A common precursor for CD4+ T cells producing IL-2 or IL-4.

To investigate whether CD4+ T cells are predetermined to produce a given pattern of lymphokines, we have used a culture system that allows the controlled induction of either IL-2- or IL-4-producing CD4+ T cells. Single, freshly isolated murine CD4+ T cells were activated with Con A, rIL-2, and APC; the developing clones were split and then cultured for an additional 14 days with either rIL-2 alone or with rIL-2 and anti-CD3 stimulation. Subclones expanded in the presence of rIL-2 alone produced predominantly IL-2, although subclones derived from the same precursor and expanded in the presence of rIL-2 and a mitogenic antibody to CD3 released predominantly IL-4. Subclones expanded for 2 wk in the presence of rIL-2 plus a mitogenic mAb to CD3 released up to 60 times more IL-4 but only 1/90 the amount of IL-2 released by subclones derived from the same precursor cell and expanded with rIL-2. Both phenotypes can be derived from IL-2-producing precursor cells. These results demonstrate that IL-2-producing clones can be derived from the same cells as IL-4-producing clones and are most consistent with the view that the IL-2-producing Th1 or the IL-4-producing Th2 phenotype of a T cell clone is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations that are predetermined to produce a specific cytokine pattern.     

Independent regulation of IFN-gamma and tumor necrosis factor by IL-1 in human T helper cells

The lymphokines IL-2 and IL-4 promoted the growth of human PHA-triggered T cells, but only IL-2 induced the production of IFN-gamma and TNF. The addition of purified monocytes strongly enhanced the production of IFN-gamma in IL-2-stimulated T cell cultures but did not influence the production of TNF or the level of T cell proliferation. The addition of IL-1 to T cells activated by PHA and optimal concentrations of IL-2 resulted in a strong induction of IFN-gamma production but had no influence on TNF production or T cell proliferation. IL-6 did not influence IFN-gamma or TNF production or T cell proliferation induced by PHA-IL-2 and did not modulate IL-1-induced IFN-gamma production. The production of IFN-gamma by CD4+ 45R+ Th cells was strongly enhanced by IL-1, whereas CD8+ T cells were less responsive to IL-1 and CD4+ 45R+ T cells were unresponsive to IL-1. We demonstrate, at the clonal level, that the optimal production of IFN-gamma by human Th cells requires both IL-1 and IL-2, whereas the production of TNF and T cell proliferation are induced by IL-2 alone. We suggest that IL-1 acts as a second signal for IFN-gamma production and that it may have an important function in regulating the pattern of lymphokines produced by T cell subsets during activation.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Central role for TCR/CD3 ligation in the differentiation of CD4+ T cells toward A Th1 or Th2 functional phenotype.

Activated CD4+ T cells can be classified into distinct subsets; the most divergent among them may be considered to be the IL-2 and IFN-gamma-producing Th1 clones and the IL-4 and IL-5-producing Th2 clones. Because Th1 and Th2 clones can usually be detected only after several months of culture, we used conditions that modulate the IL-2 and IL-4 production in short term culture. Here we show that freshly isolated and subsequently in vitro-activated CD4+ T cells that were cultured for 11 days with rIL-2 and restimulated showed a IFN-gamma+ IL-2+ IL-3+ IL-4- IL-5- pattern. Because these cells were not capable of providing B cell help for IgG1, IgG2a, or IgE in an APC- and TCR-dependent T-B cell assay, they expressed a phenotype typical for most Th1 clones. In contrast, activated T cells that were cultured for 11 days with IL-2 plus a mAb to CD3 and then restimulated produced a IFN-gamma- IL-2- IL-3+ IL-4+ IL-5+ pattern. These cells were capable of providing B cell help for IgG1, IgG2a, and IgE synthesis and thus presented a phenotype typical for Th2 clones. Similar results were observed when mitogenic mAb to Thy-1.2 or to framework determinants of the alpha beta TCR were used. The induction of Th1- and Th2-like cells did not depend on the relative expression of CD44 or CD45 by the T cells before activation in vitro. Because the incubation of activated T cells with anti-CD3/TCR mAb induced high unrestricted lymphokine production, the latter might be responsible for the Th2-like lymphokine pattern observed after restimulation. To address this point, TCR V beta 8+ and V beta 8- T cell blasts were co-cultured in the presence of mAb to V beta 8. After restimulation, V beta 8+ cells had a IL-4high IL-2low phenotype and V beta 8- cells had a IL-4low IL-2high phenotype. This demonstrates that TCR ligation but not lymphokines alone are capable of inducing Th2-like cells, and this points out a central role for the TCR in the generation of T cell subsets.     

p38 alpha mitogen-activated protein kinase is activated by CD28-mediated signaling and is required for IL-4 production by human CD4+CD45RO+ T cells and Th2 effector cells.

T cell proliferation and cytokine production usually require stimulation via both the TCR/CD3 complex and the CD28 costimulatory receptor. Using purified human CD4+ peripheral blood T cells, we show that CD28 stimulation alone activates p38 alpha mitogen-activated protein kinase (p38 alpha). Cell proliferation induced by CD28 stimulation alone, a response attributed to CD4+CD45RO+ memory T cells, was blocked by the highly specific p38 inhibitors SB 203580 (IC50 = 10-80 nM) and RWJ 67657 (IC50 = 0.5-4 nM). In contrast, proliferation induced by anti-CD3 plus anti-CD28 mAbs was not blocked. Inhibitors of p38 also blocked CD4+ T cell production of IL-4 (SB 203580 IC50 = 20-100 nM), but not IL-2, in response to CD3 and CD28 stimulation. IL-5, TNF-alpha, and IFN-gamma production were also inhibited, but to a lesser degree than IL-4. IL-4 production was attributed to CD4+CD45RO+ T cells, and its induction was suppressed by p38 inhibitors at the mRNA level. In polarized Th1 and Th2 cell lines, SB 203580 strongly inhibited IL-4 production by Th2 cells (IC50 = 10-80 nM), but only partially inhibited IFN-gamma and IL-2 production by Th1 cells (<50% inhibition at 1 microM). In both Th1 and Th2 cells, CD28 signaling activated p38 alpha and was required for cytokine production. These results show that p38 alpha plays an important role in some, but not all, CD28-dependent cellular responses. Its preferential involvement in IL-4 production by CD4+CD45RO+ T cells and Th2 effector cells suggests that p38 alpha may be important in the generation of Th2-type responses in humans.     

Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines.

PGE2 is known to inhibit IL-2 and IFN-gamma production from Th cells and is widely viewed as a general immunosuppressant. However, PGE2 was found not to inhibit IL-4 production from Th2 clones, and IL-5 production from these clones was slightly enhanced. The same results were obtained with short term T cell lines, which indicates that the lack of inhibition of IL-4 and IL-5 production by PGE2 is a general phenomenon. PGE2 functions by increasing cAMP levels through activation of adenylate cyclase. Despite its failure to inhibit lymphokine release, PGE2 was capable of increasing cAMP levels in Th2 cells, and forskolin, a direct activator of adenylate cyclase, also did not inhibit IL-4 or IL-5 production. These data indicate that the failure of PGE2 to inhibit IL-4 and IL-5 production was not due to an inability of PGE2 to induce an increase in intracellular cAMP, and suggested instead that the expression of IL-4 and IL-5 in Th2 cells is insensitive to elevated cAMP levels. When Th0 clones were examined, PGE2 was again found to differentially affect IL-2 and IL-4 production in three of five clones tested. In two additional Th0 clones, both IL-2 and IL-4 production were inhibited. These data suggest that lymphokine production may be regulated on two different levels. First, Th1- and Th2-associated lymphokines may be differentially sensitive to intracellular signals such as cAMP. Second, T cell subsets may exist, including subsets of Th0 cells, with different signaling pathways. In addition, our data suggest that PGE2 may play an important role in regulating the development of a response dominated by Th1- or Th2-associated lymphokines.     

Heterogeneity of helper/inducer T lymphocytes. III. Responses of IL-2- and IL-4-producing (Th1 and Th2) clones to antigens presented by different accessory cells

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.     

Characterization of T helper 1 and 2 cell subsets in normal mice. Helper T cells responsible for IL-4 and IL-5 production are present as precursors that require priming before they develop into lymphokine-secreting cells

We have shown that the requirements for the production of IL-4 and IL-5 by normal L3T4+T cells from murine spleen are very different from those for the production of IL-2. Secretion of detectable quantities of IL-4 and IL-5 and induction of the mRNA for each lymphokine occurs in vitro only after cells are primed and re-stimulated. This priming can be achieved by mitogens (Con A), by antibodies to the TCR (anti-T3) or by stimulation with alloantigen. In contrast, requirements for induction of lymphokine production after priming resemble those for initial production of IL-2. Thus the majority of T cells of helper phenotype that have the potential to become IL-4- and IL-5-secreting T cells, exist in the form of precursors requiring stimulation and several days of culture as well as re-stimulation with mitogen or Ag before they become detectable as lymphokine-secreting cells. In contrast, among fresh CD4+T cells, secretion of IL-2, IL-3, granulocyte/macrophage CSF, and IFN-gamma is easily detected within 24 h of stimulation with mitogen or Ag. These observations establish that distinct phenotypes of Th cells are found at different times after stimulation and support the concept that synthesis and secretion of different lymphokines or groups of lymphokines are regulated independently. Furthermore the patterns of lymphokines secreted by fresh vs primed Th cells, which largely correspond to the patterns that have been used to define the Th1 and Th2 subsets among Th cell lines, provides evidence that different subsets of normal T cells exist that may correspond to these designations. Secretion of different lymphokines by two subsets of Th cells at different times in an immune response, and perhaps in different places, suggests a model in which the ratio of the two T cell subsets (Th1 vs Th2) and state of differentiation of each (precursor vs effector), influence or determine the direction of the response, with variations in these parameters leading to differing responses.     

Anti-CD3 antibody induces unresponsiveness to IL-2 in Th1 clones but not in Th2 clones

Culture of murine T cells with immobilized (platebound) anti-CD3 antibody results in autocrine growth factor secretion in both Th1 (IL-2 producing) and Th2 (IL-4 producing) cells. Using a panel of murine T cell clones, we demonstrate that the IL-2-induced proliferation of Th1 clones is dramatically inhibited by immobilized anti-CD3 antibody, whereas that of Th2 clones is not. This unresponsiveness of Th1 clones to IL-2 is not due to decreases in IL-2R expression. Supernatants from Th1 or Th2 cell cultures fail to alter the effects of anti-CD3 on the two types of clones, suggesting that unresponsiveness induced in Th1 clones or the lack thereof in Th2 clones is not mediated by a stable cytokine(s). Accessory cells enhance the proliferation of Th1 cells exposed to low concentrations of anti-CD3, but the unresponsiveness induced by high concentrations of anti-CD3 is not prevented by accessory cells. Finally, soluble anti-CD4 antibody prevents the induction of the unresponsive state even at high concentrations of anti-CD3. These experiments demonstrate that two subsets of cloned CD4+ T cells differ in their responses to anti-CD3, and that CD4 molecules may play a critical role in regulating the outcome of receptor-mediated stimulation.     

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.     

Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones

In the present study, we have investigated the ability of human T cells to secrete IL-2, IL-4, and IFN-gamma. IL-4 and IFN-gamma were quantified with enzymatic immunoassays and IL-2 with a biologic assay by using the murine IL-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted IL-2, IL-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced IL-2, IL-4, and IFN-gamma and another (HY640), that produced only IL-2 and IFN-gamma. Significant IL-2, IL-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of IL-2 and IL-4 were found 20 h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The IL-2, IL-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to 20 h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce IL-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted IL-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for IL-2, IL-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted IL-2, IL-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted IL-2 and IFN-gamma, but not IL-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and IL-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of IL-4 from IFN-gamma by using HPLC, the IL-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-IL-4 antiserum. Finally, the partly purified IL-4, that was devoid of IL-2, promoted the growth of the clone GEO-2.     

Modulation of IL-2- and IL-4-induced cytotoxicities in human T helper lymphocyte clones by tumor necrosis factor-alpha.

A set of alloreactive IL-2-dependent human CD4+ 45RA-w29+56- Th cell clones was divided into two groups according to their ability to respond to IL-4 by proliferation and their susceptibility to inhibition by TNF-alpha. The latter cytokine blocked proliferative responses to IL-2 of IL-4-nonresponsive clones, but did not affect proliferation of IL-4-responsive clones. In the present communication, it is demonstrated that exposure of apparently non-cytotoxic Th cells to IL-4 resulted in the dose-dependent induction of allospecific CTX in clones previously shown to be capable of responding to IL-4 by proliferation. In contrast, IL-2 induced both allospecific and MHC-unrestricted "NK-like" CTX in both IL-4 responder and nonresponder TCC. However, coculture with IL-4 in addition to IL-2 down-regulated this induction of NK-like CTX by the IL-2 (in those clones capable of responding to IL-4). Acquisition of these two types of CTX by the same TCC was additionally modulated by TNF-alpha, which also blocked the induction of NK-like CTX but had no effect on the induction of allospecific CTX by either IL-2 or IL-4. In contrast, IFN-gamma was unable to block induction of either type of CTX in this model system. These data suggest that even at the clonal level, the relative availability of a number of different up- and down-regulatory cytokines influences the outcome of an immune response. In the present model, IL-2 up-regulates specific and NK-like CTX, the latter component of which is down-regulated by TNF-alpha or IL-4, whereas IL-4 itself can up-regulate specific but not NK-like CTX.     

Identification of a novel cytokine, ML-1, and its expression in subjects with asthma

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4(+) T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 +/- 19.1 pg/ml; for IL-8, 1724.2 +/- 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 +/- 55.6 pg/ml; for IL-8, 4371.4 +/- 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 +/- 4.39 vs baseline, MFI = 12.26 +/- 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 +/- 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.     

High levels of IL-2 alter signal transduction in cloned IL-4-producing CD4 T cells.

Cloned CD4 T cells of the Th2 type make IL-4 and related cytokines upon receptor cross-linking, whereas cloned CD4 T cells of the Th1 type make IL-2, IFN-gamma, and TNF-beta. These two types of CD4 T cell are also reported to use distinct mechanisms of signal transduction. It has been reported that Th1 cells flux Ca2+ upon receptor cross-linking, whereas Th2 cells do not. We have noted that when cloned Th2 cells are exposed to high levels (20 U/ml) of IL-2, they show an altered phenotype. Such cells are much more sensitive to activation by certain antireceptor antibodies, they flux calcium upon receptor ligation without additional cross-linking with anti-Ig antibodies, and they make much larger amounts of IL-4. In addition, the organization of their TCR is altered, with increased levels of the TCR-eta chain and an increase in the extent of association of CD4 with CD3 and CD45, changes similar to those found in Th1 cells. These results suggest that there is no fundamental difference in the signal transduction apparatus of Th1 and Th2 cells; rather, the IL-2 made by Th1 cells may create similar phenotypic changes in these cells and thus create the impression of altered signal transduction mechanisms. These results do show that exposure to high levels of IL-2 can profoundly affect signal transduction in T cells. Furthermore, we found that the Ca2+ signal caused by CD3 antibodies seemed to differ in character from that caused by TCR antibodies suggesting that the use of CD3 antibodies is not always a good model for activation through the TCR.     

Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation

IL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor alpha-chain (IL-4Ralpha-/-) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Ralpha-/-mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Ralpha-/- DO11.10/scid mice secreted approximately 50 pg of IL-4 per10(6) cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-gamma and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Ralpha-/- mice gave rise to a minor proportion (approximately 2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-gamma and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-gamma and IL-12, this IL-4 can induce Th2 polarization.     

IL-2, IL-6, and IFN-gamma have distinct effects on the IL-4 plus PMA-induced proliferation of thymocyte subpopulations

We have previously reported complex effects of cytokine-containing T cell supernatants on the interleukin (IL)4 plus phorbol 12-myristate 13-acetate (PMA)-induced proliferative response of murine thymocytes. Here we show that recombinant murine IL-2, IL-6, and IFN-gamma each differentially regulate the IL-4/PMA-driven growth of thymocyte subpopulations. Thymocytes fractionated into four subpopulations on the basis of CD4 and CD8 expression were stimulated to proliferate by IL-4/PMA. Interferon-gamma (IFN-gamma) caused almost complete inhibition of the CD4+/CD8- response but had no measurable effect on the growth of CD4-/CD8+ or CD4-/CD8- populations. This inhibitory effect was also observed on splenic CD4+/CD8- T cells. In contrast, IL-6 strongly enhanced the proliferative response of CD4+/CD8- thymocytes, but showed no effect on peripheral CD4+/CD8- T cells, suggesting that IL-6 may be an important regulator of growth in the thymus. IL-2 also enhanced the proliferation of both CD4-/CD8+ and CD4-/CD8- thymocytes to IL-4 and PMA. To test whether the IL-4/PMA stimulus provided all the signals required to initiate growth in each subpopulation, we titrated cell number and examined the relationship between cell dose and cell response. Growth of CD8+/CD4- cells was cell density independent, indicating that IL-4/PMA is sufficient stimulus to induce growth of these cells. In contrast, growth of CD4-/CD8- and CD4+/CD8- cells is cell density dependent, suggesting a requirement for another signal provided by the cells themselves. These observations suggest that more signals remain to be identified in this thymocyte growth system.     

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.     

IL-4-responsive human helper T cell clones are resistant to growth inhibition by tumor necrosis factor-alpha

Positive and negative signals for clonal expansion of preactivated human CD4+ alloreactive Th cells have been examined. Fifteen T cell clones tested 3 days after Ag-specific stimulation proliferated with IL-2 but only five of these responded to IL-4. The remaining 10 also failed to respond to IL-4 in the presence of IL-1 and/or autologous B-LCL. The response of the latter five to IL-4 was independent of IL-2 as shown by the inability of IL-2R mAb to prevent proliferation. In contrast, transferrin R mAb blocked responses to both IL-2 and IL-4. IL-4 responder but not nonresponder clones demonstrated IL-4-enhanced responses to suboptimal concentrations of IL-2 (1 U/ml), but none of the clones showed enhanced responses with 1 U/ml IL-2 plus IL-1, IL-3, IL-5, or granulocyte-macrophage-CSF. IFN-gamma did not enhance or inhibit responses to either IL-2 or IL-4. TNF-alpha did not block proliferation supported by IL-4. In contrast, TNF-alpha did block proliferative responses to IL-2, but only by those clones which were incapable of responding to IL-4. Thus, proliferation of the IL-4-responder clones was not blocked by TNF-alpha when optimal or even supraoptimal concentrations of IL-2 were used as growth factor. Because all T cell clones themselves secreted TNF-alpha after specific stimulation, these results suggest a novel autocrine negative regulatory pathway, whereby IL-4-reactive helper cells would have a selective advantage over IL-4-nonreactive cells during the evolution of an immune response.