Interleukin 4 (IL-4) or IL-7 Prevents the Death of Resting T Cells: Stat6 Is Probably Not Required for the Effect of IL-4

Although much is known about the activation, proliferation, and function of CD4⁺ T cells, little is known about how they survive as resting T cells in animals. Resting T cells have a half-life in animals of more than a week; however, when they are removed from animals and placed in tissue culture their half-life falls to ~24 h. In this paper, we show that the survival of resting T cells in vitro is promoted by two cytokines, interleukins 4 and 7 (IL-4, IL-7). They may do this in part by maintaining levels of survival-promoting proteins such as Bcl-2 in the cells, because the levels of Bcl-2 and Bcl-X_l in resting T cells fall rapidly after the cells are isolated from animals, and are maintained by culture in IL-4. Because the IL-4 receptor is known to signal through the JAK1 and JAK3/Stat6 pathway, we tested whether Stat6 was required for IL-4– dependent T cell survival. Surprisingly, we found that IL-4 rescued T cells from apoptosis in what appeared to be a Stat6-independent manner. These results demonstrate that the survival of resting T cells is an active process that can be affected by signals delivered by cytokines and also suggest that the IL-4 receptor on resting T cells may use a novel signaling pathway to facilitate T cell viability.     

Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth

This study examines the potential role of transforming growth factor beta (TGF-beta) in the regulation of human T lymphocyte proliferation, and proposes that TGF-beta is an important autoregulatory lymphokine that limits T lymphocyte clonal expansion, and that TGF-beta production by T lymphocytes is important in T cell interactions with other cell types. TGF-beta was shown to inhibit IL-2-dependent T cell proliferation. The addition of picograms amounts of TGF-beta to cultures of IL-2-stimulated human T lymphocytes suppressed DNA synthesis by 60-80%. A potential mechanism of this inhibition was found. TGF-beta inhibited IL-2-induced upregulation of the IL-2 and transferrin receptors. Specific high-affinity receptors for TGF-beta were found both on resting and activated T cells. Cellular activation was shown to result in a five- to sixfold increase in the number of TGF-beta receptors on a per cell basis, without a change in the affinity of the receptor. Finally, the observations that activated T cells produce TGF-beta mRNA and that TGF-beta biologic activity is present in supernatants conditioned by activated T cells is strong evidence that T cells themselves are a source of TGF-beta. Resting T cells were found to have low to undetectable levels of TGF-beta mRNA, while PHA activation resulted in a rapid increase in TGF-beta mRNA levels (within 2 h). Both T4 and T8 lymphocytes were found to make mRNA for TGF-beta upon activation. Using both a soft agar assay and a competitive binding assay, TGF-beta biologic activity was found in supernatants conditioned by T cells; T cell activation resulted in a 10-50-fold increase in TGF-beta production. Thus, TGF-beta may be an important antigen-nonspecific regulator of human T cell proliferation, and important in T cell interaction with other cell types whose cellular functions are modulated by TGF-beta.     

Tyrosine phosphorylation of CD6 by stimulation of CD3: augmentation by the CD4 and CD2 coreceptors

When T cells are activated via the T cell receptor (TCR) complex a number of cellular substrates, including some cell surface proteins, become phosphorylated on tyrosine (Tyr) residues. Phosphorylation of cytoplasmic Tyr renders these cell surface receptors competent to interact with proteins that link cell surface receptors to protein in the intracellular signaling pathways. Here we show that Tyr residues in the cytoplasmic domain of CD6 become phosphorylated upon T cell activation via the TCR complex. Tyr phosphorylation was observed when the T cells were activated by crosslinking CD3 or by cocrosslinking CD3 with CD2 or CD4, but not when the cells were stimulated by crosslinking CD2, CD4, or CD28 alone. Unlike other Tyr kinase substrates, such as the phospholipase C gamma 1-associated pp35/36 protein, whose level of Tyr phosphorylation is highest when T cells are activated by cocrosslinking CD3 with CD2, the levels of CD6 Tyr phosphorylation are highest when T cells were activated by cocrosslinking CD3 with CD4.     

Interleukin 4 reduces expression of inhibitory receptors on B cells and abolishes CD22 and Fc gamma RII-mediated B cell suppression

Inhibitory receptors CD22, Fc gamma RII (CD32), CD72, and paired immunoglobulin-like receptor (PIR)-B are critically involved in negatively regulating the B cell immune response and in preventing autoimmunity. Here we show that interleukin 4 (IL-4) reduces expression of all four on activated B cells at the level of messenger RNA and protein. This reduced expression is dependent on continuous exposure to IL-4 and is mediated through Stat6. Coligation of Fc gamma RII to the B cell receptor (BCR) via intact IgG increases the B cell activation threshold and suppresses antigen presentation. IL-4 completely abolishes these negative regulatory effects of Fc gamma RII. CD22 coligation with the BCR also suppresses activation -- this suppression too is abolished by IL-4. Thus, IL-4 is likely to enhance the B cell immune response by releasing B cells from inhibitory receptor suppression. By this coordinate reduction in expression of inhibitory receptors, and release from CD22 and Fc gamma RII-mediated inhibition, IL-4 is likely to play a role in T cell help of B cells and the development of T helper cell type 2 responses. Conversely, B cell activation in the absence of IL-4 would be more difficult to achieve, contributing to the maintenance of B cell tolerance in the absence of T cell help.     

Constitutive expression of the B7h ligand for inducible costimulator on naive B cells is extinguished after activation by distinct B cell receptor and interleukin 4 receptor-mediated pathways and can be rescued by CD40 signaling

The recently described ligand-receptor pair, B7h-inducible costimulator (ICOS), is critical for germinal center formation and antibody responses. In contrast to the induced expression of the related costimulatory ligands B7.1 and B7.2, B7h is constitutively expressed on naive B cells and is surprisingly extinguished after antigen engagement and interleukin (IL)-4 cytokine signaling. Although signaling through both B cell receptor (BCR) and IL-4 receptor (R) converge on the extinction of B7h mRNA levels, BCR down-regulation occurs through Ca2+ mobilization, whereas IL-4R down-regulation occurs through a distinct Stat6-dependent pathway. During antigen-specific B cell activation, costimulation through CD40 signaling can reverse both BCR- and IL-4R-mediated B7h down-regulation. These data suggest that the CD40-CD40 ligand signaling pathway regulates B7h expression on activated B cells and may control whether antigen-activated B cells can express B7h and costimulate cognate antigen-activated T cells through ICOS.     

Receptor-induced death in human natural killer cells: involvement of CD16

Propriocidal regulation of T cells refers to apoptosis induced by interleukin 2 (IL-2) activation with subsequent antigen receptor stimulation. We examined whether natural killer (NK) cells exhibited cytokine- and ligand-induced death similar to activated T cells. Peripheral NK cells were examined for ligand-induced death using antibodies to surface moieties (CD2, CD3, CD8, CD16, CD56), with and without prior activation of IL-2. Only those NK cells stimulated first with IL-2 and then with CD16 exhibited ligand-induced death; none of the other antibody stimuli induced this phenomenon. Next we examined various cytokines (IL-2, IL-4, IL-6, IL-7, IL-12, IL-13, interferon alpha and gamma) that can activate NK cells and determined if CD16- induced killing occurred. Only IL-2 and IL-12 induced NK cell death after occupancy of this receptor by aggregated immunoglobulin or by cross-linking with antireceptor antibody. The CD16-induced death was inhibited by herbimycin A, indicating that cell death was dependent upon protein tyrosine kinases. Identical to T cells, the form of cell death for NK cells was demonstrated to be receptor-induced apoptosis. Overall these data indicate that highly activated NK cells mediate ligand-induced apoptosis via signaling molecules like CD16. Whereas the propriocidal regulation of T cells is antigen specific, this is not the case for NK cells due to the nature of the receptor. The clinical implications of this finding are considered.     

Partially phosphorylated T cell receptor zeta molecules can inhibit T cell activation

The T cell receptor complex (TCR) zeta chain is constitutively tyrosine phosphorylated specifically at two of the six zeta immunoreceptor tyrosine-based activation motif (ITAM) tyrosine residues in resting peripheral T cells. Further phosphorylation of zeta is induced by both agonist and antagonist ligands of the TCR, with agonists inducing complete phosphorylation of the zeta ITAM tyrosines. After antagonist stimulation, zeta phosphorylation is incomplete and generates discrete forms of partially phosphorylated ITAMs. Here, we mutate specific tyrosines in chimeric human CD8-zeta molecules to reflect phosphorylation in resting T cells as well as phosphorylation induced by agonist and antagonist ligands. We demonstrate that such partially phosphorylated TCR-zeta species can inhibit IL-2 production in T cell hybridomas and proliferation in T cell clones. This reveals a previously unrecognized, inhibitory function of partially phosphorylated ITAMs. These findings support the concept that TCR antagonism can arise through the generation of an inhibitory signal within the TCR complex and that constitutive zeta phosphorylation in resting T cells is an inhibitory signaling environment.     

Antigen-independent activation of naive and memory resting T cells by a cytokine combination

We investigated whether human resting T cells could be activated to proliferate and display effector function in the absence of T cell receptor occupancy. We report that combination of interleukin 2 (IL-2), tumor necrosis factor alpha, and IL-6 activated highly purified naive (CD45RA+) and memory (CD45RO+) resting CD4+ T cells to proliferate. Under this condition, memory resting T cells could also display effector function as measured by lymphokine synthesis and help for immunoglobulin production by B cells. This novel Ag-independent pathway of T cell activation may play an important role in vivo in recruiting effector T cells at the site of immune response and in maintaining the clonal size of memory T cells in the absence of antigenic stimulation. Moreover, cytokines can induce proliferation of naive T cells without switch to memory phenotype and this may help the maintenance of the peripheral pool of naive T cells.     

Impaired Interleukin 4 Signaling in T Helper Type 1 Cells

Cluster of differentation (CD)4⁺ T helper cells (Th)1s fail to produce interleukin (IL)-4. Even if restimulated in the presence of IL-4, a condition that induces IL-4–producing capacity in naive CD4⁺ T cells, Th1s fail to become IL-4 producers. We report that Th1 cells have a major impairment in IL-4 signaling. When compared to both Th2s and naive T cells, they display a striking diminution in phosphorylation of Stat6. They also show reduced phosphorylation of Janus kinase (JAK)-3 and insulin receptor substrate (IRS)-2 when compared to Th2s. Stat6 and JAK-3 are present in equivalent amounts in Th1s and Th2s, but IRS-2 protein levels are much lower in Th1s than in Th2s. Altered sensitivity to IL-4, the major inducer of the Th2 phenotype, may explain the stability of the Th1 state.     

B cells drive lymphocyte activation and expansion in mice with the CD45 wedge mutation and Fas deficiency

CD45 and Fas regulate tyrosine phosphorylation and apoptotic signaling pathways, respectively. Mutation of an inhibitory wedge motif in CD45 (E613R) results in hyperresponsive thymocytes and B cells on the C57BL/6 background, but no overt autoimmunity, whereas Fas deletion results in a mild autoimmune disease on the same genetic background. In this study, we show that these two mutations cooperate in mice, causing early lethality, autoantibody production, and substantial lymphoproliferation. In double-mutant mice, this phenotype was dependent on both T and B cells. T cell activation required signaling in response to endogenous or commensal antigens, demonstrated by the introduction of a transgenic T cell receptor. Genetic deletion of B cells also prevented T cell activation. Similarly, T cells were necessary for B cell autoantibody production. However, B cells appeared to be intrinsically activated even in the absence of T cells, suggesting that they may drive the phenotype of these mice. These results reveal a requirement for careful control of B cell signaling and cell death in preventing inappropriate lymphocyte activation and autoimmunity.     

Interleukin 2 receptor gamma chain expression on resting and activated lymphoid cells

The interleukin 2 receptor (IL-2R) is known to be comprised of at least three genetically distinct subunits termed alpha, beta, and gamma. These chains can be expressed individually or in various combinations resulting in distinct receptors with different affinities for IL-2. In contrast to alpha and beta, the cell surface expression of the gamma chain protein previously has not been well-characterized. To examine cell surface expression of IL-2R gamma on hematopoietic cells, we developed two new monoclonal antibodies (mAbs) specific for this protein. Both 1A11 (immunoglobulin [IgG1]) and 3G11 (IgM) specifically reacted with murine cells transfected with IL-2R gamma cDNA, and immunoprecipitation studies indicated that both antibodies precipitated a protein of approximately 62-65 kD. Scatchard analysis of IL-2 binding to murine cells transfected with cDNA-encoding combinations of IL-2R components demonstrated that neither beta nor gamma chain bind IL-2 with measurable affinity, but coexpression of both beta and gamma is sufficient to form an intermediate affinity receptor. In the absence of gamma chain, beta chain interacts with alpha chain to form a "pseudo- high" affinity receptor. In contrast, gamma chain does not appear capable of interacting with alpha in the absence of beta chain. Thus, gamma chain appears to interact only with beta, but beta chain is capable of interacting with both alpha and gamma. Using the newly developed mAbs to examine cell surface expression by immunofluorescence, resting T cells were found to express low levels of gamma chain without detectable alpha or beta. Early after mitogen stimulation, T cells expressed higher levels of alpha, beta, and gamma. However, at later time points, T cells expressed alpha and gamma in marked excess over beta. Thus, formation of high affinity IL-2R on activated T cells was primarily limited by beta chain expression. In contrast, resting natural killer (NK) cells constitutively expressed IL- 2R beta without detectable alpha or gamma. After activation with either IL-2 or IL-12, expression of both alpha and gamma transiently increased and then returned to very low levels. Expression of functional IL-2R on resting and activated NK cells, therefore, appeared to be primarily limited by the expression of gamma chain. IL-2 binding studies with resting NK cells confirmed the results of immunofluorescence studies indicating the presence of very low numbers of intermediate affinity (beta gamma) receptors for IL-2 on these cells.(ABSTRACT TRUNCATED AT 400 WORDS)