Inducible Costimulator Ligand Research Articles

The TH2-polarizing function of atopic interleukin 17 receptor B–positive dendritic cells up-regulated by lipopolysaccharide

BackgroundRecent studies suggest that epithelial cell (EC)-derived cytokines contribute to allergic airway disease exacerbation. ObjectiveTo confirm our hypothesis that atopic dendritic cells (DCs) are activated to up-regulate the receptors of cytokines that mainly derived from ECs and enhance TH2 responses. MethodsThe expressions of interleukin 17 receptor B (IL-17RB) (IL-25 receptor), membrane-bound ST2 (IL-33 receptor), thymic stromal lymphopoietin receptor (TSLPR), granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR), and several functional markers on CD1c+ monocyte-derived DCs (mo-DCs) were detected by flow cytometry. Lipopolysaccharide (LPS)-activated mo-DCs were cocultured with autologous CD4+ T cells, and cytokine production by these T cells was determined by intracellular flow cytometry. ResultsLPS activated both nonatopic and atopic mo-DCs to express a higher level of GM-CSFR but only activated atopic mo-DCs to express increased IL-17RB, which was subsequently activated by IL-25 involved with signal transducer and activator of transcription 5 phosphorylation. In addition, LPS increased the expression of the OX40 ligand (OX40L) but decreased inducible costimulator ligand on atopic CD86+ mo-DCs. More importantly, IL-25 further up-regulated OX40L on atopic CD86+ mo-DCs. After coculturing with LPS-activated mo-DCs from atopic individuals, CD4+ T cells had enhanced inflammatory responses by increased production of IL-4, IL-5, IL-13, and interferon γ (IFN-γ). In contrast, further addition of IL-25 led CD4+ T cells to produce higher level of IL-4 but lower level of IFN-γ. ConclusionAtopic IL-17RB+ DCs can be up-regulated by LPS and promote a TH2-type response, implying that the IL-25/IL-17RB pathway may represent a potential molecular mechanism underlying the regulation of ECs on DCs in allergic airway disease.

TNF-α Regulates Mast Cell Functions by Inhibiting Cell Degranulation

Background/Aims: The aim of this study was to investigate the involvement of inducible co-stimulatory ligand (ICOSL) expression in stimulation of mast cells (MCs) by TNF-α and the ability of TNF-α stimulation of MCs to influence CD4+ T cell differentiation and function. The mechanisms underlying TNF-α stimulation of MCs were also explored. Methods: Mast cells and CD4⁺ T cells were prepared from C57BL/6 mice (aged 6–8 weeks). ICOSL expression by MCs was measured by real-time PCR and flow cytometry, and levels of IL-4, IL-10 and IFN-γ were measured by ELISA. Results: ICOSL expression by MCs was increased by TNF-α stimulation, and resulted in interaction with CD4+ T cells. The IL-4 and IL-10 levels in the co-culture system increased, while IFN-γ levels decreased. Furthermore, CD4+CD25+Foxp3+ T cell proliferation was induced by co-culture with TNF-α-stimulated MCs. The mechanism by which TNF-α stimulated MCs was dependent on the activation of the MAPK signaling pathway. Conclusion: TNF-α upregulated the expression of ICOSL on mast cells via a mechanism that is dependent on MAPK phosphorylation. TNF-α-treated MCs promoted the differentiation of regulatory T cells and induced a shift in cytokine expression from a Th1 to a Th2 profile by up-regulation ICOSL expression and inhibition of MC degranulation. Our study reveals a novel mechanism by which mast cells regulate T cell function.

Protective Role of ICOS and ICOS Ligand in Corneal Transplantation and in Maintenance of Immune Privilege

The interaction between the inducible costimulatory molecule (ICOS) and ICOS ligand (ICOSL) has been implicated in the differentiation and functions of T cells. The purpose of the present study was to determine the role of ICOS-ICOSL in the immune privilege of corneal allografts. Expression of ICOS and ICOSL mRNA from mouse eyes was assessed by RT-PCR. Corneas of C57BL/6 mice were orthotopically transplanted into the eyes of ICOS-/- BALB/c recipients and BALB/c wild-type (WT) recipients treated with anti-ICOSL mAb, and graft survival was assessed. A separate set of WT and ICOS-/- BALB/c mice received an anterior chamber injection of C57BL/6 splenocytes, and induction of allospecific anterior chamber-associated immune deviation (ACAID) was assessed. In vitro, cornea was incubated with T cells from WT and ICOS-/- BALB/c mice, and destruction of corneal endothelial cells (CECs) and the population of Foxp3+ CD25+ CD4+ T cells was assessed. Inducible costimulatory molecule ligand mRNA was constitutively expressed in the cornea, iris-ciliary body, and retina. Allograft survival in ICOS-/- recipients and WT recipients treated with anti-ICOSL mAb was significantly shorter than in control recipients. Anterior chamber-associated immune deviation was induced less efficiently in ICOS-/- mice. Destruction of CECs by alloreactive ICOS-/- T cells was enhanced compared with WT T cells. After coincubation with allogeneic corneal tissue, the proportion of regulatory T cells was significantly greater among WT T cells than in ICOS-/- T cells. The expression of ICOSL in the cornea and the ICOS-mediated induction of Foxp3+ CD4+ regulatory T cells may contribute to successful corneal allograft survival.

Type 2 innate lymphoid cell suppression by regulatory T cells attenuates airway hyperreactivity and requires inducible T-cell costimulator–inducible T-cell costimulator ligand interaction

Atopic diseases, including asthma, exacerbate type 2 immune responses and involve a number of immune cell types, including regulatory T (Treg) cells and the emerging type 2 innate lymphoid cells (ILC2s). Although ILC2s are potent producers of type 2 cytokines, the regulation of ILC2 activation and function is not well understood. In the present study, for the first time, we evaluate how Treg cells interact with pulmonary ILC2s and control their function. ILC2s and Treg cells were evaluated by using invitro suppression assays, cell-contact assays, and gene expression panels. Also, human ILC2s and Treg cells were adoptively transferred into NOD SCID γC-deficient mice, which were given isotype or anti-inducible T-cell costimulator ligand (ICOSL) antibodies and then challenged with IL-33 and assessed for airway hyperreactivity. We show that induced Treg cells, but not natural Treg cells, effectively suppress the production of the ILC2-driven proinflammatory cytokines IL-5 and IL-13 both invitro and invivo. Mechanistically, our data reveal the necessity of inducible T-cell costimulator (ICOS)-ICOS ligand cell contact for Treg cell-mediated ILC2 suppression alongside the suppressive cytokines TGF-β and IL-10. Using a translational approach, we then demonstrate that human induced Treg cells suppress syngeneic human ILC2s through ICOSL to control airway inflammation in a humanized ILC2 mouse model. These findings suggest that peripheral expansion of induced Treg cells can serve as a promising therapeutic target against ILC2-dependent asthma.

Functional exhaustion of CD4+ T cells induced by co-stimulatory signals from myeloid leukaemia cells.

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T-cell exhaustion. T-cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co-stimulatory molecules to T-cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia-derived co-stimulatory signals on CD4+ T helper (Th) cell exhaustion, which may limit anti-tumour immunity. Here, CD86 and inducible T-cell co-stimulator ligand (ICOS-LG) co-stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co-cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro-generated exhausted Th cells were defined by up-regulation of programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG3) and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) inhibitory receptors. They were reluctant to proliferate upon re-stimulation and produced reduced amounts of interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). Nonetheless, IL-2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co-stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD-1, TIM-3 and/or LAG3 was identified together with CD86+ and/or ICOS-LG+ myeloid blasts. Collectively, co-stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.

Abstract LB-092: Induction of IL-17-mediated cellular immunity targeting MUC1 through vaccination

Abstract Prophylactic vaccination targeting tumor-associated antigens (TAA) holds the potential to prevent the development of cancer. Clinical evidence has demonstrated a protective effect of cellular immunity against the mucin 1 (MUC1) TAA, expressed by ∼90% of mammary tumors. Nevertheless, conventional vaccination strategies have failed to prevent breast cancer due to the induction of terminally differentiated, short-lived Th1 cells of limited functionality. By contrast, IL-17-producing T cells, including CD4+ Th17 and CD8+ Tc17 subtypes, account for pathogenic agents that escape Th1-mediated control and are thus endowed with specialized, non-redundant mechanisms to promote inflammation at the cell and tissue levels. Recent studies have confirmed that signaling through the inducible T-cell co-stimulator (ICOS) pathway is sufficient to polarize T cells towards IL-17-expression in vitro, providing a novel mechanism to induce Th17/Tc17-mediated immunity against tumors in vivo. To generate IL-17-mediated antitumor immunity through vaccination, we examined the immunogenicity of combinations of adenoviral vectors encoding MUC1, ICOS ligand (ICOS-L) and the cytokines IL-6 and IL-23, implicated in Th17 development and expansion, respectively. Vaccination against MUC1, in the presence or absence of IL-17-polarizing conditions, overcame immune tolerance in transgenic mice recognizing MUC1 as a “self” antigen, inducing robust MUC1-specific intracellular IFNγ production from CD8+ T cells detected by FACS. Co-administration of vectors encoding MUC1 and ICOS-L, with or without vector-derived IL-6 and/or IL-23, induced significant IL-17 production in response to mitogen from both CD4+ and CD8+ T cells compared to MUC1 vaccination alone. Interestingly, ICOS-L augmentation resulted in the upregulation of the master Th17 transcription factor RORγt in CD4+, but not CD8+ T cells. Additionally, administration of ICOS-L, with or without IL-6 and/or IL-23, increased levels of granzyme B and CD107a in both CD4+ and CD8+ T cell populations following MUC1 vaccination, suggesting IL-17-polarization enhances T cell cytotoxicity. Studies are currently ongoing to evaluate the efficacy of IL-17-polarizing or conventional MUC1-targeting vaccination to protect MUC1-tolerant, transgenic mice against an orthotopic challenge of MUC1-expressing EO771 cells and findings will be presented at the AACR Annual Meeting. To the best of our knowledge, this study represents the first demonstration that IL-17-polarized immunity may be induced through vaccination, providing a novel platform for the generation of MUC1-specific immunity for the prevention of breast cancer. Citation Format: Amanda C. LaRue, Rebecca Carrell, Lindsay T. McDonald, Dayvia A. Russell, Stephen P. Ethier, Adam C. Soloff. Induction of IL-17-mediated cellular immunity targeting MUC1 through vaccination. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-092.

Transcriptomic Signature of the CD24hi CD38hi Transitional B Cells Associated With an Immunoregulatory Phenotype in Renal Transplant Recipients.

The role of B cells after transplant regarding allograft rejection or tolerance has become a topic of major interest. Recently, in renal transplant recipients, a B cell signature characterized by the overexpression of CD19+ CD38hi CD24hi transitional B cells has been observed in operationally tolerant patients and in belatacept-treated patients with significantly lower incidence of donor-specific antibodies. The phenotypic and functional characterization of these transitional B cells is far from exhaustive. We present the first transcriptomic and phenotypic analysis associated with this cell phenotype. Three populations were studied and compared: (i) transitional CD24hi CD38hi , (ii) CD24+ CD38- , and (iii) CD24int CD38int B cells. Transcriptome bioinformatic analysis revealed a particular signature for the CD24hi CD38hi population. Phenotypic analysis showed that CD24hi CD38hi transitional B cells also expressed CD9, CD10, CD1b and inducible T cell costimulator ligand (ICOS-L) markers. In addition, we found enrichment of IL-10+ cells among CD24hi CD38hi cells expressing ICOS-L and CD1b, the latter showing regulatory properties. Renal transplant recipients treated with belatacept exhibited significant expression of CD1b. Our results show that transitional CD24hi CD38hi B cells exhibit a distinct and specific profile, and this could be helpful for understanding of immune-regulatory mechanisms and immune monitoring in the field of organ transplant and autoimmune disease.

ICOS Promotes the Function of CD4+ Effector T Cells during Anti-OX40-Mediated Tumor Rejection.

ICOS is a T-cell coregulatory receptor that provides a costimulatory signal to T cells during antigen-mediated activation. Antitumor immunity can be improved by ICOS-targeting therapies, but their mechanism of action remains unclear. Here, we define the role of ICOS signaling in antitumor immunity using a blocking, nondepleting antibody against ICOS ligand (ICOS-L). ICOS signaling provided critical support for the effector function of CD4(+) Foxp3(-) T cells during anti-OX40-driven tumor immune responses. By itself, ICOS-L blockade reduced accumulation of intratumoral T regulatory cells (Treg), but it was insufficient to substantially inhibit tumor growth. Furthermore, it did not impede antitumor responses mediated by anti-4-1BB-driven CD8(+) T cells. We found that anti-OX40 efficacy, which is based on Treg depletion and to a large degree on CD4(+) effector T cell (Teff) responses, was impaired with ICOS-L blockade. In contrast, the provision of additional ICOS signaling through direct ICOS-L expression by tumor cells enhanced tumor rejection and survival when administered along with anti-OX40 therapy. Taken together, our results showed that ICOS signaling during antitumor responses acts on both Teff and Treg cells, which have opposing roles in promoting immune activation. Thus, effective therapies targeting the ICOS pathway should seek to promote ICOS signaling specifically in effector CD4(+) T cells by combining ICOS agonism and Treg depletion. Cancer Res; 76(13); 3684-9. ©2016 AACR.

Imperatorin exerts antiallergic effects in Th2-mediated allergic asthma via induction of IL-10-producing regulatory T cells by modulating the function of dendritic cells

Imperatorin is a furanocoumarin compound which exists in many medicinal herbs and possesses various biological activities. Herein, we investigated the antiallergic effects of imperatorin in asthmatic mice and explored the immunomodulatory actions of imperatorin on immune cells. We used a murine model of ovalbumin (OVA)-induced asthma to evaluate the therapeutic potential of imperatorin. Additionally, bone marrow-derived dendritic cells (DCs; BMDCs) were used to clarify whether imperatorin exerts an antiallergic effect through altering the ability of DCs to regulate T cells. Oral administration of imperatorin to OVA-sensitized and −challenged mice decreased serum OVA-specific immunoglobulin E (IgE) production, attenuated the airway hyperresponsiveness (AHR), and alleviated airway inflammation in a dose-dependent manner. Notably, secretions of Th2 cytokines and chemokines were reduced, and numbers of interleukin (IL)-10-producing regulatory T cells (Tregs) increased in imperatorin-treated mice. Imperatorin inhibited proinflammatory cytokines and IL-12 production but enhanced IL-10 secretion by lipopolysaccharide (LPS)-stimulated BMDCs. Compared to fully mature DCs, imperatorin-treated DCs expressed high levels of the inducible costimulatory ligand (ICOSL) and Jagged1 molecules, and had the regulatory capacity to promote the generation of IL-10-producing CD4+ T cells in vitro. Additionally, imperatorin directly suppressed activated CD4+ T-cell proliferation and cytokine production. Imperatorin may possess therapeutic potential against Th2-mediated allergic asthma not only via stimulating DC induction of Tregs but also via direct inhibition of Th2 cell activation. These findings provide new insights into how imperatorin affects the Th2 immune response and the development of imperatorin as a Treg-type immunomodulatory agent to treat allergic asthma.

Development of a novel monoclonal antibody to human inducible co-stimulator ligand (ICOSL): Biological characteristics and application for enzyme-linked immunosorbent assay

ICOSL (B7-H2, CD275), a co-stimulatory molecule of the B7 superfamily, functions as a positive signal in immune response. To investigate whether ICOSL could be released into sera and the possible biological function of soluble ICOS (sICOSL), we generated and characterized a functional anti-human ICOSL monoclonal antibody (mAb), 20B10, and developed a novel enzyme-linked immunosorbent assay (ELISA) based on two anti-human ICOSL antibodies with different epitope specificities. Using the ELISA system, we found that sICOSL in the serum of healthy donors increases in an age-dependent manner and that the matrix metalloproteinase inhibitor (MMPI) could suppress sICOSL production. Together, these data demonstrate that the existence of circulating sICOSL in human serum might play an important role in immunoregulation.

Inducible T-cell co-stimulator ligand (ICOSL) blockade leads to selective inhibition of anti-KLH IgG responses in subjects with systemic lupus erythematosus

ObjectivesTo evaluate the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of single-dose and multiple-dose administration of AMG 557, a human anti-inducible T cell co-stimulator ligand (ICOSL) monoclonal antibody, in subjects...

Abstract IA08: Mechanisms and signaling pathways involved in regulation of activating and inhibitory T cell responses

Abstract IA08: Mechanisms and signaling pathways involved in regulation of activating and inhibitory T cell responses

Immunomodulating effects of the anti-viral agent Silibinin in liver transplant patients with HCV recurrence.

BackgroundSilibinin has been shown to have anti-HCV activity and immune-modulating properties by regulating dendritic cell (DC) function. DCs are antigen-presenting cells that, together with regulatory T cells (Treg), play a pivotal role in controlling alloimmune, as well as anti-HCV immune responses.MethodsTwelve liver transplant patients with HCV recurrence received iv infusion of Silibinin (iv-SIL) for 14 consecutive days. Using flow cytometry, before and at the end of treatment, we determined the frequencies of circulating myeloid (m) and plasmacytoid (p) DC and Treg and the expression of costimulatory/coregulatory molecules by the DC subsets and Treg. Statistical analysis was performed using the paired Student’s t test and Pearson correlation test.ResultsAfter iv-SIL treatment, we observed an elevated plasmacytoid dendritic cell (pDC)/myeloid dendritic cell (mDC) ratio, while pDC displayed lower HLA-DR and higher immunoglobulin-like transcript 4 (ILT4), CD39, and HLA-G expression compared to the pretreatment baseline. In addition, after iv-SIL, mDC showed increased inducible costimulator ligand (ICOSL) expression. No changes were detected in Treg frequency or programed death (PD)-1 expression by these cells. Moreover, several correlations between DC/Treg markers and clinical parameters were detected.ConclusionsThis descriptive study, in liver transplant patients with HCV recurrence, reveals the impact of iv-SIL on DC and Treg. The changes observed in circulating pDC and mDC that have previously been associated with tolerogenic conditions shed new light on how iv-SIL may regulate anti-viral and alloimmunity. We have also observed multiple clinical correlations that could improve the clinical management of liver transplant patients and that deserve further analysis.

Insights into Group 2 Innate Lymphoid Cells in Human Airway Disease.

Recent discoveries have led to the identification of a novel group of immune cells, the innate lymphoid cells (ILCs). The members of this group are divided into three subpopulations: ILC1s, ILC2s, and ILC3s. ILC2s produce Th2 cytokines, IL-4, IL-5, and IL-13, upon activation by epithelial cell-derived cytokines, lipid mediators (cysteinyl leukotrienes and prostaglandin D2), and TNF family member TL1A and promote structural and immune cell responses in the airways after antigen exposure. In addition, ILC2 function is also influenced by inducible T cell costimulator (ICOS)/ICOS-ligand (ICOS-L) interactions via direct contact between immune cells. The most common airway antigens are allergens and viruses which are highly linked to the induction of airway diseases with underlying type 2 inflammation including asthma and allergic rhinitis. Based on recent findings linking ILC2s and airway Th2 responses, there is intensive investigation into the role of ILC2s in human disease with the hope of a better understanding of the pathophysiology and the discovery of novel potential therapeutic targets. This review summarizes the recent advances made in elucidating ILC2 involvement in human Th2 airway disease.

IMPS-16MESENCHYMAL TYPE GLIOMA STEM CELL EXPRESS ICOS LIGAND AND UPREGULATE ICOS+IL-10+T CELLS

Tumor heterogeneity of high-grade glioma (HGG) is recognized by four clinically relevant subtypes based on core gene signatures. Glioma stem cells, which is isolated from patient-derived glioma sphere cultures, also resemble either the proneural (PN) or mesenchymal (MES) transcriptomal subtypes differ significantly in their biological characteristics. The aims of this study were the immunobiological characterization of MES and PN GSCs. GSCs were generated and examined by microarray, and flow cytometry for expression of B7 family. The B7 family ligands and their receptors play a critical role in activation, expansion, as well as contraction of T cell populations. Only MES GSCs express Inducible Co-stimulatory Ligand (ICOSLG). In vitro analysis, we found MES GSC have the potential to prime CD4+ T-cells to differentiate into IL-10-producing T regulatory cells through ICOSLG. Moreover, we found that a subset of the PN GSCs undergoes differentiation to a MES state in a TNF-alfa-dependent manner with an associated upregulation of CD44 and ICOSLG. We further show that the MES signature, CD44 expression, and ICOSLG expression correlate with shorter survival in patients with GBM.

Activated Conventional T-Cells Are Present in Langerhans Cell Histiocytosis Lesions Despite the Presence of Immune Suppressive Cytokines.

Langerhans cell histiocytosis (LCH) lesions are characterized by neoplastic CD1a(+)/Langerin(+) histiocytes (LCH-cells) and display many features of chronic inflammation. Cancer cells can escape immune-surveillance through intra-tumoral secretion of immune-suppressive cytokines. We therefore studied by immunohistochemistry the local cytokine milieu and phenotypic characteristics of T-cells and LCH-cells present in LCH lesions collected from 25 therapy naïve patients. LCH biopsies predominantly expressed interleukin-10 (IL-10) (10/25), transforming growth factor-beta (TGF-β) (9/25), or both cytokines (6/25). The absolute number of CD3(+)T-cells and the CD3(+)FOXP3(-) conventional cell (T-CONV) versus the CD3(+)FOXP3(+) regulatory T-cell (T-REG) was comparable for each suppressive cytokine profile (5:1). IL-10-expressing lesions contained, however, a higher proportion of T-CONV expressing the activation markers CD25 98% (38%-100%) and inducible costimulatory molecule (ICOS) 86% (47%-100%) than lesions wherein solely TGF-β was detected (CD25(+) 20% (6%-54%); ICOS(+) 29% (7%-51%)). Virtually all T-REG expressed CD25 and ICOS in IL-10 lesions, whereas TGF-β(+) lesions contained a lower proportion of ICOS(+) T-REG (P=0.05). IL-10(+) lesions contained more LCH-cells expressing high intensity of ICOS ligand (ICOSL) compared with TGF-β(+) lesions (P=0.03). ICOS expression by lesion-infiltrating T-CONV and T-REG positively correlated to the extent of ICOSL expression by LCH-cells (P=0.004). Our study points out that the combined detection of interlesional IL-10 and ICOSL expression by LCH-cells is associated with the highest prevalence of activated T-CONV. Immune profiling of LCH-affected tissues obtained at the time of diagnosis may set the stage for the development of new types of therapies, which aim at local boosting of immune cells that recognize and eliminate neoplastic LCH-cells.

Abstract 3184: Tumor-entrained dendritic cells promote ICOS/ICOSL-dependent Th17-like responses in pancreatic adenocarcinoma

Abstract BACKGROUND: Ranking 4th among cancer-related deaths worldwide, pancreatic adenocarcinoma (PDAC) boasts a dismal prognosis. The robust immune infiltrate that comprises its tumor microenvironment (TME) can influence disease progression. Tumor rejection by T lymphocytes relies on proper guidance by dendritic cells (DCs). This antigen-education is manifested by the DCs’ expression of co-stimulatory factors, such as ICOS-ligand (ICOSL). In this study, we plan to elucidate the mechanisms used by PDAC-entrained DC to modify T cell effector function and differentiation. METHODS: To establish our in vivo model, either PBS or 2.5e+5 “FC1242” murine PDAC cells derived from the tumor of a KPC mouse (LSL-KrasG12D; Trp53R172H; pdxCre/+) were injected into the distal pancreata of C57BL/6J (WT) mice. After 20 days, mice were euthanized and the spleen and tumor were assayed. DC phenotype was determined by flow cytometry (FC). DC function was assessed by mixed-leukocyte reactions (MLR) and peptide-specific proliferation assays, +/- neutralizing mAbs against ICOSL or ICOS. Supernatant cytokines were assessed at 84h using cytometric bead assays (CBA). Intracellular cytokines were analyzed by FC after 5h re-stimulation with PMA/IMN. To assess in vivo proliferation, 2.5e+6 of both OT1 and OT2 T cells labeled with CFSE or CPD, respectively, were co-injected into WT mice and challenged with 5e+5 OVA-loaded DC after 24h. Proliferation was assessed 4 days post-DC inoculation. RESULTS: Splenic FC1242-DC had lower expression of MHC2 and CD83, while tumor-infiltrating DC (TME-DC) displayed higher levels of each, relative to controls. TME-DC also exhibited higher expression of ICOSL, CD80, and CD86. Despite no difference in ICOSL expression between splenic groups, MHC2 and CD83 were substantially lower on ICOSL+ DC of FC1242 spleen, while highest on that of the TME, relative to controls. We found a higher fraction of CD11b+ myeloid DC (mDC) and a lower fraction of cross-presenting CD8α+ DC (xDC) in both tumor-bearing groups relative to controls. FC1242-mDC expressed higher CD80 and PDL1 than sham-mDC, while FC1242-xDC displayed lower CD80 and no difference in PDL1. Also, tumor-entrained DC had a diminished propensity for CD8+ T cell expansion in vitro and in vivo, resulting in lower Granzyme B, IFNγ, and TNFα. Interestingly, DC tumor-entrainment improved CD4+ T cell expansion and augmented IL-17A, IL-6, G-CSF secretion, while diminishing IFNγ, TNFα, IL-13, and IL-2. Blockade of the ICOS-pathway hindered IL17 production, and amplified production of IFNγ and TNFα by T cells. CONCLUSION: This study suggests that DC possess a vital role as liaisons between the TME and cell-mediated immunity. Furthermore, Th17 cells in the TME can accelerate PDAC progression. This makes the ICOS-pathway especially attractive, as it can modify anti-tumor immunity while circumventing the barriers of drug-delivery to the TME. Citation Format: Rocky M. Barilla, Raul Caso, Antonina Avanzi, Anjlee Panjwani, Xiaopei L. Zeng, Steve Matthews, Daniel M. Tippens, Lena Tomkoetter, Elliot M. Levie, Alejandro Torres-Hernandez, Donnele Daley, George Miller. Tumor-entrained dendritic cells promote ICOS/ICOSL-dependent Th17-like responses in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3184. doi:10.1158/1538-7445.AM2015-3184

Effects of histamine and its antagonists on murine T-cells and bone marrow-derived dendritic cells.

We determined the effects of histamine and its antagonists on the surface marker expression of dendritic cells (DCs) and the influence of lipopolysaccharide (LPS), histamine, and histamine receptor antagonists on DCs and T-cells. The bone marrow was extracted from the femurs and tibiae of 6- to 8-week-old female Balb/c mice and cultured in medium containing penicillin, streptomycin, L-glutamine, fetal calf serum, or granulocyte macrophage colony-stimulating factor (GM-CSF) alone or with interleukin (IL)-4. The cells received three different doses of LPS and histamine, plus three different doses of descarboethoxyloratadine (DCL). We assayed the supernatant for various cytokines. The spleen cells of DO11.10 mice were examined by flow cytometry, which included labeling and sorting CD4+ T-cells, as well as coculture of DCs and T-cells with ovalbumin (OVA)323–339 peptide. Histamine or histamine plus DCL did not affect the expression of major histocompatibility complex class II, CD11c, CD11b, CD86, and CD80. However, GM-CSF increased the expression of all markers except CD80. Histamine increased interferon-γ production in GM-CSF + IL-4-cultured cells; it also enhanced IL-10 production, but suppressed IL-12 production in LPS-stimulated DCs with no DCL. Cimetidine inhibited IL-10 production and restored IL-12 secretion in LPS-treated DCs. LPS increased IL-10 and decreased IL-12 levels. GM-CSF + IL-4-generated DCs had a stronger stimulatory effect on DO11.10 T-cell proliferation than GM-CSF-generated DCs. Inducible costimulator ligand expression was higher in GM-CSF + IL-4- than in GM-CSF-generated DC groups after 2 days of coculture, but decreased 4 days later. IL-13 production was higher in bone marrow DCs generated with GM-CSF than in those generated with GM-CSF + IL-4. OVA-pulsed DCs and OVA-plus-DCL DCs showed increased IL-12 levels. OVA plus LPS increased both IL-10 and interferon-α. Although histamine or histamine receptor-1 antagonists did not influence DC LPS-driven maturation, they influenced cytokine production. LPS and GM-CSF influenced surface marker expression and cytokine production.

Aberrant germinal center formation, follicular T-helper cells, and germinal center B-cells were involved in chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is an important complication after allogeneic hematopoietic stem cell transplantation (HSCT). To define the roles of T-cells and B-cells in cGVHD, a murine minor histocompatibility complex-mismatched HSCT model was used. Depletion of donor splenocyte CD4(+) T-cells and B220(+) B-cells alleviated cGVHD. Allogeneic recipients had significantly increased splenic germinal centers (GCs), with significant increases in follicular T-helper (Tfh) cells and GC B-cells. There were increased expressions in Tfh cells of inducible T-cell co-stimulator (ICOS), interleukin (IL)-4 and IL-17, and in GC B-cells of B-cell activating factor receptor and ICOS ligand. Depletion of donor splenocyte CD4(+) T-cells abrogated aberrant GC formation and suppressed Tfh cells and GC B-cells. Interestingly, depletion of donor splenocyte B200(+) B-cells also suppressed Tfh cells in addition to GC B-cells. These results suggested that in cGVHD, both Tfh and GC B-cells were involved, and their developments were mutually dependent. The mammalian target of rapamycin (mTOR) inhibitor everolimus was effective in suppressing cGVHD, Tfh cells, and GC B-cells, either as a prophylaxis or when cGVHD had established. These results implied that therapeutic targeting of both T-cells and B-cells in cGVHD might be effective. Signaling via mTOR may be another useful target in cGVHD.

ICOSL-mediated signaling is essential for the survival and functional maturation of germinal center B cells through the classical NF-κB pathway (IRM10P.611)

Abstract The molecular mechanisms by which helper T-cells support B-cell activation, survival and functional differentiation in the germinal center (GC) are not well-defined. To date, signaling through CD40 by CD40L is the only established receptor-ligand signaling pathway initiated by helper T-cells. Here we show that helper T-cells provide helper signal to B-cells through the inducible T-cell co-stimulator (ICOS)-ICOS ligand (ICOSL) interaction. We found that reverse ICOSL signaling on GC B-cells is essential and sufficient to promote GC B-cell survival and functional maturation; whereas CD40-mediated signaling plays a minor role during GC reaction, although critical in initiating the GC response. In vivo ICOSL engagement by ICOS-Ig fusion protein can rescue impaired GC formation and antibody production in ICOS-deficient mice. Various lines of ICOSL mutant mice, including pan-B cell-specific ICOSL-deficient mice, GC B cell-specific ICOSL-deficient mice, and ICOSL intra-cellular domain mutant mice, all show defective humoral response and impaired GC reaction. ICOSL reverse signaling in GC B cells is dependent on activation of the classical NF-kB signal pathway, resulting in increased BCL2, c-MYC and FOXO1 expression that supports GC B cell survival and differentiation. Thus, CD40-CD40L interaction mainly supports the initiation of GC response, whereas ICOS-ICOSL collaboration provides bi-directional signals that support the maintenance and functional maturation of GC B cells.