Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: NK cells are a main source of XCL1 but not a critical one upon MCMV infection (A and B) Analysis of mTFP1 expression in splenocytes of Wt (dark grey) and Xcl1-mTfp1 fl/fl (white) mice at steady state (A) or 40 h after infection (B). Cells were gated as follow: NK cells (TCRβ - CD19 - NK1.1 + ), ILC1 (NK1.1 + TCRβ - CD19 - CD127 + ), NKT cells (CD19 - CD1d + ), CD8 + T (CD1d - NK1.1 - TCRβ + CD19 - CD8 + ), CD4 + T (CD1d - NK1.1 - TCRβ + CD19 - CD4 + ), γδ T cells (CD1d - NK1.1 - CD3ε + TCRβ - TCRγδ + ) and B cells (NK1.1 - TCRβ - CD19 + ). One representative experiment of 4 independent ones with at least three mice per group is shown. (C and D) Proportion of immune populations within mTFP1-positive cells at steady state (C) and 40 h after MCMV infection (D). Others: sum of all the other cell subsets not detailed in the pie charts. One representative experiment of 4 independent ones with at least three mice per group is shown. (E) IFN-γ production by NK cells in Wt, Xcr1 −/− , Xcl1 fl/fl , and Nkp46 Cre ;Xcl1 fl/fl mice 40 h after infection. One representative experiment of two independent ones with at least four mice per group is shown. A one-way ANOVA statistical analysis with a Tukey's multiple comparison test was applied. ∗, p < 0.05; ∗∗, p < 0.01, n.s., nonsignificant; ANOVA, analysis of variance. See also Figure S3 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Infection, Expressing, Comparison

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: CCR7-mediated cDC1 relocalization into the T cell zone promotes MCMV-specific CD8+ T cell responses and host resistance to MCMV infection (A) Analysis of m45-specific CD8 + T cell response in Xcr1 −/− mice and littermate controls 6 days after MCMV infection. One representative experiment of two independent ones with at least 4 mice per group is shown. ∗, p < 0.05. (B) Analysis of m45-specific CD8 + T cell response in Wt : Xcr1 - Dta, Wt : Ccr7 −/− and Xcr1 - Dta : Ccr7 −/− BM chimera mice 6 days after MCMV infection (10 4 PFU). Two independent experiments with at least 4 mice per infected group were pooled. A one-way ANOVA statistical analysis with a Tukey's multiple comparison test was applied. ∗∗, p < 0.01; ∗, p < 0.05, n.s., nonsignificant. Statistical analysis between all other groups is nonsignificant. (C) MCMV titers in spleens and livers of Wt and Xcr1 −/− mice 5 days after MCMV infection (10 4 PFU). Two independent experiments with at least 3 mice per infected group were pooled. NI, noninfected. ∗∗∗, p < 0.001. See also Figure S6 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Infection, Comparison

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet: Dermal cDC1 make cell/cell contacts with Xcl1 -expressing DETCs (A) Analysis of the lymphocyte heterogeneity within mTFP1 + cells in the skin of Xcl1 mTfp1 fl/fl mice at steady state. One representative experiment of 2 independent ones with at least three mice per group is shown. (B) DETC staining in the skin at steady state. DETCs are CD3ε + TCRγδ + lymphocytes localized in the epidermis. The dotted line delineates the basal layer separating the dermis (D) from the epidermis (EP). C, cartilage. Scale bar: 50 μm (C–E) Analysis of cDC1 localization and contacts with DETCs in the skin, at steady state in Karma Cre ;Rosa26 tdRFP ;Xcr1 +/− mice (C), and at 40 h after MCMV infection in Karma Cre ;Rosa26 tdRFP ;Xcr1 +/− (D) and Karma Cre ;Rosa26 tdRFP ;Xcr1 −/− (E) mice. The dotted line delineates the basal layer separating the dermis from the epidermis. Skin sections (scale bar: 50 μm) were stained for tdRFP (red; cDC1), CD3ε (cyan), TCRγδ (yellow), IE-1 (purple), and Avidin (green; mast cell). EP, epidermis; D, dermis; C, cartilage. (F) 3D visualization of DETC/cDC1 contacts identified in (D). (G) Kinetics of cDC1/DETC contacts occurring in the epidermis and through the epidermis-dermis border per mm of skin length, during MCMV infection. Data are represented as mean (+/− SEM). p.i., postinfection. (H) Quantification of cDC1 nuclear bodies inside the epidermis per mm of skin length 40 h after skin infection. For (G-H), two independent experiments with at least 4 mice per infected group were pooled. n.s., nonsignificant; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (I) Proportion of CD103 + migcDCs in the ear-draining LN of Xcr1 −/− and littermate controls 48 h after skin infection. (J) Analysis of CCR7 expression on migcDCs in ear-draining LN 48 h after skin infection of Xcr1 −/− and Wt mice. Two independent experiments with at least 3 mice per infected group were pooled. ∗∗, p < 0.01. (K and L) Analysis of m45-specific CD8 + T cell response in Xcl1 −/− mice (K), Xcr1 −/− mice (L) and their respective controls 6 days after MCMV infection of the ear. CD8 + T cells from ear-draining LN were stimulated in vitro for 4 h with m45 peptide. Two independent experiments with at least 3 mice per infected group were pooled for (K), and one experiment with at least 4 mice per group is shown for (L). ∗, p < 0.05∗∗, p < 0.01. See also Figure S7 .

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Expressing, Staining, Infection, Avidin-Biotin Assay, In Vitro

Journal: iScience

Article Title: Natural killer cells and dendritic epidermal γδ T cells orchestrate type 1 conventional DC spatiotemporal repositioning toward CD8 + T cells

doi: 10.1016/j.isci.2021.103059

Figure Lengend Snippet:

Article Snippet: CD8 + Dendritic Cell Isolation Kit, mouse , Miltenyi Biotec , #130-091-169.

Techniques: Purification, Avidin-Biotin Assay, Virus, Recombinant, High Molecular Weight, Cell Isolation, Microarray, Software

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Regnase-1 downregulation promotes pancreatic cancer through myeloid-derived suppressor cell-mediated evasion of anticancer immunity.

doi: 10.1186/s13046-023-02831-w

Figure Lengend Snippet: Fig. 6 Suppression of CTLs by CD11b+ MDSCs is responsible for the acceleration of tumor progression by Regnase-1 downregulation. A-D Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1 KO murine pancreatic cancer cells. Representative macro images of pancreatic tumors (A). Relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (B) (N = 6 per group). Representative images of HE (C, left panel) and CD8a immunostaining (C, right panel) and the number of CD8-positive cells (C, right) (N = 6 per group). Dot plots of CD3+CD8+ cells evaluated by flow cytometry (D, left) and the proportion of CD8 + cells among CD45+ cells (D, right) (N = 3 per group). E–H Evaluation of phenotypes of orthotopic syngeneic tumors of WT or Regnase-1-KO murine pancreatic cancer cells with or without depletion of CD8+ cells upon anti-CD8a antibody or IgG treatment. Experimental schematic (E). Dot plots of CD3+ and CD8.+ cells in WT or Regnase-1-KO syngeneic tumors upon anti-CD8a antibody or IgG treatment evaluated by flow cytometry (F). Tumor weights (G) (N = 6 per group). The relative mRNA levels of Cd8a, Ifng, Fasl, and Gzmb (H) (N = 6 per group). Student’s t test was used to evaluate differences between 2 groups. One-way ANOVA with Tukey’s post hoc test was used to compare differences among 4 groups. *P < 0.05, scale bars: 100 μm (insets)

Article Snippet: BE0061, a fully neutralizing mAb recognizing CD8a, and control IgG were obtained from Bioxcell.

Techniques: Immunostaining, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Tumor-intrinsic RRBP1 inhibition triggers antitumor immunity. ( A ) Representative images of IHC staining for RRBP1 and CD8 + T cells in BC samples. ( B ) The correlation between RRBP1 expression and CD8 + T-cell infiltration was analyzed based on 96 patients from in-house BC cohort. Scale bar: 50 µm. ( C ) Representative images of IHC staining for RRBP1 expression in PD, SD, PR, and CR samples. Scale bar: 50 µm. ( D ) Bar plot showed the response rates of anti-PD-L1 therapy. Blue bars represent CR/PR, Red bars represent PD/SD. ( E ) Volcano plot of RNA-seq data for shNC or shRRBP1 tumors (n=3). Differentially expressed genes were identified with the threshold of |log2 (fold change) | >1 and FDR<0.05. ( F ) GSEA for DEGs showed the activation of immune-associated pathways in shRRBP1 tumors in the RNA-seq data. ( G ) Representative images of IHC and mIHC staining for RRBP1 and CD8 + T cells in shNC, shRRBP1, control or radezolid tumor tissues. Expression levels of the indicated proteins were displayed. Scale bar: 20 µm. ( H, I ) Flow cytometry showed the percentages of CD8 + T cells in CD3 + cells in shNC, shRRBP1, control or radezolid tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using Spearman correlation analysis ( B ), unpaired two-tailed t-test ( I ). ****p<0.0001. BC, bladder cancer; CR, complete response; FDR, false discovery rate; progressive disease; PR, partial response; PD-L1, programmed death-ligand 1; RNA-seq, RNA sequencing; RRB1, ribosomal-binding protein 1; SD, stable disease; IHC, immunohistochemistry; GSEA, gene set enrichment analysis; DEGs, differentially expressed genes; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Immunohistochemistry, Expressing, RNA Sequencing, Activation Assay, Staining, Control, Flow Cytometry, Two Tailed Test, Binding Assay, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: Single-cell RNA sequencing reveals the difference of CD8 + T-cell subgroup. The UMAP plot of CD8 + T cells subpopulation, color-coded by cell cluster and cell type. ( A ) The expression of markers in each CD8 + T cells subpopulation. ( B ) Bar plot showed the proportion of CD8 + T cells subpopulation in the shNC and shRRBP1 groups. ( C ) The percentage of each CD8 + T-cell clusters in shNC and shRRBP1 groups. ( D ) Heatmap showed the differentially activated pathway among all the CD8 + T-cell clusters. ( E ) The differentially expressed genes in CD8 + T cells between shNC and shRRBP1 groups. ( F ) KEGG analysis for differentially expressed genes showed the enrichment of immune-associated pathways. ( G, H ) mIHC and flow cytometric analysis displayed the tumor-infiltrating IFN-γ + or GZMB + CD8 + T cells in shNC or shRRBP1 tumor tissues. Scale bar: 20 µm. ( I–K ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Isotype control (IgG) or anti-mouse CD8 antibody administered on days –6, –3, and –1 before tumor challenge, with the same dose repeated on days 7, 9 and 11 after tumor challenge. Tumor sizes ( I ), volumes ( J ), and weight ( K ) were measured. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( H, K ) and two-way ANOVA with Tukey’s multiple comparison test ( J ). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; GZMB, Granzyme B; IFN, interferon; TEX, exhausted T cells; UMAP, Uniform Manifold Approximation and Projection; mIHC, multiplex immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Single Cell, RNA Sequencing, Expressing, Injection, Control, Two Tailed Test, Comparison, Multiplex Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition promotes antitumor immunity via the CXCL10-CXCR3 axis in BC. ( A ) ScRNA-seq data showed the CXCR3 expression of CD8+T cells in shNC and shRRBP1 groups. ( B ) The correlation between CXCR3 expression and CXCL10 expression or activated CD8 + T cell based on 571 patients from TCGA-BLCA cohort and GSE13507 cohorts. ( C ) MB49 cells were co-cultured with CD8 + T cells, and tumor cells were stained with crystal violet. ( D ) Evaluation of the effect of genetic inhibition of RRBP1 on the cytotoxicity of CD8 + T cells in vitro conditioned culture model. ( E ) Schematic diagram of in vitro CD8 + T-cell migration assays. ( F ) The number of CD8 + T cells passing through the membrane of a Transwell system was analyzed by flow cytometry. ( G–I ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice received intraperitoneal injection of either vehicle or anti-CXCL10 when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( G ), volumes ( H ), and weights ( I ) were measured. ( J ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( K ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( D, F, I, K ) and two-way ANOVA with Tukey’s multiple comparison test ( H ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; RRBP1, ribosomal-binding protein 1; scRNA-seq, single-cell RNA sequencing; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry; BLCA, bladder urothelial carcinoma.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Cell Culture, Staining, In Vitro, Migration, Membrane, Flow Cytometry, Injection, Two Tailed Test, Comparison, Binding Assay, Single Cell, RNA Sequencing, Immunohistochemistry, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: RRBP1 inhibition enhances response to anti-PD-L1 therapy in BC. ( A–D ) The protein expression of surface PD-L1 was analyzed in BC cells or tumor tissues by flow cytometry after RRBP1 inhibition and was shown as the mean fluorescence intensity. ( E–G ) C57BL/6 mice were subcutaneously injected with 5×10 5 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice were received intraperitoneal injection of either vehicle or anti-PD-L1 antibody when the tumor volume reached a calculated average of 100 mm 3 . The tumor sizes ( E ), volumes ( F ), and weights ( G ) were measured. ( H ) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. ( I ) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, CXCR3 + CD8 + T cells, IFN-γ + CD8 + T cells or GZMB + CD8 + T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test ( B, D, G, I ) and two-way ANOVA with Tukey’s multiple comparison test ( F ). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; PD-L1, programmed death-ligand 1; RRBP1, ribosomal-binding protein 1; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Injection, Staining, Two Tailed Test, Comparison, Binding Assay, Immunohistochemistry, Multiplex Assay

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 3 Lipo-MP-LPS induces immunological changes in both primary tumours and lung metastases. a, c Representative immunohis- tochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-CD8a antibodies. Scale bar = 50 μm. b, d Quantification of CD8a positive cells in xenograft tumours and lung metastases. Data are represented as means ± SD; *p < 0.05, using Mann–Whitney U test. e, g Representative immunohistochemistry images of xenograft tumours and lung metastases stained with mouse specific anti-F4/80 antibodies. Scale bar = 50 μm. f, h Quantification of F4/80 positive cells in xenograft tumours and lung tissues. Data are represented as means ± SD; n = 6 mice per group, *p < 0.05, using Mann–Whitney U test. (i) iNOS, MHC II, TNF-α, CD206, and IL10 gene expression in xenograft tumours and lung metastases determined by RT-qPCR. Data are presented as means ± SD; n = 6 mice per group, *p < 0.05, **p < 0.01, using two-tailed t-test. NS not significant.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Staining, MANN-WHITNEY, Immunohistochemistry, Gene Expression, Quantitative RT-PCR, Two Tailed Test

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 4 The tumour suppressive effect of Lipo-MP-LPS depends on CD8+ T cells. a Representative images of IHC staining for CD8a positive cells of xenograft tumours from mice treated with anti-CD8a antibody or IgG2b isotype. Scale bar = 100 μm. b CD8a expression in spleen tissues determined by RT-qPCR. Data are represented as means ± SD; n = 4. ***p < 0.001, using two-tailed t-test. c Tumour growth curve in C3H/HeN mice treated with empty liposome or Lipo-MP-LPS in addition to injection with anti-CD8a or isotype IgG2b antibodies (n = 6 mice per group). Data are shown as mean tumour volume ± SD. *p < 0.05, using Mann–Whitney U test. d Kaplan–Meier survival curves. p < 0.05 was considered significant using the log-rank test. Representative images of H&E staining of xenograft tumours (e) and lungs (g) for each group. Scale bar = 200 μm. f Quantification of necrotic areas in tumour tissues. h Quantification of lung metastasis areas. Data are presented as means ± SD; *p < 0.05, **p < 0.01; NS not significant, using Mann–Whitney U test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques: Immunohistochemistry, Expressing, Quantitative RT-PCR, Two Tailed Test, Injection, MANN-WHITNEY, Staining

Journal: BJC reports

Article Title: A detoxified TLR4 agonist inhibits tumour growth and lung metastasis of osteosarcoma by promoting CD8+ cytotoxic lymphocyte infiltration.

doi: 10.1038/s44276-024-00120-3

Figure Lengend Snippet: Fig. 6 Survival analysis of patients with osteosarcoma according to immune cell infiltration levels estimated by consensus TME. Of the 84 patients with osteosarcoma, the top third (28 patients) and bottom third (28 patients) were classified into high and low score groups, respectively. Kaplan–Meier curves of (a) CD8+ T cells (b) macrophage, (c) M1 macrophage, and (d) M2 macrophage infiltration for OS (left) and PFS (right) are presented. p < 0.05 was considered significant using the log-rank test.

Article Snippet: In vivo depletion of CD8+ T cells Mice were depleted of CD8+ T cells by the intraperitoneal administration of an anti-CD8a antibody (YTS169.4, rat IgG2b; BioXCell, West Lebanon, NH, USA).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: IST delivers both the TCR and CD28 co-stimulatory signals required for expansion and differentiation of naïve MART-1–specific CD8 + T cells. (A) Schematic of the MART-1–specific IST proteins. MART-1-ISTs are constructed as c-pMHC-Fc fusion proteins that deliver an HLA-A*0201 restricted antigenic peptide (MART-1) alongside an anti-CD28 (αCD28) or alone. (B) Representative methods and timeline. Flow cytometric analysis was performed to identify live, CD8 + , CD3 + , MART-1-tetramer + single cells, which were characterized as T EM (CD45RO + CD62L − ), T CM (CD45RO + CD62L + ), orT N (Naive T cell, CD45RO − TCD62L + ). (C) Representative flow cytometry plots on day 17 following stimulation with no IST or indicated IST (1 nM) (gated on live cells, single cells, CD8 + ). (D) Selective expansion kinetics of MART-1–specific CD8 + T cells determined as percentage of CD8 + population for 1 nM treated conditions and no IST controls (E) Representative flow cytometry plots displaying memory markers for CD3 + MART-1–tetramer + cells on day 17 following stimulation with no IST or indicated IST (1 nM). Data are from independent treatment replicates ( n = 3) for all IST-treated samples and n = 2 for no IST samples from one experiment representative of multiple independent experiments. Line and bars depict mean ± SEM. Two-way AN OVA was performed, and significant P values are displayed for comparison to no IST control. ** P < 0.01.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Construct, Flow Cytometry, Comparison, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: Delivery of the TCR signal alone by IST induces selective CMV (NLV)–specific memory CD8 + T-cell activation and expansion comparable to combined TCR and co-stimulatory signaling. (A) Schematic of the NLV-specific IST structure. NLV-ISTs are constructed as c-pMHC-Fc fusion proteins that deliver an HLA-A*0201 restricted antigenic peptide (NLV from CMV or SL9 from HIV) alongside an anti-CD28 (αCD28), a 4-1BBL agonistic domain, or a nonstimulatory FLAG-tag domain. (B) Experimental protocol and timeline. All IST treatments used a concentration of 0.01 nM. (C) Representative flow cytometry plots of NLV-tetramer + and tetramer − populations on day 7 following treatment from a single donor. Data are representative of 4 independent experiments each using unique donor PBMCs. Gated on single, 7-AAD − , CD8 + cells. (D) Growth of CD8 + NLV-tetramer + or CD8 + NLV-tetramer − cells were calculated by the fold change of cell count on days 1, 5, 7, and 14 following treatment in the presence of IL-2 (100 U/mL) relative to the cell count of the day 1 no IST treatment condition. Growth kinetics (left) and day 7 growth (right). (E) Growth of CD8 + NLV-tetramer − cells as in (D). (F) Memory phenotype was determined for CD8 + NLV-tetramer + cells on day 14 following treatment in the presence of 100 U/mL IL-2. Effector memory (T EM ) denotes CD45RO + CD62L − , central memory (T CM ) denotes CD45RO + CD62L + , stem cell-like memory (T SCM ) denotes CD45RO − CD62L + , and effector (T EMRA ) denotes CD45RO − CD62L − Expression of markers (G) 4-1BB, (H) LAG-3, and (I) CD38 were evaluated on days 1, 5, 7, and 14 following treatments in the presence of IL-2 (100 U/mL), and the percentage of CD8 + NLV-tetramer + T cells that expressed each marker was determined by flow cytometry. Lines and bars depict mean ± SEM of independent experiments each using unique donor PBMCs with n = 5 donors up to day 5 and n = 4 donors up to day 14 with each shape denoting an independent donor. Repeated measurement one-way ANOVA with Tukey multiple comparisons test was performed on all samples for log 10 -transformed fold changes and % positive marker expression. All P values <0.1 are depicted. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Activation Assay, Construct, FLAG-tag, Concentration Assay, Flow Cytometry, Cell Characterization, Expressing, Marker, Transformation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: Delivery of the TCR signal alone by IST also induces equivalent selective activation and expansion of the HIV SL9-specific memory CD8 + T cells as combined TCR and co-stimulatory signaling. (A) Experimental protocol and timeline. All IST treatments were at a concentration of 0.01 nM. (B) Growth of CD8 + NLV-tetramer + T cells or were calculated by the fold change of cell count on days 1, 5, 7, and 14 following treatment in the presence of IL-2 (100 U/mL) compared to the cell count of the cognate tetramer + day 1 no IST treatment condition. Flow cytometry gating on single, 7-AAD − , CD8 + , NLV-tetramer + cells. (C) Growth of SL9-tetramer + as in (B). Growth kinetics (left) and day 7 growth (right). (D) Growth of CD8 + tetramer − cells as in (B). (E) Memory phenotype was determined for CD8 + SL9-tetramer + cells on day 14 following treatment in the presence of 100 U/mL. Effector memory (T EM ) denotes CD45RO + CD62L − , central memory (T CM ) denotes CD45RO + CD62L + , stem cell-like memory (T SCM ) denotes CD45RO − CD62L + , and effector (T EMRA ) denotes CD45RO − CD62L − . (F) Representative histograms for NLV-specific CD8 + T cells on day 4 following NLV-IST treatment. The color histograms represent treated NLV-tetramer+ cells and black outline represents untreated NLV-tetramer+ cells. (G) Representative histograms for SL9-specific CD8+ T cells on day 4 following SL9-IST treatment. Color histogram represents treated SL9-tetramer+ cells and black outline represents untreated SL9-tetramer+ cells. Lines and bars depict mean ± SEM of independent experiments each using unique donor PBMCs ( n = 3 donors). Each shape denotes an independent donor. Histograms are representative of 2 independent experiments using a single donor. Repeated measurements one-way ANOVA with Tukey multiple comparisons test was performed on all samples. All P values <0.1 are depicted. ** P < 0.01, *** P < 0.001.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Activation Assay, Concentration Assay, Cell Characterization, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: Combined TCR and co-stimulatory stimulation of resting memory NLV-specific CD8 + T cells induce a different transcriptome compared to TCR stimulation alone. (A) Experimental protocol and timeline. (B) Hierarchical clustering and heat map for relative expression of all DEGs identified between IST treatments and no IST control. DEGs were classified as genes with adjusted P ≤.05 and log 2 fold change ≥1.5 as determined using limma. Each column represents a technical replicate that was treated and processed independently using PBMCs from a single donor, pooled from 2 sequencing runs ( n = 3). (C) Venn diagram of DEGs identified for the comparison of each IST treatment against the no IST control. (D) Top 15 IPA downstream functions predicted for the 196 DEGs shared between all IST treatments, (E) 176 shared between only NLV-αCD28 and NLV-4-1BBL, and (F) 507 unique to NLV-αCD28 alone from the comparisons between each IST treatment and the no IST treatment control. Predictions are ordered from lowest P value on top to highest on bottom with z score plotted as identified using Fisher exact test. All functions have P < 0.05.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Expressing, Control, Sequencing, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: Gene expression by TCR-activated memory NLV-specific CD8 + T cells is modified by TCR restimulation and is further modulated by combined TCR and 4-1BBL signaling. (A) Experimental protocol and timeline. (B) Hierarchical clustering and heat map for relative expression of DEGs identified between NLV-4-1BBL retreatment (NLV-FLAG > NLV-4-1BBL) and NLV-FLAG retreatment (NLV-FLAG > NLV-FLAG). DEGs were classified as genes with adjusted P ≤ .05 and log 2 fold change ≥1 as determined using limma. Each column represents an independent treatments replicate ( n = 2) using PBMCs from a single donor. (C) Venn diagram of shared and unique DEGs identified for NLV-4-1BBL retreatment (NLV-FLAG > NLV-4-1BBL) and NLV-FLAG retreatment (NLV-FLAG > NLV-FLAG) in comparison to a single NLV-FLAG treatment (NLV-FLAG > no retreatment). DEGs identified for the comparison of each IST second treatment against the no second treatment control. (D) Top 30 IPA downstream functions predicted for the DEGs unique to NLV-4-1BBL retreatment from the Venn diagram. Predictions are ordered from lowest P value on top to highest P value on bottom with z score plotted as identified using Fisher exact test. All functions have P < 0.05.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Gene Expression, Modification, Expressing, Comparison, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: TCR-mediated activation and expansion of CMV-specific memory CD8 + T cells occur independently of CD28 or 4-1BB co-stimulation requiring only low levels of IL-2. (A) Experimental protocol and timeline. (B) Representative flow cytometry plots on day 7 following treatment. Data are representative of 4 independent experiments each using unique donor PBMCs. Gated on single, 7-AAD − , CD8 + cells. (C) Growth of CD8 + NLV-tetramer + was calculated by the fold change of cell count on days 1, 5, 7, and 14 following treatments compared to the cell count of day 1 for no IST with IL-2 (5 U/mL). Growth kinetics (left) and day 7 growth (right). (D) Growth of CD8 + NLV-tetramer + cells with no added IL-2 as in (C). (E) Growth of CD8 + NLV-tetramer − cells with IL-2 (5 U/mL) as in (C). (F) Growth of CD8 + NLV-tetramer − cells with no added IL-2 as in (C). Expression of markers (G) 4-1BB, (H) LAG-3, and (I) CD38 were evaluated on days 1, 5, 7, and 14 following treatment in the presence of IL-2 (5 U/mL) and the percentage of CD8 + NLV-tetramer + that expressed each marker was determined by flow cytometry. Lines and bars depict mean ± SEM of independent experiments each using unique donor PBMCs with n = 5 donors up to day 5 and n = 4 donors up to day 14. Each shape denotes an independent donor. Repeated measurements one-way ANOVA with Tukey multiple comparisons test was performed. All P values <0.1 are depicted. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Activation Assay, Flow Cytometry, Cell Characterization, Expressing, Marker

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: NLV-specific memory CD8 + T cells expanded by IST are responsive to subsequent restimulation. (A) Experimental protocol and timeline. (B) The percentage of CD8 + NLV-tetramer + T cells that expressed CD25 and LAG-3 on day 35 was determined by flow cytometry. Gated on single, 7-AAD − , CD8 + , NLV-tetramer + cells (C) Summary plot of the normalized count of CD8 + NLV-tetramer + cells for all indicated treatments following retreatment or no retreatment on day 42. Bars depict independent treatment replicates ( n = 3) with the mean displayed from one independent experiment. Ordinary 2-way ANOVA with Šidák multiple comparisons test performed between nonrestimulated and restimulated for (B) and (C). All P values <0.1 are depicted. *** P < 0.001, **** P < 0.0001.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: HIV- and cytomegalovirus-specific human memory CD8 + T cells are activated and expanded independent of co-stimulatory signaling by T-cell receptor-specific immunotherapeutics

doi: 10.1093/jimmun/vkaf329

Figure Lengend Snippet: CD8 + T cells engineered to express an SL9-specific TCR display enhanced expansion and cytotoxic output following stimulation with IST-delivered SL9-specific TCR and CD28 signals. (A) Experimental protocol and timeline. (B) Representative flow cytometry plot of transduced cells. Gated on single, 7-AAD – cells. (C) TNF-α expression in GFP + and (D) GFP – cells following overnight stimulation with the indicated concentrations of each IST or the no IST control. (E) The frequency of GFP + cells within the total live populations and (F) GFP + cell count/well on days 7, 14, and 21 following treatments with 100 nM of the indicated IST or no IST control. All GFP + and GFP − cells previously gated on single-cell, 7-AAD − populations. (G) Elimination of T2 cells pulsed with SL9- or NLV-peptide following co-culture with T cells containing SL9-specific TCR-engineered T cells collected on day 21 following stimulation with 100 nM of the indicated IST or no IST control. %T2 Elimination is calculated as the percent reduction in T2 cells recovered in each experimental sample relative to the NLV-peptide–loaded T2 cells co-cultured with the no IST effector T cells. All data represent mean ± SD of 3 independent treatment wells from one of 2 independent experiments. For (C, D, and G), 2-way ANOVA with Dunnett multiple comparisons test was performed For (E and F), one-way ANOVA with Tukey multiple comparisons test was performed and significance reported for day 21 data. *** P <0.001, **** P <0.0001.

Article Snippet: On day −1, PBMCs were thawed, and naïve CD8 + T cells were isolated by immunomagnetic sorting using the Naïve CD8 + T Cell Isolation Kit (Miltenyi Biotec, Cat# 130-093-244) according to the manufacturer’s directions and were rested overnight in I-10 media supplemented with IL-7 (5 ng/mL, National Institutes of Health [NIH]).

Techniques: Flow Cytometry, Expressing, Control, Cell Characterization, Single Cell, Co-Culture Assay, Cell Culture

Journal: Nature Communications

Article Title: Increased SOAT2 expression in aged regulatory T cells is associated with altered cholesterol metabolism and reduced anti-tumor immunity

doi: 10.1038/s41467-025-56002-w

Figure Lengend Snippet: A Schematic design for screening the old mice with SOAT2 high or SOAT2 low , and the young mice with SOAT2 low (Created in BioRender. Major, Z. (2023) https://BioRender.com/l79u279 ). B The mRNA expression of SOAT2 in the young and old mice PBMCs was monitored by quantitative RT-PCR using gene-specific primers and probes ( n = 20). C , D The protein expression of SOAT2 in the young and old mice PBMCs was monitored by western blotting analysis ( C ); quantitation of SOAT2 protein concentrations was shown ( n = 20) ( D ). E – G The KLN-205 cells allograft-bearing model ( n = 5). The tumor growth curves ( E ), representative images of subcutaneous tumors ( F ), and tumor weight ( G ) in indicated groups. H Representative immunohistochemical staining of ki67, SOAT2, FOXP3, and CD8 in the tumor nodules in indicated groups. scale bars: 2000 and 50 μm. I , J quantitation of ki67 ( I ); the correlation of SOAT2 with ki67 expression was shown ( n = 5) ( J ). K – M Quantitation of FOXP3 integratedoption density (IntDen) and FOXP3-positive cells ( K ); the correlation of FOXP3-IntD and FOXP3-positive cells with SOAT2 expression were shown ( n = 5) ( L , M ). N , O Quantitation of FOXP3 average optical density (AOD) ( N ); the correlation of FOXP3-AOD with SOAT2 expression was shown ( n = 5) ( O ). P – R Quantitation of CD8-IntDen and FOXP3-positive cells ( P ); the correlation of CD8-IntD and CD8-positive cells with SOAT2 expression were shown ( n = 5) ( Q , R ). S , T Quantitation of CD8 average optical density (AOD) (S); the correlation of FOXP3-AOD with SOAT2 expression were shown ( n = 5) (T). Data represented means ± SD. Statistical difference was evaluated by unpaired two-tailed student’s t -test ( B , D ), and one-way ANOVA test ( E , G , I , K , N , P , S ). Source data are provided as a Source Data file. (** P < 0.01; *** P < 0.001; ns no significance).

Article Snippet: For human samples, single-cell suspensions from human peripheral blood were gathered and extracted with CD4 + CD25 + CD45RA + Regulatory T Cell Isolation Kit (#130-093-631, Miltenyi Biotec) or CD8 + CD45RA + Effector T Cell Isolation Kit (#130-094-485, Miltenyi Biotec).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay, Immunohistochemical staining, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Increased SOAT2 expression in aged regulatory T cells is associated with altered cholesterol metabolism and reduced anti-tumor immunity

doi: 10.1038/s41467-025-56002-w

Figure Lengend Snippet: A – C Treg cells were co-cultured with CFSE-labeled CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and CD8 + T lymphocytes proliferation expansion was detected by flow cytometry assay ( A ); quantitation of CFSE Low (%) was shown ( n = 3) ( B , C ). D – I Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and the release of CD8 + T lymphocytes for granzyme B and perforin were detected by ELISPOT assay ( D , G ); quantitation of granzyme B ( E , H ) and perforin ( F , I ) were shown (biological replicate, n = 3). J , K Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and the release of CD8 + T lymphocytes for granzyme B and perforin were detected by ELISA assay; quantitation of granzyme B and perforin were shown ( n = 3). L , M Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and Teff-mediated cytoxicity was determined by LDH release assay using a cytotoxicity detection kit ( n = 5). Data represented means ± SD. Statistical difference was evaluated by one-way ANOVA test ( B , C , E , F , H , I , J , K , L , M ). Source data are provided as a Source Data file. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: For human samples, single-cell suspensions from human peripheral blood were gathered and extracted with CD4 + CD25 + CD45RA + Regulatory T Cell Isolation Kit (#130-093-631, Miltenyi Biotec) or CD8 + CD45RA + Effector T Cell Isolation Kit (#130-094-485, Miltenyi Biotec).

Techniques: Cell Culture, Labeling, Flow Cytometry, Quantitation Assay, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: Nature Communications

Article Title: Increased SOAT2 expression in aged regulatory T cells is associated with altered cholesterol metabolism and reduced anti-tumor immunity

doi: 10.1038/s41467-025-56002-w

Figure Lengend Snippet: A – C Representative images of Teffs stained for β-galactosidase (arrows) after co-cultured with Treg cells ( n = 3) ( A ), scale bars: 50 μm; quantitation of β-galactosidase-positive Teffs were shown ( B , C ). D – F The protein expression of P16, P21, P53 in Teff cells in the young and old individuals were monitored by western blotting analysis in indicated groups. The experiment was repeated 3 times independently with similar results. G – J , Treg cells were co-cultured with CD8 + T lymphocytes at a 0.2:1 ratio in indicated groups, and the expression of CD27 or CD28 in human Teffs ( G , H ) and murine Teffs ( I , J ) were detected by flow cytometric analysis ( n = 3). K Schematic representation of Treg cells exerts immunosuppressive path on Teffs (Created in BioRender. Major, Z. (2024) https://BioRender.com/s68z662 ). L , M The content of cholesterol components were measured by the Cholesterol Assay Kit in indicated groups ( n = 5). N – P Representative images of Teffs stained for β-galactosidase (arrows) after co-cultured with Treg cells (N), scale bars: 50 μm; quantitation of β-galactosidase-positive Teffs were shown ( n = 3) ( O , P ). Data represented means ± SD. Statistical difference was evaluated by one-way ANOVA test ( B , C , L , M , O , P ). Source data are provided as a Source Data file. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: For human samples, single-cell suspensions from human peripheral blood were gathered and extracted with CD4 + CD25 + CD45RA + Regulatory T Cell Isolation Kit (#130-093-631, Miltenyi Biotec) or CD8 + CD45RA + Effector T Cell Isolation Kit (#130-094-485, Miltenyi Biotec).

Techniques: Staining, Cell Culture, Quantitation Assay, Expressing, Western Blot, Cholesterol Assay