Simultaneous measurement of transferrin receptor and DNA content of human IL 2 dependent T cells by flow cytometry.

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child’s clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

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Estimation of nuclear DNA content by flow cytometry in fishes of the genus Xiphophorus

Comparative Biochemistry and Physiology Part B Comparative Biochemistry ◽

10.1016/0305-0491(89)90182-x ◽

1989 ◽

Vol 94 (3) ◽

pp. 465-468 ◽

Cited By ~ 14

Author(s):

Terrence R. Tiersch ◽

Robert W. Chandler ◽

Klaus D. Kallman ◽

Stephen S. Wachtel

Keyword(s):

Flow Cytometry ◽

Dna Content ◽

Nuclear Dna ◽

Nuclear Dna Content

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POS0776 CORRELATION OF PERIPHERAL CD4+GRANZB+CTLS WITH DISEASE SEVERITY IN PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.3450 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 641.3-642

Author(s):

Q. Wang ◽

L. Gu ◽

M. Zhang

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Sjögren’S Syndrome ◽

Mhc Class Ii ◽

Sjögren's Syndrome ◽

Sjogren’S Syndrome ◽

Sjogren's Syndrome ◽

Healthy Controls ◽

Extraglandular Manifestations ◽

Sjögren‘S Syndrome

Background:Sjögren’s syndrome (SS) is a chronic autoimmune disorder. The major histopathologic lesion of it is a focal lymphocytic infiltrate around ductal and acinar epithelial cells, which include a majority of CD4+T. Several studies have shown that the epithelial cells in SS present diverse phenomena, such as MHC class II overexpression. CD4+T cells with cytotoxic activity (CD4 CTL) have been detected in various immune responses. They are characterized by their ability to secrete perforin and granzyme B to kill the target cells in an MHC class II-restricted fashion.Objectives:So this study was to investigate the correlation of peripheral CD4+GranzB+CTLs with disease severity and organ involvement in patients with primary Sjögren’s syndrome.Methods:We recruited 116 pSS patients and 46 healthy controls using flow cytometry to examine proportion of CD4+GranzB+CTLs in their peripheral blood, and immunofluorescence to test the expression of CD4+GranzB+CTLs in labial gland. The correlations of CD4+GranzB+CTLs and the relevant clinical data were analyzed.Results:We analyzed the percentage of CD4+GranzB+cytotoxic T cells in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Frequency of peripheral CD4+GranzB+CTLs were measured in 116 patients with pSS and 46 healthy controls matched for age and sex. The percentage of CD4+GranzB+CTLs were significantly up-regulated in pSS patients than healthy controls (7.1%±4.9% vs 3.1%±1.9%, p <0.0001) and positive correlation with ESSDAI in pSS patients(r = 0.6332, p<0.001). The percentage of CD4+GranzB+CTLs were markedly higher in pSS patients with extraglandular manifestations. Moreover, CD4+GranzB+CTLs were observed in the lymphocytic foci and periductal areas of the LSGs and were elevated with increased foci index (FI). After excluding the other risk factors associated with pSS, CD4+GranzB+CTLs were still related to ESSDIA and extraglandular manifestations independently(p<0.05). ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS, and 0.851 to presume extraglandular manifestations. The best diagnostic cut-off point was 4.865 for pSS patients.Conclusion:In this study, We provide new evidence indicating involvement of CD4+GranzB+CTLs over activation in the disease pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.References:[1]Takeuchi A, Saito T. Front Immunol. (2017) 23:194.[2]Brown DM, et al. Front Immunol. (2016) 9:93.[3]Polihronis M, et al. Clin Exp Immunol. (1998) 114:485-90.[4]Xanthou G, et al. Clin Exp Immunol. (1999) 118:154-63.[5]Maehara T, et al. Ann Rheum Dis. (2017) 76:377-385.[6]Goules AV, et al. Clin Immunol. (2017) 182:30-40.[7]Hashimoto K, et al. Proc Natl Acad Sci U S A. (2019) 116:24242-24251.[8]Croia C, et al. Arthritis Rheumatol. (2014) 66:2545-57.[9]Schmidt D,et al. J Clin Invest. (1996) 97:2027–37.[10]Pandya JM, et al. Arthritis Rheum. (2010) 62:3457–66.[11]Moosig F, et al. Clin Exp Immunol. (1998) 114:113–8.[12]Peeters LM, et al. Front Immunol. (2017) 20:1160.Table 1.Multivariate analysis of CD4+GranzB+CTLs influenced by pSS-related factorsregression coefficientstandard errort-statisticsp value95%CICD8+GranzB+CTLs(%)0.1440.0334.3346.9E-50.077, 0.211ESSDAI0.2560.1222.0950.0410.011, 0.502extraglandular manifestations2.6121.2682.0590.0450.065, 5.158Figure 1.Receiver operating characteristic (ROC) curve of the frequency of CD4+GranzB+CTLs to predict ESSDAI and extraglandular manifestations responseDisclosure of Interests:None declared

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588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0588 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A623-A623

Author(s):

Yannick Rakké ◽

Lucia Campos Carrascosa ◽

Adriaan van Beek ◽

Valeska de Ruiter ◽

Michael Doukas ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Colorectal Carcinoma ◽

Liver Metastases ◽

Mismatch Repair ◽

Immune Checkpoint ◽

Ex Vivo ◽

Human Tumour ◽

Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

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Evaluation of CD40 ligand (CD154) expression on neonatal cord blood T cells using a rapid whole blood flow cytometry procedure

Clinical Immunology Newsletter ◽

10.1016/s0197-1859(97)80300-7 ◽

1997 ◽

Vol 17 (6) ◽

pp. 90

Author(s):

M. Paniagua ◽

D. Zaas ◽

M.R.G. O'Gorman

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Blood Flow ◽

Cord Blood ◽

Whole Blood ◽

Cd40 Ligand

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Brief communication. First evaluation of nuclear DNA content in Setaria genus by flow cytometry

Journal of Heredity ◽

10.1093/jhered/89.6.556 ◽

1998 ◽

Vol 89 (6) ◽

pp. 556-559 ◽

Cited By ~ 20

Author(s):

M Le Thierry d'Ennequin ◽

O Panaud ◽

S Brown ◽

S Siljak-Yakovlev ◽

A Sarr

Keyword(s):

Flow Cytometry ◽

Dna Content ◽

Nuclear Dna ◽

Nuclear Dna Content

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109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0109 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A121-A121

Author(s):

Nina Chu ◽

Michael Overstreet ◽

Ryan Gilbreth ◽

Lori Clarke ◽

Christina Gesse ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Dominant Negative ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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Diltiazem inhibits transferrin receptor expression and causes G1 arrest in normal and neoplastic T cells

Molecular and Cellular Biology ◽

10.1128/mcb.6.12.4244-4250.1986 ◽

1986 ◽

Vol 6 (12) ◽

pp. 4244-4250

Author(s):

L M Neckers ◽

S Bauer ◽

R C McGlennen ◽

J B Trepel ◽

K Rao ◽

...

Keyword(s):

T Cells ◽

Transferrin Receptor ◽

Interleukin 2 ◽

Receptor Expression ◽

Calcium Flux ◽

G1 Arrest ◽

Receptor Mrna ◽

Interleukin 2 Receptor ◽

Transferrin Receptor Expression ◽

Malignant T Cells

Transferrin receptor expression is essential for the proliferation of both normal and malignant T cells. While transferrin receptor expression in normal T cells is tightly coupled to interleukin-2 receptor expression, transferrin receptor expression in malignant cells is usually constitutive and is released from this constraint. Temporally, the appearance of these membrane receptors is preceded by changes in the expression of the proto-oncogenes c-myc and c-myb. In addition, although an increase in the level of intracellular free calcium occurs early in the sequence of T-cell activation, the activation events dependent on this calcium flux have not been resolved. In the present study we report that diltiazem, an ion channel-blocking agent that inhibits calcium influx, arrested the growth in vitro of both normal and malignant human T cells in the G1 phase of the cell cycle. However, diltiazem did not inhibit the expression of c-myc or interleukin-2 receptor mRNA and protein in normal mitogen-activated T cells or the constitutive expression of c-myc and c-myb mRNA in malignant T cells (T acute lymphoblastic leukemia cells). In contrast, diltiazem prevented the induction of transferrin receptor (mRNA and protein) in normal T cells and caused a progressive loss of transferrin receptor (mRNA and protein) in malignant T cells. These data demonstrate that diltiazem can dissociate several growth-related processes normally occurring in G1 and thereby disrupt the biochemical cascade leading to cell proliferation.

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