Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Analysis of monocyte subsets in colorectal cancer patients: Marker potential in diagnosis, cancer therapy and tissue regeneration

Humane Monozyten stellen eine heterogene Population außerordentlich vielfältiger Immunzellen dar. Sie werden anhand unterschiedlicher Expression des Endotoxin Co-Rezeptors CD14 und des Fc Rezeptors CD16 in die Hauptpopulation CD14++CD16- “klassischer” sowie in die zwei kleineren Subpopulationen CD14++CD16+ “intermediärer” und CD14+CD16++ “nicht-klassischer” Monozyten eingeteilt, welche unterschiedliche genetische, phänotypische und funktionelle Eigenschaften aufweisen. Klassische Monozyten haben das größte Phagozytosepotential, nicht-klassische Monozyten produzieren die größten Mengen proinflammatorischer Zytokine und intermediäre Monozyten sind spezialisiert auf Antigenpräsentation und exprimieren die größten Mengen proangiogenetischer Moleküle wie TIE2. Die TIE2-exprimierenden Monozyten (TEMs) sind speziell innerhalb der intermediären Monozyten angereichert, stellen eine Monozyten Hauptpopulation in Tumoren dar und fördern das Tumorwachstum. In Anbetracht der unterschiedlichen Monozyten Populationen sowie ihrer Entwicklung zu Tumor-assoziierten Makrophagen haben wir die Hypothese aufgestellt, dass lokale Gegebenheiten im Tumor bereits durch Veränderungen der zirkulierenden Monozyten, entweder in ihrer Anzahl oder ihrer Genexpression, reflektiert sind und daher der Makrophageninfiltration oder Polarisierung im Tumorgewebe vorausgehen. Folglich haben wir Monozyten Subpopulationen im Blut sowie im Gewebe von Patienten mit Kolorektalkarzinom (KRK) charakterisiert. Unsere Ergebnisse zeigen, dass intermediäre Monozyten (aber nicht TEMs) signifikant im Blut der KRK Patienten erhöht sind und Potential für KRK Diagnose ausweisen. Die größte Anzahl intermediärer Monozyten wurde in Patienten mit lokalisiertem Krankheitsstadium detektiert. Diese Ergebnisse deuten auf eine Rolle der intermediären Monozyten in der Früherkennung des Tumors durch das angeborene Immunsystem. In der neoadjuvanten Chemotherapie des metastasierten KRK stieg die Anzahl der intermediären Monozyten im Blut nach zwei Therapiezyklen deutlich an und korrelierte mit dem radiologischen Therapieansprechen, während ihr Genexpressionsmuster und ihre Migrationsfähigkeit unverändert blieben. Dieser Anstieg der intermediären Monozyten ist vermutlich Ausdruck einer systemischen Reaktion auf die zytotoxische Tumorzerstörung durch die Chemotherapie. Bemerkenswerterweise korrelierten die Blutwerte der intermediären Monozyten mit der Tumorinfiltration durch CD14+CD16+ Monozyten in kolorektalen Lebermetastasen (KLM). Nach Leberresektion bei KLM stiegen die intermediären Monozyten im Blut am ersten postoperativen Tag (POT) auffallend stark an, was mit der Konzentration an C-reaktivem Protein korrelierte und als Ausdruck der Akuten-Phase-Reaktion interpretiert werden kann. Anhaltend hohe Werte intermediärer Monozyten am 3. POT korrelierten signifikant mit erhöhten Leberenzymen, die als Marker für verzögerte Leberregeneration gelten. Intermediäre Monozyten, insbesondere jedoch TEMs, akkumulierten in der subhepatischen Wundflüssigkeit am 3. POT, was eine Rolle der TEMs in der Geweberegeneration nahelegt. Abschliessend lässt sich festhalten, dass intermediäre Monozyten ein wesentliches diagnostisches sowie auch prädiktives Markerpotential in Patienten mit KRK besitzen.Human monocytes represent a heterogenous population of highly versatile immune cells. They are officially classified by their differential expression of the pattern recognition receptor CD14 and the Fc receptor CD16 into the major subset of CD14++CD16- classical and the two minor subsets of CD14++CD16+ intermediate and CD14+CD16++ non-classical monocytes, all of which have distinct genetic, phenotypical and functional properties. Classical monocytes show high phagoycytosis potential, non-classical monocytes produce highest amounts of pro-inflammatory cytokines, and intermediate monocytes are specialized in antigen presentation and express highest levels of pro-angiogenic molecules including TIE2. TIE2-expressing monocytes (TEMs) are selectively enriched in the intermediate subset and represent a major monocyte population in tumors which is proposed to promote cancer growth. Given the diverse nature of human monocytes and their development into tumor-associated macrophages, we hypothesized that the local disturbance in the tumor tissue may be reflected by circulating monocytes, either numerically or in terms of gene expression and function, and thus might precede alterations in macrophage infiltration or polarization in tumor tissue. We thus characterized monocyte subsets in blood and tissue of colorectal cancer (CRC) patients. Our results show that intermediate monocytes (but not TEMs) are significantly elevated in the blood of colorectal cancer patients and possess diagnostic potential for CRC diagnosis. Highest levels of intermediate monocytes were detected in localized disease and a stronger in vitro induction of these cells was observed in response to supernatants from primary as opposed to metastastic colon cancer cells. These findings support a role of intermediate monocytes in early tumor recognition by the innate immune system. In neoadjuvant cancer therapy for metastasized CRC, the frequency of blood intermediate monocytes rapidly increased after 2 therapy cycles and correlated with radiological treatment response, while their gene expression and migratory capacity remained unaltered. This increase of intermediate monocytes is likely to reflect the systemic response to the cytotoxic destruction of tumor tissue by chemotherapy. Notably, the blood levels of intermediate monocytes were also found to correlate with tumor infiltration by CD14+CD16+ monocytes in colorectal liver metastases (CLM). After liver resection for CLM, blood monocytes shifted further to remarkably high numbers of intermediate monocytes on postoperative day (POD) 1, which correlated with C-reactive protein values, likely reflecting the acute phase reaction. Sustained levels of intermediates on POD 3 signifcantly correlated with elevated liver enzymes, a marker of delayed liver regeneration. Intermediate monocytes, in particular TEMs, accumulated in subhepatic wound fluid by POD 3, which supports a role for TEMs in tissue regeneration. In conclusion, intermediate monocytes were found to exhibit substantial diagnostic and predictive marker potential for CRC patients.submitted by Domink SchauerAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Diss., 2017OeBB(VLID)247752

Platelet-Stored Angiogenesis Factors: Clinical Monitoring Is Prone to Artifacts

Background: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing

Myelosuppression of Thrombocytes and Monocytes Is Associated with a Lack of Synergy between Chemotherapy and Anti-VEGF Treatment1

Purpose: Chemotherapeutic agents that have shown improved patient outcome when combined with anti-vascular endothelial growth factor (VEGF) therapy were recently identified to induce the mobilization of proangiogenic Tie-2-expressing monocytes (TEMs) and endothelial progenitor cells (EPCs) by platelet release of stromal cell-derived factor 1α (SDF-1α). VEGF blockade was found to counteract cell mobilization. We aimed to determine why agents like gemcitabine do not elicit TEM and EPC recruitment and may therefore lack synergy with anti-VEGF therapy. Experimental Design: Locally advanced pancreatic cancer patients (n = 20) were monitored during 16 weeks of neoadjuvant therapy. Treatment was based on gemcitabine with or without the addition of bevacizumab. Blood levels of proangiogenic cell populations and angiogenesis factors were determined in 2-week intervals. Results: The lack of EPC mobilization during gemcitabine therapy was associated with severe thrombocytopenia and reduced SDF-1α blood concentrations. Furthermore, myelosuppression by gemcitabine correlated significantly with loss of TEMs. With respect to angiogenic factors stored and released by platelets, plasma levels of the angiogenesis inhibitor thrombospondin 1 (TSP-1) were selectively decreased and correlated significantly with thrombocytopenia in response to gemcitabine therapy. Conclusions: A thorough literature screen identified thrombocytopenia as a common feature of chemotherapeutic agents that lack synergy with anti-VEGF treatment. Our results on gemcitabine therapy indicate that myelosuppression (in particular, with respect to thrombocytes and monocytes) interferes with the mobilization of proangiogenic cell types targeted by bevacizumab and may further counteract antiangiogenic therapy by substantially reducing the angiogenesis inhibitor TSP-1

Discrimination between Circulating Endothelial Cells and Blood Cell Populations with Overlapping Phenotype Reveals Distinct Regulation and Predictive Potential in Cancer Therapy1

AbstractBACKGROUND: Circulating endothelial cells (CECs) have been proposed to predict patient response to antiangiogenic cancer therapy. However, contradictory reports and inconsistency in the phenotypic identification of CECs have led us to compare three cell populations with partially overlapping phenotype in cancer patients receiving chemotherapy and the antiangiogenic agent bevacizumab. METHODS: Patients (n = 20) with locally advanced pancreatic cancer were monitored during 16 weeks of neoadjuvant treatment with gemcitabine and bevacizumab. Detection of circulating cell populations was based on the marker combination CD45, CD31, and CD146; levels of viable and dead (7-aminoactinomycin D-positive) cells were evaluated by flow cytometry in 2-week intervals. RESULTS: We were able to discriminate and concomitantly monitor three cell populations elevated in cancer patients. Whereas CECs were defined as CD45- CD31+ CD146+, the distinct populations of CD45- CD31- CD146+ and CD45- CD31high CD146- cells were partly positive for CD3 and CD41, respectively. CECs and CD45- CD31- CD146+ cells increased during therapy; the rise in dead cells was positively correlated with patient response or survival. Conversely, CD45- CD31high CD146- cells decreased in neoadjuvant treatment. A highly significant correlation was established for improved patient response and a minor decrease in viable cell counts. CONCLUSIONS: Flow cytometric CEC analysis based on CD45, CD31, and CD146 requires careful discrimination between blood cell populations with overlapping phenotype showing hallmarks of activated T cells and large platelets. However, these three cell populations show distinct regulation during cancer therapy, and their concomitant analysis may offer extended prognostic and predictive information

Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

<div><p>Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones.</p> </div