CD4+ subset regulation in viral infection. Preferential activation of Th2 cells during progression of retrovirus-induced immunodeficiency in mice.

Progressive lymphoproliferation and increasingly severe immunodeficiency are prominent features of a syndrome, designated mouse AIDS, which develops in susceptible strains of mice infected with the mixture of murine leukemia viruses, termed LP-BM5. Development of splenomegaly and lymphadenopathy, caused primarily by increases in B cell immunoblasts, requires the presence of CD4+ T cells and is assumed to be mediated by lymphokines produced by these cells inasmuch as progression of disease is markedly inhibited by treatment of infected mice with cyclosporin A. Studies of spleen cells from infected mice revealed spontaneous production of cytokines (IFN-gamma, IL-2, IL-4, IL-5, and IL-10) characteristic of Th0 (or a mixture of Th1 and Th2) T helper cells at 1 wk after infection. At later times, IFN-gamma and IL-2, characteristic products of Th1 helper clones, were expressed poorly, either spontaneously or after stimulation of cells with Con A. In contrast, IL-4, IL-5, IL-6, and IL-10, cytokines typically synthesized by Th2 cells, were produced in response to Con A or spontaneously through 18 wk post-infection. Increased serum IgE levels and enhanced IL-10 mRNA expression were consistent with expression of Th2 cytokines at biologically significant levels in vivo. Selective depletion of T cell subsets before stimulation with Con A showed that CD4+ T cells were the primary source of IL-2, IL-4, IL-10, and, to a lesser extent, IFN-gamma in spleens and lymph nodes of normal or infected mice. These results suggest that persistent activation of CD4+ T cells with the lymphokine profile of Th2 helper clones is responsible for chronic B cell stimulation, down-regulation of Th1 cytokines, and impaired CD8+ T cell function in mouse AIDS. This provides the first demonstration that, like many parasitic infections, viruses encoding potent antigenic stimuli can markedly affect the balance of Th subset expression.     

Enhancement of IL-2-induced T cell proliferation by a novel factor(s) present in murine spleen dendritic cell-T cell culture supernatants

The mechanism(s) underlying the potent accessory cell function of dendritic cells (DC) remains unclear. The possibility was considered that a soluble factor(s) released during the interaction of DC and T cells might contribute to the potent T cell activating function of DC. Culture supernatants were generated from mixtures of murine spleen DC and periodate-treated spleen T cells and were examined for the presence of known cytokine activities and factors capable of enhancing T cell responsiveness to IL-2. Serum-free supernatants from 24 h DC-T cell co-cultures exhibited high levels of IL-2, detectable levels of IL-3, and negligible levels of IL-1, -4, -5, -6, and TNF. A factor(s) was also identified with an apparent Mr of 12.5 to 17.0 kDa, henceforth designated IL-2 enhancing factor (IL-2EF), which enhanced the IL-2-induced proliferation of murine thymocytes, CTLL, and HT-2 cells by approximately three- to fourfold. This enhancement was also observed in the presence of neutralizing antibodies to murine IL-1 alpha, -1 beta, -3, -4, -5, -6, granulocyte-macrophage (GM)-CSF, TNF, and IFN-gamma. However, IL-2EF failed to enhance: 1) the activity of IL-1, -3, -4, -5, or -6 on cells responsive to these cytokines; 2) IL-2-augmented, IL-5-induced BCL1 proliferation; and 3) either PHA- or Con A-stimulated thymocyte proliferation. Moreover, neither IFN-gamma nor GM-CSF exhibited IL-2EF activity. When DC and T cells were cultured separately (after an initial 12 h co-culture period), IL-2EF activity resided predominantly in the T cell-derived supernatants. These and other data indicate that IL-2EF, a heat-labile T cell-derived 12.5 to 17.0 kDa protein, is distinct from IL-1 alpha, -1 beta, -2, -3, -4, -5, -6, TNF, IFN-gamma, GM-CSF, and previously described factors that co-stimulate thymocyte proliferation in the presence of Con A or PHA. It is suggested that IL-2EF functions to specifically enhance IL-2-driven T cell proliferation and contributes to the potent activation of T cells induced by DC.     

Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

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.     

Generation of B cell memory and affinity maturation. Induction with Th1 and Th2 T cell clones.

We used an adoptive transfer system and CD4+ T cell clones with defined lymphokine profiles to examine the role of CD4+ T cells and the types of lymphokines involved in the development of B cell memory and affinity maturation. Keyhole limpet hemocyanin (KLH)-specific CD4+ Th2 clones (which produce IL-4 and IL-5 but not IL-2 or IFN-gamma) were capable of inducing B cell memory and affinity maturation, after transfer into nude mice or after transfer with unprimed B cells into irradiated recipients and immunization with TNP-KLH. In addition, KLH-specific Th1 clones, which produce IL-2 and IFN-gamma but not IL-4 or IL-5, were also effective in inducing B cell memory and high affinity anti-TNP-specific antibody. The induction of affinity maturation by Th1 clones occurred in the absence of IL-4, as anti-IL-4 mAb had no effect on the affinity of the response whereas anti-IFN-gamma mAb completely blocked the response. Th1 clones induced predominantly IgG2a and IgG3 antibody, although Th2 clones induced predominantly IgG1 and IgE antibody. We thus demonstrated that some Th1 as well as some Th2 clones can function in vivo to induce Ig synthesis. These results also suggest that a single type of T cell with a restricted lymphokine profile can induce both the terminal differentiation of B cells into antibody secreting cells as well as induce B cell memory and affinity maturation. Moreover, these results suggest that B cell memory and affinity maturation can occur either in the presence of Th2 clones secreting IL-4 but not IFN-gamma, or alternatively in the presence of Th1 clones secreting IFN-gamma but not IL-4.     

Human atopen-specific types 1 and 2 T helper cell clones.

Eight representative T lymphocyte clones (TLC) randomly selected from previously described panels of CD4+ housedust mite Dermatophagoides pteronyssinus (Dp)-specific TLC from atopic and nonatopic donors were studied in more detail in a comparative investigation. The TLC from the atopic donors closely resembled murine type 2 Th (Th2) cells by secreting substantial IL-4, IL-5, IL-6, TNF-alpha, and granulocyte-macrophage (GM)-CSF, minimal IFN-gamma, and relatively little IL-2. In contrast, the nonatopic's TLC resembled murine type 1 Th (TH1) cells by secreting substantial IFN-gamma, IL-2, TNF-alpha, and GM-CSF, no IL-4, and little IL-5. A difference with murine Th1 cells was their additional secretion of IL-6. These cytokine profiles were consistent upon stimulation via different activation pathways including stimulation with specific Dp Ag, mitogenic lectins, and antibodies to CD2, CD3, or CD28. The observed differences in IL-2 secretion, however, were most evident upon stimulation with anti-CD28. If TLC cells were cultured with highly purified B cells and stimulated with anti-CD3 in the absence of exogenous IL-4, IgE synthesis was induced only in cultures with the atopics' Th2 clones, which could be completely abrogated by anti-IL-4. The mere presence of exogenous rIL-4, however, did not result in IgE synthesis, nor did unstimulated TLC cells alone. But if unstimulated TLC cells (that proved not to secrete detectable amounts of cytokines) were added together with rIL-4, again IgE synthesis was induced only in cultures with the atopics' Th2 clones, suggesting the involvement of an additional, as yet unidentified accessory helper function of the atopics' Th2 clones for IgE induction. Unstimulated Th2 clones showed a significantly higher expression of CD28 than the Th1 clones, but three days after stimulation, CD28 expression was elevated to comparable levels on both subsets. When added to B cells at this time point, together with rIL-4 and anti-IFN-gamma, still only the atopics' Th2 clones supported IgE synthesis, arguing against a role for CD28 in this accessory helper function. Whereas the atopics' Th2 clones were excellent helper cells for IgE induction, a unique property of the nonatopic's Th1 clones was their cytolytic activity toward autologous APC which could be induced by specific Dp Ag and by anti-CD3. The present data provide clear evidence for the existence of Th1 and Th2 cells in man.     

Cytokine secretion by C3H-lpr and -gld T cells. Hypersecretion of IFN-gamma and tumor necrosis factor-alpha by stimulated CD4+ T cells.

Mice homozygous for lpr and gld develop profound lymphadenopathy characterized by the expansion of two unusual T cell subsets, a predominant Ly-5(B220)+ CD4- CD8- double negative (DN) population and a minor CD4 dull+ Ly-5(B220)+ population. The mechanisms promoting lymphoproliferation are unknown, but one possibility is a abnormality in the production of cytokines that regulate T cell growth. In the present report, unfractionated LN cells and sorted T cell subsets from C3H-lpr, -gld, and -+/+ mice were compared for spontaneous and induced secretion of a spectrum of lymphokines. In addition, CD4+, CD4 dull+ Ly-5(B220)+, and DN T cells were examined for expression of CD3 epsilon, TCR-alpha/beta heterodimers, Ly-6C, and CD44 and for proliferative responses to immobilized anti-TCR mAb and cofactors. These studies revealed that sorted DN T cells did not secrete IL-3, IL-4, IL-5, IL-6, GM-CSF, TNF-alpha, or IFN-gamma spontaneously or after TCR-alpha/beta cross-linking. In contrast, stimulated unfractionated lpr and gld LN cells proliferated strongly and secreted high levels of IFN-gamma and TNF-alpha and low levels of IL-3, IL-4, and IL-6. Despite a 5- to 10-fold deficit in the frequency of CD4+ and CD8+ T cells, cytokine secretion by lpr and gld LN generally exceeded that of +/+ LN. Comparisons of cytokine secretion by stimulated CD4+ T cells revealed that +/+, lpr, and gld CD4+ Ly-5(B220)- T cells proliferated strongly, but only lpr and gld cells produced significant levels of IFN-gamma. The lpr and gld CD4+ T cells also produced higher levels of TNF-alpha and IL-2 than +/+ cells. In contrast to normal CD4+ T cells, lpr and gld CD4+ Ly-5(B220)+ T cells proliferated weakly and did not secrete TNF-alpha, IL-2, or, in most experiments, IFN-gamma after stimulation. Phenotypic studies of T cell subsets revealed that unstimulated lpr and gld CD4+ Ly-5(B220)- T cells express significantly higher levels of CD44 than +/+ CD4+ T cells. In addition, CD4 dull+ Ly-5(B220)+ cells closely resembled DN T cells in size and expression of TCR-alpha/beta, CD3epsilon, CD44, and Ly-6C. Since elevated CD44 expression is generally associated with T cell activation and only previously activated normal CD4+ T cells produce high levels of IFN-gamma in vitro, our data suggest that lpr and gld CD4+ Ly-5(B220)- T cells contain a higher than normal proportion of primed or memory T cells and thus may be polyclonally activated in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of random classes of immunoglobulins in brain tissue during persistent viral infection paralleled by secretion of interleukin-6 (IL-6) but not IL-4, IL-5, and gamma interferon

The activities of cytokines were determined in cerebrospinal fluid (CSF) and serum of mice persistently or intracerebrally acutely infected with lymphocytic choriomeningitis (LCM) virus (LCMV). In contrast to CBA/J (LCMV carrier) mice that responded with low levels of LCMV-specific antibody, high-responder NMRI (carrier) mice showed antibody production by B cells outside of lymphoid organs. The B cells that had infiltrated the brains of LCMV carrier mice exhibited no preferential immunoglobulin isotype or subtype virus-specific antibody production. Phenotypic analysis of the brain infiltrates in virus carrier mice revealed dominance of CD4+ T cells in contrast to virtual absence of CD4+ and dominance of CD8+ in mice with acute LCM. In NMRI but not in CBA/J carrier mice, significant concentrations of interleukin-6 (IL-6) were detected in CSF and serum; IL-2, IL-4, IL-5, granulocyte-macrophage CSF (GM-CSF), and gamma interferon (IFN-gamma) were not elevated. In contrast, during acute, lethal LCM, IL-6 and IFN-gamma were found at high concentrations, and IL-4, IL-5, and GM-CSF were detectable in CSF and serum, but virus-specific antibody-producing cells were not (yet) detectable in the brain. Thus, distinct cytokine patterns are found in acute versus chronic LCMV infection of the brain: in LCM carrier mice, local random-class immunoglobulin production correlated with the absence of IL-2, IL-4, IL-5, and IFN-gamma but active secretion of IL-6.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.     

CD4+ Leu-8+ T cell supernatant activity that inhibits Ig production.

The subpopulation of CD4+ T cells that expresses the Leu-8 peripheral lymph node homing receptor suppresses PWM-stimulated Ig synthesis. To determine the mechanism of this suppression, the immunoregulatory activity of culture supernatants obtained from peripheral blood CD4+ Leu-8+ T cells cultured with anti-CD3 mAb and PMA (Leu-8+ supernatant) was determined. Leu-8+ supernatant suppressed PWM-stimulated Ig synthesis in cultures containing non-T cells and CD4+ Leu-8- T cells. In contrast, the supernatant from CD4+ Leu-8- T cells did not suppress Ig synthesis. The inhibitory activity of CD4+ Leu-8+ T cell supernatants could not be accounted for by a deficiency or excess of IL-2, IL-4, IFN-gamma, IL-6, or PGE2. In studies examining the effect of CD4+ Leu-8+ supernatant on T cells, the supernatant did not alter either mitogen-induced proliferation or the helper function of CD4+ Leu-8- T cells. In studies examining the effect of CD4+ Leu-8+ supernatant on B cells, the supernatant inhibited Staphylococcus aureus Cowan I strain-induced B cell Ig secretion but not B cell proliferation. The suppressor activity of Leu-8+ supernatant was eliminated by protease treatment and was eluted by HPLC in two main peaks, with molecular sizes of 44 and 12 kDa. In summary, these studies indicate that supernatants from activated CD4+ Leu-8+ T cells directly suppress B cell Ig production.     

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.     

Induction of CD69 activation molecule on human neutrophils by GM-CSF,IFN-gamma,and IFN-alpha

The CD69 glycoprotein is an early activation antigen of T and B lymphocytes but it expression is induced in vitro on cells of most hematopoietic lineages, including neutrophils after stimulation with PMA or fMLP. In this study, we investigated whether CD69 expression on human neutrophils could be modulated by inflammatory or anti-inflammatory cytokines (IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, G-CSF, GM-CSF, TNF-alpha, TGF-beta, IFN-alpha, IFN-gamma). Resting neutrophils from healthy subjects did not express CD69 on the cell surface; moreover, a preformed intracellular pool of CD69 was not evident in these cells. CD69 was barely detectable on these cells after overnight incubation in medium while overnight incubation with GM-CSF, IFN-gamma or IFN-alpha significantly induced CD69 expression on neutrophils with GM-CSF appearing to be the most potent inducer. This induction was dependent on a new protein synthesis as it was significantly inhibited by cycloheximide (about 50% inhibition). CD69 cross-linking on GM-CSF-primed neutrophils sinergized with LPS and increased TNF-alpha production and secretion suggesting a role for CD69-positive neutrophils in the pathogenesis and maintenance of different inflammatory diseases.     

Production of lymphokine mRNA by CD45R+ and CD45R- helper T cells from human peripheral blood and by human CD4+ T cell clones

The expression of lymphokine mRNA by human CD4+CD45R+ and CD4+CD45R- Th cells was assessed after mitogen stimulation. These Ag have previously been shown to relate closely to virgin and primed T cells, respectively. CD4+CD45R+ (virgin) and CD4+CD45R- (primed) cell fractions were isolated by sorting double-labeled cells with a fluorescence-activated cell sorter. CD4+CD45R+ cells produced high levels of IL-2 mRNA when stimulated with either PMA together with calcium ionophore, or with PHA, but they expressed only trace quantities of mRNA for IL-4 or IFN-gamma. In contrast, CD4+CD45R- cells produced high levels of mRNA for IL-2, IL-4, and IFN-gamma. After 14 days of continuous culture, CD4+CD45R+ Th cells lost expression of the CD45R Ag, but gained high level expression of CDw29, such that they were indistinguishable from the cell population which originally expressed this Ag. At the same time, they acquired the ability to synthesize IL-4 mRNA. It seemed likely that the broad lymphokine profile of primed Th cells might mask clonal heterogeneity. Analysis of 122 CD4+ T cell clones showed that all of them synthesized IL-2 mRNA. One clone failed to express IL-4 mRNA, but did produce those for IL-2 and IFN-gamma. A total of 34 of the clones was investigated to determine expression of IFN-gamma mRNA; two of these clones were negative for IFN-gamma mRNA, and both expressed IL-2 and IL-4 message. These data suggest that while fresh virgin and primed peripheral blood T cells show a clear resolution of lymphokine production, a simple subdivision of human CD4+ T cell clones on the basis of their lymphokine production (such as that reported for mouse Th cell clones) is not possible.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.     

Gamma-interferon induces apoptosis of the B lymphoma WEHI-279 cell line through a CD95/CD95L-independent mechanism.

Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and natural killer (NK) cells, plays a central role in the development of humoral and cell-mediated immunity. IFN-gamma participates in the maturation and differentiation of B cells, but it has been previously reported that IFN-gamma may inhibit the early stages of B cell activation. We report that the inhibition of the B lymphoma cell WEHI-279-proliferation induced by IFN-gamma, involves the induction of typical features of apoptosis (nuclear chromatin condensation and fragmentation, cell shrinkage, phosphatidyl-serine (PS) exposure and mitochondrial membrane potential (delta psim) loss). IFN-gamma-mediated B cell apoptosis was decreased by the addition of the T helper type 2 cytokine, IL-4. WEHI-279 cells express CD95 and undergo apoptosis after treatment with either an agonistic anti-CD95 Ab or with a soluble recombinant CD95L. However, incubation with CD95-Fc or TRAIL-R1-Fc fusion proteins, did not prevent IFN-gamma-mediated apoptosis, suggesting that IFN-gamma-mediated apoptosis occurs independently of CD95/CD95L and TRAIL-R/TRAIL interactions. IFN-gamma-mediated apoptosis is associated with caspase-3 activation that can be prevented by the addition of the broad caspase inhibitor zVAD-fmk. These data indicate that IFN-gamma may play a major role in the regulation of B cell apoptosis, and suggest the involvement of an alternative pathway which is independent of the death receptors.     

Minor lymphocyte stimulatory antigen-bearing stimulator cells require lipopolysaccharide activation to induce programmed cell death in T cell hybridomas.

T cell hybridomas respond to conventional peptide Ag associated with self major histocompatibility restriction elements, as well as to alloantigens, activating lectins, and stimulatory forms of mAb by producing lymphokines and undergoing programmed cell death (PCD). We show here that the level of PCD and IL-2 production correlate well in responses to CD3 or allostimulation. The response to minor lymphocyte-stimulatory (Mls) Ag, members of the family of endogenous superantigens, however, are marked by divergence in the levels of the PCD and lymphokine responses. Specifically, PCD in response to Mls activation is achieved poorly despite vigorous IL-2 production. B lymphoma cell stimulators induced PCD in alloreactive T cell hybridomas but not in Mls-reactive T cell hybridomas. This suggests that the absence of PCD in the Mls response is a function of superantigen recognition rather than the stimulator cell type. LPS-preactivated Mls+ stimulators, either splenic B or B lymphoma cells, are shown to trigger PCD in the T cell hybridomas. These results imply that T cell interaction with Mls presented by untreated stimulator cells is not sufficient for induction of PCD and thus is distinct from interactions with conventional Ag.     

The cell biology of T-dependent B cell activation

The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell activation through coculture with T cells activated by anti-T-cell receptor or anti-CD3 antibodies suggest that cellular interaction with T cells, independent of antigen presentation or lymphokine secretion, induces or triggers B cells to become responsive to T-derived lymphokines, and that this may be an integral component of the physiological, antigen- and MHC-restricted T-dependent B cell activation that leads to antibody production.