Immune Responses after Heavy Alcohol Consumption: Cytokine Concentrations in Hangover-Sensitive and Hangover-Resistant Drinkers.

This study investigated immunological changes during an alcohol hangover, and the possible difference between hangover-resistant and hangover-sensitive drinkers in terms of immune reactivity. Using a semi-naturalistic design, N = 36 healthy social drinkers (18 to 30 years old) provided saliva samples on a control day (after drinking no alcohol) and on a post-alcohol day. Hangover severity was rated directly after saliva collection. Cytokine concentrations, interleukin (IL)-1ß, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α, and hangover severity were compared between both test days and between hangover-sensitive and -resistant drinkers. Data from N = 35 drinkers (17 hangover-sensitive and 18 hangover-resistant) were included in the statistical analyses. Relative to the control day, there were significant increases in saliva IL-6 and IL-10 concentrations on the post-alcohol day. No significant differences in cytokine concentrations were found between hangover-sensitive and hangover-resistant drinkers, nor did any change in cytokine concentration correlate significantly with hangover severity. In line with previous controlled studies assessing cytokines in blood, the current naturalistic study using saliva samples also demonstrated that the immune system responds to high-level alcohol intake. However, further research is warranted, as, in contrast to previous findings in blood samples, changes in saliva cytokine concentrations did not differ significantly between hangover-sensitive and hangover-resistant drinkers, nor did they correlate significantly with hangover severity.

Immune monitoring in mesothelioma patients identifies novel immune-modulatory functions of gemcitabine associating with clinical response.

BACKGROUND: Gemcitabine is a frequently used chemotherapeutic agent but its effects on the immune system are incompletely understood. Recently, the randomized NVALT19-trial revealed that maintenance gemcitabine after first-line chemotherapy significantly prolonged progression-free survival (PFS) compared to best supportive care (BSC) in malignant mesothelioma. Whether these effects are paralleled by changes in circulating immune cell subsets is currently unknown. These analyses could offer improved mechanistic insights into the effects of gemcitabine on the host and guide development of effective combination therapies in mesothelioma. METHODS: We stained peripheral blood mononuclear cells (PBMCs) and myeloid-derived suppressor cells (MDSCs) at baseline and 3 weeks following start of gemcitabine or BSC treatment in a subgroup of mesothelioma patients included in the NVALT19-trial. In total, 24 paired samples including both MDSCs and PBMCs were included. We performed multicolour flow-cytometry to assess co-inhibitory and-stimulatory receptor- and cytokine expression and matched these parameters with PFS and OS. FINDINGS: Gemcitabine treatment was significantly associated with an increased NK-cell- and decreased T-regulatory cell proliferation whereas the opposite occurred in control patients. Furthermore, myeloid-derived suppressor cells (MDSCs) frequencies were lower in gemcitabine-treated patients and this correlated with increased T-cell proliferation following treatment. Whereas gemcitabine variably altered co-inhibitory receptor expression, co-stimulatory molecules including ICOS, CD28 and HLA-DR were uniformly increased across CD4+ T-helper, CD8+ T- and NK-cells. Although preliminary in nature, the increase in NK-cell proliferation and PD-1 expression in T cells following gemcitabine treatment was associated with improved PFS and OS. INTERPRETATION: Gemcitabine treatment was associated with widespread effects on circulating immune cells of mesothelioma patients with responding patients displaying increased NK-cell and PD-1 + T-cell proliferation. These exploratory data provide a platform for future on treatment-biomarker development and novel combination treatment strategies.

The PD-1/PD-L1-Checkpoint Restrains T cell Immunity in Tumor-Draining Lymph Nodes.

PD-1/PD-L1-checkpoint blockade therapy is generally thought to relieve tumor cell-mediated suppression in the tumor microenvironment but PD-L1 is also expressed on non-tumor macrophages and conventional dendritic cells (cDCs). Here we show in mouse tumor models that tumor-draining lymph nodes (TDLNs) are enriched for tumor-specific PD-1+ T cells which closely associate with PD-L1+ cDCs. TDLN-targeted PD-L1-blockade induces enhanced anti-tumor T cell immunity by seeding the tumor site with progenitor-exhausted T cells, resulting in improved tumor control. Moreover, we show that abundant PD-1/PD-L1-interactions in TDLNs of nonmetastatic melanoma patients, but not those in corresponding tumors, associate with early distant disease recurrence. These findings point at a critical role for PD-L1 expression in TDLNs in governing systemic anti-tumor immunity, identifying high-risk patient groups amendable to adjuvant PD-1/PD-L1-blockade therapy.

Dendritic cell vaccination and CD40-agonist combination therapy licenses T cell-dependent antitumor immunity in a pancreatic carcinoma murine model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates. METHODS: Immune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes. RESULTS: Mesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells. CONCLUSION: These results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.

Tnfaip3 expression in pulmonary conventional type 1 Langerin-expressing dendritic cells regulates T helper 2-mediated airway inflammation in mice.

BACKGROUND: Conventional type 1 dendritic cells (cDC1s) control anti-viral and anti-tumor immunity by inducing antigen-specific cytotoxic CD8+ T-cell responses. Controversy exists whether cDC1s also control CD4+ T helper 2 (Th2) cell responses, since suppressive and activating roles have been reported. DC activation status, controlled by the transcription factor NF-κB, might determine the precise outcome of Th-cell differentiation upon encounter with cDC1s. To investigate the role of activated cDC1s in Th2-driven immune responses, pulmonary cDC1s were activated by targeted deletion of A20/Tnfaip3, a negative regulator of NF-κB signaling. METHODS: To target pulmonary cDC1s, Cd207 (Langerin)-mediated excision of A20/Tnfaip3 was used, generating Tnfaip3fl/fl xCd207+/cre (Tnfaip3Lg-KO ) mice. Mice were exposed to house dust mite (HDM) to provoke Th2-mediated immune responses. RESULTS: Mice harboring Tnfaip3-deficient cDC1s did not develop Th2-driven eosinophilic airway inflammation upon HDM exposure, but rather showed elevated numbers of IFNγ-expressing CD8+ T cells. In addition, Tnfaip3Lg-KO mice harbored increased numbers of IL-12-expressing cDC1s and elevated PD-L1 expression in all pulmonary DC subsets. Blocking either IL-12 or IFNγ in Tnfaip3Lg-KO mice restored Th2 responses, whereas administration of recombinant IFNγ during HDM sensitization in C57Bl/6 mice blocked Th2 development. CONCLUSIONS: These findings indicate that the activation status of cDC1s, shown by their specific expression of co-inhibitory molecules and cytokines, critically contributes to the development of Th2 cell-mediated disorders, most likely by influencing IFNγ production in CD8+ T cells.

Notch signaling licenses allergic airway inflammation by promoting Th2 cell lymph node egress.

Allergic asthma is mediated by Th2 responses to inhaled allergens. Although previous experiments indicated that Notch signaling activates expression of the key Th2 transcription factor Gata3, it remains controversial how Notch promotes allergic airway inflammation. Here we show that T cell-specific Notch deficiency in mice prevented house dust mite-driven eosinophilic airway inflammation and significantly reduced Th2 cytokine production, serum IgE levels, and airway hyperreactivity. However, transgenic Gata3 overexpression in Notch-deficient T cells only partially rescued this phenotype. We found that Notch signaling was not required for T cell proliferation or Th2 polarization. Instead, Notch-deficient in vitro-polarized Th2 cells showed reduced accumulation in the lungs upon in vivo transfer and allergen challenge, as Notch-deficient Th2 cells were retained in the lung-draining lymph nodes. Transcriptome analyses and sequential adoptive transfer experiments revealed that while Notch-deficient lymph node Th2 cells established competence for lung migration, they failed to upregulate sphingosine-1-phosphate receptor 1 (S1PR1) and its critical upstream transcriptional activator Krüppel-like factor 2 (KLF2). As this KLF2/S1PR1 axis represents the essential cell-intrinsic regulator of T cell lymph node egress, we conclude that the druggable Notch signaling pathway licenses the Th2 response in allergic airway inflammation via promoting lymph node egress.

T cells and ILC2s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice.

Influenza virus infection is an important cause of severe asthma exacerbations, but it remains unclear how a Th1-mediated antiviral response triggers a prototypical Th2 disease. We investigated CD4+ T cells and group 2 innate lymphoid cells (ILC2s) in influenza virus-infected mice. We found that ILC2s accumulated in the lung rapidly after influenza virus infection, but the induction of IL-5 and IL-13 secretion was delayed and concomitant with T cell activation. In an influenza-induced exacerbation of allergic airway inflammation model we noticed an initial reduction of ILC2 numbers and cytokine production in broncho-alveolar lavage compared to chronic house dust mite (HDM)-mediated airway inflammation alone. ILC2s phenotype was characterized by low T1/ST2, ICOS, KLRG1, and CD25 expression, resembling naïve ILC2s. The contribution of ILC2s to type 2 cytokine production in the early stage of the influenza-induced exacerbation was limited. In contrast, T cells showed increased IL-4 and IL-5 production when exposed to both HDM and influenza virus. Upon virus clearance, ILC2s regained an activated T1/ST2high ICOShigh KLRG1high CD25high phenotype paired with cytokine production and were major contributors to the type 2 cytokine milieu. Collectively, our data indicate that both T cells and ILC2s contribute to influenza-induced exacerbation of allergic airway inflammation, but with different kinetics.

A pathophysiological role of PDE3 in allergic airway inflammation.

Phosphodiesterase 3 (PDE3) and PDE4 regulate levels of cyclic AMP, which are critical in various cell types involved in allergic airway inflammation. Although PDE4 inhibition attenuates allergic airway inflammation, reported side effects preclude its application as an antiasthma drug in humans. Case reports showed that enoximone, which is a smooth muscle relaxant that inhibits PDE3, is beneficial and lifesaving in status asthmaticus and is well tolerated. However, clinical observations also showed antiinflammatory effects of PDE3 inhibition. In this study, we investigated the role of PDE3 in a house dust mite-driven (HDM-driven) allergic airway inflammation (AAI) model that is characterized by T helper 2 cell activation, eosinophilia, and reduced mucosal barrier function. Compared with wild-type (WT) littermates, mice with a targeted deletion of the PDE3A or PDE3B gene showed significantly reduced HDM-driven AAI. Therapeutic intervention in WT mice showed that all hallmarks of HDM-driven AAI were abrogated by the PDE3 inhibitors enoximone and milrinone. Importantly, we found that enoximone also reduced the upregulation of the CD11b integrin on mouse and human eosinophils in vitro, which is crucial for their recruitment during allergic inflammation. This study provides evidence for a hitherto unknown antiinflammatory role of PDE3 inhibition in allergic airway inflammation and offers a potentially novel treatment approach.

The Notch pathway inhibitor stapled α-helical peptide derived from mastermind-like 1 (SAHM1) abrogates the hallmarks of allergic asthma.

BACKGROUND: The Notch signaling pathway has been implicated in the pathogenesis of allergic airway inflammation. Targeting the active Notch transactivation complex by using the cell-permeable, hydrocarbon-stapled synthetic peptide stapled α-helical peptide derived from mastermind-like 1 (SAHM1) resulted in genome-wide suppression of Notch-activated genes in leukemic cells and other models. However, the efficacy of SAHM1 in allergic asthma models has remained unexplored. OBJECTIVE: We aimed to investigate the therapeutic efficacy of SAHM1 in a house dust mite (HDM)-driven asthma model. METHODS: Topical therapeutic intervention with SAHM1 or a control peptide was performed during sensitization, challenge, or both with HDM in mice. Airway inflammation was assessed by using multicolor flow cytometry, and bronchial hyperreactivity was studied. Additionally, SAHM1 therapy was investigated in mice with established allergic airway inflammation and in a model in which we neutralized IFN-γ during HDM challenge to support the TH2 response and exacerbate asthma. RESULTS: SAHM1 treatment during the challenge phase led to a marked reduction of eosinophil and T cell numbers in bronchoalveolar lavage fluid compared with those in diluent-treated or control peptide-treated mice. Likewise, T-cell cytokine content and bronchial hyperreactivity were reduced. SAHM1 treatment dampened TH2 inflammation during ongoing HDM challenge and enhanced recovery after established asthma. Additionally, in the presence of anti-IFN-γ antibodies, SAHM1 downregulated expression of the key TH2 transcription factor GATA3 and intracellular IL-4 in bronchoalveolar lavage fluid T cells, but expression of the TH17 transcription factor retinoic acid-related orphan receptor γt or intracellular IL-17 was not affected. SAHM1 therapy also reduced serum IgE levels. CONCLUSIONS: Therapeutic intervention of Notch signaling by SAHM1 inhibits allergic airway inflammation in mice and is therefore an interesting new topical treatment opportunity in asthmatic patients.

Group 2 Innate Lymphoid Cells Exhibit a Dynamic Phenotype in Allergic Airway Inflammation.

Group 2 innate lymphoid cells (ILC2) are implicated in allergic asthma as an early innate source of the type 2 cytokines IL-5 and IL-13. However, their induction in house dust mite (HDM)-mediated airway inflammation additionally requires T cell activation. It is currently unknown whether phenotypic differences exist between ILC2s that are activated in a T cell-dependent or T cell-independent fashion. Here, we compared ILC2s in IL-33- and HDM-driven airway inflammation. Using flow cytometry, we found that surface expression levels of various markers frequently used to identify ILC2s were dependent on their mode of activation, highly variable over time, and differed between tissue compartments, including bronchoalveolar lavage (BAL) fluid, lung, draining lymph nodes, and spleen. Whereas in vivo IL-33-activated BAL fluid ILC2s exhibited an almost uniform CD25+CD127+T1/ST2+ICOS+KLRG1+ phenotype, at a comparable time point after HDM exposure BAL fluid ILC2s had a very heterogeneous surface marker phenotype. A major fraction of HDM-activated ILC2s were CD25lowCD127+T1/ST2low ICOSlowKLRG1low, but nevertheless had the capacity to produce large amounts of type 2 cytokines. HDM-activated CD25low ILC2s in BAL fluid and lung rapidly reverted to CD25high ILC2s upon in vivo stimulation with IL-33. Genome-wide transcriptional profiling of BAL ILC2s revealed ~1,600 differentially expressed genes: HDM-stimulated ILC2s specifically expressed genes involved in the regulation of adaptive immunity through B and T cell interactions, whereas IL-33-stimulated ILC2s expressed high levels of proliferation-related and cytokine genes. In both airway inflammation models ILC2s were present in the lung submucosa close to epithelial cells, as identified by confocal microscopy. In chronic HDM-driven airway inflammation ILC2s were also found inside organized cellular infiltrates near T cells. Collectively, our findings show that ILC2s are phenotypically more heterogeneous than previously thought, whereby their surface marker and gene expression profile are highly dynamic.

Enhanced Bruton's Tyrosine Kinase Activity in Peripheral Blood B Lymphocytes From Patients With Autoimmune Disease.

OBJECTIVE: Bruton's tyrosine kinase (BTK) transmits crucial survival signals from the B cell receptor (BCR) in B cells. Pharmacologic BTK inhibition effectively diminishes disease symptoms in mouse models of autoimmunity; conversely, transgenic BTK overexpression induces systemic autoimmunity in mice. We undertook this study to investigate BTK expression and activity in human B cells in the context of autoimmune disease. METHODS: Using intracellular flow cytometry, we quantified BTK expression and phosphorylation in subsets of peripheral blood B cells from 30 patients with rheumatoid arthritis (RA), 26 patients with primary Sjögren's syndrome (SS), and matched healthy controls. RESULTS: In circulating B cells, BTK protein expression levels correlated with BTK phosphorylation. BTK expression was up-regulated upon BCR stimulation in vitro and was significantly higher in CD27+ memory B cells than in CD27-IgD+ naive B cells. Importantly, BTK protein and phospho-BTK were significantly increased in B cells from anti-citrullinated protein antibody (ACPA)-positive RA patients but not in B cells from ACPA-negative RA patients. BTK was increased both in naive B cells and in memory B cells and correlated with frequencies of circulating CCR6+ Th17 cells. Likewise, BTK protein was increased in B cells from a major fraction of patients with primary SS and correlated with serum rheumatoid factor levels and parotid gland T cell infiltration. Interestingly, targeting T cell activation in patients with primary SS using the CTLA-4Ig fusion protein abatacept restored BTK protein expression in B cells to normal levels. CONCLUSION: These data indicate that autoimmune disease in humans is characterized by enhanced BTK activity, which is linked not only to autoantibody formation but also to T cell activity.

Notch signaling in T cells is essential for allergic airway inflammation, but expression of the Notch ligands Jagged 1 and Jagged 2 on dendritic cells is dispensable.

BACKGROUND: Allergic asthma is characterized by a TH2 response induced by dendritic cells (DCs) that present inhaled allergen. Although the mechanisms by which they instruct TH2 differentiation are still poorly understood, expression of the Notch ligand Jagged on DCs has been implicated in this process. OBJECTIVE: We sought to establish whether Notch signaling induced by DCs is critical for house dust mite (HDM)-driven allergic airway inflammation (AAI) in vivo. METHODS: The induction of Notch ligand expression on DC subsets by HDM was quantified by using quantitative real-time PCR. We used an HDM-driven asthma mouse model to compare the capacity of Jagged 1 and Jagged 2 single- and double-deficient DCs to induce AAI. In addition, we studied AAI in mice with a T cell-specific deletion of recombination signal-binding protein for immunoglobulin Jκ region (RBPJκ), a downstream effector of Notch signaling. RESULTS: HDM exposure promoted expression of Jagged 1, but not Jagged 2, on DCs. In agreement with published findings, in vitro-differentiated and HDM-pulsed Jagged 1 and Jagged 2 double-deficient DCs lacked the capacity to induce AAI. However, after in vivo intranasal sensitization and challenge with HDM, DC-specific Jagged 1 or Jagged 2 single- or double-deficient mice had eosinophilic airway inflammation and a TH2 cell activation phenotype that was not different from that in control littermates. In contrast, RBPJκ-deficient mice did not experience AAI and airway hyperreactivity. CONCLUSION: Our results show that the Notch signaling pathway in T cells is crucial for the induction of TH2-mediated AAI in an HDM-driven asthma model but that expression of Jagged 1 or Jagged 2 on DCs is not required.

T cells are necessary for ILC2 activation in house dust mite-induced allergic airway inflammation in mice.

Allergic asthma is a chronic inflammation of the airways mediated by an adaptive type 2 immune response. Upon allergen exposure, group 2 innate lymphoid cells (ILC2s) can be rapidly activated and represent an early innate source of IL-5 and IL-13. Here, we used a house dust mite (HDM)-driven asthma mouse model to study the induction of ILC2s in allergic airway inflammation. In BALF, lungs, and lymph nodes, ILC2 activation is critically dependent on prior sensitization with HDM. Importantly, T cells are required for ILC2 induction, whereby T-cell activation precedes ILC2 induction. During HDM-driven allergic airway inflammation the accumulation of ILC2s in BALF is IL-33 independent, although infiltrating ILC2s produce less cytokines in Il33(-/-) mice. Transfer of in vitro polarized OVA-specific OT-II Th2 cells alone or in combination with Th17 cells followed by OVA and HDM challenge is not sufficient to induce ILC2, despite significant eosinophilic inflammation and T-cell activation. In this asthma model, ILC2s are therefore not an early source of Th2 cytokines, but rather contribute to type 2 inflammation in which Th2 cells play a key role. Taken together, ILC2 induction in HDM-mediated allergic airway inflammation in mice critically depends on activation of T cells.