unconjugated rabbit anti human cd8α  (Cell Signaling Technology Inc)

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: SMARCA4 deficiency impairs CD8 + T cell function and confers resistance to PD-1 blockade in NSCLC (A) Progression-free survival (PFS) of eight patients with SMARCA4-deficient (BRG1-deficient) NSCLC treated with ICIs. (B) Representative axial CT images of a patient (P6) with SMARCA4-deficient NSCLC before and after ICIs treatment. (C) Change in the sum of target lesion diameters for six evaluable patients with SMARCA4-deficient NSCLC from baseline to first radiographic assessment. (D) Schematic of the experimental design for the orthotopic lung cancer model ( n = 8/group). (E) Representative in vivo bioluminescence images of mice from the indicated treatment groups at different time points. (F) Quantification of total bioluminescence flux from mice in each group over time. (G) Kaplan-Meier survival curves of mice from the four treatment groups. (H) Individual tumor growth curves, as measured by bioluminescence, for mice in each treatment group. (I) UMAP visualization of major immune cell populations within the TME, analyzed by flow cytometry. (J) Quantitative comparison of the frequencies of indicated immune cell lineages between SMARCA4-WT and -KD tumors. (K and L) Representative flow cytometry plots showing the production of IFN-γ and TNF-α by tumor-infiltrating CD8 + T cells. (M) Quantification of the frequencies of IFN-γ + and TNF-α + cells among tumor-infiltrating CD8 + T cells. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one- or two-way ANOVA where appropriate.

Article Snippet: CD8 + T cell dependency was assessed by co-administering a depleting anti-human CD8α antibody (BioXcell, 15 μg/mouse) with PD-1/IL-2 bsAb in the SMARCA4-KD model. Depletion (initiated one day before PD-1/IL-2 bsAb and maintained twice weekly) was confirmed by flow cytometry (>90% reduction in circulating human CD3 + CD8 + T cells).

Techniques: Cell Function Assay, In Vivo, Flow Cytometry, Comparison

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: Attenuated IL-2/STAT5 signaling and an enhanced exhaustion phenotype in CD8 + T cells within SMARCA4-deficient tumors (A) Schematic of the workflow for transcriptomic profiling of CD8 + T cells. (B) Volcano plot displaying differentially expressed genes in CD8 + T cells from SMARCA4-KD versus WT tumors. (C–E) Pathway enrichment analyses of genes downregulated in CD8 + T cells from SMARCA4-KD tumors, including Gene Ontology (GO) terms, KEGG pathways, and Reactome pathways. (F) The gene set enrichment analysis (GSEA) plot. (G) Correlation matrix (pie chart) showing the association between IL-2 receptor subunits expression and key T cell exhaustion marker genes in tumor-infiltrating CD8 + T cells. (H) Radar plot comparing the normalized expression levels of genes encoding IL-2 receptor subunits and exhaustion markers in CD8 + T cells. (I) Quantification by flow cytometry of the expression frequencies of PD-1, TIGIT, and TIM-3 on tumor-infiltrating CD8 + T cells. (J and K) Representative multiplex immunofluorescence (mIF) images of SMARCA4-WT and -KD tumor sections stained for PanCK (cyan), CD8 (green), GZMB (white), PD-1 (red), TIGIT (orange), and TIM-3 (yellow). Scale bars, 20 μm. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: CD8 + T cell dependency was assessed by co-administering a depleting anti-human CD8α antibody (BioXcell, 15 μg/mouse) with PD-1/IL-2 bsAb in the SMARCA4-KD model. Depletion (initiated one day before PD-1/IL-2 bsAb and maintained twice weekly) was confirmed by flow cytometry (>90% reduction in circulating human CD3 + CD8 + T cells).

Techniques: Expressing, Marker, Flow Cytometry, Multiplex Assay, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: SMARCA4 loss in tumor cells attenuates CD8 + T cell function via NF-κB-mediated suppression of ICAM1 (A) Flow cytometry analysis of surface expression of the exhaustion markers PD-1, TIGIT, and TIM-3 on human CD8 + T cells following co-culture. (B and C) Representative flow cytometry plots showing the production of IFN-γ and TNF-α and the surface expression of IL-2Rα (CD25) by CD8 + T cells following co-culture. (D) Quantification of the frequencies of IFN-γ + , TNF-α + , and IL-2Rα + cells among co-cultured CD8 + T cells. (E) Integrated single-nucleus RNA sequencing (snRNA-seq) analysis comparing IL2-STAT5 signaling activity. y axis: IL2-STAT5 signaling score. (F and G) Incoming and outgoing signaling patterns between major cell types in the TME, as inferred from snRNA-seq. (H) Specific cell-cell communication network illustrating the ICAM signaling pathway from tumor cells to CD8 + T cells in patients with SMARCA4-WT NSCLC. (I) Correlation analysis between SMARCA4 and ICAM1 mRNA expression in TCGA cohorts. (J) Immunohistochemistry staining and quantification of ICAM1 protein expression in tumor tissues from SMARCA4-WT ( n = 10) and -deficient ( n = 10) NSCLC patients. (K) Schematic illustrating the proposed link between SMARCA4 deficiency and impaired NF-κB activation. (L) Immunoblot analysis of ICAM1 and p65 protein levels in SMARCA4-WT H2122 cells treated with the NF-κB inhibitor PTDC or vehicle control. (M) ChIP-qPCR analysis showing NF-κB (p65) binding to a specific site within the ICAM1 promoter in SMARCA4-WT H2122 cells ( n = 3). (N) Dual-luciferase reporter assay in SMARCA4-WT H2122 cells ( n = 3). Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: CD8 + T cell dependency was assessed by co-administering a depleting anti-human CD8α antibody (BioXcell, 15 μg/mouse) with PD-1/IL-2 bsAb in the SMARCA4-KD model. Depletion (initiated one day before PD-1/IL-2 bsAb and maintained twice weekly) was confirmed by flow cytometry (>90% reduction in circulating human CD3 + CD8 + T cells).

Techniques: Cell Function Assay, Flow Cytometry, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Activity Assay, Immunohistochemistry, Staining, Activation Assay, Western Blot, Control, ChIP-qPCR, Binding Assay, Luciferase, Reporter Assay

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: The PD-1/IL-2 bsAb exerts potent anti-tumor efficacy in SMARCA4-deficient models (A) Schematic of the therapeutic experiment in humanized mice ( n = 6/group). (B) Representative images of excised tumors from each treatment group at the endpoint. (C) Tumor growth curves of individual mice in the indicated treatment groups. (D) Kaplan-Meier survival curves of mice from the four treatment groups. (E) Quantification by flow cytometry of tumor-infiltrating CD3 + CD8 + T cells. (F) Quantification of the percentage of tumor-infiltrating CD8 + T cells expressing PD-1, TIGIT, and TIM-3. (G) Frequency of polyfunctional (TNF-α + IFN-γ + ) CD8 + T cells among tumor-infiltrating lymphocytes. (H) Schematic of the humanized patient-derived xenograft (PDX) model ( n = 5/group). (I) Representative images of excised PDX tumors from each treatment group. (J) Tumor growth curves of individual PDX-bearing mice. (K) Kaplan-Meier survival curves of PDX-bearing mice from the three treatment groups. (L) Quantification of tumor-infiltrating CD3 + CD8 + T cells in PDX tumors. (M) Quantification of the percentage of CD8 + T cells from PDX tumors expressing PD-1, TIGIT, and TIM-3. (N) Frequency of polyfunctional (TNF-α + IFN-γ + ) CD8 + T cells in PDX tumors. (O) Representative multiplex immunofluorescence (mIF) images of PDX tumor sections from different treatment groups, stained for PanCK (cyan), CD8 (green), GZMB (white), PD-1 (red), TIGIT (orange), and TIM-3 (yellow). Scale bars, 40 μm. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA or two-way ANOVA where appropriate.

Article Snippet: CD8 + T cell dependency was assessed by co-administering a depleting anti-human CD8α antibody (BioXcell, 15 μg/mouse) with PD-1/IL-2 bsAb in the SMARCA4-KD model. Depletion (initiated one day before PD-1/IL-2 bsAb and maintained twice weekly) was confirmed by flow cytometry (>90% reduction in circulating human CD3 + CD8 + T cells).

Techniques: Flow Cytometry, Expressing, Derivative Assay, Multiplex Assay, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: STAT5 activation mediates the therapeutic effect of the PD-1/IL-2 bsAb in SMARCA4-deficient NSCLC (A) Representative flow cytometry plots showing the expression of the exhaustion markers PD-1, TIGIT, and TIM-3 on activated human CD8 + T cells. (B) Quantification of the mean fluorescence intensity (MFI) of the exhaustion markers PD-1, TIGIT, and TIM-3 on CD8 + T cells from the experiment in (A) ( n = 3). (C) Representative flow cytometry plots showing the production of IFN-γ and TNF-α by CD8 + T cells under the conditions described in (A). (D) Quantification of the frequencies of IFN-γ + and TNF-α + cells among CD8 + T cells ( n = 3). (E) Schematic of the adoptive T cell transfer experiment ( n = 8/group). (F) Representative in vivo bioluminescence images of mice from the indicated groups at different time points. (G) Tumor growth curves, as measured by bioluminescence, for mice in each treatment group. (H) Kaplan-Meier survival curves of mice from the four treatment groups. (I) Quantification of the frequency of donor-derived CD45.2 + CD8 + T cells among total tumor-infiltrating lymphocytes. (J) Flow analysis of donor-derived CD45.2 + CD8 + T cells expressing PD-1, TIGIT, and TIM-3. (K–M) Frequency of IFN-γ + (K–L) and TNF-α + (M) cells among donor-derived CD45.2 + CD8 + T cells. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA or two-way ANOVA where appropriate.

Article Snippet: CD8 + T cell dependency was assessed by co-administering a depleting anti-human CD8α antibody (BioXcell, 15 μg/mouse) with PD-1/IL-2 bsAb in the SMARCA4-KD model. Depletion (initiated one day before PD-1/IL-2 bsAb and maintained twice weekly) was confirmed by flow cytometry (>90% reduction in circulating human CD3 + CD8 + T cells).

Techniques: Activation Assay, Flow Cytometry, Expressing, Fluorescence, In Vivo, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: PD-1/IL-2 bsAb protects CD8 + T cells from macrophage phagocytosis via STAT5-mediated upregulation of CD47 (A) Schematic of the Cleavage Under Targets and Tagmentation (CUT&Tag) assays workflow. (B) Distribution of STAT5 binding signals relative to transcription start sites (TSS) in CD8 + T cells treated with or without the PD-1/IL-2 bsAb. (C) Genomic annotation of differentially enriched STAT5 binding peaks in the bsAb-treated group. (D) KEGG pathway enrichment analysis of genes associated with STAT5 binding peaks. (E and F) Strategy and Venn diagram for identifying potential STAT5 downstream genes. (G) ChIP-qPCR analysis of STAT5 binding to the promoter regions of selected candidate genes ( n = 3). (H) Genome browser tracks showing STAT5 binding signals at the CD47 locus in control and PD-1/IL-2 bsAb-treated CD8 + T cells. (I) Schematic of the macrophage phagocytosis assay. (J) Representative confocal microscopy images showing macrophages (red) engulfing CD8 + T cells (green). Scale bars, 20 μm. (K) Flow cytometry quantification of the percentage of macrophages that had phagocytosed CD8 + T cells under the indicated conditions ( n = 3). Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by unpaired Student’s t test or one-way ANOVA where appropriate.

Article Snippet: CD8 + T cell dependency was assessed by co-administering a depleting anti-human CD8α antibody (BioXcell, 15 μg/mouse) with PD-1/IL-2 bsAb in the SMARCA4-KD model. Depletion (initiated one day before PD-1/IL-2 bsAb and maintained twice weekly) was confirmed by flow cytometry (>90% reduction in circulating human CD3 + CD8 + T cells).

Techniques: Binding Assay, ChIP-qPCR, Control, Phagocytosis Assay, Confocal Microscopy, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Dual PD-1/IL-2Rα targeting restores CD8 + T cell fitness via STAT5/CD47 axis in SMARCA4-deficient NSCLC

doi: 10.1016/j.xcrm.2026.102633

Figure Lengend Snippet: CD47 protects CD8 + T cells from macrophage clearance to boost antitumor immunity in SMARCA4-deficient NSCLC (A) Schematic of the adoptive T cell therapy experiment ( n = 8/group). (B) Representative in vivo bioluminescence images of mice from the indicated treatment groups at different time points. (C) Tumor growth curves, as measured by bioluminescence, for mice in each treatment group. (D) Individual tumor growth curves for mice in each treatment group. (E) Kaplan-Meier survival curves of mice from the four treatment groups. (F) Quantification by flow cytometry of donor-derived CD45.2 + CD8 + T cells among total tumor-infiltrating lymphocytes. (G) Representative flow cytometry plots for donor-derived CD45.2 + CD8 + T cells expressing the exhaustion markers PD-1, TIGIT, and TIM-3. (H) Quantification of the percentage of CD45.2 + CD8 + T cells expressing PD-1, TIGIT, and TIM-3. (I) The production of TNF-α by donor-derived CD45.2 + CD8 + T cells. (J) The production of IFN-γ by donor-derived CD45.2 + CD8 + T cells. (K) Representative immunofluorescence images of tumor sections. White: CD8, green: CD47, red: F4/80. Scale bars, 70 μm. (L) Schematic model depicting the proposed mechanism of action. Data are represented as mean ± SD (error bars) from biological replicates. Statistical analyses, n.s., no significance. Statistical significance was determined by one-way ANOVA.

Article Snippet: CD8 + T cell dependency was assessed by co-administering a depleting anti-human CD8α antibody (BioXcell, 15 μg/mouse) with PD-1/IL-2 bsAb in the SMARCA4-KD model. Depletion (initiated one day before PD-1/IL-2 bsAb and maintained twice weekly) was confirmed by flow cytometry (>90% reduction in circulating human CD3 + CD8 + T cells).

Techniques: In Vivo, Flow Cytometry, Derivative Assay, Expressing, Immunofluorescence

Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) Heatmaps depict the enrichment of immune and non-immune cell types in the immediate neighborhood of CCR7 + DCs in NSCLC spatial transcriptomic data ( n = 4). (B) (Left) Representative FOV displaying CCR7 + DCs (HLA-DR + LAMP3 + ; yellow) located near BVs (CD31 + PDPN − ; magenta) and Tregs (CD4 + FOXP3 + ; white) in one HNSCC sample using high-plex whole-tissue imaging. Scale bar represents 20 μm. (Right) Box plots display the frequencies of BV-associated, LV-associated, and non-vessel-associated CCR7 + DCs close (<5 μm) to Tregs among all tumor CCR7 + DCs with nearby Tregs. Wilcoxon test, whiskers represent min to max; * p < 0.05. (C) Correlations between CCR7 + DCs and Tregs within CD45 + cells, as determined by scRNA-seq in multiple human cancer types. Spearman rank correlation; significant correlations are shown with a fitted red line. (D) (Left) Scheme outlining the analyses of CCR7 + DCs and Tregs in NSCLC samples. Patients with numerous (>5) CCR7 + DC clusters ( n = 12) were selected for downstream analyses. (Right) Frequency of CCR7 + DCs (CD11c + LAMP3 + ) with at least one nearby (<50 μm) Treg (CD4 + FOXP3 + ) in each individual patient. Numbers of FOVs analyzed per sample are as follows: NR01, n = 126; NR06, n = 455; NR09, n = 180; NR12, n = 79; NR26, n = 293; R11, n = 122; R15, n = 205; R35, n = 175; R37, n = 459; R45, n = 276. (E) (Left) Scheme outlining the analysis of tumor biopsies from HNSCC patients before immunotherapy (pre-IO). Patients were divided into non-responders (NR, n = 5) and responders (R, n = 5) based on the assessment of clinical response at 6 months. (Right) CCR7 + DC shortest distance to Tregs, T CONV , and CD8 + T cells in NR versus R tumors. Data are shown for all CCR7 + DCs compiled (NR tumors, n = 1,457 cells; R tumors, n = 1,324 cells). Unpaired t test, whiskers represent min to max; **** p < 0.0001. Numbers of FOVs analyzed per sample as in (D). (F) (Left) Scheme outlining the analyses of CCR7 + DC-CD8 + T cell niches. (Right) Frequencies of CCR7 + DC-CD8 + T cell niches with or without Tregs in their proximity (<100 μm). Two-way ANOVA with multiple comparisons, whiskers represent min to max; * p < 0.05. Numbers of FOVs analyzed per sample as in (D). (G) Representative FOV displaying CCR7 + DCs (FSCN1 + cells; FSCN1 in yellow) located near BVs (CD31 + LYVE-1 − cells; CD31 in magenta) and Tregs (FOXP3 + cells; FOXP3 in white) in untreated MC38 tumors. Scale bar represents 50 μm. (H) Correlations between the numbers of CCR7 + DCs and Tregs per mg of tumor tissue, as determined by fluorescence-activated cell sorting (FACS) analyses of MC38 and D4M3. A tumors. Spearman rank correlation; significant correlations are shown with a fitted red line. (I) Box plots show the frequencies of tumor CCR7 + DCs close (<5 μm) to Tregs that are associated to BVs or LVs in MC38 tumors ( n = 7). Whole-tumor sections were analyzed. Paired t test, whiskers represent min to max; **** p < 0.0001. See also and .

Article Snippet: Unconjugated rabbit anti-human CD8α (Clone D8A8Y) , Cell Signaling Technology , Cat#85336.

Techniques: Imaging, Fluorescence, FACS

Journal: Immunity

Article Title: Positioning and reversible suppression of CCR7 + dendritic cells in perivascular tumor niches shape cancer immunity

doi: 10.1016/j.immuni.2025.11.020

Figure Lengend Snippet: (A) (Left) Scheme outlining the experimental setup for bulk RNA-seq analyses of tumor-derived CCR7 + DCs. (Right) GO pathway enrichment analyses performed on differentially expressed genes (DEGs) in CCR7 + DCs in MC38 tumors ( n = 4) from Treg-depleted ( FoxP3 -DTR) compared with Treg-sufficient (WT) mice. Bar plot indicates the −log 10 raw binomial p -values of the top 10 most enriched pathways in CCR7 + DCs. (B) (Left) Experimental setup for ex vivo stimulation of OT-I CD8 + T cells with tumor CCR7 + DCs. (Right) Percentage of OT-I CD8 + T cells that proliferated after 5-day culture with OVA 257–264 peptides-loaded CCR7 + DCs isolated from WT or Treg-depleted tumors. As a control, CCR7 + DCs without OVA 257–264 peptides were used. Two-way ANOVA with multiple comparisons, whiskers represent min to max; ** p < 0.01. (C) (Left) Relative gene expression levels analyzed by bulk RNA-seq. Each dot represents one mouse ( n = 4), whiskers represent mean to max. Unpaired t test with multiple comparisons; * p < 0.05. (Right) Representative histogram of CD40 protein expression and relative mean fluorescence intensity (MFI) measured by FACS and expressed both as normalized values and absolute MFI. Each dot represents one mouse ( n = 18), whiskers represent min to max. Unpaired t test; ** p < 0.01. (D) Analyses of cDCs in tumor-draining lymph nodes. Absolute cell counts (left, n = 10) and MFI of CD40 expression (right, n = 18) measured by FACS in migratory cDCs (CCR7 + CD8α − ) from WT or Treg-depleted mice. Whiskers represent mean to max. (E) (Left) Experimental setup for ex vivo analyses of tumor CCR7 + DCs isolated from anti-PD-1-treated mice that received or not αCD25 NIB mAbs. (Right) CD40 protein expression measured by FACS and expressed both as normalized values and absolute MFI. Each dot represents one mouse ( n = 4 WT and n = 6 FoxP3-DTR), whiskers represent min to max. Unpaired t test; ** p < 0.01. (F) (Left) Overall survival analyses of MC38 tumor-bearing mice treated, or not treated, with αPD-1 and αCD25 NIB mAbs, and in which CD4 + or CD8 + cells were depleted or not ( n = 8 or 9 mice/group). Log-rank Mantel-Cox test; * p < 0.05, *** p < 0.001, and *** p < 0.0001. (Right) Percentage of tumor-free mice on day 60 in the indicated treatment groups. (G) (Left) Experimental setup for ex vivo stimulation of OT-I CD8 + T cells with tumor CCR7 + DCs as in (B). The DCs were obtained from mice receiving anti-PD-1 immunotherapy and that were treated or not with αCD25 NIB mAbs. (Right) Percentage of OT-I CD8 + T cells that proliferated after 5-day culture with OVA 257–264 peptide-loaded CCR7 + DCs. Each dot represents one mouse ( n = 8 and n = 7), whiskers represent min to max. Two-way ANOVA with multiple comparisons; * p < 0.05. (H) (Left) Scheme outlining bone marrow chimeras with inducible Cd40 -deficiency in cDCs and the treatment schedule. (Right) Growth curves of MC38 tumors inoculated in zDC iDTR : Cd40 WT and zDC iDTR : Cd40 KO bone marrow chimeras treated with αPD-1, αCD25 NIB , or αPD-1 + αCD25NIB combination ( n = 8–10 mice/group). Mean with SEM. Two-way ANOVA with multiple comparisons; * p < 0.05 and **** p < 0.0001. See also and .

Article Snippet: Unconjugated rabbit anti-human CD8α (Clone D8A8Y) , Cell Signaling Technology , Cat#85336.

Techniques: RNA Sequencing, Derivative Assay, Ex Vivo, Isolation, Control, Gene Expression, Expressing, Fluorescence

Journal: Scientific Reports

Article Title: Immune mobilising T cell receptors redirect polyclonal CD8 + T cells in chronic HIV infection to form immunological synapses

doi: 10.1038/s41598-022-23228-3

Figure Lengend Snippet: Phenotypic analysis of CD8 + T cells from people living with HIV and HIV-naïve donors. CD8 + T cells from a total of 26 virologically suppressed PLWH (PLWH, blue) and ten HIV-naïve donors (HD, red) were analysed for phenotype and/or production of granzyme B. ( a ) CD39 and PD-1 in naïve, T CM , T EM and T EMRA subpopulations and plotted as percentage positive of gated (PLWH, n = 10; HD, n = 10). ( b ) T-bet, Eomes, TIGIT, granzyme B and perforin in the CD3 + CD8 + population (ex vivo) and plotted as percentage positive in each population (PLWH, n = 9; HD, n = 10). ( c ) Granzyme B-producing cells in ex vivo PBMC (PLWH, n = 11; HD, n = 6) were quantified by ELISpot after culture in triplicate with SL9 peptide-pulsed T2 cells for 48 h (E:T = 1:1), together with m121 ImmTAV (0.5 nM), a non-binding control TCR fusion protein, m231 (1 nM) or no ImmTAV (mock); PHA stimulation was used as a positive control for cell viability. Negative control wells (no TCR) yielded spot-forming cell counts < 3/well in all subjects, for both peptide-pulsed and unpulsed conditions. Results are plotted as mean ± SD for each condition. Groups were analysed by Mann–Whitney test or 1-way ANOVA with Sidak’s multiple comparisons.

Article Snippet: CD8 + T cells were identified using a mouse anti-human CD8α (clone 37,006, R&D Systems) or rabbit anti-human CD8α (Abcam).

Techniques: Ex Vivo, Enzyme-linked Immunospot, Binding Assay, Control, Positive Control, Negative Control, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Immune mobilising T cell receptors redirect polyclonal CD8 + T cells in chronic HIV infection to form immunological synapses

doi: 10.1038/s41598-022-23228-3

Figure Lengend Snippet: Time course of immunological synapse (IS) formation by ImmTAV-redirected HIV-naïve donor CD8 + T cells. SL9-peptide pulsed T2s were ( a ) stained for surface epitopes using a labelled TCR and imaged by TIRF microscopy for epitope/cell quantification (brightfield and maximum intensity projection (MIP) of labelled TCR shown; one dot represents one cell). These targets were then co-cultured with HIV-naïve donor CD8 + T cells (E:T of 1:1) for 5, 15 or 30 min before confocal microscopy imaging and analysis. ( b ) % of target cells in conjugates with CD8 + T cells over time in the presence of HIV-specific ImmTAV (m121, 0.5 nM) or irrelevant TCR-anti-CD3 fusion protein (ImmTAX, m232, 1 nM). C-F all show IS formation in the presence of the HIV ImmTAV, m121. ( c ) Representative confocal images of conjugates showing CD8 + T cell Zap70 (green) localisation in the cytoplasm (left) or in the plasma membrane (right). The percentage of conjugates with Zap70 localised to the cytoplasm (grey) or membrane (black) was calculated at different conjugation times. ( d ) The percentage Zap70 localised at the IS in CD8 + T cells. ( e ) Confocal images of α-tubulin (green) to define MTOC location (indicated with white arrow) and distance from IS to MTOC (μM). ( f ) Confocal microscopy images of perforin staining (summed Z stack shown in white) and % conjugates with distal, dispersed or docked perforin in the CD8 + T cells. For D & E each dot represents one T2-CD8 + T cell conjugate (0.5 nM m121 for c – f ); 1 slide (representing 1 donor) was analysed per time-point; magenta = CD8 on differential interference contrast image (DIC). Means shown and data were analysed by one-way ANOVA.

Article Snippet: CD8 + T cells were identified using a mouse anti-human CD8α (clone 37,006, R&D Systems) or rabbit anti-human CD8α (Abcam).

Techniques: Staining, Microscopy, Cell Culture, Confocal Microscopy, Imaging, Membrane, Conjugation Assay

Journal: Scientific Reports

Article Title: Immune mobilising T cell receptors redirect polyclonal CD8 + T cells in chronic HIV infection to form immunological synapses

doi: 10.1038/s41598-022-23228-3

Figure Lengend Snippet: Comparison of ImmTAV-mediated immunological synapses formed by CD8 + T cells from HIV-naïve subjects and PLWH. SL9-pulsed T2 cells were cultured with HIV-naïve donor (HD) CD8 + T cells (n = 3) or SL9 peptide pre-stimulated PLWH CD8 + T cells (n = 3) at a ratio of 1:1 in the presence or absence of m121 ImmTAV (0.5 nM). ( a ) Quantification of conjugates formed after 15 min with pre-stimulated PLWH ± ImmTAV (n > 50 targets counted/slide, blinded analysis) and plotted as mean ± SD). ( b ) Quantification of MTOC distance from the IS (µM; data were analysed by one-way ANOVA with correction for multiple comparisons) and ( c ) % conjugates with distal, dispersed or docked expression of perforin in HD and PLWH CD8 + T cells. At least 10 conjugates per subject and per condition were analysed and data were pooled.

Article Snippet: CD8 + T cells were identified using a mouse anti-human CD8α (clone 37,006, R&D Systems) or rabbit anti-human CD8α (Abcam).

Techniques: Comparison, Cell Culture, Expressing

Journal: Scientific Reports

Article Title: Immune mobilising T cell receptors redirect polyclonal CD8 + T cells in chronic HIV infection to form immunological synapses

doi: 10.1038/s41598-022-23228-3

Figure Lengend Snippet: ImmTAV redirection of CD8 + T cells to HIV-infected primary CD4 + T cells. ( a ) Gag p24 total corrected cellular fluorescence (TCCF; as measured from confocal microscopy images) of activated or resting HIV-infected primary CD4 + T cells in conjugates with ImmTAV-redirected HIV-naïve donor CD8 + T cells (bottom); each dot represents a conjugate (n > 20/condition). Horizontal lines indicate median value. Groups were analysed by Mann Whitney test. Representative images of conjugates with activated infected CD4 + T cell targets shown as Gag expression in resting infected cells was not visible (top). ( b ) Confocal microscopy images of Zap70 localisation to the IS (examples of activated and resting, top), % Zap70 localisation at IS (bottom left) or cytoplasmic vs. membrane distribution (bottom right). ( c ) Confocal microscopy of α-tubulin (MTOC shown with white arrow; examples of activated and resting, left) and distance from MTOC to the synapse (µM, right). ( d ) Confocal microscopy of perforin localisation (docked example, left) and % of conjugates with distal, dispersed or docked perforin (right) in the CD8 + T cell. All synapse markers: at least 8 conjugates analysed per condition. Red = p24*, magenta = CD8 on DIC image, green = synapse molecule. For ( b )–( d ), horizontal lines indicate mean values. Groups were analysed by unpaired t test. *As p24 intensity in resting infected cells is very low and thus difficult to visualize, only DIC & CD8 signal images are shown.

Article Snippet: CD8 + T cells were identified using a mouse anti-human CD8α (clone 37,006, R&D Systems) or rabbit anti-human CD8α (Abcam).

Techniques: Infection, Fluorescence, Confocal Microscopy, MANN-WHITNEY, Expressing, Membrane

Journal: Scientific Reports

Article Title: Immune mobilising T cell receptors redirect polyclonal CD8 + T cells in chronic HIV infection to form immunological synapses

doi: 10.1038/s41598-022-23228-3

Figure Lengend Snippet: Resting infected CD4 + T cells are susceptible to ImmTAV-mediated killing. Resting infected CD4 + T cells from HIV-naïve donors (HD) were co-cultured with ( a ) autologous HD CD8 + T cells (E:T of 1:1 or 2:1) and m121 (0.5 nM), m231 (non-CD3 binding TCR fusion protein; 1 nM) or m232 (irrelevant TCR-anti-CD3 fusion protein; 1 nM) or ( b ) autologous HD CD8 + T cells at varying E:T ratios with 0.5 nM m121 for 48 h. The proportion of Gag + cells remaining after ImmTAV exposure was used to determine the % elimination (normalised to % Gag + cells in infected CD4 + T cells cultured alone). Horizontal lines indicate means. Groups were analysed by one-way ANOVA with Dunnett’s multiple comparisons test. Data shown are representative of two donors and four independent experiments.

Article Snippet: CD8 + T cells were identified using a mouse anti-human CD8α (clone 37,006, R&D Systems) or rabbit anti-human CD8α (Abcam).

Techniques: Infection, Cell Culture, Binding Assay

Journal: Scientific Reports

Article Title: Immune mobilising T cell receptors redirect polyclonal CD8 + T cells in chronic HIV infection to form immunological synapses

doi: 10.1038/s41598-022-23228-3

Figure Lengend Snippet: Elimination of resting infected CD4 + T cells by PLWH CD8 + T cells. Resting infected HD CD4 + T cells were co-cultured with CD8 + T cells from eight PLWH (E:T of 1:1 or 2:1) with and without m121 ImmTAV (0.5 nM) for 48 h. ( a ) Percent elimination was normalised to the percentage of Gag + cells in infected CD4 + T cell cultures alone. For technical reasons, the no ImmTAV condition at an E:T ratio of 2:1 was not included. ( b ) The percent elimination data from ( a ) for six of the PLWH with phenotyping data was plotted against CD39 expression in the T EM population with and without m121 ImmTAV. Mean values are indicated by horizontal bars. Groups were analysed by one-way ANOVA with Tukey’s multiple comparisons test or Spearman’s correlation.

Article Snippet: CD8 + T cells were identified using a mouse anti-human CD8α (clone 37,006, R&D Systems) or rabbit anti-human CD8α (Abcam).

Techniques: Infection, Cell Culture, Expressing

Journal: Oncoimmunology

Article Title: Therapeutic Targeting of MYC in Head and Neck Squamous Cell Carcinoma

doi: 10.1080/2162402X.2022.2130583

Figure Lengend Snippet: MYC inhibition induces expression of CD8 + T cell-attracting chemokines in HNSCC cells. (a-b) The mRNA expression of IFNβ, CXCL9, CXCL10 , and CXCL11 in HN6 and CAL27 cells were induced by MYCi975. Means ± SD are shown. * P < .05 and ** P < .01 by one-way ANOVA. (c-d) The mRNA expression of IFNβ, CXCL9, CXCL10 , and CXCL11 in HN6 and CAL27 cells were induced by MYC knockdown. Means ± SD are shown. * P < .05 and ** P < .01 by one-way ANOVA.

Article Snippet: Sections were incubated with the following primary antibodies at 4°C overnight: anti-MYC (Cell Signaling Technology, Danvers, MA, USA; Cat#18583; 1:100), anti-human CD8α (Cell Signaling Technology; Cat#85336; 1:100), and anti-mouse CD8α (Cell Signaling Technology; Cat#98941; 1:200).

Techniques: Inhibition, Expressing, Knockdown

Journal: Oncoimmunology

Article Title: Therapeutic Targeting of MYC in Head and Neck Squamous Cell Carcinoma

doi: 10.1080/2162402X.2022.2130583

Figure Lengend Snippet: MYC inhibition promotes CD8 + T cell infiltration in vivo. (a) Immunostaining showing that MYCi975 inhibits MYC expression and promotes CD8 + T cell infiltration in the same view of the slice. Scale bar, 50 μm. (b) Quantification of the percentage of MYC + or CD8 + T cells in vivo after MYCi975 treatment. Means ± SD are shown. n = 3, ** P < .01 by an unpaired Student’s t test. (c) Experimental design for MYCi975 and anti-CXCR3 treatment in vivo . MSCC1 cells were injected into the dorsal subcutaneous region of C57BL/6 J mice. Four weeks after injection, tumor-bearing mice were randomly divided into four experimental groups (n = 5 per group): control vehicle, MYCi975, anti-CXCR3, and MYCi975 plus anti-CXCR3. Mice were sacrificed after treatment for 4 weeks. (d) Representative image of tumor samples harvested from subcutaneous tumor models in C57BL/6 J mice. (e) Tumor weights of subcutaneous tumor models in C57BL/6 J mice after 4 weeks of treatment. * P < .05 and ** P < .01 by one-way ANOVA. (f) Tumor volume growth curve of subcutaneous tumor models. * P < .05 and ** P < .01 by one-way ANOVA. (g) Representative images of CD8 + T cell infiltration in vivo after treatment. MYCi975 promoted CD8 + T cell infiltration and anti-CXCR3 reversed the immune activation by MYCi975. Scale bar, 50 μm. (h) Quantification of the percentage of CD8 + T cells in vivo after treatment. Values are the mean ± SD. ** P < .01 by one-way ANOVA.

Article Snippet: Sections were incubated with the following primary antibodies at 4°C overnight: anti-MYC (Cell Signaling Technology, Danvers, MA, USA; Cat#18583; 1:100), anti-human CD8α (Cell Signaling Technology; Cat#85336; 1:100), and anti-mouse CD8α (Cell Signaling Technology; Cat#98941; 1:200).

Techniques: Inhibition, In Vivo, Immunostaining, Expressing, Injection, Control, Activation Assay

Journal: Oncoimmunology

Article Title: Therapeutic Targeting of MYC in Head and Neck Squamous Cell Carcinoma

doi: 10.1080/2162402X.2022.2130583

Figure Lengend Snippet: CD8 + T cell infiltration density is negatively associated with the MYC protein levels in patients with HNSCC. (a) Immunostaining of human HNSCC samples showing that high MYC levels correlated with low CD8 + T cell infiltration density and low MYC levels correlated with high CD8 + T cell infiltration density in the same view of the slice. Scale bar, 50 μm. (b) CD8 + T cell infiltration density correlates negatively with MYC protein level in human HNSCC samples (n = 121). The Pearson correlation coefficient of linear regression was used to determine the correlation between different proteins. (c) Kaplan–Meier curves with log-rank tests showing that patients with high CD8 + T cell infiltration density had longer survival ( P = .002). (d) Kaplan–Meier curves with log-rank tests showing that patients with “high expression of MYC and low CD8 + T cell infiltration” had significantly shorter survival than those with “low expression of MYC or high CD8 + T cell infiltration” ( P < .001). (e) A diagram of the mechanism by which MYC inhibition eliminates tumors and activates antitumor immunity.

Article Snippet: Sections were incubated with the following primary antibodies at 4°C overnight: anti-MYC (Cell Signaling Technology, Danvers, MA, USA; Cat#18583; 1:100), anti-human CD8α (Cell Signaling Technology; Cat#85336; 1:100), and anti-mouse CD8α (Cell Signaling Technology; Cat#98941; 1:200).

Techniques: Immunostaining, Expressing, Inhibition

Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: a, Four cohorts were used to assess adaptive immunity in AD. b, Representative SPADE trees of PBMCs from healthy individuals and patients with MCI or AD in cohort 1 show an increased abundance of a CD8+ cluster (cluster 63) in patients with MCI or AD. Background tree nodes are sized according to cell counts. Insets are coloured according to CD8 expression. c, Quantification of cluster 63 as a percentage of total PBMCs. The percentage of cluster 63 cells is significantly higher in patients with MCI or AD than healthy control individuals. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. d, Marker expression analysis of cluster 63 corresponds to a CD3+CD8+CD45RA+CD27− TEMRA population. Data in c, d were pooled from seven independent experiments with similar results. e, Linear regression showing the inverse correlation between cognitive score and the percentage of CD8+ TEMRA cells in individuals from cohort 2. Pearson’s correlation r values are shown for each group. The significance of the difference between the two data sets was measured by ANCOVA. f, Stimulation with PMA and ionomycin (stim.) induces increased expression of IFN-γ in CD8+ T cells from patients with MCI or AD. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. g, Differential expression analysis (scRNA-seq) of CD8+ Temra cells from healthy individuals (n = 7) and patients with MCI or AD (n = 6) shows upregulated TCR signalling. Model-based analysis of single-cell transcriptomics (MAST) differential expression test with Benjamini-Hochberg correction. h, Pathway analysis of differentially expressed genes in CD8+ TEMRA cells from patients with MCI or AD (n = 6 subjects) versus healthy individuals (n = 7 subjects) shows increased antigenic stimulation of CD8+ TEMRA cells in patients with MCI or AD. Fisher’s exact test with Benjamini-Hochberg correction. Pathways (circles) with positive z-scores are coloured red; those with negative z-scores are coloured blue. The size of the circle corresponds to the size of the z -score (two-sided).

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Expressing, Control, Marker, Quantitative Proteomics, Single-cell Transcriptomics

Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: a, Confocal imaging of cerebral amyloid angiopathy (CAA) in the post-mortem brain of a patient with AD from cohort 3 shows CD8+ T cells in the perivascular space of Aβ+ blood vessels with cerebral amyloid angiopathy in three AD-affected hippocampi. Arrowheads indicate CD8+ T cells; asterisks indicate blood vessel lumen. Scale bars, 20 μm. b, Higher numbers of CD8+ T cells were detected in AD-affected than control hippocampi. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction. c, A CD8+ T cell is shown associated with MAP2+ neuronal processes. d, CD8+ T cells are localized to the leptomeninges and adjacent to hippocampal AP plaques. Scale bar, 100 μm. Data in c, d were replicated in three independent experiments.

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Imaging, Control

Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: a, Plate-seq and drop-seq methods used for scTCR-seq and scRNA-seq of immune cells of the CSF in patients from cohort 4. b, CD8+ TCRαβ clonality (plate-seq) in the CSF of patients with AD and healthy control individuals. c, The top (most expanded) clone in AD had a marker expression profile of CD8+CD45RA+CD27− TEMRA cells. Data were replicated in two independent experiments. d, CSF cells analysed by drop-seq and clustered by multidimensional reduction with t-SNE, showing populations of immune cells that include CD8+ TEMRA cells (n = 9 healthy control individuals (10,876 cells); n = 9 patients with MCI or AD (10,391 cells)). e, Marker expression of CSF clusters, including CD8+ TEMRA cells. CD62L is also known as SELL; CD11c is also known as ITGAX. Data were pooled from three independent experiments. f, Concentration of clonal cells in locations of CD8+ T cell clusters (n = 9 subjects per group). g, Representative plots of CD8+ TCRαβ clonality (drop-seq) in age-matched subjects shows enhanced clonal expansion and more highly expanded clones in AD. Clones are coloured by proportion of the total TCRαβ sequences. h, Quantification of maximum clones (% TCRαβ sequences) shows a higher percentage in patients with MCI or AD than healthy control individuals (n = 9 subjects per group). Samples lacking clonal cells were scored as zero. Box plots show median and 25th to 75th percentiles, and whiskers indicate the minimum and maximum values. Unpaired two-sided f-test with Welch’s correction. i, Differential expression of highly expanded clones (clonal TCRαβ > 5) revealed increased expression of cytotoxic effector genes. MAST differential expression test with Benjamini-Hochberg correction (n = 9 patients with MCI or AD). j , Quantification of highly expanded clones (clonal TCRαβ > 5) showed that 49.13% of them are CD8+ TEMRA cells (n = 9 patients with MCI or AD). k, Increased expression of B2M, NKG7and GZMA in clonal CD8+ T cells from patients with MCI or AD. MAST differential expression test with Benjamini-Hochberg correction (n = 9 subjects/group). l, A hippocampal CD8+ T cell in an AD-affected brain (cohort 3) shows expression of GZMA adjacent to MAP2+ neuronal processes. Scale bar, 5 pm. Data were replicated in three independent experiments. m, Percentages of CD8+ T cells that express GZMA in control and AD-affected hippocampi. Mean ± s.e.m.; unpaired two-sided t-test with Welch’s correction.

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Control, Marker, Expressing, Concentration Assay, Clone Assay, Quantitative Proteomics

Journal: Nature

Article Title: Clonally expanded CD8 T cells patrol the cerebrospinal fluid in Alzheimer’s disease

doi: 10.1038/s41586-019-1895-7

Figure Lengend Snippet: a, Unweighted network analysis of CD8 T CRαβ sequences combined from plate-seq and drop-seq experiments. Group node IDs with individual TCRαβ clones are depicted as circles and sized according to the proportion of total sequences of each clone. Arrow indicates a shared clonal T CRaP sequence with specificity for EBV EBNA3A. Note that several healthy control (HC) subjects have no clones. b, Shared TCRαβ sequences among patients with MCI or AD. Three patients had identical TCRβ chains with specificity for EBV EBNA3A. The antigen specificity of T CRβ is shown below19. c, Differential expression of EBV-specific clones in MCI and AD (from n = 3 subjects) versus all CSF T cells shows enhanced expression of cytotoxic effector genes. MAST differential expression test with Benjamini-Hochberg correction. d, Workflow for antigen identification of CSF TCRs. GLIPH was applied to TCR sequencing to derive homologous TCR sequences between patients. GLIPH identified two patients with AD who had identical TCRβ chains and a third patient with a similar sequence. The T CRαβ sequences derived from GLIPH were introduced into SKW-3 cells. e, Autologous fibroblasts were used to present antigens to TCRαβ 1 and TCRαβ 2 cells. Only TCRαβ 1 cells showed significant upregulation of CD69 following antigen presentation. Data are averages from three separate experiments performed in triplicate.Mean ± s.e.m.; one-way analysis of variance (ANOVA) (F(3, 8) = 1,050,P = 1.01 × 10−10) with Tukey’s multiple comparisons test. f, Peptide 7 (RAKFKQLL) of the EBV trans-activator BZLF1 protein activates TCRαβ 1 but not TCRαβ 2 cells. Two-way ANOVA (F(14, 60) = 14.06, P =4.8 × 10−14) followed by Sidak’s multiple comparisons test. Data were pooled from n = 3 independent experiments. The P value shown is from comparing peptide 7 values for each cell line. Mean ± s.d. g, A fluorescent dextramer composed of H LA-B*08:01 presenting the BZLF1 peptide RAKFKQLL shows nearly 100% positivity with TCRαβ 1 but no positivity with TCRαβ 2 cells. Unpaired two-sided t-test with Welch’s correction (n = 6 per group).

Article Snippet: Primary antibodies included rat anti-human CD3 (Abcam), rabbit anti-human CD8α (Cell Signaling), mouse anti-Aβ (Cell Signaling), chicken anti-human MAP2 (Abcam), mouse anti-human granzyme-A (Abcam), rat anti-mouse CD8a (eBioscience) and rabbit anti-mouse NEFH (Abcam).

Techniques: Clone Assay, Sequencing, Control, Quantitative Proteomics, Expressing, Derivative Assay, Immunopeptidomics