Multi-channel Flow Cytometry Research Articles

(058) Detection and Transcriptional Profiling of Mesenchymal Stem Cells in Peyronie’s Plaque Explants

Abstract Introduction The mechanisms behind the pathogenesis of Peyronie’s disease largely remain a mystery. Plaques have largely been thought to be derived from epithelial to mesenchymal transformation of fibroblasts to myofibroblasts. Objective We sought to transcriptomically profile explanted cells from Peyronie’s plaques and evaluate their profiles with transforming growth factor B (TGF-B) stimulation. Methods Peyronie’s plaque were excised from 3 patients undergoing penile incision and grafting surgery. Cells were explanted from biopsy tissue and propagated. Cells were profiled using single cell RNA sequencing (scRNAseq). These cells were then exposed to TGF-□ to simulate the active phase of Peyronie’s disease, and profiled with scRNAseq at baseline and 24 hours following TGF-□□ stimulation. We used principle component analysis (PCA) to reduce the dimension of data and built the nearest-neighbors graph using PCA weights. We performed clustering over the nearest-neighbor graph and visualized the clusters using UMAP algorithm. Differential gene expression and pathway analyses were performed using the top 50 genes with increased expression between 0- and 24-hour time points. Multi-channel flow cytometry and immunofluorescence were used for mesenchymal stem cell (MSC) phenotypic validation. Results Assessment of cell clustering identified profiles of fibroblasts, myofibroblasts, smooth muscle, and mesenchymal stem-like cells. We further confirmed rare presence of mesenchymal stem cells using multi-channel flow cytometry in our cultured cells and plaque biopsies. Differential gene expression of the 50 genes showing the greatest induction showed that genes involved in cellular binding and catalytic activity were most commonly upregulated following introduction of TGF-B. Conclusions This analysis provides unique insight into the expression profiles of Peyronie’s plaque cell populations explanted in culture and stimulated via TGF-B to activate myofibroblasts akin to the active phase of Peyronie’s disease. Using the information gained from this single-cell RNA sequencing analysis ongoing efforts can be performed to identify therapeutic targets for active phase Peyronie’s disease. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast; Boston; Teumo Health Technologies.

PXPermute reveals staining importance in multichannel imaging flow cytometry

Imaging flow cytometry (IFC) allows rapid acquisition of numerous single-cell images per second, capturing information from multiple fluorescent channels. However, the traditional process of staining cells with fluorescently labeled conjugated antibodies for IFC analysis is time consuming, expensive, and potentially harmful to cell viability. To streamline experimental workflows and reduce costs, it is crucial to identify the most relevant channels for downstream analysis. In this study, we introduce PXPermute, a user-friendly and powerful method for assessing the significance of IFC channels, particularly for cell profiling. Our approach evaluates channel importance by permuting pixel values within each channel and analyzing the resulting impact on machine learning or deep learning models. Through rigorous evaluation of three multichannel IFC image datasets, we demonstrate PXPermute's potential in accurately identifying the most informative channels, aligning with established biological knowledge. PXPermute can assist biologists with systematic channel analysis, experimental design optimization, and biomarker identification.

Uncovering mechanisms of response of pembrolizumab and lenvatinib for the treatment of platinum-resistant high grade serous ovarian cancer.

TPS5619 Background: Despite initial response to conventional platinum-containing therapies, the majority of high grade serous ovarian cancer (HGSOC) patients will eventually develop treatment resistance. Despite interest for immune checkpoint inhibitor alternatives, pembrolizumab monotherapy is poor with objective response rates (ORR) ranging between 4.1 - 10%. Poor response may be linked to higher expression of immunosuppressive factors (e.g. VEGF, TGF-β) in the tumor microenvironment (TME). In an immunosuppressive TME, monotherapy with PD-1 inhibitors can induce T-cell dysfunction and apoptosis, leading to innate resistance to pembrolizumab. Preclinical studies of other tumors have demonstrated that lenvatinib can improve T-cell activation and reduce the tumor-associated macrophage population and TGF-β/VEGF/FGF-signaling. In HGSOC, mechanisms of resistance to pembrolizumab and whether this resistance can be overcome by the addition of lenvatinib remain unknown. The objective of this investigation is to uncover the immunologic basis and synergistic mechanisms of action underlying clinical efficacy of pembrolizumab and lenvatinib in HGSOC. Specifically, we will evaluate the dynamic changes in proliferative and dysfunctional T-cell populations in the peritoneal TME (p-TME) of HGSOC patients receiving monotherapy with pembrolizumab or lenvatinib followed by combination therapy. Methods: All patients will receive a cycle of monotherapy (pembrolizumab 200 mg IV every 3 weeks or lenvatinib 20 mg po daily) followed by combination therapy (both) up to 35 cycles. Enrolled patients will receive intra-peritoneal port placement for serial peritoneal fluid evaluations (cycle 0 day 1, cycle 0 day 8, cycle 1 day 8, cycle 2 day 1, cycle 3 day 1, cycle 5 day 1, then day 1 of every 3rd cycle) for multichannel flow cytometry and single-cell RNA sequencing. Our primary translational objective is to estimate both the individual and combined effects of pembrolizumab and lenvatinib on T-cell dysfunction (PD1+ CD38+) and proliferation (ki67+) in the p-TME using multichannel flow cytometry on the peritoneal fluid. Secondary clinical objective is ORR. Secondary translational objectives include individual and synergistic effects of monotherapy and combination therapy on T-cell effector function, T-cell memory, and myeloid cell subpopulations. Exploratory objectives include evaluating the dynamic changes in immune and non-immune cells in the p-TME for exceptional responders and non-responders using single cell RNA sequencing. Key eligibility criteria include 1) histologic diagnosis of high grade serous ovarian, peritoneal, or fallopian tube cancer 2) platinum-resistant disease and 3) no prior lenvatinib therapy. The trial is open with 10 patients enrolled at the time of submission. Clinical trial information: NCT05114421 .

Molecular Markers of Blood Cell Populations Can Help Estimate Aging of the Immune System.

Aging of the immune system involves functional changes in individual cell populations, in hematopoietic tissues and at the systemic level. They are mediated by factors produced by circulating cells, niche cells, and at the systemic level. Age-related alterations in the microenvironment of the bone marrow and thymus cause a decrease in the production of naive immune cells and functional immunodeficiencies. Another result of aging and reduced tissue immune surveillance is the accumulation of senescent cells. Some viral infections deplete adaptive immune cells, increasing the risk of autoimmune and immunodeficiency conditions, leading to a general degradation in the specificity and effectiveness of the immune system in old age. During the COVID-19 pandemic, the state-of-the-art application of mass spectrometry, multichannel flow cytometry, and single-cell genetic analysis have provided vast data on the mechanisms of aging of the immune system. These data require systematic analysis and functional verification. In addition, the prediction of age-related complications is a priority task of modern medicine in the context of the increase in the aged population and the risk of premature death during epidemics. In this review, based on the latest data, we discuss the mechanisms of immune aging and highlight some cellular markers as indicators of age-related immune disbalance that increase the risk of senile diseases and infectious complications.

Myeloid-Derived Suppressor Cells (MDSC) in Melanoma Patients Treated with Anti-PD-1 Immunotherapy.

Myeloid-derived suppressor cells (MDSC) are a subset of immature myeloid cells with suppressive activity well described in the context of cancer. They inhibit anti-tumour immunity, promote metastasis formation and can lead to immune therapy resistance. In a retrospective study, blood probes of 46 advanced melanoma patients were analysed before the first administration of anti-PD-1 immunotherapy and in the third month of treatment for MDSC, immature monocytic (ImMC), monocytic MDSC (MoMDSC) and granulocytic MDSC (GrMDSC) by multi-channel flow cytometry. Cell frequencies were correlated with response to immunotherapy, progression-free survival (PFS) and lactate dehydrogenase (LDH) serum level. Responders to anti-PD-1 therapy had higher MoMDSC levels (4.1 ± 1.2%) compared to non-responders (3.0 ± 1.2%) (p = 0.0333) before the first administration of anti-PD-1. No significant changes in MDSCs frequencies were observed in the groups of patients before and in the third month of therapy. The cut-off values of MDSCs, MoMDSCs, GrMDSCs and ImMCs for favourable 2- and 3-year PFS were established. Elevated LDH level is a negative prognostic factor of response to the treatment and is related to an elevated ratio of GrMDSCs and ImMCs level compared to patients' LDH level below the cut-off. Our data may provide a new perspective for more careful consideration of MDSCs, and specially MoMDSCs, as a tool for monitoring the immune status of melanoma patients. Changes in MDSC levels may have a potential prognostic value, however a correlation with other parameters must be established.

The tumor neomatrix component fibrillin-1 (FBN1) is a druggable oncologically relevant target: Proof-of-principle in vitro using an FDA-approved medicine as pioneer inhibitor of protein hydroxylation (274)

<b>Objectives:</b> Fibrillins <i>(FBN1-4)</i> determine the structure and function of the extracellular matrix. In malignancies, they drive the cytokine-loaded microenvironment and neoangiogenesis that promote tumor growth. In high-grade serous ovarian cancer (HGSOC), <i>FBN1</i> ranks among the top five genes with robust prognostic signature for survival [Ann. Oncol 31, 1240-50 (2020)]. Fibrillin-1 displays ≈60 globules aligned as ‘string-of-pearls,' ≈45 being epidermal growth factor domains (EGFDs); ≈40 EGFDs are hydroxylated at Asp/Asn residues by AspH, a 2-oxoacid utilizing dioxygenase (2OUD). AspH forms a high-affinity Ca²⁺ binding site in-between adjacent EGFDs, stabilizing the elastic alignment of ‘pearls' within fibrillin. We hypothesized that AspH inhibition causes misfolding of fibrillin due to failed Ca²⁺ binding, thus flawed alignment of EGFDs and intracellular retention of this intentionally defective fibrillin, similar to intracellular retention of genetically defective fibrillin in Marfan. We aimed to Identify a pioneer fibrillin-1 inhibitor that disrupts fibrillin synthesis preferentially in cancer cells, blocks any fibrillin contribution to the biology of solid tumors and has potential for clinical testing in ovarian serous and uterine serous cancers (USC). <b>Methods:</b> Crystal structure-guided modeling; cultured cell lines with high genomic fidelity to tissue and validation in drug discovery (MRC5 [normal fibroblasts, NF], KURAMOCHI [HGSOC], ARK1 [USC]); RNA-seq analysis; multi-channel flow cytometry. <b>Results:</b> To identify a medically actionable AspH suppressor for testing our hypothesis, we measured the inhibition-pertinent 3D parameters of the HAG mechanism, known to resolve 2OUD catalysis at the scale of atomic orbitals (https://www.researchgate.net/publication/7621997). We applied this HAG parameter grid to the library of FDA-approved drugs and selected deferiprone (DEF) as a candidate AspH suppressor. Globally, thousands of thalassemic children use DEF for a lifetime to alleviate transfusional iron overload. <i>In silico,</i> we found DEF accesses the catalytic pocket of AspH and blocks the two coordination sites at its iron atom that are essential for utilizing molecular oxygen during EGFD hydroxylations in fibrillins. <i>In vitro,</i> ARK1 transcribes <i>FBN1, FBN2,</i> and <i>FBN3.</i> MRC5, KURAMOCHI, and ARK1 produce fibrillin-1, detected by the FITC-labeled monoclonal 11C1.3, specific for a unique epitope within residues 723-909 of fibrillin-1 (exons 18-20 of <i>FBN1).</i> Employed at clinically relevant concentrations (≤ 150 µM), DEF treatment caused dose-dependent intracellular accumulation of 11C1.3-reactive fibrillin-1 material in all cell lines, exceeding untreated controls by up to five-fold after 48 hrs (p≤0.05). After 96 hours on DEF, such accumulation resolved in non-malignant MRC5 but persisted in malignant KURAMOCHI (p=0.04) and increased even further in malignant ARK1 (p≤0.02). Measured simultaneously, cleaved <i>PARP-1</i>, a signature of apoptotic death, was downregulated in DEF-treated MRC5 yet was elevated up to four-fold over controls in DEF-treated KURAMOCHI (p<0.03) and DEF-treated ARK1 (p<0.006). These increases correlated directly with the accumulation of 11C1.3-reactive material (Pearson's r: 0.97 in Kuramochi, 0.99 in ARK1) <b>Conclusions:</b> DEF causes intracellular accumulation of fibrillin-1, mis- or unfolded by deficient hydroxylation, and triggers apoptotic death in HGSOC and USC, but not in NF cells. Our results encourage a DEF pilot trial in HGSOC/USC patients.

The immune checkpoint controller C1q (C1QA/B/C) is a druggable oncologically relevant target: Proof-of-concept in vitro with an FDA-approved medicine, used as pioneer inhibitor of protein hydroxylation (273)

<b>Objectives:</b> C1q, a key initiator of immune reactions (adaptive, innate), is the single most important protein mediating tolerogenic clearance of apoptotic debris (‘self-waste') and functions in tissue homeostasis, e.g., angiogenesis. Cancer cells employ locally secreted C1q to secure growth and immune escape [Cancer Immunol. Res. 7.7, 1091-1195 (2019)]. In the tissue of high-grade serous ovarian cancer (HGSOC), expression of <i>C1</i>QA, or <i>C1QB,</i> or <i>C1QC</i> is a negative prognostic indicator [OSFPreprint 10.31219/osf.io/bcv9a]. C1q consists of ABC monomers, each comprising two domains, globule, and stem, the latter aligning six of these monomers into a ‘bouquet-of-tulips.' Every ABC monomer requires multiple Pro/Lys hydroxylations by specific 2-oxoacid utilizing dioxygenases (2OUDs) to form its stem as a triple helix identical to collagen. This collagenous stem (CS) mediates all C1q functions. Several experimental agents, read off the HAG mechanism that defines the principally possible inhibitors of 2OUDs (https://pubmed.ncbi.nlm.nih.gov/6281585/), are long known to prevent CS formation in C1q, to cause intracellular accumulation of underhydroxylated collagen and thus CS-defective C1q, and to block the secretion of any C1q that is dysfunctional due to CS mis-or unfolding [Biochem. J. 248, 625-33 (1987)]. We hypothesized that HGSOC cells generate their own C1q, which is rendered CS-defective and thus intracellularly retained by the Pro/Lys hydroxylation-inhibiting medicine deferiprone (DEF), discovered per the HAG mechanism. We aimed to Identify a medicine that disrupts all C1q support for malignancy. <b>Methods:</b> Cell line cultures (KURAMOCHI [HGSOC], ARK1 [uterine serous cancer, USC], MRC5 [normal fibroblasts, NF]); RNA-seq; multi-channel flow cytometry. <b>Results:</b> KURAMOCHI, which is unique among 47 HGSOC cell lines for displaying superior genetic similarity to HGSOC tumors [Nat. Commun. 4.1, 1-10 (2013)], synthesized C1q within 48 hours of plating, as detected intracellularly by the FITC-labeled monoclonal antibody JL-1 that specifically recognizes the CS domain of C1q. At 96 hours, the flow-cytometric JL-1 signal indicated further C1q increase by at least half and mirrored C1q kinetics in MRC5, a model of NF, the major source of C1q in non-malignant tissues. DEF, ranged at its concentrations in serum during multi-year treatment of iron overload conditions, e.g., transfusion-dependent thalassemia, caused all cell lines to accumulate JL-1 reactive C1q material, even at the residual 50 µM that patients on the FDA-approved DEF regimen may maintain for most or all of 24 hours [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154842]. After 96 hours on the drug, this intracellular accumulation exceeded the JL-1 signal of C1q within untreated controls by a factor of 3 in KURAMOCHI and by a factor of 10 in ARK1 (p<0.02), whereas JL-1 reactivity in treated MRC5 had, even at 150 µM, reverted to that of untreated MRC5. In KURAMOCHI, the accumulated JL-1 reactivity coincided with a 25-fold increase in active caspase 3 at 96 hours, a level of apoptotic activity reached in ARK1 after 48 hours already. In MRC5, active caspase-3 was suppressed at 96 hours on DEF. <b>Conclusions:</b> DEF at clinically relevant concentrations disrupts C1q synthesis selectively in HGSOC and USC cells and triggers their apoptotic self-destruction; however, NF remain unaffected. Our results encourage a DEF trial in HGSOC/USC patients and the further HAG mechanism-guided development of improved 2OUD inhibitors for oncological indications.

Characterization of Oral Squamous Cell Carcinoma Associated Inflammation: A Pilot Study.

Background: Oral squamous cell carcinoma (OSCC) is a devastating disease that is usually associated with a dense associated inflammatory infiltrate. Characterizing tumor-associated inflammation is critical to understand the pathogenies of tumor development and progression.Methods: We have tested a protocol to analyze tissue and salivary immune cells and mediators of 37 patients with OSCC at different stages and compared to eight chronic periodontitis patients and 24 healthy controls. Tissue analysis was based on fluorescent immunohistochemistry (FIHC) and inflammatory mediators were analyzed using a Luminex-based 30-Plex panel. Immune cells were analyzed using multichannel flow cytometry including CD45, CD66b, CD3, CD4, CD8, CD25, CD56, CD68, CD138, PD-1, and PD-L1.Results: We show an increase in OSCC-associated inflammation characterized by increased pro-inflammatory cytokines including IL-6, IL-8, TNFα, and GMCSF and increased salivary immune cells.Conclusion: We described a new method to analyze salivary inflammatory markers that can be used in future studies to monitor disease progression and prognosis.

Curauá-derived carbon dots: Fluorescent probes for effective Fe(III) ion detection, cellular labeling and bioimaging

This study reports the generation of curauá-derived carbon dots (C-dots) and their suitability for Fe(III) detection, bioimaging and FACS analysis. C-dots were generated from curauá (Ananas erectifolius) fibers by a facile one-step hydrothermal approach. They exhibited graphite-like structure with a mean diameter of 2.4 nm, high water solubility, high levels of carboxyl and hydroxyl functional groups, excitation-dependent multicolor fluorescence emission (in the range 450 nm - 560 nm) and superior photostability. C-dots were highly selective and effective for the detection of ferric Fe(III) ion in an aqueous medium with a detection limit of 0.77 μM in the linear range of 0–30 μM, a value much lower than the guideline limits proposed by the World Health Organization (WHO). In biological cell systems, C-dots were very well tolerated by B16F1 mouse melanoma and J774.A1 mouse macrophages cell lines, both of which effectively internalized C-dots in their cytoplasmic compartment. Finally, C-dots were effective probes for long-term live cell imaging experiments and multi-channel flow cytometry analysis. Collectively, our findings demonstrate that curauá-derived C-dots serve as versatile and effective natural products for Fe(III) ion sensing, labeling and bioimaging of various cell types. This study adds novel C-dots to the library of carbon-based probes and paves the way towards a sustainable conversion of a most abundant biomass waste into value-added products.

Generation and Application of T-Cell Lymphoma Patient Derived Tumor Xenograft Models

Introduction:Although pivotal “driver mutations” have been described in T-cell lymphomas (T-NHL), targetable dependences are lacking for many entities. As result, T-NHL patients experience dismal therapeutic responses and have a poor survival. Therapeutic improvements have been hindered by the lack of informative models. Patient-Derived-Tumor-Xenografts (PDTX), recapitulating many of the biological features of primary cancers, represent a novel and powerful platform. Here, we constructed a library of T-NHL PDTX to foster drug discovery programs and new patient-tailored approaches.Methods:Fresh and/or viable cryopreserved samples from primary T-NHL were obtained from Turin, WCM, and MSKCC hospitals. For the initial tumorgraft implants, lymphoma fragments, PBMC or bone marrow cells were implanted in NSG-B2m mice. Seeds from arising tumors were then serially transplanted. MRI was used to follow tumor growth and dissemination. The lymphoma phenotype was assessed by multi-channel flow cytometry and immunohistochemistry. We used TCR gene rearrangement analyses, whole exome sequencing (WES), total RNAseq, and CHIP-Seq (STAT3, methylation marks) to annotate primary and PDTX samples. For the in vitro studies, PDTX were digested and stromal cells were excluded by panning. Enriched Patient-Derived Tumor Cells (PDTC) were cultured in either RPMI/20% FBS or StemSpan/10% KSR media. High-throughput drug screening was performed using a library of 432 compounds (1 µM for 72hrs). Cell titer glo/blue, trypan blue cell count, and annexin-V/7AAD were used to assess cell viability, apoptosis, and proliferation.Results:We implanted 123 fresh T-NHL samples, resulting in the generation of 41 PDTX lines (Fig.1). In addition, 14 implants led to the growth of lymphoblastoid EBV+ populations. All PDTX lines were successfully propagated (5-26 serial passages) with the exception of angioimmunoblastic T-cell lymphoma (AITL) PDTX, which displayed a delay in engraftment over time, and eventually ceased growing (passage 3-7). Serially propagated PDTX showed distinct immunophenotypic profiles, which closely reflected their corresponding primary samples. TCR gene rearrangement studies confirmed the faithful correspondence between donor and PDTX over different generations. WES and total RNA-Seq on primary and serially propagated PDTX (at least T1-T5) yielded a rich landscape of mutations shared by primary tumors and PDTX derivatives. On going mutations were also defined along propagations. CHIP-Seq assay defined a STAT3 classifier in STAT+ PDTC.In vitro, all PDTC were shortly maintained (<7days), with some surviving for 6-8 weeks. To overcome their limited growth in vitro, we tested whether stromal cells (i.e. E4-Endothelial cells, E4-EC) could enhance their survival and/or growth. Indeed, co-culturing of PDTC with E4-ECs rescued cell death induced by ex vivo long-term propagation (peripheral T-cell lymphoma not otherwise specified and NK/T-Cell Lymphoma, PTCL-NOS and NK) or starvation (anaplastic large cell lymphoma, ALCL).PDTC were also used to perform a drug screening targeting major signaling pathways (>3x replicates of 8 ALCL, 1 NK, and 2 PTCL-NOS), demonstrating that each PDTX exhibited a unique drug sensitivity profile, mostly dictated by its genomic landscape. This was epitomized by two PDTC (IL-2 and IL-19) derived from the same PTCL-NOS patient bearing an activating V658F JAK1 somatic mutation. These two PDTC, once exposed to JAK inhibitors, displayed down-regulation of pSTAT3 (24hr) and massive cell death (T3-T6, >95% at 72hrs). Similar results were seen with ALK inhibitors in ALK+ ALCL PDTC (n=5). Moreover, specific drugs were identified to bypass refractory phenotypes in selective PDTC (i.e. proteasome and topoisomerase inhibitors).Conclusions:We built up a large and representative PDTX library from major T-NHL entities. Immunophenotyping, genomics and functional studies showed a close correspondence between primary samples and correspondent PDTX derivatives. Our in vitro models provide a starting point for elucidating stromal:tumor cell interactions that contribute to lymphoma growth and survival in vivo, and evidence that a drug screening can be successfully executed on PDTCs. We predict that this cohort may be a valuable tool for testing novel therapeutic compounds and fostering personalized therapeutic approaches in T-NHL patients. [Display omitted] DisclosuresThompson:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees. Aster:Eli Lilly and Company: Membership on an entity's Board of Directors or advisory committees. Ruan:Cell Medica: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Dogan:Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Peer Review Institute: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche Pharmaceuticals: Consultancy. Cerchietti:Celgene: Research Funding; Lymphoma Research Foundation: Research Funding; Leukemia and Lymphoma Society: Research Funding; Weill Cornell Medicine - New York Presbyterian Hospital: Employment. Horwitz:Celgene: Consultancy, Research Funding; Kyowa-Hakka-Kirin: Consultancy, Research Funding; Infinity/Verastem: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; BMS: Consultancy; HUYA: Consultancy; Aileron Therapeutics: Research Funding; ADCT Therapeutics: Research Funding; Millenium/Takeda: Consultancy, Research Funding; Mundipharma: Consultancy; Forty-Seven: Consultancy, Research Funding.

Bone Marrow CD8 T Cells Express High Frequency of PD-1 and Exhibit Reduced Anti-Leukemia Response in AML Patients

Acute myeloid leukemia (AML) is a devastating blood cancer with 5- year survival of only 25%. Optimizing anti-leukemia immune response to improve clinical outcome is a promising strategy for novel effective leukemia therapeutics. However due to limited accessibility, the majority of studies have been restricted to evaluate samples from peripheral blood. The immune response in bone marrow of AML is poorly understood. In this study, we aim to investigate the T cell immune response in bone marrow samples derived from AML patients.Samples collected from a cohort of patients with newly diagnosed AML (n=19) were used in this study. Mononuclear cells of bone marrow and peripheral blood were isolated from clinical samples before multi-channel flow cytometry analysis. We observed a comparable frequency of CD3+ T cells among lymphocytes in bone marrow and peripheral blood. Similarly there is no difference of CD4, CD8 T cell frequency, or CD4/CD8 ratio. However, when T cell subsets for each differentiation stage were analyzed based on the expression of CD45RA and CCR7, we found significantly increased effector memory T cells (CD45RA-CCR7-) among CD8 T cells in bone marrow compared with that in peripheral blood (43.88±5.012% vs. 35.15±5.104%, P =0.0088). Whereas the percentage of terminally differentiated effector cells (CD45RA+CCR7-) was significantly lower in bone marrow (20.36±5.025% vs. 28±5.947%, P =0.0481). No difference was found in the frequency of naive (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) subsets.Several studies including ours have demonstrated an involvement of the inhibitory receptors PD-1, TIM-3 and TIGIT in AML progression. Most of the studies were performed using clinical samples from peripheral blood. To evaluate whether the effect of inhibitory pathways is different between bone marrow and peripheral blood, we assessed the expression of these inhibitory receptors on T cells derived from both sites. We observed comparable expression of TIGIT and TIM-3 in bone marrow and peripheral blood. The frequency of PD-1 expression on CD8 T cells was however significantly higher in bone marrow (39.55±3.809% vs. 32.8±3.512%, P =0.0058). We further dissected the intracellular expression of Eomesodermin (Eomes) and T-bet, the two T box transcription factors that are crucial in regulating T cell function. We found that the frequency of Eomes+T-betint CD8 T cells was significantly higher in bone marrow than in peripheral blood (17.01±1.843% vs. 14.95±1.682%, P =0.0405), indicating a predominance for late stage of T cell exhaustion status in bone marrow. This result suggests a relatively more suppressive environment in bone marrow compared with that of peripheral blood.We next examined the functional status of CD8 T cells. Intracellular IFN-γ and TNF-α production was assessed upon in vitro stimulation with anti-CD3/CD28. Intriguingly no significant difference between bone marrow and peripheral blood was detected. Consistently we observed no difference between the two sites in term of the expression of Ki67, Granzyme B, and Perforin, suggesting a comparable proliferation and killing capacity of CD8 T cells between bone marrow and peripheral blood. We further investigated the function of leukemia-reactive CD8 T cells by testing T cell response against a WT-1 peptide. WT-1 is a well known tumor-associated antigen, in which HLA-A*0201 restricted WT-1126-134 is one of the mostly studied epitope in AML. Purified CD8 T cells derived from bone marrow or blood of newly diagnosed HLA-A*0201 AML patients were co-cultured with T2 cells (used as antigen presenting cells) pulsed with WT-1126-134. We found a significantly lower production of IFN- γ by CD8 T cells from bone marrow compared with that from peripheral blood (0.4325±0.1612% vs, 0.7425±0.2809%, P =0.0472). These data demonstrate that although the total CD8 T cells in bone marrow remain functional, the leukemia-reactive CD8 T cells are less functional in bone marrow compared with that in peripheral blood.Collectively, our data demonstrate that bone marrow CD8T cells from AML patients express higher frequency of PD-1 and exhibit reduced anti-leukemia response. Our study suggests that bone marrow is a unique anatomical location fostering negative immune response in AML, thus targeting the negative regulatory pathways in bone marrow is of great potential to develop novel effective leukemia treatment. DisclosuresNo relevant conflicts of interest to declare.

Crosstalk between nonclassical monocytes and alveolar macrophages mediates transplant ischemia-reperfusion injury through classical monocyte recruitment.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R–dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β–dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

A Novel Experimental Approach for In Vivo Analyses of the Salivary Gland Microvasculature.

Microvascular dysfunction plays a fundamental role in the pathogenesis of salivary gland disorders. Restoring and preserving microvascular integrity might therefore represent a promising strategy for the treatment of these pathologies. The mechanisms underlying microvascular dysfunction in salivary glands, however, are still obscure, partly due to the unavailability of adequate in vivo models. Here, we present a novel experimental approach that allows comprehensive in vivo analyses of the salivary gland microvasculature in mice. For this purpose, we employed different microscopy techniques including multi-photon in vivo microscopy to quantitatively analyze interactions of distinct immune cell subsets in the submandibular gland microvasculature required for their infiltration into the surrounding parenchyma and their effects on microvascular function. Confocal microscopy and multi-channel flow cytometry in tissue sections/homogenates complemented these real-time analyses by determining the molecular phenotype of the participating cells. To this end, we identified key adhesion and signaling molecules that regulate the subset- and tissue-specific trafficking of leukocytes into inflamed glands and control the associated microvascular leakage. Hence, we established an experimental approach that allows in vivo analyses of microvascular processes in healthy and diseased salivary glands. This enables us to delineate distinct pathogenetic factors as novel therapeutic targets in salivary gland diseases.

CD8 and CD4 T Cell Populations in Human Kidneys.

Background: At border sites, and in internal organs, tissue resident memory T cells (TRM) contribute to the immune barrier against pathogens like viruses, bacteria, fungi, and cancer. However, information on the presence and function of these cells in the human kidney is scant. In order to better understand the T cell-mediated immunological defense in this organ, we aimed to determine phenotypic and functional aspects of CD8 and CD4 T cells present in healthy and allograft kidney tissue. Methods: Using multichannel flow cytometry, we assessed the phenotype and function of T cells in healthy renal tissue samples (n = 5) and kidney allograft tissue (n = 7) and compared these aspects to T cells in peripheral blood from healthy controls (n = 13). Results: Kidney tissue samples contained substantial amounts of CD8 and CD4 T cells. In contrast to the circulating cells, kidney T cells frequently expressed CD69 and CD103, and were more often actively cycling. Furthermore, nearly all kidney T cells expressed CXCR3, and often expressed CXCR6 compared to T cells in the circulation. Markedly, kidney T cells produced greater quantities of IFNγ than circulating cells and were frequently polyfunctional. Conclusion: Functional T cells with the characteristic traits of TRM reside in human kidney tissues. These cells are more often actively cycling and frequently express CXCR3 and CXCR6.

Precise determination of input-output mapping for multimodal gene circuits using data from transient transfection.

The mapping of molecular inputs to their molecular outputs (input/output, I/O mapping) is an important characteristic of gene circuits, both natural and synthetic. Experimental determination of such mappings for synthetic circuits is best performed using stably integrated genetic constructs. In mammalian cells, stable integration of complex circuits is a time-consuming process that hampers rapid characterization of multiple circuit variants. On the other hand, transient transfection is quick. However, it is an extremely noisy process and it is unclear whether the obtained data have any relevance to the input/output mapping of a circuit obtained in the case of a stable integration. Here we describe a data processing workflow, Peakfinder algorithm for flow cytometry data (PFAFF), that allows extracting precise input/output mapping from single-cell protein expression data gathered by flow cytometry after a transient transfection. The workflow builds on the numerically-proven observation that the multivariate modes of input and output expression of multi-channel flow cytometry datasets, pre-binned by the expression level of an independent transfection reporter gene, harbor cells with circuit gene copy numbers distributions that depend deterministically on the properties of a bin. We validate our method by simulating flow cytometry data for seven multi-node circuit architectures, including a complex bi-modal circuit, under stable integration and transient transfection scenarios. The workflow applied to the simulated transient transfection data results in similar conclusions to those reached with simulated stable integration data. This indicates that the input/output mapping derived from transient transfection data using our method is an excellent approximation of the ground truth. Thus, the method allows to determine input/output mapping of complex gene network using noisy transient transfection data.

Circulating mucosal-associated invariant T cells in subjects with recurrent urinary tract infections are functionally impaired.

BackgroundUrinary tract infection recurrence is common, particularly in women and immunocompromised patients, such as renal transplant recipients (RTRs). Mucosal‐associated invariant T (MAIT) cells play a role in the antibacterial response by recognizing bacterial riboflavin metabolites produced by bacteria such as Escherichia coli. Here, we investigated whether MAIT cells are involved in the pathogenesis of recurrent urinary tract infections (RUTIs).MethodsUsing multichannel flow cytometry, we characterized the MAIT cell phenotype and function in blood from immunocompetent adults with (n = 13) and without RUTIs (n = 10) and in RTRs with (n = 9) and without RUTIs (n = 10).ResultsThere were no differences in the numbers of MAIT cells between the study groups. MAIT cells in patients with RUTI expressed T‐bet more often than those in controls. MAIT cells from immunocompetent RUTI participants required more antigen‐presenting cells coincubated with E. coli to evoke a similar cytokine and degranulation response than those from controls. This effect was absent in the RTR with RUTI vs RTR control groups, where the overall percentage of MAIT cells that responded to stimulation was already reduced.ConclusionCirculating MAIT cells in immunocompetent individuals with RUTIs respond to bacterial stimuli with reduced efficacy, which suggests that they are involved in the pathogenesis of RUTIs.

DETERMINING THE INflAMMATORY RESPONSE IN ORAL SQUAMOUS CELL CARCINOMA BY SALIVA ANALYSIS

Objectives Oral squamous cell carcinoma (OSCC) often shows a pronounced inflammatory infiltrate and there is accumulating evidence that this inflammatory infiltrate plays an active role in tumor development and progression. Analyses of saliva may provide a non-invasive approach to study the inflammatory response in OSCC. Our aim is to develop a protocol for collection and analysis of saliva in OSCC patients and to use it to characterize both the inflammatory cell profile and cytokine profile in the saliva of patients across all stages of OSCC. Methods 42 patients undergoing treatment for OSCC at the Toronto Odette Cancer Centre (stages I to IV), 25 healthy patients, and 9 patients with periodontitis were enrolled. Saliva samples were obtained by rinsing with 3 ml of saline for 30 sec. The samples were kept on ice and stabilized with protease inhibitor until filtration using a 20 μm membrane filter. Cell pellets were separated by centrifugation and supernatants were analyzed using a BioFLex 30-Plex inflammatory panel (BioRad) and Luminex® detection technology. Cell pellets were fixed in 4% PFA and analyzed using multichannel flow cytometry. The fluorescent markers included CD45, CD66b, CD3, CD4, CD8, CD25, CD56, CD68, CD138, Siglec8, PD1 and PDL1. Findings Distinctive, reproducible changes were observed in salivary cytokines and inflammatory cell profile of patients with OSCC compared to control and periodontal disease patients. Using our protocol, we were able to describe specific patterns of inflammation for oral cancer, including altered CD4/CD8 ratio and marked increase in IL-1b, IL-6 and TNF-a. Conclusion We created a reproducible protocol to collect saliva and perform high-throughput analysis of inflammatory profile of saliva. This technology can be used to develop non-invasive, early detection/prognostic tests for OSCC, new adjuvant therapies and new techniques to monitor response to treatment.

TIGIT Expression Positively Associates with NK Cell Function in AML Patients

Natural killer (NK) cells are essential innate immune effectors with promising anti-leukemia activity in acute myeloid leukemia (AML). However, clinical success of applying NK cells in AML treatment has not been achieved. A better understanding of the regulatory mechanisms for NK cell function is important to optimize this therapeutic strategy. T cell immunoglobulin and ITIM domain (TIGIT) is a recently identified inhibitory receptor expressed on T cells and NK cells. Multiple studies including ours have demonstrated its suppressive effect in anti-tumor CD8 T cell response. However whether and how TIGIT impacts NK cells in AML is unknown. Here we performed phenotypic and functional studies on NK cells derived from patients with newly diagnosed AML (n=30). Cells collected from healthy individuals (n=18) were used as controls. TIGIT expression and their contributions to NK cell function in AML were assessed.Peripheral blood samples were first examined by flow cytometry for the frequency of NK cells (defined as CD56+CD3-). The percentage of NK cells among peripheral blood mononuclear cells (PBMCs) in AML patients is comparable with that of healthy controls. In contrast, when we performed functional analysis to assess NK cells for cytokine release upon in vitro stimulation with a human leukemia cell line K562, we observed significantly lower intracellular production of IFN-γ in cells from AML patients compared with that of healthy controls. Consistently NK cells from AML patients expressed less Perforin, indicating a compromised killing capacity. We next evaluated the expression of TIGIT on CD56+CD3- NK cells. As some AML blasts and monocytes also express CD56, we performed multichannel flow cytometry and carefully gated out other cell components when assessing TIGIT expression. To our surprise, we observed a significantly lower frequency of TIGIT-expressing NK cells in AML compared with that of healthy controls (36.82 ±4.543% vs. 48.9±3.818%, P=0.0463). This data indicated that low-TIGIT expression associates with impaired NK cell function and AML progression.We further examined the phenotype and functional status of TIGIT+ NK cells. Expression of activating receptors (CD16 and CD160) and inhibiting receptors (KIR and NKG2A) on TIGIT+ vs. TIGIT- NK cells were analyzed. We observed a significant higher expression of CD16 (51.27±9.009% vs. 20.63±5.334%, P=0.0001) and CD160 (39.84±6.447% vs. 21.24±4.287%, P=0.0103) on TIGIT+ NK cells compared with that of TIGIT- NK cells. By contrast, TIGIT+ NK cells expressed lower KIR (24.06±3.796% vs. 43.59±6.96%, P=0.0046) and NKG2A (7.658±1.717% vs. 18.68±4.256%, P=0.0167) than TIGIT- NK cells. Importantly, functional studies demonstrated an elevated expression of Granzyme B and increased cytokine (IFN-γ and TNF-α) production by TIGIT+ NK cells compared with TIGIT- NK cells (IFN-γ, P=0.0283; TNF-α P=0.0347; Granzyme B, P=0.0493). These data suggest that TIGIT expression on NK cells associated with activated and high functional status.Collectively, our study demonstrates that 1) in line with lower capacity to produce IFN-γ, NK cells from AML patients express less frequency of TIGIT compared with healthy individuals; 2) TIGIT+ NK cells from AML patients express high levels of activating receptors and are highly functional manifested by more cytokine production and enhanced expression of Granzyme B compared with TIGIT- NK cells. These results indicate that in AML patient, TIGIT may contribute to the upregulation of NK cell function. This is in contrast to the observations of CD8 T cells in which TIGIT plays a suppressive role. Targeting TIGIT for cancer treatment is currently under active development. Our findings bring a call for caution on the TIGIT-targeted therapeutic strategy in AML as TIGIT might be a double-edged sword in anti-leukemia immune regulation. DisclosuresNo relevant conflicts of interest to declare.

Fluorescence Observation of Single-Cell cAMP Signaling by G Protein-Coupled Receptors.

We present complementary flow cytometric and microscopic imaging methods, both utilizing a membrane-targeted cAMP sensor protein ICUE3, to examine hormone-dependent signaling by the luteinizing hormone (LH) receptor in individual cells. This receptor, a seven transmembrane domain protein belonging to the GPCR family, signals by activating adenylate cyclase to increase cAMP levels. The ICUE3 sensor protein exhibits fluorescence energy transfer between its CFP and YFP moieties and the ratio of CFP emission to YFP sensitized emission (YFPSE) increases with cAMP concentration. We used multichannel flow cytometry to compare CFP emission and YFPSE from each cell and hence measure that cell's cAMP level. This technique measured changes in cAMP levels in CHO cells expressing LH receptors and stimulated by forskolin or the hormone human chorionic gonadotropin (hCG) and showed that significant cell-to-cell variations exist in such cAMP responses. Because LH receptor behavior may reflect receptor expression levels, we developed a procedure to measure numbers of particular fluorescent cell proteins from measurements of MESF bead standards for slightly different fluorophores. We find that basal cAMP levels increase substantially in cells expressing high numbers mCherry-LH receptors per cell. This suggests activation through increased inter-receptor interactions at high concentrations. We then explored a microscope-based method for single cell measurements so that responses could be correlated with specific cell morphology and with time after treatments. This showed that cell responses to hCG are fully-developed after ~100s. Taken together, these results demonstrate the utility of fluorescence methods in exploring cAMP signaling in individual cells.

Peripheral T Cell Depletion by FTY720 Exacerbates Hypoxic-Ischemic Brain Injury in Neonatal Mice.

Hypoxic-ischemic injury to the developing brain remains a major cause of significant long-term morbidity and mortality. Emerging evidence from neonatal brain injury models suggests a detrimental role for peripheral lymphocytes. The immunomodulatory substance FTY720, a sphingosine-1-phosphate receptor agonist, was shown to reduce adult ischemia-induced neurodegeneration through its lymphopenic mode of action. In the present study, we hypothesized that FTY720 promotes neuroprotection by reducing peripheral lymphocytes and their infiltration into the injured neonatal brain. Term-born equivalent postnatal day 9 C57BL/6 mice were exposed to hypoxia ischemia (HI) followed by a single injection of 1 mg/kg FTY720 or vehicle (0.9% sodium chloride). Brain injury, microglia, and endothelial activation were assessed 7 days post HI using histology and western blot. Peripheral and cerebral leukocyte subsets were analyzed by multichannel flow cytometry. Whether FTY720s’ effects could be attributed to its lymphopenic mode of action was determined in T cell-depleted mice. In contrast to our hypothesis, FTY720 exacerbated HI-induced neuropathology including loss of gray and white matter structures. While microglia and endothelial activation remained unchanged, FTY720 induced a strong and sustained depletion of peripheral T cells resulting in significantly reduced cerebral infiltration of CD4 T cells. CD4 T cell subset analysis revealed that circulating regulatory and effector T cells counts were similarly decreased after FTY720 treatment. However, since neonatal HI per se induces a selective infiltration of Foxp3 positive regulatory T cells compared to Foxp3 negative effector T cells effects of FTY720 on cerebral regulatory T cell infiltration were more pronounced than on effector T cells. Reductions in T lymphocytes, and particularly regulatory T cells coincided with an increased infiltration of innate immune cells, mainly neutrophils and inflammatory macrophages. Importantly anti-CD3-mediated T cell depletion resulted in a similar exacerbation of brain injury, which was not further enhanced by an additional FTY720 treatment. In summary, peripheral T cell depletion by FTY720 resulted in increased infiltration of innate immune cells concomitant to reduced T cell infiltration and exacerbation HI-induced brain injury. This study indicates that neonatal T cells may promote endogenous neuroprotection in the term-born equivalent hypoxic-ischemic brain potentially providing new opportunities for therapeutic intervention.