Journal: mBio

Article Title: Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques

doi: 10.1128/mBio.01503-21

Figure Lengend Snippet: Experimental design and T cell depletions. (a) T cell subset-depleting antibodies were administered on days −7, 0, and +4, as indicated by blue arrows. Infections were done on days 0 and 42, as indicated by red arrows. Blood withdrawals were performed on the days indicated by the black arrows, and flow cytometry was used to determine the lymphocyte subset numbers over time. The flow cytometry gating strategies are shown in Fig. S1b . Each symbol represents a single animal throughout. All CD4-depleted animals except CD4-5 were still more than 90% depleted of CD4 + T cells at 7 dpi. CD4-5 was 78% depleted. CD4 + Th numbers excluded FoxP3 + cells. At 7 days after reinfection (49 dpi), the animals averaged 81% depletion. All CD8-depleted animals were >99% depleted at 7 dpi and remained 78% depleted at 49 dpi. The differences between subset numbers at 0 dpi and 7 dpi were calculated by a two-way paired t test. ns, not significant. P values are shown. Numbers of B cells (d, g, j, and m) were determined by flow cytometry using CD45 and CD20 as markers. The numbers of B cells in the CD4-depleted group were significantly lower over time than those in the controls, as determined by mixed-effects analysis ( P = 0.0118).

Article Snippet: Specific staining was detected using SARS-CoV/SARS-CoV-2 nucleocapsid antibody (Sino Biological cat#40143-MM05) at a 1:1,000 dilution, CD4 antibody (Abcam catalog no. ab133616) at a 1:100 dilution, CD8 antibody (Sino Biological catalog no. 10980-T24) at a 1:500 dilution, and CD20 (Thermo Scientific catalog no. RB-9013) at a 1:250 dilution.

Techniques: Flow Cytometry

Journal: mBio

Article Title: Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques

doi: 10.1128/mBio.01503-21

Figure Lengend Snippet: Virus detection from nasal swabs and Broncho-alveolar lavages. (a to d) Each symbol represents the value of viral RNA copies from an individual animal at each time point. The brackets delineate comparisons of cumulative values from the first 2 weeks after infection with those from the 2 weeks after reinfection, and numbers P values from two-way paired t tests showing significantly reduced virus levels following the second infection (b to d) except in the control group (a), for which the difference was marginally nonsignificant. The cumulative RNA titers from the CD4- and CD8-depleted groups for the first 2 weeks after initial infection were not significantly different from those from the controls, but the CD4/CD8-depleted group had significantly higher titers ( P = 0.0362 by one-way analysis of variance (ANOVA) with a Dunnett’s posttest). (e to h) Mean values comparing the total viral RNA data from panels a to d (black lines) with sgRNA results (blue lines). (i to l) Bronchoalveolar lavage fluids were taken at 1 day after infection and reinfection. sgRNA was measured from each animal and showed significantly reduced virus replication upon second infection ( P values from paired t tests are shown).

Article Snippet: Specific staining was detected using SARS-CoV/SARS-CoV-2 nucleocapsid antibody (Sino Biological cat#40143-MM05) at a 1:1,000 dilution, CD4 antibody (Abcam catalog no. ab133616) at a 1:100 dilution, CD8 antibody (Sino Biological catalog no. 10980-T24) at a 1:500 dilution, and CD20 (Thermo Scientific catalog no. RB-9013) at a 1:250 dilution.

Techniques: Infection

Journal: mBio

Article Title: Recovery from Acute SARS-CoV-2 Infection and Development of Anamnestic Immune Responses in T Cell-Depleted Rhesus Macaques

doi: 10.1128/mBio.01503-21

Figure Lengend Snippet: Immunohistochemical staining of cervical lymph nodes for CD4 + and CD8 + T cells and B cells. Representative animals from each experimental group are shown. (a to d) Cervical lymph nodes stained with anti-CD4 antibodies. (e to h) Cervical lymph nodes stained with anti-CD8 antibodies. (i to l) Cervical lymph nodes stained with anti-CD20 antibodies to detect B cells. None of the tissues stained positive for the presence of SARS-CoV-2 at 56 dpi.

Article Snippet: Specific staining was detected using SARS-CoV/SARS-CoV-2 nucleocapsid antibody (Sino Biological cat#40143-MM05) at a 1:1,000 dilution, CD4 antibody (Abcam catalog no. ab133616) at a 1:100 dilution, CD8 antibody (Sino Biological catalog no. 10980-T24) at a 1:500 dilution, and CD20 (Thermo Scientific catalog no. RB-9013) at a 1:250 dilution.

Techniques: Immunohistochemical staining, Staining

Journal: Oncogene

Article Title: Tissue factor overexpression promotes resistance to KRAS-G12C inhibition in non-small cell lung cancer

doi: 10.1038/s41388-023-02924-y

Figure Lengend Snippet: A TCGA datasets were analyzed for TF and KRAS mRNA expression. B OS and PFS of TF high and TF low groups were calculated by Kaplan-Meier method. C The infiltration levels of immune cells in TF high vs TF low groups in TCGA cohort. D KRASmut lung cancer tissue microarrays were subjected to IHC staining. TF expression was analyzed versus the indicated clinical parameters. E OS and PFS in negative/low ( n = 36) and moderate/high ( n = 42) TF expression groups were calculated by Kaplan-Meier method. F IHC was performed for the indicated biomarkers, quantified as described in methods and plotted as expression level heatmap in KRASmut LUAD patients of stage III and IV. G The levels of P-ERK, P-AKT and immune cell markers in TF high/low KRASmut LUAD patients of stage III and IV ( n = 29). H Comparing the correlation between P-ERK, P-AKT, CD206, CD8A + GZMB + and TF expression in KRASmut LUAD patients of stage III and IV. I IHC profile of representative TF-high/low tumors are shown. Loss of tissue during staining was not included in the analysis. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The primary antibodies are as follows: TF (E9M6T, CST), P-ERK1/2 (4370, CST), P-AKT (Ser473) (ab81283, Abcam), CD68 (ab955, Abcam), (24595 S, CST), CD8A (50389-T26, Sino Biological) and GZMB (255598, Abcam).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement

doi: 10.1016/j.apsb.2021.10.028

Figure Lengend Snippet: Mechanism of action of TF-TCB. (A) Cross-linking of CD3 + Jurkat cells and TF + AsPC-1 cells by TF-TCB measured through flow cytometry. Jurkat cells were labeled with PKH26 (PE) and AsPC-1 cells were labeled with CFSE (FITC), the two cells were mixed at equal ratio in the presence of TF TCB (or TF-011) and incubated for 30 min. (B) and (C) T cell activation and cytokine release mediated by TF-TCB. T cell activation (B) was assessed by measuring percentage of CD69 + cells within CD4 + and CD8 + T cells after 20 h of incubation of human peripheral blood mononuclear cells (PBMC) with AsPC-1 cells (E:T, 10:1) and TF-TCB. IFN γ and IL-2 released into culture supernatant were also determined (C). TF-011 was used as control. (D) Percentage of tumor cell lysis detected after incubation of AsPC-1 (36 h), MDA-MB-231 (24 h) and SKOV-3 cells (36 h) with PBMC and TF-TCB. TF-011 and CD22-TCB were used as controls. (E) T cell proliferation evaluated through the decrease in CFSE labeling on T cells. Purified T cells were labeled with CFSE (FITC) and incubated with or without AsPC-1 cells, in the presence of TF-TCB or not for 96 h, and the CFSE labeling on T cells was evaluated by flow cytometry. Data are mean ± SD, n = 3.

Article Snippet: Effector cells were washed 1 time using FACS buffer and stained with FITC-labeled anti-CD8 (Sino Biological), PE-labeled anti-CD4 (Sino Biological) and APC-labeled anti-CD69 antibodies (BD Biosciences, San Jose, CA, USA) under manufacturer's instructions.

Techniques: Flow Cytometry, Labeling, Incubation, Activation Assay, Lysis, Purification

Journal:

Article Title: Distinct Dacryoadenitides Autoadoptively Transferred to Rabbits by Different Subpopulations of Lymphocytes Activated Ex vivo

doi: 10.1097/ICO.0b013e3181d0090e

Figure Lengend Snippet: Rabbit Specific Primer and Probe Sequences for Real Time RT-PCR

Article Snippet: Cell surface staining was done with PBLs before and after mixed cell reaction with mAB specific for rabbit T cell, B cell, CD4 and CD8 (Antigenix America) antibodies and analyzed by FACS.

Techniques:

Journal:

Article Title: Distinct Dacryoadenitides Autoadoptively Transferred to Rabbits by Different Subpopulations of Lymphocytes Activated Ex vivo

doi: 10.1097/ICO.0b013e3181d0090e

Figure Lengend Snippet: (A) Mononuclear cells were stained with an anti-T cell PE- labeled antibody before (unstimulated) and after (stimulated) culture in an AMCR. (B) Same as for (A) except the cells were stained with an anti-CD4 PE-labeled antibody. (C) Same as for (A) except the cells were stained with an anti-CD8 FITC-labeled antibody.

Article Snippet: Cell surface staining was done with PBLs before and after mixed cell reaction with mAB specific for rabbit T cell, B cell, CD4 and CD8 (Antigenix America) antibodies and analyzed by FACS.

Techniques: Staining, Labeling

Journal:

Article Title: Distinct Dacryoadenitides Autoadoptively Transferred to Rabbits by Different Subpopulations of Lymphocytes Activated Ex vivo

doi: 10.1097/ICO.0b013e3181d0090e

Figure Lengend Snippet: Immunohistochemical analysis of inflammatory cells in lacrimal glands.

Article Snippet: Cell surface staining was done with PBLs before and after mixed cell reaction with mAB specific for rabbit T cell, B cell, CD4 and CD8 (Antigenix America) antibodies and analyzed by FACS.

Techniques: Immunohistochemical 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: 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