scholarly journals Multiscale fluorescent tracking of immune cells in the liver with a highly biocompatible far-red emitting polymer probe

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Malo Daniel ◽  
Laurence Dubreil ◽  
Romain Fleurisson ◽  
Jean-Paul Judor ◽  
Timothée Bresson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Cell Tracking ◽  
Classical Model ◽  
Stokes Shift ◽  
Water Soluble ◽  
Two Photon Microscopy ◽  
Two Photon

Abstract The development of innovative immune cell therapies relies on efficient cell tracking strategies. For this, multiscale fluorescence-based analyses of transferred cells into the host with complementary techniques, including flow cytometry for high-throughput cell analysis and two-photon microscopy for deep tissue imaging would be highly beneficial. Ideally, cells should be labelled with a single fluorescent probe combining all the properties required for these different techniques. Due to the intrinsic autofluorescence of most tissues and especially the liver, far-red emission is also an important asset. However, the development of far-red emitting probes suitable for two-photon microscopy and compatible with clearing methods to track labelled immune cells in thick samples, remains challenging. A newly-designed water-soluble far-red emitting polymer probe, 19K-6H, with a large Stokes shift, was thus evaluated for the tracking of primary immune CD8 T cells. These cells, prepared from mouse spleen, were efficiently labelled with the 19K-6H probe, which was internalized via endocytosis and was highly biocompatible at concentrations up to 20 μM. Labelled primary CD8 T cells were detectable in culture by both confocal and two-photon microscopy as well as flow cytometry, even after 3 days of active proliferation. Finally, 19K-6H-labelled primary CD8 T cells were injected to mice in a classical model of immune mediated hepatitis. The efficient tracking of the transferred cells in the liver by flow cytometry (on purified non-parenchymal cells) and by two-photon microscopy on 800 μm thick cleared sections, demonstrated the versatility of the 19K-6H probe.

756 Assessment of the immune checkpoint landscape in head and neck squamous cell carcinoma by single-cell RNA sequencing and multispectral imaging

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A804-A805
Author(s):  
Lazar Vujanovic ◽  
Aditi Kulkarni ◽  
Cornelius Kürten ◽  
Anthony Cillo ◽  
Patricia Santos ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Squamous Cell Carcinoma ◽  
T Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Immune Checkpoint ◽  
Multispectral Imaging ◽  
Cell Types ◽  
Cell Type ◽  
Single Cell Rna Sequencing

BackgroundResistance to the current generation of immunotherapies is mediated by complex relations between stromal, cancer and immune cells found within the tumor microenvironment (TME). Development of more efficacious drugs is predicated on improved understanding of these multi-spatial interactions. With emergence of new immune checkpoint receptor (ICR)-targeting therapies, a better understanding of topological expression of immune checkpoint ligand (ICL) on suppressive cell types in the TME may allow for improved strategies to treat cancer patients.MethodsSingle cell RNA sequencing (scRNAseq) was performed from head and neck squamous cell carcinoma (HNSCC) specimens (n=18) with matched blood from treatment-naïve patients. Immune and non-immune cells were enriched from tumor cell suspensions. Novel transcriptomic cell-to-cell interactions were predicted between heterogeneous cell populations. Histologic inflammation was corroborated with scRNAseq and multiplex flow cytometry. Cell type-specific PD-L1 contributions within the TME were quantified using multispectral imaging.ResultsMajor cell type clusters (immune, epithelial, fibroblast and endothelial cells) were identified. Expression patterns for PD-1, TIGIT, LAG-3 and TIM-3 ligands were evaluated on these suppressive TME cell types. By modeling receptor-ligand interactions between CD8+ T cells and the rest of the major TME cell types, CD8+ T cells were predicted to form more ICR-ICL interactions with tumor-associated macrophages (TAMs) than with any other cell type. With focus on LGALS9/galectin-9 and CD274/PD-L1, flow cytometric analyses validated the scRNAseq observation that both ligands were expressed on TAMs from both inflamed and non-inflamed tumors. Furthermore, flow cytometry and multispectral imaging analyses implicated macrophages as one of the major contributors of CD274/PD-L1 within the TME.ConclusionsOur data suggest that in the setting of HNSCC, TAMs are one of the major contributors of ICL in the HNSCC TME. Strategies that selective target this immunosuppressive population may be necessary to break tolerance to PD-1-targeting therapies.Ethics ApprovalThe study was approved by the UPMC Hillman Cancer’s Ethics Board, approval number 99-069.

scholarly journals Direct and Indirect endocrine-mediated suppression of human endometrial CD8+T cell cytotoxicity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Z. Shen ◽  
M. Rodriguez-Garcia ◽  
M. V. Patel ◽  
C. R. Wira
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cytotoxic Activity ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Cd8 T Cell ◽  
Gynecological Cancers ◽  
Protective Strategies ◽  
T Cell Cytotoxicity

AbstractRegulation of endometrial (EM) CD8+T cells is essential for successful reproduction and protection against pathogens. Suppression of CD8+T cells is necessary for a tolerogenic environment that promotes implantation and pregnancy. However, the mechanisms regulating this process remain unclear. Sex hormones are known to control immune responses directly on immune cells and indirectly through the tissue environment. When the actions of estradiol (E2), progesterone (P) and TGFβ on EM CD8+T cells were evaluated, cytotoxic activity, perforin and granzymes were directly suppressed by E2 and TGFβ but not P. Moreover, incubation of polarized EM epithelial cells with P, but not E2, increased TGFβ secretion. These findings suggest that E2 acts directly on CD8+T cell to suppress cytotoxic activity while P acts indirectly through induction of TGFβ production. Understanding the mechanisms involved in regulating endometrial CD8+T cells is essential for optimizing reproductive success and developing protective strategies against genital infections and gynecological cancers.

Engineered immunostimulatory cells can convert PBMCs from chronic lymphocytic leukemia (CLL) patients into potent tumor killing immune cells.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 7517-7517
Author(s):  
Joshua W. Keegan ◽  
Frank Borriello ◽  
Stacey M. Fernandes ◽  
Jennifer R. Brown ◽  
James A. Lederer
Keyword(s):  
T Cells ◽  
B Cells ◽  
Tumor Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Killing Activity ◽  
Tumor Killing ◽  
Tumor Cell Killing ◽  
Potent Tumor

7517 Background: Alloplex Biotherapeutics has developed a cellular therapeutic that uses ENgineered Leukocyte ImmunoSTimulatory cell lines called ENLIST cells to activate and expand populations of tumor killing effector cells from human peripheral blood mononuclear cells (PBMCs). This process leads to a 300-fold expansion of NK cells, CD8+ T cells, NKT cells, and TCRγδ T cells that are called SUPLEXA cells, which will be cryopreserved and transferred back into patients as an autologous immune cell therapy for cancer. In this study, PBMCs from CLL patients were used to generate SUPLEXA cells as a first approach to comparatively profile SUPLEXA cells from cancer patients and normal healthy volunteers (NHVs). Methods: ENLIST cell lines were engineered by expressing curated immunomodulatory proteins in the SK-MEL-2 melanoma cell line. Two million (M) PBMCs from 10 CLL patients or 2 NHVs were incubated with 0.4 M freeze/thaw killed ENLIST cells for 5 days in XVIVO-15 medium with 2% heat-inactivated human AB serum (XAB2) and then split 1:15 in XAB2 containing IL-7 and IL-15 to expand. After 9 days, SUPLEXA cells were harvested and cryopreserved. Results: Original PBMCs and matched SUPLEXA cells from each donor were thawed and characterized by mass cytometry (CyTOF) using a 47-marker antibody panel. CyTOF staining results of PBMCs from CLL patients demonstrated approximately 95% leukemia cells and few T cells, NK cells, B cells, and monocytes. CyTOF staining of SUPLEXA cells from all 10 CLL patients showed expansion of NK cells (17%), CD8 T cells (11%), and CD4 T cells (7.5%) that were similar in phenotype to SUPLEXA cells from NHVs showing high expression of granzymes and perforin that are indicative of potent tumor cell killing activity. Cancer cells in the original CLL PBMC samples were reduced to 0.78%. However, a population of non-T/non-B cells (60% ± 9.5%) was detected in SUPLEXA cells from all CLL patients that require further characterization. Next, SUPLEXA cells from CLL and NHV patients were comparatively tested for tumor cell killing activity at 2:1, 1:1, and 1:2 effector to target cell (MEL-14 melanoma cells expressing RFP) ratios. Percent killing of tumor cells by SUPLEXA cells prepared from CLL patients (77.8% ± 2.6% at 2:1) and NHVs (81.5% ± 0.3% at 2:1) were nearly identical at all effector to target ratios. Conclusions: We demonstrate for the first time that PBMCs from CLL patients can be converted into SUPLEXA cells despite low numbers of normal immune cells at baseline and the known immunologic impairment present in CLL patients. Importantly, SUPLEXA cells derived from CLL patients acquire potent tumor killing activity that is indistinguishable from SUPLEXA cells prepared from NHVs. Taken together, these findings support the feasibility of converting PBMCs from CLL patients with low percentages of NK and T cells into an autologous cellular therapy for cancer.

scholarly journals Identification of Immune-Related Prognostic mRNA and lncRNA in Patients with Hepatocellular Carcinoma

2022 ◽  
Vol 2022 ◽  
pp. 1-16
Author(s):  
Dan Chen ◽  
Xiaoting Li ◽  
Hui Li ◽  
Kai Wang ◽  
Xianghua Tian
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
Regression Analysis ◽  
Survival Analysis ◽  
Immune Cells ◽  
Cox Regression ◽  
Differentially Expressed ◽  
Cox Regression Analysis ◽  
Immune Related Genes

Background. As the most common hepatic malignancy, hepatocellular carcinoma (HCC) has a high incidence; therefore, in this paper, the immune-related genes were sought as biomarkers in liver cancer. Methods. In this study, a differential expression analysis of lncRNA and mRNA in The Cancer Genome Atlas (TCGA) dataset between the HCC group and the normal control group was performed. Enrichment analysis was used to screen immune-related differentially expressed genes. Cox regression analysis and survival analysis were used to determine prognostic genes of HCC, whose expression was detected by molecular experiments. Finally, important immune cells were identified by immune cell infiltration and detected by flow cytometry. Results. Compared with the normal group, 1613 differentially expressed mRNAs (DEmRs) and 1237 differentially expressed lncRNAs (DElncRs) were found in HCC. Among them, 143 immune-related DEmRs and 39 immune-related DElncRs were screened out. These genes were mainly related to MAPK cascade, PI3K-AKT signaling pathway, and TGF-beta. Through Cox regression analysis and survival analysis, MMP9, SPP1, HAGLR, LINC02202, and RP11-598F7.3 were finally determined as the potential diagnostic biomarkers for HCC. The gene expression was verified by RT-qPCR and western blot. In addition, CD4 + memory resting T cells and CD8 + T cells were identified as protective factors for overall survival of HCC, and they were found highly expressed in HCC through flow cytometry. Conclusion. The study explored the dysregulation mechanism and potential biomarkers of immune-related genes and further identified the influence of immune cells on the prognosis of HCC, providing a theoretical basis for the prognosis prediction and immunotherapy in HCC patients.

scholarly journals 708 Novel ways to exploit IL-21 to augment adoptive T cell transfer therapy against tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Anna Cole ◽  
Guillermo Rangel RIvera ◽  
Aubrey Smith ◽  
Megan Wyatt ◽  
Brandon Ware ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Striking Difference ◽  
T Cell Therapy ◽  
Cell Immunity ◽  
Emory University

BackgroundIL-21 enhances the anti-tumor capacity of adoptively transferred CD8+ T cells, while IL-2 and IL-15 impair T cell immunity by driving their expansion to a more differentiated status. Yet, these cytokines can act on many different immune cells. Given the potency of IL-21, we tested if this cytokine directly augments T cells or rather if it enhances other immune cells in the culture that indirectly improves T cell therapy.MethodsTo test this question, splenocytes from pmel-1 transgenic mice were used, as all CD8+ T cells express a transgenic TCR specific for tumor-antigen gp10025–33 overexpressed on melanoma. We then peptide activated naïve CD8+ T cells enriched or not from the spleen of pmel-1 mice and expanded them in the presence of IL-21 or IL-2 (10 ng/mL) for four days. Expanded pmel-1 from these various cultures were then restimulated with irradiated splenocytes pulsed with gp10025–33 and grown an additional seven days with IL-2 (10 ng/mL), irrespective of their initial cytokine condition. The in vitro memory phenotype, exhaustion profile, and cytokine secretion of these cultures were then assayed. Furthermore, mice bearing B16KVP melanoma tumors were infused with pmel-1 T cells expanded via these various approaches and compared for their relative capacity to engraft, persist, and regress tumor in vivo.ResultsInterestingly, we discovered that IL-21-treated T cells generated from bulk splenocytes are phenotypically and functionally distinct from IL-21-treated isolated T cells. Upon restimulation, IL-21-treated T cells from bulk splenocytes exhibited an exhausted phenotype that was like anergic IL-2-treated T cells. Moreover, few cells expressed CD62L but expressed heightened markers of suppression, including TIM3, PD-1, and EOMES. Moreover, they produced more effector molecules, including granzyme B and IFN-gamma. In vivo IL-21-treated T cells expanded from bulk splenocytes engrafted and persisted poorly, in turn mediating suboptimal regression of melanoma. Conversely, IL-21 dramatically bolstered the engraftment and antitumor activity of T cells only if they were first isolated from the spleen prior to their expansion and infusion into the animal.ConclusionsCollectively, our data shows that IL-21 may improve ACT therapy best when used directly on antitumor CD8+ T cells. Further studies will illuminate the mechanism behind this striking difference and determine whether other cell subsets reactive to IL-21 cause T cell dysfunction and/or reduced bioavailability. These findings are important for defining the best culture conditions in which to use IL-21 for ACT.AcknowledgementsWe would like to acknowledge Emory University, The Winship Cancer Institute, and the Pediatrics/Winship Flow Cytometry Core.Ethics ApprovalAll animal procedures were approved by the Institutional Animal Care and Use Committee of Emory University, protocol number 201900225.

scholarly journals ILDR1 Is a Prognostic Biomarker and Associated With Immune Infiltration in Gastric Cancer

2021 ◽  
Author(s):  
Yanling Ma ◽  
WenBo Qi ◽  
BaoHong Gu ◽  
XueMei Li ◽  
ZhenYu Yin ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
T Cells ◽  
Dendritic Cells ◽  
Cancer Patients ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Copy Number ◽  
Sequencing Data ◽  
Number Variation ◽  
Gastric Cancer Patients

Abstract Objective: To investigate the association between ILDR1 and prognosis and immune infiltration in gastric cancer. Methods: We analyzed the RNA sequencing data of 9736 tumor tissues and 8587 normal tissues in the TCGA and GTEx databases through the GEPIA2 platform. The expression of ILDR1 in gastric cancer and normal gastric mucosa tissues with GEPIA and TIMER. Clinical subgroup analysis was made through Kaplan-Meier analysis. Analyzed the correlation between ILDR1 and VEGFA expression in gastric cancer, through the gene sequencing data of gastric cancer in TCGA. Explored the relationship between ILDR1 methylation and the prognosis of gastric cancer patients through the MethSurv database. The correlation between ILDR1 and immune cells and the correlation of copy number variation were explored through the TIMER database. Results: ILDR1-high GC patients had a lower PFS and OS. High ILDR1 expression was significantly correlated with tumor grade. There was a negative correlation between the ILDR1 expression and the abundances of CD8+ T, Macrophages and DC and etc. The methylation level of ILDR1 is associated with a good prognosis of gastric cancer. ILDR1 copy number variation was correlated with immune cells, IDLR1 arm-loss was associated with the infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells, and arm-duplication was associated with the infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils and dendritic cells. Conclusion: The increased expression of ILDR1 is associated with poor prognosis in patients with gastric cancer. ILDR1 can be used as a novel predictive biomarker to provide a new therapeutic target for gastric cancer patients.

scholarly journals APOC1 is a Potential Prognostic Biomarker and Correlated With Immune Infiltration in ESCC

2020 ◽  
Author(s):  
Jia-yi XIE ◽  
Ming Liu ◽  
Yaxin Luo ◽  
Zhen Wang ◽  
Zhenghong Lu ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Poor Prognosis ◽  
Killer Cell ◽  
Gene Set Enrichment Analysis ◽  
Immune Infiltration ◽  
Kaplan Meier ◽  
Kaplan Meier Plotter

Abstract PurposeEsophageal cancer (EC) is the sixth leading cause of cancer death worldwide. Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of EC. Identifying diagnostic biomarkers for ESCC is necessary for cancer practice. Increasing evidence illustrates that apolipoprotein C-1 (APOC1) participates in the carcinogenesis. However, the biological function of APOC1 in ESCC remains unclear. Patients and methodsWe investigated the expression level of APOC1 using TIMER2.0 and GEO databases, the prognostic value of APOC1 in ESCC using Kaplan-Meier plotter and TCGA databases. We used LinkedOmics to identify co-expressed genes with APOC1 and perform GO and KEGG pathway analysis. The target networks of kinases, miRNAs and transcription factors were predicted by gene set enrichment analysis (GSEA). The correlations between APOC1 and immune infiltration were calculated using TIMER2.0 and CIBERSORT databases. We further performed the prognostic analysis based on APOC1 expression levels in related immune cells subgroups via Kaplan-Meier plotter database. ResultsAPOC1 was found overexpressed in tumor tissues in multiple ESCC cohorts and high APOC1 expression was related to a dismal prognosis. Multivariate analysis confirmed that APOC1 overexpression was an independent indicator of poor OS. Functional network analysis indicated that APOC1 might regulate the natural killer cell mediated cytotoxicity, phagosome, AMPK and hippo signaling through pathways involving some cancer-related kinases, miRNA and transcription factors. Immune infiltration analysis showed that APOC1 was significantly positively correlated with M0 macrophages cells, M1 macrophages cells and activated NK cells, negatively correlated with regulatory T cells, CD8 T cells, neutrophils and monocytes. High APOC1 expression had a poor prognosis in server immune cells subgroups in ESCC, including decreased CD8+ T cells subgroups. ConclusionThese findings suggest that increased expression of APOC1 is related to poor prognosis and immune infiltration in ESCC. APOC1 holds promise for serving as a valuable diagnostic and prognostic marker in ESCC.

scholarly journals Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

2020 ◽  
Vol 8 (2) ◽  
pp. e001250
Author(s):  
Benson Chellakkan Selvanesan ◽  
Kiran Meena ◽  
Amanda Beck ◽  
Lydie Meheus ◽  
Olaya Lara ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Cells ◽  
Mouse Tumor ◽  
Epitope Spreading ◽  
Stromal Compartment ◽  
Positive Effects

BackgroundTreatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B3), in mice with pancreatic cancer.MethodsVarious mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (KrasG12D, p53R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope.ResultsA significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading.ConclusionThis study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

scholarly journals Distinct changes of in BTLA, ICOS, PD-1, and TIGIT expression on peripheral blood and decidual CD8+ T cells in women with unexplained recurrent spontaneous abortion†

2020 ◽  
Vol 103 (5) ◽  
pp. 1012-1017
Author(s):  
Qianqian Liang ◽  
Lingxia Tong ◽  
Liping Xiang ◽  
Sujuan Shen ◽  
Chenhuan Pan ◽  
...  
Keyword(s):  
T Cells ◽  
Spontaneous Abortion ◽  
Cd8 T Cells ◽  
Immune Cells ◽  
Peripheral Blood ◽  
Immune Escape ◽  
Recurrent Spontaneous Abortion ◽  
Decidual Tissue ◽  
Unexplained Recurrent Spontaneous Abortion ◽  
The Difference

Abstract The two-way communication between the mother and the fetus is accomplished by immune cells. CD8+ T cells of normal pregnant (NP) women express progesterone receptor (PR). Binding of PR to progesterone (P) and the production of progesterone-induced blocking factor (PIBF) can aid immune escape, which is an important factor in the maternal immune response. We detected the proportion of CD8+ T cells and the expression of the surface costimulatory molecules BTLA, TIGIT, ICOS, and PD-1 in peripheral blood and decidual tissues of women with unexplained recurrent spontaneous abortion (URSA) and in NP women. All patients were at 8 -10 weeks of gestation. The results showed that there was no change in the proportions of CD8+ T cells in peripheral blood and decidual tissues of URSA patients compared to those of NP women. In peripheral blood, compared with the NP group, the URSA group showed decreased expression of BTLA + CD8+ T cells and the difference was statistically significant, but there was no difference between the groups in terms of TIGIT + CD8+, PD-1 + CD8+, and ICOS + CD8+ T cells. There was no change in the levels of TIGIT + CD8+, PD-1 + CD8+, ICOS + CD8+, and BTLA + CD8+ T cells in decidual tissue. These data confirm that the number of CD8+ T cells in peripheral blood and decidual tissue is not the main factor leading to the pathogenesis of URSA, and other immune cells may play an important role in URSA, but this hypothesis needs further exploration and research.

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