Cell Markers CD133 Research Articles

Heparan Sulfate Glycosaminoglycan Is Predicted to Stabilize Inflammatory Infiltrate Formation and RANKL/OPG Ratio in Severe Periodontitis in Humans.

Since chronically inflamed periodontal tissue exhibits extracellular matrix (ECM) degradation, the possible alternative to standard periodontitis treatment is to restore ECM by supplementing its components, including heparan sulfate glycosaminoglycan (HS GAG). Supplementation of the degraded ECM with synthetic derivatives of HS GAGs has been shown to be effective for periodontal tissue regeneration in experimental animal models of periodontitis. However, the potential of HS GAG supplementation for the treatment of periodontal disease in humans is still unknown. Here, we used a statistical model to investigate the role of HS GAG on inflammatory infiltrate formation and alveolar bone resorption in humans with severe periodontitis. The model was based on data from immunofluorescence staining (IF) of human gingiva samples, and reconstruction of a subset of HS GAG -related proteins from STRING reactome database. According to predictions, increased expression of native HS GAG might stabilize the accumulation of gingival inflammatory infiltrate (represented by the general inflammatory cell marker CD45) and alveolar bone resorption (represented by Receptor Activator of Nuclear ΚΒ ligand (RANKL) and osteoprotegerin (OPG) ratio) but could not restore them to healthy tissue levels. Therefore, supplementation of native HS GAG may be of limited benefits for the treatment of sever periodontitis in humans.

Microfluidic chip with reversible interface for noninvasive remission status monitoring and prognosis prediction of acute myeloid leukemia

Acute myeloid leukemia (AML) requires close monitoring of remission status for timely disease management. Liquid biopsy serves as a noninvasive approach for evaluating treatment response and guiding therapeutic modifications. Herein, we designed a non-invasive Leukemic stem cell Specific Capture Chip (LSC-Chip) with reversible recognition interface for AML remission status monitoring and prognosis prediction. A stem cell marker CD34 antibody coated herringbone chip with disulfide linkers was designed to capture and release leukemic stem cells (LSCs) in peripheral blood for efficient LSC enumeration and downstream single-cell analysis. Samples from 32 AML patients and 10 healthy donors were recruited for LSC enumeration and prognosis-associated subtyping with panels of official LSC markers (CD34+/CD123+/CD38-) and (Tie2+/CD34+/CD123+/CD38-), respectively. A cutoff value of 2.5 LSCs per milliliters of peripheral blood can be used to precisely distinguish non-remission AML patients from complete remission group. Moreover, single-cell RNA-seq of LSCs was performed to check different transcriptional profiles of LSC subtypes. Overall, the LSC-Chip with reversible recognition interface enabled reliable detection of LSCs from AML patient samples for noninvasive remission status monitoring and prognosis prediction in clinical AML management.

Contribution of CD4+ cells in the emotional alterations induced by endometriosis in mice.

Endometriosis is a disease defined by the presence of endometrial tissue in extrauterine locations. This chronic condition is frequently associated with pain and emotional disorders and has been related with altered immune function. However, the specific involvement of immune cells in pain and behavioral symptoms of endometriosis has not been yet elucidated. Here, we implement a mouse model of non-surgical endometriosis in which immunocompetent mice develop abdomino-pelvic hypersensitivity, cognitive deficits, anxiety and depressive-like behaviors. This behavioral phenotype correlates with expression of inflammatory markers in the brain, including the immune cell marker CD4. Depletion of CD4 + cells decreases the anxiety-like behavior of mice subjected to the endometriosis model, whereas abdomino-pelvic hypersensitivity, depressive-like behavior and cognitive deficits remain unaltered. The present data reveal the involvement of the immune response characterized by CD4 + white blood cells in the anxiety-like behavior induced by endometriosis in mice. This model, which recapitulates the symptoms of human endometriosis, may be a useful tool to study the immune mechanisms involved in pain and behavioral alterations associated to endometriosis.

Evaluation the role of CD34 in patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common leukemia among the adult population and accounts for about 80% of all cases, and its characterized by clonal expansion of immature “blast cells” in the peripheral blood and bone marrow. The stem cell marker CD34 is expressed by leukemia blasts only for a subset of patients with acute myelogenous leukemia (AML). The aim of this study is to determine the role of CD34 in AML and its relationship with the parameters of complete blood (CBC). A total of (60) venous blood sample from patients and (30) control their ages ranged between 18 and 66 years, the samples was collected from Baghdad medical city hospital. Sera from all participants were tested to assessment CD34 level by ELISA. In the current study, CD34 in patients and control groups show a significant difference in relation with AML patients at p-value (<0.001). The levels of CD34 in the patients were 0.55 ± 0.03, whereas it's significantly increased to 0.81 ± 0.05 in the control. Also we found the majority of patients presented with subtype M3 38.3% of total patients.

Evaluation the role of CD34 in patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is the most common leukemia among the adult population and accounts for about 80% of all cases, and its characterized by clonal expansion of immature “blast cells” in the peripheral blood and bone marrow .The stem cell marker CD34 is expressed by leukemia blasts only for a subset of patients with acute myelogenous leukemia (AML).The aim of this study is to determine the role of CD34 in AML and its relationship with the parameters of complete blood (CBC). A total of (60) venous blood sample from patients and (30) control their ages ranged between 18 and 66 years, The samples was collected from Baghdad medical city hospital. Sera from all participants were tested to assessment CD34 level by ELISA.In the current study, CD34 in patients and control groups show a significant difference in relation with AML patients at p-value (< 0.001). The levels of CD34 in the patients were 0.55 ± 0.03, whereas it’s significantly increased to 0.81 ± 0.05 in the control. also we found The majority of patients presented with subtype M3 38.3% of total patients.

Weipiling decoction alleviates N-methyl-N-nitro-N′-nitrosoguanidine-induced gastric precancerous lesions via NF-κB signalling pathway inhibition

Aim of the studyWe aimed to explore how weipiling (WPL) decoction WPL alleviates gastric precancerous lesions (GPLs) and uncover its anti-inflammatory roles in GPL treatment.Materials and methodsThe anti-GPL action mechanisms of WPL were analysed using a network pharmacological method. The WPL extract was prepared in a traditional way and evaluated for its major components using high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS). BALB/c mice were exposed to N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (150 μg/mL) for 6 weeks to induce GPLs. GPL mice were administered WPL (3.75 g/kg/day and 15 g/kg/day) for an additional 8 weeks. Haematoxylin and eosin (H&E) staining was used to investigate histological alterations in gastric tissues. Expression of the T helper 1 (Th1) cell markers CD4+ and interferon-gamma (INF-γ) were tested using immunohistochemistry (IHC). Inflammatory protein and mRNA levels in the nuclear factor kappa B (NF-κB) pathway were detected using western blotting and a quantitative reverse transcription polymerase chain reaction (RT-qPCR), respectively.ResultsWe identified and selected 110 active compounds and 146 targets from public databases and references. Four representative components of WPL were established and quantified by HPLC–MS/MS analysis. WPL attenuated MNNG-induced GPLs, including epithelial shedding, cavity fusion, basement membranes with asymmetrical thickness, intestinal metaplasia, dysplasia, pro-inflammatory Th1-cell infiltration, and INF-γ production, indicating that WPL prevents inflammation in the gastric mucosa. Furthermore, WPL reversed MNNG-induced activation of the IκB/NF-κB signalling pathway and subsequently attenuated the upregulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase (NOX)) family members NOX2 and NOX4.ConclusionWPL attenuated GPLs by controlling the generation of pro-inflammatory elements and inhibiting the NF-κB signalling pathway in vivo.

216.13: Spatial Protein Profiling of Leukocyte Infiltrates in Renal Biopsies After Cellular Therapy

Background: Regulatory T cells (Tregs) are powerful modulators of immune function, and are of increasing interest as an adoptive cellular therapy for the treatment of transplant rejection. Elucidate the impact of Treg therapeutics in vivo is crucial to identify the therapeutic efficacy. This study aimed to examine the infiltrated leukocytes to provide a clearer picture of the dynamic process that occurs in the transplanted tissue after cellular therapy compared to rejection. Methods: Cellular infiltrates were assessed by GeoMx digital spatial profiling (DSP) platform in tissue biopsies from three transplant patients who received Treg therapy and two control patients with confirmed rejection, with biopsies taken at similar time points after transplantation (6-12 months). Various regions of interest (ROIs) were selected (9 -12 regions) from each patient with diverse infiltration types (Figure 1). In addition, FOXP3hi CD4+ and FOXP3low/neg CD4+ regions were selected from both groups to assess the protein signatures of these cellular infiltrates. Results: The FOXP3 signal was highly enriched in regions from cell therapy compared to rejection patients, suggesting that infused Tregs are likely homing to the allograft. The B cell marker CD20 was highly expressed in cell therapy ROIs than in rejection ROIs, suggesting a link between infused Tregs and allograft infiltration by B cells. Conversely, the rejection ROIs have a high expression of Ki67, indicating the presence of highly proliferative cells. The monocyte/macrophage markers CD14, CD68, and CD163 were also enriched in the rejection ROIs, suggesting that macrophages may be associated with graft rejection in these patients. Further analysis revealed interesting differences between the cellular infiltrates in FOXP3hi segments from cell therapy patients compared with rejection group such as enrichment in proteins related to signalling, including STAT3 and p-STAT3, and coinciding with enrichment in regulatory molecules, including CTLA-4 and PD-1. VISTA protein was found to be highly enriched in FOXP3hi segments from cell therapy patients, suggesting that VISTA might play a regulatory role in suppressing the alloimmune response. Our analysis suggests that FOXP3hi cells that are present in regions collected from rejecting patients are likely suppressive Tregs, since FOXP3hi cells coincide with enrichment of Tregs-associated markers. Nevertheless, these Tregs failed to control the alloresponse, as demonstrated by the enrichment of CD8 T cells, which were notably represented in FOXP3hi regions in rejection tissues. Conclusion: The GeoMx DSP allowed for an in-depth analysis of leukocyte infiltrates and provided comprehensive information about the molecular processes associated with stable renal allograft in patients receiving Treg therapy, which in turn may assist in understanding the mechanisms of therapeutic immune regulation and optimal timing for immunosuppression minimisation.

T Cell Transcriptome in Chromosome 22q11.2 Deletion Syndrome.

Phenotypic variations of chromosome 22q11.2 deletion syndrome (22qDS) have unclear explanations. T cell lymphopenia in 22qDS related to varying degrees of thymic hypoplasia contributes to the phenotypic heterogeneity. No phenotype correlation with genotype or deletion size is known for lymphopenia. We investigated gene expression in human T cells of participants with and without 22qDS and T cells of participants with 22qDS with low or normal T cells. Peripheral blood was collected from participants aged 5-8 y. Immune function was checked. RNA sequencing was completed on isolated T cells, and differential gene expression profiles of T cells between 22qDS and healthy control subjects were established. A total of 360 genes were differentially expressed (q < 0.05) between T cells of patients with 22qDS (n = 13) and healthy control subjects (n = 6) (log2 fold change range, -2.0747, 15.6724). We compared gene expression between participants with 22qDS with low (n = 7) and normal T cell counts (n = 6), finding 94 genes that were differentially expressed (q < 0.05) (log2 fold change range, -4.5445, 5.1297). Twenty-nine genes correlated with T cell counts and markers CD3, CD4, CD8, and CD45RA+CD4 (R ≥ 0.8). We found significantly differentially expressed genes in participants with 22qDS compared with healthy control subjects and in participants with 22qDS with low T cell counts compared with those with normal T cell counts. Several enriched pathways suggest a role of T cells in defective communication between T cells and the innate immune system in 22qDS. Among these, the liver X receptor/retinoid X receptor pathway was noted to show several differentially expressed genes affecting participants with 22qDS compared with healthy control subjects and more so those with low T cell counts than in those with normal T cell counts.

TNFAIP3 mediates FGFR1 activation-induced breast cancer angiogenesis by promoting VEGFA expression and secretion.

To investigate the role and mechanism of TNF-inducible protein 3(TNFAIP3) in breast cancer angiogenesis induced by fibroblast growth factor receptor1 (FGFR1) activation. The immunohistochemical assay was used to detect the expression of vascular endothelial cell marker CD31 and CD105 in mice DCIS.COM-iFGFR1 transplanted tumor (previously established by our group). The effects of TNFAIP3 knockout/knockdown breast cancer cell lines on angiogenesis, migration, and invasion of Human Umbilical Vein Endothelial Cells (HUVEC) were detected by the tubulogenesis and Trewells assay. RNA-seq analysis of TNFAIP3 downstreams differential genes after TNFAIP3 knockdown. The expression and secretion of VEGFA after FGFR1 activation in breast cancer cells were detected by qPCR, Western blot, and ELISA. Immunohistochemistry showed that TNFAIP3 knockout inhibited the expression of CD31 and CD105 in DCIS grafted tumors promoted by FGFR1 activation. Tubulogenesis and Trewells experiments showed that TNFAIP3 gene knockout/knockdown inhibited the angiogenesis, migration, and invasion of HUVEC cells promoted by FGFR1 activation. qPCR assay showed that VEGFA mRNA level in the TNFAIP3 knockdown cell line was significantly down-regulated (p < 0.05). qPCR, Western blot and ELISA results showed that TNFAIP3 gene knockout/knockdown could inhibit the expression and secretion of VEGFA in breast cancer cells induced by FGFR1 activation. TNFAIP3 promotes breast cancer angiogenesis induced by FGFR1 activation through the expression and secretion of VEGFA.

Comparison of Sources and Methods for the Isolation of Equine Adipose Tissue-Derived Stromal/Stem Cells and Preliminary Results on Their Reaction to Incubation with 5-Azacytidine.

Simple SummaryThe function of the equine heart is different from that in other species, and a species-specific in vitro model would simplify investigations in the field of equine cardiology. The recent advances in stem cell research and the availability of adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point for the development of such an in vitro model. In order to test the hypothesis that equine ASCs can be differentiated into cells resembling heart cells, we isolated ASCs from abdominal, retrobulbar, and subcutaneous adipose tissue after collagenase digestion or from direct cultivation of explants. Both techniques resulted in similar yields of cells displaying morphological, immunophenotypical, and molecular biological characteristics of mesenchymal stem cells. Abdominal adipose tissue was found to be most suitable for ASC isolation in equines. However, contrasting earlier studies performed with ASCs from other species, equine ASCs were refractory to 5-azacytidine-induced upregulation of markers characteristic for the differentiation into heart cells. Hence, further studies are required to establish equine cardiomyocyte induction.Physiological particularities of the equine heart justify the development of an in vitro model suitable for investigations of the species-specific equine cardiac electrophysiology. Adipose tissue-derived stromal/stem cells (ASCs) could be a promising starting point from which to develop such a cardiomyocyte (CM)-like cell model. Therefore, we compared abdominal, retrobulbar, and subcutaneous adipose tissue as sources for the isolation of ASCs applying two isolation methods: the collagenase digestion and direct explant culture. Abdominal adipose tissue was most suitable for the isolation of ASCs and both isolation methods resulted in comparable yields of CD45-/CD34-negative cells expressing the mesenchymal stem cell markers CD29, CD44, and CD90, as well as pluripotency markers, as determined by flow cytometry and real-time quantitative PCR. However, exposure of equine ASCs to 5-azacytidine (5-AZA), reportedly inducing CM differentiation from rats, rabbits, and human ASCs, was not successful in our study. More precisely, neither the early differentiation markers GATA4 and NKX2-5, nor the late CM differentiation markers TNNI3, MYH6, and MYH7 were upregulated in equine ASCs exposed to 10 µM 5-AZA for 48 h. Hence, further work focusing on the optimal conditions for CM differentiation of equine stem cells derived from adipose tissue, as well as possibly from other origins, are needed.

Abstract P3113: RAF 'Paradox Breaker' Inhibitor, PLX8394, Enhances Inflammation And Apoptosis In Murine Hypertension In Vivo

Introduction: The ERK1/2 cascade, activated by RAF kinases, is a key pathway for cardiac remodelling and cytoprotection. Due to activating oncogenic mutations in BRAF, small molecule RAF inhibitors have been developed. However, owing to inhibitor resistance or paradoxical ERK1/2 pathway activation, a new generation of ‘paradox breaking’ drugs are currently in clinical trials. Our previous work characterised RAF targeting in hypertension, where it is instrumental in driving cardiac remodelling. Here we explored whether use of the RAF paradox breakers (e.g. PLX8394) are viable modalities for targeting RAF in hypertension. Hypothesis: We hypothesize that RAF ‘paradox breakers’ will protect cardiac function by inhibiting maladaptive hypertensive remodelling. Methods &amp; Results: To assess the role of RAF inhibition in cardiac adaptation to hypertension in vivo , C57Bl/6J mice were treated with angiotensin-II (AngII; 0.8mg/kg/d, 7d n=8;) without/with 5mg/kg/d PLX8394 (n=11). PLX8394 alone had no detrimental effect on cardiac function or remodelling. AngII promoted cardiac hypertrophy, by increasing left ventricular (LV) wall thickness (WT) and decreasing LV internal diameter (ID; assessed by echocardiography). PLX8394 had no impact on AngII-induced hypertrophy (WT:ID, p=0.45), or abundance of AngII-induced hypertrophic Nppa (p=0.77) or Nppb (p=0.49) mRNAs. Additionally, PLX8394 did not affect AngII-induced perivascular fibrosis (p=0.69; picrosirius red histology) or moderate Col1a1 (p=0.22) and Col3a1 (p=0.82) mRNAs. However, PLX8394 use in AngII-hypertension was pro-inflammatory, with increased pro-inflammatory cytokines ( Il1b , p=0.02; Il6 , p=0.008) and the inflammatory cell marker CD45 (p=0.001) mRNAs; a response echoed in hearts labelled by immunohistochemistry for CD45 (p=0.02). Moreover, western blotting revealed elevated PARP cleavage (p=0.005), indicating increased apoptosis with PLX8394 use. Conclusion: Despite no adverse effect on cardiac function or global hypertensive remodelling at 7 days, RAF inhibition by PLX8394 appears to be pro-inflammatory and pro-apoptotic. Thus, long-term use of RAF ‘paradox breakers’ in hypertensive individuals may be cardiotoxic and promote adverse cardiac events.

Cancer Stem Cell Markers in Rhabdomyosarcoma in Children.

(1) Background: The aim of the present study was to assess the cancer stem cell (CSC) markers CD24, CD44, CD133, and ALDH1A1 in rhabdomyosarcoma (RMS) in children and to define their prognostic role in this group of patients. (2) Methods: The study material was archival tissue specimens collected from 49 patients under 18 years of age and who had been diagnosed with RMS. Immunohistochemistry (IHC) was used to evaluate the expression of the selected CSC markers in the tumor tissue. Expression was evaluated using a semiquantitative IRS scale based on the one developed by Remmele and Stenger and was correlated with the clinical and pathomorphological parameters of prognostic importance in RMS. (3) Results: Expression of the selected CSC markers CD24, CD44, CD133, and ALDH1A1 was demonstrated in 83.7%, 55.1%, 81.6%, and 100% of the RMS patients, respectively. The expression of all of the assessed CSC markers was statistically significantly higher in the study group versus the control group. No significant correlation was found between the expression of the selected CSC markers and clinical and pathological prognostic factors that were analyzed. The expression of the CSC markers did not have a significant influence on RMS survival rates. (4) Conclusions: The results of the conducted study confirm the expression of selected CSC markers in rhabdomyosarcoma tissue in children. The study did not support the prognostic relevance of the expression of any of the assessed CSC markers. However, further studies are needed to fully understand the relevance of the selected CSC markers in RMS carcinogenesis.

Plasmablastic lymphoma with MYC::IGH fusion and BCL2 rearrangement.

A 50-year-old male with history of nodal stage III diffuse large B-cell lymphoma (DLBCL), refractory to R-CHOP, presented with vomiting, abdominal pain, and an umbilical mass. Abdominal computed tomography (CT) scan showed bulky retroperitoneal adenopathy and possible bowel obstruction. Histologic examination of the umbilical mass needle biopsy demonstrated diffuse atypical cells with medium to large nuclei, prominent central nucleoli, and eccentrically placed amphophilic cytoplasm (Figure 1, top left panel, objective 100×). The original lymphoma was of germinal center B-cell phenotype, was MYC-negative by immunohistochemistry (IHC), and had BCL2 rearrangement (BCL2-R) without MYC-R by interphase fluorescence in situ hybridization (FISH). In contrast, the umbilical mass neoplastic cells expressed plasma cell markers CD138 (Figure 1, top right, objective 60×) and MUM1 (Figure 1. Bottom left, objective 60×) and MYC (90%), were lambda light chain restricted, had a very high Ki-67 proliferation index (>90%), and were negative for all B-cell markers, germinal center cell markers, and Epstein-Barr virus. Interphase FISH studies identified BCL2-R and IGH::MYC fusion (Figure 1, bottom right, Ch 8 centromere: aqua probe, MYC [8q24]: red probe, IGH [14q32]: green probe; yellow fusion indicates IGH::MYC) without BCL6-R. These findings are characteristic of plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. This likely represents transformation of the patient's prior DLBCL, with clonal evolution characterized by acquisition of MYC-R. Transformation of follicular lymphoma to plasmablastic lymphoma with MYC-R and BCL2-R has been previously described, but to our knowledge this is the first reported case of transformation of DLBCL to plasmablastic lymphoma with MYC::IGH fusion and BCL2-R. The “double-hit” cytogenetic features may portend an aggressive clinical course, similar to what has been observed in “high-grade B-cell lymphomas with MYC-R and BCL2-R and/or BCL6-R” (so-called double-hit lymphoma). The authors would like to thank the Mayo Clinic Genomics Laboratory for their continuous assistance and support of our service. The authors declare they have no conflict of interest. The authors received no specific funding for this work. Mohammad Barouqa wrote the manuscript. Patricia Greipp reviewed the article and cytogenetic probes. Rebecca King reviewed the article and stains. Ellen D. McPhail supervised the manuscript and finalized it.

Calreticulin Identified as One of the Androgen Response Genes That Trigger Full Regeneration of the Only Capable Mammalian Organ, the Deer Antler

Deer antlers are male secondary sexual characters that develop to become bone; they are unique appendages that, once lost, can fully regenerate from the permanent bony protuberances or pedicles. Pedicle periosteum (PP) is the tissue that gives rise to the regenerating antlers with three differentiation stages, namely, dormant (DoPP), potentiated (PoPP), and activated (AcPP). Thus far, the transition from the PoPP to the AcPP has not been studied. Our results showed that the AcPP cells maintained their original stem cell features by expressing mesenchymal stem cell (MSC) markers CD73, CD90, and CD105, although they had entered the proliferation mode. The differentially expressed genes (DEGs) in the AcPP compared with those of the PoPP were mainly involved in protein processing, cell cycle, and calcium signaling pathways. Calreticulin (CALR), an androgen response gene, was significantly differentially upregulated in the AcPP cells, and its expression level was negatively regulated by androgens, in contrast to the currently known model systems where all regulation is positive. The downregulation of CALR expression in the AcPP cells in vitro inhibited cell proliferation, induced apoptosis, and inhibited cell cycle progression at G1-S transition. Therefore, CALR is likely a downstream mediator of androgen hormones for triggering initiation of antler regeneration. We believe that the identification of CALR has not only discovered “one critical piece” of the “jigsaw puzzle” in the initiation of antler regeneration but also helps in revealing the mechanism underlying this unique mammalian epimorphic regeneration and has also opened a new avenue for the study of the nature of CALR regulation by androgen (putative binding partners), thus facilitating the identification of potential molecule(s) for investigation as targets for clinical evaluation.

A novel microbial-derived peptide therapeutic vaccine (EO2463) as monotherapy and in combination with lenalidomide and rituximab, for treatment of patients with indolent non-Hodgkin lymphoma (SIDNEY).

TPS7586 Background: EO2463 is a therapeutic vaccine designed to activate existing commensal bacteria-specific memory T cells that cross-react with B cell markers in order to drive anti-tumor immune activity against B-cell malignancies. The four microbial-derived, synthetically produced peptides contained in EO2463 (OMP72, OMP64, OMP65, and OMP66), correspond to cytotoxic CD8 T cell HLA-A2 restricted epitopes, and exhibit molecular mimicry with the B cell markers CD20, CD22, CD37, and CD268 (BAFF-receptor), respectively. In pre-clinical models, these peptides can generate strong immune responses and specifically stimulate cross-reactive cytotoxic CD8 T cells to recognize the chosen B cell targets. EO2463 also contains a CD4 helper peptide referred to as universal cancer peptide 2, derived from the human telomerase reverse transcriptase catalytic subunit. The present study is a first-in-human clinical trial of this microbiome-derived peptide therapeutic cancer vaccine approach in patients with follicular lymphoma (FL) and marginal zone lymphoma (MZL). Methods: This four-cohort phase 1/2 trial will investigate EO2463 monotherapy, and combinations of EO2463/lenalidomide (EL), EO2463/rituximab (ER), and EO2463/lenalidomide/rituximab (ER2), for treatment of patients with FL and MZL. Cohort 1 is a safety lead-in dose-finding in patients with relapsed/refractory (RR) disease, with a 3-by-3 design to establish the recommended phase 2 dose (RP2D) for EO2463 monotherapy and to confirm the safety of the RP2D for combination schedules of EL, and ER2. After the recommended EO2463 monotherapy dose is established, cohorts 2, 3, and 4 will open to accrual. Cohort 2 will investigate EO2463 monotherapy in patients with newly diagnosed FL/MZL who are not in need of treatment; cohort 3 will investigate EO2463 monotherapy, followed by ER in patients with limited tumor burden who need treatment, and cohort 4 will further investigate EL, followed by ER2 in the RR setting. EO2463 will be administered SC 4 times at 2-week intervals, followed by continued booster administrations every 4 weeks for 9 (Cohorts 2 and 3) or 12 (Cohorts 1 and 4) months. Inclusion/exclusion criteria, and the design and schedule of the intense immune and safety monitoring will be presented. The safety lead-in dose-finding is currently ongoing, and no safety concerns have been observed thus far. Clinical trial information: NCT04669171.

Validation of an Accurate Automated Multiplex Immunofluorescence Method for Immuno-Profiling Melanoma.

Multiplex immunofluorescence staining enables the simultaneous detection of multiple immune markers in a single tissue section, and is a useful tool for the identification of specific cell populations within the tumour microenvironment. However, this technology has rarely been validated against standard clinical immunohistology, which is a barrier for its integration into clinical practice. This study sought to validate and investigate the accuracy, precision and reproducibility of a multiplex immunofluorescence compared with immunohistochemistry (IHC), including tissue staining, imaging and analysis, in characterising the expression of immune and melanoma markers in both the tumour and its microenvironment. Traditional chromogenic IHC, single-plex immunofluorescence and multiplex immunofluorescence were each performed on serial tissue sections of a formalin-fixed paraffin-embedded (FFPE) tissue microarray containing metastatic melanoma specimens from 67 patients. The panel included the immune cell markers CD8, CD68, CD16, the immune checkpoint PD-L1, and melanoma tumour marker SOX10. Slides were stained with the Opal™ 7 colour Kit (Akoya Biosciences) on the intelliPATH autostainer (Biocare Medical) and imaged using the Vectra 3.0.5 microscope. Marker expression was quantified using Halo v.3.2.181 (Indica Labs). Comparison of the IHC and single-plex immunofluorescence revealed highly significant positive correlations between the cell densities of CD8, CD68, CD16, PD-L1 and SOX10 marker positive cells (Spearman’s rho = 0.927 to 0.750, p < 0.0001). Highly significant correlations were also observed for all markers between single-plex immunofluorescence and multiplex immunofluorescence staining (Spearman’s rho >0.9, p < 0.0001). Finally, correlation analysis of the three multiplex replicates revealed a high degree of reproducibility between slides (Spearman’s rho >0.940, p < 0.0001). Together, these data highlight the reliability and validity of multiplex immunofluorescence in accurately profiling the tumour and its associated microenvironment using FFPE metastatic melanoma specimens. This validated multiplex panel can be utilised for research evaluating melanoma and its microenvironment, such as studies performed to predict patient response or resistance to immunotherapies.

Prominent B-Cell Signature Differentiates Discoid from Subacute Cutaneous Lupus Erythematosus

Although B cells account for a significant proportion of the lymphocytic infiltrate in discoid lupus erythematosus (DLE), their contribution to pathogenesis is unknown. In this study, we compare the immune landscape of 17 subjects with DLE with that of 21 subjects with subacute cutaneous lupus erythematosus using transcriptomic and histologic analyses of lesional skin. A few of the subjects (3 of 17 subjects with DLE, and 5 of 21 subjects with subacute cutaneous lupus erythematosus) had concomitant systemic lupus erythematosus. Using a modified Autoimmune Profiling Panel (NanoString Technologies, Seatle, WA), we show that B-cell‒specific genes, including canonical pan‒B cell markers CD19 (P= 0.0060), MS4A1(CD20) (P= 0.0047), and CD79a (P= 0.0201), are among the most upregulated genes in DLE. Numerous other genes encoding B-cell‒associated proteins, including Igs, BAFF receptors, and FCRL family members, are similarly enriched. Relative cell type scoring reveals that among various inflammatory cell types, only B cells are more prevalent in DLE. Digital whole-image slide analysis of immunohistochemistry for B cells (CD20) and T cells (CD3) supports our gene expression findings of a disproportionately greater B-cell infiltrate in DLE lesions. Overall, this study identifies a B-cell‒predominant signature unique to DLE and highlights the importance of studying the role of cutaneous B cells in DLE pathogenesis.

Minocycline Alleviates White Matter Injury following Intracerebral Hemorrhage by Regulating CD4+ T Cell Differentiation via Notch1 Signaling Pathway.

Neuroinflammation is a major reason for white matter injury (WMI) after intracerebral hemorrhage (ICH). Apart from microglia/macrophage activation, T cells also play an important role in regulating immune responses after ICH. In a previous study, we have revealed the role of minocycline in modulating microglia/macrophage activation after ICH. However, the exact mechanisms of minocycline in regulating T cells differentiation after ICH are still not well understood. Hence, this study explored the relationship between minocycline and CD4+ T cell differentiation after ICH. Piglet ICH model was used to investigate naive CD4+ T cell differentiation and T cells signal gene activation after ICH with immunofluorescence and whole transcriptome sequencing. Naive CD4+ T cells and primary oligodendrocyte coculture model were established to explore the effect and mechanism of minocycline in modulating CD4+ T cell differentiation after ICH. Flow cytometry was used to indicate CD4+ T cell differentiation after ICH. The mechanism of minocycline in modulating CD4+ T cell differentiation was demonstrated with immunofluorescence and western blot. Double immunostaining of representative CD4+ T cell marker CD3 and different subtype CD4+ T cell assisted proteins (IL17, IL4, Foxp3, and IFNγ) demonstrated naive CD4+ T cell differentiation in piglet after ICH. Whole transcriptome sequencing for perihematomal white matter sorted from piglet brains indicated T cell signal gene activation after ICH. The results of luxol fast blue staining, immunofluorescent staining, and electron microscopy showed that minocycline alleviated white matter injury after ICH in piglets. For our in vitro model, minocycline reduced oligodendrocyte injury and neuroinflammation by regulating CD4+ T cell differentiation after ICH. Moreover, minocycline increased the expression of NOTCH1, ACT1, RBP-J, and NICD1 in cultured CD4+ T cell when stimulated with hemoglobin. Hence, minocycline treatment could modulate naive CD4+ T cell differentiation and attenuate white matter injury via regulating Notch1 signaling pathway after ICH.

Eight Weeks of Daily Cannabidiol Ingestion Does Not Alter Peripheral Blood Mononuclear Cell Profile or Natural Killer Cell Cytotoxicity

ObjectiveThe purpose of this randomized placebo‐controlled trial was to determine the effects of an 8‐week cannabidiol (CBD) intervention on peripheral blood mononuclear cells (PBMCs) profile and natural killer cell (NKC) cytotoxicity.HypothesisFollowing an 8‐week CBD intervention, participants consuming CBD will have an increased percentage of NKC in peripheral blood, as well as decreased NKC cytotoxicity determined by a measure of K562 cell viability.MethodsPhysically active men and women (18‐45 y), currently meeting the American College of Sports Medicine (ACSM) guidelines for physical activity were randomly assigned to placebo (CN, n=5) or CBD (CB, n=9) groups. Participants consumed capsulized control; coconut derived medium chain triglycerides (MCT; 225mg/day) or CBD (50mg/day with 175mg of MCT) daily for 8 weeks. Participants completed measures of body size, body composition (BodPod) and fitness (VO2max), and peripheral blood was drawn before and after the intervention. Ficoll‐isolated PBMCs were co‐incubated for 4h with K562 human leukemia cells before cytotoxicity analysis by flow cytometry. Before co‐culture, PBMCs were stained with T cell marker CD3, and NKC marker, CD56, for immune profiling. K562 cells were pre‐stained with cell‐specific CD71 (transferrin receptor) and the cell viability dye Calcein AM. Data are presented as mean ± standard deviation with significance set at a=0.05. At the pre intervention time point, data was not normally distributed, so a Welch’s t‐test was preformed to determine group differences. A 2 (group) x 2 (time) analysis of variance (ANOVA) was used to identify any interactions or main effects.ResultsAt the pre‐intervention time points, there were no significant differences between groups with respect to participant characteristics (age: 27.1 ± 6.1 y; height: 169 ± 9.7 cm; weight: 73.8 ± 15.5 kg; lean body mass: 57.5 ± 12.0 kg; body fat: 21.5 ± 8.3%; VO2Peak: 45.2 ± 7.6 ml/kg/min). There were no significant differences in percentage of NKC (Pre: 8.8 ± 5.8%; Post:7.4 ± 5.7%), natural killer T‐cells (NKT; Pre: 1.7 ±1.1; Post: 1.5 ±1.1%), or T‐cells (Pre: 47.37±13.3; Post: 46.29±10.17%) when CN was compared to CB following the intervention. Furthermore, there were no differences in K562 cell mean or median fluorescence intensity of Calcein AM when CN was compared to CB following the intervention.ConclusionEight weeks of CBD (50mg/day) did not alter NKC, NKT, or T‐cell distribution in PBMCs. In line with these findings, NKC showed similar cytolytic function through no change in mean or median fluorescence intensity of Calcein AM expression of K562 human leukemia cells, indicating no change in cell viability following CBD supplementation. This preliminary study suggests that CBD does not alter the distribution of circulating mononuclear immune cells or NKC cytolytic function.

Abstract 217: PCSK6 Is A Key Regulator Of Immune Status In Mice And Its Ablation Increases Atherosclerotic Plaque Burden In A Bone Marrow Transplant Model

Introduction: Proprotein convertase subtilisins/kexins (PCSKs) activate cytokines and growth factors and have been implicated in various cancers. We have previously shown that PCSK6 is a key protease modulating smooth muscle cell response in atherosclerosis, but its expression also correlated positively with typical markers of T lymphocytes and macrophages in plaques and it was localised in the proximity of these cells. Here, we hypothesized that PCSK6 may be involved in modulating inflammatory responses and aimed to elucidate its role in a hyperlipidemic mice model. Methods: Detailed immunophenotyping using histology, FACS-, OLINK- and ELISA-based analyses and primary cell cultures was used to compare Pcsk6 -/- and littermate controls. Atherosclerosis was evaluated in Ldlr -/- mice upon bone marrow transplant with Pcsk6 -/- or wild-type bone-marrow. Results: At baseline Pcks6 -/- mice showed an enrichment of pro-inflammatory cytokines Ccl2, Ccl3, Ccl20, Cxcl1 and in particular Il17a and Il17f in plasma compared controls. Spleens of Pcsk6 -/- mice had an increased number of germinal centres and FACS analysis showed that they contained significantly more CD8+ T cells. Microarray analysis of spleens from Pcsk6 -/- vs. controls confirmed that T cell markers CD4, CD3E and CD3G were upregulated. In vitro , splenocytes isolated from Pcsk6 -/- mice secreted more IFN-γ, IL-2 and IL-10 than controls upon stimulation with α-CD3 and α-CD28 antibodies. Moreover, peritoneal macrophages from Pcsk6 -/- mice secreted more TNF-α, MCP-1, IL-6 and IL-10 compared to control mice upon LPS stimulation. Interestingly, bone marrow derived macrophages from Pcsk6 -/- mice were also more prone to lipid uptake. Finally, in vivo transplantation of Pcsk6 -/- bone marrow to Ldlr -/- mice led to increased atherosclerotic plaque burden compared to controls, as quantified in the aortic root. Conclusions: PCSK6 ablation led to increased number of CD8+ T cells, as well as macrophage and cytokine activation. Transplantation of Pcsk6 -/- bone marrow resulted in an increase in atherosclerotic plaque burden compared to controls. Taken together, these results indicate that PCSK6 is a key regulator of the immune system, though the exact mechanisms involved require further investigation.