Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions

Antibody-secreting cells are terminally differentiated B cells that play a critical role in humoral immunity through immunoglobulin secretion along with possessing the potential to be long-lived. It is now appreciated that antibody-secreting cells regulate multiple aspects of biology through the secretion of various cytokines. In this regard, intracellular flow cytometry is a key tool used to assess the presence of intracellular proteins such as cytokines and transcription factors. Here, we showed that the use of phycoerythrin-containing antibody conjugates led to a false interpretation of antibody-secreting cell intracellular protein expression compared to other cell types. This was mainly due to the inappropriate retention of these antibodies specifically within antibody-secreting cells. Furthermore, we demonstrated how to reduce this retention which allowed for a more accurate comparison of intracellular protein expression between antibody-secreting cells and other cell types such as B lymphocytes. Using this methodology, our data revealed that spleen antibody-secreting cells expressed Toll-like receptor 7 as well as the pro-form of the inflammatory cytokine interleukin-1β.

878 Novel microbial immunotherapy approach for the treatment of bladder cancer

BackgroundMicrobial immunotherapy, in the form of intravesical Bacillus Calmette-Guérin (BCG), is the standard-of-care for non-muscle invasive bladder cancer (NMIBC). BCG therapy is associated with significant side-effects, high disease recurrence and progression rates, and product supply shortages, leaving a significant unmet medical need for bladder cancer patients.1 2 We sought to establish the preclinical safety and efficacy of live-attenuated Salmonella enterica Typhi strain ZH9 (ΔaroC, ΔssaV) as a novel microbial immunotherapy.MethodsTherapeutic efficacy of intravesical ZH9 was established in the murine orthotopic, syngeneic MB49 bladder tumor model. Tumor-bearing animals were treated with a single intravesical dose of ZH9 or OncoTice BCG and long-term survival comparisons were evaluated by log-rank (Mantel-Cox) test. ZH9 interaction with urothelial cancer cells was investigated using in vitro invasion assays and flow cytometry staining for intracellular Salmonella common antigen (CSA-1) and propidium iodide for cell death. Local immune responses were analyzed by flow cytometry staining of disaggregated mouse bladders.ResultsMice treated with a single intravesical dose of ZH9 2 days after MB49 tumor inoculation demonstrated significant survival benefit compared to vehicle treated (median survival 49.5 vs. 31 days, p=0.003) and BCG treated animals (median survival 49.5 vs. 27.5 days, p<0.001). A second, stringent model setup with intravesical treatment 4 days after tumor inoculation showed significant efficacy of ZH9 versus vehicle and BCG (median survival 30 vs. 20.5 (p=0.003) and 23.5 (p=0.025) days, respectively). Surviving ZH9 treated animals demonstrated 100% protection from tumor take following repeated intravesical challenge with MB49 tumor cells, suggesting lasting anti-tumor immunity resulting from ZH9 treatment. In vitro, intracellular flow cytometry in human (UMUC3, T24, RT4, 5637) and mouse (MB49) urothelial cancer cell lines showed that ZH9 invaded and induced cell death in all cell lines. In vivo, a single treatment with intravesical ZH9 resulted in strong cellular immune responses characterized by recruitment of monocytes, NK cells, CD4+ and CD8+ T cells, and dendritic cells with an activated, cross-presenting (Ly6C+, CD103+) phenotype. Intravesical ZH9 resulted in a greater magnitude and duration of immune cell recruitment in the urothelium compared to a single equivalent dose of intravesical BCG.ConclusionsLive-attenuated Salmonella strain ZH9 demonstrated a significant survival benefit over the standard-of-care OncoTice BCG in an orthotopic bladder cancer model. ZH9 demonstrated direct tumour cell killing in vitro and induction of robust cellular immune responses in vivo. Preclinical studies indicate significant therapeutic potential of intravesical ZH9 as a novel microbial immunotherapy in bladder cancer.ReferencesLiu Y, Lu J, Huang Y, Ma L. Clinical spectrum of complications induced by intravesical immunotherapy of Bacillus Calmette-Guérin for bladder cancer. J Oncol 2019 March 10;2019:6230409.vanRhijn BW, Burger M, Lotan Y, et al. Recurrence and progression of disease in non-muscle-invasive bladder cancer: from epidemiology to treatment strategy. Eur Urol 2009 September;56(3):430–42.Ethics ApprovalThe studies were conducted under approval from Pennsylvania State College of Medicine Institutional Animal Care and Use Committee approval number 47682, or with approval of the Cantonal Veterinary Office of Canton de Vaud, Switzerland.

Bicentric evaluation of stabilizing sampling tubes for assessment of monocyte HLA-DR expression in clinical samples

Background. Diminished expression of human leukocyte antigen DR on circulating monocytes (mHLA-DR), measured by standardized flow cytometry procedure, is a reliable indicator of immunosuppression in severely injured intensive care unit patients. As such, it is used as stratification criteria in clinical trials evaluating novel immunostimulating therapies. Pre-analytical constraints relative to the short delay between blood sampling and flow cytometry staining have nevertheless limited its use in multicentric studies. The objective of the present work was to compare mHLA-DR expression between whole blood samples simultaneously drawn in EDTA or Cyto-Chex BCT tubes. Methods. In 2 university hospitals, mHLA-DR was assessed in fresh whole blood from septic patients (n = 12) and healthy donors (n = 6) simultaneously sampled on EDTA and Cyto-Chex BCT tubes. Staining was performed immediately after sampling and after blood storage at room temperature. Results. We observed the remarkable stability of mHLA-DR results when blood was collected in Cyto-Chex BCT tubes (until 48-72 h). On baseline values, despite good correlation between tubes (r = 0.98, p< 0.001), mHLA-DR expression was systematically lower with Cyto-Chex BCT. Conclusion. The present reports confirms the great potential of Cyto-Chex BCT tubes to delay mHLA-DR staining in centers without rapid access to flow cytometry facilities. However, a 30 % gap exists between results obtained with EDTA and Cyto-Chex BCT tubes. As current thresholds for clinical decisions were obtained with EDTA samples, further studies are needed to confirm clinical thresholds with Cyto-Chex BCT tubes.

Reticulated Platelets—Which Functions Have Been Established by In Vivo and In Vitro Data?

Reticulated platelets (RP) are the youngest platelet fraction released into the circulation. These immature platelets have increased RNA content, a larger cell volume, more dense granules, higher levels of surface activation markers and are thought to be more reactive compared to their mature counterparts. RP have been associated with cardiovascular disease, diabetes and increased mortality. Yet only a few animal studies investigating RP have been conducted so far and further investigations are warranted. Established methods to count RP are flow cytometry (staining with thiazole orange or SYTO13) or fully automated hematology analyzers (immature platelet fraction, IPF). IPF has been established as a diagnostic parameter in thrombocytopenia, cardiovascular disease and, in particular, the response to antiplatelet therapy. This review seeks to provide an overview of the key features of RP as well as preanalytical and analytical aspects that need to be considered when working with this platelet population.

Computer-Aided Design, Synthesis, and Antiviral Evaluation of Novel Acrylamides as Potential Inhibitors of E3-E2-E1 Glycoproteins Complex from Chikungunya Virus

Chikungunya virus (CHIKV) causes an infectious disease characterized by inflammation and pain of the musculoskeletal tissues accompanied by swelling in the joints and cartilage damage. Currently, there are no licensed vaccines or chemotherapeutic agents to prevent or treat CHIKV infections. In this context, our research aimed to explore the potential in vitro anti-CHIKV activity of acrylamide derivatives. In silico methods were applied to 132 Michael’s acceptors toward the six most important biological targets from CHIKV. Subsequently, the ten most promising acrylamides were selected and synthesized. From the cytotoxicity MTT assay, we verified that LQM330, 334, and 336 demonstrate high cell viability at 40 µM. Moreover, these derivatives exhibited anti-CHIKV activities, highlighting the compound LQM334 which exhibited an inhibition value of 81%. Thus, docking simulations were performed to suggest a potential CHIKV-target for LQM334. It was observed that the LQM334 has a high affinity towards the E3-E2-E1 glycoproteins complex. Moreover, LQM334 reduced the percentage of CHIKV-positive cells from 74.07 to 0.88%, 48h post-treatment on intracellular flow cytometry staining. In conclusion, all virtual simulations corroborated with experimental results, and LQM334 could be used as a promising anti-CHIKV scaffold for designing new drugs in the future.

SAT0011 COMBINED INHIBITION OF AUTOPHAGY AND GLUTAMINE METABOLISM SUPPRESSES CELL GROWTH OF RA SYNOVIOCYTES AND AMELIORATES ARTHRITIS IN SKG MICE

Background:Immunometabolism is now recognaized to be crucial in the pathogenesis of rheumatoid arthritis (RA). We have recently shown that the expression of glutaminase 1 (GLS1), a key enzyme in glutaminolysis, is upregulated in fibroblast-like synoviocytes from RA patients (RA-FLS) and that GLS1 inhibition suppresses RA-FLS proliferation (1). However, glutaminolysis has been known to suppress autophagy by activating mTORC1 or counteracting ROS production (2). Given the possibility of autophagy upregulation following glutamiolysis inhibition, therapies targeting both autophagy and glutaminolysis may be more effective in suppressing cell growth of RA-FLS, yet the relation between glutaminolysis and autophagy in RA-FLS has not been investigated.Objectives:To examine the effects of inhibiting both glutaminolysis and autophagy on RA-FLS and autoimmune arthritis in SKG mice.Methods:GLS1 inhibitor, compound 968 (C968), was used to suppress glutaminolysis, and Chloroquine (CQ) was used to inhibit autophagy. To detect autophagy, the expression of ATG5 and LC3B was measured by real-time PCR and the production of LC3-II was analyzed by Western blotting. The formation of autophagic vacuoles was identified by immunfluorescense. Cell growth was evaluated by BrdU assay. Apoptosis was analyzed by flow cytometry staining with Annexin V-FITC and PI. C968 and CQ were administered subcutaneously to Zymosan A-injected SKG mice.Results:C968 upregulated the expression of ATG5 and LC3B, and increased the protein level of LC3-II in RA-FLS. C968 also facilitated autophagosome formation. These results suggested that inhibition of glutaminolysis promoted autophagy in RA-FLS. The combined treatment with C968 and CQ significantly suppressed cell proliferation of RA-FLS more strongly than did C968 or CQ alone. In addition, C968 combined with CQ increased the apoptosis rate, whereas either C968 or CQ alone did not. Furthermore, combination of C968 and CQ significantly attenuated the degree of arthritis in SKG mice, while C968 or CQ monotherapy did not (Figure).Conclusion:The GLS1 inhibitor C968 promotes autophagy in RA-FLS. C968 in combination with CQ reduces proliferation and enhances apoptosis in RA-FLS, and ameliorates the arthritis in SKG mice. Suppressing C968-induced autophagy may be a promising therapy for arthritis.References:[1] Takahashi S., et al. Arthritis Res Ther. 2017 Apr 11;19(1):76.[2] Villar VH., et al. Autophagy. 2015;11(8):1198-208.Acknowledgments :NoneDisclosure of Interests:None declared