Novel IL-15 dendritic cells have a potent immunomodulatory effect in immunotherapy of multiple myeloma

Dendritic cells (DCs) are the most potent antigen-presenting cells, and have thus been used in clinical cancer vaccines. However, the effects of DC vaccines are still limited, leading researchers to explore novel ways to make them effective. In this study, we investigated whether human monocyte-derived DCs generated via the addition of interleukin 15 (IL-15) had a higher capacity to induce antigen-specific T cells compared to conventional DCs. We isolated CD14⁺ monocytes from peripheral blood from multiple myeloma (MM) patients, and induced immature DCs with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 in the presence or absence of IL-15 for 4–6 days. Then we generated mature DCs (mDCs) with lipopolysaccharide for another 2 days [IL-15 mDCs (6 days), IL-15 mDCs (8 days), and conventional mDCs (8 days)]. IL-15 mDCs (6 days) showed higher expression of MHC I and II, CD40, CD86, and CCR7, and the secretion of IFN-γ was significantly higher compared to conventional mDCs. IL-15 mDCs (6 days) showed superior polarization of naïve T cells toward Th1 cells and a higher proportion of activated T cells, cytokine-induced killer (CIK) cells, and natural killer (NK) cells for inducing strong cytotoxicity against myeloma cells, and lower proportion of regulatory T cells compared to conventional mDCs. These data imply that novel multipotent mDCs generated by the addition of IL-15, which can be cultivated in 6 days, resulted in outstanding activation of T cells, CIK cells and NK cells, and may facilitate cellular immunotherapy for cancer patients.     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions

Introduction

The aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)–primed CD1c myeloid dendritic cells (mDCs).

Methods

Expression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression.

Results

PD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation.

Conclusion

SF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation.     

Cutting edge: IL-4-induced protection of CD4+CD25- Th cells from CD4+CD25+ regulatory T cell-mediated suppression

CD4(+)CD25(+) T regulatory (Treg) cells are a CD4(+) T cell subset involved in the control of the immune response. In vitro, murine CD4(+)CD25(+) Treg cells inhibit CD4(+)CD25(-) Th cell proliferation induced by anti-CD3 mAb in the presence of APCs. The addition of IL-4 to cocultured cells inhibits CD4(+)CD25(+) Treg cell-mediated suppression. Since all cell types used in the coculture express the IL-4Ralpha chain, we used different combinations of CD4(+)CD25(-) Th cells, CD4(+)CD25(+) Treg cells, and APCs from wild-type IL-4Ralpha(+/+) or knockout IL-4Ralpha(-/-) mice. Results show that the engagement of the IL-4Ralpha chain on CD4(+)CD25(-) Th cells renders these cells resistant to suppression. Moreover, the addition of IL-4 promotes proliferation of IL-4Ralpha(+/+)CD4(+)CD25(+) Treg cells, which preserve full suppressive competence. These findings support an essential role of IL-4 signaling for CD4(+)CD25(-) Th cell activation and indicate that IL-4-induced proliferation of CD4(+)CD25(+) Treg cells is compatible with their suppressive activity.     

Interleukin 10 (IL-10)-mediated Immunosuppression: MARCH-I INDUCTION REGULATES ANTIGEN PRESENTATION BY MACROPHAGES BUT NOT DENDRITIC CELLS*

Efficient immune responses require regulated antigen presentation to CD4 T cells. IL-10 inhibits the ability of dendritic cells (DCs) and macrophages to stimulate antigen-specific CD4 T cells; however, the mechanisms by which IL-10 suppresses antigen presentation remain poorly understood. We now report that IL-10 stimulates expression of the E3 ubiquitin ligase March-I in activated macrophages, thereby down-regulating MHC-II, CD86, and antigen presentation to CD4 T cells. By contrast, IL-10 does not stimulate March-I expression in DCs, does not suppress MHC-II or CD86 expression on either resting or activated DCs, and does not affect antigen presentation by activated DCs. IL-10 does, however, inhibit the process of DC activation itself, thereby reducing the efficiency of antigen presentation in a March-I-independent manner. Thus, IL-10 suppression of antigen presenting cell function in macrophages is March-I-dependent, whereas in DCs, suppression is March- I-independent.     

The cathepsin B inhibitor, z-FA-FMK, inhibits human T cell proliferation in vitro and modulates host response to pneumococcal infection in vivo

The cathepsin B inhibitor, benzyloxycarbonyl-phenyl-alanyl-fluoromethylketone (z-FA-FMK) at nontoxic doses was found to be immunosuppressive and repressed human T cell proliferation induced by mitogens and IL-2 in vitro. We showed that z-FA-FMK suppresses the secretion of IL-2 and IFN-gamma as well as the expression of IL-2R alpha-chain (CD25) in activated T cells, whereas the expression of the early activated T cell marker, CD69, was unaffected. Furthermore, z-FA-FMK blocks NF-kappaB activation, inhibits T cell blast formation, and prevents cells from entering and leaving the cell cycle. z-FA-FMK inhibits the processing of caspase-8 and caspase-3 to their respective subunits in resting T cells stimulated through the Ag receptor, but has no effect on the activation of these caspases during Fas-induced apoptosis in proliferating T cells. When administered in vivo, z-FA-FMK significantly increased pneumococcal growth in both lungs and blood, compared with controls, in a mouse model of intranasal pneumococcal infection. Because host response to bronchopneumonia in mice is T cell dependent, our collective results demonstrated that z-FA-FMK is immunosuppressive in vitro and in vivo.     

2B4 (CD244) signaling via chimeric receptors costimulates tumor-antigen specific proliferation and in vitro expansion of human T cells

Regulatory NK cell receptors can contribute to antigen-specific adaptive immune responses by modulating T cell receptor (TCR)-induced T cell activation. We investigated the potential of the NK cell receptor 2B4 (CD244) to enhance tumor antigen-induced activation of human T cells. 2B4 is a member of the CD2 receptor subfamily with both activating and inhibitory functions in NK cells. In T cells, its expression is positively associated with the acquisition of a cytolytic effector memory phenotype. Recombinant chimeric receptors that link extracellular single-chain Fv fragments specific for the tumor-associated surface antigens CD19 and G_D2 to the signaling domains of human 2B4 and/or TCRζ were expressed in non-specifically activated peripheral blood T cells by retroviral gene transfer. While 2B4 signaling alone failed to induce T cell effector functions or proliferation, it significantly augmented the antigen-specific activation responses induced by TCRζ. 2B4 costimulation did not affect the predominant effector memory phenotype of expanding T cells, nor did it increase the proportion of T cells with regulatory phenotype (CD4+CD25^hiFoxP3+). These data support a costimulatory role for 2B4 in human T cell subpopulations. As an amplifier of TCR-mediated signals, 2B4 may provide a powerful new tool for immunotherapy of cancer, promoting sustained activation and proliferation of gene-modified antitumor T cells.     

Mycophenolic acid inhibits IL-2-dependent T cell proliferation,but not IL-2-dependent survival and sensitization to apoptosis

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5'-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5'-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27(Kip1). Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G(1) phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-x(L) up-regulation by IL-2 or other cytokines whose receptors share the common gamma-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.     

Dendritic cells post-maturation are reprogrammed with heightened IFN-gamma and IL-10

Mature dendritic cells (mDCs) undergo "exhaustion" in producing cytokines. Nevertheless, whether this "exhaustion" of mDCs is selective to certain cytokines, or whether mDCs have specific cytokine-producing profiles has yet to be defined. Herein, we investigated the cytokine production in vitro by immature DCs (iDCs) and LPS-induced mDCs. Compared to iDCs, mDCs produced comparable levels of IL-6 and TNF-alpha. Strikingly, mDCs produced significantly higher IFN-gamma and IL-10. IL-12 production of mDCs was suppressed. Kinetic studies of the responses of iDCs and mDCs to LPS or CD40L showed that mDCs acquired progressively heightened activity in producing IFN-gamma and IL-10. TNF-alpha-, IL-6-producing capability of mDCs was maintained. Nevertheless, IL-12 production by mDCs was not recovered at any time point. Mature DCs were potent in priming both Th1 and Th2 cells. In conclusion, upon maturation, DCs are reprogrammed with a distinct cytokine-secreting profile, which may play an important role in regulating T cell functions.     

Immunosuppression induced by immature dendritic cells is mediated by TGF-beta/IL-10 double-positive CD4+ regulatory T cells.

Dendritic cells (DC) have important functions in T cell immunity and T cell tolerance. Previously, it was believed that T cell unresponsiveness induced by immature DC (iDC) is caused by the absence of inflammatory signals in steady-state in vivo conditions and by the low expression levels of costimulatory molecules on iDC. However, a growing body of evidence now indicates that iDC can also actively maintain peripheral T cell tolerance by the induction and/or stimulation of regulatory T cell populations. In this study, we investigated the in vitro T cell stimulatory capacity of iDC and mature DC (mDC) and found that both DC types induced a significant increase in the number of transforming growth factor (TGF)-beta and interleukin (IL)-10 double-positive CD4(+) T cells within 1 week of autologous DC/T cell co-cultures. In iDC/T cell cultures, where antigen-specific T cell priming was significantly reduced as compared to mDC/T cell cultures, we demonstrated that the tolerogenic effect of iDC was mediated by soluble TGF-beta and IL-10 secreted by CD4(+)CD25(-)FOXP3(-) T cells. In addition, the suppressive capacity of CD4(+) T cells conditioned by iDC was transferable to already primed antigen-specific CD8(+) T cell cultures. In contrast, addition of CD4(+) T cells conditioned by mDC to primed antigen-specific CD8(+) T cells resulted in enhanced CD8(+) T cell responses, notwithstanding the presence of TGF-beta(+)/IL-10(+) T cells in the transferred fraction. In summary, we hypothesize that DC have an active role in inducing immunosuppressive cytokine-secreting regulatory T cells. We show that iDC-conditioned CD4(+) T cells are globally immunosuppressive, while mDC induce globally immunostimulatory CD4(+) T cells. Furthermore, TGF-beta(+)/IL-10(+) T cells are expanded by DC independent of their maturation status, but their suppressive function is dependent on immaturity of DC.     

IL-21 counteracts the regulatory T cell-mediated suppression of human CD4+ T lymphocytes

High expression of IL-21 and/or IL-21R has been described in T cell-mediated inflammatory diseases characterized by defects of counterregulatory mechanisms. CD4(+)CD25(+) regulatory T cells (Treg) are a T cell subset involved in the control of the immune responses. A diminished ability of these cells to inhibit T cell activation has been documented in immune-inflammatory diseases, raising the possibility that inflammatory stimuli can block the regulatory properties of Treg. We therefore examined whether IL-21 controls CD4(+)CD25(+) T cell function. We demonstrate in this study that IL-21 markedly enhances the proliferation of human CD4(+)CD25(-) T cells and counteracts the suppressive activities of CD4(+)CD25(+) T cells on CD4(+)CD25(-) T cells without affecting the percentage of Foxp3(+) cells or survival of Treg. Additionally, CD4(+)CD25(+) T cells induced in the presence of IL-21 maintain the ability to suppress alloresponses. Notably, IL-21 enhances the growth of CD8(+)CD25(-) T cells but does not revert the CD4(+)CD25(+) T cell-mediated suppression of this cell type, indicating that IL-21 makes CD4(+) T cells resistant to suppression rather than inhibiting CD4(+)CD25(+) T cell activity. Finally, we show that IL-2, IL-7, and IL-15, but not IL-21, reverse the anergic phenotype of CD4(+)CD25(+) T cells. Data indicate that IL-21 renders human CD4(+)CD25(-) T cells resistant to Treg-mediated suppression and suggest a novel mechanism by which IL-21 could augment T cell-activated responses in human immune-inflammatory diseases.     

Costimulation via glucocorticoid-induced TNF receptor in both conventional and CD25+ regulatory CD4+ T cells

The glucocorticoid-induced TNF receptor (GITR), which is a member of the TNF receptor family, is expressed preferentially at high levels on CD25+CD4+ regulatory T cells and plays a key role in the peripheral tolerance that is mediated by these cells. GITR is also expressed on conventional CD4+ and CD8+ T cells, and its expression is enhanced rapidly after activation. In this report we show that the GITR provides a potent costimulatory signal to both CD25+ and CD25- CD4+ T cells. GITR-mediated stimulation induced by anti-GITR mAb DTA-1 or GITR ligand transfectants efficiently augmented the proliferation of both CD25-CD4+ and CD25+CD4+ T cells under the limited dose of anti-CD3 stimulation. The augmentation of T cell activation was further confirmed by the enhanced cell cycle progression; early induction of the activation Ags, CD69 and CD25; cytokine production, such as IL-2, IFN-gamma, IL-4, and IL-10; anti-CD3-induced redirected cytotoxicity; and intracellular signaling, assessed by translocation of NF-kappaB components. GITR costimulation showed a potent ability to produce high amounts of IL-10, which resulted in counter-regulation of the enhanced proliferative responses. Our results highlight evidence that GITR acts as a potent and unique costimulator for an early CD4+ T cell activation.     

Possible mechanism for the alpha subunit of the interleukin-2 receptor (CD25) to influence interleukin-2 receptor signal transduction

The receptors for interleukin 2 (IL-2) and interleukin 15 (IL-15) in T cells share the IL-2R beta subunit (CD122) and gamma(C) subunit but have private alpha subunits. Despite utilizing the same receptor chains known to be necessary and sufficient to transduce IL-2 signals the two cytokines manifest different cellular effects. It is commonly held that the alpha subunit of the IL-2R (CD25) is involved solely in the generation of a high affinity receptor complex. This is questioned by the development of autoimmune diseases in instances where the expression of CD25 is absent. The timely expression of CD25 in the thymus has been linked with clonal deletion. Evidence from peripheral T cells indicates that survival signals arising from the intermediate affinity IL-2R (lacking CD25) do not require the activation of Janus kinase 3 (Jak3) but do require the presence of the membrane proximal region of the gamma(C) chain. This particular signalling pathway is not observed in the high affinity receptor complex where Jak3 is activated. Recent data point to CD25 having a surface distribution consistent with it being localized within membrane microdomains. Here we suggest that in the absence of CD25 expression, IL-2R activation occurs within the soluble membrane fraction. This membrane environment and the absence of CD25 promotes Jak3 independent signal transduction and induction of antiapoptotic mechanisms. T cell antigen receptor (TCR) signalling leads to the induction of CD25 expression, which localizes to membrane microdomains. There is a dynamic pre-association of CD25 and CD122 leading to the loose association of the heterodimer with membrane microdomains. High affinity IL-2R signalling in the context of CD25 and the microdomain environment is characterized by Jak3 activation. The relative levels of high to intermediate affinity receptor signalling determines whether a cell proliferates or undergoes activation induced cell death dependent upon cell status.     

Cooperation of invariant NKT cells and CD4+CD25+ T regulatory cells in the prevention of autoimmune myasthenia

CD1d-restricted NKT cells and CD4+CD25+ regulatory T (Treg) cells are thymus-derived subsets of regulatory T cells that have an important role in the maintenance of self-tolerance. Whether NKT cells and Treg cells cooperate functionally in the regulation of autoimmunity is not known. We have explored this possibility in experimental autoimmune myasthenia gravis (EAMG), an animal model of human myasthenia gravis, induced by immunization of C57BL/6 mice with the autoantigen acetylcholine receptor. We have demonstrated that activation of NKT cells by a synthetic glycolipid agonist of NKT cells, alpha-galactosylceramide (alpha-GalCer), inhibits the development of EAMG. alpha-GalCer administration in EAMG mice increased the size of the Treg cell compartment, and augmented the expression of foxp3 and the potency of CD4+CD25+ cells to inhibit proliferation of autoreactive T cells. Furthermore, alpha-GalCer promoted NKT cells to transcribe the IL-2 gene and produce IL-2 protein. Depletion of CD25+ cells or neutralization of IL-2 reduced the therapeutic effect of alpha-GalCer in this model. Thus, alpha-GalCer-activated NKT cells can induce expansion of CD4+CD25+ Treg cells, which in turn mediate the therapeutic effects of alpha-GalCer in EAMG. Induced cooperation of NKT cells and Treg cells may serve as a superior strategy to treat autoimmune disease.     

T cell-dependent hapten-specific and polyclonal B cell responses require release of interleukin 5

CD4+ T cells in the mouse have recently been subdivided into two major subpopulations which differ in their functional activities and in the lymphokines they produce. Although cloned T cells lines representative of both sets will activate B cells in polyclonal responses, only the subset producing interleukin 4 (IL-4) will activate antigen-specific B cells in linked recognition assays. This suggested that IL-4 was essential for such responses. In the present experiments, the requirements were compared for B cell activation in specific as opposed to polyclonal antibody responses by T cell clones of the helper (IL-4 producing) subset. It was found that specific responses involve primarily small B cells, whereas polyclonal responses activate exclusively the large B cells. Second, polyclonal B cell responses can proceed in the absence of T:B contact, whereas specific responses require physical interaction of the two cells. Third, it was found that interleukin 5 (IL-5, formerly known as T cell replacing factor/B cell growth factor II) is essential for these polyclonal responses by inhibition of such responses with monoclonal anti-IL-5 antibody. Anti-IL-5 also inhibits specific antibody responses involving direct T:B interaction. Thus, IL-5 is clearly a critical mediator of differentiation to immunoglobulin secretion of activated B cells, whether such B cells are obtained as large B cells from freshly isolated spleen cells or are initially activated in an IL-4-dependent fashion by cognate interaction by a helper T cell clone.     

Inhibition of Breast Cancer Resistance Protein (ABCG2) in Human Myeloid Dendritic Cells Induces Potent Tolerogenic Functions during LPS Stimulation

Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c⁺ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c⁺ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4⁺ T cells and promoted expansion of CD25⁺FOXP3⁺ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.