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.     

CD8+ T cells can be primed in vitro to produce IL-4.

IL-4 production by T lymphocytes from naive mice in response to stimulation by plate-bound anti-CD3 is concentrated among CD4+ T cells. In vitro stimulation of lymph node T cells with anti-CD3 plus IL-2 and IL-4 strikingly increases the frequency of cells that produce IL-4 in response to subsequent stimulation with anti-CD3 plus IL-2. Separation of these primed cell populations into CD4+ and CD8+ T cell by cell sorting reveals that the frequency of IL-4-producing cells in both population is similar. Verification that CD8+ T cells produce IL-4 is provided by the capacity of anti-IL-4 mAb to inhibit the response of the indicator cell line to the growth factor produced by the primed cells and by detection of IL-4 by an IL-4-specific ELISA. The in vitro "priming" of CD8+ T cells to produce IL-4 is not dependent on the presence of CD4+ T cells because highly purified CD8+ T cells can be stimulated to develop into cells capable of producing IL-4 by culture with plate-bound anti-CD3 plus IL-2 and IL-4.     

The anti-T cell monoclonal antibody 9.3 (anti-CD28) provides a helper signal and bypasses the need for accessory cells in T cell activation with immobilized anti-CD3 and mitogens

CD28 is an antigen of 44 kDa which is expressed on the membrane of the majority of human T cells. The present study examines the functional effects of an anti-CD28 monoclonal antibody (mAb 9.3) on T cell activation induced with immobilized anti-CD3 mAb OKT3 or with mitogens, in the absence of accessory cells. To this end, we used blood resting T cells that were completely depleted of accessory cells (monocytes, B cells, and natural killer cells), and consequently did not respond to recombinant interleukin-2 (rIL-2), to immobilized OKT3, to PHA, or to Con A. Addition of mAb 9.3 to the cultures enhanced IL-2 receptor expression (Tac antigen) on PHA- or immobilized OKT3-stimulated T cells and induced IL-2 receptors on Con A-stimulated T cells. Moreover, addition of mAb 9.3 to cultures of T cells stimulated with PHA, Con A, or immobilized OKT3 resulted in IL-2 production. Soluble mAb 9.3 was a sufficient helper signal for T cell proliferation in response to PHA or immobilized OKT3. Crosslinking of mAb 9.3 by culture on anti-mouse IgG-coated plates enhanced the helper effect and was an essential requirement for the induction of T cell proliferation in response to Con A. No other anti-T cell mAb (anti-CD2, -CD4, -CD5, -CD7, -CD8) was found to provide a complete accessory signal for PHA or Con A stimulation of purified T cells. T cell proliferation induced by the combination of PHA and mAb 9.3 was strongly inhibited by the anti-IL-2 receptor mAb anti-Tac. In conclusion, mAb 9.3 can provide a signal bypassing monocyte requirement in T cell activation with immobilized OKT3, PHA, and Con A, resulting in an autocrine IL-2-dependent pathway of proliferation.     

IL-1 serves as a secondary signal for IL-9 expression.

IL-9 is produced in vitro by activated CD4+ T cell lines of the Th2 subtype and by naive CD4+ T cells. Here we show that T cell lines stimulated with Con A in the presence of accessory cells (AC) such as irradiated spleen cells or bone marrow-derived macrophages produced substantially more IL-9 than T cells stimulated with Con A alone. These data suggest that AC influence the production of IL-9 through accessory signals that result in an at least 10-fold increase of IL-9 secretion by the respective T cells. Addition of IL-1 to T cells activated by Con A, PHA, or anti-CD3 antibodies revealed that this monokine was responsible for the potentiation of IL-9 production. This finding was confirmed by applying anti-IL-1 antibodies. The production of other lymphokines, namely, IL-3, IL-4, and IL-6, by activated T cells was not or only marginally enhanced in the presence of AC or IL-1, thus indicating that the synthesis of IL-9 is regulated differently from that of other Th2-derived lymphokines. Furthermore, it was demonstrated by Northern blot analysis that IL-1 increases IL-9 expression at the pretranslational level. Because IL-1 alone failed to induce the production of IL-9 by T cells, this monokine acts as a costimulator in combination with a T cell receptor-mediated signal.     

Cross-linking Fc receptors on monocytes triggers IL-6 production. Role in anti-CD3-induced T cell activation

IL-6 is a multifunctional cytokine which is produced by a variety of cells. Therefore it was examined whether anti-CD3-induced T cell activation was associated with the induction of functionally relevant IL-6 in human monocyte accessory cells. Significantly increased amounts of IL-6 were detected in supernatants of anti-CD3-treated PBMC. Stimulation of FACS-sorted greater than 98% pure monocyte accessory cells, but not of highly purified T cells with anti-CD3, resulted in an increased IL-6 production. Furthermore, anti-CD3 significantly enhanced IL-6 mRNA expression in monocyte accessory cells. IL-6 production was not limited to anti-CD3, inasmuch as equivalent IL-6 stimulation could be achieved with a mouse IgG2a isotype control antibody. In contrast to solid phase-bound mouse IgG2a, the soluble form of this antibody failed to induce IL-6 secretion indicating a requirement for Fc gamma RI receptor cross-linking. Moreover, this property may be specific for the Fc gamma RI receptor inasmuch as mouse IgG1 antibodies binding to the Fc gamma RII receptor did not significantly enhance IL-6 production. The role of IL-6 being an additional signal in T cell activation was confirmed by the finding that an anti-IL-6 antiserum was able to suppress anti-CD3-induced T cell activation. These data indicate that binding of anti-CD3 to Fc gamma RI may generate an activation signal towards the monocyte accessory cell leading to the production and secretion of monocyte IL-6, which in turn augments T cell activation, and also may be relevant to a variety of antibody-mediated immune responses against viral and bacterial infections.     

IL-4/B cell stimulatory factor 1 stimulates T cell growth by an IL-2-independent mechanism

Resting T cells are stimulated to synthesize DNA by IL-4 and phorbol myristate acetate (PMA). This response of T cells to IL-4 plus PMA is independent of the action of IL-2 as judged by 1) the lack of IL-2 in supernatants of stimulated cells, 2) the failure to detect IL-2 mRNA in stimulated cells by in situ hybridization, 3) the inability of anti-IL-2R antibody and of anti-IL-2 antibody to block responses to IL-4 plus PMA, and 4) the failure of cyclosporin A to block responses. T cells also respond to anti-CD3 antibodies and IL-4 in the presence of anti-IL-2R antibodies. IL-4 stimulation of growth of the long term T cell line HT-2 also appears to be independent of the action of IL-2. No IL-2 mRNA is found in IL-4-stimulated HT-2 cells by Northern blotting; the response of HT-2 cells to IL-4 is not blocked by anti-IL-2R antibodies; the response of HT-2 cells to IL-4 is not inhibited by cyclosporin A. Although IL-4 stimulation of T cells is independent of IL-2, IL-4 plus PMA treatment of resting T cells does cause enhanced expression of IL-2R and prepares cells to proliferate to IL-2 alone. In both these properties IL-4 resembles IL-2. These experiments lead us to conclude that IL-4 can act as an alternative to IL-2 as authentic T cell growth factor.     

Costimulatory requirements of murine Th1 clones. The role of accessory cell-derived signals in responses to anti-CD3 antibody

We examined the role of accessory cell-derived signals in promoting growth and lymphokine production by murine Th1 clones. Five of six Th1 clones failed to proliferate to immobilized anti-CD3 antibody despite producing IL-2 and IFN-gamma. These clones became unresponsive to Ag after exposure to anti-CD3. With the addition of irradiated splenic accessory cells (SAC), Th1 clones proliferated to anti-CD3 and produced greater amounts of IL-2 and IFN-gamma. High doses of plate-bound anti-CD3 completely inhibited responses of these clones to IL-2 and diminished the growth-promoting activity of SAC. The costimulatory effects of SAC on growth of Th1 clones were also seen in the presence of exogenous IL-2, indicating that enhanced IL-2 production alone was not responsible for the costimulatory effect. Delivery of the costimulatory signal from SAC required their close proximity to the T cells. The costimulatory activity of SAC was not reproduced by the addition of IL-1, IL-6, or IL-1 plus IL-6. IL-7 induced weak proliferation of Th1 clones, but did not synergize with plate-bound anti-CD3. Our results suggest a model in which SAC-derived costimulatory signals regulate growth of Th1 cells primarily at the level of cell cycle progression rather than at the level of IL-2 production.     

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.     

Regulatory role of CD4/L3T4 molecules in IL-2 production by affecting intracellular Ca2+ concentration of T cell clone stimulated with soluble anti-CD3

To elucidate the role of CD4 molecule in T cell activation, the effect of anti-CD4 on T cell IL-2 production was examined by using an alloreactive Th clone. The alloreactive T cell used in the present experiments produced IL-2 in response to soluble anti-CD3 epsilon-chain (anti-CD3) without accessory cell or insoluble antibody carrier. The IL-2 production was suppressed by the addition of anti-CD4 in cultures. An intracellular free Ca2+ concentration ([Ca2+]i) of the T cell clone was elevated by anti-CD3 stimulation, but the elevation was suppressed in the presence of anti-CD4. When the clone was stimulated in Ca2(+)-free medium, the elevation of [Ca2+]i was not observed. When Ca2+ influx was induced by calcium ionophore A23187 or ionomycin, the clone produced IL-2 in response to anti-CD3 in the presence of anti-CD4. When polyclonal T cell line or several other alloreactive T cell clones were examined for their anti-CD3 response, essentially the same results as mentioned above were obtained. Taken together, these results suggest that the slow and sustained elevation of [Ca2+]i is an essential signal for IL-2 production of T cells, and that anti-CD4 suppresses the IL-2 production by interfering the [Ca2+]i elevation. The significance of CD4 molecules in murine T cell activation was discussed.     

T cell activation through TCR/-CD3 complex. IL-2 production of T cell clones stimulated with anti-CD3 without cross-linkage.

To elucidate the Th cell activation mechanism through the TCR/CD3 complex, we examined the reactivity of T cell clones to soluble monovalent and divalent anti-CD3 without accessory cells or costimulatory factor. All T cell clones tested produced IL-2 in response to monovalent anti-CD3, although reactivity to divalent anti-CD3 was variable depending upon clones. IL-2 production of T cell clones induced by monovalent anti-CD3 was suppressed by cross-linking of the antibody with anti-hamster IgG. IL-2 mRNA expression and the increment of intracellular Ca2+ concentration were consistent with the IL-2 production. When T cell clones were stimulated with monovalent anti-CD3, they increased in size, although divalent anti-CD3 stimulation did not affect their size irrespective of their IL-2 production. These results indicate that monovalent anti-CD3 is more efficient than divalent anti-CD3 in induction of IL-2 production and that the cross-linkage of the TCR/CD3 complex is not necessarily required for the T cell clone activation.     

IL-6 is an accessory signal in the alternative CD2-mediated pathway of T cell activation

Recent studies have demonstrated that IL-1 and IL-6 are synergistic accessory signals for activation of T cells. In this study, highly purified human T cells were cultured with either a stimulating pair of anti-CD2 mAb or with immobilized anti-CD3 mAb. Monocytes, a cellfree monocyte culture supernatant or IL-1 were required for anti-CD2-stimulated T cell proliferation, and they each strongly enhanced anti-CD3-induced T cell growth. IL-6 was synergistic with IL-1 as a helper factor for T cell growth after activation via CD2, but we could not demonstrate any effect of IL-6 in the CD3 pathway. The mechanism of the synergistic helper activity of IL-1 and IL-6 on T cell activation in the CD2 pathway was further examined. IL-1 (but not IL-6) was required for induction of IL-2 production. Both IL-1 and IL-6 enhanced IL-2R (p55) expression and the proliferative response to IL-2. T cell proliferation after stimulation with anti-CD2 and IL-1 or IL-1/IL-6 proceeded through an autocrine IL-2-dependent pathway. Moreover we found that, in the absence of IL-1, IL-6 still supported a transient and limited proliferation of anti-CD2- (but not of anti-CD3-) stimulated T cells, which apparently was independent of the autocrine growth factors IL-2 or IL-4. Our data suggest that IL-6 is important as an accessory signal for T cell growth in the CD2 pathway of T cell activation.     

Freshly isolated, murine neonatal T cells produce IL-4 in response to anti-CD3 stimulation.

In previous studies of chimeric animals, we found that fetal intrathymic T cell precursors give rise to phenotypically abnormal peripheral T cell populations. Because most peripheral T lymphocytes in newborn mice are the progeny of fetal T cell precursors, this result led to the hypothesis that neonatal and adult T cells differ in their functional capacities. To investigate this issue, the responses of neonatal and adult T cells to anti-CD3 antibody and TCR-independent stimulation were compared. When stimulated with soluble anti-CD3 antibody in the presence of adult accessory cells, neonatal T cell proliferation was markedly decreased compared with that of adult T cells. This reduction in proliferation was associated with both quantitative and qualitative differences in lymphokine production. At 48 h of stimulation with anti-CD3 antibody, neonatal T cells produced at least 10-fold less IL-2 than adult T cells. This apparently accounted for their reduced proliferation because the addition of exogenous IL-2 restored their proliferation to the levels achieved by adult T cells. In striking contrast to adult T cells, neonatal T cells secreted large amounts of IL-4 upon primary stimulation in vitro. The differences between neonatal and adult T cells in proliferation and lymphokine production were shown to be specific for CD3-mediated stimulation. In the presence of phorbol ester and calcium ionophore, neonatal and adult T cells showed equivalent proliferation and IL-2 production. Under these conditions, IL-4 production by neonatal or adult T cells was essentially undetectable. Thus, in response to TCR-independent stimulation, freshly isolated neonatal and adult T cells show similar functional responses. However, when stimulation occurs via the CD3 components of the TCR, the responses of neonatal T cells resemble those of primed T cells from adult animals.     

Induction of competence to respond to IL-4 by CD4+ T helper type 1 cells requires costimulation.

Rested murine CD4+ Th1 clones do not produce IL-4, but have previously been shown to be capable of responding to IL-4 if they are first activated with Ag and APC. In this study, we have examined the activation requirements for induction of competence to respond to IL-4 in these clones. TCR occupancy alone (given either as chemically fixed APC and Ag, anti-CD3, Con A, or ionomycin and PMA) was inadequate, but the addition of a source of costimulation to any of these stimuli resulted in complete induction of competence to respond to IL-4. Pretreatment of the Th1 clones with TCR occupancy alone induced an anergic state from which subsequent full stimulation with Ag and APC failed to give IL-4 responsiveness. Pretreatment of the cells with IL-2 alone was an inadequate signal to induce IL-4 responsiveness and only a partial response was obtained when TCR occupancy was combined with IL-2. Addition of anti-IL-2 and anti-IL-2R antibodies during full activation with APC and Ag gave a 50% inhibition of competence induction. These results demonstrate that costimulation, in addition to its role in IL-2 production, is an important second signal for inducing T cells to become competent to respond to IL-4.     

Enhancement of T helper2 response in the absence of interleukin (IL-)6; an inhibition of IL-4-mediated T helper2 cell differentiation by IL-6

Functional roles of interleukin (IL-)6 in T cell response were investigated. Mice deficient in IL-6 and wild mice were immunized with antigens (myelin oligodendrocyte glycoprotein or methylated BSA) and production of IL-4 and interferon (IFN)-gamma by regional lymph nodes was measured. IL-6 deficiency led to an enhancement of IL-4 and an inhibition of IFN-gamma production. Moreover, polyclonal stimulation of spleen T cells from unimmunized IL-6-deficient mice with anti-CD3 plus anti-CD28 antibodies (Abs) demonstrated an enhancement of T helper (Th)(2)responses. The presence of IL-6, however, augmented IL-4 production but it inhibited IFN-gamma expression by spleen T cells in response to polyclonal stimulation and by antigen-primed spleen T cells in response to re-challenge with the antigen. In contrast, the induction of spleen CD4-positive T cells into Th(2)cells in vitro by the anti-CD3 plus IL-4 was completely suppressed by exogenously added IL-6, whereas Th(1)differentiation of T cells by the anti-CD3 plus IL-12 was not inhibited by the presence of IL-6. Thus, these results indicate that IL-6 physiologically could modulate qualitative T cell response and suggest that it augments Th(1)responses partly through its inhibitory capability of IL-4-induced Th(2)differentiation of naive T cells.     

Isolated peripheral T cells from GvHR recipients exhibit defective IL-2R expression, IL-2 production, and proliferation in response to activation stimuli

Graft-vs-host reactions (GvHR) following the injection of class I/II MHC disparate parental cells into unirradiated F1 recipient mice result in the development of marked immune dysfunction. Following negative selection using adherence and antibody and complement depletion, highly purified T cells were examined to determine their ability to undergo activation. Three weeks after GvHR initiation, unstimulated splenic T cells from GvHR mice displayed normal CD3 and IL-2R expression but elevated expression of class I MHC and Ly-6A/E antigens. Despite culture with normal F1 accessory cells, both CD4+ and CD8+ GvHR T cells exhibited low levels of proliferation to both Con A and anti-CD3 mAb. Although following exposure for 12 h to either of these stimuli, GvHR T cells expressed normal levels of IL-2R, expression was greatly decreased vs normal T cells between 24 and 48 h. In addition, at no timepoint was detectable IL-2 produced by GvHR T cells. Importantly, mixing experiments did not demonstrate detectable suppressive activity in the purified GvHR T cell subsets. GvHR T cells were also tested for their ability to respond to stimuli in the absence of any accessory cell population. These cells again did not proliferate to levels equivalent to normal T cells. Incubation with PMA and either cytokines (Con A supernatant, rIL-7) or anti-CD3 mAb resulted in only low levels of proliferation in GvHR T cells. Notably, at high ionomycin concentrations together with PMA, GvHR T cells did proliferate to equivalent levels as normal cells. However, with decreasing concentrations of ionophore, these cells failed to proliferate as well as normal cells. In total, these findings demonstrate that GvHR T cells are phenotypically and functionally distinct from normal T cells. The results suggest that GvHR T cells themselves may contribute to the well-characterized immune depression occurring in recipients undergoing GvHR.     

Requirement for delivery of signals by physical interaction and soluble factors from accessory cells in the induction of receptor-mediated T cell proliferation. Effectiveness of IFN-gamma modulation of accessory cells for physical interaction with T cells

We analyzed the mechanism by which accessory cells support the induction of the proliferation of human peripheral blood T cells by a monoclonal anti-CD3 antibody, OKT3. Cross-linking of T cell receptor/CD3 complex by anti-CD3 coupled to latex beads and the addition of IL-1 are not enough to induce the IL-2 production and proliferation of T cells extensively depleted of accessory cells, while the addition of both the culture supernatant of macrophages or a monoblastic cell line, U937 cells, and the paraformaldehyde-fixed macrophages or U937 cells which had been precultured with interferon-gamma before fixation into the culture of the T cells with anti-CD3-latex did induce the T cell proliferation. Lack of the addition of either one of these did not induce the response. These results indicate that the signal(s) delivered by soluble factors released from the accessory cells and that delivered by the physical interaction between accessory cells and T cells are both required for the induction of IL 2 production and proliferation of T cells by anti-CD3-latex. Importantly, the macrophages or U937 cells had to be cultured with Con A-stimulated lymphocyte culture supernatant or IFN-gamma prior to fixation with paraformaldehyde, suggesting that a molecule(s) inducible on accessory cells surface by IFN-gamma or other lymphokine is necessary for the effective accessory cell-T cell interaction to induce the T cell response. It was further revealed that the activity of the culture supernatant of accessory cells may be mediated synergistically by IL 1 and a certain other factor(s) and was actually shown to be replaced by the combined addition of rIL-1 and rIL-6 but not by rIL-1 alone. The experimental system described here will be very useful for dissecting the accessory functions for T cell activation.     

Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation

Transforming growth factor-beta1 is essential to maintain T cell homeostasis, as illustrated by multiorgan inflammation in mice deficient in TGF-beta1 signaling. Despite the physiological importance, the mechanisms that TGF-beta1 uses to regulate T cell expansion remain poorly understood. TGF-beta1 signals through transmembrane receptor serine/threonine kinases to activate multiple intracellular effector molecules, including the cytosolic signaling transducers of the Smad protein family. We used Smad3(-/-) mice to investigate a role for Smad3 in IL-2 production and proliferation in T cells. Targeted disruption of Smad3 abrogated TGF-beta1-mediated inhibition of anti-CD3 plus anti-CD28-induced steady state IL-2 mRNA and IL-2 protein production. CFSE labeling demonstrated that TGF-beta1 inhibited entry of wild-type anti-CD3 plus anti-CD28-stimulated cells into cycle cell, and this inhibition was greatly attenuated in Smad3(-/-) T cells. In contrast, disruption of Smad3 did not affect TGF-beta1-mediated inhibition of IL-2-induced proliferation. These results demonstrate that TGF-beta1 signals through Smad3-dependent and -independent pathways to inhibit T cell proliferation. The inability of TGF-beta1 to inhibit TCR-induced proliferation of Smad3(-/-) T cells suggests that IL-2 is not the primary stimulus driving expansion of anti-CD3 plus anti-CD28-stimulated T cells. Thus, we establish that TGF-beta1 signals through multiple pathways to suppress T cell proliferation.     

CD40 stimulation provides an IFN-gamma-independent and IL-4-dependent differentiation signal directly to human B cells for IgE production.

IgE induction from human cells has generally been considered to be T cell dependent and to require at least two signals: IL-4 stimulation and T cell/B cell interaction. In the present study we report a human system of T cell-independent IgE production from highly purified B cells. When human cells were co-stimulated with a mAb directed against CD40 (mAb G28-5), there was induction of IgE secretion from purified blood and tonsil B cells as well as unfractionated lymphocytes. Anti-CD40 alone failed to induce IgE from blood mononuclear cells or purified B cells. The effect of the combination of anti-CD40 and IL-4 on IgE production was very IgE isotype specific as IgG, IgM, and IgA were not increased. Furthermore, anti-CD40 with IL-5 or PWM did not co-stimulate IgG, IgM, or IgA and in fact strongly inhibited PWM-stimulated IgG, IgM and IgA production from blood or tonsil cells. IgE synthesis induced by anti-CD40 plus IL-4 was IFN-gamma independent as is the in vivo production of IgE in humans; the doses of IFN-gamma that profoundly suppressed IgG synthesis induced by IL-4, or IL-4 plus IL-6, had no inhibitory effect on anti-CD40-induced IgE production. Anti-CD23 and anti-IL-6 also could not block anti-CD40 plus IL-4-induced IgE production, but anti-IL-4 totally blocked their effect. IgE production via CD40 was not due to IL-5, IL-6 or nerve growth factor as none of these synergized with IL-4 to induce IgE synthesis by purified B cells. Finally, we observed that CD40 stimulation alone could enhance IgE production from in vivo-driven IgE-producing cells from patients with very high IgE levels; cells that did not increase IgE production in response to IL-4. Taken together, our data suggest that the signals delivered for IgE production by IL-4 and CD40 stimulation may mimic the pathway for IgE production seen in vivo in human allergic disease.     

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.     

Athymic nude CD4+8- T cells produce IL-2 but fail to proliferate in response to mitogenic stimuli

A comparative study of immune functions of CD4+8- T cells isolated from normal and athymic nude mice by electronic cell sorting was performed. Athymic nude CD4+8- T cells expressed the TCR-associated CD3 molecule but the level of expression was significantly lower than that of normal CD4+8- T cells. Proliferative responses were studied upon stimulation by 1) the T cell mitogen Con A; 2) anti-CD3 mediated cross-linking of the CD3:TCR complex, and 3) the combined action of PMA + ionomycin. All three mitogenic stimuli caused readily detectable cell division in normal (euthymic) CD4+8- T cells. In marked contrast, none of the mitogenic stimuli induced significant proliferation in athymic nude CD4+8- T cells. The failure of athymic nude CD4+8- T cells to proliferate occurred over a wide range of mitogen concentrations and over a 4-day observation period. Neither exogenously supplied rIL-2 or mixed lymphocyte culture supernatant had any effect on the impaired proliferative response by athymic nude CD4+8- T cells. Although IL-2 was produced by athymic nude CD4+8- T cells at a reduced level when compared to normal CD4+8- T cells, it was nevertheless readily detected upon stimulation with either Con A or anti-CD3. Furthermore, stimulation of athymic nude CD4+8- T cells by anti-CD3 induced the expression of the p55 chain of IL-2R on the cell surface. Therefore, despite production of IL-2 and induced expression of IL-2R, athymic nude CD4+8- T cells failed to undergo cell division.