Activation via TCR or IL-2 receptor of a CD8+ suppressor T cell clone: Effect on IL-10 production and on proliferation of the suppressor T cell

In a previous study, we established CD8⁺ suppressor T cell (Ts) clone 13G2 which produced the suppressive lymphokine, interleukin-10 (IL-10). In this study, we examined what physiological activator could induce both production of IL-10 from 13G2 and the proliferation of 13G2. Both the antigenic stimulation mimicked by the anti-CD3 antibody and the T cell growth factor interleukin-2 (IL-2) induced IL-10 production from the 13G2 clone equally well. 13G2 cells proliferated remarkably with IL-2 stimulation, while anti-CD3 only slightly induced proliferation of the clone. 13G2 cells also produced IL-10 in the presence of hydroxyurea which blocked transit of cells from G₁ to S phase. However, cycloheximide blocked the production of IL-10 from the Ts clone. The study demonstrates that both the anti-CD3 antibody and IL-2 induced IL-10 synthesis of the Ts clone equally well, and the proliferative response of Ts cells was induced more by IL-2 than by anti-CD3. IL-2 proved to be a good stimulator for Ts cells to produce suppressive lymphokine and to multiply their population.Abbreviation Ts suppressor T cell - Th helper T cell - Ag antigen - APC antigen presenting cell - IL interleukin - TCR T cell receptor - mAb monoclonal antibody     

Lymphoma models for B cell activation and tolerance. VI. Reversal of anti-Ig-mediated negative signaling by T cell-derived lymphokines

We have recently described three "immature" B cell lymphomas which are exquisitely sensitive to growth inhibition by anti-Ig reagents and may serve as models for tolerance induction in normal B cells. These cells are inhibited from cell cycle progression into S after receiving a negative signal in early G1. In this paper, we demonstrate that the growth inhibition by anti-Ig can be prevented and reversed by the addition of supernatants from T cell lines. One such line, called Tova, produces factors which restore normal levels of DNA synthesis in the presence of concentrations of anti-Fab or anti-kappa immunoglobulins which cause up to a 90% inhibition of thymidine incorporation in a 2- to 3-day culture period. This factor is at least partially effective when added up to 24 hr after anti-Ig to unsynchronized lymphoma cells and it does not alter the growth of control cultures. Studies using synchronized lymphoma cells indicated that the T cell factor permitted cycle progression into S when added during the early G1 exposure to anti-kappa and was less effective when added late in G1. Preliminary characterization suggests that both B cell growth factor II (interleukin 5) and B cell stimulatory factor 1 (interleukin 4) have additive activity in this system, although another unidentified lymphokine may also be involved. The relevance of T cell reversal of Ig receptor-mediated negative signaling to neonatal B cell tolerance is emphasized.     

A polyclonal model for B-cell tolerance. II. Linkage between signaling of B-cell egress from G0, class II upregulation and unresponsiveness.

Overnight exposure of adult splenic B cells to anti-Ig, a surrogate for antigen/tolerogen, can result in a hyporesponsive state in terms of antibody synthesis. Since B cells treated with either intact of F(ab')2 fragments of anti-Ig will exit the G0 phase of the cell cycle and enter G1 or S, respectively, we examined which steps in B-cell activation were required for this form of hyporesponsiveness. We found that B-cell hyporesponsiveness could be induced under conditions leading to either abortive or productive B-cell cycle progression, depending on the immunogenic challenge employed. Thus, PMA + ionomycin, concanavalin A, PMA alone, or ionomycin alone induced hyporesponsiveness. Each of these reagents is able to drive B-cell exit from G0 into G1 and cause class II hyperexpression. We next examined the effect of cyclosporin A (CSA), a reagent that blocks anti-Ig but not by PMA-induced class II hyperexpression. Interestingly, CSA only interfered with the induction of B-cell hyporesponsiveness with anti-Ig. These results suggest that upregulation of MHC class II may be coincident with a CSA-sensitive tolerance pathway in B cells stimulated by anti-Ig. Finally, IL-4 pretreatment was found to ablate hyporesponsiveness induced by either intact anti-Ig or PMA. These results parallel the Fc-dependent induction of hyporesponsiveness reported earlier (G. Warner and D. W. Scott, J. Immunol. 146, 2185, 1991). We propose that crosslinking of surface Ig, leading to cell cycle progression out of G0 as well as class II hyperexpression, in the absence of a cognate T cell signal, leads to B-cell hyporesponsiveness.     

Anti-immunoglobulin treatment of murine B-cell lymphomas induces active transforming growth factor beta but pRB hypophosphorylation is transforming growth factor beta independent.

Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. When WEHI-231 lymphoma cells were selected for growth in TGF-beta, the majority of the TGF-beta-resistant clones remained sensitive to anti-Ig-mediated growth inhibition. In these clones, the retinoblastoma gene product was found to be in the underphosphorylated form after 24-h treatment with anti-Ig, but not with TGF-beta. These data show that anti-Ig treatment of murine B-cell lymphomas stimulates the production of active TGF-beta but that a TGF-beta-independent pathway may be responsible for the pRB underphosphorylation and cell cycle blockade.     

The role of CD4 in antigen-independent activation of isolated single T lymphocytes

The membrane molecule CD4 (L3T4) is thought to facilitate activation of Class II H-2-restricted T cells by binding to Ia determinants on antigen-presenting cells. Recent reports suggest that CD4 can also contribute to antigen-independent activation by anti-T cell receptor (TCR) antibodies. An assay which measures the secretion of two lymphokines, granulocyte-macrophage colony-stimulating factor and interleukin 3 (IL-3), by single T cells activated with an anti-TCR antibody, F23.1, was used to analyze the effects of anti-CD4 antibodies on antigen-independent T cell activation. Single cells of a CD4+F23.1+ clone were micromanipulated into wells to which F23.1 had been immobilized, and their lymphokine secretion was measured 24 hr later. The frequency of lymphokine-secreting cells was consistently reduced up to 10-fold in the presence of soluble anti-CD4 antibody (GK1.5) but only up to 2.5-fold by an antibody to the cell adhesion molecule, LFA-1. In both bulk and single-cell cultures, responses to suboptimal concentrations of F23.1 were more susceptible to inhibition by GK1.5 than responses to optimal F23.1. The failure of GK1.5 to inhibit IL-2-stimulated lymphokine synthesis in bulk cultures suggested that CD4 ligation did not deliver a negative signal to the clone. By contrast, when either anti-CD4 or anti-LFA-1 was immobilized on the same surface as F23.1, the frequency of lymphokine-secreting cells could be increased up to 10-fold. It is concluded that anti-CD4 antibodies can act directly on the responding T cell to affect TCR-dependent activation, in the absence of interaction with antigen-presenting cells or any other cell type.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.     

Activation of human B cells. Comparison of the signal transduced by IL-4 to four different competence signals

The effects of the cytokine IL-4 on resting and activated human B cells were compared with the effects of known "competence" signals able to drive resting B cells into the cell cycle, including anti-Ig, PMA, anti-CD20, and a recently described competence signal, anti-Bgp95. In proliferation assays, IL-4 was costimulatory with anti-Ig and anti-Bgp95 but not with anti-CD20 or PMA. IL-4 alone triggered increases in expression of class II DR/DQ and CD40, but it did not trigger increases in intracellular free calcium [Ca2+]i in resting B cells or induce resting B cells to leave G0 and enter the G1 phase of the cell cycle. Although IL-4 has some characteristics of competence signals, it was most effective if added to B cells up to 12 h after anti-Ig or anti-Bgp95 rather than before, and thus, in this respect, works more like a progression signal. Like IL-4, all four competence signals for B cells triggered increases in class II and CD40, but only IL-4 consistently induced increases in CD23 surface levels. IL-4 was costimulatory only with anti-Ig and anti-Bgp95, each of which can trigger increases in [Ca2+]i and new protein synthesis of the proto-oncogene c-myc, and can increase attachment of protein kinase C to the plasma membrane. IL-4 was not costimulatory with signals that 1) did not affect [Ca2+]i yet induced c-myc protein synthesis (anti-CD20), 2) only stimulated the translocation of protein kinase C (PMA), or 3) only stimulated increases in [Ca2+]i (calcium ionophore). These results suggest that resting human B cells require at least two intracytoplasmic signals before IL-4 can effectively promote B cell proliferation.     

IL-4 production by T cells from naive donors. IL-2 is required for IL-4 production

Utilizing a sensitive and selective assay for IL-4, it was shown that lymph node T cells from naive mice could produce small amounts of this lymphokine in response to anti-CD3 antibodies adsorbed to culture dishes. The capacity of these cells to produce IL-4 in response to plate-bound anti-CD3 was substantially enhanced by the addition of IL-2 to the culture and was strikingly inhibited by monoclonal anti-IL-2 antibody. Thus, IL-2 appears to be essential for IL-4 production by anti-CD3 antibody-stimulated T cells from naive mice. The effect of IL-2 was not mediated either by preferential proliferation or survival of precursors of IL-4 producing cells, indicating that IL-2 regulates T cell production of IL-4. IL-4 producing capacity of T cells from naive mice was found mainly among CD4+ T cells. Large T cells produced much more IL-4, on a per cell basis, than did small T cells. In contrast, small T cells appeared to be equal or superior to large T cells in producing IL-2. The superiority of large T cells in IL-4-producing capacity was not accounted for by a lack of an accessory cell population from the small T cells as addition of large spleen cells depleted of both B and T cells did not enhance IL-4 production by small lymph node T cells. These results suggest that the bulk of IL-4 production by T cell populations, from normal mice, in response to anti-CD3 depends upon cells that are already activated and that IL-2 is required for such production.     

Regulation of antigen presentation. II. Anti-Ig and IL-2 induce IL-1 production by murine splenic B cells

Murine splenic B cells did not constitutively express IL-1 activity. After culture with anti-Ig and T cell-conditioned media and then fixation, B cells expressed membrane IL-1 and were able to stimulate growth of the IL-1-dependent T cell clone D10. Expression of membrane IL-1 required stimulation of B cells for 2 days before fixation. Significant IL-1 activity was detectable in freeze-thaw lysates of identical B cell preparations by 12 h. B cells also released IL-1 into the culture media. In situ hybridization studies by using probes to murine IL-1 alpha and IL-1 beta genes supported these observations. Thus, messenger RNA for IL-1 alpha and IL-1 beta rose in parallel, were detected between 6 and 24 h of culture, and declined to low levels by 30 h. Despite the presence of mRNA for IL-1 alpha and IL-1 beta, only IL-1 alpha had functional activity as determined by the use of a mAb to IL-1 alpha. IL-2 was found to be an essential component of the T cell-derived supernatant. Although IL-4 or TNF did not induce significant B cell IL-1 expression, they both caused a modest, but reproducible enhancement when added in combination with IL-2. IFN-gamma, by contrast, partially inhibited IL-1 induction.     

The role of human interleukin-6 in B-cell isotype regulation and differentiation

Human Interleukin-6 (IL-6) is a cytokine secreted by T cells, as well as a variety of other cell types, which exhibits B-cell differentiating activity. The recent cloning of the gene that codes for this molecule has allowed us the opportunity to study the function of this molecule alone and in conjunction with other lymphokines in human B-cell isotype-regulation and differentiation. Recombinant human IL-6 enhances immunoglobulin (Ig) M and G secretion by B-cells activated by Staphylococcal A Cowan strain (SAC) and enhances IgM, IgG, and IgA secretion by B-cells activated by pokeweed mitogen. IL-6 also augments immunoglobulin secretion of differing isotypes from various Epstein-Barr Virus transformed B-cell lines. However, IL-6 does not alter the secreted isotype of naive surface IgM-positive B-cells. As human T-cells secrete other lymphokines in association with IL-6 after activation we examined the interaction of Interleukin-2 (IL-2) gamma-interferon (IFN-gamma) and Interleukin-4 (IL-4) with IL-6 on B-cell immunoglobulin secretion. IL-2 and IL-4 synergized with IL-6 in augmenting immunoglobulin secretion by SAC-activated B-cells. IFN-gamma significantly inhibited the Ig secretion of SAC-activated B-cells cocultured with IL-6 alone or in combination with IL-2. These results demonstrate that human recombinant IL-6 augments immunoglobulin secretion of isotype-committed B-cells but it does not induce a change in the isotype secreted. In addition, this lymphokine synergizes with IL-2 and IL-4 in supporting Ig secretion. However, IFN-gamma significantly inhibits IL-6 induced Ig secretion.     

Stimulation of a T helper cell class 2 clone with immobilized anti-T cell receptor antibody activates a Ca2+ and protein kinase C-independent lethal signaling pathway.

Stimulation of an IL-2-dependent variant of the Th2 clone D10.G4.1 with antibodies (Ab) specific for CD3 epsilon or the TCR-alpha beta caused either activation of the clone to secrete the autocrine lymphokine IL-4, or lethal activation in which the cells secreted high quantities of IL-4 but then died within 2 days. High densities of immobilized Ab delivered a lethal signal, whereas soluble forms of Ab and low densities of immobilized Ab caused productive activation in which cell viability was maintained. Lethal activation was not prevented by accessory cells, IL-1, or IL-2, or by co-cross-linkage of CD4 and TCR. The lethal signal was not mediated via a soluble effector from the activated cells. Lethal signaling was insensitive to cyclosporin A or dexamethasone. Studies with activators of protein kinase C (PKC), and PKC inhibitors, indicated that direct activation of PKC was not sufficient for lethal signaling. Nor could direct activation of PKC prevent the lethal signal. The lethal signal was not caused by Ca2+ mobilization mediated by Ca2+ ionophore and there was no evidence of apoptosis. The combination of a PKC activator and Ca2+ ionophore was not lethal, thereby showing that together these events are not sufficient. That these signal pathways were not necessary for lethal activation was evidenced by their inability to lower the density of immobilized anti-CD3 required to cause cell death. In this model, ligation of the TCR specifically activates a Ca2+/PKC-independent lethal signal transduction pathway.     

Regulation of antigen-presentation-I. IFN-gamma induces antigen-presenting properties on B cells

B cells require activation to efficiently present Ag to T cells. In agreement with this earlier observation we show that live, mitomycin C-treated B cells, but not B cells fixed in paraformaldehyde, stimulated the growth of allogeneic T cells in the primary MLR. However, if B cells were cultured with anti-Ig antibodies and IFN-gamma before fixation they acquired excellent T cell stimulatory activity. Neither reagent alone conferred this novel co-stimulatory function on the B cell surface. The activity induced by both stimuli was not attributed to an increase expression of class II-MHC molecules or IL-1. IL-2 or IL-4, in combination with anti-Ig, also induced B cell stimulatory activity, but were less effective than IFN-gamma. TNF failed to stimulate B cells, but synergized with IFN-gamma in the induction of this activity. These studies therefore demonstrate an important role for lymphokines in modulating B cell Ag-presenting activity as well as the acquisition by B cells of a novel co-stimulatory surface activity.     

Altered IFN-gamma and IL-4 pattern lymphokine secretion in mice partially depleted of CD4 T cells by anti-CD4 monoclonal antibody.

Complete elimination of CD4 cells by in vivo treatment with anti-CD4 mAb may result in B cell polyclonal activation. Additionally, mice treated with doses of anti-CD4 that eliminate half the CD4 cells produced higher anti-SRBC antibody responses than controls. This suggests that partial CD4 depletion enhances Th2-like function. To test this hypothesis we examined Th1 and Th2 lymphokine potential in mice partially depleted of CD4 cells. We measured IL-4 and IFN-gamma secretion by stimulated unfractionated spleen cells and analyzed activated, purified CD4 cells by RNA in situ hybridization to determine the percentage of IFN-gamma- or IL-4-producing cells. Unfractionated splenocytes from partially CD4-depleted mice secreted more IL-4 and less IFN-gamma than splenocytes from control mice. In situ hybridization proved that CD4 cells from partially depleted mice contained a higher percentage of IL-4 and a lower percentage of IFN-gamma-producing cells than controls. These results indicate that treatment with a dose of mAb resulting in partial CD4 depletion may permit increased Th2-like lymphokine expression. This study also provides evidence that cells committed to Th2-like function exist in vivo in mice.     

Frequency analysis of lymphokine-secreting CD4+ and CD8+ T cells activated in a graft-versus-host reaction

Lymphokine secretion by in vivo-activated T cells was analyzed at the population and single-cell levels in lymphocytes from mice undergoing an acute allogeneic graft-vs-host reaction (GVHR). Three observations were made. First, constitutive lymphokine production by these cells was very low but could be dramatically up-regulated by TCR ligation. Thus, even when harvested at the peak of the GVHR, fewer than 0.1% of lymphocytes secreted detectable granulocyte-macrophage (GM)-CSF, IFN-gamma, or IL-3 in the first 24 h in vitro, and average production of these lymphokines in bulk cultures was less than 10(-5) U/cell. However, when cultured for 24 h with anti-CD3 antibody under conditions which activated less than 0.1% of normal cells, about 30% of GVHR T cells secreted GM-CSF, IFN-gamma, and/or IL-3, and average production levels were increased by 10(3)- to 10(4)-fold. Together with evidence that host alloantigen-induced lymphokine secretion was 10 to 100 times lower than the anti-CD3 response, these data suggest that physiologic lymphokine synthesis by most T cells is low (less than 10(-18) mol of IL-3 per cell) but can be raised above the threshold of detection by TCR cross-linking. Second, individual GVHR lymphocytes varied markedly in their total and relative production of different lymphokines in response to anti-CD3 stimulation, with some cells secreting IL-3 alone, some secreting IL-3 accompanied by other lymphokines (GM-CSF and/or IFN-gamma), and some secreting other lymphokines without detectable IL-3. Finally, both CD4+ and CD8+ T cells from GVHR mice responded to anti-CD3 antibody by secreting IL-3 and other lymphokines: purified CD4+ cells contained an average of 16% and CD8+ cells an average of 10% anti-CD3-inducible lymphokine-secreting cells. By contrast, only 2 to 3% of cells of either subset formed clones in cultures with host allogeneic cells and IL-2, suggesting that clonogenic alloreactive cells were a minority of the T cells activated in the GVHR.     

Antigen presentation by hapten-specific B lymphocytes. V. Requirements for activation of antigen-presenting B cells

Splenic B cells specific for the haptens, 2,4,6-trinitrophenyl (TNP) or fluorescein isothiocyanate (FITC) were cultured with a range of concentrations of unmodified or TNP- or FITC-conjugated conalbumin and the conalbumin + I-Ak-specific, interleukin (IL) 1-dependent helper T cell clone, D10 . G4, in the presence and absence of IL-1. Lymphokine secretion, T cell proliferation, and antibody secretion by B cells all exhibited identical antigen dose responses. Thus, hapten-binding B cells presented low concentrations of haptenated conalbumin for activation of both the T and the antigen-presenting B cells. Whereas proliferation of D10 . G4 required the addition of IL-1, both lymphokine production and stimulation of B cells to antibody secretion occurred without exogenous IL-1. These results demonstrate that when B lymphocytes function as presenting cells for antigens that bind to their immunoglobulin receptors, activation of the responding T cells and the B cells themselves occur at similar concentrations of antigen. Moreover, for functional T-B interactions, antigen-presenting B and responding T lymphocytes constitute a complete system that requires no other accessory stimuli, whereas clonal expansion of T cells is dependent on accessory factors such as IL-1. Finally, since D10 . G4 secretes IL-4 but neither IL-2 nor interferon-gamma, our results demonstrate that differentiation of B cells as a consequence of direct ("cognate") interactions with helper T cells as well as of bystander B cells can occur in the absence of IL-1, IL-2, and interferon-gamma.