IL-6, in synergy with IL-7 or IL-15, stimulates TCR-independent proliferation and functional differentiation of CD8+ T lymphocytes

Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8(+) T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8(+) T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8(+) T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8(+) T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8(+) T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8(+) T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.     

Distinct IL-2 receptor signaling pattern in CD4+CD25+ regulatory T cells

Despite expression of the high-affinity IL-2R, CD4(+)CD25(+) regulatory T cells (Tregs) are hypoproliferative upon IL-2R stimulation in vitro. However the mechanisms by which CD4(+)CD25(+) T cells respond to IL-2 signals are undefined. In this report, we examine the cellular and molecular responses of CD4(+)CD25(+) Tregs to IL-2. IL-2R stimulation results in a G(1) cell cycle arrest, cellular enlargement and increased cellular survival of CD4(+)CD25(+) T cells. We find a distinct pattern of IL-2R signaling in which the Janus kinase/STAT pathway remains intact, whereas IL-2 does not activate downstream targets of phosphatidylinositol 3-kinase. Negative regulation of phosphatidylinositol 3-kinase signaling and IL-2-mediated proliferation of CD4(+)CD25(+) T cells is inversely associated with expression of the phosphatase and tensin homologue deleted on chromosome 10, PTEN.     

The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands

The programmed death (PD)-1 molecule and its ligands (PD-L1 and PD-L2), negative regulatory members of the B7 family, play an important role in peripheral tolerance. Previous studies have demonstrated that PD-1 is up-regulated on T cells following TCR-mediated activation; however, little is known regarding PD-1 and Ag-independent, cytokine-induced T cell activation. The common gamma-chain (gamma c) cytokines IL-2, IL-7, IL-15, and IL-21, which play an important role in peripheral T cell expansion and survival, were found to up-regulate PD-1 and, with the exception of IL-21, PD-L1 on purified T cells in vitro. This effect was most prominent on memory T cells. Furthermore, these cytokines induced, indirectly, the expression of PD-L1 and PD-L2 on monocytes/macrophages in PBMC. The in vivo correlate of these observations was confirmed on PBMC isolated from HIV-infected individuals receiving IL-2 immunotherapy. Exposure of gamma c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by gamma c cytokines. However, PD-1 ligand-IgG dramatically inhibited anti-CD3/CD28-driven proliferation and Lck activation. Furthermore, following restimulation with anti-CD3/CD28, cytokine secretion by both gamma c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that gamma c cytokine-induced PD-1 does not interfere with cytokine-driven peripheral T cell expansion/survival, but may act to suppress certain effector functions of cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host.     

Impaired TCR-mediated induction of Ki67 by naive CD4+ T cells is only occasionally corrected by exogenous IL-2 in HIV-1 infection

Perturbations in naive T cell homeostasis and function may play a major role in the immunodeficiency that accompanies HIV infection. By examining naive CD4(+) T cell function on a single cell basis, we provide evidence that these cells have significant qualitative defects in HIV disease. Ki67, a molecule expressed during cell cycle progression, is induced less efficiently among naive CD4(+) T cells from HIV-infected individuals following activation with anti-TCR Ab. The impairment in Ki67 expression is evident even when a separate function, CD62L down-modulation, is within normal ranges. Moreover, the defects in Ki67 induction are only sometimes corrected by the addition of rIL-2 to cell cultures. An initial assessment of IL-2 unresponsiveness in cells from selected HIV-infected individuals suggests that the defect is not a consequence of impaired IL-2R expression or IL-2R signaling capability. Qualitative defects in naive T cells that cannot be routinely corrected by IL-2 have significant implications for disease pathogenesis and for strategies using IL-2 as a vaccine adjuvant in HIV disease.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.     

Uncoupling of promitogenic and antiapoptotic functions of IL-2 by Smad-dependent TGF-beta signaling

TGF-beta opposes proliferative signaling by IL-2 through mechanisms that remain incompletely defined. In a well-characterized CD8(+) T cell model using wild-type and mutated IL-2 receptors, we examined the effects of TGF-beta on distinct IL-2 signaling events in CD8(+) T cells. IL-2 induces c-myc, cyclin D2, and cyclin E in a redundant manner through the Shc and STAT5 pathways. TGF-beta inhibited the ability of either the Shc or STAT5 pathway to induce these genes, as well as T cell proliferation. The inhibitory effects of TGF-beta were reversed by expression of a dominant-negative form of Smad3. TGF-beta did not impair proximal signaling by Shc or STAT5, and induction of some downstream genes, including cytokine-inducible Src homology-2-containing protein (CIS), bcl-x(L), and bcl-2, was spared. Experiments with c-fos, cyclin D2, and CIS reporter genes revealed that promoter-proximal regulatory elements dictate the sensitivity of IL-2 target genes to inhibition by TGF-beta. By leaving the Shc and STAT5 pathways functional while inhibiting their target genes selectively, TGF-beta was found to uncouple the proliferative and antiapoptotic functions of IL-2. Thus, TGF-beta is not a simple antagonist of IL-2, but rather serves to qualitatively modify the IL-2 signal to create a unique pattern of gene expression that neither cytokine can induce independently.     

Uncoupling of IL-2 signaling from cell cycle progression in naive CD4+ T cells by regulatory CD4+CD25+ T lymphocytes

Prior reports have shown that CD4(+)CD25(+) regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4(+)CD25(+) T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G(0)-G(1). Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4(+)CD25(+) regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4(+)CD25(+) T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4(+)CD25(+) regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.     

Late expression of granulysin by microbicidal CD4+ T cells requires PI3K- and STAT5-dependent expression of IL-2Rbeta that is defective in HIV-infected patients

Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4(+) T cells with IL-2 occurs before granulysin is produced. Unfortunately, the mechanisms responsible for this delay are unknown. We have recently demonstrated that granulysin-mediated killing of Cryptococcus neoformans by CD4(+) T cells is defective during HIV infection. This is because CD4(+) T cells from HIV-infected patients fail to produce granulysin in response to IL-2 activation. The present studies examined the mechanism of delayed production of granulysin and the mechanism of the defect in HIV patients. We demonstrate that IL-2 initially requires both STAT5 and PI3K activation to increase expression of IL-2Rbeta, produce granulysin, and kill C. neoformans. The increased expression of IL-2Rbeta precedes granulysin, and preventing the increased expression of IL-2Rbeta using small interfering RNA knockdown abrogates granulysin expression. Moreover, following the increased expression of IL-2Rbeta, blocking subsequent signaling by IL-2 using IL-2Rbeta-specific blocking Abs abrogates expression of granulysin. Finally, CD4(+) T cells from HIV-infected patients, who are defective in both STAT5 and PI3K signaling, fail to express IL-2Rbeta and fail to produce granulysin. These results suggest that IL-2 signals via PI3K and STAT5 to increase expression of IL-2Rbeta, which in turn is required for production of granulysin. These results provide a mechanism to explain the "late" production of granulysin during normal T cell responses, as well as for defective granulysin production by CD4(+) T cells in HIV-infected patients.     

A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.     

Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells

Coincident production of IL-2 and induction of high-affinity IL-2R upon TCR engagement has precluded a clear distinction for the biological outcome of signaling through TCR/costimulatory molecules vs the IL-2R. Using a novel transgenic mouse on the IL-2Rbeta(-/-) genetic background, this study has separated the relative outcome of signaling through the TCR and IL-2R. We show that stimulation through the TCR and CD28 or CD40 ligand directly leads to T cell activation and several rounds of proliferation in an IL-2-independent fashion. However, this stimulation is insufficient for extended T cell growth to multiple cytokines or differentiation into CTL or IFN-gamma-secreting effector T cells. IL-2 is required for these functions in part by regulation of cyclin D3 and granzyme B. Somewhat less efficiently, IL-4 stimulation of these transgenic T cells redundantly rescued many of these activities. These data demonstrate a fundamental requirement for IL-2 and perhaps other common gamma-chain-dependent cytokines to promote selective gene expression by Ag-activated T cells for their subsequent growth and differentiation into effector T lymphocytes.     

IL-21 can supplement suboptimal Lck-independent MAPK activation in a STAT-3-dependent manner in human CD8(+) T cells

Although both MHC class II/CD8α double-knockout and CD8β null mice show a defect in the development of MHC class I-restricted CD8(+) T cells in the thymus, they possess low numbers of high-avidity peripheral CTL with limited clonality and are able to contain acute and chronic infections. These in vivo data suggest that the CD8 coreceptor is not absolutely necessary for the generation of Ag-specific CTL. Lack of CD8 association causes partial TCR signaling because of the absence of CD8/Lck recruitment to the proximity of the MHC/TCR complex, resulting in suboptimal MAPK activation. Therefore, there should exist a signaling mechanism that can supplement partial TCR activation caused by the lack of CD8 association. In this human study, we have shown that CD8-independent stimulation of Ag-specific CTL previously primed in the presence of CD8 coligation, either in vivo or in vitro, induced severely impaired in vitro proliferation. When naive CD8(+) T cells were primed in the absence of CD8 binding and subsequently restimulated in the presence of CD8 coligation, the proliferation of Ag-specific CTL was also severely hampered. However, when CD8-independent T cell priming and restimulation were supplemented with IL-21, Ag-specific CD8(+) CTL expanded in two of six individuals tested. We found that IL-21 rescued partial MAPK activation in a STAT3- but not STAT1-dependent manner. These results suggest that CD8 coligation is critical for the expansion of postthymic peripheral Ag-specific CTL in humans. However, STAT3-mediated IL-21 signaling can supplement partial TCR signaling caused by the lack of CD8 association.     

The IL-7Rα pathway is quantitatively and functionally altered in CD8 T cells in multiple sclerosis

The IL-7Rα single nucleotide polymorphism rs6897932 is associated with an increased risk for multiple sclerosis (MS). IL-7Rα is a promising candidate to be involved in autoimmunity, because it regulates T cell homeostasis, proliferation, and antiapoptotic signaling. However, the exact underlying mechanisms in the pathogenesis of MS are poorly understood. We investigated whether CD4 and CD8 lymphocyte subsets differed in IL-7Rα expression and functionality in 78 MS patients compared with 59 healthy controls (HC). A significantly higher frequency of IL-7Rα(+) CD8 effector memory (CD8EM) was found in MS. Moreover, IL-7Rα membrane expression was significantly increased in MS in naive and memory CD8 (all p < 0.05) with a similar trend in CD8EM (p = 0.055). No correlation was found between the expression level or frequency of IL-7Rα(+)CD8(+) and rs6897932 risk allele carriership. Upon IL-7 stimulation, MS patients had stronger STAT5 activation in CD8EM compared with HC. IL-7 stimulation had a differential effect on both mRNA and protein expression of granzyme A and granzyme B between MS and HC. Stainings of different lesions in postmortem MS brain material showed expression of IL-7 and CD8(+)IL-7Rα(+) in preactive, but not in active, demyelinating MS lesions, indicating involvement of IL-7Rα(+) lymphocytes in lesion development. The intralesional production of IL-7 in combination with the lower threshold for IL-7-induced cytotoxicity in MS may enhance the pathogenicity of these CD8 T cells. This is of special interest in light of the established demyelinating and cytotoxic actions of granzyme A.     

Selective reduction of post-selection CD8 thymocyte proliferation in IL-15Rα deficient mice

Peripheral CD8(+) T cells are defective in both IL-15 and IL-15Rα knock-out (KO) mice; however, whether IL-15/IL-15Rα deficiency has a similar effect on CD8 single-positive (SP) thymocytes remains unclear. In this study, we investigated whether the absence of IL-15 transpresentation in IL-15Rα KO mice results in a defect in thymic CD8 single positive (SP) TCR(hi) thymocytes. Comparison of CD8SP TCR(hi) thymocytes from IL-15Rα KO mice with their wild type (WT) counterparts by flow cytometry showed a significant reduction in the percentage of CD69(-) CD8SP TCR(hi) thymocytes, which represent thymic premigrants. In addition, analysis of in vivo 5-bromo-2-deoxyuridine (BrdU) incorporation demonstrated that premigrant expansion of CD8SP TCR(hi) thymocytes was reduced in IL-15Rα KO mice. The presence of IL-15 transpresentation-dependent expansion in CD8SP TCR(hi) thymocytes was assessed by culturing total thymocytes in IL-15Rα-Fc fusion protein-pre-bound plates that were pre-incubated with IL-15 to mimic IL-15 transpresentation in vitro. The results demonstrated that CD8SP thymocytes selectively outgrew other thymic subsets. The contribution of the newly divided CD8SP thymocytes to the peripheral CD8(+) T cell pool was examined using double labeling with intrathymically injected FITC and intravenously injected BrdU. A marked decrease in FITC(+) BrdU(+) CD8(+) T cells was observed in the IL-15Rα KO lymph nodes. Through these experiments, we identified an IL-15 transpresentation-dependent proliferation process selective for the mature CD8SP premigrant subpopulation. Importantly, this process may contribute to the maintenance of the normal peripheral CD8(+) T cell pool.     

TLR2 activation enhances HIV nuclear import and infection through T cell activation-independent and -dependent pathways

TLR2 activation plays a crucial role in Neisseria gonorrheae-mediated enhancement of HIV infection of resting CD4(+) T cells. We examined signaling pathways involved in the HIV enhancing effect of TLR2. TLR2 but not IL-2 signals promoted HIV nuclear import; however, both signals were required for the maximal enhancing effect. Although TLR2 signaling could not activate T cells, it increased IL-2-induced T cell activation. Cyclosporin A and IkBα inhibitor blocked TLR2-mediated enhancement of HIV infection/nuclear import. PI3K inhibitor blocked HIV infection/nuclear import and T cell activation and exerted a moderate inhibitory effect on cell cycle progression in CD4(+) T cells activated by TLR2/IL-2. Blockade of p38 signaling suppressed TLR2-mediated enhancement of HIV nuclear import/infection. However, the p38 inhibitor did not have a significant effect on T cell activation or TCR/CD3-mediated enhancement of HIV infection/nuclear import. The cell cycle arresting reagent aphidicolin blocked TLR2- and TCR/CD3-induced HIV infection/nuclear import. Finally, cyclosporin A and IκBα and PI3K inhibitors but not the p38 inhibitor blocked TLR2-mediated IκBα phosphorylation. Our results suggest that TLR2 activation enhances HIV infection/nuclear import in resting CD4(+) T cells through both T cell activation-dependent and -independent mechanisms.     

Signaling events involved in interleukin 27 (IL-27)-induced proliferation of human naive CD4+ T cells and B cells

IL-27 induces stronger proliferation of naive than memory human B cells and CD4(+) T cells. In B cells, this differential response is associated with similar levels of IL-27 receptor chains, IL-27Rα and gp130, in both subsets and stronger STAT1 and STAT3 activation by IL-27 in naive B cells. Here, we show that the stronger proliferative response of CD3-stimulated naive CD4(+) T cells to IL-27 is associated with lower levels of IL-27Rα but higher levels of gp130 compared with memory CD4(+) T cells. IL-27 signaling differs between naive and memory CD4(+) T cells, as shown by more sustained STAT1, -3, and -5 activation and weaker activation of SHP-2 in naive CD4(+) T cells. In the latter, IL-27 increases G0/G1 to S phase transition, cell division and, in some cases, cell survival. IL-27 proliferative effect on naive CD4(+) T cells is independent of MAPK, but is dependent on c-Myc and Pim-1 induction by IL-27 and is associated with induction of cyclin D2, cyclin D3, and CDK4 by IL-27 in a c-Myc and Pim-1-dependent manner. In BCR-stimulated naive B cells, IL-27 only increases entry in the S phase and induces the expression of Pim-1 and of cyclins A, D2, and D3. In these cells, inhibition of Pim-1 inhibits IL-27 effect on proliferation and cyclin induction. Altogether, these data indicate that IL-27 mediates proliferation of naive CD4(+) T cells and B cells through induction of both common and distinct sets of cell cycle regulators.