Absence Of pDCs Research Articles

Plasmacytoid dendritic cells are central regulators of the humoral ChAdOx1 nCoV-19 vaccine response in a human tonsil organoid model

Abstract Studying human vaccine responses is limited by the difficulty of access to secondary lymphoid tissues. To overcome this, we have optimized a human tonsil organoid model to investigate the processes that regulate the humoral responses to the ChAdOx1 nCoV-19 vaccine. Stimulation of tonsil cultures with ChAdOx1 nCoV-19 induced broad innate immune activation and cytokine secretion within 24 hours. This induced (global and antigen-specific) B cell activation and spike-specific antibody secretion over the following 14-day culture period. Stimulation with an irrelevant ChAdOx1 GFP vector did not induce spike-specific antibody secretion. Plasmacytoid dendritic cells (pDCs) showed the highest rate of transduction and activation. Critically, pDC-depletion decreased both innate and humoral responses. IFN-⍺ blockade phenocopied pDC depletion while supplementing IFN-⍺ to pDC-depleted cultures rescued humoral responses. IL-6 was also critical for optimal humoral responses, and its production was modulated in an IFN-⍺-dependent manner even in the absence of pDCs, suggesting cross-talk of multiple key signaling pathways. Overall, this model reveals a critical role for pDCs in inducing humoral responses to ChAdOx1 nCoV-19, primarily through IFN-⍺. IFN-⍺ appears to act directly and indirectly through IFN-induced IL-6 secretion to augment the humoral response. This model has the potential for studying other vaccine platforms.

Identification of Metabolic Enzymes ENO1 and KMO As Novel Immunotherapeutic Targets in Myeloma

Introduction. Our previous studies showed an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs are present in increased numbers in MM BM, promote tumor growth and survival, protect tumor from therapy-induced cytotoxicity; and suppress immune responses (Chauhan et al, Cancer Cell 2009, 16:309-323) . Aberrant pDC function is evidenced from their interactions with MM tumor and T/NK effector cells in the BM milieu (Ray et al, Leukemia 2015, 29:1441-1444) . Direct interruption of pDC-MM and pDC-T cells interactions and their sequelae utilizing novel targeted agents is necessary to further enhance both anti-tumor immunity and cytotoxicity. Based on DNA microarray analysis, we identified several novel targets on MM cells that are upregulated after coculture with pDCs, and focused on metabolic pathway enzymes that are upregulated both in pDC and MM cells. We identified alpha-Enolase (ENO1) and Kynurenine 3-monooxygenase (KMO) as the two most promising therapeutic targets, based on their roles in normal and cancer metabolism. ENO1 is a glycolytic enzyme, and its surface localization is a unique feature of the tumor cells. Moreover, ENO1 promotes tumor metastasis. KMO is one of the two rate-limiting enzymes involved in eukaryotic tryptophan catabolic kynurenine pathway; and importantly, its overexpression has been reported to inhibit functional immune responses in cancer. Here, we utilized our pDC/MM or pDC/T cells co-culture model to show that targeting ENO1 or KMO generates MM-specific cytotoxic T lymphocyte activity.MethodsDNA MicroArray Analysis: MM.1S cells were cocultured with pDCs for 72h, separated using CD138 Microbeads, and harvested. Poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM.1S cells cultured in the presence vs absence of pDCs were compared, and heat map was generated (>1.5-fold change in transcript was considered significant, CI > 95%). pDC-induced overexpression of target proteins : MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in target protein expression. CD138+ tumor cells were gated out, and KMO-FITC or ENO1-AlexaFluor488-stained cells were analyzed. C TL activity assays : MM patient BM CD8+ T cells were cocultured with autologous pDCs at 1:10 (pDC:T) ratio in the presence or absence of KMO inhibitor Ro 61-8048 (10 nM) or ENO1 inhibitor ENOblock (0.1-0.2 µM) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T:MM), followed by quantification of viable MM cells by FACS. Statistical parameters were calculated using GraphPad Prism. Pharmacological inhibitors were obtained from Selleck Chemicals.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of both ENO1 and KMO transcripts in MM cells (1.772 and 2.153-fold, respectively, vs MM.1S alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high KMO and surface ENO1 expression. Coculture of MM cells with pDCs further increased ENO1 surface expression levels in MM cells (5-6-fold increase; n=3; p = 0.003), as well as the ENO1-positive MM cell population (3-4-fold versus MM alone; mean ± SD; n=3; p = 0.005). 3) Like ENO1, pDCs also increased both KMO expression on MM cells and KMO-positive MM cells (MFI: 2-3-fold in the presence vs absence of pDCs; p = 0.006). 4) Functional studies showed that treatment of pDC-T cell cocultures with ENO1 antagonists triggers a robust MM-specific CD8+ CTL activity against autologous CD138-positive MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2-fold vs control; p = 0.008). Finally, 6) KMO inhibitor Ro 61-8048 activates patient BM-CD8+T cells in autologous pDCs/T cell cocultures, and triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0-fold vs control; p = 0.009).Conclusions Our study shows that pDC-MM interactions upregulate tumor promoting and immunosuppressive metabolic enzymes in MM. Blockade of ENO1 or KMO enzymes tiggers MM-specific CD8-positive CTL activity. These data therefore identify novel immune-based therapeutic strategies targeting ENO1 and KMO to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Preclinical Validation of Alpha-Enolase (ENO1) As a Novel Immunometabolic Target in Multiple Myeloma

Introduction. Bone marrow plasmacytoid dendritic cells (pDCs) in patients with multiple myeloma (MM) both promote tumor growth, survival, and drug resistance, as well as induce decreased T/NK effector cell function and immune suppression. Delineation of the mechanism(s) mediating pDCs-MM-T-NK cells interactions may therefore identify novel therapeutic targets to enhance anti-MM immunity. Using gene expression profiling, we show that pDC-MM interactions trigger significant upregulation of the immunosuppressive metabolic enzyme alpha-Enolase (ENO1) in both pDCs and MM cells. ENO-1 functions as a glycolytic enzyme and plasminogen receptor which is overexpressed on the surface of tumor cells and MM pDCs. Here, we utilized our coculture models of patient autologous pDC-T-NK-MM cells to examine whether targeting ENO1, either alone or in combination can enhance anti-MM immunity in the BM milieu. Methods Gene expression profiles of MM cells cultured in the presence vs absence of pDCs were compared, and a heat map was generated (>1.5-fold change was considered significant, CI > 95%). MM cells were co-cultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in ENO1 expression. Cytotoxic T lymphocyte (CTL) and NK cell activity assays: MM patient BM CD8+ T or NK-cells were cocultured with autologous pDCs (pDC1:T/NK10 ratio) in the presence or absence of ENO1 inhibitor/ENO1i ENOblock (0.2 µM) or anti-ENO1 Ab for 5 days; MM pre-stained cells were added for 24h (10T/NK:1MM), followed by quantification of viable MM cells by FACS. Anti-PD-L1 Ab (5 ug/ml) or HDAC 6 selective inhibitor ACY-241 (0.2 uM) were utilized for combination studies with ENO1i. CD107a expression was quantified in a degranulation assay. Results GEP analysis showed that pDCs induce upregulation of ENO1 transcript in MM (1.8-fold vs MM alone; n = 3; CI > 95%). Protein expression analysis showed that ENO1 is expressed in both pDC and MM cells; and importantly, that pDC-MM coculture further increases ENO1 expression in MM cells (5-6-fold; n=3; p = 0.003). The ENO+ MM cell population is also increased after pDC-MM cell coculture (3-4-fold vs MM; mean ± SD; n = 3; p = 0.005). Blockade of ENO1 with ENO inhibitor (ENOi) activates pDCs, as evidenced by increase in pDCs maturation/activation markers (CD80/CD83/CD86). Importantly, ENOi restores the ability of pDCs to trigger T cell activation and proliferation (n = 3; p = 0.018). Above all, ENOi increases pDC-induced MM-specific CD8+ CTL activity (p = 0.006), as well as NK cell-mediated cytolytic activity against autologous tumor cells (p = 0.005). Moreover, pDC-mediated MM-specific CD8+ CTL activity was effective even against allogeneic HLA-A2+ U266 MM cells (p = 0.008). Consistent with CTL and NK cell activation, ENO1i increases expression of surface CD107a on CD8+ T and NK cells (p = 0.006 and p= 0.0112, respectively; n = 7). The combination of ENO1i and anti-PD-L1 Ab induces more robust allogeneic and autologous MM-specific CD8+ CTL activity than ENO1i alone (% MM lysis: ENO1i: 34%; ENO1i + anti-PD-L1 Ab: 54%; n = 7; p = 0.0013). Finally, the combination of ENOi and ACY-241 also enhances anti-MM immune responses (% MM lysis: ENO1i: 35%; ENO1i + ACY-241: 54%; n = 8; p = 0.0044). Conclusions Our preclinical data provide the basis for novel immune-based therapeutic approaches targeting immunosuppressive metabolic alpha-enolase enzyme ENO1 to restore anti-MM immunity and improve patient outcome. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Anderson:Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau.

Distinguishing pustular psoriasis and acute generalized exanthematous pustulosis on the basis of plasmacytoid dendritic cells and MxA protein.

Distinguishing acute generalized exanthematous pustulosis (AGEP) and pustular psoriasis (PS) can be challenging. Staining for plasmacytoid dendritic cells, or PDCs (producer of IFN-α/β), and MxA (an IFN-α/β inducible protein) may help discriminate these entities. Forty-three cases of AGEP and PS were compiled from two academic institutions. All cases were examined for CD123+ PDCs, eosinophils, acanthosis, papillomatosis, suprapapillary plate thinning, tortuous dilated capillaries, single necrotic keratinocytes, papillary dermal edema, vasculitis, eosinophil exocytosis, intraepidermal pustules, and subcorneal pustules. A subset of cases (n = 26) was stained for MxA. Perivascular and intraepidermal PDCs, dilated tortuous vessels, and MxA expression in the dermal inflammatory infiltrate were significantly (P < 0.05) in favor of a diagnosis of PS. The absence of PDCs and presence of eosinophils favored a diagnosis of AGEP (P < 0.05). We found compelling evidence for the use of CD123 to highlight PDCs in these cases. The presence of PDCs and expression of MxA in dermal inflammatory infiltrate, as well as absence of eosinophils and presence of tortuous dilated capillaries favored a diagnosis of PS. Expression of MxA in the dermal infiltrate corresponds with a Th1 pathway in PS and may indicate a Th1 component in the early initial phase of AGEP.

Identification of Alpha-Enolase (ENO1) As a Novel Immunometabolic Target in Multiple Myeloma

Introduction. Our previous studies demonstrated an integral role of bone marrow (BM) plasmacytoid dendritic cells (pDCs) in MM pathogenesis. pDCs interactions with tumor cells and T/NK effector cells in the MM-BM milieu induce immune suppression and MM cell proliferation (Chauhan et al, Cancer Cell 2009, 16:309-323;Ray et al, Leukemia 2015, 29:1441-1444). Here we performed DNA microarray analysis to identify and functionally validate the aberrant moleculer mechanisms mediating pDC-MM interactions with a goal to restore anti-tumor immunity and increase cytotoxicity. Our data shows that pDC-MM interactions trigger significant upregulation of the metabolic enzyme alpha-Enolase (ENO1) in both pDCs and MM cells. ENO1 is known to play a role in metabolic pathways in normal and cancer cells. It functions as a glycolytic enzyme and plasminogen receptor on the surface of tumor cells and MM pDCs. The surface localization of overexpressed ENO1 is a unique feature of tumor cells. We utilized our pDC/MM or pDC/T cells coculture models to show that targeting ENO1 generates MM-specific CTL activity and enhances NK-cell mediated lytic activity. Additionally, we examined whether the combination of ENO1 antagonist with anti-PD-L1 Ab or HDCAi ACY-241 enhances anti-tumor immunity and cytotoxicity in MM.MethodsMicroArray: MM cells were co-cultured with pDCs for 72h, and then separated using CD138 Microbeads; poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared, and heat map was generated ( >1.5-fold change was considered significant, CI > 95%). pDC-induced overexpression of target proteins: MM cells were cocultured with pDCs for 24h, followed by multicolor flow analyses to determine the pDC-induced change in ENO1 expression. CTL or NK-lytic activity assays: MM patient BM CD8+ T or NK-cells were cocultured with autologous pDCs (pDC:T/NK; 1:10 ratio) in the presence or absence of ENO1 inhibitor/ENO1i ENOblock (0.1-0.2 µM; Selleck Chemicals) or anti-ENO1 Ab for 5 days; pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T/NK:MM), followed by quantification of viable MM cells by FACS. Combination studies were performed using ENO1 antagonists and anti-PD-L1 Ab (5 ug/ml) or ACY-241 (0.1-0.2 uM). Degranulation assay was done to quantify cell surface CD107a. Osteoclast (OCL) function was measured using TRAP staining. Osteoblast (OBL) formation and subsequent bone mineralization were assessed using Alizarin Red staining.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of ENO1 transcript in MM (1.772-fold vs MM alone; n=3; CI > 95%). 2) Both pDC and MM cells showed high surface ENO1 expression, and pDC-MM coculture further increased surface ENO1 expression in MM cells (5-6 fold; n=5; p = 0.003), as well as the ENO1+ MM cell population (3-4 fold vs MM; mean ± SD; n=5; p = 0.005). 3) ENO1 antagonists directly activate pDCs and upregulate functional markers CD80 (1.2-fold) and CD83 (1.3-fold). 4) Treatment of pDC-T cell co-cultures with ENO1i triggers a robust MM-specific CD8+ CTL activity against autologous CD138+ MM cells (2 fold in treatment vs control; p = 0.005). 5) pDC-mediated MM-specific CD8+ CTL activity was also confirmed using allogeneic HLA-A2+ U266 MM cells (1.8-2 fold; p = 0.008). 6) In concert with above findings, ENO1 blockade also increased surface CD107a expression on NK and CD8+T cells (CD107a+ NK: 1.4-fold; p = 0.003; CD107a+ CD8+T: 1.3 fold; p= 0.001). 7) Importantly, combination of ENO1i and anti-PD-L1 Ab triggers a more robust allogeneic and autologous MM-specific CD8+ CTL activity than ENO1i alone (%MM lysis: ENO1i: 20%; ENO1i + anti-PD-L1 Ab: 54%; n=7; p = 0.006). Combination of ENOi and ACY-241 also enhances the anti-tumor immunity in MM vs ENOi alone. Finally, 8) ENO1i blocked monocyte-derived osteoclast formation, as well as restored MM patient BM-derived osteoblast formation.Conclusions pDC-MM interactions upregulate immunosuppressive metabolic alpha-enolase enzyme ENO1. Importantly, blockade of ENO1 tiggers MM-specific CD8+ CTL and NK-cell-medaited cytoxicity against MM cells. Our findings therefore identify novel immune-based therapeutic strategies targeting ENO1 to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership.

Targeting Tryptophan Catabolic Kynurenine Pathway Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Introduction. Plasmacytoid dendritic cells (pDCs) play an integral role in MM pathogenesis (Chauhan et al, Cancer Cell 2009, 16:309-323). pDCs interactions with tumor cells and T/NK effector cells in the MM-BM milieu induce immune suppression and MM cell proliferation (Chauhan et al, Cancer Cell 2009, 16:309-323;Ray et al, Leukemia 2015, 29:1441-1444). A direct interruption of pDC-MM and pDC-T cells interactions with novel agents will enhance cytotoxicity and anti-tumor immunity. In this context, our DNA microarray analysis identified upregulation of tryptophan catabolic kynurenine (Kyn) pathway during pDC-MM interactions. To date, the role of Kyn pathway in MM is unclear, but two rate-limiting enzymes of Kyn pathway, kynurenine 3-monooxygenase (KMO) and indoleamine 2,3-dioxygenase 1 (IDO1), have been implicated in various cancers. Overexpression of these enzymes causes hyper-accumulation of terminal metabolites, thereby triggering immune suppression via disruption of bidirectional signaling axes between antigen-presenting cells and T cells. In our study, we found that KMO and IDO1 are increased in both pDCs and MM cells during pDC-MM interactions. To assess the functional significance of these findings, we used our pDC/MM or pDC/T cells co-culture models, and show that targeting Kyn pathway generates MM-specific CTL activity. Moreover, the combination of KMO/IDO1 inhibitors with anti-PD-L1 Ab enhances anti-tumor immunity and cytotoxicity in MM.MethodsMicroArray: MM cells were cocultured with pDCs for 72h; separated using CD138 Microbeads; poly RNA was subjected to microarray analysis using HG-U133 plus 2.0 plus Affymetrix chip. Gene expression patterns for MM cells cultured in the presence vs absence of pDCs were compared ( >1.5-fold change was considered significant, CI > 95%). pDC-induced overexpression of target proteins: MM cells were cocultured with pDCs for 24h; analyzed by flow to detect KMO/IDO1 expression. CTL or NK activity assays: Minimally cytotoxic concentrations of inhibitors were used to assess immune functions. MM-BM CD8+ T or NK- cells were cultured with autologous pDCs (pDC:T/NK; 1:10 ratio) in the presence or absence of KMO (Ro 61-8048: 100 nM) or IDO1 (INCB 024360: 0.1 µM) inhibitors (Selleck Chemicals) for 5 days; drug washed, pre-stained MM cells were added for 24-48h (E/T ratio 10:1, T/NK:MM), followed by quantification of viable MM cells by FACS. Degranulation assay was quantified using cell surface CD107a. Osteoclast and osteoblast formations were assessed from TRAP and Alizarin Red staining, respectively.Results. 1) Normalized microarray expression profile showed pDC-induced upregulation of KMO in MM cells (2.153 fold vs MM alone; n=3; CI > 95%). 2) Both pDCs and MM cells expressed KMO and IDO1; pDC-MM coculture further increased KMO expression in MM cells (2-3-fold vs MM; p = 0.006); KMO+ MM cell populations also increased after coculture (~1.2 fold; p <0.05). Importantly, IDO1 expression in MM cells is also enhanced after co-culture; validating the importance of Kyn pathway in the context of pDC-MM interactions (MFI: 1.5-2 fold; p=0.006); IDO1+ MM cell populations increased by ~1.5 fold after coculture (p <0.05). 3) KMOi directly activates pDCs and upregulates functional markers on pDCs (CD80: 1.19 fold; CD83: 1.4 fold; p <0.05). 4) KMOi activates patient BM-CD8+T cells in autologous pDCs/T cell coculture; triggers robust allogeneic and autologous MM-specific CD8+ CTL activity (~2.0 fold vs control; p = 0.009). 5) In agreement with above findings, KMOi also increased surface CD107a expression on NK and CD8+T cells (CD56+NK: 1.41 fold; p = 0.004; CD8+T: 1.6 fold; p= 0.001). 6) Importantly, the combination of KMOi or IDO1i and anti-PD-L1 Ab (5 µg/ml) triggers a more robust MM-specific CD8+ CTL activity than single agent (%MM viability: KMOi: 72%; KMOi + anti-PD-L1 Ab: 57%; IDOi: 82%; IDOi + anti-PD-L1 Ab: 70%; n=7; p = 0.01). Finally, 7) Both KMOi and IDO1i blocked monocyte-derived osteoclast formation, as well as restored MM patient BM-derived osteoblast formation.Conclusions pDC-MM interactions upregulate immunosuppressive enzymes in Kyn pathway; and importantly, blockade of Kyn pathway enzymes induces MM-specific CD8+ CTL activity. Our preclinical data therefore provides provides the basis for novel immune-based therapeutic approaches targeting kynurenine (Kyn) pathway enzymes KMO and IDO1 to enhance MM cytotoxicity and restore anti-MM immunity. DisclosuresAnderson:C4 Therapeutics: Equity Ownership; Oncopep: Equity Ownership; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Takeda Millennium: Consultancy.

Conventional Dendritic Cells Impair Recovery after Myocardial Infarction.

Ischemic myocardial injury results in sterile cardiac inflammation that leads to tissue repair, two processes controlled by mononuclear phagocytes. Despite global burden of cardiovascular diseases, we do not understand the functional contribution to pathogenesis of specific cardiac mononuclear phagocyte lineages, in particular dendritic cells. To address this limitation, we used detailed lineage tracing and genetic studies to identify bona fide murine and human CD103+ conventional dendritic cell (cDC)1s, CD11b+ cDC2s, and plasmacytoid DCs (pDCs) in the heart of normal mice and immunocompromised NSG mice reconstituted with human CD34+ cells, respectively. After myocardial infarction (MI), the specific depletion of cDCs, but not pDCs, improved cardiac function and prevented adverse cardiac remodeling. Our results showed that fractional shortening measured after MI was not influenced by the absence of pDCs. Interestingly, however, depletion of cDCs significantly improved reduction in fractional shortening. Moreover, fibrosis and cell areas were reduced in infarcted zones. This correlated with reduced numbers of cardiac macrophages, neutrophils, and T cells, indicating a blunted inflammatory response. Accordingly, mRNA levels of proinflammatory cytokines IL-1β and IFN-γ were reduced. Collectively, our results demonstrate the unequivocal pathological role of cDCs following MI.

Combination of a Novel HDAC 6 Inhibitor ACY-241 with Anti-PD-L1 Antibody Enhances Anti-Tumor Immunity and Cytotoxicity in Multiple Myeloma

Introduction Histone deacetylases (HDACs) are attractive therapeutic targets, and selective HDAC inhibitors (HDACi), alone or in combination with other anti-cancer agents are useful treatment strategies in multiple myeloma (MM). HDACs are also involved in immune regulation in hematological malignancies. For example, blockade of HDACs upregulate immunecheckpoints such as PD-1 ligand (PD-L1). Furthermore, combination of HDACi with PD-1/PD-L1 checkpoint blockade significantly improves immunotherapy in a murine B16F10 model [Woods et al, Cancer Immunol Res. 2015; 3:1375-85]. Here we utilized an orally bioavalable HDAC6 selective inhibitor ACY-241 currently under clinical trials to examine whether combination of ACY-241 with anti-PD-L1 antibody enhances anti-MM immunity. For these studies, we utilized our previously published co-culture models of immune effector cells (plasmacytoid dendritic cells, pDCs, T cells, NK cells) and MM cells (Chauhan et al, Cancer Cell 2009, 16:309-323; Ray et al, Leukemia 2015, 29:1441-1444). Specifically, in MM we have shown that: 1) PD-1 is highly expressed on T and NK cells, and both pDCs and MM cells express PD-L1; 2) MM-pDCs confer T- and NK cell immune suppression via PD-L1/PD-1 immune checkpoints and 3) blockade of PD-L1-PD-1 signaling axis by anti-PD-L1 Ab generates MM-specific CD8+ CTL activity, as well as enhances NK-cell-mediated MM cell cytolytic activity. We here examined whether combination of ACY-241 with anti-PD-L1 Ab increases anti-tumor immunity and cytotoxicity in MM.Methods Minimally cytotoxic concentrations of ACY-241 (0.1-0.2 µM) or anti-PD-L1 Ab (5 µg/ml) were utilized to assess immune function. In T cell proliferation assay, MM patient pDCs were co-cultured with autologous T cells (pDC:T ratio; 1:10) in the presence of ACY-241, anti-PD-L1 Ab, or a combination of ACY-241 and anti-PD-L1 Ab for 5 days; proliferation was then quantified using CellTrace Violet proliferation kit for flow cytometry. CTL activity assays MM CD8+ T cells were cultured with autologous pDCs (1:10 pDC/T ratio) with ACY-241, anti-PD-L1 Ab, or both for 5 days; cells were then washed to remove drug, and pre-stained target MM cells (20:1 E/T ratio) were then added for another 2-3 days, followed by quantification of viable MM cells using FACS. NK-cell mediated cytotoxicity was assessed using flow-based CFSE-stained target lysis assays, as well as degranulation assay quantifying cell surface CD107a. Statistical parameters were calculated using GraphPad Prism.Results. 1) Treatment of RPMI-8226, U266, and MM.1S cells with ACY-241 increases PD-L1 expression; and blockade of PD-L1 using anti-PD-L1 Ab increases the anti-MM activity of ACY-241. 2) Combination of ACY-241 and anti-PDL1 Ab enhances MM-patient pDC-induced NK cell-mediated cytolytic activities against allogenic MM cells (p < 0.008; 2-5 fold increase for combination versus anti-PD-L1 Ab alone). NK cell mediated lysis was insignificant in the absence of pDCs. 3) ACY-241, anti-PD-L1 Ab or their combination, activates patient BM-CD8+T cells in autologous pDCs/T cell co-cultures. Importantly, combination of ACY-241 and anti-PD-L1 Ab triggers a more robust allogenic and autologous MM-specific CD8+ CTL activity than anti-PD-L1 Ab alone (1.5-2.0 fold increase in combination regimen vs anti-PD-L1 Ab; p = 0.01). 4) Treatment of pDC-T cell cocultures with ACY-241 plus anti-PD-L1 Ab enhanced cytotoxic activity of CD8+ T cells against HLA-A2+ U266 cells (1.5-2 fold in combinations vs anti-PD-L1 Ab; p = 0.003). 5) In concert with above findings, ACY-241 plus anti-PD-L1 Ab increases CD107a expression in NK and CD8+T cells (CD107a+ NK: 5 fold; p = 0.003; CD107a+ CD8+T: 5 fold; p= 0.001; vs anti-PD-L1 Ab alone). Finally, 6) pDC-mediated CD8+ CTL activity was also confirmed using HLA-A2+ HCT116 colon cancer cells (~2 fold loss in viability in combination vs anti-PD-L1 Ab; p = 0.006)Conclusions The combination of HDAC6 inhibitor ACY-241 and anti-PD-L1 Ab enhances pDC-induced T and NK cell-mediated cytolytic acitvities in MM. Our study provides the basis for combining ACY-241 with anti-PD-L1 Ab to restore immune function, and enhance MM cytotoxicity, and improve patient outcome in MM. DisclosuresHideshima:Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Chauhan:C4 Therapeutics: Equity Ownership; Epicent Rx: Consultancy; Oncopeptide AB: Consultancy; Stemline Therapeutics, Inc.: Consultancy. Anderson:Celgene Corporation: Consultancy; Millennium Pharmaceuticals: Consultancy; Novartis AG: Consultancy; Bristol-Myers Squibb:: Consultancy; Oncopep: Other: Scientific Founder; Acetylon: Other: Scientific Founder.

HIV-1 infection induces a type I IFN response in primary CD4+ T cells

Background: Production of type 1 interferon (IFN) in response to viral infection requires the detection of viral nucleic acids or proteins by at least one cellular pattern recognition receptor. HIV-1 is capable of inducing an elaborate IFN response in plasmacytoid dendritic cells (pDCs) through Toll-like receptor mediated recognition of the entering viral RNA genome. However in T cells and macrophages, the main HIV target cells, IFN induction by HIV-1 is considered to be weak or undetectable. Here, we re-evaluate the occurrence of an innate immune response upon HIV-1 infection of primary CD4+ T cells (PBLs). Methods and results: Type 1 IFN induction was evaluated in activated purified PBLs during productive infection with HIV-1. In cells from several donors we observed a clear increase in IFNβ and IFNα mRNA levels, as well as induction of several interferon stimulated genes (ISGs) upon HIV-1 infection. The levels of induction progressed concurrently with the levels of HIV infection in the culture. Secreted type 1 IFN protein biological activity was furthermore detected in supernatants of HIV-1 infected cultures. To rule out residual contaminating pDCs as the source of IFN production in these cultures, we performed additional depletion of these cells from CD4+ T cell populations prior to infection and found that type 1 IFN induction was maintained in the absence of pDCs. Furthermore, we evaluated the biological relevance of the detected levels of type 1 IFN by addition of neutralizing antibodies to IFNβ and IFNα during infection. In presence of the both antibodies, we observed an increase in HIV-1 infection, indicating that the levels of HIV-induced IFN in the T cell cultures are sufficient to have an antiviral effect. To gain more insight in the mechanism of IFN induction by HIV-1, we used inhibitors of reverse transcription, integration and of the HIV-1 protease during single-cycle infection of VSV-pseudotyped HIV-1. We found that integration of the HIV provirus is required for full IFN induction in PBLs, indicating that newly expressed HIV RNA or newly produced HIV proteins are important for evoking an innate immune response in T cells. Conclusion: These data show that activated PBLs are capable of producing relevant levels of type 1 IFN in response to HIV-1 infection and suggest recognition of newly expressed HIV RNA or proteins as a main trigger of the innate response in these cells. Characterization of the responsible innate immune pathway and pattern recognition receptors will be subject of further investigation.

In Vivo Ablation of Plasmacytoid Dendritic Cells Inhibits Autoimmunity through Expansion of Myeloid-Derived Suppressor Cells

Autoimmunity ensues upon breakdown of tolerance mechanism and priming of self-reactive T cells. Plasmacytoid dendritic cells (pDCs) constitute a unique cell subset that participates in the activation of autoreactive T cells but also has been shown to be critically involved in the induction of self-tolerance. However, their functional importance during the priming phase of an organ-specific autoimmune response remains unclear. In this study, we demonstrate that absence of pDCs during myelin antigenic challenge resulted in amelioration of experimental autoimmune encephalomyelitis and reduced disease severity. This was accompanied by significantly decreased frequency of myelin-specific T cells in the draining lymph nodes and inhibition of Th1 and Th17 immune responses. Unexpectedly, in vivo ablation of pDCs increased myelopoiesis in the bone marrow and specifically induced the generation of CD11b(hi)Gr1(+) myeloid-derived suppressor cells (MDSCs). Furthermore, we demonstrate that pDC depletion enhanced the mobilization of MDSCs in the spleen, and that sorted MDSCs could potently suppress CD4(+) T cell responses in vitro. Importantly, pDC-depleted mice showed increased levels of MCP-1 in the draining lymph nodes, and in vivo administration of MCP-1 increased the frequency and absolute numbers of MDSCs in the periphery of treated mice. Together, our results reveal that absence of pDCs during the priming of an autoimmune response leads to increased mobilization of MDSCs in the periphery in an MCP-1-dependent manner and subsequent amelioration of autoimmunity.

Pivotal role of plasmacytoid dendritic cells in inflammation and NK-cell responses after TLR9 triggering in mice

AbstractThe physiologic role played by plasmacytoid dendritic cells (pDCs) in the induction of innate responses and inflammation in response to pathogen signaling is not well understood. Here, we describe a new mouse model lacking pDCs and establish that pDCs are essential for the in vivo induction of NK-cell activity in response to Toll-like receptor 9 (TLR9) triggering. Furthermore, we provide the first evidence that pDCs are critical for the systemic production of a wide variety of chemokines in response to TLR9 activation. Consequently, we observed a profound alteration in monocyte, macrophage, neutrophil, and NK-cell recruitment at the site of inflammation in the absence of pDCs in response to CpG-Dotap and stimulation by microbial pathogens, such as Leishmania major, Escherichia coli, and Mycobacterium bovis. This study, which is based on the development of a constitutively pDC-deficient mouse model, highlights the pivotal role played by pDCs in the induction of innate immune responses and inflammation after TLR9 triggering.

CpG and poly(I:C) stimulation of dendritic cells and fibroblasts limits herpes simplex virus type 1 infection in an IFNβ-dependent and -independent way

Viral activation of toll-like receptors (TLRs) on dendritic cells (DCs) leads to production of various cytokines, including antiviral type I interferons (IFNs). Synthetic ligands specific for TLRs are also able to induce the production of type I IFNs (IFNα/β) by DCs, suggesting that these ligands have potential as antiviral drugs. In this in vitro study we extensively investigated the antiviral activity of various TLR ligands. Mouse bone marrow (BM) cells were differentiated into plasmacytoid and conventional DCs (pDCs and cDCs), stimulated with various TLR ligands and tested the antiviral abilities of collected supernatants in an in vitro herpes simplex virus type 1 (HSV-1) infection model. We observed a significant IFNβ-, (but not IFNα-) dependent reduction in HSV-1 infection when a mixed pDC/cDC population was stimulated with the TLR9 ligand CpG. In the absence of pDCs, TLR stimulation resulted in less pronounced antiviral effects. The most pronounced antiviral effect was observed when both DC subsets were stimulated with poly(I:C). A similar noticeable antiviral effect was observed when fibroblasts (L929 cells) were stimulated directly with poly(I:C). These poly(I:C)-mediated antiviral effects were only partially IFNβ-mediated and probably TLR independent. These data demonstrate that TLR ligands are not only able to produce type I IFN but can indeed act as antiviral drugs. In particular poly(I:C), which exerts its antiviral effects even in the absence of DCs, may become a promising drug e.g. to prevent respiratory infections by topical intranasal application.

Cutting Edge: Plasmacytoid Dendritic Cells Provide Innate Immune Protection against Mucosal Viral Infection In Situ

Dendritic cells (DCs) are powerful APCs capable of activating naive lymphocytes. Of the DC subfamilies, plasmacytoid DCs (pDCs) are unique in that they secrete high levels of type I IFNs in response to viruses but their role in inducing adaptive immunity remains divisive. In this study, we examined the importance of pDCs and their ability to recognize a virus through TLR9 in immunity against genital HSV-2 infection. We show that a low number of pDCs survey the vaginal mucosa at steady state. Upon infection, pDCs are recruited to the vagina and produce large amounts of type I IFNs in a TLR9-dependent manner and suppress local viral replication. Although pDCs are critical in innate defense against genital herpes challenge, adaptive Th1 immunity developed normally in the absence of pDCs. Thus, by way of migrating directly into the peripheral mucosa, pDCs act strictly as innate antiviral effector cells against mucosal viral infection in situ.