An examination of the TRF assay reveals a heterogeneity of TRF-like activities

Supernatant from the cloned Mlsa,d-reactive helper T cell line L2V is a potent source of T cell replacing factor (TRF) activity. To determine whether L2V SF was representative of TRF-active supernatants, it was compared with supernatant from Con A-stimulated spleen cells (CASF) by using TRF assays. This analysis, facilitated by the use of four different antigens (R36a, TNP-R36a, SRBC, and HRBC), produced the following results. 1) L2V SF and CASF allowed responses of similar magnitude against either R36a or TNP-R36a; however, CASF always allowed responses of fivefold to 30-fold greater magnitude against SRBC than L2V SF. 2) B cells from xid and "normal" mice will give equally large responses to TNP-R36a when CASF is used as the source of TRF; however, L2V SF will only effect the responses by "normal" B cells. 3) L2V SF-driven responses to R36a and SRBC, and CASF-driven responses to R36a, follow single-hit kinetics and are IL 2 independent, whereas CASF-driven responses to SRBC follow multi-hit kinetics and are IL 2 dependent. These results indicate that the TRF assay measures a heterogeneity of TRF-like activities that can be distinguished according to the supernatant, antigen, and/or B cell responders used. The determination of whether this heterogeneity is due to the number of molecular components of a single "type" of TRF required for each antigen-induced PFC response or to the existence of distinct "types" of TRF is examined.     

Immunoregulation in the rat: cellular and molecular requirements for B cell responses to types 1, 2, and T-dependent antigens

The requirements for primary in vitro plaque-forming cell (PFC) development in cultures of purified rat splenic B cells have been examined. Rat B cells were directly responsive to the type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA), but both T cells and adherent accessory cells were required for B cell responses to the type 2 antigen TNP-Ficoll and the T cell-dependent (TD) antigen sheep erythrocytes (SRBC). However, the cellfree supernatants from concanavalin A-induced spleen cells of rat or mouse origin replaced the requirement for T cells and macrophages, and resulted in PFC development in response to TNP-Ficoll and SRBC and augmented PFC numbers in response to TNP-BA. Culture supernatants from induced murine T cell and macrophage cell lines were used to partially deduce the molecular requirements for the support of PFC development by rat B cells to these three antigens. Supernatants from the EL-4 (EL-4 sup) and B151 K12 (B15 sup) T cell lines augmented TNP-BA responses, suggesting that B cell growth factor II (BCGF-II) mediated this effect. An admixture of purified interleukin 2 (IL 2) and B15 sup supported PFC development to SRBC; indicating that IL 2, BCGF-II, and the T cell-replacing factor in B15 sup (B15-TRF) were sufficient to support this response. In addition, the IL 2 plus B15 sup-supported anti-SRBC PFC response was increased by the addition of an interleukin 1-containing fraction from the supernatant of the macrophage line P388D1. PFC development in response to TNP-Ficoll had the most stringent requirements and only occurred in the presence of EL-4 sup and B15 sup (IL 2, BCGF-I, BCGF-II, EL-TRF, B15-TRF). These data indicate that different cellular and molecular requirements exist for PFC development in response to types 1, 2, and TD antigens by rat B cells.     

Purification and physicochemical characterization of murine T cell replacing factor (TRF)

Murine T cell replacing factor (TRF) was purified from a cellfree supernatant of a T cell hybridoma (B151K12) that constitutively produces TRF. Two assay systems for TRF activity were employed: 1) induction of anti-DNP IgG PFC responses in cultures of splenic B cells from DNP-KLH-primed BALB/c mice, and 2) induction of IgM PFC in chronic B cell leukemic cells (BCL1). The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, gel permeation with fast protein liquid chromatography (FPLC), and disc polyacrylamide gel electrophoresis. Overall, TRF was purified approximately 34,000-fold with a maximum 3.8% recovery of activity, and the specific activity of the purified TRF was approximately 9.6 X 10(4) U/mg. The TRF that is active in these systems is distinct from the other lymphokines such as IL 1, IL 2, BCGFI (now known as BSFp1), and gamma-interferon. The TRF is extremely hydrophobic, with an apparent m.w. of 50,000 to 60,000 on gel permeation chromatography and 18,000 on SDS-PAGE under reducing conditions. Highly purified B151-TRF abrogated the activity by treatment with trypsin but not with RNase. Moreover, it bound to lima bean agglutinin-Sepharose specific for N-acetylgalactosamine residues, indicating that B151-TRF is a glycosylated glycoprotein containing N-acetylgalactosamine residues. The role of N-acetylgalactosamine residues on TRF activity was additionally substantiated by the fact that the addition of appropriate amounts of N-acetylgalactosamine in the assay systems for TRF preferentially induced a profound suppression for TRF-mediated PFC responses.     

Synergy between T cell-replacing factor and bacterial lipopolysaccharides (LPS) in the primary antibody response in vitro: a model for lipopolysaccharide adjuvant action.

Unfractionated spleen cells, B cells from normal mice, and nu/nu spleen cells respond to the addition of bacterial lipopolysaccharide (LPS) and T-cell-replacing factor (TRF) by production of plaque-forming cells (PFC) in excess of the number expected from the addition of LPS and TRF separately. This synergistic activity is dependent on the presence of the antigen, SRBC. Supernatants of both allogeneic spleen cell mixtures and spleen cells cultured with Con A are effective and synergize best at concentrations suboptimal for their ability to act as TRF alone. Culture supernatants of unstimulated normal or fractionated cell populations are ineffective. Synergy is not dependent on the presence of macrophages in the cultures. Purified LPS free from active contaminants, as well as commercially available LPS, show synergy with TRF. Synergy was seen when TRF was added at initiation of culture or 24 hr later. It is suggested that synergy is the equivalent of LPS adjuvant activity, that the role of T cells in LPS adjuvanticity is that of a conventional cooperating cell, and the LPS acts as an adjuvant by inducing B cells to become more sensitive to T cell helper factors.     

Alloreactive cloned T cell lines. VI. Multiple lymphokine activities secreted by helper and cytolytic cloned T lymphocytes

Culture supernatants generated by alloantigenic or lectin stimulation of a cloned helper T lymphocyte, designated L2, contain interleukin 2 (IL 2), granulocyte/macrophage colony-stimulating factor (CSF), B cell stimulating factor (BCSF), macrophage (Ia+)-recruiting factor (MIRF), (Ia+)-inducing activity, gamma-interferon, Fc receptor-enhancing activity, macrophage migration inhibitory factor (MIF), macrophage activation factor (MAF), interleukin 3 (IL 3), and a factor responsible for prolonging the synthesis and secretion of the fourth and second components of complement by guinea pig peritoneal macrophages. Erythropoietin was not detected. A spontaneously arising variant of L2, designated L2V, produces much lower quantities of macrophage-stimulating activities, IL 2, and interferon. However, when compared to L2, L2V produces much higher levels of BCSF, equivalent amounts of IL 3, and slightly smaller amounts of CSF. Unlike L2V, a cytolytic clone, designated L3, secretes lymphokines that primarily affect macrophage function. The time course of lymphokine production by L2 cells indicates that for the six lymphokine activities studied there are three different times at which maximal or near maximal levels are reached, as follows: 1) IL 2, 12 to 24 hr; 2) IL 3 and CSF, 24 to 48 hr; and 3) (Ia+)-inducing activity, MAF, and interferon, 48 hr or later. Only IL 2 activity disappears during the 8-day culture cycle. The time course data and the differential production of activities by the three types of lymphocyte clones suggest that at least four terminal effector lymphokine molecules account for the ten biologic activities tested.     

Cloned T cells with "co-helper" activity. I. Biologic activities of the clone

Clones of sheep erythrocyte-(SRBC) specific helper T cells with the surface phenotype Thy-1+, Ly-1+, Ly-2- have been derived that grow in vitro in the absence of exogenous antigen or added growth factors. The IL 2-independent clone, 101.6 has been shown to produce a supernatant factor that augments the primary anti-SRBC but not anti-burro RBC responses of whole spleen cells or Ly-1 T plus B cell cultures. The supernatant does not help B cells directly. This augmenting activity is terminated "co-helper" because the enhancement requires the presence of normal Ly-1 T cells. The supernatant of 101.6 was not shown to contain IL 2; co-helper activity was distinguishable from IL 2 activity by absorption with SRBC but not with Con A blasts, and we observed that co-helper activity does not act on spleen cells that differ at the major histocompatibility complex.     

T cells produce TRF in response to Con A and factors in T cell hybridoma supernatants

In recent experiments, a soluble factor (TRF) that mediates the differentiation of anti-immunoglobulin (Ig)-activated B cells to Ig-secreting cells has been identified. TRF works in concert with a growth factor, probably IL 2, in the induction of activated B cells. In previous studies, TRF was identified in culture supernatants of activated T cells and accessory cells, and thus the cellular source (T cell or accessory cell) of the factor was not determined. In the present studies, we succeeded in inducing the production of TRF by T cell populations from which accessory cells had been vigorously depleted. Lymph node cells were depleted of accessory cells by nylon wool adherence and anti-Ia and complement treatment; these cells were activated with Con A and a T cell hybridoma supernatant that contains IL 2. Supernatants from these activated T cell cultures supported the differentiation of anti-Ig-activated B cells to Ig secreting cells. These results show that T cells produce the differentiation factor, and further that they do so in response to ligand (Con A) plus a T cell-derived factor.     

Effect of passive administration of alloantiserum containing antibody against putative acceptor(s) for T cell-replacing factor (TRF) in the neonatal stage on development of B cell activity responsive to TRF

Previously, we showed that the antiserum raised in male (DBA/2Ha X BALB/c)F1(DCF1) mice (T cell-replacing factor [TRF]-low response animals) by immunizing them with activated B cells from BALB/c mice (TRF-high-responders) contained antibodies against putative TRF-acceptor site(s). We have now evaluated the hypothesis that neonatal treatment of mice with the above antiserum suppresses the development of B cells responsive to TRF. Male DCF1 mouse anti-BALB/c B-cell antiserum or normal DCF1 mouse serum as a control was injected into BALB/c mice within 24 hr after birth. In the antiserum-treated mice, no augmented primary immunoglobulin M (IgM) antibody responses to sheep red blood cells (SRBC) were observed under the conditions in which markedly augmented IgM anti-SRBC responses were induced in control BALB/c mice, suggesting that development of B cells reacting with male DCF1 mouse anti-BALB/c B-cell antiserum is suppressed by the neonatal treatment with the antiserum. Furthermore, the development of B cell activity responsible for helper factors derived from T cells, such as TRF, was markedly suppressed in the neonatally antiserum-treated mice, whereas activity of B cells capable of interacting directly with helper T cells through antigen-bridges was not significantly affected by the same treatment. Such suppression of the B cell activity could be induced only when the antiserum was administered within 48 hr after birth. Moreover, neonatal treatment of mice with the antiserum induced suppressed responsiveness of B cells to a T-independent type 2 antigen, TNP-Ficoll. Neither serum-borne suppressive serum components nor suppressor cells were detected by the system employed. These results support the hypothesis that TRF responsive B cells constitute a subpopulation distinct from the other B cells capable of cooperating with helper T cells via cognate interaction.     

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.     

T-cell help for the IgA response: the function of T cells from different lymphoid organs in regulating the proportions of plasma cells expressing various isotypes

Differential distribution of IgA-specific primed Lyt 2- T cells (TH) in favor of gut-associated lymphoid tissue (GALT) has been proposed to account for the high proportion of IgA-producing plasma cells at mucosal versus nonmucosal sites. We find, however, that GALT TH primed enterically with sheep red blood cells (SRBC) contain no more help for IgA responses than peripheral lymph node (PN) TH primed subcutaneously. Moreover, GALT TH are only poorly primed by enterically administered soluble protein antigen and therefore provide less help for all isotypes than PN TH primed subcutaneously with the same antigen. On the other hand, supernatants of GALT TH stimulated with concanavalin A (Con A) in vitro do help higher IgA:IgG plaque-forming cell (PFC) ratios in cultures with 2,4, 6-trinitrophenyl-SRBC (TNP-SRBC) than supernatants from PN and spleen, indicating that, when appropriately stimulated, GALT TH are capable of promoting relatively higher IgA responses than TH from other sources. Responses elicited by either SRBC-primed TH or splenic Con A supernatants in the presence of TNP-SRBC contained higher IgA:IgG PFC ratios than those elicited by linked recognition in the presence of haptenated soluble protein carrier.     

"Nonspecific" immunoenhancing T cells in tumor-bearing mice include anti-idiotypic subsets

Splenocytes from DBA/2 mice inoculated 3 wk earlier with syngeneic P815 mastocytoma tumor cells produce increased numbers of antibody plaque-forming cells (PFC) when stimulated with either sheep red blood cells (SRBC) or phosphorylcholine (PC) on Streptococcus pneumoniae R36a in vitro. The nature of this nonspecific hyperreactivity was investigated in mixed cultures of purified splenic T and B cells. The addition of T cells from P815 tumor-bearing mice (TP815) into the cultures of normal B cells produced a significant enhancement of the PFC response to both SRBC and PC, when compared with the effect of normal T cells added to control cultures. The idiotypic profile of the enhanced anti-PC response was studied by a PFC-inhibition assay with monoclonal antibodies against two distinct idiotopic determinants (Id) of the T15 family. Normal B cells produced greater than 90% of T15 Id-positive (Id+) PFC. Addition of normal T cells diminished the proportion of T15 Id+ PFC to approximately 60%, whereas the rest of PFC were Id-. Addition of the immunoenhancing TP815 cells into the normal B cells cultures elevated the number of both T15 Id+ and Id- PFC responses, proportionally. However, when TP815 cells were first incubated on T15 protein-coated dishes and the non-adherent fraction was added to B cell cultures, the anti-PC PFC response remained enhanced but consisted of predominently T15 Id- PFC. These observations suggest that the early stage of P815 tumor growth activates various populations of specific helper/amplifier T cells including subsets with anti-idiotypic activity and that the generalized increase of antibody response to various antigens in tumor-bearing mice may be regarded as a polyclonal activation of specific T cells.     

The immunoregulatory role of bone marrow. IV. Role of an immunoenhancing glycoprotein derived from murine bone marrow

Bone marrow-enhancing factor (B-EF) is the spontaneous product of whole bone marrow cells cultured in serum-free medium for a short term (24-48 hr). The factor is prepared by ultrafiltration of BMC supernates to yield a preparation with a MW of greater than 10,000. Production of the factor is not dependent upon antigenic or mitogenic stimulation of BMC, but is inhibited by treatment of BMC with cycloheximide. B-EF augments the in vitro primary PFC response to SRBC, as well as in vitro secondary IgM and IgG PFC responses to SRBC. Enhancement by B-EF is antigen dependent, genetically nonrestricted, and maximal when present at the initiation of culture. B-EF cannot induce a polyclonal antibody response like the polyclonal activator LPS. B-EF is directly mitogenic for thymocytes, bone marrow, and whole spleen cells, but fails to act as a costimulator of thymocyte proliferation in the presence of Con A. B-EF cannot support the growth of the IL-2-dependent cell line CTLL-2. Since B-EF has not been purified, the supernatant may contain more than one activity. The factor is heat labile at 65 degrees C and is sensitive to enzymatic digestion with trypsin and neuraminidase; this implies that B-EF may be a glycoprotein.     

Isotype-specific immunoregulation. Systemic antigen induces splenic T contrasuppressor cells which support IgM and IgG subclass but not IgA responses

In the present study, we have isolated and characterized the Lyt-1+, -2- T contrasuppressor (Tcs) cells from mice systemically primed with SRBC. Adoptive transfer of splenic Tcs cells from these mice abrogates oral tolerance and supports IgM and IgG anti-SRBC plaque-forming cell (PFC) responses; however, unlike the responses seen after transfer of Tcs cells derived from orally primed mice, low IgA responses were seen. Mice systemically primed with lower SRBC doses (0.01 to 1%) exhibited contrasuppression only within the L3T4- T cell subset, whereas mice primed with a high dose of SRBC (10%), harbored Lyt-1+, -2- Tcs cells in both the L3T4+ and L3T4- subsets. Both the L3T4- and L3T4+ Tcs cell subsets supported IgM and IgG responses when adoptively transferred to orally tolerized mice, and when added to tolerized spleen cell cultures. Splenic Tcs cells from systemically primed mice supported mainly IgG1 and IgG2b subclass anti-SRBC PFC responses, a pattern also seen with Tcs cells derived from orally primed mice. Both L3T4+ and L3T4- Tcs cells from systemically primed mice exhibited well established characteristics of contrasuppressor cells including binding to Vicia villosa lectin and expression of I-J. The splenic effector Tcs cells which support IgM, IgG1 and IgG2b anti-SRBC PFC responses are antigen-specific, since both L3T4- and L3T4+ Tcs cells from spleens of mice primed with 10% SRBC reverse tolerance to SRBC, but not to horse erythrocytes (HRBC). Further, both L3T4- and L3T4+ Tcs cells from HRBC-primed mice reverse tolerance to IgM and IgG anti-HRBC, but not to anti-SRBC responses. Isolation of T3-positive Lyt-1+, -2- and L3T4- Tcs cell subsets by flow cytometry followed by adoptive transfer, showed that effector Tcs cells express T3 and presumably contain an Ag-R (TCR-T3 complex). These studies show that systemic priming with heterologous RBC induces splenic Ag specific Tcs cells in a dose-dependent manner, which support IgM and IgG subclass responses, but not IgA responses.     

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)     

Regulation of immune response in allogeneic mixed spleen cell cultures. I. Influence of I-region on the generation of suppressor cells

The immune responses of allogeneic mixed spleen cell cultures (MLC) to the T-dependent antigen, SRBC, and to the T-independent antigen, DNP-PAA, were investigated. The immune response to DNP-PAA in MLC with certain strain combinations was always suppressed as compared with the expected PFC response calculated from the PFC responses of the individual strains. This suppression was eliminated by treating the spleen cells with RAMB antiserum plus complement before the incubation of the MLC with DNP-PAA. It can be concluded that the suppression in the PFC response to the T-independent antigen DNP-PAA in MLC is due to the generation of suppressor T-cells. The PFC response to the T-dependent antigen, SRBC, in MLC showed either suppression, no change, or rarely augmenation, suggesting that the allogeneic mixed spleen cell cultures can generate both suppressor and helper T cells and that the balance between helper and suppressor activity regulates the PFC response to a T-dependent antigen. Suppressor activity was also generated in a one-way MLC, but the degree of suppression depended upon which of the two strains was responding. Similar amounts of thymidine were incorporated in the one-way MLR irrespective of which strains was responding. Thus, the extent of proliferation in one-way MLR is not related to the degree of suppressor activity generated. The results further indicate that a difference between two strains in the I-C, S, and G regions of the major histocompatibility complex is required to generate suppressor activitiy that can depress the response to a T-independent antigen, MLC between strains differing in K, I-A, I-B, I-J, I-E, and D regions generate little or no suppressor activity in this system.     

Heterologous antibody responses in mice with chronic T. cruzi infection: depressed T helper function restored with supernatants containing interleukin 2

Antibody production to sheep erythrocytes (SRBC) or hapten-conjugated SRBC (TNP-SRBC) was studied in mice with chronic Trypanosoma cruzi infections. Studies in vivo demonstrated that both IgM and IgG anti-SRBC responses were suppressed during chronic infection. Secondary IgG responses were suppressed regardless of whether the primary immunization was given before or after infection. The ability of cells from infected mice to provide help for antibody production was examined in vitro. Anti-SRBC responses were restored to cultures of whole spleen cells from infected mice by the addition of interleukin 2 (IL 2)-rich supernatants, indicating that these cells were capable of antibody production when sufficient help was provided. T cells from SRBC-primed infected mice were unable to provide significant help to normal B cell/M phi cultures for in vitro anti-TNP or anti-SRBC responses. The percentages of Thy-1+, Lyt-1+, and Lyt-2+ spleen cells were not significantly different between normal and infected mice. Anti-TNP and anti-SRBC responses were restored to cultures that contained T cells from infected mice and normal B cell/M phi by the addition of IL 2-rich spleen cell supernatants. The suppression of in vitro antibody responses in mice with chronic T. cruzi infections was associated with a lack of T cell help, which was provided by exogenous spleen cell supernatant.     

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.     

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.     

BCGFII activity on activated B cells of a purified murine T cell-replacing factor (TRF) from a T cell hybridoma (B151K12)

Experiments were performed to examine a growth-promoting activity on B cells or B leukemic cells of T cell-replacing factor (TRF) produced by a murine T cell hybridoma (B151K12) which constitutively produces TRF. The cellfree supernatant (CFS) from B151K12 cells (B151-CFS) could induce terminal differentiation of pre-activated B cells or in vivo passaged chronic B leukemia cells, BCL1, into immunoglobulin-secreting cells, while it did not exert a nominal lymphokine activity such as BCGFI (now known as BSFpl), IL 2, or gamma-interferon. However, it promoted [3H]thymidine uptake of dextran sulfate (DXS)-stimulated normal B cells and in vivo passaged BCL1 cells, suggesting that it also has BCGFII activity. We tried extensively to purify and to separate the TRF active molecule from the BCGFII active molecule by using many types of purification procedures. The purification scheme consisted of ammonium sulfate precipitation, DEAE-cellulose chromatography, Blue-Sepharose chromatography, hydroxylapatite chromatography, and gel permeation with fast protein liquid chromatography (FPLC). It was revealed that the BCGFII active molecule was hardly separable from the TRF during the entire purification procedure. The TRF as well as BCGFII active materials were glycoprotein with an apparent m.w. of 50 to 60 Kd on gel permeation chromatography and 18 Kd on SDS-PAGE under reducing conditions. The BCGFII active materials were hardly separable from the TRF active one, even after a reverse-phase FPLC, in which both BCGFII and TRF activities were recovered in the fractions eluted at 44 to 48% acetonitrile in 0.1% trifluoroacetic acid (TFA). Furthermore, the absorption of TRF and BCGFII active materials by using BCL1 cells removed not only TRF but also BCGFII activity. Moreover, B cell-specific monoclonal antibody (9T1), which can preferentially block TRF-dependent plaque-forming cell responses, also inhibited the expression of BCGFII activity to BCL1 cells. Taking all of the results together, we conclude that the TRF from B151K12 cells promotes growth of appropriately activated, such as DXS-stimulated normal cells and BCL1 tumor cells. These results suggest that B151-TRF may act on B cells as B cell growth and differentiation factors.     

Suppression of humoral and delayed hypersensitivity responses by distinct T cell subpopulations.

Immunization of mice with a supraoptimal dose of sheep red blood cells (SRBC) results in splenic T cell populations capable of specifically suppressing recipients' plaque forming cell (PFC) and delayed-type hypersensitivity (DTH) responses to SRBC when tested in an adoptive transfer system. By localization on discontinuous bovine serum albumin (BSA) gradients and relative sensitivity to Cytoxan, two distinct T cell subpopulations suppressing DTH reactivity were identified. One population could not be distinguished from T cells capable of inhibiting direct and indirect PFC responses. However, another population appeared quite distinct and capable of inhibiting DTH, but not PFC responses.