The ontogeny of thymic independent antibody responses in vitro in normal mice and mice with an X-linked B cell defect.

The primary in vitro antibody response of neonatal spleen cells to three thymic independent antigens has been examined. The time of onset of responsiveness to TNP-Brucella abortus and TNP-lipopolysaccharide was significantly earlier than the onset of responsiveness to TNP-Ficoll. This ontologic sequence was not affected by T cell depletion or antigen presentation on adult macrophages. In neonatal mice bearing the X-linked CBA/N defect, the response to TNP-Brucella abortus and TNP-lipopolysaccharide was much delayed and no response to TNP-Ficoll developed. We conclude that different thymic independent antigens address different subpopulations of B cells, one of which appears earlier in ontogeny than the other.     

Evidence for existence of two distinct TNP-Ficoll-responsive B cell subpopulations preferentially reactive either to a T cell-replacing factor (B151-TRF) or to a signal from accessory cells

The ability of B cells to respond to TNP-Ficoll has been shown to correlate with their ability to respond to T cell-replacing factor (TRF). The present study analyzed the relationship of TNP-Ficoll-responsive B cells to a TRF-responsive B cell subpopulation. The B cells from normal, unprimed mice responded to TNP-Ficoll in the presence of accessory cells. Such responses were notably augmented by the addition of TRF derived from a monoclonal T cell hybridoma, B151K12(B151-TRF). Interestingly, B cells of mutant X-linked immunodeficient DBA/2Ha which failed to respond to B151-TRF gave anti-TNP PFC responses to TNP-Ficoll comparable to those of normal mice, depending on the presence of accessory cells. However, under this condition, the addition of B151-TRF did not augment the TNP-Ficoll responses. One explanation of the augmentation of TNP-Ficoll response by TRF for the B cells from nondefective mice was that two distinct B cell subpopulations exist which differ in their respective activation requirement for TRF and accessory cells. To examine this possibility, syngeneic accessory cells were pulsed with TNP-Ficoll and were assayed for their ability to activate normal B cells in the presence or absence of B151-TRF. The results revealed that TNP-Ficoll-pulsed accessory cells were able to induce primary anti-TNP PFC responses in normal B cells to the same magnitude as soluble TNP-Ficoll. However, these B cell responses induced by the TNP-Ficoll-pulsed accessory cells were not augmented by the addition of B151-TRF to the culture. These results support the notion that two distinct TNP-Ficoll-responsive B cell subpopulations exist; one requires accessory cell-B cell interaction to be activated by TNP-Ficoll but fails to respond to TRF, and the other can be activated by TRF in a totally accessory cell-independent manner.     

Role of accessory cells in B cell activation. IV. Ia+ accessory cells are required for the in vitro generation of thymic independent type 2 antibody responses to polysaccharide antigens

It has been previously shown that the in vitro antibody response to TNP-Ficoll requires the presence of adherent accessory cells. In order to determine if this characteristic was unique to TNP-Ficoll or a general feature of the TI-2 antibody responses, responses to the polysaccharide antigens TNP-Levan and TNP-Dextran were studied. Also, it was determined if the functionally relevant accessory cell expresses Ia determinants. Passage of spleen cells over Sephadex G-10 abrogated the response to TNP-Levan and TNP-Dextran as well as to TNP-Ficoll. Addition of adherent accessory cells to the G-10 passed spleen cells reconstituted the response to all 3 antigens. Pretreatment of the adherent accessory cells with a specific anti-Ia serum plus complement abrogated the ability of these cells to provide accessory cell function in the responses to all 3 antigens. Thus, an Ia-positive adherent accessory cell is required for the generation of TI-2 antibody responses to these polysaccharide antigens. This raises the possibility that genetic restrictions may exist between the Ia-positive accessory cell and the lymphocytes involved in the responses to TNP-Ficoll, TNP-Dextran, and TNP-Levan.     

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.     

Interleukin-10 enhances immune responses to pneumococcal polysaccharides and sheep erythrocytes in young and aged mice.

Antibody responses to pneumococcal polysaccharides are decreased in aged mice. Using a system to measure murine antibody responses to the Pnu-Imune vaccine, here we demonstrate that interleukin-10 (IL-10) has an adjuvant effect in enhancing the vaccine response in the aged. IL-10 increased the vaccine responses of B cells from aged mice in vitro only if either T cells or macrophages were also present. The need for T cells or macrophages could be substituted by cytokines such as IL-1 or IL-5, which are normally made by these accessory cells. Thus, IL-10 appeared to act on B cells directly but it worked in conjunction with other cytokines to induce an antigen specific response. In vivo studies showed that IL-10 administration enhanced antibody responses not only to thymic independent antigens but also to thymic-dependent antigens such as sheep erythrocytes. These data suggest that IL-10 may be useful in enhancing vaccine-specific responses in situations in which the host is immunocompromised.     

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.     

Antigen presentation by human B cells: T cell proliferation induced by Epstein Barr virus B lymphoblastoid cells

T cell proliferation in response to antigen requires the presence of an antigen-presenting accessory cell. The accessory cell most commonly associated with antigen presentation has been the macrophage (Mo). This study demonstrates that B cells in the form of Epstein Barr virus-transformed B lymphoblasts (EBV-B) are able to present tetanus toxoid (TT) to human T cells and induce proliferation of these cells. T cells freshly isolated from peripheral blood were shown to undergo blast transformation and proliferation in response to TT and irradiated EBV-B cells. Furthermore, TT-reactive T cell blasts were shown to proliferate in the presence of EBV-B cells and TT, but not with other antigens. There was a progressive increase to a plateau in T blast proliferation with increasing numbers of EBV-B cells added to the culture. The concentration of TT required for optimal antigen presentation was similar for EBV-B cells and for Mo. TT-specific cloned T cells also proliferated in response to TT and EBV-B cells and could be continuously grown in culture with TT, interleukin 2-containing supernatant, and EBV-B cells in place of autologous Mo. EBV-B cells pulsed with TT could also act as antigen-presenting cells. The proliferative response of T cell clones to TT in the presence of EBV-B cells was inhibited by antiserum to human p29,34 glycoprotein but not by anti-beta 2-microglobulin. This inhibition was shown to result from interaction with Ia-like determinants on EBV-B cells. These results indicate that B lymphoblastoid cells in man are able, like Mo, to present antigen to T cells in the context of Ia-like determinants.     

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.     

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.     

T lymphocyte heterogeneity in the rat. III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4+ autoreactive T cell lines (ATs)2 in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia+ and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.     

Qualitative and quantitative studies of antigen-presenting cell function by using I-A-expressing L cells

I-A-expressing transfected murine L cells were analyzed as model antigen-presenting cells. Four features of accessory cell function were explored: antigen processing, interaction with accessory molecules (LFA-1, L3T4), influence of Ia density, and ability to stimulate resting, unprimed T lymphocytes. I-A+ L cells could present complex protein antigens to a variety of T cell hybridomas and clones. Paraformaldehyde fixation before but not subsequent to antigen exposure rendered I-A+ L cells unable to present intact antigen. These results are consistent with earlier studies that made use of these methods to inhibit "processing" by conventional antigen-presenting cells. The ability of anti-L3T4 antibody to inhibit T cell activation was the same for either B lymphoma or L cell antigen-presenting cells. In striking contrast, anti-LFA-1 antibody, which totally blocked B lymphoma-induced responses, had no effect on L cell antigen presentation, measured as interleukin 2 (IL 2) release by T hybridomas, proliferation, IL 2 release, or IL 2 receptor upregulation by a T cell clone. I-A+ L cell transfectants were found to have a stable level of membrane I-A and I-A mRNA, even after exposure to interferon-gamma-containing T cell supernatants. In agreement with earlier reports, a proportional relationship between the (Ia) X (Ag) product and T cell response was found for medium or bright I-A+ cells. However, dull I-A+ cells had a disproportionately low stimulatory capacity, suggesting that there may be a threshold density of Ia per antigen-presenting cell necessary for effective T cell stimulation. Finally, I-A-bearing L cells were shown to trigger low, but reproducible primary allogeneic mixed lymphocyte responses with the use of purified responder T cells, indicating that they are capable of triggering even resting T cells. These studies confirm the importance of antigen processing and I-A density in antigen-presenting cell function, but raise questions about the postulated role of the LFA-1 accessory molecule in T cell-antigen-presenting cell interaction. They also illustrate the utility of the L cell transfection model for analysis and dissection of antigen-presenting cell function.     

Role of antigen-specific B cells in the induction of SRBC-specific T cell proliferation

In this study, we ask whether antigen presentation can be effected by antigen-activated B cells. Antigen-dependent in vitro proliferation of T cells from mice primed with SRBC or HoRBC occurs in the presence of B cells primed to the relevant antigen. B cells prepared from lymph nodes of mice primed with irrelevant antigens are not effective antigen-presenting cells for RBC-specific T cell proliferation over a wide range of SRBC doses. This is true even when both RBC and the antigen to which the B cells are primed are included in the culture. In contrast, B cells specific for a hapten determinant coupled to SRBC are able to support proliferation of T cells specific for SRBC determinants. We conclude from these data that antigen-specific B cells play a role in the induction of T cell proliferative responses to SRBC and HoRBC antigens. Two models are proposed: either B cells, upon antigen interaction with surface antibody, are able to act as accessory cells to induce Ia-dependent proliferation of immune T cells; or B cells augment the T cell proliferative response by secretion of antibody, leading to opsonization of the antigen for macrophage uptake and presentation.     

Two signals are required for accessory cells to induce B cell unresponsiveness. Tolerogenic Ig and prostaglandin

We previously demonstrated that a lymphoid dendritic cell-like tumor line (P388AD.2) presented a normally tolerogenic signal, fluoresceinated sheep gamma-globulin (FL-SGG), as an immunogenic one. In contrast, macrophages derived from the peritoneal cavity potentiated the ability of FL-SGG to induce B cell unresponsiveness. In this paper we examined whether two different Ia+ splenic accessory cells differentially presented tolerogen to spleen cells or fluorescein (FL)-binding B cells. Interestingly, lymphoid dendritic cells presented FL-SGG to spleen cells and elicited augmented anti-FL antibody responses, whereas splenic macrophages presented this same moiety and elicited hapten-specific B cell unresponsiveness. The mechanism of splenic macrophage-elicited B cell negative signaling was investigated, and it was found that B cell unresponsiveness was abrogated in the presence of the cyclooxygenase inhibitor indomethacin. This observation suggested a crucial role for PG in B cell negative signaling. The addition of 10 nM PGE2 restored unresponsiveness in cultures treated with indomethacin and tolerogen-pulsed macrophages, even though this dose of PG had no effect on the ability of B cells to be triggered by an immunogenic signal. A role for T cells was excluded, inasmuch as purified hapten-specific B cells were specifically tolerized by FL-SGG-pulsed macrophages. Lymphoid dendritic cells pulsed with FL-SGG did not deliver a tolerogenic or immunogenic signal to FL-specific B cells. However, when PGE2 was supplied, B cell unresponsiveness was induced. Finally, we tested whether "non-tolerogenic" doses of FL-SGG could render hapten-specific B cells unresponsive in the presence of PGE2, but in the absence of accessory cells. Interestingly, the combination of non-tolerogenic amounts (10 to 1000 pg/ml) of FL-SGG in conjunction with PGE2 induced unresponsiveness, whereas neither moiety alone was effective. These results suggest that splenic macrophages and lymphoid dendritic cells exert opposing effects on the immune system as evidenced by the induction of negative or positive B cell signaling. Our observations suggest that one of the key factors in controlling whether an accessory cell delivers a tolerogenic signal is the ability to secrete PG.     

Requirement of Ia-positive accessory cells in the MLR response against class II antigen on human B cell tumor line

In this report we have made a comparative study of the capacity of normal human stimulator cells and Epstein-Barr virus-transformed human B cell line Wa (EBV-Wa) cells to stimulate alloreactive T cells. Class II antigen (presumably HLA-DR4 determinant) on EBV-Wa cells was shown to act as a stimulating molecule in the mixed lymphocyte reaction (MLR) through a blocking study by using anti-Ia antibodies. Furthermore, it was found that HLA-DR-positive accessory cells in the responder population were required to elicit MLR responses against HLA-DR antigen on EBV-Wa cells. In contrast, HLA-DR-positive accessory cells in the responding cell population were not essential for elicitation of MLR responses against HLA-DR antigen on normal allogeneic peripheral blood mononuclear cells, as reported. The cell-cell interaction between responder HLA-DR-positive accessory cells and responding T cells in a major histocompatibility complex (MHC)-restricted manner was required for eliciting MLR responses against class II antigen on EBV-Wa cells such as antigen-presenting cell-T cell interaction in soluble antigen-specific T cell proliferative responses. The function of HLA-DR-positive accessory cells in the responder population could not be substituted for by the presence of interleukin 1. Furthermore, there was no obvious correlation between the degree of surface HLA-DR antigen expression on EBV-Wa cells and its stimulating ability. Thus, two distinct types of allo-class II, antigen-specific T cell activation between normal human stimulator cells and EBV-Wa cells were shown to exist.     

Immunoregulation in the rat: ontogeny of B cell responses to types 1, 2, and T-dependent antigens

The development of rat B cells has been examined in neonatal and adult Fischer rats through the use of type 1 (TNP-Brucella abortus), type 2 (TNP-LPS(Ph), TNP-Ficoll) and T cell-dependent (TD) (SRBC) antigens. In vivo splenic PFC responses to TNP-Brucella abortus could be induced in newborn rats and by 12 days of age had reached adult levels. In contrast, the responses to the type 2 and TD antigens were 30% and 70%, respectively, of the adult levels at 30 days of age. Adoptive transfer of the B cells from neonatal and young rats into irradiated adult hosts demonstrated that the kinetics in the development of responses to these antigens (early for type 1, intermediate for TD, and late for type 2) were not due to limiting accessory cell or T cell help in immature rats. In vitro cultures of purified B cells from neonatal and adult rats were responsive to TNP-BA and TNP-LPS(Ph) but not to TNP-Ficoll and SRBC. However, the addition of spleen cell-derived Con A supernatant to the B cell cultures resulted in responses to all four antigens, which arose as a function of B cell age, with kinetics that were identical to those observed in vivo. Fluorescent staining of B cells from rats of various ages for cell surface IgM and analysis on the fluorescence-activated cell sorter (FACS) revealed that all splenic B cells from rats 4 days of age expressed a relatively high level of sIgM, and that a subpopulation that expressed a relatively low level of sIgM increased with age until it represented approximately 50% of the adult splenic B cells. Challenging Con A supernatant-supplemented cultures of FACS-prepared low sIgM+ and high sIgM+ cells revealed that B cells responsive to TNP-Ficoll were confined to the ontogenically late-arising low sIgM+ subpopulation but that B cells responsive to TNP-BA, TNP-LPS(Ph), and SRBC were present in both subpopulations.     

T cell development in B cell-deficient mice. IV. The role of B cells as antigen-presenting cells in vivo

B cell-deficient, rabbit anti-mouse IgM-treated mice were compared with normal or normal rabbit immunoglobulin-treated controls in their ability to develop proliferative T cell responses, delayed hypersensitivity, and primary or secondary cytotoxic T cell responses. Immunization with hapten-coupled autologous spleen cells resulted in anti-mu-treated mice generating only marginal T cell responses. This decreased responsiveness was shown to be attributable not to an intrinsic T cell defect or to changes in the ability of macrophages from anti-mu-treated mice to present soluble antigen, but rather to the greatly diminished capacity of B cell-deficient spleen cells to present antigen. The results support the concept that B cells play a significant role in antigen presentation required for T cell activation.     

Adherent cell function in murine T lymphocyte antigen recognition. I. A. macrophage-dependent T cell proliferation assay in the mouse.

The data in this report describe a T cell proliferation assay with nylon wool column-purified murine lymph node lymphocyte from animals immunized by footpad injection of antigen in CFA. It was found that the in vitro immune response of sensitized T cells to soluble protein antigens was functionally dependent on the presence of adherent cells, more specifically macrophages, at all concentrations of in vitro antigen challenge. The response was due to T cells in that cytotoxic treatment of the immune lymphocyte cells with anti-Thy 1.2 serum and complement effectively eliminated the antigen-specific DNA synthetic responses. The antigen-specific proliferation of murine lymphocytes depleted of adhereent cells could not be reconstituted with either guinea pig macrophages nor murine fibroblasts, indicating the existence of species and cell type specificity. In contrast to previous observations in the guinea pig, soluble products of cultured adherent cells could at least partially replace the function of intact macrophages in the response to antigen.     

Mouse splenic macrophage cell lines with different antigen-presenting activities for CD4+ helper T cell subsets and allogeneic CD8+ T cells.

A panel of seven mouse splenic macrophage cell lines, derived from cloned progenitors, was compared for their ability to present antigen to Th1 or Th2 helper T cell lines and hybridomas, as well as to naive T cells, and to provide accessory cell function for the synthesis of antibody from primed B cells. One of the cell lines expressed MHC class II molecules and was the only line with constitutive antigen-presenting activity for Th1 cells. It may represent a subset of splenic macrophages responsible for the activation of naive Th1 helper cells in situ. The remaining six cell lines responded to INF-gamma by up-regulating their class II expression and acquiring Th1 antigen-presenting activity. They may represent cells which, in situ, lack constitutive antigen-presenting activity but are promoted to presenting status by Th1-derived INF-gamma. Five of the cell lines provided accessory cell function to Th2 cells, as indicated by antibody synthesis in suspensions of spleen cells from primed mice depleted of their antigen-presenting cells. One of the cell lines lacking accessory cell activity had constitutive antigen-presenting activity for Th1 cells. This reciprocal expression of antigen-presenting activity supports the idea that Th1 and Th2 helper cells are activated by different antigen-presenting cells. Finally, the cell lines differed in their ability to constitutively induce an allogeneic response; a response that was limited to CD8+ T cells occurred in a CD4+ helper cell-independent manner and was unaffected by the addition of INF-gamma. The alloantigen-presenting macrophage cell lines also possessed the most efficient accessory cell activity for antibody synthesis. These cell lines, which represent a spectrum of antigen-presenting activities in the spleen afford models for defining the roles of macrophages in the induction of immune responses and for resolving issues concerning their development.     

B cell activation: role of dendritic and T cell factors in the response to thymic-independent and -dependent antigens

We described a cloned dendritic cell, clone Den-1, that is a potent accessory cell for some B cell responses. Clone Den-1 produces a novel lymphokine that is distinct from previously described factors produced by T cells. In the present study, we compare the role of nonspecific helper factors produced by Den-1 (Den-1 SN) or the T cell thymoma EL4 (EL4-SN) in promotion of B cell plaque-forming cell (PFC) responses to a variety of antigens. We find that the antigen in culture determines the B cell requirement for dendritic and/or T cell factors. B cell PFC responses to TNP-Brucella abortus (BA) and TNP-lipopolysaccharide (LPS) are greatly increased by EL4-SN but show little, if any, enhancement with Den-1 SN. Responses to TNP-polyacrylamide are reconstituted by either Den-1 SN or EL4-SN, whereas responses to TNP-Ficoll, TNP-dextran and TNP-levan are reconstituted by Den-1 SN and are much less sensitive to factors present in EL4-SN. Responses to SRBC require the presence of both Den-1 SN and EL4-SN. We also show that the time at which Den-1 SN must be provided to the B cell is dependent on the antigen in culture. Our findings are discussed in terms of present classification of antigens based on their ability to stimulate various B cell subpopulations.     

Dissection of the functions of antigen-presenting cells in the induction of T cell activation

The functions of antigen-presenting cells (APC) in the initiation of T cell activation was examined by culturing antigen-bearing guinea pig macrophages (M phi) with T cells obtained from antigen-primed animals. Although such antigen-bearing M phi stimulated primed syngeneic T cell DNA synthesis, as assessed by tritiated thymidine incorporation, paraformaldehyde fixation (0.15% for 1 min at 37 degrees C) abolished this capacity. Analysis with acridine orange staining indicated that fixed antigen-bearing M phi could not trigger primed syngeneic T cells to progress from the G0 to the G1 phase of the cell cycle. The addition of control non-antigen-bearing syngeneic or allogeneic M phi but not interleukin 1 or 2 to cultures of T cells and fixed APC permitted a proliferative response. Although the interaction between fixed antigen-bearing M phi and responding T cells was genetically restricted, there was no similar restriction for the supplemental control M phi. In fact, completely Ia-negative endothelial cells (EC) and fibroblasts (FB) could restore antigen responsiveness to cultures of fixed antigen-bearing M phi and syngeneic responding T cells, although they could not directly present antigen. Moreover, metabolically intact accessory cells, including Ia-negative EC and FB, could take up and process antigen to an immunogenic moiety, which fixed Ia-positive M phi could present to primed T cells. These data indicate that recognition of the antigen-Ia complex on an APC is necessary but not sufficient to trigger proliferation of freshly obtained primed T cells. The results additionally support the conclusion that APC carry out at least two separate functions necessary for the initiation of antigen-induced T cell activation. Not only must the APC display the antigen-Ia complex, but it must also convey another required effect. This influence, which apparently involved the establishment of cell to cell contact, was neither Ia nor antigen dependent and could only be provided by a metabolically intact cell. By contrast, genetically restricted antigen presentation could be accomplished by a fixed Ia-positive cell. Only when both the antigen-Ia complex and the influence of an intact accessory cell were provided by the same or different accessory cell were T cells triggered to enter the cell cycle.