cd127 microbead kit  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: NK-like and networked CD8 + T cell immunity mediates exceptional HIV control

doi: 10.64898/2026.07.02.735504

Figure Lengend Snippet: a , Representative gating strategy for the CFSE-based HIV-specific CD8⁺ T cell proliferation assay. Sequential gates are applied to identify lymphocytes (FSC-A vs. SSC-A), live cells (SSC-A vs. viability dye), CD3⁺ T cells (SSC-A vs. CD3), and CD3⁺CD8⁺ T cells (CD8 vs. CD3). CFSE dilution in the CD8⁺ T cell gate is shown for DMSO negative control (0.13% CFSEᴸᴼᵂ) and HIV peptide-stimulated conditions (35.6% CFSEᴸᴼᵂ), illustrating the proliferative CD8⁺ T cell population. b, Representative gating strategy for the HIV-specific CD8⁺ T cell recognition assay (IFN-γ/CD107a co-expression). Sequential gates are applied to identify lymphocytes, singlets, live CD3⁺CD8⁺ T cells. HIV-specific CD8⁺ T cells are identified as dual IFN-γ⁺CD107a⁺ in the CD8⁺ T cell gate (representative unstimulated value: 0.27%).

Article Snippet: After 6 days of expansion, CD8 + T cells were isolated by positive selection using the REAlease CD8 MicroBead Kit following the manufacturer’s instructions (REAlease CD8 MicroBead Kit; Miltenyi, Cat# 130-117-036).

Techniques: Proliferation Assay, Negative Control, Expressing

Journal: bioRxiv

Article Title: NK-like and networked CD8 + T cell immunity mediates exceptional HIV control

doi: 10.64898/2026.07.02.735504

Figure Lengend Snippet: a , Stacked bar plots showing the proportion of cells from each donor (EEC unstimulated, EEC stimulated, and ART stimulated) within each of the 17 transcriptional clusters, confirming that no single cluster is dominated by cells from a single individual. b, Cluster enrichment analysis (log₂ odds ratio, EEC versus ART, stimulated cells) showing the relative enrichment of each cluster in EEC or ART conditions. FDR-adjusted P values are indicated for significantly enriched clusters. c, Volcano plot of differential gene expression analysis comparing cluster 3 with all other clusters, identifying memory and effector T cell markers (up in cluster 3, shown in blue). d, Volcano plot of differential gene expression analysis comparing cluster 14 with all other clusters, identifying canonical stem-like memory T cell markers (up in cluster 14, shown in blue). e, Cytotoxicity module score ( PRF1 , GZMB , NKG7 , GNLY ) across all 17 clusters shown as a violin plot, demonstrating significant enrichment of cytotoxic gene expression in cluster 16 relative to all other clusters (Kruskal-Wallis test, χ² = 8571.8, df = 16, p < 2.2 x 10 - ). Cluster 16 displayed the highest cytotoxicity score (median = 2.10), significantly exceeding all other clusters (pairwise Wilcoxon tests with Benjamini-Hochberg correction, all adjusted p < 2 x 10 - ), consistent with a highly cytotoxic CD8 + T cells population. f, Gene set enrichment analysis (GSEA) bubble plot for cluster 16 versus all other clusters showing top enriched and depleted Hallmark pathways (normalized enrichment scores); IFN-α response and TNF-α signaling pathways are among the top enriched pathways in cluster 16, while cell cycle pathways (mitotic spindle, G2M checkpoint, E2F targets) are depleted. g, GSEA bubble plot for cluster 16 cells from EEC versus ART (pseudobulk), showing further enrichment of IFN-α and IFN-γ response gene sets in EEC-derived cluster 16 cells relative to ART-derived cluster 16 cells.

Article Snippet: After 6 days of expansion, CD8 + T cells were isolated by positive selection using the REAlease CD8 MicroBead Kit following the manufacturer’s instructions (REAlease CD8 MicroBead Kit; Miltenyi, Cat# 130-117-036).

Techniques: Gene Expression, Protein-Protein interactions, Derivative Assay

Journal: bioRxiv

Article Title: NK-like and networked CD8 + T cell immunity mediates exceptional HIV control

doi: 10.64898/2026.07.02.735504

Figure Lengend Snippet: a , Representative gating strategy for sorting pan-KIR⁺ CD8⁺ T cells for global KIR⁺ CD8⁺ T cell scRNAseq. Sequential gates identify lymphocytes (FSC-A vs. SSC-A), singlets (FSC-H vs. FSC-A), live cells (7-AAD⁻), CD3⁺ T cells, CD8⁺ T cells (CD8 vs. CD4), and pan-KIR⁺ CD8⁺ T cells using the APC-conjugated KIR antibody cocktail (5.22% of CD8⁺ T cells in a representative donor). b, Violin plots showing expression levels of individual KIR transcript family members ( KIR2DL1 , KIR2DL3 , KIR3DL1 , KIR3DL2 ) across all 13 transcriptional clusters within the global KIR⁺ CD8⁺ T cell UMAP, confirming that each cluster individually expresses KIR transcripts. Clusters are colored by identity as shown in .

Article Snippet: After 6 days of expansion, CD8 + T cells were isolated by positive selection using the REAlease CD8 MicroBead Kit following the manufacturer’s instructions (REAlease CD8 MicroBead Kit; Miltenyi, Cat# 130-117-036).

Techniques: Expressing

Journal: bioRxiv

Article Title: Mapping the immune landscape in small cell lung cancer unveils a distinct tumor-reactive CD8+ T cell molecular signature

doi: 10.64898/2026.06.29.735200

Figure Lengend Snippet: (a) CD8+ T cells from SCLC biopsies are subsetted from the main UMAP shown in . (b-c) Cytotoxic, exhaustion and proliferation gene markers expressed by different CD8+ effector states are depicted in a dotplot (b) and a feature plots (c). (d) The bar plot represents the distribution and clonality of TCRs on T cells from the SCLC tumor microenvironment. Clonality is quantified as clone size across different T cell states. Clone size refers to the number of cells expressing a specific TCR. (e) The heat map visualization shows the gene-set variation analysis of CD8+ TILs from SCLC patients. The y-axis displays the applied gene signatures, while the x-axis represents the selected CD8+ T cell subsets within the SCLC tumor microenvironment. In addition to known T cell phenotypic gene panels, the NeoTCR8 signature from Lowery et al. (Science, 2022) was applied to the CD8+ TILs.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were enriched using the StraightFrom® Whole Blood CD8 MicroBead Kit (Miltenyi Biotec, Cat# 130-090-878), following the manufacturer’s protocol for positive magnetic selection.

Techniques: Expressing

Journal: bioRxiv

Article Title: Mapping the immune landscape in small cell lung cancer unveils a distinct tumor-reactive CD8+ T cell molecular signature

doi: 10.64898/2026.06.29.735200

Figure Lengend Snippet: (a) The schematic representation summarizes the stepwise prioritization and validation process, narrowing down from an initial pool of 15,627 TCRs to 6 functionally validated tumor-reactive TCRs. (b) Experimental setup for the functional validation of TCR candidates, shortlisted using the NeoTCR8 signature, is depicted. Endogenous TCRs from CD8+ T cells from healthy donors are disrupted using CRISPR followed by transfection with mRNA encoding for TCR candidates. Upon co-culture of T cells and autologous cell lines T cell reactivity was measured in ELISpot and killing assays. Image created with BioRender.com. (c) Flow cytometry data illustrates the expression of candidate TCRs on CD8+ T cells from healthy donors. (d) Flow cytometry data shows MHCI expression on patient-derived xenografts (PDX) from SCLC patient 5, 6 and 7 before and after IFN-γ treatment. (e) IFN-γ ELISPOT assay results demonstrating the immune response elicited by CD8+ T cells from healthy donors transfected with our shortlisted TCR candidates from Patients 5, 6 and 7. (f) Images from the 3D tumor spheroid killing assay for TCR16 from patient 6, where tumor cells are stained in infrared dye (blue), demonstrate disintegration of tumor spheroids when tumor-reactive TCR-expressing T cells are introduced (lower panel), compared to control T cells with non-specific TCRs (upper panel). (g) Tumor spheroid integrity was followed over time in a 3D tumor-killing assay for five TCRs from patient 6. The y-axis shows normalized spheroid intensity (size of the largest object in each well), and the x-axis represents the assay duration. Different colors represent each TCR, as indicated in the legend on the right.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were enriched using the StraightFrom® Whole Blood CD8 MicroBead Kit (Miltenyi Biotec, Cat# 130-090-878), following the manufacturer’s protocol for positive magnetic selection.

Techniques: Biomarker Discovery, Functional Assay, CRISPR, Transfection, Co-Culture Assay, Enzyme-linked Immunospot, Flow Cytometry, Expressing, Derivative Assay, Staining, Control

Journal: bioRxiv

Article Title: Mapping the immune landscape in small cell lung cancer unveils a distinct tumor-reactive CD8+ T cell molecular signature

doi: 10.64898/2026.06.29.735200

Figure Lengend Snippet: (a) The volcano plot shows genes differentially expressed between reactive T cells and non-reactive CD8+ T cells from SCLC biopsies. CD8+ effector_2 cluster was excluded from analysis to avoid bias from potential untested tumor-reactive TCRs. Upregulated genes in reactive T cells appear on the right side of the volcano plot, while downregulated genes are on the left. The legend on the bottom right depicts the colors used to represent gene regulation, and specifically in black, genes related to the TCR alpha and beta chains (filtering criteria for DGEA: p_val_adj < 0.05 & log2FC > 0.5). (b) The dot plot visualizes the expression of molecular signature genes from significantly upregulated genes in reactive T cells and removal of non-significant genes between reactive and non-reactive T cells (p_val_adj < 0.05 & log2FC > 0.35). Expression of signature genes is compared across reactive T cells, non-reactive T cells, and other CD8+ subsets (excluding CD8+ effectors_2). The color gradient represents the magnitude of average expression, ranging from low (blue) to high (red). The size of the circles reflects the fraction of cells expressing the gene in percentage. The genes are ordered according to function. (c) The feature plot shows the spatial distribution across the UMAP of subsetted CD8+ T cells from SCLC patient biopsies enriched in the SCLC_TR signature of tumor-reactive T cells. (d) Representative high-plex immunohistochemistry (IHC) staining illustrates the spatial distribution of mature TLS structures within a SCLC tumor sample with a magnified view of a representative TLS characterized by a core of B cells (CD20+) and follicular dendritic cells (CD21+) surrounded by a ring of CD4+ T reg cells (CD3+ FOXP3+ CD8-), T helper cells (CD3+ FOXP3− CD8−) and cytotoxic CD8+ T cells (CD3+ CD8+) in a peritumoral region (Ki67+) (scale bar: 1.5 mm; inset scalebar: 50 µm). (e) Cell-cell communication analysis showcases elevated predicted chemokine and cytokine signaling between reactive and non-reactive T cells on the bottom as source cells and pDCs, monocytes/macrophages, activated cDC2, cDC2, and B cells on the top as target cells in all SCLC patients. (f-g) Cell-cell communication analysis showcases elevated predicted immunomodulatory ligand-receptor interactions between CD4+ T reg cells, reactive and non-reactive T cells as target cells and pDCs, monocytes/macrophages, activated cDC2, cDC2, and B cells as source cells in all SCLC patients. The circos plot depict either co-stimulatory signaling (f) or co-inhibitory signaling (g), respectively (same legend as in (e)). (h) Violin plots depict the expression of inhibitory receptor expression across tumor-reactive and non-reactive CD8+ T cell subsets. (i) Representative high-plex IHC staining shows the spatial proximity of CD4+ T reg cells (CD3+ FOXP3+ CD8-) and CD8+ T cells (CD3+ CD8+) within SCLC tumor sections from four different patients (scale bar: 50 µm). (j) Faceted box plots illustrate the distribution of average shortest distances (µm) from each cell phenotype to target cell phenotype CD4+ T reg cells (upper panel) and CD8+ T cells (lower panel), respectively.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were enriched using the StraightFrom® Whole Blood CD8 MicroBead Kit (Miltenyi Biotec, Cat# 130-090-878), following the manufacturer’s protocol for positive magnetic selection.

Techniques: Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) UMAP plot of 109,914 HNSCC TILs, including 106,667 CD8 T cells and 3,247 natural killer (NK) cells. (B) Heatmap displaying average Z-scored RNA expression per cluster. Notable genes, including ENTPD1 (CD39), CCR6 , and KLRC1 (NKG2A) are highlighted and boxed in red. (C) Representative plots of NKG2A and CCR6 expression on the total Tex population (CD39+ KLRG1-) for an HPV- and HPV+ HNSCC tumor. (D) Percentage of each Tex subset within the total Tex population (CD39+ KLRG1-) for Tex-Conv ( left , NKG2A- CCR6-), Tex-CCR6 ( middle , NKG2A- CCR6+), and Tex-KLR ( right , NKG2A+ CCR6-; n = 15 HPV-, 16 HPV+; Student’s t test). (E) Mean fluorescence intensity (MFI) of CD39 within each Tex subset and non-Tex cells (CD39-KLRG1-/+; n = 31; Tukey’s multiple comparisons test). (F) Ternary plot of the proportion of cells of each expanded Tex clonotype among the Tex clusters. Each dot represents an expanded Tex clonotype and is sized by the total cells in the clone. The left axis is the proportion of cells in the Tex-KLR cluster with 0 to 1 oriented from bottom to top. The right axis is the proportion of cells in the Tex-CCR6 cluster with 0 to 1 oriented from top to bottom. The bottom axis is the proportion of cells in the Tex-Conv cluster with 0 to 1 oriented from right to left. (G) Density plots of the individual cells fro each biased Tex clonotype category.

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: RNA Expression, Expressing, Fluorescence

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) UMAP plot demonstrating the transfer of cluster annotations from the reference CD8 T cell atlas ( left , Xue et al., 2024) to our HNSCC CD8+ TIL dataset ( right ). (B) Stacked bar plots showing the proportions of the transferred annotations within each of our annotated HNSCC CD8+ TIL clusters. (C) Dot plot of the top five marker genes per cluster (log 2 fold change > 1 and minimum percent of cells in which gene was detected > 50%). Genes of interest, including KLRC1 , CCR6 , and ENTPD1 , are highlighted in red.

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Marker

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Flow cytometry gating strategy for HNSCC patient tumor samples. (B-D) Percentage of (B) CD4 T cells of total T cells, (C) CD8 T cells among of T cells, and (D) exhausted CD8 T cells of CD8 T cells (n = 15 HPV-, 16 HPV+; Student’s t test; box plot whiskers show the full range).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Total number of CD8+ TILs analyzed per HNSCC patient sample. The fill of the bar indicates number of cells with a TCR captured by scTCR-seq. (B) Number of expanded Tex clonotypes biased towards each Tex cluster per patient. (C-D) Cellular composition of the top expanded clonotypes among the (C) Tex population or (D) the total CD8+ TIL population across clusters for all patients with > 600 cells with a TCR.

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques:

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Representative low-magnification images of pan-cytokeratin (PanCK) and TP63 ( top ) in addition to CD8 and CD3E ( bottom ). (B) High-magnification views of the boxed region in (A) for CD8 and NKG2A ( top left ), CD8 ( middle left ), NKG2A ( bottom left ), PanCK and TP63 ( top right ), TOX and PD-1 ( middle right ), and GZMB ( bottom right ). NKG2A+ Tex cells are indicated with a filled arrow, and NKG2A- Tex cells are indicated with an empty arrow. The circle indicates a region of interest. (C) Percentage of non-exhausted ( left ) and exhausted ( right ) cells of the total cells in each 10 µm bin spanning -250 µm to +250 µm relative to the tumor-stroma interface. The lines are the patient-specific locally estimated scatterplot smoothing fits, and shaded ribbons are the 95% confidence intervals for the fit of the line. (D) Percentage of NKG2A+ cells within each CD8 T cell subset (n = 11 samples; Wilcoxon rank-sum test). (E) Percentage of NKG2A+ Tex-KLR cells of total Tex cells in the intra-tumoral and peri-tumoral regions (n = 10 tumors with ≥ 100 cells per region; Wilcoxon signed-rank test).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques:

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Schematic of the avidity-modulated UM-SCC47 cell line models, including HLA-A2 OE with peptide pulsing (500 nM peptide) or natural E7 11-20 presentation ( left ) and the wildtype and mutant E7 11-20 SCT cell lines ( right ). (B) Representative histograms of CD3, CD137, and NKG2A expression on TCR-transduced CD8 T cells, normalized to the mode, following 48 h co-culture with each UM-SCC47 cell line. Data for donor LRS071724 is shown. (C) Representative flow cytometry plots of NKG2A and CD137 expression on TCR-transduced CD8 T cells following 48 h co-culture with each UM-SCC47 cell line. (D-F) Quantification of (D) CD3 mean fluorescence intensity (MFI) relative to T cells alone, (E) CD137 MFI relative to T cells alone, and (F) percentage of NKG2A+ cells among TCR-transduced CD8 T cells (n = 4 donors; repeated-measures Sidak’s multiple comparisons test; donors LRS050924, LRS071724, LRS081224, and LRS081524 were used).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Expressing, Co-Culture Assay, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Flow cytometry gating strategy for analysis of TCR-transduced CD8 T cells following in vitro co-culture. (B) HLA-A2 expression on the UM-SCC47 cell lines displayed as histograms ( left ) and quantified as mean fluorescence intensity (MFI; right ). (C) IL-12p70 concentration (pg mL -1 ) following 48 h of culture in the indicated condition (n = 4 donors; donors 85, 267, 481, and 948 were used).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, In Vitro, Co-Culture Assay, Expressing, Fluorescence, Concentration Assay

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Schematic of the avidity-modulated HEK293T cell line (naturally HLA-A2+) models, including HLA-A2 OE, HPV16 E7 11-20 peptide pulsing (500 nM peptide), and HPV16 E7 OE ( left ), and the wildtype and mutant E7 11-20 SCT cell lines ( right ). (B) Representative histograms of CD3, CD137, and NKG2A expression on TCR-transduced CD8 T cells, normalized to the mode, for each co-culture condition. Data for donor LRS071724 is shown. (C) Representative flow cytometry plots of NKG2A and CD137 expression on TCR-transduced CD8 T cells following 48 h co-culture with each HEK293T cell line. (D-F) Quantification of (D) CD3 mean fluorescence intensity (MFI) relative to T cells alone, (E) CD137 MFI relative to T cells alone, and (F) percentage of NKG2A+ cells among TCR-transduced CD8 T cells (n = 3 donors; repeated-measures Sidak’s multiple comparisons test; donors LRS072323, LRS050924, and LRS071724 were used).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Expressing, Co-Culture Assay, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Representative flow cytometry plots of NKG2A and CD137 expression on TCR-transduced CD8 T cells following 48 h culture in the indicated condition. FK506 was used at 5 nM, and IL-12p70 was used at 10 ng mL -1 . The vehicle control was 0.0005% ethanol. Data for donor LRS081324 is shown. (B-C) Percentage of CD137+ ( left ) and NKG2A+ ( right ) TCR-transduced CD8 T cells in the indicated treatments for (B) T cells alone and stimulation with anti-CD3/anti-CD28 and (C) co-culture with UM-SCC47 target cell lines (n = 3 donors, repeated-measures Tukey’s multiple comparisons test). (D) Percentage of UM-SCC47 target cells killed after 48 h co-culture. Values were mean-centered across experiments for visualization (n = 7 replicates from 4 unique donors, repeated-measures Tukey’s multiple comparisons test).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Expressing, Control, Co-Culture Assay

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Principal component analysis (PCA) plot of the bulk RNA-seq data from isolated TCR-transduced CD8 T cells following 48 hours of co-culture with each UM-SCC47 cell line and treatment condition (n = 4 donors per condition). (B-C) Log 2 normalized counts for (B) TNFRSF9 and (C) KLRC1 . (D) Heatmap of activation-induced genes defined as genes upregulated in the wildtype E7 11-20 SCT or HLA-A2 OE + IL-12p70 condition relative to the empty vector control. Genes are sorted by the log 2 fold change of gene expression in the comparison of HLA-A2 OE + IL-12p70 over wildtype E7 11-20 SCT. (E) Log 2 fold change of the differentially expressed genes for HLA-A2 OE + IL-12p70 versus HLA-A2 OE ( y-axis ) and wildtype E7 11-20 SCT versus HLA-A2 OE ( x-axis ). Points are colored by their respective k-means cluster from the heatmap in (D) . The diagonal lines indicate the 0.75 delta log 2 fold change threshold between the two comparisons. Colored overlays and labels denote the genes selected for the IL-12-specific, shared, and avidity-specific gene sets.

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: RNA Sequencing, Isolation, Co-Culture Assay, Activation Assay, Plasmid Preparation, Control, Gene Expression, Comparison

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Module scores of the bulk RNA-seq gene sets. Datapoints are the median module score per patient among the cells in the cluster (n = 30-32 patients per cluster; Dunnett’s multiple comparisons test). (B) Mean Z-scored RNA expression per cluster of the genes in the bulk RNA-seq gene sets. Genes are sorted by the delta Z-score of the Tex-KLR cluster minus the mean of the Tex-Conv and Tex-CCR6 clusters. Only genes detected in > 5% of cells in at least one cluster are shown. (C) Avidity-specific gene set module score of the cells within representative expanded Tex clonotypes from each quartile (Q). The score distribution of all cells of the expanded Tex clones is overlaid. (D) Mean Z-scored RNA expression for the pseudo-bulked expanded Tex clonotypes of each quartile for selected general Tex genes and Tex-KLR marker genes. The sign and significance of the Spearman correlation between the avidity-specific gene set module score and each gene are indicated in the quartile 1 (Q1) column. (E) RNA expression for the pseudo-bulked expanded Tex clonotypes following variance stabilized transformation (VST). The large points represent the mean expression for the quartile, and smaller points represent individual clonotypes (n = 82 clonotypes for Q1, 81 each for the other quartiles; DESeq2 Wald test). (F) Expression of the top avidity-specific gene set ( Methods ) and the Tex-KLR signature in FACS-sorted CD8+ TIL subsets from human melanoma samples as quantified by UCell (n = 364 clonotypes from 5 patients for singlet vs. cancer cell doublet and n = 137 clonotypes from 3 patients for singlet vs. APC doublet). Statistical significance was tested using linear regression with patient, clonotype, and log10(clonotype cell count) as fixed-effect covariates.

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: RNA Sequencing, RNA Expression, Clone Assay, Marker, Transformation Assay, Expressing, Cell Characterization

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A–D) Smoothed histograms showing the normalized cell density among all expanded Tex clonotypes for each quartile ( solid line ) with the inter-quartile range ( ribbon ). Data are displayed for the expanded Tex clonotypes in our HNSCC CD8+ TIL dataset (A, B) and the treatment-naïve samples in the CD8 T cell atlas dataset (C, D) , organized by the IL-12-specific gene set ( A, C ) and shared gene set ( B, D ) module scores. Only the genes that were present in > 5% of the cells in at least one cluster were used to apply the module score. (E–H) Heatmaps of the mean Z-scored average RNA expression for the pseudo-bulked Tex clonotypes of each quartile. The sign and significance of the Spearman correlation between the respective module score and each gene are indicated in the quartile 1 (Q1) column. Panels display our HNSCC CD8+ TIL dataset ( E, F ) and CD8 T cell atlas dataset ( G, H ) for the IL-12-specific gene set ( E, G ) and shared gene set ( F, H ) module scores.

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: RNA Expression

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) UMAP plot of the CD8 T cell atlas (Xue et al., 2024). (B) Feature plots showing module scores for each bulk RNA-seq gene set. Only the genes that were present in > 5% of the cells in at least one cluster were used to apply the module score. (C) Violin plots of the module score for each bulk RNA-seq gene set. Datapoints indicate the median module score for each sample in the cluster. (D) Avidity-specific gene set module score of the cells within representative expanded Tex clonotypes from each quartile. The distribution of all cells of expanded Tex clones is shown as an overlaid black line. (E) Smoothed histogram of the normalized avidity-specific gene set module score distributions for the cells in the expanded Tex clonotypes ( solid line ) with the inter-quartile range ( shaded ribbon ). (F) Heatmap of the mean Z-scored RNA expression for the pseudo-bulked expanded Tex clonotypes of each quartile for selected general Tex genes and Tex-KLR marker genes. The sign and significance of the Spearman correlation between the avidity-specific gene set module score with each gene are indicated in the quartile 1 (Q1) column. (G) Pseudo-bulked RNA expression of expanded Tex clonotypes following variance stabilized transformation (VST). The large points connected by the line represent the mean expression, and small points represent individual clonotypes (n = 80 clonotypes for Q1-Q3, 79 for Q4; DESeq2 Wald test).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: RNA Sequencing, Clone Assay, RNA Expression, Marker, Transformation Assay, Expressing

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) UMAP plot of an scRNA-seq dataset of murine CD8 T cells collected during the acute and chronic lymphocytic choriomeningitis virus (LCMV) infection models from the spleen, lung, and liver (Daniel et al., 2022). (B) Feature plots of the module score for each bulk RNA-seq gene set. Genes were converted to the equivalent mouse gene, and only the genes within the gene set that were present in > 5% of the cells in at least one cluster were used to apply the module score. (C-E) Module scores of the (C) avidity-specific, (D) IL-12-specific, and (E) shared gene sets in the CD8 T cell clusters from day 21 post-infection with the chronic LCMV strain. (F) Spearman correlation of Klr genes in the dataset with the avidity-specific gene set module score at day 21 post-infection with the chronic LCMV strain. Bars are colored by -log 10 (FDR). (G) Dot plot of selected activation-associated genes among the Tex clusters from day 21 post-infection with the chronic LCMV model.

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Virus, Infection, RNA Sequencing, Activation Assay

Journal: bioRxiv

Article Title: High-avidity TCR signaling induces a distinct KLR-positive exhaustion state in human tumor-infiltrating CD8 T cells associated with immunotherapy response

doi: 10.64898/2026.05.29.728765

Figure Lengend Snippet: (A) Schematic of clinical trial design. Pathologic treatment response was assessed on the surgical specimen. (B) Percent of Tex-Conv ( left , NKG2A-) and Tex-KLR ( right , NKG2A+) cells of all cells within the peri-tumoral region (200 µm into the stromal region from the tumor-stroma interface) and intra-tumoral region in pre-treatment Cohort 1 samples of the tissue microarray (TMA). A Wilcoxon signed-rank test was used in the comparison between the peri-tumoral and intra-tumoral cell densities within each the non-responders (NR) and responders (R; n = 15 NR and 9 R). A Wilcoxon rank-sum test was used in the comparisons between non-responders and responders in the intra-tumoral region (n = 15 NR and 11 R) and peri-tumoral region (n = 16 NR and 9 R). The p values were not adjusted because the comparisons were pre-determined. (C) Representative images of the TMA IF data of Cohort 1 pre-treatment samples. Selected Tex-KLR cells are indicated with a white arrow. (D) Representative images of the whole tissue section IF data of patient OC36 of Cohort 2 at the pre- and post-treatment timepoints. Selected Tex-KLR cells are indicated with a white arrow. (E-G) Percent of each cell type of total cells within the indicated region at the pre- and post-treatment timepoints for (E) Tex-Conv, (F) Tex-KLR, and (G) Non-Tex CD8 T cells (n = 2 non-responders and 2 responders).

Article Snippet: CD8+ TILs were isolated with the REAlease CD8 (TIL) MicroBead Kit (Miltenyi Biotec, 130-121-560) according to the manufacturer’s protocol.

Techniques: Microarray, Comparison

Journal: Nature Communications

Article Title: Tumour-associated high endothelial venules drive portal-specific immune evasion in lymph nodes via ALOX12

doi: 10.1038/s41467-026-72412-w

Figure Lengend Snippet: a Intravital multiphoton microscopy of GFP-EO771 cells and HEVs in the LNs of Chst4-tdTomato mice. Arrowheads indicate HEV-intravasating tumour cells. Orthogonal views (XY, XZ, YZ) of single EO771 cells intravasating blood vessels are shown. Scale bar, 50 μm. b LNs from GFP-EO771-bearing Chst4-tdTomato mice were stained for CD31. Confocal images were tiled to reconstruct a 3D LN vascular network. Yellow arrowheads indicate tumour-associated HEVs and white arrowheads indicate tumour-associated non-HEV blood vessels. Scale bar, 500 μm. Each dot represents data pooled from three sections from each mouse. c The schematic of iterative in vivo selection of Blood Vessel Metastatic (BVM) and HEV Metastatic (HEVM) tumour cells using photoconversion of TDLNs. Representative immunofluorescence images ( d ) and bioluminescent images ( e ) of lungs harvested from mice inoculated with the indicated EO771 derivatives via fat pad injection. Scale bars, 50 μm. Quantitation in Supplementary Fig. and . f Representative flow cytometry plots for CD8 + T cell-mediated cytotoxicity to indicated EO771 derivatives. The unstimulated T cell control and quantitation in Supplementary Fig. , f. g The design of the sLP-mCherry labelling system. h Flow cytometry gating strategy for the isolation of lung metastatic tumour cells disseminating via HEVs (GFP + mCherry + ) and non-HEV vessels (GFP + mCherry − ). i Representative flow cytometry plots for CD8 + T cell-mediated cytotoxicity to indicated tumour cells. The unstimulated T cell control and quantitation in Supplementary Fig. . Immune profiling in mediastinal LNs of BVM3 and HEVM3-bearing mice, including CD40 and CD80 expression in CD103 + DCs ( j , k ), Granzyme B + CD8 + T cells ( l , m ), CD8 + T cell-DC interaction ( n ), MDSCs ( o ) and Treg cells ( p ). Scale bar, 10 μm. Gate strategy, isotype controls and fluorescence minus one (FMO) controls are provided in Supplementary Fig. . n = 6 mice per group ( a , b , j , k , m – p ). Mean ± SD ( b , j , k , m – p ). P -values were calculated by a two-tailed unpaired t -test ( b , j , k , m – p ).

Article Snippet: CD8 + T cells were then isolated using a CD8 + T Cell Isolation Kit (Miltenyi Biotec, #130-117-044).

Techniques: Microscopy, Staining, In Vivo, Selection, Immunofluorescence, Injection, Quantitation Assay, Flow Cytometry, Control, Isolation, Expressing, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Tumour-associated high endothelial venules drive portal-specific immune evasion in lymph nodes via ALOX12

doi: 10.1038/s41467-026-72412-w

Figure Lengend Snippet: a UMAP plots showing cell types identified from paired EO771 tumours and metastatic LNs from 6 mice. ECs, endothelial cells. b Heatmap showing relative expression of marker genes across cell clusters in scRNA-seq of primary tumours and LNs. c UMAP plots showing the expression levels of indicated genes for HECs in LNs. HECs are identified as an endothelial cell subpopulation expressing Glycam1 , Chst4 and Fut7 . d Representative immunofluorescence images show the colocalisation of CD31, PNAd and ALOX12 in human primary tumours, LN without (LN − ) or with metastases (LN + ). Scale bars, 50 μm. Isotype control staining and quantitation in Supplementary Fig. . e Construction diagram of Alox12 fl/fl ; Chst4-CreERT2 mice. f Representative immunofluorescence images showing ALOX12 levels in HECs in LNs from wild-type and Alox12 fl/fl ; Chst4-CreERT2 mice. Scale bars, 20 μm. g Relative content of 12-HETE in LNs from wild-type and Alox12 fl/fl ; Chst4-CreERT2 mice. h Representative bioluminescent images of lungs harvested from indicated mice inoculated with luciferase-expressing EO771 cells in mammary fat pads. Quantitation in Supplementary Fig. . i Representative images (left) and quantitation (right) of immunohistochemistry staining for cleaved caspase-3 (Cl. caspase-3) in the TDLNs and lung metastases of indicated mice inoculated with EO771 cells. Scale bar, 100 μm. Each dot represents data pooled from 5 distinct areas from a slice and 3 slices per mouse. j Representative flow cytometry plots (left) and quantitation (right) for CD8 + T cell-mediated cytotoxicity to vehicle-treated and 12-HETE-treated EO771 cells. Unstimulated T cells were used as a negative control. k Representative bioluminescent images (left) and quantitation (right) of lungs harvested from Alox12 fl/fl ; Chst4-CreERT2 mice inoculated with vehicle-treated and 12-HETE-treated EO771 cells via intravenous injection. n = 3 independent experiments ( g ); n = 6 mice per group ( i , k ); n = 4 independent experiments ( j ). Mean ± SD ( g , i – k ). P -values were calculated by a two-tailed unpaired t -test ( g , i – k ).

Article Snippet: CD8 + T cells were then isolated using a CD8 + T Cell Isolation Kit (Miltenyi Biotec, #130-117-044).

Techniques: Expressing, Marker, Immunofluorescence, Control, Staining, Quantitation Assay, Luciferase, Immunohistochemistry, Flow Cytometry, Negative Control, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: Tumour-associated high endothelial venules drive portal-specific immune evasion in lymph nodes via ALOX12

doi: 10.1038/s41467-026-72412-w

Figure Lengend Snippet: a Recombinant ADAR1 p150 was mixed with dsRNA in the presence or absence of 12-HETE, and the fraction of edited dsRNA was quantified. b ADAR1 editing activity in EO771 cells with indicated treatment was measured as the ratio between luminescence from Nano luciferase/Firefly luciferase. c Quantitation for CD8 + T cell-mediated cytotoxicity to EO771 cells with indicated treatment. Representative bioluminescent images of lungs ( d ) and immunohistochemistry staining for CD8α and NK1.1 in the lung metastases ( e ) from the indicated EO771-bearing mouse with the indicated treatment. Scale bars, 100 μm. Quantitation in the Supplementary Fig. . f ADAR1 editing activity in Adar1 -knockout EO771 cells expressing indicated ADAR1 variants with or without 12-HETE treatment was measured as the ratio between luminescence from Nano luciferase/Firefly luciferase. Quantitation of ADAR1 p150 and dsRNA concentrations in indicated phases ( g ) and volume-normalised editing rate in indicated phases and droplets equivalent concentration (DEC) ( h ). i Representative images of droplets formed by 100 nM Cy5-dsRNA with 100 nM wild-type ADAR1 p150-GFP or mutant ADAR1 p150-GFP (E861A) with or without 100 nM 12-HETE. Scale bars, 5 μm. Quantitation in the Supplementary Fig. . j Indicated recombinant ADAR1 p150 proteins were mixed with dsRNA in the presence or absence of 12-HETE, and the fraction of edited dsRNA was quantified. The blue P -values for (wild-type ADAR1 p150 + 12-HETE) versus (wild-type ADAR1 p150 + vehicle), the violet P -values for (ADAR1 p150 (E861A) + 12-HETE) versus (wild-type ADAR1 p150 + 12-HETE) and the red P -values for (ADAR1 p150 (E861A) + vehicle) versus (wild-type ADAR1 p150 + vehicle). k Representative flow cytometry plots (left) and quantitation (right) for CD8 + T cell-mediated cytotoxicity to indicated EO771 cells with indicated treatment. Unstimulated T cell control in Supplementary Fig. . n = 4 independent experiments ( a – c , f – h , j , k ). Mean ± SD ( a – c , f – h , j , k ). P -values were calculated by two-tailed unpaired t -test ( a , f , g , j , k ) and one-way ANOVA with Tukey’s multiple comparisons test ( b , c , h ).

Article Snippet: CD8 + T cells were then isolated using a CD8 + T Cell Isolation Kit (Miltenyi Biotec, #130-117-044).

Techniques: Recombinant, Activity Assay, Luciferase, Quantitation Assay, Immunohistochemistry, Staining, Knock-Out, Expressing, Concentration Assay, Mutagenesis, Flow Cytometry, Control, Two Tailed Test