Inhibition of normal B-cell function by human immunodeficiency virus envelope glycoprotein, gp120.

Despite the occurrence of hypergammaglobulinemia in human immunodeficiency virus (HIV) infection, specific antibody production and in vitro B-cell differentiation responses are frequently impaired. In this study, we have examined the effects of HIV envelope glycoprotein gp120 on T-helper cell function for B cells. In the culture system used, B-cell functional responses were dependent on T-B-cell contact, since separation of T and B cells in double chambers by Transwell membranes rendered the B cells unresponsive in assays of antigen-induced B-cell proliferation and differentiation. Cytokines secreted by T cells were also essential, since anti-CD3 monoclonal antibody (mAb)-activated, paraformaldehyde-fixed T-cell clones failed to induce B-cell proliferation and differentiation. Pretreatment of the CD4+ antigen-specific T cells with gp120 was found to impair their ability to help autologous B cells, as determined by B-cell proliferation, polyclonal IgG secretion, and antigen-specific IgG secretion. The gp120-induced inhibition was specific in that it was blocked by soluble CD4. Furthermore, only fractionated small B cells (which are T-cell-dependent in their function) manifested impaired responses when cultured with gp120-treated T cells. Antigen-induced interleukin (IL)-2 and IL-4, but not IL-6, secretion were markedly reduced in gp120-treated T-cell clones. Addition of exogenous cytokines failed to compensate for defective helper function of gp120-treated T cells. The findings in this study indicate that gp120 impairs helper functions of CD4+ T cells by interfering with T-B-cell contact-dependent interaction; the inhibitory effects of soluble envelope proteins of HIV may contribute to the immunopathogenesis of the HIV-associated disease manifestations.     

Interleukin 4 mediates autocrine growth of helper T cells after antigenic stimulation.

The role of interleukin 4 (IL-4) (previously called B-cell stimulatory factor 1) in the response of T helper (TH) cells to antigen presented by antigen-specific B cells or splenic adherent cells was investigated. Antigenic stimulation of either a keyhole-limpet-hemocyanin-specific TH-cell line or two keyhole-limpet-hemocyanin-specific T-cell clones resulted in the secretion of IL-4 but not interleukin 2 (IL-2). The secretion of IL-4 was first detected in the culture supernatant 6-8 hr after antigenic stimulation. Induction of IL-4 secretion was antigen specific and major histocompatibility complex restricted. Antigenic stimulation also resulted in increased responsiveness of the TH cells to exogenously added or endogenously produced IL-4. The antigen-induced proliferation of the TH cells could be inhibited by an anti-IL-4 antibody but not by an anti-IL-2-receptor antibody. These results suggest that IL-4 mediates the proliferation of some TH cells by an antigen-induced autocrine mechanism. Taken together with past results, these data indicate that, during T-cell-B-cell interactions involving some soluble protein antigens, IL-4 and not IL-2 is the critical lymphokine for activating resting B cells and inducing proliferation of the TH cells.     

Dissociation of two signals required for activation of resting B cells.

Cellular interactions involved in the T cell-dependent activation of B cells were analyzed by using lines and clones of helper T cells specific for determinants expressed on the B cell surface. Activation of male antigen-, M locus-, and H-2-specific T cells was shown to support polyclonal Ig production by a population of B cells that did not require T-cell-B-cell interaction for induction/amplification. However, these T cells alone did not activate gradient-purified small (resting) B cells. The activation of small B cells was shown to require not only a signal derived through an antigen-specific T-helper cell-B cell interaction but in addition a second signal that could be provided by anti-Ig antibodies.     

Stimulation of B-cell proliferation by membrane-associated molecules from activated T cells

Activation of B cells to proliferation and antibody secretion is dependent on soluble lymphokines secreted by activated T cells. Activation of T cells results from physical contact between B cells and T cells through binding of the T-cell antigen receptor to a complex of antigen and class II major histocompatibility complex (MHC) molecules. To determine whether this interaction also contributes to B-cell activation by mechanisms other than those mediated by soluble T cell-derived lymphokines, I examined the ability of isolated T-cell plasma membranes to stimulate proliferation in cultures of unfractionated B cells. Membranes prepared from a cloned antigen-specific helper T-cell line induced substantial proliferation provided that the T cells had been mitogen-activated before isolation of membranes. Membranes from splenic Con A-treated blasts also stimulated B-cell proliferation, suggesting that this activity may be a common property of some subsets of activated T cells. Induction of B-cell proliferation was not found to be antigen-dependent or MHC-restricted, indicating no significant contribution by the T-cell receptor for antigen. The presence of interleukins 4 and 5 in membrane fractions was indicated by proliferation of lymphokine-sensitive cell lines, although culture supernatants from mitogen-activated T cells proved to be far more potent sources of these activities. The combined effect of membranes and lymphokine-containing culture supernatants in B-cell cultures was greater than their added effects in separate cultures. This observation suggests that lymphokines or molecules with mitogenic activity for B cells other than those found in abundance in culture supernatants may be present on activated T-cell membranes.     

Role of cell surface immunoglobulin in B-lymphocyte activation.

The role of cell surface immunoglobulin in helper T-cell-dependent B-cell activation was analyzed using a B-cell lymphoma, CH12, with known antigen specificity and activation properties similar to those of a resting B cell. Two sources of helper T cells were used, both selected such that they interact with H-2-encoded determinants on CH12 in the absence of the specific B-cell antigen, sheep erythrocytes. By this dissociation of the specificity of the T cells from that of the B cells, the requirement for antigen in the induction of CH12 to antibody secretion could be studied. The results show that both helper T-cell-B-cell interactions and surface immunoglobulin-antigen binding are involved in inducing B-cell differentiation, thus establishing a signalling function for the antigen receptor on B lymphocytes. Our data also show that the requirement for surface immunoglobulin-ligand interactions in B-cell activation can, under certain conditions, be circumvented, notably when high (nonphysiologic) multiplicities of T-cell help are used.     

Impaired T-cell regulation of B-cell growth in Helicobacter pylori--related gastric low-grade MALT lymphoma.

BACKGROUND & AIMS: Neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma are responsive to T helper cells and sensitive to withdrawal of H. pylori-induced T-cell help. METHODS: The clonal progeny of T cells from the gastric mucosa of 5 patients with MALT lymphoma was compared with that of T-cell clones obtained from 5 H. pylori-infected patients with chronic gastritis. RESULTS: T-cell clones were assessed for specificity to H. pylori, cytokine profile, help for B-cell proliferation, and perforin- or Fas-mediated cytotoxic regulation of B-cell growth. Twenty-eight of 165 CD4(+) gastric clones from MALT lymphoma and 33 of 178 CD4(+) clones from chronic gastritis recognized H. pylori antigens. Cytokine production was similar in the 2 series of clones. All MALT lymphoma-derived clones dose-dependently increased their B-cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1 because of concomitant cytolytic killing of B cells. T-cell clones from MALT lymphoma had both reduced perforin-mediated cytotoxicity and poor ability to induce Fas-mediated apoptosis. These defects were limited to gastric T cells. CONCLUSIONS: H. pylori-induced T cell-dependent B-cell activation and deficient cytotoxic control of B-cell growth may link H. pylori infection, local T-cell response, and genesis of low-grade gastric MALT lymphoma.     

Age-dependent appearance of non-major histocompatibility complex-restricted helper T cells.

T cells which recognize antigen in association with self major histocompatibility complex (MHC) molecules are positively selected within the thymus. This results in skewing of the T-cell repertoire toward the recognition of antigenic peptides presented by self MHC molecules. While the thymus gland involutes at a relatively young age, bone marrow function and the size of the peripheral T-cell pool are maintained with age. We have investigated the MHC restriction of helper T-cell function for B-cell production of specific antibody in mice of different ages. Splenic helper T cells from 2- to 3-month old mice immunized with phosphocholine-keyhole limpet hemocyanin conjugate were MHC-restricted as defined by their capacity to induce phosphocholine-specific antibody synthesis by syngeneic but not by allogeneic B cells. In contrast, splenic T cells from immunized 18- to 20-month-old mice induced specific anti-phosphocholine antibodies by both syngeneic and allogeneic B cells. No evidence of polyclonal immunoglobulin synthesis was detected. The ability of T cells from old mice immunized with phosphocholine-keyhole limpet hemocyanin to induce phosphocholine-specific antibody synthesis by B cells from allogeneic mice was inhibited by T cells from immunized young mice. These findings suggest that non-MHC-restricted T-cell helper activity in old mice results from the loss of T cells, present in young mice, which suppress non-MHC-restricted helper cells.     

The specific direct interaction of helper T cells and antigen-presenting B cells.

Cell couples have been formed by mixing an antigen- and Ia-specific cloned helper T-cell line with a B-cell hybridoma presenting the antigen. By immunofluorescence observations, we have shown that the microtubule-organizing center (MTOC) inside the helper T cell, but not in the bound antigen-presenting cell, becomes oriented to face the area of specific cell-cell contact. This MTOC orientation is antigen- and Ia-specific, and thus provides direct evidence for the specific interaction of a helper T cell with a B cell. It is presumed that the function served by this MTOC orientation, which is accompanied by the coordinate reorientation of the Golgi apparatus, is to target Golgi apparatus-derived secretory vesicles, containing putative lymphokines and/or growth factors, from the helper T cell directly to the antigen-presenting cell.     

Differential ability of B cells specific for external vs. internal influenza virus proteins to respond to help from influenza virus-specific T-cell clones in vivo.

When a helper T-cell (TH) clone specific for the hemagglutinin, neuraminidase, matrix protein, or nucleoprotein of influenza strain A/PR/8/34 is adoptively transferred to athymic mice 1 day after virus infection the anti-viral antibody response of the mouse is enhanced. This response is directed predominantly to the hemagglutinin and requires associative T-cell-B-cell interactions. Delaying transfer of the TH clone has three consequences: (i) the onset of the anti-hemagglutinin antibody response is delayed; (ii) the titer of the anti-hemagglutinin response is reduced; and (iii) the titer of the antibody in the response against the internal proteins, matrix protein and nucleoprotein, is enhanced upon transfer of matrix protein- or nucleoprotein-specific, but not hemagglutinin- or neuraminidase-specific, TH clones. Thus, there is a hierarchy of help: B cells recognizing viral surface components, hemagglutinin or neuraminidase, can receive help from TH clones specific for any of the major structural viral proteins. In contrast, B cells responding to internal viral components, matrix protein or nucleoprotein, are restricted to receiving help almost exclusively from TH clones with the same protein specificity. These observations suggest that, upon B-cell surface immunoglobulin-antigen interaction and uptake of intact virus, B cells specific for viral surface proteins process and present all major structural viral antigens, enabling the B cells to interact with TH clones specific for any virion protein. B cells recognizing internal viral components, which may be accessible to interaction with B-cell immunoglobulin receptors mainly as free proteins, would present only the protein for which they are specific and, thereby, receive help only from the TH clones of the same protein specificity.     

Two separate functions of class II (Ia) molecules: T-cell stimulation and B-cell excitation.

We have evaluated the role of major histocompatibility complex-encoded class II (Ia) molecules as transmembrane signaling receptors in the T helper cell-dependent activation of B lymphocytes. For these studies, we utilized the murine B-cell lymphoma CH12, which expresses both I-A and I-E class II molecules. In addition, CH12 cells carry IgM of known antigen specificity and require both specific antigen and Ia-restricted T-cell help for the induction of antibody secretion. In this respect, they resemble normal resting B cells. We have studied the ability of antigen-specific or alloreactive T helper cells reactive with either the I-A or the I-E molecules on CH12 to be activated and their ability to stimulate antibody production by CH12. The results show that, although CH12 cells present antigen to T helper cells that interact with either the I-A or the I-E molecules, CH12 cells are stimulated to secrete antibody only by T helper cells reactive with their I-E molecules. Our data demonstrate that class II molecules are transducers of signals for B-cell excitation in addition to serving a restricting function for helper T-cell stimulation. Moreover, the data demonstrate that these two functions, T-cell stimulation and B-cell excitation, are discrete and need not be expressed by the same Ia molecule.     

Antigen-specific helper T-cell clone supernatant is sufficient to induce both polyclonal proliferation and differentiation of small resting B lymphocytes.

Gradient-purified resting B lymphocytes can be polyclonally stimulated by antigen-specific major histocompatibility complex (MHC)-restricted helper T lymphocytes as well as by antigen-activated helper T-cell supernatant. In contrast to what has been described so far, we show that helper T-cell supernatant (in the absence of any other added stimulus, such as that provided by anti-mu antibodies) is sufficient to induce both proliferation of resting B cells and their differentiation into IgM-secreting cells. The stimulation induced by the helper T-cell supernatant takes place in serum-free medium and is not MHC-restricted. Our findings strongly support the existence of a B-cell activating factor acting on the resting B cell and causing it to enter the G1 phase of the cell cycle in a MHC-unrestricted manner.     

Balanced expression of CXCR5 and CCR7 on follicular T helper cells determines their transient positioning to lymph node follicles and is essential for efficient B-cell help

The production of high-affinity antibodies to T-dependent antigens requires the interaction of B cells and T helper cells expressing receptors specific for the same antigen. Although several mechanisms have been elucidated that regulate B-cell trafficking within lymphoid organs, less is known about molecular cues that guide the small subpopulation of CD4+ follicular T helper cells to B-cell follicles. Using adoptive transfer of transgenic T cells in mice, we demonstrate that antigen-induced activation leads to a finely tuned positioning of T cells either to the T-cell area or the B-cell follicle. We show that expression of CXCR5 is indispensable for T cells to enter B-cell follicles, whereas expression of CCR7 provides a counteracting signal to retain activated T cells in the T-cell area. Although only few T cells transiently migrate from the T-cell area to the B-cell follicle of peripheral lymph nodes following antigenic challenge, this step is essential to provide the help B cells require to produce antibodies efficiently. Thus, we demonstrate that the balanced expression of CCR7 and CXCR5 determines the positioning and proper function of follicular T helper cells.     

A single antigen-specific B cell can conjugate to either a type 1 or a type 2 helper T cell.

Keyhole-limpet-hemocyanin-specific I-Ad-restricted T helper cells type 1 (TH1 cells) and type 2 (TH2 cells) were studied for their ability to physically conjugate to trinitrophenyl-specific antigen-binding B cells (TNP-ABCs). The same TNP-ABCs (which had already processed TNP-keyhole limpet hemocyanin) formed conjugates with one or the other T helper (TH) cell type, and conjugation was antigen-specific and major histocompatibility complex-restricted. The conjugation of a fixed number of one type of TH cell to the TNP-ABCs was inhibited by co-incubation with increasing numbers of cells of the other TH cell type. Thus, the vast majority of TNP-ABCs can conjugate to either type of TH cell. A similar pattern of inhibition of the conjugation of one TH cell type to the TNP-ABCs was seen when cells were co-incubated with increasing numbers of cells from an alloreactive T-cell clone. In all cases, conjugates contained only one T cell bound to one B cell, suggesting that interaction of an antigen-presenting B cell with one TH cell prevents the simultaneous binding of another TH cell.     

T cell-dependent activation of resting B cells: requirement for both nonspecific unrestricted and antigen-specific Ia-restricted soluble factors.

Cloned murine helper T cells, restricted to the Iab antigens of the major histocompatibility locus and specific for horse erythrocytes as a foreign antigen, produce, in cooperation with antigen and histocompatible adherent cells, soluble factors that replace the helper T cells in their action on B cells. Three types of factors can be distinguished on the basis of molecular weight: proteins having apparent Mr 30,000 (p30) that act antigen- and Ia-nonspecifically as replication- and maturation-inducing factors and proteins having apparent Mr 55,000 (p55) and 125,000 (p125) that act on resting B cells in an Ia-specific, restricted fashion. Neither horse erythrocytes (a T-cell specific antigen) nor p55 and p125, alone or together, stimulate resting B cells to proliferation and maturation. Double occupancy by antigen and p55 or p125, however, renders Ia-compatible, but not Ia-incompatible, resting B cells susceptible to stimulation. The subsequent addition of p30 to these "excited" B cells then results in the proliferation and maturation of clones of horse erythrocyte-specific resting B cells, which then replicate under the stimulatory action of p30. p30 do not bind antigen, nor do they bind anti-Ia or anti-immunoglobulin antibodies. p55 are bound by anti-heavy chain variable region antibodies. p55 are bound by anti-heavy chain variable region antibodies, but not by anti-heavy or anti-light chain constant region antibodies or anti-Ia antibodies. p125 molecules bind horse but not sheep erythrocytes and are bound by anti-heavy chain variable region, but not by anti-heavy or light chain constant region or anti-Ia antibodies. p55 and p125 are likely to be soluble analogues of the antigen-specific, Ia-restricted T-cell receptors of the cloned helper T cells.     

Immunoregulation in man.

The process during which a B lymphocyte, after ligation of the antigen receptor, differentiates into a high-rate antibody-secreting plasma cell is under strict regulatory control of T lymphocytes and their soluble products. For most so-called T-cell-dependent (protein) antigens, antigen-specific CD4-positive T cells or T-cell factors participate in the transition of resting into activated B cell. Terminal B-cell differentiation, including immunoglobulin class switch, is regulated by a variety of (antigen non-specific) cytokines which act in a highly coordinated fashion. The other major class of naturally occurring antigens, the so-called T-cell independent (polysaccharide) antigens, apparently can induce B-cell activation in the absence of antigen-specific CD4-positive T cells. Terminal B-cell differentiation, however, also depends on the action of cytokines, although the relative contribution of individual cytokines remains to be determined. Downregulation of both T-cell-dependent and T-cell independent antigens is mediated by specific suppressor T cells. Next to this "internal control" by regulatory T cells, it is important to realize that components of the (neuro-)endocrine system (such as the endorphins and the family of insulin-like growth factors) and the complement system also exert important immunoregulatory functions.     

Functional helper activity of monoclonal T cell populations: antigen-specific and H-2 restricted cloned T cells provide help for in vitro antibody responses to trinitrophenyl-poly(LTyr,Glu)-poly(DLAla)--poly(LLys).

The ability of long-term cultured and monoclonal T cell populations to provide antigen-specific help was assessed in a system of Ir gene-controlled in vitro antibody responses to soluble antigens. T-cell colonies and monoclonal T-cell lines were generated which proliferated specifically in response to poly(LTyr,Glu)-poly(DLAla)--poly(LLys) [(T,G)-A--L] and were I-A restricted in these proliferative responses. These (T,G)-A--L-specific T-cell populations were evaluated for their ability to help unprimed and T-cell depleted spleen cell populations in the generation of antibody responses to trinitrophenyl (TNP)-(T,G)-A--L in vitro. It was found that long-term T-cell lines, including monoclonal T-cell populations derived by limiting dilution, were highly efficient helper cells for IgM responses to TNP-(T,G)-A--L. These helper T cells were both antigen-specific and I-A restricted in their ability to be activated and to cooperate with T-cell depleted spleen cell populations. Once specifically activated, however, these clones provided help that was antigen nonspecific. These studies have thus demonstrated the ability of antigen-specific and H-2-restricted monoclonal T-cell populations to provide help for responses to soluble antigens in vitro.     

T cells and human autoimmune thyroid disease: emerging data show lack of need to invoke suppressor T cell problems.

Human T cells recognize self and foreign antigens when such antigens are processed into small peptides and bound to molecules coded for by genes of the HLA region on chromosome 6. The part of the T-cell surface which is responsible for such recognition is a set of molecules coded for by a variety of genes and known as the T-cell-receptor complex. In animal models, T cells are able to transfer autoimmune thyroiditis and T cells have, therefore, long been implicated in the etiology of human autoimmune thyroid disease (AITD). Information gained from the study of intrathyroidal T cells and thyroid antigen-specific T-cell clones has shown that in patients with Graves' disease, mainly helper T-cell clones have been obtained, whereas in autoimmune (Hashimoto's) thyroiditis cytolytic T-cell clones may be predominant. Such thyroid antigen-specific T cells have now been shown to recognize one or other of the three major thyroid-specific antigens; thyroglobulin, thyroid peroxidase, or the TSH receptor and efforts are currently in progress to characterize the T-cell epitopes of these major thyroid autoantigens. Recent findings of restricted T-cell receptor V gene use amongst intrathyroidal T cells confirm the primary role of T cells in human thyroid autoimmune processes leading to AITD. However, the mechanisms whereby such autoreactive T cells escape deletion and anergy, and how they become activated, remain uncertain. There is compelling evidence that the thyroid cell itself, by expressing HLA molecules, and presenting antigen directly to the T cells, may initiate disease, perhaps after an external insult.     

Toll-like receptor 4-activated B cells out-compete Toll-like receptor 9-activated B cells to establish peripheral immunological tolerance

B-cell-induced peripheral T-cell tolerance is characterized by suppression of T-cell proliferation and T-cell-dependent antibody production. However, the cellular interactions that underlie tolerance induction have not been identified. Using two-photon microscopy of lymph nodes we show that tolerogenic LPS-activated membrane-bound ovalbumin (mOVA) B cells (LPS B cells) establish long-lived, highly motile conjugate pairs with responding antigen-specific OTII T cells but not with antigen-irrelevant T cells. Treatment with anti-CTLA-4 disrupts persistent B-cell-T-cell (B-T) contacts and suppresses antigen-specific tolerance. Nontolerogenic CpG-activated mOVA B cells (CpG B cells) also form prolonged, motile conjugates with responding OTII T cells when transferred separately. However, when both tolerogenic and nontolerogenic B-cell populations are present, LPS B cells suppress long-lived CpG B-OTII T-cell interactions and exhibit tolerogenic dominance. Contact of LPS B cells with previously established B-T pairs resulted in partner-swapping events in which LPS B cells preferentially migrate toward and disrupt nontolerogenic CpG mOVA B-cell-OTII T-cell pairs. Our results demonstrate that establishment of peripheral T-cell tolerance involves physical engagement of B cells with the responding T-cell population, acting in a directed and competitive manner to alter the functional outcome of B-T interactions.     

T-cell-dependent B-cell stimulation is H-2 restricted and antigen dependent only at the resting B-cell level.

Cloned lines of helper thymus-derived (T) cells produce help for bone marrow-derived (B) cell growth and Ig secretion in the presence of histocompatible adherent cells and of specific antigen. This help stimulates histocompatible as well as histoincompatible B-cell blasts polyclonally. Thus, neither antigen nor histocompatibility, but antigen-unspecific factor(s) for growth and Ig secretion are required to stimulate a B-cell blast through the next round of division. On the other hand, only histocompatible, resting, small B cells, and only those binding their specific antigen, can be stimulated by antigen-activated T-cell help to initiate growth and Ig secretion. The preference of the resting B cells for such collaboration with T-cell help is mapped to the K end of the H-2 histocompatibility locus, and probably constitutes the antigen expressed on B cells by the immune response (I) region. It appears that a resting B cell is excited by the binding of specific antigen to surface Ig and by the interaction of its surface Ia antigen with helper T cells. After this dual recognition the excited B cell can be stimulated by the antigen-unspecific factor(s) generated by the interaction of helper T-cells, adherent cells, and antigen to initiate replication.     

NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells

We have developed a new method that allows detection and isolation of viable, antigen-specific, human T cells from a heterogeneous pool of T cells. We have engineered a self-inactivating retroviral vector containing multiple (3 or 6) nuclear factor of activated T-cell (NFAT)-binding sites, followed by the minimal IL2 promoter and the reporter gene GFP. Jurkat cells, primary T-cell blasts, and T-cell clones were transduced with high efficiency (20%-40%). Stimulation of the transduced cells with phorbol myristate acetate (PMA) and ionomycin resulted in a high level expression of GFP that was maximal after 12 to 14 hours and remained stable for another 12 hours. Activation of T cells carrying the construct containing 6 NFAT-binding sites resulted in the highest mean fluorescence intensity. Cyclosporin-A and FK506 were able to block the activation-dependent GFP expression. Activation of transduced T-cell blasts with the superantigen staphylococcal enterotoxin B or of transduced antigen-specific T-cell clones with cognate antigen resulted in GFP expression. After an overnight stimulation of a heterogeneous T-cell bulk culture with an HLA mismatched stimulator cell (JY), the GFP expressing cells were cloned. As expected, the cloning frequency of the antigen-specific GFP(+) cells was considerably higher than that of the total T-cell population. Most of the T-cell clones were either cytolytic, or proliferative toward JY stimulator cells. Interestingly, we also isolated T-cell clones that were noncytolytic and nonproliferative toward JY cells, but specifically up-regulated GFP after an overnight stimulation with JY. (Blood. 2000;96:459-466)