Transgene-enforced co-stimulation of CD4+ T cells leads to enhanced and sustained anti-tumor effector functioning

Background The role of co-stimulation in CD4+ T cell activation by professional APC is well established, while less is known of the role co-stimulation plays when CD4+ T cells interact directly with tumor cells. Methods Through genetic engineering of human CD4+ T cells, we tested the hypothesis that integration of co-stimulatory signaling domains within a tumor-targeting chimeric Ag receptor (CAR), the IL-13Ralpha2-specific IL-13-zetakine (IL13zeta), would enhance CD4+ T cell mediated responses against tumors that fail to express ligands for co-stimulatory receptors. Results Compared with CD3zeta-mediated activation alone, CD4+ effector T cells expressing the IL13-CD28-41BBzeta CAR exhibited augmented/sustained MAPK and AKT activity, up-regulated Th1 cytokine production, and enhanced cytolytic potency against tumor targets. Moreover, upon recursive stimulation with tumor, the IL13-CD28-41BBzeta+ cells retained/recycled their lytic function, whereas IL-13zeta+ CD4+ cells became anergic/exhausted. These in vitro observations correlated with enhanced in vivo control of established orthotopic CNS glioma xenografts in immunodeficient mice mediated by adoptively transferred ex vivo-expanded CD4+ T cells expressing the co-stimulatory CAR. Discussion Together these studies demonstrate the importance of integrating co-stimulation with CD3zeta signaling events to activate fully CD4+ anti-tumor effector cells for sustained function in the tumor microenvironment.     

Interleukin-25 induces type 2 cytokine production in a steroid-resistant interleukin-17RB+ myeloid population that exacerbates asthmatic pathology

Interleukin-25 (IL-25) is a cytokine associated with allergy and asthma that functions to promote type 2 immune responses at mucosal epithelial surfaces and serves to protect against helminth parasitic infections in the intestinal tract. This study identifies the IL-25 receptor, IL-17RB, as a key mediator of both innate and adaptive pulmonary type 2 immune responses. Allergen exposure upregulated IL-25 and induced type 2 cytokine production in a previously undescribed granulocytic population, termed type 2 myeloid (T2M) cells. Il17rb(-/-) mice showed reduced lung pathology after chronic allergen exposure and decreased type 2 cytokine production in T2M cells and CD4(+) T lymphocytes. Airway instillation of IL-25 induced IL-4 and IL-13 production in T2M cells, demonstrating their importance in eliciting T cell-independent inflammation. The adoptive transfer of T2M cells reconstituted IL-25-mediated responses in Il17rb(-/-) mice. High-dose dexamethasone treatment did not reduce the IL-25-induced T2M pulmonary response. Finally, a similar IL-4- and IL-13-producing granulocytic population was identified in peripheral blood of human subjects with asthma. These data establish IL-25 and its receptor IL-17RB as targets for innate and adaptive immune responses in chronic allergic airway disease and identify T2M cells as a new steroid-resistant cell population.     

The histamine H4 receptor mediates allergic airway inflammation by regulating the activation of CD4+ T cells

Histamine is an important inflammatory mediator that is released in airways during an asthmatic response. However, current antihistamine drugs are not effective in controlling the disease. The discovery of the histamine H4 receptor (H4R) prompted us to reinvestigate the role of histamine in pulmonary allergic responses. H4R-deficient mice and mice treated with H4R antagonists exhibited decreased allergic lung inflammation, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in Th2 responses. Ex vivo restimulation of T cells showed decreases in IL-4, IL-5, IL-13, IL-6, and IL-17 levels, suggesting that T cell functions were disrupted. In vitro studies indicated that blockade of the H4R on dendritic cells leads to decreases in cytokine and chemokine production and limits their ability to induce Th2 responses in T cells. This work suggests that the H4R can modulate allergic responses via its influence on T cell activation. The study expands the known influences of histamine on the immune system and highlights the therapeutic potential of H4R antagonists in allergic conditions.     

Ribavirin exerts differential effects on functions of cd4(+) Th1, th2, and regulatory T cell clones in hepatitis C

Ribavirin improves outcomes of therapy in chronic hepatitis C but its mode of action has still remained unclear. Since ribavirin has been proposed to modulate the host's T cell responses, we studied its direct effects on CD4(+) T cell clones with diverse functional polarization which had been generated from patients with chronic hepatitis C. We analysed in vitro proliferation ([(3)H] thymidine uptake) and cytokine responses (IL-10, IFN-gamma) at varying concentrations of ribavirin (0-10μg/ml) in 8, 9 and 7 CD4(+) TH1, TH2 and regulatory T cell (Treg) clones, respectively. In co-culture experiments, we further determined effects of ribarivin on inhibition of TH1 and TH2 effector cells by Treg clones. All clones had been generated from peripheral blood of patients with chronic hepatitis C in the presence of HCV core protein. Ribavirin enhanced proliferation of T effector cells and increased production of IFN-gamma in TH1 clones, but had only little effect on IL-10 secretion in TH2 clones. However, ribavirin markedly inhibited IL-10 release in Treg clones in a dose dependent fashion. These Treg clones suppressed proliferation of T effector clones by their IL-10 secretion, and in co-culture assays ribavirin reversed Treg-mediated suppression of T effector cells. Our in vitro data suggest that - in addition to its immunostimulatory effects on TH1 cells - ribavirin can inhibit functions of HCV-specific Tregs and thus reverses Treg-mediated suppression of T effector cells in chronic hepatitis C.     

4-1BB ligand induces cell division, sustains survival, and enhances effector function of CD4 and CD8 T cells with similar efficacy

A costimulatory member of the TNFR family, 4-1BB, is expressed on activated T cells. Although some reports have suggested that 4-1BB is primarily involved in CD8 T cell activation, in this report we demonstrate that both CD4 and CD8 T cells respond to 4-1BB ligand (4-1BBL) with similar efficacy. CD4 and CD8 TCR transgenic T cells up-regulate 4-1BB, OX40, and CD27 and respond to 4-1BBL-mediated costimulation during a primary response to peptide Ag. 4-1BBL enhanced proliferation, cytokine production, and CTL effector function of TCR transgenic T cells. To compare CD4 vs CD8 responses to 4-1BBL under similar conditions of antigenic stimulation, we performed MLRs with purified CD4 or CD8 responders from CD28(+/+) and CD28(-/-) mice. We found that CD8 T cells produced IL-2 and IFN-gamma in a 4-1BBL-dependent manner, whereas under the same conditions the CD4 T cells produced IL-2 and IL-4. 4-1BBL promoted survival of CD4 and CD8 T cells, particularly at late stages of the MLR. CD4 and CD8 T cells both responded to anti-CD3 plus s4-1BBL with a similar cytokine profile as observed in the MLR. CD4 and CD8 T cells exhibited enhanced proliferation and earlier cell division when stimulated with anti-CD3 plus anti-CD28 compared with anti-CD3 plus 4-1BBL, and both subsets responded comparably to anti-CD3 plus 4-1BBL. These data support the idea that CD28 plays a primary role in initial T cell expansion, whereas 4-1BB/4-1BBL sustains both CD4 and CD8 T cell responses, as well as enhances cell division and T cell effector function.     

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.     

Peripheral immature CD2-/low T cell development from type 2 to type 1 cytokine production

Immature myeloid and NK cells exist, and undergo cytokine-induced differentiation, in the periphery. In this study, we show that also immature CD2(-/low) T cells exist in peripheral blood. These cells produce the type 2 cytokines IL-13, IL-4, and IL-5, but not IFN-gamma or IL-10, and, upon culture with IL-12- and TCR-mediated stimuli, differentiate to IL-13(+)IFN-gamma(+) cells producing high IL-2 levels, and finally IL-13(-)IFN-gamma(+) cells. The monokine combination IL-12, IL-18, and IFN-alpha substitutes for TCR-mediated stimulation to induce the same differentiation process in both immature CD2(-/low) and primary mature CD2(+) IL-13(+) T cells. IFN-alpha is needed to maintain high level IL-2 production, which is confined to type 2 cytokine-producing cells and lost in the IFN-gamma(+) ones. Upon TCR-mediated stimulation, IFN-gamma(+) cells are then induced to produce IL-10 as they undergo apoptosis. These data indicate that peripheral type 2 cytokine(+) T cells are immature cells that can differentiate to effector IFN-gamma(+) cells following a linear monokine-regulated pathway identical with that previously described for NK cells. They define the cellular bases to support that cell-mediated immune responses are regulated not only via Ag-induced activation of mature effector cells, but also via bystander monokine-induced maturation of immature T cells.     

Potential role of melatonin in autoimmune diseases

Autoimmune diseases are a broad spectrum of disorders involved in the imbalance of T-cell subsets, in which interplay or interaction of Th1, Th17 and Tregs are most important, resulting in prolonged inflammation and subsequent tissue damage. Pathogenic Th1 and Th17 cells can secrete signature proinflammatory cytokines, including interferon (IFN)-γ and IL-17, however Tregs can suppress effector cells and dampen a wide spectrum of immune responses. Melatonin (MLT) can regulate the humoral and cellular immune responses, as well as cell proliferation and immune mediators. Treatment with MLT directly interferes with T cell differentiation, controls the balance between pathogenic and regulatory T cells and regulates inflammatory cytokine release. MLT can promote the differentiation of type 1 regulatory T cells via extracellular signal regulated kinase 1/2 (Erk1/2) and retinoic acid-related orphan receptor-α (ROR-α) and suppress the differentiation of Th17 cells via the inhibition of ROR-γt and ROR-α expression through NFIL3. Moreover, MLT inhibits NF-κB signaling pathway to reduce TNF-α and IL-1β expression, promotes Nrf2 gene and protein expression to reduce oxidative and inflammatory states and regulates Bax and Bcl-2 to reduce apoptosis; all of which alleviate the development of autoimmune diseases. Thus, MLT can serve as a potential new therapeutic target, creating opportunities for the treatment of autoimmune diseases. This review aims to highlight recent advances in the role of MLT in several autoimmune diseases with particular focus given to novel signaling pathways involved in Th17 and Tregs as well as cell proliferation and apoptosis.     

Increased nonobese diabetic Th1:Th2 (IFN-gamma:IL-4) ratio is CD4+ T cell intrinsic and independent of APC genetic background

Autoreactive CD4(+) T cells play a major role in the pathogenesis of autoimmune diabetes in nonobese diabetic (NOD) mice. We recently showed that the non-MHC genetic background controlled enhanced entry into the IFN-gamma pathway by NOD vs B6.G7 T cells. In this study, we demonstrate that increased IFN-gamma, decreased IL-4, and decreased IL-10 production in NOD T cells is CD4 T cell intrinsic. NOD CD4(+) T cells purified and stimulated with anti-CD3/anti-CD28 Abs generated greater IFN-gamma, less IL-4, and less IL-10 than B6.G7 CD4(+) T cells. The same results were obtained in purified NOD.H2(b) vs B6 CD4(+) T cells, demonstrating that the non-MHC NOD genetic background controlled the cytokine phenotype. Moreover, the increased IFN-gamma:IL-4 cytokine ratio was independent of the genetic background of APCs, since NOD CD4(+) T cells generated increased IFN-gamma and decreased IL-4 compared with B6.G7 CD4(+) T cells, regardless of whether they were stimulated with NOD or B6.G7 APCs. Cell cycle analysis showed that the cytokine differences were not due to cycle/proliferative differences between NOD and B6.G7, since stimulated CD4(+) T cells from both strains showed quantitatively identical entry into subsequent cell divisions (shown by CFSE staining), although NOD cells showed greater numbers of IFN-gamma-positive cells with each subsequent cell division. Moreover, 7-aminoactinomycin D and 5-bromo-2'-deoxyuridine analysis showed indistinguishable entry into G(0)/G(1), S, and G(2)/M phases of the cell cycle for both NOD and B6.G7 CD4(+) cells, with both strains generating IFN-gamma predominantly in the S phase. Therefore, the NOD cytokine effector phenotype is CD4(+) T cell intrinsic, genetically controlled, and independent of cell cycle machinery.     

Chemotactic responses of IL-4-, IL-10-, and IFN-gamma-producing CD4+ T cells depend on tissue origin and microbial stimulus

Th1- and Th2-polarized immune responses are crucial in the defense against pathogens but can also promote autoimmunity and allergy. The chemokine receptors CXCR3 and CCR4 have been implicated in differential trafficking of IFN-gamma- and IL-4-producing T cells, respectively, but also in tissue and inflammation-specific homing independent of cytokine responses. Here, we tested whether CD4+ T cells isolated from murine tissues under homeostatic or inflammatory conditions exhibit restricted patterns of chemotactic responses that correlate with their production of IFN-gamma, IL-4, or IL-10. In uninfected mice, IL-4-producing T cells preferentially migrated to the CCR4 ligand, CCL17, whereas IFN-gamma-expressing T cells as well as populations of IL-4+ or IL-10+ T cells migrated to the CXCR3 ligand, CXCL9. All cytokine-producing T cell subsets strongly migrated to the CXCR4 ligand, CXCL12. We assessed chemotaxis of T cells isolated from mice infected with influenza A virus or the nematode Nippostrongylus brasiliensis, which induce a strong Th1 or Th2 response in the lung, respectively. Unexpectedly, the chemotactic responses of IL-4+ T cells and T cells expressing the immunosuppressive cytokine IL-10 were influenced not only by the strongly Th1- or Th2-polarized environments but also by their anatomical localization, i.e., lung or spleen. In contrast, IFN-gamma+ T cells exhibited robust chemotaxis toward CXCL9 and had the most consistent migration pattern in both infection models. The results support a model in which the trafficking responses of many effector and regulatory T cells are regulated as a function of the infectious and tissue environments.     

Modulation of Fas-dependent apoptosis: a dynamic process controlling both the persistence and death of CD4 regulatory T cells and effector T cells

We have previously shown that regulatory CD25(+)CD4(+) T cells are resistant to clonal deletion induced by viral superantigen in vivo. In this work we report that isolated CD25(+)CD4(+) T cells activated in vitro by anti-CD3 Ab are resistant to Fas-induced apoptosis, in contrast to their CD25(-)CD4(+) counterparts. Resistance of CD25(+)CD4(+) T cells to Fas-dependent activation-induced cell death is not linked to their inability to produce IL-2 or to their ability to produce IL-10. The sensitivity of both populations to Fas-induced apoptosis can be modulated in vitro by changing the CD25(+)CD4(+):CD25(-)CD4(+) T cell ratio. The sensitivity of CD25(-)CD4(+) T cells to apoptosis can be reduced, while the sensitivity of CD25(+)CD4(+) T cells can be enhanced. Modulation of Fas-dependent apoptosis is associated with changes in cytokine production. However, while CD25(-)CD4(+) T cell apoptosis is highly dependent on IL-2 (production of which is inhibited by CD25(+)CD4(+) T cells in coculture), modulation of CD25(+)CD4(+) T cell apoptosis is IL-2 independent. Taken together, these results suggest that CD25(+)CD4(+) and CD25(-)CD4(+) T cell sensitivity to Fas-dependent apoptosis is dynamically modulated during immune responses; this modulation appears to help maintain a permanent population of regulatory T cells required to control effector T cells.     

Attenuation of immunological symptoms of allergic asthma in mice lacking the tyrosine kinase ITK

Allergic asthma patients manifest airway inflammation and some show increases in eosinophils, T(H)2 cells, and cytokines, increased mucous production in the lung, and elevated serum IgE. This T(H)2-type response suggests a prominent role for T(H)2 cells and their cytokines in the pathology of this disease. The Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) has been shown to play a role in the differentiation and/or function of T(H)2-type cells, suggesting that ITK may represent a good target for the control of asthma. Using a murine model of allergic asthma, we show here that ITK is involved in the development of immunological symptoms seen in this model. We show that mice lacking ITK have drastically reduced lung inflammation, eosinophil infiltration, and mucous production following induction of allergic asthma. Notably, T cell influx into the lung was reduced in mice lacking ITK. T cells from ITK(-/-) mice also exhibited reduced proliferation and cytokine secretion, in particular IL-5 and IL-13, in response to challenge with the allergen OVA, despite elevated levels of total IgE and increased OVA-specific IgE responses. Our results suggest that the tyrosine kinase ITK preferentially regulates the secretion of the T(H)2 cytokines IL-5 and IL-13 and may be an attractive target for antiasthmatic drugs.