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BackgroundMany 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.MethodWe measured cytokine/chemokine content of 252 malignant pleural effusion (MPE) samples across multiple cancers using a 40-analyte panel and Luminex multiplexing technology.ResultsEleven 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), TGFβ1 (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.ConclusionThe 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.

Background:. Fat grafting is an effective treatment for craniofacial deformities. Stromal vascular fraction (SVF) is a concentrated form of adipose derived stem cells that can be isolated from fat. The aim of this clinical trial was to assess the impact of SVF enrichment on craniofacial fat grafting. Methods:. Twelve subjects with at least two regions of craniofacial volume deficit were enrolled, and they underwent fat grafting with SVF-enriched or standard fat grafting to each area. All patients had bilateral malar regions injected with SVF-enriched graft on one side and control standard fat grafting to the contralateral side. Outcome assessments included demographic information, volume retention determined by CT scans, SVF cell populations assessed by flow cytometry, SVF cell viability, complications, and appearance ratings. Follow-up was 9 months. Results:. All patients had improvement in appearance. There were no serious adverse events. There was no significant difference in volume retention between the SVF-enriched and control regions overall (50.3% versus 57.3%, P = 0.269) or comparing malar regions (51.4% versus 56.7%, P = 0.494). Patient age, smoking status, obesity, and diagnosis of diabetes did not impact volume retention. Cell viability was 77.4% ± 7.3%. Cellular subpopulations were 60.1% ± 11.2% adipose derived stem cells, 12.2 ± 7.0% endothelial cells, and 9.2% ± 4.4% pericytes. A strong positive correlation was found between CD146+ CD31-pericytes and volume retention (R = 0.863, P = 0.027). Conclusions:. Autologous fat transfer for reconstruction of craniofacial defects is effective and safe, leading to reliable volume retention. However, SVF enrichment does not significantly impact volume retention.

IntroductionTreatment options for patients with malignant pleural effusions (MPE) are limited due, at least in part, to the unique environment of the pleural space, which drives an aggressive tumor state and governs the behavior of infiltrating immune cells. Modulation of the pleural environment may be a necessary step toward the development of effective treatments. We examine immune checkpoint molecule (ICM) expression on pleural T cells, the secretomes of pleural fluid, pleural infiltrating T cells (PIT), and ability to activate PIT ex vivo.MethodsICM expression was determined on freshly drained and in vitro activated PIT from breast, lung and renal cell cancer. Secretomics (63 analytes) of activated PIT, primary tumor cultures and MPE fluid was determined using Luminex technology. Complementary digital spatial proteomic profiling (42 analytes) of CD45+ MPE cells was done using the Nanostring GeoMx platform. Cytolytic activity was measured against autologous tumor targets.ResultsICM expression was low on freshy isolated PIT; regulatory T cells (T-reg) were not detectable by GeoMx. In vitro activated PIT coexpressed PD-1, LAG-3 and TIGIT but were highly cytotoxic against autologous tumor and uniquely secreted cytokines and chemokines in the > 100 pM range. These included CCL4, CCL3, granzyme B, IL-13, TNFα, IL-2 IFNγ, GM-CSF, and perforin. Activated PIT also secreted high levels of IL-6, IL-8 and sIL-6Rα, which contribute to polarization of the pleural environment toward wound healing and the epithelial to mesenchymal transition. Addition of IL-6Rα antagonist to cultures reversed tumor EMT but did not alter PIT activation, cytokine secretion or cytotoxicity.DiscussionDespite the negative environment, immune effector cells are plentiful, persist in MPE in a quiescent state, and are easily activated and expanded in culture. Low expression of ICM on native PIT may explain reported lack of responsiveness to immune checkpoint blockade. The potent cytotoxic activity of activated PIT and a proof-of-concept clinical scale GMP-expansion experiment support their promise as a cellular therapeutic. We expect that a successful approach will require combining cellular therapy with pleural conditioning using immune checkpoint blockers together with inhibitors of upstream master cytokines such as the IL-6/IL-6R axis.

Objective: The goal of this study was to determine whether antiIL-6Rα block (tocilizumab) will alter the pleural secretome and will diminish tumor-specific immune responses. Methodology: Pleural T cells were isolated from freshly drained pleural effusions (n=6). Autologous pleural tumor was expanded in vitro using the Mammary Epithelial Growth Medium (Lonza). Pleural T cells were stimulated using anti-CD3/CD28 Dynal beads and low dose IL-2 (60 Cetus U/ml) for 2,4,7, 14 or 21 days in the presence of tocilizumab for the last 48h (0, 0.35, 0.72, 1.43, 2.86 and 5.72ug/ml). Pleural T cell effectors were counted and plated on tumor targets at 12.5:1 E:T in the presence of tocilizumab. Results: Ex vivo expanded pleural T cells were effector-memory phenotype (CD45RA-CD27-) and were highly cytotoxic against autologous tumor (89-100%). The majority of CD8+ T cells were central memory (CD45RA-/CD27-) or effector memory (CD45RA+/CD27-); the majority co-expressed granzyme B, perforin, 20-60% expressed PD-1. Most CD4+ co-expressed granzyme B and perforin and were PD-1+, suggesting cytotoxic CD4+ T cells. The presence of tocilizumab reversed tumor EMT but did not alter cytotoxicity. Conclusion: We show that the IL-6/IL-6Rα axis is prominent in MPE, drives tumor growth and inhibits anti-tumor immunity. Pleural T cells are neither exhausted nor dysfunctional but are suppressed by the pleural environment. Ex vivo expanded MPE CD8 and CD4 T cells are highly cytotoxic against autologous tumor. Anti-IL-6Rα block reverses tumor EMT but does not inhibit effector responses.

ABSTRACT Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

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), TGFβ1 (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. [ABSTRACT FROM AUTHOR]

Adoptive Cellular Therapy (ACT) is transitioning from experimental to standard of care. Limited specificities of chimeric antigen receptor T cells (CAR-T) are licensed drugs, with commercial products selling for ∼400,000 USD. Tumor infiltrating lymphocytes (TIL) can target multiple cell surface and intracellular tumor antigens. TIL have not been commercialized, due to complex logistics and cost. Our objective is to leverage a single standardized platform (Miltenyi Prodigy) for in-house ACT.We currently manufacture CD19 CAR-T under an FDA IND and are developing a virtually identical TIL protocol minus the lentiviral vector. TIL are manufactured from malignant pleural effusions rich in immune infiltrates. CD4+ and CD8+ cells are enriched and activated with anti-CD3/CD28 beads. CAR-T are transduced, and both CAR-T and TIL are expanded (IL-7/IL-15) for 5 days. The product is infused fresh, avoiding losses associated with cryopreservation and thawing.Developing and validating release tests (absence of replication competent virus, vector copy number, chimeric antigen receptor expression, endotoxin testing) posed an initial challenge. Having completed assay development, our manufacturing process, including release testing, can be performed for less than 1/10 the cost of commercial CAR-T.We expect that the platform that we have validated will be easily transitioned to new chimeric antigen receptor designs and specificities and will likewise be adaptable to TIL manufacture for the wide variety of cancers that metastasize to the pleura of peritoneum. Standardized in-house ACT manufacture may greatly reduce the cost of cellular immunotherapies, making it more widely available to patients.

Metastasis to the pleural and peritoneal cavities is a common terminal pathway for a wide variety of cancers. This article explores how these unique environments both promote aggressive tumor behavior and suppresses anti-tumor immunity, and ways in which local delivery of protein therapeutics can leverage the contained nature of these spaces to a therapeutic advantage, achieving high intra-cavital concentrations while minimizing systemic toxicity.

Abstract Malignant pleural effusions (MPE) are a common terminal pathway for many cancers, with an estimated United States incidence of more than 150,000 cases per year. MPE is an aggressive disease with a uniformly fatal prognosis and a life expectancy of only 3 to 12 months. The development of an effective targeted therapy represents a pressing unmet need. This commentary focuses on how cellular and humoral components condition the pleural space as a tumor-promoting, wound-healing environment. Despite an abundance of potential antigen presenting and effector cells in the pleura, their physical isolation by the mesothelial barrier, the concentration of cytokines and chemokines driving the epithelial to mesenchymal transition (EMT) and M2 /Th-2 polarization, suppress tumor-specific immune effector responses. We argue that local immune repolarization must precede either immune checkpoint or cellular therapy to successfully eradicate pleural tumor. We further hypothesize that, because of its cellular content, a repolarized pleural space will provide an effective immune environment for generation of systemic anti-tumor response.