Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals.

Polycyclic aromatic hydrocarbons (PAHs) are toxic chemicals released into the environment by fossil fuel combustion. Moreover, a primary route of human exposure to PAHs is tobacco smoke. Oocyte destruction and ovarian failure occur in PAH-treated mice, and cigarette smoking causes early menopause in women. In many cells, PAHs activate the aromatic hydrocarbon receptor (Ahr), a member of the Per-Arnt-Sim family of transcription factors. The Ahr is also activated by dioxin, one of the most intensively studied environmental contaminants. Here we show that an exposure of mice to PAHs induces the expression of Bax in oocytes, followed by apoptosis. Ovarian damage caused by PAHs is prevented by Ahr or Bax inactivation. Oocytes microinjected with a Bax promoter-reporter construct show Ahr-dependent transcriptional activation after PAH, but not dioxin, treatment, consistent with findings that dioxin is not cytotoxic to oocytes. This difference in the action of PAHs versus dioxin is conveyed by a single base pair flanking each Ahr response element in the Bax promoter. Oocytes in human ovarian biopsies grafted into immunodeficient mice also accumulate Bax and undergo apoptosis after PAH exposure in vivo. Thus, Ahr-driven Bax transcription is a novel and evolutionarily conserved cell-death signaling pathway responsible for environmental toxicant-induced ovarian failure.

An idiotype-specific helper population that bears immunoglobulin, Ia, and Lyt-1 determinants.

A helper cell population with phenotypic characteristics of both B and T cells is described. This helper population, called BH, is present in normal unprimed C57BL/6 mice and preferentially helps the expression of NPb idiotype-bearing plaque-forming B cells in the absence of T helper cells. Its surface phenotype is Lyt-1.2+, Ig+, Lyb-3+, Thy-1.2-, Lyt-2.2-. The helper activity of the BH population is IgH restricted and BH cells selectively bind NPb idiotypic determinants. Collectively the data demonstrate that this unique subpopulation can regulate the response of antibody-secreting B cells through specific recognition of idiotypic determinants.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. IX. Characterization of Idiotype-specific effector-phase suppressor cells on plaque-forming cell responses in vitro.

The ability of T suppressor cells, induced by the intravenous injection of 4-hydroxy-3-nitrophenyl acetyl (NP)-modified syngeneic spleen cells, to affect an ongoing B cell response was studied in vitro. It was found that the expression of NPb idiotype-positive B cells could be selectively inhibited by the addition of antigen-induced suppressor cells in the last 24 h of the in vitro culture. This effector-phase suppression of B cell responses was antigen specific and mediated by an Lyt 1-, Lyt 2+, idiotype-binding, T cell population whose suppressive function was restricted by genes linked to the Igh locus.

Ontogeny of B-lymphocyte function. V. Thymus cell involvement in the functional maturation of B-lymphocytes from fetal mice transferred into adult irradiated hosts.

Lethally irradiated mice reconstituted with adult thymus cells and neonatal or fetal liver cells produce an anti-2,4-dinitrophenyl or anti-bovine gamma globulin response of restricted heterogeneity of affinity in comparison with the response of mice reconstituted with B cells from adult donors. In addition, mice reconstituted with day 15 fetal B cells and adult thymus cells produce relatively few indirect plaque-forming cells (PFC). It was found that B cells acquire the capacity to produce a heterogeneous response, of predominantly indirect PFC within 7 days of transfer only when thymus cells are transferred along with the B cells. B cells from fetal or neonatal donors transferred without young adult thymus cells develop the capacity to generate indirect PFC within 13 days after transfer to adult recipients, but continue to produce a response of restricted heterogeneity of affinity for up to 28 days after transfer. Thus, it has been shown that cells present in the thymus facilitate, or are necessary for the functional maturation of B lymphocytes. Furthermore, the data suggest that maturation of the B-cell population to produce a heterogeneous response is controlled independently of its maturation to be capable of producing indirect PFC.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XII. Fine specificity of anti-idiotypic suppressor T cells (Ts2).

The fine specificity of anti-idiotypic, effector-phase suppressor T cells (Ts2) induced by the intravenous injection of syngeneic spleen cells covalently coupled with the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten was studied in an in vitro plaque-forming cell system. By comparing the ability of these suppressor cells to bind monoclonal anti-NP antibodies that express different levels of serologically detected NPb idiotypic determinants, it was shown that anti-idiotypic suppressor T cells do not recognize the predominant NPb idiotypic determinants that are defined by serologic analysis. The implications for the possible expression and/or recognition of different sets of idiotypic determinants on T and B cells are discussed.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XI. Pseudogenetic restrictions of hybridoma suppressor factors.

Suppressor factor derived from three different murine T cell hybridomas were characterized . They specifically inhibited 4-hydroxy-3-nitrophenyl acetyl cutaneous sensitivity responses. The factors bind antigen and bear I-J and idiotypic determinants, but lack conventional immunoglobulin constant-region determinants. The factors function during the induction phase of the immune response, by inducing a second population of suppressor cells (Ts(e)). Suppressor factor can inhibit both cellular and plaque-forming cell responses in appropriate strains of mice. These hybridoma suppressor factors directly suppress strains of mice that are Igh-V homologous with the strain producing the factor. Thus, there is an apparent Igh-V restriction in the activity of these factors. However, this is a pseudogenetic restriction because these factors generate second order suppressor cells (Ts(e)) in Igh-incompatible mice, but in order to express the suppressive activity, the cells must be adoptively transferred into recipients that are Igh compatible with the strain producing the suppressor factor. Finally, it was shown that the factor-induced Ts(e) population is under an apparent dual genetic restriction. Thus, Igh and H-2 homology is required in order for the Ts(e) population to express its suppressive activity.

Analysis of T cell hybridomas. III. Distinctions between two types of hapten-specific suppressor factors that affect plaque-forming cell responses.

The ability of two cloned T cell hybridomas and their products to specifically suppress the in vitro plaque-forming cell (PFC) response to the 4-hydroxy-3-nitrophenyl acetyl hapten (NP) was studied. Supernatant from one hybridoma (TS1) was shown to suppress in the induction but not the effector phase of the immune response. Supernatant from the TS1 hybridoma was capable of inducing second-order (TS2) effector-phase suppressor cells in vitro but did not suppress the response of anti-I-J plus C-treated responder cells. In contrast, supernatant from a second hybridoma (TS3) was capable of suppressing PFC responses when added either in the induction or the effector phase of the response. TS3 supernatant was unable to induce effector-phase suppressor cells but was capable of suppressing the response of anti-I-J plus C-treated responder cells. In addition, specific suppressor factors isolated from supernatants of the TS1 and TS3 hybridomas were shown to bind to NP, bear NPb idiotypic and I-J-encoded but not immunoglobulin-constant region determinants. The factor secreted by the TS3 hybridoma appears to act directly on B cell targets. Mild reduction of this factor results in two separable moieties, only one of which binds NP. Reconstitution experiments suggest that both chains are required for function. The collective data indicate that these hybridomas represent cells from first- and third-order suppressor T cell populations described previously in contact sensitivity and in vitro PFC systems. The implications of the ability of these hybridoma products to affect both T and B cell-mediated immune responses are discussed.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. VII. Idiotype-specific suppression of plaque-forming cell responses.

The ability of suppressor cells induced by the intravenous administration of 4-hydro-3-nitrophenyl acetyl (NP)-modified syngeneic cells to reduce an idiotypic B cell response was studied in both an in vivo and an in vitro system. Idiotype-positive B cells were assayed by the ability of guinea pig anti-idiotypic antiserum to specifically inhibit idiotype-positive plaque formation. It was found that up to 57% of the PFC response in vivo and 100% of the PFC response in vitro was inhibitable with antiidiotypic antiserum. The expression of these idiotype-positive B cells could be suppressed by the transfer of spleen cells form mice treated 7 d previously with NP coupled syngeneic cels. T cells are both required and sufficient for the transfer of idiotype specific suppression. The induction of these idiotype-specific T suppressor cells directly with antigen suggests that recognition of unique determinants on cell surfaces is important for regulation of lymphoid cell interactions. The role of idiotype-specific suppressor cells in the network of lymphoid interactions is discussed.

Inhibition of c-myc expression induces apoptosis of WEHI 231 murine B cells.

Treatment of WEHI 231 immature B-lymphoma cells with an antibody against their surface immunoglobulin (anti-Ig) induces apoptosis and has been studied extensively as a model of B-cell tolerance. Anti-Ig treatment of exponentially growing WEHI 231 cells results in an early transient increase in c-myc expression that is followed by a decline to below basal levels; this decrease in c-myc expression immediately precedes the induction of cell death. Here we have modulated NF-kappaB/Rel factor activity, which regulates the rate of c-myc gene transcription, to determine whether the increase or decrease in c-Myc-levels mediates apoptosis in WEHI 231 cells. Addition of the serine/threonine protease inhibitor N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), which blocks the normally rapid turnover of the specific inhibitor of NF-kappaB/Rel IkappaBalpha in these cells, caused a drop in Rel-related factor binding. TPCK treatment resulted in decreased c-myc expression, preventing the usual increase seen following anti-Ig treatment. Whereas inhibition of the induction of c-myc expression mediated by anti-Ig failed to block apoptosis, reduction of c-myc expression in exponentially growing WEHI 231 cells induced apoptosis even in the absence of anti-Ig treatment. In WEHI 231 clones ectopically expressing c-Myc, apoptosis induced by treatment with TPCK or anti-Ig was significantly diminished and cells continued to proliferate. Furthermore, apoptosis of WEHI 231 cells ensued following enhanced expression of Mad1, which has been found to reduce functional c-Myc levels. These results indicate that the decline in c-myc expression resulting from the drop in NF-kappaB/Rel binding leads to activation of apoptosis of WEHI 231 B cells.

The RelA NF-kappaB subunit and the aryl hydrocarbon receptor (AhR) cooperate to transactivate the c-myc promoter in mammary cells.

NF-kappaB/Rel transcription factors regulate many genes involved in control of cellular proliferation, neoplastic transformation, and apoptosis, including the c-myc oncogene. Recently, we have observed that levels of NF-kappaB and aryl hydrocarbon receptor (AhR), which mediates malignant transformation by environmental carcinogens, are highly elevated and appear constitutively active in breast cancer cells. Rel factors have been found to functionally interact with other transcription factors. Here we demonstrate a physical and functional association between the RelA subunit of NF-kappaB and AhR resulting in the activation of c-myc gene transcription in breast cancer cells. RelA and AhR proteins were co-immunoprecipitated from cytoplasmic and nuclear extracts of non-malignant MCF-10F breast epithelial and malignant Hs578T breast cancer cells. In transient co-transfection, RelA and AhR gene products demonstrated cooperation in transactivation of the c-myc promoter, which was dependent on the NF-kappaB elements, and in induction of endogenous c-Myc protein levels. A novel AhR/RelA-containing NF-kappaB element binding complex was identified by electrophoretic mobility shift analysis of nuclear extracts from RelA and AhR co-transfected Hs578T cells. Thus, the RelA and AhR proteins functionally cooperate to bind to NF-kappaB elements and induce c-myc gene expression. These findings suggest a novel signaling mechanism whereby the Ah receptor can stimulate proliferation and tumorigenesis of mammary cells.

The aryl hydrocarbon receptor, a basic helix-loop-helix transcription factor of the PAS gene family, is required for normal ovarian germ cell dynamics in the mouse.

The aryl hydrocarbon receptor (AhR), so-designated based on the ability of the protein to bind with and be activated by polycyclic aromatic hydrocarbons (PAH) and related halogenated hydrocarbons, is part of an emerging family of ligand-activated transcriptional regulators that are distinct from the steroid-thyroid hormone receptor superfamily. Once bound by ligand, the AhR interacts with the AhR nuclear translocator (ARNT) protein to form the aryl hydrocarbon receptor complex (AHRC). Both subunits of the AHRC contain sequences corresponding to basic helix-loop-helix domains, a motif that is shared by a number of other dimeric transcription factors. Although the natural ligand(s) for the AhR remains to be elucidated, to date over fifteen genes, including enzymes, growth factors and other transcription factors, have been identified as potential targets for transcriptional regulation by the chemically-activated AHRC. In the ovary, PAH exposure is known to cause destruction of oocytes within immature follicles, implying that one function of the AhR is to mediate cell death signaling in the female germ line. To assess this possibility, we explored AhR expression patterns in the murine ovary, and then determined the impact of AhR-deficiency (gene knockout) on female germ cell dynamics. Immunohistochemical analysis of ovaries of wild-type female mice indicated that AhR protein was abundantly and exclusively expressed in oocytes and granulosa cells of follicles at all stages of development. Histomorphometric analysis of serial ovarian sections revealed a two-fold higher number of primordial follicles in Ahr-null versus wild-type females at day 4 postpartum. This phenotype likely results from a cell-intrinsic death defect in the developing germ line since AhR-deficiency attenuated the magnitude of oocyte apoptosis in fetal ovaries cultured without hormonal support for 72 h. We propose that the AhR, activated by an as yet unknown endogenous ligand(s), serves to regulate the size of the oocyte reserve endowed at birth by affecting germ cell death during female gametogenesis.

A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation.

Apoptosis, a metabolically active process of programmed cell death characterized by DNA fragmentation, is believed to play an important role in development of lymphocyte repertoires and in embryogenesis. Studies of this phenomenon would be greatly facilitated by the development of a simple assay capable of identifying and isolating intact apoptotic cells. A rapid fluorescence assay which identifies relatively small, intact cells containing fragmented DNA is described in this report. Thymocytes in which DNA fragmentation is induced by culture with or without dexamethasone are readily identified by their bright blue fluorescence after a 15 min treatment with Hoechst 33342, a DNA binding fluorochrome which diffuses through cell membranes. Since Hoechst 33342 staining does not require destruction of the cell membrane, it is possible to directly phenotype cell surface antigen expression on Hoechst 33342bright lymphocytes by conventional immunofluorescence techniques and to evaluate membrane integrity of Hoechst 33342bright cells by dye exclusion criteria. The advantages of this system are that it: (1) is rapid and simple, (2) quantitates the percentage of cells fragmenting their DNA and presumably undergoing apoptosis, (3) permits standard immunofluorescence staining of cell surface markers to identify even minor cell subsets of presumably apoptotic cells within heterogeneous populations, (4) provides the tools (fluorescence activated cell sorting) for purifying intact cells containing fragmented DNA for further biochemical studies, and (5) provides a means for identifying cells which exclude vital dyes and in which DNA fragmentation will eventually result in cell death.

Fluoranthene induces programmed cell death and alters growth of immature B cell populations in bone marrow cultures.

Many studies have demonstrated that polycyclic aromatic hydrocarbons adversely affect mature mammalian immune systems. However, little is known about the cellular mechanism(s) mediating this immunosuppression or the potential for these ubiquitous environmental chemicals to similarly compromise lymphocyte development (lymphopoiesis). Murine bone marrow cultures were exploited in the present studies to evaluate the potential for fluoranthene, a mutagenic, cocarcinogenic, polycyclic aromatic hydrocarbon, to modulate B cell lymphopoiesis. In this well characterized system, interactions between immature bone marrow-derived precursor B (preB) cells and bone marrow-derived stromal cells closely mimic preB-stromal cell interactions in vivo and resemble interactions between other bone marrow-derived hematopoietic cells and their supporting stroma. Data presented herein indicate that: (i) fluoranthene suppresses B lymphopoiesis within 2 days in bone marrow cultures; (ii) fluoranthene suppresses lymphopoiesis at least in part by direct interactions with preB cells; (iii) fluoranthene lymphotoxicity is mediated by rapid induction of DNA fragmentation characteristic of programmed cell death (apoptosis) and (iv) preB cell populations surviving the initial death signal or preB cell populations exposed to lower doses of fluoranthene (0.5-5 micrograms/ml) exhibit altered growth and survival characteristics. The data suggest several levels at which fluoranthene could compromise B lymphopoiesis.

Characterization of anti-idiotypic suppressor T cells (Tsid) induced after antigen priming.

The induction and fine specificity of idiotype-specific suppressor T cells (Tsid) were studied. Spleen cells from C57BL/6 mice, immunized 4 wk previously with NP-KLH, failed to express NPb3 idiotype-bearing PFC when challenged in vitro with NP-Ficoll or NP-Brucella abortus. After treatment of NP-primed responder cultures with anti-Thy-1.2 anti-serum + C, NPb idiotype-bearing B cells could be detected. This B cell subset was preferentially suppressed by the addition of T cells from NP-primed mice. With this reconstitution protocol, it was determined that suppression of the NPb idiotype-bearing portion of the B cell response was mediated by a specifically induced T cell population (Tsid) that directly suppressed NPb-bearing B cells. As with a previously described suppressor population induced with hapten-modified syngeneic spleen cells (Ts2), the Tsid population bound and was lysed by NPb idiotype-bearing serum antibodies. However, the Tsid could be distinguished from the Ts2 population because it lacked I-J determinants and functioned as an effector T cell, not an intermediary suppressor cell. Furthermore, fine specificity studies with monoclonal NP-specific antibodies expressing various levels of serologically detectable NPb idiotypic determinants indicated that unlike the Ts2, the Tsid population reacts with conventional, serologically detected members of the NPb family. The combined idiotype binding studies for the Tsid and Ts2 populations demonstrate that the fine specificity of suppressor T cell populations reflects their independent mechanisms of regulation.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XIII. Characterization of a third T cell population involved in suppression of in vitro PFC responses.

The involvement of a third-order suppressor T cell population (Ts3) in the suppression of in vitro PFC responses was analyzed. It was shown that Ts2 effector-phase suppressor cells, induced by the i.v. injection of NP-coupled syngeneic spleen cells, require a third suppressor T cell population to effect NPb idiotype-specific suppression of an in vitro B cell response. This Ts3 population was shown to be present in NP-primed but not unprimed donors. The Ts3 population specifically binds NP and is Lyt-1-, Lyt-2+, I-J+ and bears NPb idiotypic determinants. The involvement of the Ts3 population in a suppressor pathway that requires recognition of idiotypic determinants is discussed.

H-2-restricted helper activity mediated by immunoglobulin-bearing lymphocytes.

The genetic requirements for helper activity mediated by a unique, Ig-bearing lymphocyte population were studied. This Lyt-1+, I-A+, Thy-1- population, called BH, preferentially helps expression of NPb idiotypic plaque-forming cells when added to T cell-depleted responder cultures. Furthermore, the BH population can directly bind NPb idiotypic determinants. Using H-2 congenic mice, we show that BH helper activity can be expressed only when BH cells share I-A subregion alleles with responder B cell populations. This H-2 restriction is not a result of thymic influences, because the activity of BH cells from athymic mice are also H-2 restricted. Macrophages present in the BH population do not contribute to the H-2 restriction. Results are presented that definitively rule out the possible role for T lymphocytes in BH activity and demonstrate that a single helper population expresses both Lyt-1 and I-A determinants. These results indicate that Ig-bearing cells serve a regulatory as well as an effector role in immune responses and that, like other regulatory lymphoid subsets, their activity is regulated in part by MHC-encoded determinants.

Idiotype-specific helper cells (BH) express immunoglobulin markers and employ T cell function-associated molecules.

An Lyt-1+ population, distinct from T cell subsets, that helps expression of B cell responses to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten was characterized. This lymphoid population, called BH, is present in the spleens of normal and athymic mice and preferentially helps the expression of plaque-forming B cells that carry NPb idiotypic determinants. To define the mechanism by which this cell population functions, the roles of T and B lymphocyte function associated antigens were studied. The data indicate that BH cells express immunoglobulin receptor components, i.e., IgM, IgD heavy chain, and lambda light chain immunoglobulin markers as well as the J11d marker associated with immature B cells. BH cells may also express determinants identical to or cross-reactive with the T cell-associated antigens L3T4a, L3T4b, and LFA-1 as defined by treatment with monoclonal antibodies specific for these antigens. In addition, L3T4a- and LFA-1- but not Lyt-1-like antigens appear to be functionally involved in BH-dependent helper activity, since augmentation of NPb idiotypic PFC responses was blocked with anti-L3T4a or anti-LFA-1 monoclonal antibodies. Further analysis of BH-containing populations indicates that T cells are probably not involved in BH cell function and therefore are not responsible for the presence of Lyt-1, L3T4a, or L3T4b determinants in this T-independent system. The relationship of this helper cell subset to conventional T and B cell populations is discussed.

Mechanisms by which benzo[a]pyrene, an environmental carcinogen, suppresses B cell lymphopoiesis.

The capacity for polycyclic aromatic hydrocarbons (PAH) to suppress immune cell function has been well documented. Nevertheless, mechanisms responsible for PAH immunosuppression and potential effects of PAH on lymphocyte development (lymphopoeisis) remain poorly defined. Murine bone marrow cultures were used in the present studies to determine if and by what mechanism(s) benzo[a]pyrene (B[a]P), a prototypic and highly carcinogenic PAH, suppresses B cell lymphopoiesis. Emphasis was placed on similarities between the processes leading to transformation and immunosuppression and on a possible role for programmed cell death (apoptosis) in B[a]P lymphotoxicity. Data presented herein indicate that: (1) B[a]P suppresses B cell lymphopoiesis in bone marrow cultures at extremely low concentrations (10(-8) M); (2) benzo[e]pyrene, the relatively noncarcinogenic congener of B[a]P, is approximately 1000 times less potent than B[a]P in suppressing B cell lymphopoiesis; (3) bone marrow cells from PAH-resistant DBA/2 mice are less sensitive to B[a]P than cells from C57BL/6 mice; (4) B[a]P induces preB cell apoptosis; and (5) alpha-naphthaflavone, an inhibitor of Ah-receptor dependent, P450 isoenzyme activity, blocks B[a]P-mediated preB cell apoptosis and inhibits B[a]P-dependent suppression of lymphopoiesis. The results support the hypothesis that B[a]P suppression of B cell lymphopoiesis is mediated at least in part by the induction of programmed cell death and that the Ah receptor and/or P450 isoenzymes are involved in this process. The results suggest the potential for PAH to affect development of the B lymphocyte repertoire.

The role of peritoneal stromal cells in the survival of sIgM+ peritoneal B lymphocyte populations.

Peritoneal (PE) B cells, a large fraction of which belong to the B-1 subset (i.e., the CD5+ B cell lineage), exhibit unusual growth and/or survival characteristics. To evaluate if these characteristics reflect intrinsic properties of PE B cells or the support provided by a potentially unique peritoneal microenvironment, B cells from various organs were cultured on PE stromal cell monolayers. It was determined that sIgM+ PE B cell populations survive for notably long periods of time (at least 4 weeks) when cultured with PE stromal cells. Contact of PE B cells with stromal cell monolayers optimized B cell survival. Although expressing a similar spectrum of adhesion molecules as peritoneal stromal cells, splenic and bone marrow stromal cells were significantly inferior at supporting PE B cells. Increased survival of PE B cells was characterized by a significant but transient increase in proliferation and by an increase in the percentage of B220/CD45high/CD5+ PE B cells. PE stromal cell support of B cell populations did not extend to all B or even to all B-1 cell populations since survival of splenic B cells, only a minority of which belong to the B-1 lineage, or thymic B cells, a majority of which belong to the B-1 lineage, was not enhanced by culture on PE stromal cells. Results demonstrate: (a) a system for relatively long-term maintenance of mature B cells, (b) that the growth/survival of PE B cells is preferentially supported by PE stromal cells, and (c) that growth on PE stromal cells is a characteristic of peritoneal B cells but not necessarily of splenic B cells or B-1 cells from other organs. The suggestion that the local microenvironment contributes to unusual growth, survival, or anatomic distribution of B-1 cells is discussed.

Idiotype-specific Ly-1 B cell-mediated helper activity: hybridomas that produce anti-idiotype antibody and nonimmunoglobulin lymphokine(s)1.

Previous work has shown that the expression of a predominant family of idiotypic determinants (NPb) in the in vitro response to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten is dependent on helper activity provided by Ly-1- and Ig-bearing B cells called BH. The ability of these BH cells to perform this idiotype-specific, genetically controlled helper function is related to the NPb idiotype specificity of their cell surface receptors. However, the means by which BH cells communicate with and stimulate NPb idiotypic B cell subsets is unknown. In this paper, an Ly-1- and immunoglobulin-bearing B helper cell hybridoma is described. Supernatants from the hybridoma or its subclones were shown to specifically help the response of NPb idiotypic PFC to NP-Ficoll when added to responder cell cultures depleted of Thy-1 and Ly-1 regulatory cell populations. Under these experimental conditions hybridoma supernatants functioned in much the same fashion as populations of Ly-1- and Ig-bearing BH helper populations described previously. NPb idiotype-specific helper activity was mediated by two separable activities elaborated by the hybridoma, an anti-NPb idiotype antibody and a non-Ig (lymphokine) activity. It was shown that both the Ig and the lymphokine components were required for helper activity. Kinetics experiments showed that the anti-idiotype antibody must be added early in the response to NP-Ficoll, whereas the lymphokine fraction could be added at least as late as day 3 of a 4-day culture in order to observe NPb idiotype-specific help. The data suggest that Ly-1 B cell hybridomas may affect the responsiveness of B cell subsets initially by interaction of anti-idiotype antibody with NPb idiotypic B cell surface receptors, followed by growth or maturation signals mediated by non-Ig lymphokine(s). The possibility that the helper activity of these Ly-1 B cell hybridomas represents the combined effects of an idiotype-specific network system and nonspecific growth or maturation factor activity in direct B cell-B cell interactions is discussed.