Immunoregulation in the rat: requirements for in vitro B cell responses to classical TI-1 and TI-2 antigens

The presence of suppressor cells and their mediators has made it difficult to induce B cell mitogenic or immune responses in rat spleen cell cultures. In the present study, we have defined culture conditions required for induction of in vitro thymic independent (TI) immune responses in the rat. Rat spleen cell cultures support low responses to various trinitrophenyl (TNP) haptenated antigens including TNP-Brucella abortus (TNP-BA), TNP-lipopolysaccharide [LPS; either phenol (Ph)- or butanol (Bu)-water extracted], TNP-Ficoll, and TNP-dextran. However, all of these antigens induced good splenic anti-TNP PFC responses when given at appropriate doses in vivo. When spleen cells were depleted of adherent cells and cultured with TI antigens in vitro, good anti-TNP PFC responses were seen with TNP-BA, whereas, lower responses were induced by TNP-LPS (Ph or Bu). No responses were observed in cultures incubated with either TNP-Ficoll or TNP-dextran. Purified splenic B cell cultures [prepared by panning on plates coated with anti-rat F (ab')2] supported good responses to TNP-LPS (Ph or Bu) and TNP-BA. The addition of irradiated splenic adherent cells (macrophages, M phi) to either M phi-depleted or purified B cell cultures completely abrogated in vitro responses to TNP-BA or TNP-LPS (Ph or Bu). Purified splenic B cell cultures generally responded poorly to TNP-Ficoll or TNP-dextran. Addition of indomethacin (IM) to spleen cell cultures abrogated suppression and allowed anti-TNP PFC responses to TNP-BA, TNP-LPS (Ph or Bu), TNP-Ficoll, and TNP-dextran. Furthermore, nude spleen cell cultures treated with IM, also allowed significant TNP-Ficoll and TNP-dextran immune responses; however, untreated cultures did not respond to these antigens. Our studies indicate that rat splenic B cell cultures are responsive to TI antigens, and highest responses occur with the murine TI-1 class, e.g., TNP-BA and TNP-LPS. Inhibition of suppression with IM restored splenic B cell responses to the murine TI-2 class, i.e., TNP-Ficoll and TNP-dextran.     

Delayed maturation of the antibody response to type 2 thymus-independent antigens in a partially inbred strain of chicken

The ontogeny of antibody responses to trinitrophenylated (TNP) thymus-independent (TI) antigens was compared in two partially inbred strains of chicken: the SC strain (B2/B2 genotype) and the FP strain (B15/B22 genotype). In the SC chicken, maturation of both the splenic anti-TNP plaque-forming cell (PFC) response and the 19S hemagglutinating antibody response to TI type 2 (TI-2) antigens, TNP-Ficoll and TNP-dextran, were delayed to a significantly later time in ontogeny (20 wk of age) than in the FP chickens (9 wk of age). Four- to 6-wk-old SC chickens were virtually immunologically unresponsive to stimulation with TI-2 antigens. The TI-1 antigen TNP-Brucella abortus was equally immunogenic in both FP and SC chickens of different age groups tested. Kinetic studies of the primary PFC response to TNP-Ficoll in immunologically mature chickens of the SC and FP strains demonstrated a peak PFC response 4 days after antigen injection, followed by a rapid decline in numbers of splenic PFC/spleen on day 6. The results of these studies are discussed in relation to earlier observations that suggested there may be a delay or a defect in the ontogeny of the thymus in the SC chicken.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Ontogeny of murine B-cell responses to thymus-independent trinitrophenyl antigens.

The ontogeny of B-cell responsiveness to three thymus-independent trinitrophenyl (TNP) antigens has been examined in BALB/c mice in vivo and in vitro. When in vivo splenic plaque-forming cell (PFC) responses to TNP-conjugated lipopolysaccharide (TNP-LPS), Ficoll (TNP-Ficoll), and Brucella abortus (TNP-Brucella) were measured in neonatal and adult mice, a defined sequence of responsiveness was observed. Newborn mice responded well to TNP-LPS, but not to TNP-Ficoll or TNP-Brucella. Neonates injected at 1 day of age responded to TNP-LPS and TNP-Ficoll and mice 5 to 14 days of age responded to TNP-LPS, TNP-Ficoll, and TNP-Brucella. Furthermore, the antigen-reactive populations increased at different rates for the three antigens in the first 2 weeks of life. In vitro experiments confirmed the results obtained in vivo although slightly earlier responsiveness to TNP-Brucella was observed in vitro. PFC inhibition assays with free TNP hapten were performed so that avidity profiles could be examined in neonatal and adult anti-TNP PFC responses. The results clearly demonstrate that once a response becomes detectable in neonatal mice immunized with any of the three TI TNP antigens, fully heterogeneous or “adult-like” responses are found. In addition, experiments comparing avidity profiles in athymic (nu/nu) BALB/c mice and their normal (nu/+) littermates demonstrate that T cells are not required for the generation of fully heterogeneous anti-TNP PFC responses. These results indicate that B cells responsive to different TI TNP antigens mature at different times and at different rates during ontogeny. Late maturation events of such B cells do not include the acquisition of additional V-region specificities as detected in a PFC inhibition assay.     

Distinct subpopulations of IgG memory B cells respond to different molecular forms of the same hapten.

Spleen cells from mice primed with the thymus-dependent antigen trinitrophenyl keyhold limpet hemocyanin several months earlier can be cultured in vitro to give vigorous IgG antihapten PFC responses to thymus-dependent (TD) and thymus-independent (TI) forms of the same hapten. Here we show that the IgG memory precursors that respond to these two forms of the hapten constitute functionally distinct subpopulations. We have designated these subpopulations as B1gamma and B2gamma to represent secondary precursor cells responding to TI and TD antigens, respectively. Three types of evidence for these subpopulations are presented: 1) In vitro secondary IgG responses to TD and TI forms of the TNP hapten are additive when both forms are added to the same culture. 2) The precursor frequencies for the TD and TI antigens are additive, but addition is not observed between two TD or two TI antigens. 3) Each population can be selectively eliminated by BUdR and light treatment without affecting the other population. The ontogenetic relationships between these subpopulations are discussed in relation to all presently proposed subpopulations B1mu, B2mu, B1gamma, and B2gamma.     

Streptococcal serotype carbohydrate represents a novel class of type 2 antigen which is T-independent

Our studies reported here, fully characterize two unique type 2 antigens trinitrophenol (TNP)-M1 serotype carbohydrates (TNP-M1 g and TNP-M1 c) derived from streptococci, which fail to induce antibody responses in xid or neonatal mouse splenic cultures. These antigens generate brisk responses in normal spleen and Peyer's patch cell cultures of xid mice, all of which suggest that responses are elicited in the Lyb-3+, 5+ B subpopulation. The antibody responses to TNP-M1 g (and TNP-M1 c) are not dependent upon T cells. Furthermore, TNP-M1 carbohydrates induce anti-TNP plaque-forming (PFC) responses in cultures of small, resting splenic B cell populations without an added T cell requirement. Thus two categories of type 2 antigens are distinguished, one which requires T cells or derived factors, e.g., TNP-Ficoll, and a second TNP-carbohydrate antigen TNP-M1 that does not. Studies of the mitogenic and polyclonal B cell activation properties of M1 carbohydrates indicated that B cell proliferation is induced in both xid (Lyb-3-, 5-) and normal (Lyb-3-, 5- and Lyb-3+, 5+) splenic B cell subpopulations, but that differentiation to IgM synthesis fails to occur in the Lyb-3-, 5- B cell subpopulation. Thus M1 carbohydrates are unique probes that allow the selective induction of proliferation and differentiation of mature B cells that are presumably Lyb-3+, 5+. Because the M1 serotype carbohydrates induce polyclonal IgM synthesis and antigen-specific responses in only the mature B cell population in the absence of T cells, whereas TNP-Ficoll and other type 2 antigens require T cells or their derived factors, the Lyb-3+, 5+ B cell subpopulation may consist of a T cell-dependent and a T cell-independent compartment for responses to different carbohydrate type 2 antigens.     

Hormonal modulation of responses to thymus-independent and thymus-dependent antigens in autoimmune NZB/W mice

Previous work suggested that gonadal steroids influence immunity through the thymus, but the mechanisms were unclear. To investigate the effects of these hormones on immune responses to T1 and TD antigens in autoimmune mice, we studied hybrid NZB/W mice and the nonautoimmune DBA/2 strain. Mice castrated at 14 days of age were implanted with Silastic capsules releasing, in adults, physiologic levels of E2 in males or Te in females. Sham-operated controls received empty capsules. Splenic PFC were quantified 4 to 5 days after challenge with the TI2 antigen TNP-Ficoll, the TI1 antigen TNP-LPS, or the TD antigen SRBC. Young castrated NZB/W males implanted with E2 had striking enhancement of IgM responses to TNP-Ficoll when compared to castrated Te-treated females and comparable sham-operated controls of both sexes. E2 also stimulated responses to TNP-LPS. In response to challenge with SRBC, young E2-treated NZB/W males had a consistent trend to increased IgM PFC, and the stimulatory effect of E2 on IgG plaques was variable. Physiologic doses of Te had no consistent effect on responses in young mice. In old female NZB/W mice, Te caused PFC response after immunization with TNP-Ficoll to resemble age-matched NZB/W males. As sham-operated NZB/W females grew older, PFC responses to SRBC fell. This age-related phenomenon was delayed, however, in female castrates implanted with Te. In contrast, Te clearly suppressed responses to TNP-LPS. Implantation of E2 did not alter responses to TNP-Ficoll, TNP-LPS, or SRBC in nonautoimmune DBA/2 males. This finding suggested that exogenous E2 given in physiologic doses did not influence immunologic responsiveness in a normal strain to the degree seen in hormone-sensitive NZB/W mice. It was concluded that E2 enhanced responses to a variety of exogenous antigens in autoimmune NZB/W mice. The most consistent E2-induced increase in PFC response was observed with TI antigens, suggesting that E2 exerted its effects on B cells or Ts.     

Functions of accessory cells in B cell responses to thymus-independent antigens

The functions of adherent accessory (A) cells in thymus-independent (TI) B cell activation were investigated using homogeneous A cell lines with distinct cell surface and functional characteristics, as well as inhibitors of antigen processing and interleukin 1 (IL 1) secretion. B cell responses to both type 1 and type 2 TI antigens were found to be strictly A cell dependent. Only A cells capable of IL 1 secretion could restore responsiveness in A cell-depleted spleen cells, regardless of Ia expression or antigen-processing capability. Moreover, recombinant IL 1 completely replaced A cell function in B cell responses to both TI 1 and TI 2 antigens. Finally, T cell depletion did not diminish the reconstitution by IL 1. Thus in contrast to T cell activation, IL 1 secretion is the only A cell function required in TI B cell activation, and the data are consistent with a direct role for IL 1 in B cell activation.     

A requirement for nonspecific T cell factors in antibody responses to "T cell independent" antigens

Using murine splenic B cell preparations depleted of macrophages and rigorously depleted of T cells, we studied the role of nonspecific helper factors in in vitro antibody responses to T cell-independent (TI) type 1 and type 2 antigens. TNP-lipopolysaccharide, TNP-Brucella abortus, and DNP-liposomes containing lipid A were chosen as examples of TI type 1 antigens. DNP-Ficoll and DNP-liposomes without lipid A were chosen as TI type 2 antigens. Only the type 1 antigens were able to elicit significant, albeit very weak, responses without added helper factors. Both type 1 and 2 antigens required factors present in supernatants from concanavalin A-stimulated spleen cells (Con A SN) to stimulate optimum antibody responses. Interleukin 2- (IL 2) containing supernatant from the T cell hybridoma FS6-14.13 supported suboptimal responses to varying degrees with each TI antigen, in contrast to its lack of effect on responses to sheep red blood cells in the absence of additional factors. This activity of the FS6-14.13 supernatant was removed by absorption with the IL 2-dependent T cell line HT-2, suggesting that IL 2 was the active component. Another factor, IL-X, which is distinct from both IL 1 and IL 2 and is also found in Con A SN, was required in addition to IL 2 to achieve optimal responses with both types of TI antigens. These results clearly establish a role for factors derived from T cells in the activation of B cells by both type 1 and type 2 TI antigens.     

In vivo allogeneic effects: shift in the isotype profile of primary TI-2 responses in mice undergoing graft-vs-host reaction

We showed previously that primary responses to T-dependent (TD) and T-independent type 2 (TI-2) antigens were differentially affected by allogeneic effects induced in vivo during a graft-vs-host reaction (GVH). TD responses were greater than or equal to 80% suppressed, whereas the TI-2 responses were greatly enhanced, particularly the IgG component, which normally is very low. We have analyzed the IgG subclass distribution in primary responses of normal and GVH F1 mice in order to determine whether the strong T cell signals that occur during GVH reactions also induce shifts in the isotype profile. The effect of GVH on responses to TI-2 antigens was of particular interest because they are usually dominated by IgM and IgG3 classes in normal mice. We found a threefold to 10-fold increase in the PFC numbers of all four IgG subclasses in the response to TI-2 antigens, with an apparent shift from the usual IgG3 dominance to IgG1 in GVH mice. This IgG1 dominance was not found in serum antibodies where IgG3, IgG1, and IgG2b were equally expressed, although total IgG was increased greater than 20-fold. No isotype shift was found in either the TNP-KLH response, which was greater than or equal to 75% suppressed (IgG1 dominance was retained), or in the TI-1 response to TNP-Ba. The latter response was reduced (25 to 50%) in GVH mice and continued to be dominated by IgG2b/2a and IgG3. Unlike the unique isotype patterns found in primary responses, TNP-KLH primed mice challenged with TD, TI-1, or TI-2 antigens gave memory responses with identical isotype profiles that were dominated by IgG1 PFC. The role of T cells in B cell differentiation and isotype expression is discussed.     

Acute hepatotoxin exposure effects lymphoid and accessory cell types in inbred mice

The effect of acute hepatotoxin exposure on in vivo and in vitro immune responses were investigated in inbred mice. Splenic anti-SRBC PFC responses were slightly enhanced by carbon tetrachloride or galactosamine administration 5 hr prior to immunization. Whereas splenic anti-SRBC PFC responses were slightly enhanced in euthymic mice exposed to carbon tetrachloride 5 hr prior to immunization, immune responses to the TI antigens, Fl-LPS, Fl-Ficoll, and TNP-LPS, were significantly suppressed. Athymic mice receiving similar hepatotoxin exposure elicited enhanced immune responses to the TI immunogens, thereby suggesting that the activities of B cells and macrophages are enhanced in treated animals and in euthymic mice, T suppressor cells are also activated. By admixture of purified B- and T-cell and macrophage populations from either carbon tetrachloride-treated or control animals, it was demonstrated that hepatotoxin exposure also induces suppressor T cells regulating immune responses to the T-dependent antigen, SRBC, and that macrophages from treated animals are more functional. Further, B-cell responsiveness is enhanced. In addition to these observations, an active factor could be demonstrated in sera from hepatotoxin-treated animals which augments immune responses to SRBC in normal mice and promotes immune responses to this antigen in athymic mice. These findings indicate that the effects of acute hepatotoxin exposure are multifocal, influencing the activity of lymphoid and accessory cells.     

Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro antibody responses of channel catfish leukocytes

Three functionally distinct leukocyte subpopulations were isolated from the peripheral blood of channel catfish. Surface immunoglobulin-positive (sIg+) and sIg- lymphocytes were isolated by an indirect "planning" procedure employing monoclonal antibodies specific for channel catfish Ig. A third population composed of macrophages was isolated by adherence to baby hamster kidney cell microexudate-coated surfaces. Functional features of these three cell types were established by assessing their role(s) in primary in vitro anti-hapten PFC responses to known murine thymus-dependent (TD) and thymus-independent (TI) antigens. The results indicated that anti-hapten PFC responses to a TI antigen required the presence of sIg+ lymphocytes and macrophages. In contrast, all three cell types were required for responses to TD antigens. Furthermore, the results of studies involving the depletion of antigen-reactive lymphocytes demonstrated that both hapten-specific sIg+ cells and carrier-specific sIg- cells were required for anti-hapten responses to TD antigens. These studies provide direct evidence that catfish have separable B cells (sIg+ lymphocytes), T helper cells (sIg- lymphocytes), and accessory cells (macrophages) quite similar to those seen in higher animals.     

Idiotypic properties of the murine anti-arsonate antibody response: B- and T-cell influences

In a previous report characterizing the arsonate (ABA)-specific plaque-forming cell (PFC) responses of A/J mice induced by ABA-KLH, two interesting characteristics of the idiotypic (Id) profile were noted: (1) an apparent Id selectivity in the isotype switch since the earliest appearing IgG PFC in the primary response were significantly more "cross-reactive Id" (CRI)-dominant than the IgM PFC population, and, (2) a temporal waning of CRI dominance with time among IgG PFC, from 75-100% CRI+ PFC to about 25-45% CRI+ PFC in secondary responses. Experiments were performed to determine whether these effects are largely attributable to T or to B cells. Mice were immunized with a T-independent (TI) form of ABA (ABA-Brucella abortus) and apparent Id selectivity was observed; the earliest IgG PFC averaged 75% CRI+ while IgM PFC were only 39% CRI+. Due to the TI nature of the Ag, this provides suggestive, but not conclusive, evidence that the Id asymmetry in the isotype switch may be attributable to the direct interaction of Ag with B cells. Other studies addressed the temporal shift in CRI dominance. First, it was found that preexposure of mice to either KLH or to ABA (on an irrelevant carrier) resulted in diminished CRI dominance in subsequent "primary" responses to ABA-KLH. Secondly, adoptive transfer experiments with B and T cells from virgin mice (Bv, Tv) or ABA-KLH-primed mice (Bp, Tp) showed that recipients of Bv + Tp or Bp + Tv generated anti-ABA PFC responses with intermediate CRI levels. The Tv cells had some preferential tendency to activate CRI+ clones in the Bp population. The results demonstrate that CRI levels are jointly determined by the immune status of both B and T cells. A simple model is offered which accounts for early Id dominance and its gradual decline and has as its central postulate the assumption that CRI+ B cells in the virgin ABA-specific repertoire have an affinity advantage over CRI- clones.     

Immunopotentiation by SGP and Quil A. II. Identification of responding cell populations

The adjuvants SGP (a starch-acrylamide polymer) and Quil A (purified saponin) were shown to markedly augment antibody responses to T-independent (TI) antigens, suggesting that their adjuvant effects may be at least partially mediated through B cells. The ability of both adjuvants to augment primary responses to trinitrophenyl (TNP)-Ficoll (TI-2 antigen) in athymic nude mice further suggested these adjuvants affect B cells. SGP, however, did not induce a response to the T-dependent (TD) antigen dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH) in athymic nude mice, indicating it was unable to replace the requirement for T-helper cells for responses to TD antigens. Responses to TNP-lipopolysaccharide (LPS) were augmented by SGP in CBA/N X Balb/c immune defective (xid) mice. However, SGP was unable to induce a response to TNP-Ficoll in xid mice. The SGP and Quil A augmented responses to TNP-Ficoll were completely inhibited by the mitotic inhibitor, Velban, indicating that SGP and Quil A increased the plaque-forming cell (PFC) response primarily by stimulating cell proliferation, and not by recruitment of antigen-reactive cells. The effects of the adjuvants on secondary responses were investigated using adoptive transfer experiments. SGP and A1(OH)3 both increased the induction of hapten-specific memory B cells in mice primed with DNP-KLH. SGP, Quil A, and A1(OH)3 also increased priming of carrier specific T cells. Priming of memory B cells with DNP-KLH and either A1(OH)3 or SGP was prevented when T cells were depleted with anti-lymphocyte serum (ALS) at the time of antigen priming, indicating that the augmentation of memory B-cell priming by SGP and A1(OH)3 was dependent on the presence of functional T cells. SGP and Quil A were both unable to augment memory cell induction to the TI antigen, TNP-Ficoll, even though both adjuvants markedly augmented primary IgM and IgG responses to this antigen. Based on these results, it is suggested that SGP and Quil A can mediate their adjuvant effects primarily by a direct or indirect effect on B cells although the adjuvants may also affect T cells to some extent.     

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.     

Axon growth and guidance genes identify T-dependent germinal centre B cells

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4alpha. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4alpha, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.     

Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax?23

Protective immunity against T cell independent (TI) antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ) B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS)-based vaccine Pneumovax^®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA⁺ but not IgM⁺ or IgG⁺ memory B cells and a predominant generation of PnPS-specific IgA⁺ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax^®23. Our results demonstrate that a characteristic TI response induced by Pneumovax^®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response.     

Genetic restrictions in the development of antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 by nude mice implanted with semiallogeneic thymus glands

Athymic nude mice implanted with F1 thymus glands were used to investigate genetic restrictions regulating T cell-macrophage (M phi) interactions in the development of antibody responses to GAT. Spleen cells from conventional mice developed comparable primary plaque-forming cell (PFC) responses when stimulated by syngeneic and allogeneic GAT-M phi. However, spleen cells from strain A nude mice implanted with (A X B)F1 thymus glands were tolerant of strain B alloantigens and developed GAT-specific PFC responses to strain A GAT-M phi and allogeneic strain C GAT-M phi, but failed to respond to strain B GAT-M phi. The lack of primary GAT-specific PFC responses by spleen cells from (A X B)thy----A nude mice stimulated by strain B GAT-M phi was not due to detectable suppressor mechanisms. However, an allogeneic effect stimulated by H-2- or non-H-2-disparate GAT-pulsed or unpulsed M phi was able to overcome the inability of spleen cells from (A X B)F1 thy----A nude mice to respond to strain B GAT-M phi. Furthermore, the inability to respond to strain B GAT-M phi was overcome by the addition of supernatant fluids from independent cultures of H-2-disparate cells. These results 1) demonstrate that T cells from A nude mice implanted with (A X B)F1 thymus glands did not recognize nominal antigen in the context of B MHC antigens, and 2) suggested that the T cell repertoire was altered in strain A nude mice implanted with (A X B)F1 thymus glands, such that T cells that could recognize GAT in association with strain B MHC antigens were functionally deleted.     

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.     

T cell dependence and factor reconstitution of in vitro antibody responses to TNP-B. Abortus and TNP-Ficoll: restoration of depleted responses with chromatographed fractions of a T cell-derived factor

In vitro anti-trinitrophenyl (TNP) antibody responses to TNP conjugates of killed Brucella abortus organisms (TNP-BA), an antigen previously designated as a type 1 thymus-independent (TI-1) antigen, are markedly diminished after vigorous depletion of T cells, as are the responses to the type 2 TI (TI-2) antigen, TNP-Ficoll. We, therefore, propose that these antigens be redesignated as type 1 and type 2, respectively, to reflect their T cell dependence but to differentiate them from classical T cell-dependent (TD) antigens. T cell-depleted responses to type 1 and type 2 antigens can be restored by the addition of a) EL4 supernatant, b) phenyl-sepharose-purified fractions of EL4 supernatant that are rich in interleukin 2(IL2), and c) pl 4.5-5.5 isoelectric focused (IEF) fractions of EL4 supernatant which are also rich in IL2 activity. Removal of IL2 activity from EL4 supernatant by absorption on IL2-dependent T cells substantially reduced its restorative ability. Whether the active principle in EL4 supernatant activity responsible for restoring responses to type 1 and type 2 antigens is IL2, and whether it acts directly on B cells or by acting on contaminating T cells, is unresolved.