Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Differential gene expression in HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic individuals. ( a ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP individuals. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 and VP11/12 702–710 epitopes were sorted from HLA-A*0201-positive SYMP and ASYMP individuals, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 25,638 genes. ( b ) Frequencies of CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 and VP11/12 702–710 epitopes detected by FACS in SYMP vs. ASYMP individuals. ( c ) Heatmap is showing 772 differentially expressed genes among SYMP and ASYMP individuals. ( d ) Heatmap showing statistically significant pathways that are affected in HSV-specific CD8 + T cells from SYMP vs. SYMP individuals. Parametric Gene Set Enrichment Analysis (PSGEA) method was applied based on data curated in Gene Ontology and KEGG. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied. ( e ) Bulk RNA heatmap comparing differentially expressed CAM pathway associated T cell co-stimulatory and T cell exhaustion genes in HSV-specific CD8 + T cells from SYMP vs. SYMP individuals.

Article Snippet: Sections were washed with 1× PBS, permeabilized using 0.05% Triton X 100 in 1× PBS for 15 min, and blocked using 10% FBS in 1× PBS for 1 h. Sections were stained using anti-rabbit CD8 + , CXCR3, CXCL9, CXCL10, CXCL11 antibodies (1:200) overnight at 4 °C BD Pharmingen, Inc., San Diego, CA, USA).

Techniques: Expressing, Infection, RNA Sequencing Assay

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Single-cell RNA sequencing of trigeminal ganglia-resident CD45 + leukocytes from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Illustration of the experimental design and validation of differentially expressed genes in CD45 + leukocytes sorted on day 15 p.i. from the trigeminal ganglia (TG) of SYMP and ASYMP HLA Tg rabbits. ( b ) Heatmap expression of the most significant 140 differentially expressed genes among eight different clusters detected in TG-resident CD45 + leukocytes from HSV-1 infected SYMP and ASYMP HLA Tg rabbits (top two heatmap panels). Each cluster represents an individual immune cell population, determined on the basis of specific molecular markers: CD8 + T cells (CD8A), CD4 + T cells (CD4), NK cells (NKG7), B cells (CD19), macrophages (CD68), monocytes (CD14), granulocytes (FUT4) and dendritic cells (CD1c). The t-SNE dimensionality reduction, applied to single-cell RNA sequencing data revealed eight distinct clusters of immune cell populations among CD45 + leukocytes for the TG of HSV-1 infected ASYMP HLA Tg rabbits (middle panels). The total number of differentially expressed genes within each immune cell clusters (nCount) (lower panels). ( c ) Average frequencies of different immune cell populations detected within TG-resident CD45 + leukocytes of SYMP and ASYMP HLA Tg rabbits. ( d ) Volcano plot illustrates the total copy number reads observed for all the genes within one single cell (nFeature).

Article Snippet: Sections were washed with 1× PBS, permeabilized using 0.05% Triton X 100 in 1× PBS for 15 min, and blocked using 10% FBS in 1× PBS for 1 h. Sections were stained using anti-rabbit CD8 + , CXCR3, CXCL9, CXCL10, CXCL11 antibodies (1:200) overnight at 4 °C BD Pharmingen, Inc., San Diego, CA, USA).

Techniques: RNA Sequencing Assay, Infection, Expressing

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Differential gene expression in HSV-specific CD8 + T cells from trigeminal ganglia of HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Experimental design and validation of differentially expressed genes in CD8 + T cells sharing the same HSV-1 epitope-specificities, from SYMP and ASYMP HLA Tg rabbits. CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 , VP11/12 702–710, and gD 53–61 epitopes were sorted from TG of HLA-A*0201-positive SYMP and ASYMP HLA Tg rabbits, using specific tetramers. Total RNA was extracted from each clone of epitope-specific CD8 + T cells, and whole transcriptome analysis was performed using bulk RNA sequencing to determine the levels of expression of 23,669 rabbit genes (OryCun2.0 (GCA_000003625.1). ( b ) Frequencies of CD8 + T cells specific to HLA-A*0201-restricted HSV-1 gB 561–567 , VP11/12 702–710, and gD 53–61 epitopes detected by FACS in TG of HLA-Tg rabbits. ( c ) The heatmap is showing the most significant 2,879 differentially expressed genes among SYMP and ASYMP HLA Tg rabbits. Genes with minimum count per million (CPM) ≥ 0.5 were used for obtaining the transformed counts data for clustering using regularized log (rlog). ( d ) Bulk RNA heatmap shows the pathways that are different among ASYMP and SYMP HLA Tg rabbits. Genes differentially expressed in both single-cell RNA sequencing and bulk RNA sequencing were considered for pathway analyses. Parametric gene set enrichment analysis (PSGEA) method based on data curated in Gene Ontology and KEGG was applied. Pathway significance cut-off with a false discovery date (FDR) ≥ 0.2 was applied.

Article Snippet: Sections were washed with 1× PBS, permeabilized using 0.05% Triton X 100 in 1× PBS for 15 min, and blocked using 10% FBS in 1× PBS for 1 h. Sections were stained using anti-rabbit CD8 + , CXCR3, CXCL9, CXCL10, CXCL11 antibodies (1:200) overnight at 4 °C BD Pharmingen, Inc., San Diego, CA, USA).

Techniques: Expressing, Infection, RNA Sequencing Assay, Transformation Assay

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Activation and exhaustion genes differentially expressed in trigeminal ganglia-resident HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Expression of T cell activation genes ( CD69 , CD62L , CD44 , CD107 , and IFN-γ ) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of T cell exhaustion genes ( PD-1 , LAG-3 , CTLA4 , ICOS , and BLIMP1 ) detected by single-cell RNA sequencing from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). ( b ) Average frequencies of specific genes representing memory CD8 + T CM , CD8 + T EM , and CD8 + T RM cell subsets from TG of SYMP vs. ASYMP HLA Tg rabbits (left panel). Average frequencies of CD8 + T RM cells expressing various exhaustion genes in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (top right panel). Average frequencies of functional CD107 a/b+ IFN- γ + CD8 + T RM cells in HSV-1 infected TG of in SYMP vs. ASYMP HLA Tg rabbits (lower right panel). ( c ) Bulk RNA sequencing showing expression of T-cell activation (left panel) and T-cell exhaustion genes (right panel) in HSV-specific T RM cells from SYMP vs. ASYMP HLA Tg rabbits. ( d ) Frequencies of memory CD8 + T CM , CD8 + T EM , and CD8 + T RM cell subsets detected by FACS in HSV-1 infected TG of SYMP vs. ASYMP HLA Tg rabbits. ( e ) Fluorescence microscopy images showing infiltration of CD8 + T cells in HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits. TG sections were co-stained using DAPI and mAb specific to rabbit CD8 + T cells (magnification, × 20). Blue, DAPI: DNA, green: CD8 + T cells.

Article Snippet: Sections were washed with 1× PBS, permeabilized using 0.05% Triton X 100 in 1× PBS for 15 min, and blocked using 10% FBS in 1× PBS for 1 h. Sections were stained using anti-rabbit CD8 + , CXCR3, CXCL9, CXCL10, CXCL11 antibodies (1:200) overnight at 4 °C BD Pharmingen, Inc., San Diego, CA, USA).

Techniques: Activation Assay, Infection, Expressing, RNA Sequencing Assay, Functional Assay, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: Genes of cytokines/chemokines and receptors differentially expressed in trigeminal ganglia-resident HSV-specific CD8 + T cells from HSV-1 infected symptomatic vs. asymptomatic HLA Tg rabbits . ( a ) Expression of genes of chemokines and chemokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8 + T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (top panels). Expression of genes of cytokines and cytokine receptors detected by single-cell RNA sequencing from TG-resident HSV-specific CD8 + T cells from SYMP vs. ASYMP HLA Tg rabbits is represented through t-SNE plots (lower panels). ( b ) Average frequencies of CD8 + T RM cells expressing genes of cytokines/chemokines and receptors in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. ( c ) Bulk RNA sequencing showing expression of genes of chemokines/chemokine receptors (left panel) and genes of cytokines/cytokine receptors (right panel) in HSV-specific T RM cells from SYMP vs. ASYMP HLA Tg rabbits. ( d ) Representative (left panels) and average (right panels) frequencies of CCR3 + CD8 + T RM cells in TG of HSV-1 infected SYMP vs. ASYMP HLA Tg rabbits. ( e ) Fluorescence microscopy images showing infiltration of HSV-1 infected TG from SYMP vs. ASYMP HLA Tg rabbits by CD8 + T cells expressing T cell-attracting chemokines and receptors (i.e., CXCR3, CXCL9, CXCL10, and CXCL11) (magnification, × 20). Blue: DAPI (DNA stain); red: CXCR3 cells.

Article Snippet: Sections were washed with 1× PBS, permeabilized using 0.05% Triton X 100 in 1× PBS for 15 min, and blocked using 10% FBS in 1× PBS for 1 h. Sections were stained using anti-rabbit CD8 + , CXCR3, CXCL9, CXCL10, CXCL11 antibodies (1:200) overnight at 4 °C BD Pharmingen, Inc., San Diego, CA, USA).

Techniques: Infection, Expressing, RNA Sequencing Assay, Fluorescence, Microscopy, Staining

Journal: Scientific Reports

Article Title: Unique molecular signatures of antiviral memory CD8 + T cells associated with asymptomatic recurrent ocular herpes

doi: 10.1038/s41598-020-70673-z

Figure Lengend Snippet: TG-resident HSV-specific memory CD8 + T RM cells downregulate the T cell exhaustion associated pathway and confer protection from ocular herpes in HSV-1 infected asymptomatic humans and HLA transgenic rabbits. ( 1 ) Upon exposure to stressors, the HSV-1 enters into the cornea and travels through neurons to Trigeminal ganglia. ( 2 ) Following primary HSV-1 infection, the vast majority (up to 95%) of antiviral effector CD8 + T cells die, leaving behind only about 5% of CD8 + T cells destined to differentiate into a heterogeneous pool of memory CD8 + T cells. ( 3 ) The effector memory (T EM ) and tissue-resident memory (T RM ) CD8 + T-cell subsets are found mainly in the HSV-infected but "naturally protected" asymptomatic subjects, whereas the lymphoid organ-resident central memory (T CM ) CD8 + T cell subsets are mainly present in non-protected Symptomatic subjects. ( 4 ) Reduced viral reactivation was observed among asymptomatic subjects possessing a higher frequency of CD8 + T RM cells resulting in a less severe herpes disease. ( 5 ) The findings study suggests that by blocking immune checkpoints, there is a reduced expression of T cell exhaustion molecules ( PD-1 , LAG-3 , PSGL-1 , CTLA-4 , TIM3 , and TIGIT ) and T cell exhaustion associated Cell Adhesion Molecule pathway and increased retention of CD8 + T RM cell population in asymptomatic subjects. This memory CD8 + T cell population mediates recall responses and halts attempts of virus reactivation in the infected TG, thus accelerating viral clearance. More-so, reduced expression of T cell exhaustion pathway also gives rise to higher expression of genes associated with T cell function ( CD107 , IFN-γ ), T cell homing ( CXCR3 , CCR7 ), and T-cell keeping ( IL7R , IL15R ). This helps in reducing the ocular herpes infection and recurrent herpetic disease.

Article Snippet: Sections were washed with 1× PBS, permeabilized using 0.05% Triton X 100 in 1× PBS for 15 min, and blocked using 10% FBS in 1× PBS for 1 h. Sections were stained using anti-rabbit CD8 + , CXCR3, CXCL9, CXCL10, CXCL11 antibodies (1:200) overnight at 4 °C BD Pharmingen, Inc., San Diego, CA, USA).

Techniques: Infection, Transgenic Assay, Blocking Assay, Expressing, Cell Function Assay

Journal: Oncology Letters

Article Title: Prognostic significance of CD8 + tumor-infiltrating lymphocytes and CD66b + tumor-associated neutrophils in the invasive margins of stages I–III colorectal cancer

doi: 10.3892/ol.2022.13333

Figure Lengend Snippet: Analysis of CD8+ TILs and CD66b+ TANs in the invasive margin of CRC. (A) The ‘invasive margin’ is defined as the region centered on the border separating the host tissue from the malignant nets, with an extent of 1 mm (40×). (B) Representative images of high-density and low-density CD8+ TILs and CD66b+ TANs in the invasive margin of CRC (200×). (C) CD8+ TILs and CD66b+ TANs counts in the invasive margins of all patients (n=103). (D) Correlation between CD8+ TILs and CD66b+ TANs in CRC. Correlation analysis was performed with Spearman's rank correlation coefficient. TILs, tumor-infiltrating lymphocytes; TANs, tumor-associated neutrophils; CRC, colorectal cancer.

Article Snippet: The primary antibodies used were a monoclonal rabbit anti-human CD8 (clone: EP1150, dilution 1:1,000; GeneTex, San Antonio, TX, USA) and a monoclonal mouse anti-human CD66b (clone: G10F5, dilution 1:200; Biolegend, San Diego, CA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Prognostic significance of CD8 + tumor-infiltrating lymphocytes and CD66b + tumor-associated neutrophils in the invasive margins of stages I–III colorectal cancer

doi: 10.3892/ol.2022.13333

Figure Lengend Snippet: Association between the infiltration of immune cells and clinicopathological characteristics of patients with CRC.

Article Snippet: The primary antibodies used were a monoclonal rabbit anti-human CD8 (clone: EP1150, dilution 1:1,000; GeneTex, San Antonio, TX, USA) and a monoclonal mouse anti-human CD66b (clone: G10F5, dilution 1:200; Biolegend, San Diego, CA, USA).

Techniques: Mutagenesis

Journal: Oncology Letters

Article Title: Prognostic significance of CD8 + tumor-infiltrating lymphocytes and CD66b + tumor-associated neutrophils in the invasive margins of stages I–III colorectal cancer

doi: 10.3892/ol.2022.13333

Figure Lengend Snippet: Prognostic significance of CD8+ TILs and CD66b+ TANs in the invasive margin of stages I–III CRC. (A and B) Kaplan-Meier analysis of OS and DFS designed according to tumor infiltration of CD8+ TILs high/low infiltration. (C and D) Kaplan-Meier analysis of OS and DFS designed according to tumor infiltration of CD66b+ TANs high/low infiltration. Statistical analysis of the survival was performed using the log-rank test. TILs, tumor-infiltrating lymphocytes; TANs, tumor-associated neutrophils; CRC, colorectal cancer; OS, overall survival; DFS, disease-free survival.

Article Snippet: The primary antibodies used were a monoclonal rabbit anti-human CD8 (clone: EP1150, dilution 1:1,000; GeneTex, San Antonio, TX, USA) and a monoclonal mouse anti-human CD66b (clone: G10F5, dilution 1:200; Biolegend, San Diego, CA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Prognostic significance of CD8 + tumor-infiltrating lymphocytes and CD66b + tumor-associated neutrophils in the invasive margins of stages I–III colorectal cancer

doi: 10.3892/ol.2022.13333

Figure Lengend Snippet: Combined assessment of CD8+ TILs and CD66b+ TANs densities (Model 1) in the invasive margin. (A and B) Kaplan-Meier analysis of OS and DFS among subgroups identified by the combination of CD8+ TILs and CD66b+ TANs in patients with stage I–III CRC. (C and D) Kaplan-Meier analysis of OS and DFS among subgroups identified by the combination of CD8+ TILs and CD66b+ TANs in the invasive margin of stages II–III CRC. Statistical analysis of the survival was performed using the log-rank test. TILs, tumor-infiltrating lymphocytes; TANs, tumor-associated neutrophils; CRC, colorectal cancer; OS, overall survival; DFS, disease-free survival.

Article Snippet: The primary antibodies used were a monoclonal rabbit anti-human CD8 (clone: EP1150, dilution 1:1,000; GeneTex, San Antonio, TX, USA) and a monoclonal mouse anti-human CD66b (clone: G10F5, dilution 1:200; Biolegend, San Diego, CA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Prognostic significance of CD8 + tumor-infiltrating lymphocytes and CD66b + tumor-associated neutrophils in the invasive margins of stages I–III colorectal cancer

doi: 10.3892/ol.2022.13333

Figure Lengend Snippet: Multivariate analysis for OS of patients with stages I–III CRC.

Article Snippet: The primary antibodies used were a monoclonal rabbit anti-human CD8 (clone: EP1150, dilution 1:1,000; GeneTex, San Antonio, TX, USA) and a monoclonal mouse anti-human CD66b (clone: G10F5, dilution 1:200; Biolegend, San Diego, CA, USA).

Techniques:

Journal: Oncology Letters

Article Title: Prognostic significance of CD8 + tumor-infiltrating lymphocytes and CD66b + tumor-associated neutrophils in the invasive margins of stages I–III colorectal cancer

doi: 10.3892/ol.2022.13333

Figure Lengend Snippet: Multivariate analysis of DFS of patients with stage I–III CRC.

Article Snippet: The primary antibodies used were a monoclonal rabbit anti-human CD8 (clone: EP1150, dilution 1:1,000; GeneTex, San Antonio, TX, USA) and a monoclonal mouse anti-human CD66b (clone: G10F5, dilution 1:200; Biolegend, San Diego, CA, USA).

Techniques:

Journal: Materials Today Bio

Article Title: GTKO rabbit: A novel animal model for preclinical assessment of decellularized xenogeneic grafts via in situ implantation

doi: 10.1016/j.mtbio.2022.100505

Figure Lengend Snippet: Analysis of lymphocyte subsets in rabbit PBMCs. A) Proportions of B cells; B) CD4 + cells; C) CD8 + cells; and D) CD4+/CD8+. Experiments were performed in triplicate. WT, wild type; GTKO, α1,3-galactosyltransferase gene-knockout; Con, control; NBM, natural bone material; PCB, porcine cancellous bone.

Article Snippet: CD4 (Anti-Rabbit CD4 Purified, MRB4020; Antingenix America Inc., Melville, NY, USA), CD8 (Anti-Rabbit CD8 Purified, MRB8020, Antigenix America Inc.) purified antibodies coupled with goat anti-mouse fluorescent secondary antibody (Alexa Fluor®647, ab150115, Abcam), and B-PE (Anti-Rabbit B Cells-PE, MRB9997; Antigenix America Inc.) were used to identify lymphoid subsets in PBMCs.

Techniques: Gene Knockout, Control

Journal: Oncology Letters

Article Title: Inhibitory effects of human lactoferrin on U14 cervical carcinoma through upregulation of the immune response

doi: 10.3892/ol.2013.1776

Figure Lengend Snippet: Collected anticoagulated blood from each group was diluted to 1×10 6 cell/ml, labeled with anti-mouse monoclonal antibodies and then incubated for 30 min with rabbit anti-mouse FITC-IgG monoclonal antibodies to CD4 + and CD8 + . Stained cells were examined with an EPICS-XL FACSCalibur flow cytometer using Expo 32 ADC software. (A) Normal mice; (B) PBS-treated mice; (C) Ad-GFP-treated mice; (D) Ad-hLF-treated mice. (E) The percentages of CD4 + T cells in PBS-, Ad-GFP- and Ad-hLF-treated mice and normal mice were 12.39±2.53, 12.85±3.62, 27.38±2.12 and 32.25±1.87%, respectively. (F) The percentages of CD8 + T cells in PBS-, Ad-GFP- and Ad-hLF-treated mice and normal mice were 10.24±2.17, 9.25±3.95, 19.52±3.14 and 25.79±3.14%, respectively. Different letter superscripts between values indicate a significant difference (P<0.05) and same letter superscripts between values indicate no significant difference (P>0.05). PBS, phosphate-buffered saline; Ad-GFP, adenovirus carrying green fluorescent protein; Ad-hLF, adenovirus carrying human lactoferrin.

Article Snippet: To investigate the effect of Ad-hLF on peripheral blood T-lymphocyte subpopulations of tumor-bearing mice, the collected anticoagulated blood from the Ad-hLF-treated group was diluted to 1×10 6 cells/ml, labeled with anti-mouse monoclonal antibodies (4A Biotech Co., Ltd., Beijing, China) for 30 min and then incubated for 30 min at 4°C with rabbit anti-mouse FITC-IgG monoclonal antibodies to CD4 + and CD8 + (4A Biotech Co., Ltd.).

Techniques: Labeling, Bioprocessing, Incubation, Staining, Flow Cytometry, Software, Saline