Neonatal infection with mouse thymic virus: effects on cells regulating the antibody response to type III pneumococcal polysaccharide.

Mice infected neonatally with mouse thymic virus (TA) were evaluated at different ages with respect to their ability to give a plaque-forming cell (PFC) response to type III pneumococcal polysaccharide (SSS-III), as well as the degree of amplifier and suppressor thymus-derived (T) cell activity present. B cell activity matured rapidly from 2 to 4 weeks of age and was not affected by TA infection. Amplifier T cell activity matured progressively over the first 8 weeks of life and was transiently suppressed in TA-infected mice at 4 weeks of age. Suppressor T cell activity measured at 2,4, and 6 weeks of age was unaffected by TA. The findings suggest that TA is highly tropic for T cells and has selective effects on subpopulations of T cells.     

Effect of concanavalin A on lymphocyte interactions involved in the antibody response to type III pneumococcal polysaccharide. II. Ability of suppressor T cells to act on both B cells and amplified T cells to limit the magnitude of the antibody response.

When administered 2 days after immunization with 0.5 microgram Type III pneumococcal polysaccharide (SSS-III), the T lymphocyte mitogen concanavalin A (Con A) stimulates a 2.6-to 7-fold enhancement of the plaque-forming cells (PFC) response to SSS-III in vivo. This enhancement requires the presence of amplified T cells, which act by driving PFC or their precursors to extra rounds of proliferation. The extra proliferation that can be stimulated by Con A is not seen in the normal primary response to SSS-III; but treatment with anti-lymphocyte serum (ALS) to remove suppressor T cells will permit the additional proliferation to occur. This indicates that in the primary response to SSS-III, suppressor T cells act on amplifier T cells to limit the magnitude of the antibody response. Only suppression of B cells can account for the further suppression induced by Con A given at the time of immunization or by low-dose paralysis of the SSS-III response. The relatively late development of amplified activity compared to suppressor activity appears to account for the absence of amplifier activity after primary immunization with SSS-III. It is apparent that one can explain the regulatory effects observed during the development of an immune response to SSS-III only by considering both T cell- B cell and T cell- T cell interactions, together with the temporal relationships involved in those interactions.     

Effect of splenectomy on the expression of regulatory T cell activity.

The effect of adult splenectomy on the expression of suppressor and amplifier T cell activity was examined with respect to the serum antibody response to Type III pneumococcal polysaccharide (SSS-III) by using a sensitive radioimmunoassay. Suppressor T cell activity, as measured by the degree of low-dose paralysis induced, was not impaired in the least by splenectomy; however, amplifier T cell activity was almost completely eliminated within 7 days after splenectomy. These findings indicate that suppressor T cell activity is not confined solely to the spleen, the major site of antibody synthesis after immunization with SSS-III, and that the spleen may be an important site for the generation and/or maintenance of amplifier T cell activity.     

Characteristics of amplifier T cells involved in the antibody response to the capsular polysaccharide of type III Streptococcus pneumoniae

Amplifier T cell activity can be transferred by spleen cells harvested 72 hr after priming with type III pneumococcal polysaccharide (SSS-III) and can be abolished by treating the transferred cells with monoclonal anti-Lyt-1, or anti-Thy-1 antibodies in the presence of complement; thus, amplifier cells represent a distinct subpopulation of T cells. Amplifier T cells were found to be sensitive to irradiation but not to treatment with cyclophosphamide. When amplifier cells were transferred to athymic nude (nu/nu) mice, the enhancement obtained was much greater than that produced in thymus-bearing (nu/+) mice; this is presumably due to the lack of suppressor T cell activity in nu/nu mice that enables amplifier T cell activity to be expressed more fully. Amplifier T cells also were found to be present in peripheral blood; these amplifier T cells were Lyt-2- in phenotype. Although the induction and activation of amplifier T cells appear to be antigen-specific, the product made by amplifier T cells may not be antigen specific in its mode of action. Because amplifier T cells can be induced and activated by exposure to immune B cells, specificity is presumably due in whole or in part to the ability of amplifier T cells to recognize the idiotypic determinants of B cell-associated antibody specific for SSS-III.     

Cell surface antigens and other characteristics of T cells regulating the antibody response to type III pneumococcal polysaccharide

The administration of a subimmunogenic dose of type III pneumococcal polysaccharide (SSS-III) produces an antigen-specific T cell-dependent phenomenon termed low-dose paralysis (immunologic unresponsiveness). This form of unresponsiveness can be transferred by spleen cells obtained 5 to 24 hr after priming, and the suppressive activity of the transferred cells is abolished by prior treatment with monoclonal anti-Lyt-2 and anti-I-J antibody in the presence of complement, indicating that suppression is mediated by a distinct subset of T cells (suppressor T cells). If primed spleen cells are transferred 24 to 72 hr after immunization with SSS-III, however, the resulting antibody response of immunized recipients is enhanced. Greater enhancement is noted when transferred cells, pretreated with monoclonal anti-Lyt-2 antibody plus complement to remove suppressor T cells, are used; such enhancement is attributed to amplifier T cells. These findings indicate suppressor T cells regulate the antibody response to SSS-III by influencing the expansion of SSS-III-specific clones of B cells as well as the expression of amplifier T cell activity; the latter causes B cells to proliferate further in response to SSS-III.     

Immune response of mice exposed to cis-diamminedichloroplatinum

Summary The effects of cis-diamminedichloroplatinum (CDDP) on lymphoid organs and the immune response of young and older adult mice were studied histologically and by functionally assessing the activity of various subpopulations of immune cells. Young adult mice (6–8 weeks old) treated with 2 mg/kg CDDP mounted an enhanced splenic plaque-forming cell (PFC) response to both sheep erythrocytes, a helper T-cell-dependent antigen (HD), and pneumococcal polysaccharide type III a helper T-cell-independent antigen (HI). Older adult mice (18–22 weeks old) treated in the same way exhibited an equally enhanced PFC response to HD antigen and even a more pronounced response to HI antigen. Treatment of mice with 12 mg/kg CDDP resulted in immunosuppression. Thymus, lymph nodes, and spleen of animals treated with the higher dose of CDDP showed a marked cell depletion from both T and B areas, confirming that the immunosuppression was due to an indiscriminate elimination of both T and B lymphocytes. The immunosuppression and the cell depletion from lymphoid organs were more pronounced in younger mice. Thus, the effects of CDDP on the lymphoid organs and the immune response depend both on the age of the animals and on the dose of the drug. CDDP given in small doses enhances the PFC response, whereas a reduced PFC response is obtained following high-dose treatment. Abbreviations used: CDDP, cis-diamminedichloroplatinum; PFC, plaque-forming cell; HD, helper T-cell dependent; HI, helper T cell-independent; SIII, pneumococcal polysaccharide type III; SRBC, sheep red blood cells; TNP, trinitrophenyl; KLH, keyhole limpet hemocyanin; TNBS, 2, 4, 6-trinitrobenzene sulfonic acid; BBS, borate-buffered saline     

Effect of concanavalin A on lymphocyte interactions involved in the antibody response to type III pneumococcal polysaccharide I. Comparison of the suppression induced by con A and low dose paralysis.

Concanavalin A (Con A) administered at the time of immunization induces suppression of the in vivo splenic plaque-forming cell (PFC) response to type III pneumococcal polysaccharide (SSS-III). As with low dose paralysis of the PFC response to SSS-III, Con A-induced suppression could not be demonstrated in congenitally athymic (nu/nu) mice and could be eliminated partially by treatment with anti-lymphocyte serum (ALS). The kinetics for Con A-induced suppression paralleled those for low dose paralysis of the antibody response to SSS-III. These findings support the view that Con A-induced suppression is produced in vivo by suppressor T cells and that this form of suppression shares with low dose paralysis a common pathway through which suppression is mediated.     

Two defects in old New Zealand Black mice are involved in the loss of low-dose paralysis to type III pneumococcal polysaccharide

Treatment of normal mice with a subimmunogenic dose of type III pneumococcal polysaccharide (SSS-III) results in the development of an antigen-specific state of unresponsiveness termed low-dose paralysis. This unresponsiveness is mediated by T suppressor cells and can be transferred by Lyt-2+ T cells, but not by L3T4+ T cells, obtained 18 hr after priming. As autoimmune New Zealand Black (NZB) mice age, there is a progressive decrease in low-dose paralysis to SSS-III. The defect in older NZB mice resulting in decreased suppressive activity was investigated by transferring primed Lyt-2+ T cells from young into old mice, and vice versa. Enlarged Lyt-2+ T cells from old NZB mice could not suppress the SSS-III response of young recipients. However, Lyt-2+ T cells of normal cell size were efficient in inhibiting the antibody response upon transfer. Primed Lyt-2+ T cells from young NZB mice did not affect the response of old recipients, but effectively suppressed the response of young mice. These results suggest that there are two defects involved in the decline of low-dose paralysis to SSS-III in aging NZB mice: Enlarged Lyt-2+ T cells may lose their ability to function as mediators of suppression; and B cells may become resistant to T cell-mediated suppression.     

The effects of age on the immune response to type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) in BALB/c, SJL/J, and C3H mice.

Type III pneumococcal polysaccharide (SIII) and bacterial lipopolysaccharide (LPS) were used to evaluate B cell and T cell regulatory functions in BALB/c, SJL/J, and C3H mice of various ages. It was found that the BALB/c and C3H mice could mount high level plaque-forming cell (PFC) responses to SIII at various ages through 110 weeks whereas the levels of the SJL/J PFC responses had begun to decline by the age of 42 weeks through the age of 80 weeks. BALB/c mice were also capable of producing strong PFC responses to LPS at various ages through 110 weeks whereas the comparable SJL/J PFC responses to LPS had declined by 80 weeks of age. By using anti-lymphocyte serum (ALS) and low-dose paralysis to SIII, it was shown that suppressor T cell activity was apparently greater in young BALB/c mice than in older BALB/c mice. It was also found that paralysis to SIII in BALB/c mice was easier to achieve at an early age. SJL/J mice were found to have the necessary B cell activity to respond to SIII through 80 weeks of age and the PFC responses could be greatly enhanced by ALS. Implications of the roles of regulatory T cells in aging are discussed.     

Selective sensitivity to hydrocortisone of regulatory functions that determine the magnitude of the antibody response to type III pneumococcal polysaccharide.

Previous studies on the basis for the immunosuppressive potential of adrenal corticosteroids have stressed that the effects of these agents on immune functions depend on the animal species being considered, as well as the subpopulations of lymphocytes involved in the expression of immune functions examined. In the present work, we have evaluated the effect of a single dose of hydrocortisone on three different immunoregulatory functions that can influence the magnitude of an antibody response to Type III pneumococcal polysaccharide (SSS-III) in mice; these functions include suppressor, amplifier, and helper activity that are dependent upon the presence of distinct subpopulations of thymus-derived (T) cells. The results obtained show that a single injection of a relatively large dose of hydrocortisone, when given at the time of priming with carrier, eliminated all evidence of carrier-specific helper T cell activity; hydrocortisone was also found to eliminate a significant amount of helper T cell activity when given after such activity had been generated. But, under the same experimental conditions, suppressor and amplifier T cell activities were unaffected, even in this steroid-sensitive species. Such selective sensitivity may account for some of the immunosuppressive potency of steroids.     

Generation of low-dose paralysis in the absence of the ability to secrete antibody.

(CBA/N female x BALB/c male)F1 male mice carry an X-linked defect, originating from CBA/N mice, which renders them unable to generate an antibody response to SSS-III. Histocompatible (BALB/c female x CBA/N male) reciprocal F1 male hybrids do not carry the X-linked defect and therefore generate a readily detectable PFC response to SSS-III, which can be adoptively transferred into nonresponding reciprocal F1 male mice. In the present work, we show that this adoptive response could be inhibited in recipient (CBA/N female x BALB/c male)F1 male nonresponding mice in which low dose paralysis had been induced. Evidence is presented which indicates that such suppression is of host rather than donor cell origin. The capacity to develop low-dose paralysis, a phenomenon that is antigen specific and has been attributed to the action of suppressor T cells, indicates that nonresponding (CBA/N female x BALB/c male) F1 males (and presumably the CBA/N progenitor strain) have the ability to recognize this antigen. Furthermore, since these animals fail to make a serum antibody response to SSS-III, the signal that activates suppressor T cells cannot be circulating antibody or antigen-antibody complexes. These findings are most consistent with the view that low-dose paralysis of the response to SSS-III is not dependent on antibody-mediated feedback inhibition; rather, it is an active process mediated by suppressor T cells.     

Production of soluble suppressor factor by spleen cells from mice immunized with type III pneumococcal polysaccharide

Supernatant fluid (SF) derived from spleen cell cultures, obtained from mice 16 hr after immunization with 0.5 microgram of Type III pneumococcal polysaccharide (SSS-III), suppressed the antibody response when SF was given (i.v.) 3 hr before immunization with SSS-III. Such suppression was antigen specific and could be reproduced by SF derived from cultures of T cells from mice immunized with SSS-III (0.5 microgram) or by SF derived from cultures of spleen cells from mice primed with a subimmunogenic dose of SSS-III (0.005 microgram). Adsorption of SF with SSS-III covalently bound to a Sepharose 4B column did not alter the ability of SF to suppress the SSS-III-specific antibody response. However, adsorption of SF with Ig+ (B) cells from mice immunized with 0.5 microgram SSS-III completely removed the suppressive activity. Significant (p less than 0.05) suppression of the antibody response was observed only when SF was administered (i.v.) 24 hr before to 24 hr after immunization with 0.5 microgram of SSS-III. These results suggest that suppressor T cells generated in response to SSS-III function by releasing a soluble factor(s) that binds to determinants on B cells rather than antigen; this soluble factor(s) acts directly on antigen-stimulated B cells or inhibits the induction of amplifier T cells.     

The role of antigen in the activation of regulatory T cells by immune B cells

The transfer of B cells from mice immunized with Type III pneumococcal polysaccharide (SSS-III) results in the activation of suppressor and amplifier T cells that control the magnitude of the antibody response in recipient mice, immunized subsequently with SSS-III. Prior treatment of transferred B cells with an excess of enzyme (polysaccharide depolymerase) capable of hydrolyzing SSS-III, does not alter the capacity of these cells to activate regulatory T cells. These findings indicate that the activation of regulatory T cells by immune B cells is not mediated by residual antigen on the surface of transferred cells.     

Transferred B cells from autoimmune NZB/N mice fail to activate T suppressor cells

T-cell-mediated suppression of the antibody response of autoimmune NZB/N mice to Type III pneumococcal polysaccharide (SSS-III) can readily be induced in situ by priming with a subimmunogenic dose of SSS-III; however, the transfer of either "young" (8 weeks old) or "old" (42 weeks old) SSS-III-primed B cells, which activates suppressor T cells in normal BALB/cByJ mice, fails to induce suppression of the antibody response in recipient NZB/N mice, regardless of the number of cells transferred or the time interval between transfer and immunization. Transfer of 51Cr-labeled B cells demonstrated that syngeneic primed B cells home to the spleens of NZB/N mice in somewhat lower numbers than in BALB/cByJ mice, although the differences observed may not be sufficient to explain the complete absence of activation of suppressor T cells. These findings suggest that B cells from autoimmune NZB/N mice are unable to activate T suppressor cells upon transfer; this disorder in a normal regulatory mechanism may be important in the pathogenesis of disease.     

Age restriction in antigen-specific immunosuppression

Age-related alterations of antigen-specific T cell-mediated suppression have been examined in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. Inducer suppressor T cells (Tsi) were activated in mice at the age of 3 mo (young) or 18 mo (old) by i.v. injection of NP-conjugated syngeneic spleen cells (SC). Spleen cells from the NP-SC-injected mice were subcultured in vitro with spleen cells from normal young or old mice to generate transducer suppressor T cells (Tst). Four days later subcultured cells were added to responder cell cultures 1 day before the PFC assays to trigger effector suppressor T cells (Tse). Responder cell cultures, containing NP-conjugated horse red blood cells (HRBC) and spleen cells from HRBC-primed young or old mice, were assayed on day 4 for anti-NP and anti-HRBC PFC. Suppression was found to be antigen specific and age restricted. NP-specific suppressor cells are easily induced in subculture if the Tsi and Tst cell populations are both derived from young or old mice. Conversely, if Tsi cells from young or old mice are subcultured with Tst cells from mice of a different age, suppression of the anti-NP PFC response is hardly observed. Age restriction was also found to operate in the interactions between subcultured and responder cell populations, indicating that age-matching is required for effective triggering of Tse cells by Tst cells. These results altogether suggest that aging may affect the recognition repertoire expressed in suppressor T cell subsets. Moreover, the finding that suppression is less efficient when exerted on responder spleen cells from old than from young mice provides an explanation for the increased frequency of autoimmune disorders in aging.     

Measurement and comparison of the proliferative and antibody response of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens.

Mouse spleen cell antigenic responses to the thymic-dependent antigen sheep red blood cells (SRBC), and the thymic-independent antigens, E. Coli lipopolysaccharide (LPS) and pneumococcal polysaccharides Type I and II (SI, SII) were studied as as a function of age, employing both in vitro spleen cell stimulation and plaque-forming cell (PFC) assay systems. Primary spleen cell proliferative and PFC responses to SRBC, were either absent or meager in comparison to adult (8–12 weeks) values for the first 3 weeks of life. Thereafter responses rose achieving adult values between 4 and 8 weeks of age. The inability of young mice to respond to SRBC was not because of a different immunizing dose requirement for SRBC, since immunization with SRBC over a 200-fold range did not enhance their capability to respond. Also, addition of adherent cells or macrophages from adult mice did not enhance the immune responses of young mice. Furthermore, immunization of 2–4 week old mice with SRBC inhibited the secondary response to SRBC. In contrast, young murine spleen cell proliferative and PFC responses to SI, SII, and LPS were approximately the same as the adult by 7–14 days of life. These data suggest that B-cell immunologic activity, as measured by immunologic assays utilized in this study, develops much earlier than does T-cell responsiveness.     

Expression of GAT-715 idiotype by GAT-specific plaque-forming cells from various strains of mice

The splenic plaque-forming-cell (PFC) response of mice to immunization with pneumococcal capsular polysaccharide (SSS-III), coupled with T-cell activation by phytohemagglutinin (PHA), is characterized by enhanced numbers of IgG-producing cells, largely restricted to the IgG2a and IgG2b subclasses. In contrast, immunization with SSS-III alone results in low numbers of IgG-producing cells, fairly evenly distributed among the subclasses IgG1, IgG2a, IgG2b, and IgG3. The enhanced IgG response and a concomitantly enhanced IgM response are T-cell dependent and occur only if PHA is given 2 days after SSS-III immunization. The absence of immunologic memory to SSS-III in mice previously immunized and treated with PHA implies that enhanced IgG production results from the activation of amplifier T cells and not the helper T cells which are required for memory.     

LPS-induced autoantibody response. I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes

Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro. The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.     

Regulation of the secondary in vitro antibody response by endogenous natural killer cells: kinetics, isotype preference, and non-identity with T suppressor cells

Our previous studies had demonstrated that depletion of endogenous natural killer (NK) cells resulted in an augmented primary antibody response in vivo and in vitro. We have now examined the effect of NK cell depletion on the in vitro secondary response to antigen. Treatment of primed murine spleen cells with anti-NK-1.1 allo-antibody and complement before culture resulted in a significant increase in the magnitude of the antigen-specific plaque-forming cell (PFC) response. This treatment did not affect the proportions of Lyt-2+, L3T4+, or sIg+ cells in the population, however, indicating that the augmentation in PFC was not due to changes in the ratio of T to B cells. Removal of endogenous NK cells had a greater effect on the IgG (indirect) PFC response (100 to 200% increase) than on the IgM (direct) PFC response (25 to 50% increase). In contrast, removal of Lyt-2+ cells before culture affected the IgM and IgG responses similarly. Moreover, the kinetics of augmentation differed between cultures depleted of Lyt-2+ cells and those depleted of NK-1.1+ cells. NK cells appeared to act earlier in the response than did T suppressor cells. The NK-1.1+ cells involved in antibody regulation were not involved in the generation of the in vitro derived T suppressor cells. The conclusion that the regulation of the antibody response by NK-1.1+ cells is distinct from that involving T suppressor cells was confirmed in experiments in which removal of both regulatory cell populations resulted in an increase in PFC that was greater than in cultures depleted of either NK or T suppressor cells.     

Sensitivity of amplifier T cells involved in the antibody response to type III pneumococcal polysaccharide to anti-lymphocyte serum.

Amplifier T cells responsible for enhancement of the antibody response to type III pneumococcal polysaccharide have been shown to be resistant to the effects of antilymphocyte serum (ALS) given at the time of immunization, a treatment that eliminates suppressor T cell activity. The resistance of amplifier T cells to ALS can be attributed to the fact that their activity develops after that of suppressor T cells. ALS given 1 or 2 days after immunization does abrogate amplifier T cell activity, independent of the mode by which that activity is elicited. The data emphasize the importance of kinetic considerations in understanding the effects produced by immunologically active agents such as ALS.