Heleen Vroman

Mode of dendritic cell activation: the decisive hand in Th2/Th17 cell differentiation. Implications in asthma severity?

Asthma is a heterogeneous chronic inflammatory disease of the airways, with reversible airflow limitations and airway remodeling. The classification of asthma phenotypes was initially based on different combinations of clinical symptoms, but they are now unfolding to link biology to phenotype. As such, patients can suffer from a predominant eosinophilic, neutrophilic or even mixed eosinophilic/neutrophilic inflammatory response. In adult asthma patients, eosinophilic inflammation is usually seen in mild-to-moderate disease and neutrophilic inflammation in more severe disease. The underlying T cell response is predominated by T helper (Th) 2, Th17, or a mixed Th2/Th17 cell immune response. Dendritic cells (DCs) are professional antigen presenting cells (APCs), since their principal function is to present antigens and induce a primary immune response in resting naive T cells. DCs also drive the differentiation into distinctive Th subsets. The expression of co-stimulatory molecules and cytokines by DCs and surrounding cells determines the outcome of Th cell differentiation. The nature of DC activation will determine the expression of specific co-stimulatory molecules and cytokines, specifically needed for induction of the different Th cell programs. Thus DC activation is crucial for the subsequent effector Th immune responses. In this review, we will discuss underlying mechanisms that initiate DC activation in favor of Th2 differentiation versus Th1/Th17 and Th17 differentiation in the development of mild versus moderate to severe asthma.

CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

IntroductionPatients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA+ RA and differences in treatment outcome between these subpopulations suggest that ACPA+ and ACPA− RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA+ RA. In this context we recently identified a potential pathogenic role for CCR6+ Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status.MethodsWe performed a nested matched case–control study including 27 ACPA+ and 27 ACPA− treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4+CD45RO+ (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration.ResultsACPA status was not related to differences in total CD4+ T cell or memory Th cell proportions. However, ACPA+ patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4+ and CCR10+ Th cells were found. Within the CCR6+ cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4+CCR10−), Th17.1 (CXCR3+), Th22 (CCR4+CCR10+) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (pu2009=u20090.02), Th17.1 (pu2009=u20090.03) and CCR4/CXCR3 DP (pu2009=u20090.01) cells were present in ACPA+ patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6−CXCR3+; pu2009=u20090.90), Th2 (CCR6−CCR4+; pu2009=u20090.27) and T-regulatory (CD25hiFOXP3+; pu2009=u20090.06) cell proportions. Interestingly, CCR6+ Th cells were inversely correlated with disease duration in ACPA− patients (R2u2009=u2009−0.35; pu2009<u20090.01) but not in ACPA+ (R2u2009<u20090.01; pu2009=u20090.94) patients.ConclusionsThese findings demonstrate that increased peripheral blood CCR6+ Th cells proportions distinguish ACPA+ RA from ACPA− RA. This suggests that CCR6+ Th cells are involved in the differences in disease severity and treatment outcome between ACPA+ and ACPA− RA.

Development of eosinophilic inflammation is independent of B-T cell interaction in a chronic house dust mite-driven asthma model

Chronic exposure to environmental triggers, such as house dust mite (HDM), drives T helper 2 (Th2) cell‐mediated asthma. Recent evidence has shown that B–T cell interaction, and in particular germinal centre reactions and follicular T helper (Tfh) cells are required for the development of eosinophilic airway inflammation in HDM‐driven models containing a sensitization and challenge phase. Whether B–T cell interactions are essential for pulmonary eosinophilic inflammation following chronic allergen provocation remains unknown.

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.

TNF-α–induced protein 3 levels in lung dendritic cells instruct TH2 or TH17 cell differentiation in eosinophilic or neutrophilic asthma

Background It is currently unknown why allergen exposure or environmental triggers in patients with mild‐to‐moderate asthma result in TH2‐mediated eosinophilic inflammation, whereas patients with severe asthma often present with TH17‐mediated neutrophilic inflammation. The activation state of dendritic cells (DCs) is crucial for both TH2 and TH17 cell differentiation and is mediated through nuclear factor &kgr;B activation. Ablation of TNF‐&agr;–induced protein 3 (TNFAIP3), one of the crucial negative regulators of nuclear factor &kgr;B activation in myeloid cells and DCs, was shown to control DC activation. Objective In this study we investigated the precise role of TNFAIP3 in myeloid cells for the development of TH2‐ and TH17‐cell mediated asthma. Methods We exposed mice with conditional deletion of the Tnfaip3 gene in either myeloid cells (by using the lysozyme M [LysM] promotor) or specifically in DCs (by using the Cd11c promotor) to acute and chronic house dust mite (HDM)–driven asthma models. Results We demonstrated that reduced Tnfaip3 gene expression in DCs in either Tnfaip3CD11c or Tnfaip3LysM mice dose‐dependently controlled development of TH17‐mediated neutrophilic severe asthma in both acute and chronic HDM‐driven models, whereas wild‐type mice had a purely TH2‐mediated eosinophilic inflammation. TNFAIP3‐deficient DCs induced HDM‐specific TH17 cell differentiation through increased expression of the TH17‐instructing cytokines IL‐1&bgr;, IL‐6, and IL‐23, whereas HDM‐specific TH2 cell differentiation was hampered by increased IL‐12 and IL‐6 production. Conclusions These data show that the extent of TNFAIP3 expression in DCs controls TH2/TH17 cell differentiation. This implies that reducing DC activation could be a new pharmacologic intervention to treat patients with severe asthma who present with TH17‐mediated neutrophilic inflammation.

Dendritic cell subsets in asthma: Impaired Tolerance or exaggerated inflammation?

Asthma is a prevalent chronic heterogeneous inflammatory disease of the airways, leading to reversible airway obstruction, in which various inflammatory responses can be observed. Mild to moderate asthma patients often present with a Th2-mediated eosinophilic inflammation whereas in severe asthma patients, a Th17-associated neutrophilic or combined Th2 and Th17-mediated eosinophilic/neutrophilic inflammation is observed. The differentiation of these effector Th2 and Th17-cells is induced by allergen-exposed dendritic cells (DCs) that migrate toward the lung draining lymph node. The DC lineage comprises conventional DCs (cDCs) and plasmacytoid DCs (pDCs), of which the cDC lineage consists of type 1 cDCs (cDC1s) and cDC2s. During inflammation, also monocytes can differentiate into so-called monocyte-derived DCs (moDCs). These DC subsets differ both in ontogeny, localization, and in their functional properties. New identification tools and the availability of transgenic mice targeting specific DC subsets enable the investigation of how these different DC subsets contribute to or suppress asthma pathogenesis. In this review, we will discuss mechanisms used by different DC subsets to elicit or hamper the pathogenesis of both Th2-mediated eosinophilic asthma and more severe Th17-mediated neutrophilic inflammation.

Heterogeneity in Immune Cell Content in Malignant Pleural Mesothelioma

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with limited therapy options and dismal prognosis. In recent years, the role of immune cells within the tumor microenvironment (TME) has become a major area of interest. In this review, we discuss the current knowledge of heterogeneity in immune cell content and checkpoint expression in MPM in relation to prognosis and prediction of treatment efficacy. Generally, immune-suppressive cells such as M2 macrophages, myeloid-derived suppressor cells and regulatory T cells are present within the TME, with extensive heterogeneity in cell numbers. Infiltration of effector cells such as cytotoxic T cells, natural killer cells and T helper cells is commonly found, also with substantial patient to patient heterogeneity. PD-L1 expression also varied greatly (16–65%). The infiltration of immune cells in tumor and associated stroma holds key prognostic and predictive implications. As such, there is a strong rationale for thoroughly mapping the TME to better target therapy in mesothelioma. Researchers should be aware of the extensive possibilities that exist for a tumor to evade the cytotoxic killing from the immune system. Therefore, no “one size fits all” treatment is likely to be found and focus should lie on the heterogeneity of the tumors and TME.

A1.76 Higher proportions of pathogenic CCR6+ T cell subpopulations in ACPA+ compared to ACPA- patients with early rheumatoid arthritis

Background and Objectives Presence of serum anti-citrullinated protein antibodies (ACPAs) in patients with rheumatoid arthritis (RA) predicts worse disease course and a more erosive disease. In the pathogenesis of RA, inflammatory T cells play a central role. In this context, we recently found increased proportions of pathogenic peripheral CCR6+ memory T helper cells in patients with early RA compared to healthy individuals. However, it is unclear how T cell proportions are distributed between ACPA+ and ACPA- patients. Therefore alterations in peripheral T cell populations and their pathogenic potential were examined in ACPA+ and ACPA- patient groups. Materials and Methods A nested matched case control study was performed including 27 ACPA+ and 27 ACPA- patients with early RA. T cell profiles (Treg, Th1, Th2, Th17, Th22 and various less well classified populations) from these ACPA+ and ACPA- patients were generated based on chemokine receptor, cytokine and transcription factor expression. Differentially present T cell populations were isolated from peripheral blood and analysed for their pathologic potential in a co-culture system with RA derived synovial fibroblasts (RASF). Results In comparison to ACPA- patients, ACPA+ patients have higher proportions of regulatory T cells (Treg) and CD4+ memory CCR6+ T cells. Since the CCR6+ T cell population is still a heterogeneous population, four CCR6+ T cell subpopulations were distinguished by differential expression of CXCR3 and CCR4. All four CCR6+ subpopulations shared Th17 cell characteristics such as Rorγt and CCL20 expression, but IL-17A, IL-17F, IL-22 and IFN-γ expression differed greatly between these subpopulations. However, even the population with lowest expression of these cytokines showed high pathological potential as shown by stimulating IL-1β, IL-6, IL-8, COX-2 and MMP-3 expression upon co-culture with RASF. Indeed, despite dissimilar Th17 and Th1 characteristics between the CCR6+ subpopulations, all four showed highly increased pathological potential in co-culture compared to naive and Th1 cells. Conclusions ACPA+ and ACPA- patients can be distinguished by the distribution of Treg and CCR6+ T cell subpopulations. These CCR6+ subpopulations exhibit dissimilar Th17 and Th1 characteristics, but all possess high pathological potential, including the population that has low IL-17A/F and IL-22 expression. These findings indicate a prominent role of CCR6+ T cells in the pathogenesis of ACPA+ patients with early RA and may contribute to the worse disease outcome in ACPA+ patients.

Increased surface expression of NOTCH on memory T cells in peripheral blood from asthma patients

Using flow cytometry we found that circulating T cell subsets from asthma patients contained higher proportions of NOTCH receptor positive cells compared with healthy controls, suggesting enhanced NOTCH signaling in T cells from asthma patients.

Autologous dendritic cell therapy in mesothelioma patients enhances frequencies of peripheral CD4 T cells expressing HLA-DR, PD-1, or ICOS

Introduction: Malignant pleural mesothelioma (MPM) is a malignancy with a very poor prognosis for which new treatment options are urgently needed. We have previously shown that dendritic cell (DC) immunotherapy provides a clinically feasible treatment option. In the current study, we set out to assess the immunological changes induced by DC immunotherapy in peripheral blood of MPM patients. Methods: Peripheral blood was collected from nine patients enrolled in a phase I dose escalation study, before and after treatment with DCs that were pulsed with an allogeneic tumor lysate preparation consisting of a mixture of five cultured mesothelioma cell lines. We used immune profiling by multiplex flow cytometry to characterize different populations of immune cells. In particular, we determined frequencies of T cell subsets that showed single and combinatorial expression of multiple markers that signify T cell activation, maturation and inhibition. Therapy-induced T cell reactivity was assessed in peptide/MHC multimer stainings using mesothelin as a prototypic target antigen with confirmed expression in the clinical tumor lysate preparation. T cell receptor (TCR) diversity was evaluated by TCRB gene PCR assays. Results: We observed an increase in the numbers of B cells, CD4 and CD8 T cells, but not NK cells at 6 weeks post-treatment. The increases in B and T lymphocytes were not accompanied by major changes in T cell reactivity toward mesothelin nor in TCRB diversity. Notably, we did observe enhanced proportions of CD4 T cells expressing HLA-DR, PD-1 (at 2 weeks after onset of treatment) and ICOS (6 weeks) and a CD8 T cell population expressing LAG3 (2 weeks). Discussion: DC immunotherapy using allogeneic tumor lysate resulted in enhanced frequencies of B cells and T cells in blood. We did not detect a skewed antigen-reactivity of peripheral CD8 T cells. Interestingly, frequencies of CD4 T cells expressing activation markers and PD-1 were increased. These findings indicate a systemic activation of the adaptive immune response and may guide future immune monitoring studies of DC therapies.