoral Immune Response

The effect of ALS (I), a heterologous anti-lymphocyte serum prepared against lymph node cells from rats pre-immunised with sheep erythrocytes (SRBC), on plaque forming cells (PFC) to SRBC was studied in vitro. ALS (I) reduced the number of both IgM and IgG PFC when complement was included in the reaction. This ability of ALS (I) to inhibit FFCs in vitro was absorbed out by the IgG fraction of anti-SRBC serum. Thus ALS (I) was thought to possess an anti-idiotypic antibody directed against B-cells at a later stage of differentiation.     

Immunosuppression caused by antigen feeding. I. Evidence for the activation of a feedback suppressor pathway in the spleens of antigen-fed mice

Sheep red blood cells (SRBC) administered by the oral route to normal mice elicited no detectable splenic anti-SRBC plaque-forming cell (PFC) response until 8 weeks of antigen feeding. At this time a splenic IgA anti-SRBC PFC response was detected. On the other hand, spleen cells taken from mice given oral SRBC for 1-5 weeks showed striking changes in their in vitro anti-SRBC responsiveness as compared to spleen cells from normal mice. This was evidenced by enhanced early (days 3-4) in vitro responses, followed by suppressed late (day 5-6) in vitro responses. Both early enhancement and late suppression were T cell-mediated. Early enhancement appeared to be mediated by helper T cells of the Lyt-1+2.3- phenotype. Late suppression was also mediated by Lyt-1+2.3- cells, but Lyt-2-bearing cells had to be present in culture for suppression to occur. Lyt-2-bearing cells could be replaced with normal T cells. Furthermore, elimination of cells bearing I-J-encoded determinants from the T cell population isolated from the spleens of antigen-fed mice also partially relieved suppression. Thus, antigen feeding appears to activate a feedback suppressor pathway in which Lyt-1+2.3-, I-J subregion determinant-bearing T cells can suppress immune responses by causing normal T cells to become suppressor effectors. No evidence was found to show that antigen feeding induced Lyt-1-2.3+ suppressor cells in the spleen, nor were any serum suppressor factors detected.     

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.     

Immunomodulatory effect of a newly synthesized compound, TOK-8801 (N-(2-phenylethyl)-3,6,6-trimethyl-5,6-dihydroimidazo [2,1-b]thiazole-2-carboxamide) on antibody production in vivo and delayed-type hypersensitivity in mice

The immunopharmacological effects of a newly synthesized compound in vivo, TOK-8801 (N-(2-phenylethyl)-3,6,6-trimethyl-5,6-dihydroimidazo[2,1-b]thi azo le-2- carboxamide), on the anti-SRBC plaque-forming cell (PFC) response and delayed-type hypersensitivity (DTH) reaction were investigated. Oral administration of TOK-8801 (0.1-10 mg/kg) resulted in the suppression of the PFC responses to varying doses of antigen (5 x 10(6), 2 x 10(7), 1 x 10(8)) in C3H/He strain mice (7 W) which are high responders to SRBC antigen. On the other hand, the compound augmented the PFC response in aged mice (8-9 months) in which the PFC response was markedly depressed compared with that in young mice. In the experiment of the methylated human serum albumin-induced DTH reaction, TOK-8801 augmented the reaction in low responder (C57BL/6) mice by oral administrations of 0.1-1 mg/kg for 5 days from the sensitization, whereas suppressed the reaction in high responder (ICR) mice. These immunopharmacological actions of TOK-8801 were compared in dose and activity with those of lobenzarit and bucillamine. Thus, these results suggest that TOK-8801 may act as an immunomodulating agent and would be expected to be a useful agent for autoimmune diseases such as rheumatoid arthritis.     

Mechanisms of suppression of the immune response: I. Differences in the effect of specific inhibitory antibody on distribution of 51Cr-labelled sheep erythrocytes in different mouse strains

The fate of ⁵¹Cr-labelled sheep red blood cells (SRBC) was studied in inbred AKR and outbred Swiss Webster mice given a single dose of specific anti-SRBC inhibitory antibody 24 hours before antigen. The amount of ⁵¹Cr in spleens of untreated mice in these two strains is the same, but the amount of ⁵¹Cr differs in spleens of antibody-treated AKR mice compared with antibody-treated Swiss Webster mice. Although there is increased uptake of ⁵¹Cr-labelled SRBC by spleens of AKR mice given anti-SRBC antibody, there is decreased uptake by spleens of Swiss Webster mice given the same antibody; nevertheless, the inhibitory effects of the anti-SRBC antibody assayed by anti-SRBC haemolytic plaque-forming cell (PFC) responses are similar in the two mouse strains. These results indicate that there is a mechanism other than a mere peripheral effect of anti-SRBC antibody in this system.     

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 (T_h) 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 T_h cell activation. SRBC-“pulsed” splenic adherent cells activated T_h 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 T_h 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 by recognized by T_h 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 T_h 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.     

Cellular aspects of non-specific stimulation of antibody production by capsular polysaccharide of Klebsiella pneumoniae

Comparative studies were made of the increase in the numbers of plaque-forming cells (PFC), rosette-forming cells (RFC) and haemolytic foci for erythrocyte antigens in the spleens of mice given a non-specific stimulus (the capsular polysaccharide of Klebsiella pneumoniae (CPS-K)) and an antigenic stimulus (sheep red blood cells (SRBC)). The number of direct PFC for SRBC was increased by injection of CPS-K to as high a level as that obtained by injection of SRBC. In contrast, by injection of CPS-K the numbers of indirect PFC, RFC (probably the antibody-forming cell precursors) and haemolytic foci were not increased significantly, whereas all of them were increased markedly by injection of SRBC. The maximal number of PFC in mice injected with CPS-K was approximated to the number of background RFC of the same mice. Injection of CPS-K generated 25–130 times more direct PFC for each of three kinds of erythrocyte antigens, SRBC, rabbit red blood cells and chick red blood cells, than background PFC, whereas the total number of spleen cells was not increased significantly or increased very slightly. Repeated injections of CPS-K were not significantly more effective for increase in the number of direct PFC than a single injection of CPS-K. Injection of CPS-K could generate many direct PFC in mice which had been thymectomized, irradiated and reconstituted with foetal liver cells. In mice injected with CPS-K, increase in (or maintenance of) the numbers of direct PFC and RFC were inhibited by injection of a mitogen inhibitor, vinblastine sulphate, but their sensitivities to the drug were less than those found in mice immunized with SRBC. It has been concluded from these results that in mice injected with CPS-K a large number of antibody-forming cell precursors are differentiated to direct PFC through one division or a few divisions of the individual cells, and that the inability of CPS-K to induce sufficient cell divisions of the individual precursor cells is the cause of the lack of increase in the number of indirect PFC and in immunological memory for secondary PFC responses in mice injected with CPS-K.     

Resistance of helper T lymphocytes to cyclophosphamide

The effect of cyclophosphamide (Cy) on helper T lymphocytes using an adoptive transfer approach in athymic nude mice was investigated. The results indicated that Cy, at a dose (100 mg/kg) which virtually abolished anti-sheep erythrocyte (SRBC) antibody plaque forming cell (PFC) response of Balb/c mice, did not alter significantly the capacity of their splenic T cells to restore the anti-SRBC PFC response of nude mice. This resistance of T helper cells was observed in unimmunized mice and in mice injected with SRBC two days prior to Cy administration. It has been concluded that both resting and antigen stimulated T helper cells responsible for reconstituting anti-SRBC response of nude mice are resistant to Cy.     

Modulation of the Immune System by Neisseria meningitidis

The immunomodulatory potential of Neisseria meningitidis was investigated. Spleen cells from mice injected intraperitoneally with low to moderate doses of meningococci (10(4)-10(7)) were found to display enhanced responses to the mitogens lipopolysaccharide (LPS), phytohaemagglutinin (PHA), and concanavalin A (Con A). In contrast, high doses of meningococci (10(8)-10(9)) caused a marked decrease in mitogenic reactivity. Meningococci-injected mice also displayed a dose-dependent suppression of a primary anti-sheep red blood cell (SRBC) plaque-forming cell (PFC) response. The timing between the injection of SRBC and of meningococci appeared to play an important role in the induction of suppression by the organisms. Thus, decreased PFC responses were observed only when the bacteria were injected prior to the antigen. When meningococci were injected at the same time or after SRBC, normal or even increased PFC responses developed. Kinetic experiments showed that the onset of suppression of both mitogen and antibody responses by meningococci was very rapid, so that by 6-7 h after injection of the bacteria, mice showed markedly reduced mitogen responses and became essentially unable to mount an antibody response against SRBC. Suppression of mitogen responses was relatively transient, since reactivity returned to normal after 48 h. However, the ability of infected animals to mount a normal anti-SRBC response did not fully return until 12 days after the infection. Spleen cells from meningococci-infected mice also showed markedly depressed PFC responses when stimulated with SRBC in vitro but failed to suppress the response of normal spleen cells in mixed cultures. These observations indicate that putative suppressor cells, if they exist at all, are too insignificant in terms of numbers and/or efficiency to account for the observed immunosuppression. A more likely explanation for the inhibition, which is supported by our data, presented here and elsewhere, is that certain surface components of meningococci are capable of imparting immunosuppressive signals directly onto target lymphocytes.     

Micro-system for the primary immunization of baboon (papio cynocephalus) peripheral blood mononuclear cells with sheep erythrocytes in vitro

A micro-culture system for the stimulation of baboon peripheral blood mononuclear cells (PBMC) with sheep erythrocytes (SRBC) in vitro was established. PBMC cultures in 96-well microtiter plates were maintained in 300 μl of culture medium containing a murine mixed lymphocyte reaction (MLR) supernatant and SRBC. Cultures of 7.5 × 10⁵ PBMC and 6 × 10⁶ SRBC resulted in the highest anti-SRBC plaque-forming cell (PFC) response. It was also determined that the presence of 50 μl of murine MLR supernatant was required for optimal PFC generation and that the cultures required supplementation with nutrient cocktail 3 days post-immunization. Although PFC were detectable on days 4–8, the maximum expression of PFC occurred on day 7.     

Cellular and humoral immune responses in mice. II. Effect of intraperitoneal or subcutaneous injection of carrier of anti-hapten antibody and delayed hypersensitivity responses.

The subcutaneous (s.c.) injection of sheep red blood cells (SRBC) without adjuvant into mice preferentially induced delayed hypersensitivity (DH) reaction, as measured by footpad swelling, while intraperitoneal (i.p.) measured by the haemagglutinin test. Under these conditions, the properties of the helper activities of thymus-derived (T) cells for humoral responses were examined, in association with the features of the DH response, by measuring the anti-hapten and anticarrier antibody responses after a booster injection of trinitrophenylated (TNP) SRBC and by changing the combination of doses and injections routes of the carrier and the hapten-carrier conjugates. When mice were presensitized with i.p. injections of SRBC and boosted with i.p. injections of TNP-SRBC, the anti-TNP antibody production was maximally enhanced by presensitization with a low dose of SRBC, and gradually abolished with higher doses of SRBC for pre-sensitization. In the latter case, anti-SRBC antibody production was increases with increasing doses of SRBC..