Evaluation of PD-1 and interleukin-10-receptor expression by T lymphocytes in malignant and benign pleural effusions.

PD-1 (programmed cell death protein-1)/PD-L1 (programmed cell death ligand 1) as well as IL-10 (interleukin-10)/IL-10R (interleukin-10 receptor) interactions play a major role in tumor immune evasion in various malignancies. Several studies investigated the expression of PD-1 on T lymphocytes in pleural effusions (PE) in patients with malignant diseases. However, results in malignant pleural effusions (MPE) compared to benign PE (BPE) are underreported. In this prospective study, 51 patients (median age 66 years, IQR 54-78, 47% male) with PE of malignant or benign origin at the Medical University of Vienna between March 2021 and November 2022 were enrolled and divided into three groups according to the cytological results (group 1: MPE [n = 24, 47%]; group 2: BPE in malignant disease [n = 22, 43%]; group 3: BPE in benign disease [n = 5, 10%]). In the cytological samples, T cells were analyzed for the expression of PD-1 and IL-10R via flow cytometry. In MPE, the proportion of PD-1+ T lymphocytes on CD4+ cells was significantly lower than in BPE (40.1 vs. 56.3 in group 1 vs. 3, p = 0.019). Moreover, a significantly lower expression of PD-1+ IL-10R+ CD8+ (9.6 vs. 35.2 in group 1 vs. 2, p = 0.016; 9.6 vs. 25.0 in group 1 vs. 3, p = 0.032) and a significantly higher expression of PD-1-IL-10R-CD8+ T lymphocytes (43.7 vs. 14.0 in group1 vs. 2, p = 0.045; 43.7 vs. 23.3 in group 1 vs. 3, p = 0.032) were observed in MPE when compared to BPE. The frequency of T cells expressing PD-1 and IL-10R on CD8+ T cells is significantly lower in MPE compared to BPE regardless of the underlying disease indicating a different microenvironment in PE driven by the presence of tumor cells. Our observation spotlights the possible involvement of PD-1 and IL-10R in MPE.

A common secretomic signature across epithelial cancers metastatic to the pleura supports IL-6 axis therapeutic targeting.

Background: Many cancers metastasize to the pleura, resulting in effusions that cause dyspnea and discomfort. Regardless of the tissue of origin, pleural malignancies are aggressive and uniformly fatal, with no treatment shown to prolong life. The pleural mesothelial monolayer is joined by tight junctions forming a contained bioreactor-like space, concentrating cytokines and chemokines secreted by the mesothelium, tumor, and infiltrating immune cells. This space represents a unique environment that profoundly influences tumor and immune cell behavior. Defining the pleural secretome is an important step in the rational development localized intrapleural immunotherapy. Method: We measured cytokine/chemokine content of 252 malignant pleural effusion (MPE) samples across multiple cancers using a 40-analyte panel and Luminex multiplexing technology. Results: Eleven analytes were consistently present in concentrations ≥ 10.0 pM: CXCL10/IP10 (geometric mean = 672.3 pM), CCL2/MCP1 (562.9 pM), sIL-6Rα (403.1 pM), IL-6 (137.6 pM), CXCL1/GRO (80.3 pM), TGFß1 (76.8 pM), CCL22/MDC (54.8 pM), CXCL8/IL-8 (29.2 pM), CCL11/Eotaxin (12.6 pM), IL-10 (11.3 pM), and G-CSF (11.0 pM). All are capable of mediating chemotaxis, promotion of epithelial to mesenchymal transition, or immunosuppression, and many of are reportedly downstream of a pro-inflammatory cytokine cascade mediated by cytokine IL-6 and its soluble receptor. Conclusion: The data indicate high concentrations of several cytokines and chemokines across epithelial cancers metastatic to the pleura and support the contention that the pleural environment is the major factor responsible for the clinical course of MPE across cancer types. A sIL-6Rα to IL-6 molar ratio of 2.7 ensures that virtually all epithelial, immune and vascular endothelial cells in the pleural environment are affected by IL-6 signaling. The central role likely played by IL-6 in the pathogenesis of MPE argues in favor of a therapeutic approach targeting the IL-6/IL-6R axis.

Characterizing soluble immune checkpoint molecules and TGF-ß1,2,3 in pleural effusion of malignant pleural mesothelioma.

The clinical impact of soluble molecules in pleural effusion (PE) is unclear in patients with malignant pleural mesothelioma (MPM). In this single-center, retrospective, observational study, we assessed soluble forms of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and PD-1 ligand 1 (PD-L1) using enzyme-linked immunosorbent assays; three TGF-ß isoforms were measured via multiplex assay in PE of patients with fibrinous pleuritis (FP) or MPM, to assess relationships between the levels of six molecules, clinicopathological characteristics, and efficacy of immune checkpoint inhibitors. Soluble forms of CTLA-4, PD-L1, PD-1, TGF-ß1, TGF-ß2, and TGF-ß3 were variably produced in PE of FP (n = 34) and MPM (n = 79); we found significant relationships between the six molecules and clinicopathological features. Although none of the three soluble immune checkpoint molecules showed diagnostic or prognostic effects in patients with MPM, TGF-ß2 level in PE is a useful differential diagnostic marker between FP and MPM. Both TGF-ß1 and TGF-ß3 levels are promising prognostic markers for MPM. Moreover, we found that higher baseline levels of PD-1 soluble forms predicted the response to anti-PD1 monotherapy. Our findings identify novel diagnostic, prognostic, and predictive biomarkers for anti-PD1 therapy in patients with MPM.

Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

Exploratory pilot study to characterize the immune landscapes of malignant pleural effusions and their corresponding primary tumors from patients with breast carcinoma and lung adenocarcinoma.

INTRODUCTION: Malignant pleural effusion (MPE) is a frequent complication of advanced malignancies. In this pilot study, we characterized the immune landscapes of MPEs, compared them to their primary tumor (PT) samples from breast carcinoma (BC) and lung adenocarcinoma (LADC), and tested the utility of multiplexed image technology in cytological samples. MATERIALS AND METHODS: We evaluated the immune contexture of 6 BC and 5 LADC MPEs and their PTs using 3 multiplex immunofluorescence panels. We explored the associations between sample characteristics and pleural effusion-free survival. RESULTS: No MPE samples had positive programmed death-ligand 1 expression in malignant cells, although 3 of 11 PTs has positive programmed death-ligand 1 expression (more than 1% expression in malignant cells). Overall, in LADC samples, cluster of differentiation 3 (CD3)+ T cells and CD3+CD8+ cytotoxic T cells predominated (median percentages for MPEs versus PTs: 45.6% versus 40.7% and 4.7% versus 6.6%, respectively) compared with BC. CD68+ macrophages predominated in the BC samples (medians for MPEs 61.2% versus PTs for 57.1%) but not in the LADC samples. Generally in PTs, CD3+CD8+ forkhead box P3+ T cells and the median distances from the malignant cells to CD3+CD8+Ki67+ and CD3+ programmed cell death protein 1 + T cells correlated to earlier MPE after PT diagnosis. CONCLUSIONS: The immune cell phenotypes in the MPEs and PTs were similar within each cancer type but different between BC versus LADC. An MPE analysis can potentially be used as a substitute for a PT analysis, but an expanded study of this topic is essential.

Th17 cells and their related cytokines: vital players in progression of malignant pleural effusion.

Malignant pleural effusion (MPE) is an exudative effusion caused by primary or metastatic pleural carcinosis. Th17 cells and their cytokines are critical components in various disease including MPE. In this review, we summarize current published articles regarding the multifunctional roles of Th17 cells and their related cytokines in MPE. Th17 cells are accumulated in MPE compared with paired serum via certain manners. The upregulation of Th17 cells and the interactions between Th17 cells and other immune cells, such as Th1 cells, Th9 cells, regulatory T cells and B cells, are reported to be involved in the formation and development of MPE. In addition, cytokines, which are elaborated by Th17 cells, including IL-17A, IL-17F, IL-21, IL-22, IL-26, GM-CSF, or associated with Th17 cells differentiation, including IL-1ß, IL-6, IL-23, TGF-ß, are linked to the pathogenesis of MPE through exerting pro- or anti-tumorigenic functions on their own as well as regulating the generation and differentiation of Th17 cells in MPE. Based on these findings, we proposed that Th17 cells and their cytokines might be diagnostic or prognostic tools and potential therapeutic targets for MPE.

A meta-analysis of Th1 and Th2 cytokine profiles differentiating tuberculous from malignant pleural effusion.

To clarify the predominance of Th1 or Th2 immune responses in malignant and tuberculous pleural effusion (MPE and TPE, respectively), we performed a meta-analysis of previously published results of the levels of Th1/Th2 cytokines associated with these two types of pleural effusion to evaluate the use of Th1/Th2 cytokine profiles in distinguishing TPE from MPE. We searched the PubMed and EMBASE databases for studies indexed from 2000 to March 2021. We included studies that (a) diagnosed TPE and MPE based on culture or pleural tissue biopsy and that (b) compared levels of Th1/Th2 cytokines between TPE and MPE. Pooled data based on a random-effects model or fixed-effects model and standardized mean differences (SMDs) across studies were used to compare TPE and MPE. We also performed Egger's test to assess publication bias. Of 917 identified studies, a total of 42 studies were selected for the meta-analysis. Compared with MPE subjects, TPE subjects had a significantly higher level of TNF-α [2.22, (1.60-2.84)], an elevated level of IFN-γ [3.30, (2.57-4.40)] in pleural effusion, a situation where the Th1 immune response dominated. Conversely, the levels of interleukin-4 (IL-4) and IL-10 (Th2 cytokines) were higher in the MPE subjects than in the TPE subjects, showing statistically nonsignificant tiny effects [-0.15, (-0.94 to 0.63) and -0.04, (-0.21 to 0.12), respectively]. We confirmed that TPE, a situation in which the Th1 cytokines are predominant. The slight preponderance of Th2 cytokines in MPE, which is not convincing enough to prove.

Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

Intrapleural Injection of Anti-PD1 Antibody: A Novel Management of Malignant Pleural Effusion.

Background: Malignant tumors accompanied with malignant pleural effusion (MPE) often indicate poor prognosis. The therapeutic effect and mechanism of intrapleural injection of anti-programmed cell death protein 1 (PD1) on MPE need to be explored. Methods: A preclinical MPE mouse model and a small clinical study were used to evaluate the effect of intrapleural injection of anti-PD1 antibody. The role of immune cells was observed via flow cytometry, RNA-sequencing, quantitative PCR, western blot, immunohistochemistry, and other experimental methods. Results: Intrathoracic injection of anti-PD1 monoclonal antibody (mAb) has significantly prolonged the survival time of mice (P = 0.0098) and reduced the amount of effusion (P = 0.003) and the number of cancer nodules (P = 0.0043). Local CD8+ T cells participated in intrapleural administration of anti-PD1 mAb. The proportion of CD69+, IFN-γ+, and granzyme B+ CD8+ T cells in the pleural cavity was increased, and the expression of TNF-α and IL-1ß in MPE also developed significantly after injection. Local injection promoted activation of the CCL20/CCR6 pathway in the tumor microenvironment and further elevated the expression of several molecules related to lymphocyte activation. Clinically, the control rate of intrathoracic injection of sintilimab (a human anti-PD1 mAb) for 10 weeks in NSCLC patients with MPE was 66.7%. Local injection improved the activity and function of patients' local cytotoxic T cells (CTLs). Conclusions: Intrapleural injection of anti-PD1 mAb could control malignant pleural effusion and the growth of cancer, which may be achieved by enhancing local CTL activity and cytotoxicity.

Single-cell analysis of diverse immune phenotypes in malignant pleural effusion.

The complex interactions among different immune cells have important functions in the development of malignant pleural effusion (MPE). Here we perform single-cell RNA sequencing on 62,382 cells from MPE patients induced by non-small cell lung cancer to describe the composition, lineage, and functional states of infiltrating immune cells in MPE. Immune cells in MPE display a number of transcriptional signatures enriched for regulatory T cells, B cells, macrophages, and dendritic cells compared to corresponding counterparts in blood. Helper T, cytotoxic T, regulatory T, and T follicular helper cells express multiple immune checkpoints or costimulatory molecules. Cell-cell interaction analysis identifies regulatory B cells with more interactions with CD4+ T cells compared to CD8+ T cells. Macrophages are transcriptionally heterogeneous and conform to M2 polarization characteristics. In addition, immune cells in MPE show the general up-regulation of glycolytic pathways associated with the hypoxic microenvironment. These findings show a detailed atlas of immune cells in human MPE and enhance the understanding of potential diagnostic and therapeutic targets in advanced non-small cell lung cancer.

Higher CD4/CD8 ratio of pleural effusion predicts better survival for lung cancer patients receiving immune checkpoint inhibitors.

Pleural effusion is a rare immune-related adverse event for lung cancer patients receiving immune checkpoint inhibitors (ICIs). We enrolled 281 lung cancer patients treated with ICIs and 17 were analyzed. We categorized the formation of pleural effusion into 3 patterns: type 1, rapid and massive; type 2, slow and indolent; and type 3, with disease progression. CD4/CD8 ratio of 1.93 was selected as the cutoff threshold to predict survival. Most patients of types 1 and 2 effusions possessed pleural effusion with CD4/CD8 ratios ≥ 1.93. The median OS time in type 1, 2, and 3 patients were not reached, 24.8, and 2.6 months, respectively. The median PFS time in type 1, 2, and 3 patients were 35.5, 30.2, and 1.4 months, respectively. The median OS for the group with pleural effusion CD4/CD8 ≥ 1.93 and < 1.93 were not reached and 2.6 months. The median PFS of those with pleural effusion CD4/CD8 ≥ 1.93 and < 1.93 were 18.4 and 1.2 months. In conclusion, patients with type 1 and 2 effusion patterns had better survival than those with type 3. Type 1 might be interpreted as pseudoprogression of malignant pleural effusion. CD4/CD8 ratio ≥ 1.93 in pleural effusion is a good predicting factor for PFS.

Assessment of interferon-γ in pleural fluid as a prognostic factor of survival in malignant pleural mesothelioma.

BACKGOUND: Literature reports suggest that the host immune system may control Malignant Pleural Mesothelioma (MPM) growth, although its activity is limited by regulatory mechanisms. In this retrospective study, we analyzed the levels of pro-inflammatory (IL-1, IL-6, TNF), immune-regulatory (IL-10) and Th1/CTL-related cytokines (IL-12p70, IFN-γ) in the pleural exudate and their relationship with overall survival (OS) in MPM. METHODS: Cytokines were quantified by multiplexed immunoassay. Concentrations were dichotomized with respect to the median value. Correlation between cytokine level and OS was assessed using univariate (Kaplan-Meier curves) and multivariate (Cox regression) analyses. RESULTS: Regarding outcome, tumor histology, therapies undergone and IFN-γ were independent prognostic factors of OS in a 72 MPM training cohort. Notably, high concentrations of IFN-γ halved death probability (HR of high vs low IFN-γ concentration = 0.491, 95%CI 0.3-0.8, p = 0.007). Also in patients with epithelioid histology and those receiving at least one line of therapy, high IFN-γ level was an independent factor predictive of OS (HR of high vs low IFN-γ concentration were 0.497, p = 0.007 and 0.324, p = 0.006, respectively). However, these data were not confirmed in a 77 MPM validation cohort, possibly due to the low IFN-γ levels encountered in this population, and the heterogeneous distribution of disease stages between the training and the validation cohorts. None of the other cytokines showed any effect on survival. CONCLUSIONS: High level of IFN-γ in pleural effusion may be associated with better survival in MPM patients and potentially serve as a prognostic biomarker. Larger prospective studies are needed to ascertain this hypothesis.

Novel therapies for malignant pleural effusion: Anti­angiogenic therapy and immunotherapy (Review).

Patients with a variety of malignancies can develop malignant pleural effusion (MPE). MPE can cause significant symptoms and result in a marked decrease in quality of life and a poor prognosis. MPE is primarily considered as an immune and vascular manifestation of pleural metastases. In the present review, the existing evidence supporting the applicability of anti­angiogenic therapy and immunotherapy for the treatment of MPE was summarized. Patients with MPE have benefited from anti­angiogenic agents, including bevacizumab and endostar; however, no relevant prospective phase III trial has, thus far, specifically analyzed the benefit of anti­angiogenic therapy in MPE. Immunotherapy for MPE may be sufficient to turn a dire clinical situation into a therapeutic advantage. Similar to anti­angiogenic therapy, more clinical data on the efficiency and safety of immunotherapy for controlling MPE are urgently required. The combined use of anti­angiogenic therapy and immunotherapy may be a promising strategy for MPE, which requires to be further understood.

The M1/M2 spectrum and plasticity of malignant pleural effusion-macrophage in advanced lung cancer.

BACKGROUND: Malignant pleural effusion (MPE)-macrophage (Mφ) of lung cancer patients within unique M1/M2 spectrum showed plasticity in M1-M2 transition. The M1/M2 features of MPE-Mφ and their significance to patient outcomes need to be clarified; furthermore, whether M1-repolarization could benefit treatment remains unclear. METHODS: Total 147 stage-IV lung adenocarcinoma patients undergoing MPE drainage were enrolled for profiling and validation of their M1/M2 spectrum. In addition, the MPE-Mφ signature on overall patient survival was analyzed. The impact of the M1-polarization strategy of patient-derived MPE-Mφ on anti-cancer activity was examined. RESULTS: We found that MPE-Mφ expressed both traditional M1 (HLA-DRA) and M2 (CD163) markers and showed a wide range of M1/M2 spectrum. Most of the MPE-Mφ displayed diverse PD-L1 expression patterns, while the low PD-L1 expression group was correlated with higher levels of IL-10. Among these markers, we identified a novel two-gene MPE-Mφ signature, IL-1ß and TGF-ß1, representing the M1/M2 tendency, which showed a strong predictive power in patient outcomes in our MPE-Mφ patient cohort (N = 60, p = 0.013) and The Cancer Genome Atlas Lung Adenocarcinoma dataset (N = 478, p < 0.0001). Significantly, ß-glucan worked synergistically with IFN-γ to reverse the risk signature by repolarizing the MPE-Mφ toward the M1 pattern, enhancing anti-cancer activity. CONCLUSIONS: We identified MPE-Mφ on the M1/M2 spectrum and plasticity and described a two-gene M1/M2 signature that could predict the outcome of late-stage lung cancer patients. In addition, we found that "re-education" of these MPE-Mφ toward anti-cancer M1 macrophages using clinically applicable strategies may overcome tumor immune escape and benefit anti-cancer therapies.

Helper T cells in malignant pleural effusion.

Malignant pleural effusion (MPE) is a frequent complication of malignancies and poses a clinical problem. CD4+ T lymphocytes are the most frequent cell population in MPE. Traditionally, CD4+ T cells are classified into two subsets based on cytokine production profiles, type 1 (Th1) and type 2 (Th2) helper T cells, which exhibit distinct functions. Recently, other T-cell subsets have been added to the Th-cell "portfolio", including regulatory T, Th17, Th9, and Th22 cells. The current review focuses on summarizing the Th-cell phenotypic characteristics, mechanism of Th-cell differentiation, and their pleural space recruitment, based on recent research. We also describe the interplay in MPE among different Th cells, as well as Th cells and lung cancer cells or mesothelial cells. Future research should expand the landscape map of human MPE immune cells, explore the immuno-regulation of B cells, and investigate the communication between macrophages and Th cells in MPE, which may facilitate meaningful advancements in the diagnoses and therapeutics of MPE.

HMGB1 Promotes Myeloid Egress and Limits Lymphatic Clearance of Malignant Pleural Effusions.

Pleural effusions, when benign, are attributed to cardiac events and suffusion of fluid within the pleural space. When malignant, lymphatic obstruction by tumor and failure to absorb constitutively produced fluid is the predominant formulation. The prevailing view has been challenged recently, namely that the lymphatics are only passive vessels, carrying antigenic fluid to secondary lymphoid sites. Rather, lymphatic vessels can be a selective barrier, efficiently coordinating egress of immune cells and factors within tissues, limiting tumor spread and immune pathology. An alternative explanation, offered here, is that damage associated molecular pattern molecules, released in excess, maintain a local milieu associated with recruitment and retention of immune cells associated with failed lymphatic clearance and functional lymphatic obstruction. We found that levels of high mobility group box 1 (HMGB1) were equally elevated in both benign and malignant pleural effusions (MPEs) and that limited diversity of T cell receptor expressing gamma and delta chain were inversely associated with these levels in MPEs. Acellular fluid from MPEs enhanced γδ T cell proliferation in vitro, while inhibiting cytokine production from γδ T cells and monocytes as well as restricting monocyte chemotaxis. Novel therapeutic strategies, targeting HMGB1 and its neutralization in such effusions as well as direct delivery of immune cells into the pleural space to reconstitute normal physiology should be considered.

TSAd Plays a Major Role in Myo9b-Mediated Suppression of Malignant Pleural Effusion by Regulating TH1/TH17 Cell Response.

Emerging evidence indicates that Myo9b is a cancer metastasis-related protein and functions in a variety of immune-related diseases. However, it is not clear whether and how Myo9b functions in malignant pleural effusion (MPE). In this study, our data showed that Myo9b expression levels correlated with lung cancer pleural metastasis, and nucleated cells in MPE from either patients or mice expressed a lower level of Myo9b than those in the corresponding blood. Myo9b deficiency in cancer cells suppressed MPE development via inhibition of migration. Myo9b deficiency in mice suppressed MPE development by decreasing TH1 cells and increasing TH17 cells. CD4+ naive T cells isolated from Myo9b-/- mouse spleens exhibited less TH1 cell differentiation and more TH17 cell differentiation in vitro. mRNA sequencing of nucleated cells showed that T cell-specific adaptor protein (TSAd) was downregulated in Myo9b-/- mouse MPE, and enrichment of the H3K27me3 mark in the TSAd promoter region was found in the Myo9b-/- group. Naive T cells purified from wild type mouse spleens transfected with TSAd-specific small interfering RNAs (siRNAs) also showed less TH1 cell differentiation and more TH17 cell differentiation than those from the siRNA control group. Furthermore, downregulation of TSAd in mice using cholesterol-conjugated TSAd-specific siRNA suppressed MPE development, decreased TH1 cells, and increased TH17 cells in MPE in vivo. Taken together, Myo9b deficiency suppresses MPE development not only by suppressing pleural cancer metastasis but also by regulating TH1/TH17 cell response via a TSAd-dependent pathway. This work suggests Myo9b and TSAd as novel candidates for future basic and clinical investigations of cancer.

Characteristics of Malignant Pleural Effusion Resident CD8+ T Cells from a Heterogeneous Collection of Tumors.

While T cell-based cancer immunotherapies have shown great promise, there remains a need to understand how individual metastatic tumor environments impart local T cell dysfunction. At advanced stages, cancers that metastasize to the pleural space can result in a malignant pleural effusion (MPE) that harbors abundant tumor and immune cells, often exceeding 108 leukocytes per liter. Unlike other metastatic sites, MPEs are readily and repeatedly accessible via indwelling catheters, providing an opportunity to study the interface between tumor dynamics and immunity. In the current study, we examined CD8+ T cells within MPEs collected from patients with heterogeneous primary tumors and at various stages in treatment to determine (1) if these cells possess anti-tumor activity following removal from the MPE, (2) factors in the MPE that may contribute to their dysfunction, and (3) the phenotypic changes in T cell populations that occur following ex vivo expansion. Co-cultures of CD8+ T cells with autologous CD45- tumor containing cells demonstrated cytotoxicity (p = 0.030) and IFNγ production (p = 0.003) that inversely correlated with percent of myeloid derived suppressor cells, lactate, and lactate dehydrogenase (LDH) within the MPE. Ex vivo expansion of CD8+ T cells resulted in progressive differentiation marked by distinct populations expressing decreased CD45RA, CCR7, CD127, and increased inhibitory receptors. These findings suggest that MPEs may be a source of tumor-reactive T cells and that the cellular and acellular components suppress optimal function.

Platelet factor 4 regulates T cell effector functions in malignant pleural effusions.

Malignant pleural effusion (MPE) is defined as the presence of tumor cells in pleural fluid and it is a fatal complication of advanced lung adenocarcinoma (LAC). To understand the immune response to the tumor in MPE, we compared the concentration of immunomodulatory factors in MPE of LAC and pleural effusion of heart failure (HF) patients by ELISA, and the proliferation and cytotoxic phenotype of T cells stimulated in the presence of LAC and HF pleural fluids by cytometry. Platelet factor 4 (PF4), vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-ß) and P-selectin levels were higher in LAC than in HF pleural fluids. However, plasmatic PF4 and P-selectin levels were similar in LAC and HF. VEGF positively correlated with TGF-ß and sPD-L1 in LAC but not in HF pleural fluids. LAC pleural fluids also inhibited T lymphocyte proliferation and cytotoxicity and reduced IL-17 production. PF4 levels inversely correlated with T cell function. The high content of PF4 in MPE was associated with poor prognosis. Our findings suggest that an impaired response of T lymphocytes induced by PF4 provides a significant advantage for tumor progression.