Expression of Lyb-5 and Mls determinants by Peyer's patch B lymphocytes of X-linked immunodeficient mice

Treatment of the Peyer's patch (PP) B cells from X-linked immunodeficient (xid) (CBA/N X DBA/2)F1 male mice with anti-Lyb-5 plus complement kills approximately 75% of these cells, although xid spleen B cells are unaffected. Cytotoxic depletion of Lyb-5+ cells renders the xid PP B cell population unable to generate an in vitro direct plaque-forming cell response to the TI-2 antigen TNP-Ficoll. In addition, the B cell-enriched population from the PP of xid (CBA/N X CBA/J)F1 male mice were strong stimulators of proliferation in an M1s-defined MLR, thereby demonstrating that they also express the M1s antigen(s). Two cell surface antigens associated with mature B cells are therefore expressed by xid PP B cells, suggesting that the TNP-Ficoll responsive cells in this population are mature B cell.     

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.     

The role of complement receptor positive and complement receptor negative B cells in the primary and secondary immune response to thymus independent type 2 and thymus dependent antigens

Both complement receptor positive (CR+) and complement receptor negative (CR-) B cells have been shown to be involved in the primary immune response to PC-Hy (phosphocholine conjugated hemocyanin), a thymus dependent (TD) antigen which preferentially induces antibody secretion in Lyb-5+ B cells during a primary adoptive transfer assay. CR+ and CR- B cells also responded in a primary adoptive transfer assay to TNP-Ficoll, a thymus independent type 2 (TI-2) antigen which activates only Lyb-5+ B cells. When the secondary immune response to PC-Hy and TNP-Ficoll were analyzed, it was found that most of the immune memory to both antigens was present in the CR- B cell subset. The CR- B cell subset also dominated the secondary immune response to PC-Hy in immune defective (CBA/N X DBA/2N)F1 male mice. These data indicate that CR- B cells dominate the memory response in both the Lyb-5+ and Lyb-5- B cell subsets of normal and xid immune defective mice and suggest that Lyb-5+ and Lyb-5- B cells can be subdivided into CR+ and CR- subsets.     

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 Peyer's patch microenvironment. Cellular basis for the enhanced responses by the B cells of X-linked immunodeficient CBA/N mice

The Peyer's patches (PP) of X-linked immunodeficient (xid) CBA/N and hemizygous (CBA/N X DBA/2)F1 (CDF1) male mice contain a B cell subpopulation that expresses the Lyb-5 maturational marker and is responsive to type 2 and T cell-dependent antigens in vitro, a B cell phenotype which is absent from the spleens of xid mice. Experiments reported here show that xid spleen B cells co-cultured with B cell-depleted PP cells from xid mice differentiated into specific plaque-forming cells in response to trinitrophenyl-Ficoll (type 2) and sheep erythrocytes (T cell-dependent). Two cell types were involved in this normalization of xid B cell responses. An accessory cell activity present in the PP, but not the spleens, of both CDF1 male (xid) and CDF1 female (normal) mice was required for the response to either the type 2 or T cell-dependent antigens. In the presence of this PP accessory cell, T cells from the PP of either xid or normal mice supported responses to both classes of antigens. In contrast, T cells from the spleens of xid mice did not support the response to trinitrophenyl-Ficoll, although the splenic T cells from normal mice did synergize with PP accessory cells in allowing plaque-forming cell development by xid B cells to this type 2 antigen. The xid PP T cell activity required for the type 2 response by xid B cells was present in the Ly-1+, Lyt-2- subpopulation, and the xid PP accessory cell activity was provided by an enriched population of dendritic accessory cells. These results demonstrate the the lymphoreticular cells comprising the PP microenvironment provide effective support for the differentiation of xid B cells in response to type 2 and T cell-dependent antigens.     

Synergistic effects of the xid gene in X chromosome congenic mice. I. Inability of C3.CBA/N mice to respond to thymus-dependent antigens in adoptive transfer assays

The xid gene, which causes a B lymphocyte immune defect in CBA/N mice, has been bred onto the C3H/HeN background. The resulting X chromosome congenic mice (C3.CBA/N) exhibit immunologic defects that are much more profound than the defect exhibited by CBA/N mice; thus, the B cells from C3.CBA/N mice not only fail to respond to thymus-independent (TI) type 2 antigens such as TNP-Ficoll, but they fail to respond in vitro to TI-type 1 antigens such as TNP-Brucella abortus (BA) and B cell mitogens such as LPS and Nocardia water-soluble mitogen. In this paper we show that the synergistic defect seen in C3.CBA/N B cells is also elicited in adoptive transfer assays to thymus-dependent (TD) antigens such as TNP-KLH and PC-KLH, antigens to which both parental strains respond. Thus, the secondary adoptive transfer response of C3.CBA/N spleen cells is generally less than 5% of the immune response produced by CBA/N or C3H/HeN spleen cells. This synergistic defect is restricted to the C3.CBA/N B cells, since C3.CBA/N T cells can provide help to CBA/N B cells that is equivalent to the help obtained with CBA/N T cells. The low responsiveness of C3.CBA/N spleen cells to TD antigens, which is elicited in adoptive transfer assays, is not seen when the intact animal is immunized with antigen in CFA; this, intact C3.CBA/N mice produce anti-PC-KLH and anti-TNP-KLH responses only slightly lower than the responses of CBA/N mice to these same antigens. In contrast, when these mice are immunized with phenol-extracted LPS, a TI-type 1 antigen, their antibody responses are severely depressed. These data suggest that under conditions in which T cell help may be limiting or in which the intact physiology of the T and B cells has been disrupted, C3.CBA/N B cells demonstrate profound immunologic impairment; however, when adequate T cell help is available and the splenic architecture is not disrupted, their immune responses appear to progress in a normal fashion.     

Functional heterogeneity of the Lyb-5- B cell subpopulation: mutant xid B cells and normal Lyb-5- B cells differ in their responsiveness to phenol-extracted lipopolysaccharide

In the present study, responses stimulated by phenol-extracted lipopolysaccharide (LPS(phenol)) and butanol-extracted LPS (LPS(butanol)) were used to assess the possibility that xid B cells might not be identical to the Lyb-5- B cells present in normal mice. It was found that xid B cells responded well only to LPS(butanol) whereas normal B cells responded well to both LPS(butanol) and LPS(phenol). Thus, LPS(butanol) appeared to be a TI-1 antigen and LPS(phenol) appeared to be a TI-2 antigen. In contrast to classical TI-2 responses, however, responses stimulated by LPS(phenol) did not exhibit a stringent requirement for accessory cells. Furthermore, if LPS(phenol) were a classical TI-2 antigen, it should only activate Lyb-5+ B cells. To determine if the responsiveness of normal B cells to LPS(phenol) were due, at least in part, to the stimulation of normal Lyb-5- B cells, the responsiveness of normal neonatal B cells and normal adult B cells that had been pretreated with anti-Lyb-5.1 + C was assessed. It was found that both normal neonatal B cells and normal adult Lyb-5- B cells did respond well to LPS(phenol). Thus, even though LPS(phenol) does not stimulate xid B cells, these data demonstrate that LPS(phenol) is different from other TI-2 antigens. More importantly, these data also demonstrate that xid B cells and normal Lyb-5- B cells are not identical. It is hypothesized that the normal Lyb-5- B cell subpopulation is heterogeneous, consisting of an Lyb-5(1)- and an Lyb-5(2)-B cell subset with the xid mutation blocking the differentiation of Lyb-5(1)-B cells into Lyb-5(2)-B cells.     

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)     

Mucosal immunoregulation: environmental lipopolysaccharide and GALT T lymphocytes regulate the IgA response

In this review, we have emphasized: 1) bacterial lipopolysaccharide (LPS) involvement in IgA responses to orally administered thymic-dependent (TD) antigens; 2) characterization of Peyer's patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti-LPS immunity to infection. Gut LPS, which interacts with PP lymphoreticular cells, is a major determinant for host responses to orally administered TD antigens. Bacteroides species are the principal microflora present in the gastrointestinal tract and our studies with phenol-water LPS extracts from Bacteroides fragilis indicate that both polysaccharide and lipid A activate lymphoreticular cells. The B. fragilis lipid A moiety, like that derived from E. coli and Salmonella LPS, induces B cell mitogenic responses in cultures from LPS responsive mice, but does not stimulate C3H/ H3J B cells. The inability of lipid A to stimulate gut-associated lymphoreticular tissue (GALT) cells of C3H/HeJ mice results in the induction of greater T helper cell activity in this tissue in response to orally administered TD antigens and ultimately results in an elevated IgA response pattern. Murine PP contain accessory cells (approximately 1% dendritic cells and 6-8% macrophages) and lymphocytes T (35-38%) and B (40-42%). Recent studies with antigen-specific T cell clones from C3H/ H3J PP have resulted in the isolation of IgA isotype-specific T helper cells (PP Th A cells). PP Th A cells are antigen-specific, bear Fc alpha receptors, and require H-2 histocompatibility with B cells for helper activity. PP Th A cells most effectively collaborate with surface IgA (sIgA)-bearing B cells (IgA committed B cells) for IgA isotype responses. Other studies have shown that PP dendritic cells and T cells form clusters when stimulated in vitro with sodium periodate and that these clusters promote polyclonal IgA responses in B cell cultures. Polyclonal IgA responses in cultures containing PP cell clusters from C3H/ H3J mice are considerably higher than those in identical cultures from LPS responsive mice. In other studies, the environmental influence on GALT B cells and their resultant commitment to IgA isotype is under investigation. CBA/N, X-linked immunodeficient (xid) mice possess an immature splenic B cell population which cannot respond to thymic-independent class-2 (TI-2) or certain TD antigens. However, GALT B cells of xid mice possess a mature Lyb-5+ B cell subpopulation capable of both TI-2 and TD responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective effect of irradiation on responses to thymus-independent antigen

Low doses of ionizing radiation have a selective immunosuppressive effect on in vivo B cell responses to thymus-independent (TI) antigens. The B cell response, assayed as direct anti-trinitrophenyl (TNP)-specific plaque-forming cells (PFC), induced by type 2, TI antigens (TNP-Ficoll or TNP-Dextran), was reduced, on the average, by 10-fold in animals exposed to 200 rad of ionizing radiation 24 hr before antigen challenge. In contrast, PFC responses to type 1, TI antigens (TNP-lipopolysaccharide or TNP-Brucella abortus) are unaffected in mice exposed to the same dose of radiation. Adoptive transfers showed that this selective immunosuppression is a result of the specific inactivation of the B cell subpopulation responding to type 2, TI antigens. These experiments suggest that physiologic differences exist in the B cell subpopulations of normal mice which respond to type 1, or type 2, TI antigens.     

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.     

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.     

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.     

Immune responses to T-dependent and T-independent antigens during visceral leishmaniasis in mice: evidence for altered T-cell regulation of immune responses to non-parasite antigens

Antibody responses to T-dependent and T-"independent" antigens were studied in disease-susceptible (BALB/c and C57BL/10) and disease-resistant (A/J) mice infected with Leishmania donovani chagasi. Disease-susceptible mice but not disease-resistant mice showed a transient decrease in PFC responses to TNP on a T-dependent carrier (BGG) during the period of 4-8 weeks after infection. Infected disease-susceptible animals also showed increased responses to TNP on a type II T-independent carrier (Ficoll), which persisted until at least 14 weeks after infection. The increased responses were associated with a significant increase in anti-TNP antibody of the IgG2b subclass. When T-enriched spleen cells from infected mice and B-enriched spleen cells from uninfected mice were transferred to irradiated recipients immunized with TNP-Ficoll, increased anti-TNP PFC were observed over numbers seen in irradiated recipients which received both B and T cells from uninfected mice. Increased responses to TNP-Ficoll were also induced by prior administration of soluble leishmania extract in CFA. Infected mice immunized with TNP-LPS, a T-independent type I antigen, also had increased anti-TNP antibody responses, but had normal anti-LPS antibody responses. The elevated antibody production which occurred in response to the T-"independent" antigens could not be attributed to the relatively low polyclonal response which occurred in both disease-resistant and disease-susceptible mice infected with L. donovani chagasi. The observations are consistent with leishmania induced, transient alterations in some T-cell functions including response to haptens on T-dependent carriers, and a lack of down regulation of T-"independent" responses. Subtle lesions in immunoregulation may be important correlates of successful protozoal infection and may be responsible for some of the immunologic manifestations of the disease.     

Characterization of helper T cells induced by the type 2 antigen polyvinylpyrrolidone

Type 2 antigens are usually unable to prime for IgG memory responses or to activate helper T cells (TH) necessary for memory B cell generation. Previous studies from this laboratory have established that low doses (0.0025 microgram) of polyvinylpyrrolidone (PVP) or a T-dependent form of PVP, PVP-coupled horse red blood cells (PVP-HRBC) can activate PVP-specific TH. The present study was undertaken in order to determine some of the characteristics of the TH activated by PVP and to compare their properties with those of classical TH1 and of TH2 cells described in many T-dependent systems. TH activated with either 0.0025 microgram of PVP or PVP-HRBC were characterized with respect to cell surface antigens, Igh restriction and generation in mice expressing an X-linked immune defect (xid mice). PVP-specific TH are similar to TH1 cells in that they are required for the production of IgG subclasses absent in primary responses and have the Lyt-1+, L3T4+, I-J-surface phenotype. These TH may not be identical with TH1 cells, however, since they are I-A+ and Igh restricted. PVP-specific TH can be generated in xid mice which do not produce antibody in a primary anti-PVP response and do not develop a memory response to PVP, regardless of whether it is presented as a type 2 or T-dependent antigen.     

Dendritic and B-cell function during antibody responses in normal and immunodeficient (xid) mouse spleen cultures

Dendritic cells (DC) act as accessory cells for T-dependent antibody responses in two ways. One is to induce a class of stimulating factors (BSF) which allow B lymphocytes to respond to heterologous red cells as antigen. xid DC induce the production of these BSF, but xid B cells totally lack responsiveness. A second mechanism of DC function applies to red cell and haptenated-protein antigens. Here DC, helper T lymphocytes, and antigen-specific B cells interact in discrete clusters. Then the B cells become responsive to BSF. xid DC are fully active in this pathway, and xid B cells develop significant (10-20% of control) responses. This partial reduction in xid B-cell function could be due to the poor viability of xid lymphocytes in vitro. There is a comparable reduction in xid polyclonal responses to alloreactive helper T blasts. The other severe deficit in xid involves antibody formation to haptens on polysaccharide carriers. This response in normal mice is not influenced by DC or by BSF. The only similarity between DNP-Ficoll and RBC plus BSF responses is that both utilize B lymphocytes that do not associate with DC-T clusters, even though helper cells for DNP-Ficoll and for RBC are present in the culture. We conclude that DC function is not altered in xid. The main deficit seems to be in a B-cell activation pathway that is shared by polysaccharide carriers and some but not all BSF, and/or in a B-cell subpopulation that does not interact with carrier-specific helper cells. We speculate that this B-cell alteration primarily involves the Ig delta-poor marginal zone subpopulation of splenic B lymphocytes.     

Monoclonal helper T cells induce B cell responses to T-independent antigens: antigen-specific T cells are directly stimulated by activated B cells in the absence of antigen

Efficient B cell responses to most polysaccharide antigens such as TNP-PAA or TNP-Ficoll require factors produced by activated T cells. However, the mechanism of T cell activation during such responses has not been established, because these antigens do not activate T cells, either directly or in conjunction with I-A gene products. We used a panel of antigen-specific monoclonal helper T cells to study T cell activation during the course of such responses. We show that activated I-A-identical B cells directly stimulate these monoclonal T cells, and that this stimulation is in the absence of nominal antigen. The high frequency of inducer cells that are stimulated by activated B cells suggests a major biologic role for this novel pathway of T cell activation.     

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.     

Priming of peripheral lymph node B cells with TNP-Ficoll: role of lymphokines in B cell differentiation.

We have previously shown that peripheral lymph node (PLN) B lymphocytes of adult DBA/2J mice failed to make an antibody response to type 2 antigen TNP-Ficoll, but exhibited a good antibody response to type 1 antigen TNP-Brucella abortus. In the present study we wanted to find out whether the unresponsiveness of PLN B cells to TNP-Ficoll is due to defects in the early activation and proliferation stage or in the final differentiation stage of B cells. Therefore, we have used a two-step protocol of in vivo immunization of mice with TNP-Ficoll and the subsequent in vitro challenge with TNP-Brucella abortus and studied the anti-TNP plaque-forming cell (PFC) responses. The results indicate a three- to sixfold increase of PFC responses in PLN cell cultures derived from TNP-Ficoll-primed animals compared to saline control mice. This increased antibody response was TNP-specific as 93% of the PFC's were inhibited by TNP-lysine. Limiting dilution experiments confirm that the increase in anti-TNP PFC response from the TNP-Ficoll-primed animals was indeed due to an increase in TNP-specific precursor B cells. Further, the addition of rIL-5 or rIL-6 induced anti-TNP PFC in the TNP-Ficoll-primed and in control PLN cell cultures in the presence of antigen. However, in primed PLN cells lymphokines alone were sufficient to restore anti-TNP PFC response. In conclusion, our results show that in PLN, the TNP-Ficoll can induce proliferation of hapten-specific B cells but not final differentiation. These primed PLN B cells mature into antibody-secreting cells upon stimulation with TNP-BA or lymphokines.