Studies of the immunological capacity of germfree mouse radiation chimeras. III. In vitro reconstitution of the T-helper cell deficiency

The plaque-forming cell (PFC) response of long-term radiation induced allogeneic bone marrow chimeric (ABMC) mice has been shown to be markedly deficient. The nature of the cellular deficiency of the primary PFC response was investigated using in vitro culture techniques. Adherent spleen cells from ABMC or DBA/2 mice support equally well the development of PFC from nonadherent DBA/2 spleen cells. Nonadherent cells prepared from ABMC mice when cocultivated with DBA/2 adherent cells showed a minimal response. However, the addition of activated DBA/2 T cells to cultures containing adherent cells from DBA/2 mice and nonadherent cells from ABMC mice completely reconstituted the in vitro response to sheep erythrocytes. Therefore a cellular deficiency of the humoral immune system of ABMC mice was shown to be associated with the thymus-derived lymphocyte pool.     

Enhanced immune response late in primary cytomegalovirus infection of mice.

Murine cytomegalovirus (MCMV) infection has been previously shown to depress humoral and cell-mediated functions to non-MCMV antigens. In this report we show that in C3D2 mice undergoing nonlethal primary infection the depressed anti-sheep RBC plaque-forming cell (PFC) response is followed by an enhanced PFC response. Infected mice often generated twice the number of PFC per spleen than that of control mice. Total numbers of spleen cells as well as the recovery of virus from spleens of infected mice did not distinguish the depressed from the enhanced phase of the response. Investigation of the kinetics of the response revealed a defect in shutdown regulation. This enhanced PFC response during primary MCMV infection was not reflected in measurements of serum hemagglutinin. These findings suggest that MCMV induces an impairment of immunoregulation.     

Plaque-forming cell response in BALB/c mice to two preparations of LPS extracted from Salmonella enteritidis.

The pattern of development of antibody-forming cells in BALB/c mice after immunization with PW-LPS or TCA-LPS was shown to be different. On days 10 and 20, the primary response to PW-LPS was characterized by a low level of IgM synthesis. The plaque-forming cell (PFC) response to TCA-LPS, however, increased from day 10 to day 20. Initially, IgM was the only detectable antibody synthesized but by day 20 a significant number of IgG-producing spleen cells had developed. After a secondary immunization with the appropriate lipopolysaccharide (LPS) preparation, IgG-producing spleen cells were detected in mice immunized with either PW-or TCA-LPS. Partial removal of the LAP or TCA-LPS with phenol or trypsin and pronase significantly reduced the PFC response, suggesting that the protein moiety played an influential role in the immunogenicity of TCA-LPS. The TCA-LPS contained the same antigenic dterminants as PW-LPS, so any difference observed between PFC response was not due to any associated immunogenic moiety.     

Motheaten, an immunodeficient mutant of the mouse. II. Depressed immune competence and elevated serum immunoglobulins.

Mice homozygous for the recessive mutation motheaten (me) are deficient in capacity for immune response but show an elevated level of serum immunoglobulins. In comparison to spleen cells from normal sibs, spleen cells from me/me mice have a severely depressed 19S PFC response to SRBC. In the GVH assay, spleen and thymus cells from motheaten donors caused significantly weaker reactions than like cells from normal sibs. Serum electrophoretic patterns of motheaten mice showed increased levels of alpha-, beta-, and gamma-globulins and decreased levels of albumin. Increases in quantities of all major classes of immunoglobulins were found in serum of me/me mice 5 weeks of age and older. Elevation of serum IgM was evident by 3 weeks of age and had reached 25 times the levels in normal sibs by 6 weeks of age. Immunoelectrophoresis and Ouchterlony analysis showed motheaten serum to have both kappa and lambda2 light chains. Evidence of autoimmunity was found in motheaten mice in the granular deposition of IgM and IgG in kidney glomeruli. Motheaten mice, thus, appear to have a severe immune deficiency, but the basic nature of the deficiency is not yet known.     

Differential immunosuppressive effects of ascites fluid from mastocytoma-bearing mice on primary vs secondary immunocyte responses.

The effects of immunosuppressive ascites fluids from mastocytoma-bearing mice on the primary vs secondary immune response to sheep red blood cells (SRBC) was examined. Injection of mice with ascites fluid from tumor-bearing mice markedly depressed the primary immune response of normal syngeneic mice challenged with SRBC. However, there was a preferential depression of the 19S IgM antibody response as compared with the 7S IgG response. Injection of ascites fluid shortly before secondary immunization of mice with SRBC also resulted in depressed IgM PFC responses but only a slight to moderate depression of IgG PFC. Treatment of mice with the ascites fluid before primary immunization had little if any effect on the secondary IgG PFC response, although the IgM response was moderately depressed. These results indicate that the immunosuppressive factor(s) present in the ascites fluid of mastocytoma-bearing mice has a differential effect on distinct classes of immunocytes. Those immunocytes or their precursors involved in formation of low efficiency 7S IgG antibody are more resistant to immunodepression. Such differences appear due to different sensitivities of cells involved in the immune response system.     

Generation of cytotoxic T lymphocytes in vitro. VII. Suppressive effect of irradiated MLC cells on CTL response.

Irradiated cells obtained from MLC at the peak of the CTL response caused profound suppression of generation of CTL when added in small numbers at the initiation of primary MLC prepared with normal spleen cells. The inhibitory activity of the MLC cells was not affected by irradiation (1000 rads) but was abolished by treatment with anti-theta serum and complement. The suppression was immunologically specific. The response of A (H-2a) spleen cells toward C3H (H-2k) alloantigens was suppressed by irradiated MLC cells obtained from MLC prepared with A spleen cells and irradiated C3H-stimulating cells, whereas the response of A spleen cells toward DBA/2 (H-2d) alloantigens was affected relatively little. However, if irradiated C3H X DBA/2 F1 hybrid spleen cells were used to stimulate A spleen cells in MLC, addition of irradiated MLC cells having cytotoxic activity toward C3H antigens abolished the response to both C3H and DBA/2 antigens. The response to DBA/2 antigens was much less affected when a mixture of irradiated C3H and DBA/2 spleen cells was used as stimulating cells. Thus, the presence of MLC cells having cytotoxic activity toward one alloantigen abolished the response to another non-cross reacting antigen only when both antigens were present on the same F1 hybrid-stimulating cells. This suppression of generation of CTL by irradiated MLC cells apparently involves inactivation of alloantigen-bearing stimulating cells as a result of residual cytotoxic activity of the irradiated MLC cells. This mechanism may be active during the decline in CTL activity noted in the normal immune response in vivo and in vitro.     

In vivo immune response suppression by the supernatant from concanavalin A-activated spleen cells.

Supernatants from concanavalin A- (Con A) activated murine spleen cells have been shown to suppress the in vitro plaque-forming cell (PFC) response to sheep red blood cells (SRBC). The present study examined the effect of such Con A-activated spleen cell supernatants (herein termed CONS) on the in vivo immune response to SRBC in C57BL/6, BALB/c and CDF1 mice. CONS derived from BALB/c spleen cells suppressed direct PFC 4 and 8 days after immunization with 2 X 10(8) SRBC. CONS also suppressed indirect PFC 8 days after immunization, as well as serum hemagglutinins to SRBC. The PFC response of C57BL/6 (H-2b) mice was suppressed as much as that of BALB/c (H-2d) by CONS derived from BALB/c mice, indicating a lack of H-2 specificity of the CONS. In addition to suppression of the antibody response to SRBC, in vivo CONS administration resulted in reduction in spleen cell number. This reduction was not sufficient to explain the decreased PFC response. When the CONS was separated into less than 10,000 m.w. and greater than or equal to 10,000 m.w. fractions, the immunosuppressive activity was found in the less than 10,000 m.w. fraction. This observation suggests that intact interferon, SIRS, and MIF were not responsible for the results obtained.     

Cellular requirements for the expression of IgM. Immunological memory in vitro

In vitro culture techniques have been used to compare the direct (IgM) plaqueforming cell (PFC) response to heterologous erythrocytes (RBC) by normal mouse spleen cells and spleen cells from mice injected intravenously with 5 × 10⁴ RBC ten days previously [low dose primed (LDP)]. Although LDP mice fail to undergo a significant primary PFC response, their spleen cells are capable of a secondary or enhanced PFC response in vitro. The secondary PFC response is shown to be a function of: (A) an increase in the frequency of immunocompetent cells or units (IU) due to in vivo priming, and (B) an increased number of PFC generated per IU subsequent to in vitro stimulation. The latter increase is shown to be mediated through a shorter PFC doubling-time during logarithmic expansion of the PFC population. Analysis of nonadherent spleen cell dose response experiments indicate that two nonadherent cell types interact in the secondary response. Subsequent cocultivation experiments suggested that both of these cell types must be “primed” to allow induction of a secondary response. Although adherent cells are required for the secondary response, normal splenic adherent cells serve as equivalent substitutes for LDP adherent cells.     

Triiodothyronine improves the primary antibody response to sheep red blood cells in severely undernourished weanling mice

Three experiments were conducted in which weanling mice were fed a nutritionally complete diet either ad libitum or in restricted quantities such that they lost about 30% of their initial weight over a 14-day period. In Experiments 1 and 2, half the animals from each group received dietary triiodothyronine (T3) supplements. In Experiment 3, food-intake-restricted mice were fed graded levels of potassium iodide. Malnutrition reduced the number of nucleated cells per spleen, the number of splenic IgG plaque-forming cells (PFC) per 10(6) cells, and the serum antibody titers against sheep red blood cells. T3 supplements increased antibody titers, the number of nucleated cells per spleen, and both IgM and IgG PFC per 10(6) spleen cells in malnourished mice, but had no effect on well-nourished mice. The beneficial effect of T3 was not a result of improved protein, energy, or iodine status in the malnourished mice.     

Antibody formation in mouse bone marrow. VIII. Dependence on potentially circulating memory cells: a study with parabiotic mice.

Mouse bone marrow barely contains antibody-producing plaque-forming cells (PFC) during the primary response to sheep red blood cells (SRBC). However, during the secondary response, the number of IgM, IgG, and IgA PFC in the bone marrow can rise to a level which surpasses the number of PFC in all the other lymphoid organs together. In the present paper we investigated whether the capacity of immune mice to react upon a booster injection of SRBC with a bone marrow PFC response can be transferred from immune to nonimmune mice. Therefore, mice primed with SRBC 6 months previously and nonprimed syngeneic mice were joined for parabiosis and were separated from each other at various intervals after joining. These separated mice were subsequently immunized with SRBC. It was found that, after 3 weeks of parabiosis, the nonprimed members reacted upon an injection of SRBC with a bone marrow IgM, IgG, and IgA PFC response as high as did the previously primed members. Furthermore it could be demonstrated by means of cell transfer experiments that, after a period of parabiosis of 3 weeks, the bone marrow and spleen of the normal mice contained about as many memory cells as the bone marrow and spleen of the immune mice. These results suggest that antibody formation in mouse bone marrow is dependent on a population of potentially circulating memory cells.     

Antibody formation in mouse bone marrow. IX. Peripheral lymphoid organs are involved in the initiation of bone marrow antibody formation.

Mouse bone marrow is barely capable of plaque-forming cell (PFC) activity during the primary response to sheep red blood cells (SRBC). However, during secondary-type responses, it becomes the major organ, containing IgM, IgG, and IgA PFC. In the present paper, the influence of splenectomy (Sx) upon the secondary bone marrow PFC response to SRBC was investigated. When previously primed mice were splenectomized just before the second intravenous (iv) injection of SRBC, the effect of Sx upon the height of the bone marrow PFC response was dependent on the booster dose. Sx just before a booster of 10⁶ SRBC iv almost completely prevented bone marrow PFC activity, whereas an iv booster dose of 4 × 10⁸ SRBC evoked a normal IgM, IgG, and IgA PFC response in Sx mice. Apparently low doses of iv administered antigen require the spleen in order to evoke antibody formation in the bone marrow. Experiments with parabiotic mice, consisting of Sx and sham-Sx mice, showed that this facilitating influence of the spleen upon bone marrow antibody formation occurs via the blood stream. In a subsequent study, it was investigated whether the spleen is required throughout the bone marrow PFC response or only during the few days of the initiation phase. Therefore, mice were splenectomized at different intervals after a booster injection of 10⁶ SRBC iv. It appeared that Sx 2 days after the booster injection could still prevent the normal bone marrow PFC activity, whereas Sx at Day 4 could no longer do so. Apparently, after an iv booster injection, the spleen is only required for initiation of the bone marrow PFC response and not for the maintenance of this PFC activity thereafter.     

Dissociation of immune capacity from nutritional status by triiodothyronine supplements in severe protein deficiency

Weanling mice were fed ad libitum from age 23 to 37 days either an 18 or an 0.6% protein diet. Half the animals in each dietary group received supplemental triiodothyronine (T3, 0.2 mg/kg diet). T3 increased the primary in vivo antibody response of protein-deficient mice to sheep red blood cells, as measured by both splenic plaque-forming cells (PFC) per 10(6) nucleated spleen cells and serum hemagglutinin titers. T3 also increased PFC/spleen in well-nourished mice. The effect on protein-deficient animals was achieved although nutritional status in these animals, as estimated by weight loss and carcass composition, was further impaired by T3 supplementation. These results support the hypothesis that immune functions can be improved independently of nutritional status in severe (wasting) malnutrition. Insofar as T3 was effective in a model of malnutrition that does not reduce serum total or free T3 levels, the phenomenon appears to represent a pharmacological action of the hormone.     

The origin of antibody-forming cells detected in the bone marrow after thymus-independent antigen-stimulation and abnormality in migration of B cells of X-linked immunodeficient CBA/N mice

The anti-TNP IgM plaque-forming cells (PFC) were generated in the spleen and bone marrow of non-immunodeficient normal mice after intraperitoneal administration of TNP-LPS. Irradiation of normal mice while shielding bone marrow completely abrogated the generation of bone marrow PFC, indicating that they are derived from extramedullary sites. The bone marrow PFC, response to TNP-LPS was low in X-linked immunodeficient CBA/N strain mice, while the spleen response was comparable to that seen in the normal mice. To further study the basis of the deficient bone marrow PFC response in CBA/N mice, spleen cells were adoptively transferred to irradiated syngeneic mice stimulated with TNP-LPS. While spleen cells from normal mice generated high numbers of PFC in recipient bone marrow and spleen, those from CBA/N strain mice could not generate bone marrow PFC. This result was obtained regardless of whether normal or CBA/N recipients were used. These results indicate that TNP-LPS administration normally results in the migration of B lymphocytes from the periphery into the bone marrow and that B cells from immunodeficient CBA/N strain mice bear an inherent defect in this migratory function. This migratory defect was shown to be X-linked, as are the other previously reported B cell defects in this inbred mouse strain. The possible relationship between this migratory defect and the maturational defects of B cell lineage as reported previously in CBA/N strain mice is discussed.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

Summary A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described.With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

Latex bread technique for detection of the plaque-forming cell response to teratocarcinoma tumor antigens

A latex bead technique modified for measuring the plaque-forming cell (PFC) response to teratocarcinoma tumor antigens in syngeneic animals is described. With this method one can detect both the primary (IgM) and the secondary (IgG) immune response to tumor antigens. Optimal detection of the PFC response depends on the proper ratio of sheep red blood cells to latex beads and the dose of tumor cell antigen used for immunization. The presence of fetal calf serum interfered with immunization of animals and the coating of the latex beads with the tumor cell antigens. The plaques obtained in response to immunization with teratocarcinoma cell antigens varied in size, probably reflecting the complex immune response to more than one class of antigens on tumor cells.     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.     

Defective primary and secondary IgG responses to thymic-dependent antigens in autoimmune PN mice

Palmerston North (PN) mice spontaneously develop autoimmune disease resembling SLE. Because immune responsiveness has not been defined in this strain, a study was designed to assay primary splenic plaque-forming cell (PFC) responses to thymus-dependent (TD) and thymus-independent (TI) Ag. Initial surveys of PN mice inoculated with the TD Ag SRBC showed adequate production of IgM PFC, but small numbers of IgG PFC were developed with polyspecific antiserum. In contrast, H-2-compatible DBA/1 control mice gave the expected responses to SRBC (IgG plaques elevated twofold compared with IgM plaques). PN mice had the usual responses to Ag that are largely TI; both PN and DBA/1 mice had active IgM and modest IgG responses to TNP-LPS and TNP-Ficoll. Additional experiments determined that PN mice had similar patterns of defective IgG responses to several different TD Ag (SRBC, horse RBC, and DNP-keyhole limpet hemocyanin). In each instance, the usual predominance of IgG1 plaques was absent, and total numbers of plaques developed with antisera specific for IgG isotypes were suppressed. Defective PN IgG production was evident as early as 3 wk of age, was not influenced by aging to 43 wk, and was not corrected by increasing the antigenic challenge 10-fold. PN spleen cells treated with monoclonal anti-Thy-1.2 and C were injected with pools of DBA/1 T cells into 850-rad irradiated (DBA/1 x PN)F1 hybrids. These recipients expressed low IgG1 responses to SRBC, suggesting that the B cell-containing fraction that was not lysed by anti-Thy-1.2 transferred the PN defect. PN mice, which do not respond to TD Ag with active IgG production, contradict the proposal that autoimmunity is associated with hyper-responsiveness to TD and TI Ag.     

In vivo immune response to TNP hapten coupled to thymus-independent carrier lipopolysaccharide

Lipopolysaccharide has been utilized as a carrier for the TNP hapten, producing an antigen which induces an in vivo thymus-independent antibody response to TNP as determined using athymic nude mice and their normal littermates. The immune response to TNP-LPS was investigated at both the antibody-forming cell and the serum antibody levels.The primary response to an optimal dose of TNP-LPS (1.0 μg) exhibited unusual kinetics reaching a sharp peak on day 3 of 58,000 anti-TNP PFC/spleen. Serum antibody to TNP was first detected on day 3 and reached a maximum log₂ titer of 17.5 on day 5, an uncommonly high level for hapten-carrier conjugates and most carriers. Both the anti-TNP serum antibody and PFCs were exclusively IgM. No IgG antibody was detected in the primary response through 28 days postimmunization, nor was any detected in any experiment described in this paper. The primary PFC response to 1.0 μg of TNP-LPS was specific for TNP, producing no evidence of polyclonal antibody synthesis. The relative affinities of PFC-secreted antibody were investigated using hapten inhibition. The hapten inhibition curves for TNP-LPS and TNP-SRBC were very similar, indicating that relatively high affinity antibody was elicited by TNP-LPS. The secondary response to this dose following priming with TNP-SRBC or TNP-LPS was similar to the primary response, though the peak was less sharp in both cases. The response to the homologous secondary challenge shifted somewhat, reaching a peak on days 3–4. The effect of various doses in priming or challenging for the secondary response to TNP-LPS was investigated. Using an increased PFC response as a criterion, no dose was optimal for priming or immunological memory to TNP-LPS. While the adoptive primary response to TNP-LPS reached a low level peak on day 7, the adoptive secondary attained a maximum on day 6. This shift in kinetics in intact mice and in adoptive hosts in comparing primary to secondary responses indicated that a state of B cell priming may be induced. However, its full expression may be suppressed by endogenous factors at the time of priming, such as the high level of circulating anti-TNP antibody or residual antigen. Adoptive transfer would remove the cells from these influences, allowing such B cell priming to manifest itself fully.     

Variations in the responses of C57BL and a mice to sheep red blood cells: II. Analysis of plaque-forming cells

The number of direct and indirect plaque-forming cells (PFC) and the serum hemolytic activity was determined for A/He, C57BL/6J, and B6AF1 mice responding to multiple injections of sheep red blood cells (SRBC). Although the kinetics of the primary response differed, all mice had high numbers of both direct and indirect PFC and low-titered 2-mercaptoethanol (2-ME) sensitive serum antibody. Following multiple SRBC injections, the A/He spleens contained predominantly IgG producing PFC. Their serum antibody activity was resistant to 2-ME signifying the presence of IgG. The serum activity of both the C57BL/6J and B6AF1 mice was sensitive to 2-ME (IgM antibody) over the course of immunization, and although there was a definite IgM PFC memory response, the presence of 7S memory PFC was questionable. The results are discussed in terms of the maturation of the antibody response to SRBC and of the question of the postulated IgM and IgG switch.     

Different effects of the capsular polysaccharide of Klebsiella pneumoniae on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen.

Using the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) as a polyclonal B-cell activator (PBA) and sheep red blood cells (SRBC) as a T-dependent antigen, we studied the effects of PBA on the functions of various subpopulations of B cells in the immune response of mice to T-dependent antigen. Antibody-forming cells (AFC) of IgM and IgG types were estimated as anti-SRBC direct and indirect plaque-forming cells (PFC), and the B cells with precursor activities involving generation of AFC and supplementing new B cells as rosette-forming cells (RFC) of the B-cell type. Stimulation of normal mice by CPS-K caused a definite increase in the number of direct PFC but not in that of indirect PFC and RFC in the spleens. The responsiveness of spleen cells of CPS-K-treated mice to generate PFC and RFC responses to a subsequent injection of SRBC was lower than that of CPS-K-untreated normal mice. In this case, the responsiveness to generate RFC and indirect PFC was inhibited more strongly by CPS-K than that to generate direct PFC. When CPS-K was injected into normal mice simultaneously with SRBC, CPS-K never decreased but increased the levels of PFC and RFC responses to SRBC. In the spleens of SRBC-primed mice, the number of RFC was markedly decreased following injection of CPS-K, the number of direct PFC was increased only slightly and the number of indirect PFC was increased very slightly. The responsiveness of spleen cells of these CPS-K-treated SRBC-primed mice to generate secondary PFC and RFC responses to a subsequent injection of SRBC was much lower than that of CPS-K-untreated SRBC-primed mice. In this case, the responsiveness to generate the secondary RFC and indirect PFC responses was more strongly inhibited by CPS-K than that to generate the secondary direct PFC response. When CPS-K was injected into SRBC-primed mice simultaneously with the secondary injection of SRBC, there were marked decreases in the level of the secondary RFC response and slight decreases in that of the secondary indirect PFC response, but little change in that of the secondary direct PFC response. From these results it has been concluded that CPS-K provides the positive signal (the minor action) and the negative signal (the major action) to various subpopulations of B cells functioning at various stages of the immune response to T-dependent antigen in different ways, and acts to regulate the levels of B-cell responses to the antigen-mediated positive signal.