Regulated expression of mouse mammary tumor proviral genes in cells of the B lineage.

We evaluated the expression of mouse mammary tumor proviral (MMTV) transcripts during B cell ontogeny and compared levels of RNA in B lymphocytes and B cell lines with levels in other cells of the hematopoietic lineage and in a mammary cell line. We demonstrate that MMTV transcripts are expressed as early as the pro-B cell stage in ontogeny and are expressed at basal constitutive levels throughout most of the B cell developmental pathway. The level of MMTV expression in B cells is similar to constitutive levels in mammary tissues and two to three orders of magnitude greater than in activated T cells. Levels of MMTV transcripts in B cells are not solely due to positional effects. Transient transfection assays showed that MMTV upregulation resulted from transcriptional activation of the viral LTR, indicating that there are specific and inducible transcription factors that regulate MMTV expression in B cells. MMTV transcripts could not be upregulated in pre-B cell lines but could be induced in some mature B cell lines. There was a correlation between the ability to stimulate B cells to secrete antibody and the ability to induce upregulated MMTV expression. Evidence is presented that suggests that the principal transcription factors involved in MMTV expression do not include the B cell factors OTF-2 or NF-kappa B, but rather are likely to be novel factors that are induced during differentiation to antibody secretion. A hypothesis for why mammary tumor viruses are well adapted for expression in cells of the B lineage is proposed, and the implications of this for the documented influence of MMTV gene products on the T cell repertoire are discussed.

Regulation of B cell lymphomagenesis by a malignant Qal+ inducer T cell clone.

A series of Thy-1.2+ Ly-1+ Qa-1+ malignant T cell clones have been isolated from murine sarcoma virus-murine leukemia-Moloney (MSV-MuLV-M)-induced B cell lymphomas or from MSV-MuLV-M-infected B6 mice. These T cell clones enhance both antigen-independent and -dependent lymphocyte differentiation and function. They also induce the differentiation of granulocytes and erythrocytes in the stem cell compartment, a function that parallels the immunopathology of the disease in vivo. The malignant T cell appears to sustain B lymphoma growth in vivo by releasing a factor (BCGF) that promotes B cell proliferation.

Major histocompatibility complex-restricted recognition of retroviral superantigens by V beta 17+ T cells.

It has been established that at least some V beta 17+ T cells interact with an endogenous superantigen encoded by the murine retrovirus, Mtv-9. To analyze the role of major histocompatibility complex (MHC) class II molecules in presenting the Mtv-9 encoded superantigen, vSAG-9 to V beta 17+ hybridomas, a panel of nine hybridomas was tested for their ability to respond to A20/2J (H-2d) and LBK (H-2a) cells which had been transfected with the vSAG-9 gene. Whereas some of the hybridomas recognized vSAG-9 exclusively in the context of H-2a, other hybridomas recognized vSAG-9 exclusively in the context of H-2d or in the context of both H-2d and H-2a. These results suggest that: (a) the class II MHC molecule plays a direct role in the recognition of retroviral superantigen by T cells, rather than serving simply as a platform for presentation; and, (b) it is likely that components of the TCR other than V beta are involved in the vSAG-9/TCR/class II interaction.

Multiple pathways of T-T interaction in the generation of cytotoxic T lymphocytes to alloantigens.

The interaction of T helper (Th) cells with syngeneic and allogeneic cytotoxic T lymphocyte precursors (CTL.P) has been investigated. Unprimed and mixed lymphocyte culture-primed peripheral T cells were used as a source of Th. Thymocytes, which depend upon exogenous Th cells for activation, were used as a source of cytotoxic precursors. Data is presented that demonstrates that at least two pathways of T-T interaction can lead to the activation of cytotoxic lymphocytes. The first is an allogeneic effect, in which Th cells recognize and respond to alloantigens expressed on CTL.P. The second is the interaction of Th cells with syngeneic CTL.P, in which both cell types are thought to respond to alloantigens on stimulator cells. The latter interaction can be shown to be restricted by H-2-linked determinants when primed Th cells are used and allogeneic effects against thymocytes are minimized. Restricted interactions between unprimed Th cells and thymocyte CTL.P have never been observed. Mechanisms that may explain the difference between the interaction of unprimed and primed Th cells with CTL.P are discussed.

Endogenous superantigen expression is controlled by mouse mammary tumor proviral loci.

Superantigens are defined by their ability to stimulate T cells based predominantly on their V beta expression and ability to delete T cells in the thymus when expressed endogenously. We show here that the expression of one endogenous superantigen, Etc-1, is controlled by the expression of the open reading frame region of the 3' long terminal repeat of the mouse mammary tumor proviral gene, Mtv-9. We show that Mtv-8 controls a superantigen with similar specificity, and that both Mtv-8 and Mtv-9 stimulate some V beta 17+ T cells. A third provirus, Mtv-6, controls a superantigen with specificity for V beta 3. Data presented raise the possibility that endogenous superantigens may compete for class II molecules in a single B cell.

Lipopolysaccharide and dexamethasone induce mouse mammary tumor proviral gene expression and differentiation in B lymphocytes through distinct regulatory pathways.

Endogenous mouse mammary tumor virus (MMTV) proviral transcripts are up regulated during the normal course of B-lymphocyte differentiation. We report here that the regulatory mechanisms which lead to increased levels of MMTV transcripts in differentiating, lipopolysaccharide (LPS)-stimulated normal B cells and in the inducible B-cell lymphoma line CH12 are at least partially distinct from those controlling increases in immunoglobulin and J-chain gene expression. In studies designed to characterize the stimulatory pathways leading to MMTV expression in CH12 cells, we found that stimulation with either LPS or dexamethasone (Dex), a transcriptional activator of MMTV genes, induced not only MMTV expression but also differentiation to antibody secretion. Only Dex-induced and not LPS-induced MMTV expression and differentiation were inhibited by the glucocorticoid antagonist RU486, demonstrating that Dex and LPS stimulate B cells by distinct molecular pathways. Therefore, in B cells, MMTV expression can be regulated via either the conventional hormone receptor-dependent pathway or a hormone receptor-independent pathway. Furthermore, these results suggest that steroid stimulation of B cells can lead to alterations in the expression of other results suggest that steroid stimulation of B cells can lead to alterations in the expression of other steroid-responsive genes that can become involved in the process of B-cell differentiation.

Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.

Inducible lymphokines of T cell tumors.

T lymphocytes comprise a major class of lymphocytes and are themselves functionally heterogeneous. Some T lymphocyte functions are mediated by soluble products called lymphokines. Different lymphokines promote the activation, growth and differentiation of T and B lymphocytes, macrophages, and hemopoietic cells. Lymphokine production is associated with, but not limited to, helper T cells, and usually follows antigenic or mitogenic stimulation. The recognition that some lymphokines are produced after stimulation of neoplastic T cells has proved advantageous in the study of these molecules. T cell tumors are monoclonal, grow easily in vitro, and may produce fewer lymphokines than normal T cells. Thus, the purification and biochemical and biological characterization of some lymphokines have been facilitated by the availability of these tumors. More recently, T cell tumors have been used for evaluating the molecular structure of lymphokine-encoding genes. They have also provided information relevant to our understanding of the nature of T cell neoplasia.

Late events in assembly determine the polymeric structure and biological activity of secretory IgM.

IgM antibodies can be secreted in at least two functional polymeric forms that can be distinguished according to subunit composition. While IgM hexamers comprise six H2L2 monomeric subunits, pentamers contain an additional polypeptide, the J chain. In the presence of high abundance J chain protein, IgM pentamers are preferentially assembled at the expense of hexamers. To determine the mechanism by which J chain regulates the assembly process, we defined the point at which J chain is added to assembling polymers. We found no evidence for the presence of J chain in small IgM assembly intermediates of IgM, suggesting that it was not stably associated with these complexes. However, J chain was found associated with large polymeric IgM complexes exhibiting sedimentation properties of intracellular pentameric structures. These complexes were frequently not completely covalently assembled; however, complete covalent assembly of J chain-containing pentameric complexes did occur prior to their maturation in the Golgi. These data argue that pentameric structures are the substrate for J chain incorporation into assembling IgM and suggest that the incorporation of J chain is thermodynamically favored over the addition of a sixth monomeric subunit into an assembling polymer. We conclude that late events in IgM polymer assembly, specifically the insertion of J chain, the exclusion of an additional monomeric subunit, and the covalent closure of the pentameric IgM molecule, determine the polymeric structure and, consequently, the biological activity of secreted IgM.

T cell help in cytotoxic T lymphocyte responses. Role of the I region in helper cell induction.

We have studied the in vivo induction of T helper (TH) cells that participate in the generation of cytotoxic T (TC) lymphocytes. Helper activity was measured by the ability of the cells to help resting thymic TC cell precursors develop into effector TC cells in vitro. Direct injection of allogeneic spleen cells into the footpads of mice led to the generation of alloantigen-specific helper cells in the draining popliteal lymph nodes within 4 to 6 days. Helper activity was mediated by nylon-wool-nonadherent Lyt-1+ T lymphocytes; some activity was associated with Lyt-1,2+ cells. The genetic requirements for both the induction and restimulation of C3H anti-H-2d TH cells were investigated using cells from H-2k/H-2d recombinant mice as in vivo immunogens and in vitro stimulators. Evidence is presented that shows in a direct assay that TH cells themselves are specific for I region-coded determinants. Thus, disparity at the left side of the H-2 complex (K to I-E) but not at H-2K alone was necessary and sufficient to induce and reactivate TH cells. Proliferation in mixed lymphocyte culture was measured in combinations in which TH cells were not detectable, supporting the idea that proliferation cannot be strictly considered a measurement of helper cells.

The regulation of T cell growth: requirements for the activation and replication of antigen-specific interleukin 2 producing T cells.

The antigenic requirements for stimulating the production of interleukin 2 (IL2) and the growth of IL2 producing T cells was evaluated using antigen-(Keyhole limpet hemocyanin) specific F1 T helper cell lines. The results demonstrate that both the induction and growth of these cells requires specific antigen and antigen-presenting cells of the correct I-A type. Evidence is presented that argues that soluble macrophage derived factors released as a result of T cell-macrophage interaction are insufficient, even in the presence of antigen, to promote growth of IL2 producing cells. We thus conclude that the direct interaction of IL2 producing cells with an antigen presenting cell is obligatory for activation and growth. These results suggest that T cell proliferation, and as a consequence the magnitude of T cell mediated immune responses, is limited by the availability of "approximately" presented antigen to IL2 producing T cells. The requirement for antigen presenting cells in antigen-driven responses is in contrast with mitogen-induced T cell activation, in which soluble factors can substitute for the accessory cells.

Major histocompatibility complex-restricted and unrestricted interactions in the T cell-dependent activation of hapten-binding B cells.

The requirements for linked recognition and major histocompatibility complex-restricted interactions in helper T cell-dependent activation of purified unprimed and primed hapten-binding B cells have been investigated. The activation of unprimed hapten-binding B cells required specific antigen and restricted interactions between helper T cells and B cells. As expected, specific hapten-carrier conjugates were essential for the activation of B cells at low antigen doses. Interestingly, however, supraoptimal concentrations (125 micrograms/ml) of carrier protein alone could substitute for specific conjugates in the activation of unprimed B cells. The requirement for restricted helper T cell-B interactions was unchanged under these conditions. These results are discussed in terms of the signalling requirements for B cell activation. In contrast to the results using hapten-specific B cells from unprimed mice, the further stimulation of B cells prepared from mice primed 5 to 7 days earlier with immunogenic forms of the hapten was limited only by the requirements for helper T cell activation. The transition in the activation properties of B cells following in vivo stimulation was not solely a result of the binding of B cell membrane immunoglobulin to specific antigen, since the interaction of unprimed B cells with hapten during their purification, even for extended periods of time, did not alter the requirements for their further stimulation. These results demonstrate that the recent antigenic experience of B cells determines their activation state which, in turn, dictates the further requirements for helper T cell function in B cell stimulation. This implies that specificity of the immune response is determined in the early stages of B cell activation.

IgM accelerates affinity maturation.

Secreted IgM is a potent adjuvant that concentrates antigen into secondary lymphoid organs and initiates antibody responses, germinal centre formation and is crucial in resolving infections. The current studies investigated the influence of specific IgM on both the quantity and quality of antibody produced in response to T-dependent and T-independent antigens. The addition of IgM to either antigen had no significant effect on the titre or duration of antibody responses. However, the presence of specific IgM led to accelerated affinity maturation when mice were challenged with low doses of IgM-containing immune complexes (IgM-IC) of T-dependent antigens compared with antigen alone. Interestingly, the administration of higher concentrations of IgM-IC increased follicular deposition of antigen but did not result in accelerated affinity maturation or in higher antibody affinities. The administration of IgM complexed with T-independent antigens had no effect on antibody titre, duration or affinity maturation, despite increased antigen deposition in lymphoid follicles. Together, these results demonstrate that IgM accelerates affinity maturation in immune responses to T-dependent antigens and implies an antigen optimum exists for the generation of high affinity antibodies. The data also suggest IgM plays an important role in the induction of CD4 T cells, facilitating germinal centre formation and function.

Somatic diversification of B cells: a role for autoreactive T lymphocytes.

Although the immune system discriminates self from non-self, and autoreactivity has pathologic consequences, lymphocytes do recognize self antigens. Self-recognition involves immunoglobulin variable (Ig V) regions and major histocompatibility complex (MHC)-encoded molecules. Jerne postulated that recognition of self-idiotypes within an immune system was a necessary homeostatic mechanism, important in the regulation of the immune system both in the presence and absence of perturbation by exogenous antigen'. Support for this hypothesis has accumulated from a variety of experimental systems but the significance of autoreactive T lymphocytes which recognize MHC-encoded class II (Ia) molecules(2-5) remains unknown. Rosenberg et al. s have proposed that autoreactive T cells are involved in mediating autoimmunity when the mechanisms controlling theirfunction degenerate. Here Ronald Corley suggests that autoreactive T lymphocytes are responsible for the expansion and somatic diversification of the B-cell compartment from germ-line encoded V genes. Furthermore, he proposes that autoreactive T cells do not function autonomously but that B-cell activation follows occupation of the two relevant triggering molecules on the B-cell membrane, namely Ia molecules by the autoreactive T cells and surface immunoglobulin by internally complementary idiotypes.

Responses of alloantigen-primed lymphocytes in vitro. The contribution of increased frequencies of responding lymphocytes to differences between reactivity of normal and primed lymphocyte populations.

Sensitization of lymphocytes against allogeneic cells in vitro results in significant enrichment of alloreactive lymphocytes. Such enrichment was found to profoundly influence the conditions for measuring proliferative responses. Fewer primed lymphocytes than unprimed ones had to be cultured to favor optimal proliferation. Second, proliferative responses could be detected using 10 to 20 times fewer primed responding cells than when using unprimed responders. Finally, although responses of both unprimed and primed lymphocytes were dependent on the number of stimulator cells in culture, the primary mechanism(s) through which this dependence was expressed appeared to differ. The results demonstrate that, under the same conditions, comparisons of responses of two populations that contain different proportions of reactive lymphocytes may not be justified.

Responses of alloantigen-primed lymphocytes in vitro. The specificity of secondary responses assayed by titration of primed lymphocyte populations.

Maximal proliferative responses were observed at lower responder cell densities in alloantigen-primed lymphocyte populations when restimulated with the specific priming cell than when restimulated with other allogeneic cells. This relationship was due to the larger number of lymphocytes within the primed population which responded to the specific sensitizing cell than to unrelated stimulator cells. As a result, at high responder cell densities, stimulator cells unrelated to the specific sensitizing cells elicited a disproportionately high response relative to the response stimulated by the specific cells. Restimulation of decreasing numpacity of different cells. Because primed populations contain a larger number of lymphocytes that responde to the specific stimulator than to other stimulators, responses of primed lymphocytes could be made operationally monospecific. Limiting dilution analysis demonstrated that low numbers of primed cells were stimulated by the specific sensitizing cells but not by unrelated stimulator cells.

Constitutive nuclear translocation of NF-kappa B in B cells in the absence of I kappa B degradation.

Members of the NF-kappa B/Rel family of transcription factors are involved in many aspects of B lymphocyte development and function. NF-kappa B is constitutively active in these cells, in contrast with most other cell types. In the inactive form, NF-kappa B/Rel proteins are sequestered in the cytoplasm by members of the I kappa B family of NF-kappa B inhibitors. When activated, NF-kappa B is translocated to the nucleus, a process that involves the phosphorylation and proteasomal degradation of I kappa B proteins. Thus, NF-kappa B activation is accompanied by the rapid turnover of I kappa B proteins. We show that while this "classical" mode of NF-kappa B activation is a uniform feature of IgM+ B cell lines, all IgG+ B cells analyzed contain nuclear NF-kappa B yet have stable I kappa B alpha, I kappa B beta, and I kappa B epsilon. Furthermore, I kappa beta epsilon levels are at least 10 times lower in IgG+ B cells than in IgM+ B cells, an additional indication that the regulation of constitutive NF-kappa B activity in these two types of B cells is fundamentally different. These data imply the existence of a novel mechanism of NF-kappa B activation in IgG+ B cells that operates independently of I kappa B degradation. They further suggest that different isoforms of the B cell receptor may have distinct roles in regulating NF-kappa B activity.