Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Representative anamnestic vaccine-induced immune responses in the vaccinees. All vaccinees made CD8+ T-cell responses to several epitopes, including some restricted by the MHC-I molecule Mamu-A*02. In the interest of space, we are showing only two of the vaccinees in this figure; data from the remaining vaccinees are shown elsewhere (see Fig. S3 in the supplemental material). Whole-PBMC responses are indicated by blue bars, with responses postchallenge indicated in dark blue and responses observed immediately prior to challenge indicated in light blue. Responses observed in PBMCs depleted of CD8+ cells (red) are likely mediated by CD4+ T cells. CD8+ cell depletion was typically 99% complete (data not shown). Dark red bars indicate responses observed postchallenge, whereas light red bars indicate responses present immediately prior to the challenge. Green bars represent responses to minimal optimal peptides that bind to Mamu-A*02. As indicated in Table S1 in the supplemental material, some of these epitopes are conserved between SIVmac239 and SIVsmE660, whereas others have several substitutions, some of which could affect T-cell recognition. Again, light green bars indicate responses observed prior to the challenge, whereas dark green bars indicate anamnestic responses. Most anamnestic response analyses were performed at 14 to 15 days postinfection. For a couple of the animals (r00061, r02103), these assays were delayed to 21 days postinfection due to the very low viral loads observed.

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Frequency of anamnestic cellular immune responses in PBMC and PBMC depleted of CD8 + cells

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal:

Article Title: Vaccine-Induced Cellular Responses Control Simian Immunodeficiency Virus Replication after Heterologous Challenge ▿ †

doi: 10.1128/JVI.00272-09

Figure Lengend Snippet: Breadth of anamnestic cellular immune responses in PBMC and PBMC depleted of CD8 + cells

Article Snippet: Additionally, we examined responses by CD8-negative cells by depleting PBMCs of CD8 + cells using a CD8 MicroBead kit for nonhuman primates (Miltenyi Biotec) according to the manufacturer's instructions.

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Plasmid-based donor templates enable efficient nonviral gene editing of TRAC locus in primary T cells. (A–C) Titration of linear dsDNA donor template. (A) Diagram of linear dsDNA knock-in construct TRAC -mNG. (B) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of linear dsDNA donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (C) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (D–F) Titration of pUC57 plasmid donor template. (D) Diagram of pUC57 knock-in construct TRAC -mNG. (E) Bar graphs showing the frequency of CD8 + T cells expressing mNG, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of pUC57 plasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (F) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (G–I) Titration of nanoplasmid donor template. (G) Diagram of nanoplasmid knock-in construct TRAC -mNG. (H) Bar graphs showing the frequency of CD8 + T cells expressing mNG, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with 1, 2, 4, 6, or 8 µg of nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). (I) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. This experiment was performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Titration, Knock-In, Construct, Cell Recovery, Electroporation, Expressing

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Optimization of nonviral gene editing in primary T cells using plasmid-based donor templates. (A–F) Titration of linear dsDNA and nanoplasmid donor templates in CD8 + T cell cultures in RPMI/10% FBS medium. (A) Diagram of linear dsDNA knock-in construct TRAC -mNG. (B) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (C) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8 + T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of linear dsDNA donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (D) Diagram of nanoplasmid knock-in construct TRAC -mNG. (E) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (F) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) of CD8 + T cells cultured in RPMI/10% FBS 3 d after electroporation with 1, 2, or 4 µg of nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (G) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus in the presence of absence of PGA. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (H) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery 3 d after electroporation with 2 µg of either linear dsDNA or nanoplasmid donor template that either did or did not contain truncated Cas9 target sequences (tCTS) together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment has been performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001 in RM one-way ANOVA with Geisser–Greenhouse correction (C and F) or paired t test (G and H).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Titration, Knock-In, Construct, Expressing, Cell Recovery, Cell Culture, Electroporation

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Cytokine production and stress response induced in T cells following exposure to dsDNA donor templates. (A) IFN-α measured by Simoa and IFN-γ, TNF-α, and IL-2 measured by Luminex from CD8 + T cells 18 h after transfection with Cas9-RNP targeting the TRAC locus alone or together with nanoplasmid donor template compared with non-transfected control T cells (No RNP). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed once for Simoa and twice for Luminex. (B) GSEA from RNA-sequencing of CD8 + T cells after transfection with Cas9-RNP targeting the TRAC with nanoplasmid donor template compared with Cas9-RNP alone. Gene sets for IFN-γ response, IFN-α response, TNF-α signaling, and inflammatory response were significantly enriched. (C) GSEA from RNA-seq of CD8 + T cells after transfection with Cas9-RNP targeting the TRAC with linear dsDNA donor template compared to Cas9-RNP alone. Gene sets for IFN-γ response, IFN-α response, TNF-α signaling, and inflammatory response were significantly enriched. (B and C) The y axis represents enrichment score, and on the x axis are genes (vertical black lines) represented in gene sets. The colored band at the bottom represents the degree of differentially expressed genes (red for upregulation and blue for downregulation). (D) Gene set enrichment analysis of all 375 upregulated genes in both Nanoplasmid/Cas9-RNP and linear dsDNA/Cas9-RNP over Cas9-RNP-only using the GSEA MSigDB Hallmark 2020. (E–H) Heatmaps showing upregulated genes in Nanoplasmid/Cas9-RNP and linear dsDNA/Cas9-RNP over Cas9-RNP-only that mostly contributed to IFN-α response (E), TNF-α response (F), apoptosis (G), or inflammatory response (H; all MSigDB Hallmark). Color-coded by the normalized RNA-seq count data with variance stabilizing transformation (VST). This experiment was performed once. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 in one-way ANOVA.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Luminex, Transfection, Control, RNA Sequencing, Transformation Assay

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Optimization of CRISPR/Cas9-mediated gene knock-in with plasmid-based donor DNA in CD4 + and CD8 + T cells. (A and B) Homology arm optimization for plasmid-based donor templates. (A) Representative contour plots showing the frequency of CD8 + T cells expressing mNG. (B) Bar graphs depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation with pUC57 plasmid or nanoplasmid donor templates with homology arm lengths between 100 bp and 2,000 bp (amounts equimolar to 4 µg of the pUC57 2,000 bp construct) together with Cas9-RNP targeting the TRAC locus ( n = 2). Circles represent individual donors; bars represent median values with range. This experiment was performed three times. (C) Frequency of CD8 + T cells expressing mNG, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 3 d after electroporation after stimulating cells for 24, 36, 48, or 72 h prior to electroporation with nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus ( n = 4). Circles represent individual donors; bars represent median values with range. This experiment was performed twice. (D) Nucleofection pulse code optimization in CD8 + T cells electroporated with nanoplasmid donor template and Cas9-RNP targeting the TRAC locus. Graph shows frequency of cells expressing mNG and edited cell recovery (mNG-positive cells) 3 d after electroporation. Each circle represents a distinct pulse code. Data are representative of three independent CD8 + T cell donors. This experiment was performed twice. (E and F) Gene editing targeting the TRAC locus in CD4 + T cells. Representative contour plot showing the frequency of CD4 + T cells expressing mNG (E) and bar graphs (F) depicting knock-in efficiency, cell viability, total cell recovery, and edited cell recovery (mNG-positive cells) 5 d after electroporation of CD4 + T cells with TRAC -mNG nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus ( n = 3). Circles represent individual donors; bars represent median values with range. This experiment was performed twice. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: CRISPR, Gene Knock-In, Plasmid Preparation, Expressing, Knock-In, Cell Recovery, Electroporation, Construct

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral TCR editing using plasmid DNA donors. (A) Diagram of TCR α and β genomic loci. V gene (purple), D gene (red), J gene (blue), and constant region (green) segments. sg TRAC and sg TRBC targeting sites are indicated. (B) Diagrams of nanoplasmid knock-in constructs TRAC -1G4TCR, TRAC -TCR6-2, and TRAC -CD19CAR. (C, E, and G) Representative contour plots (left) and bar graphs (right) showing the frequencies of CD8 + T cells expressing (C) a NY-ESO-1-specific 1G4 TCR, (E) a CMV-specific pp65 6-2 TCR, and (G) a CD19-CAR 5 d after electroporation using nanoplasmid donor templates together with Cas9-RNPs targeting the TRAC locus. (D, F, and H) Bar graphs showing the cell viability, total cell recovery, and edited cell recovery 5 d after electroporation using nanoplasmid donor templates encoding (D) a NY-ESO-1–specific 1G4 TCR, (F) a CMV-specific pp65 6-2 TCR, and (H) a CD19-CAR together with Cas9-RNPs targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (I) Lactate levels in culture supernatant analyzed by luminescence using the Lactate-Glo Assay were measured 1, 3, 5, and 7 d after transfection of CD8 + T cells with sg TRAC /sg TRBC Cas9-RNP (RNP only) or sg TRAC /sg TRBC Cas9-RNP and nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) compared with non-transfected control T cells (No RNP); RLU, relative light units. (J) Number of cells recovered from cultures 7 d after transfection of CD8 + T cells with sg TRAC /sg TRBC Cas9-RNP (RNP only) or sg TRAC /sg TRBC Cas9-RNP and nanoplasmid donor template targeting the TRAC locus (RNP + nanoplasmid) compared with non-transfected control T cells (No RNP). This experiment was performed three times. *, P < 0.05 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Glo Assay, Transfection, Control

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral TCR editing in CD4 + and CD8 + T cells using plasmid DNA donors. (A) TCR expression on the cell surface by flow cytometry of CD8 + T cells 48 h after transfection with Cas9-RNP targeting the TRAC (sg TRAC ) or TRBC (sg TRBC ) loci. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (B–G) TCR editing in CD4 + T cells. Representative contour plots showing the frequencies of CD4 + T cells expressing a NY-ESO-1-specific 1G4 TCR (B), a CMV-specific pp65 6-2 TCR (D), and a CD19-CAR (F) and bar graphs showing the knock-in efficiency and cell viability 5 d after electroporation using nanoplasmid donor templates encoding a NY-ESO-1-specific 1G4 TCR (C), a CMV-specific pp65 6-2 TCR (E), and a CD19-CAR (G) together with Cas9-RNPs targeting the TRAC locus. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (H and I) Diagram depicting all possible translocation events between the TRAC , TRBC1 , and TRBC2 genomic loci (H). Bar graph (I) showing the frequencies of individual translocation events between the TRAC , TRBC1 , and TRBC2 genomic loci quantified by ddPCR in CD8 + T cells co-transfected with Cas9-RNPs targeting the TRAC and TRBC loci or in non-transfected control T cells. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (J and K) Representative histograms (J) and bar graphs (K) showing proportions of CD137-expressing pp65 TCR knock-in CD8 + T cells stimulated with indicated concentrations of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (L) Bar graphs showing IFN-γ and TNF-α production by pp65 TCR knock-in CD8 + T cells stimulated with indicated concentrations of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (M) Representative histograms showing the frequencies of CFSE-positive target cells and CFSE-negative reference cells in co-cultures with pp65 TCR knock-in CD8 + T cells in the absence or presence of the cognate peptide. (N) Graphs showing specific lysis calculated in the absence of peptide or with 0.1 µM of pp65 495–503 peptide. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed twice. (O) Bar graphs showing IFN-γ and TNF-α production by TCR6-2 (irrelevant TCR) or CD19-CAR knock-in CD4 + T cells from two donors (D1 and D2) in co-cultures with CD19-expressing B cells. Circles represent technical replicates; bars represent median values with range ( n = 9). This experiment was performed twice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction (A and K), paired t test (N), and one-way ANOVA (O).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Plasmid Preparation, Expressing, Flow Cytometry, Transfection, Knock-In, Electroporation, Translocation Assay, Control, Lysis

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: TCR-engineered T cells recognize and kill antigen-expressing target cells. (A and B) Representative histograms (A) and bar graphs (B) showing proportion of CD137 expression of 1G4 TCR knock-in CD8 + T cells stimulated with indicated concentrations of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (C and D) Bar graphs showing IFN-γ (C) or TNF-α (D) production by 1G4 TCR knock-in CD8 + T cells stimulated with indicated concentrations of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (E) Representative histograms showing the frequencies of CFSE-positive target cells and CFSE-negative reference cells in co-cultures with 1G4 TCR knock-in CD8 + T cells in the absence or presence of the cognate peptide. (F) Graphs showing specific lysis calculated in the absence of peptide or with 0.1 µM of NY-ESO-1 157–165 peptide. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (G) Bar graphs showing IFN-γ, TNF-α, and granzyme B (GzmB) production by TCR knock-out or 1G4 TCR knock-in CD8 + T cells from three donors co-cultured with A-375 cells that express the NY-ESO-1 antigen. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. (H) Representative images for A-375 cells that express the NY-ESO-1 antigen and were labeled with a cytoplasmic dye and co-cultured with TCR knock-out CD8 + T cells (left) or 1G4 TCR knock-in CD8 + T cells (right) 2 and 18 h after culture seeding in the presence of caspase 3/7-green apoptosis reagent. Scale bars indicate 300 µm distance. (I) Representative target cell killing over time as measured by the Cas3/7-positive object count in co-cultures of A-375 cells expressing the NY-ESO-1 antigen and labeled with a cytoplasmic dye and co-cultured with TCR knock-out CD8 + T cells (open circles) or 1G4 TCR knock-in CD8 + T cells (filled circles). Mean values ± SD of six technical replicates. This experiment was performed twice with three independent donors per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in RM one-way ANOVA with Geisser–Greenhouse correction (B–D); paired t test (F); one-way ANOVA (G); or Tukey’s multiple comparisons test, two-way ANOVA (I).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Expressing, Knock-In, Lysis, Knock-Out, Cell Culture, Labeling

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Kinetics of gene expression following transient transfection of linear dsDNA, plasmid, and nanoplasmid. (A) Diagram of nanoplasmid knock-in construct RAB11A -YFP. (B and C) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (B) and bar graphs (C) depicting frequency of YFP expression, cell viability, total cell recovery, and edited cell recovery 3, 5, or 7 d after electroporation with promoter-containing nanoplasmid donor template together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. (D) Diagram of linear dsDNA knock-in construct RAB11A -YFP. (E and F) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (E) and bar graph (F) depicting frequency of YFP expression 3, 5, or 7 d after electroporation with promoter-containing linear dsDNA donor templates together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed once. (G) Diagram of pUC57 plasmid knock-in construct RAB11A -YFP. (H and I) Representative histograms showing the frequencies of CD8 + T cells expressing YFP (H) and bar graph (I) depicting frequency of YFP expression 3, 5, or 7 d after electroporation with promoter-containing pUC57 plasmid donor templates together with (red) or without (blue) Cas9-RNPs targeting the RAB11A locus. Circles represent technical replicates; bars represent median values with range ( n = 3). This experiment was performed twice. *, P < 0.05; **, P < 0.05; ***, P < 0.001 in Sidak’s multiple comparisons test with RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Expression, Transfection, Plasmid Preparation, Knock-In, Construct, Expressing, Cell Recovery, Electroporation

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Generation of reporters of gene expression. (A) Diagram of nanoplasmid knock-in construct RAB11A -YFP. (B and C) Histogram overlay for YFP expression (B) and bar graphs (C) showing the frequency of YFP expression and cell viability of CD8 + T cells transfected with RAB11A -YFP nanoplasmid with or without RAB11A targeting Cas9-RNP 10 d after electroporation. Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (D) Diagram of nanoplasmid knock-in construct AAVS1- mNG. (E and F) Histogram overlay for mNG expression (E) and bar graphs (F) showing the frequency of mNG expression and cell viability of CD8 + T cells transfected with AAVS1- mNG nanoplasmid with or without AAVS1 targeting Cas9-RNP 10 d after electroporation. Circles represent individual donors, and bars represent median values with range ( n = 4). This experiment was performed three times. (G) Diagram of nanoplasmid knock-in construct CD4- mNG. (H and I) Representative contour plots (H) and bar graphs (I) showing the frequency of CD4 + and CD8 + T cells expressing mNG and cell viability 10 d after electroporation of a nanoplasmid donor template and Cas9-RNP targeting the CD4 locus. Circles represent individual donors, and bars represent median values with range ( n = 4 for CD4 + T cells, n = 3 for CD8 + T cells). This experiment was performed twice. (J) Histogram overlay for CD4 expression in CD4 + T cells transfected with CD4- mNG nanoplasmid together with a non-targeting control Cas9-RNP (sgNTC) or a Cas9-RNP targeting the CD4 locus (sg CD4 ) 10 d after electroporation. (K) Diagrams of nanoplasmid knock-in constructs TNFRSF9 -mNG and RAB11A -YFP (left) and representative contour plots (right) showing the frequency of CD8 + T cells expressing CD137 and mNG after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP either without restimulation or 6 h after restimulation with Transact. (L) Bar graphs showing the frequency of YFP (blue) and mNG (red) expressing CD8 + T cells over time after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP and restimulation with Transact at time 0 h. Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. (M) Bar graphs showing the geometric mean fluorescent intensity (gMFI) of CD137 expression in CD8 + T cells over time after electroporation with a nanoplasmid mNG reporter construct targeting the TNFRSF9 locus or a constitutive YFP expressing construct targeting the RAB11A locus together with the respective Cas9-RNP and restimulation with Transact at time 0 h ( n = 4). Circles represent individual donors; bars represent median values with range. *, P < 0.05; **, P < 0.01 in paired t test (C, F, I, and J) or in RM one-way ANOVA with Geisser–Greenhouse correction (L).

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Expression, Knock-In, Construct, Expressing, Transfection, Electroporation, Control

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Multiplexed gene knock-in in human T cells. (A–C) Diagrams of nanoplasmid knock-in constructs are provided on the top. Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 + T cells expressing mNG (A) 10 d after electroporation with a nanoplasmid TRAC -mNG donor template and Cas9-RNPs targeting the TRAC locus, mCherry (B) 10 d after electroporation with a nanoplasmid TRAC -mCherry donor template and Cas9-RNPs targeting the TRAC locus, or either mNG or mCherry (C) 10 d after electroporation with two nanoplasmid donor templates ( TRAC -mNG and TRAC -mCherry) and Cas9-RNPs targeting the TRAC locus. Graph on the right for C indicates proportion of transgene expressing cells that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 3). This experiment was performed three times. (D–F) Diagrams of nanoplasmids used in dual targeting study, RAB11A -YFP and TRAC -mCherry (D); representative contour plot (E) showing the frequency of CD8 + T cells expressing YFP, mCherry, or both; and bar graphs (F) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with nanoplasmid donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci. (G) Proportion of transgene expressing T cells co-transfected with nanoplasmid donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci that express YFP (green), mCherry (red), or both (blue). Circles represent individual donors, and bars represent median values with range ( n = 4). This experiment was performed three times. (H) Diagrams of nanoplasmids used in dual targeting study, AAVS1 -mNG and TRAC -mCherry. (I and J) Representative contour plot showing the frequency of CD8 + T cells expressing mNG, mCherry or both (I) and bar graphs (J) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with nanoplasmid donors AAVS1 -mNG and TRAC -mCherry and Cas9-RNPs targeting the AAVS1 and TRAC loci. (K) Proportion of transgene expressing cells co-transfected with nanoplasmid donors AAVS1 -mNG and TRAC -mCherry and Cas9-RNPs targeting the AAVS1 and TRAC loci that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed twice. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Knock-In, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Multiplexed gene knock-in in human T cells. (A–C) Diagrams of pUC57 plasmid knock-in constructs are provided on the top. Representative contour plots (left) and bar graphs (right) showing the frequency of CD8 + T cells expressing mNG (A) 10 d after electroporation with a pUC57 plasmid TRAC -mNG donor template and Cas9-RNPs targeting the TRAC locus ( n = 3), mCherry (B) 10 d after electroporation with a pUC57 plasmid TRAC -mCherry donor template and Cas9-RNPs targeting the TRAC locus ( n = 3), or either mNG or mCherry (C) 10 d after electroporation with two pUC57 plasmid donor templates ( TRAC -mNG and TRAC -mCherry) and Cas9-RNPs targeting the TRAC locus ( n = 3). Graph on the right for C indicates proportion of transgene expressing cells that express mNG (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range. This experiment was performed three times. (D) Diagrams of pUC57 plasmids used in dual targeting study, RAB11A -YFP and TRAC -mCherry. (E and F) Representative contour plot showing the frequency of CD8 + T cells expressing YFP, mCherry or both (E) and bar graphs (F) showing knock-in efficiency, cell viability, and total cell recovery of CD8 + T cells 10 d after electroporation with pUC57 donors RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci. (G) Proportion of transgene expressing cells co-transfected with pUC57 donor templates RAB11A -YFP and TRAC -mCherry and Cas9-RNPs targeting the RAB11A and TRAC loci that express YFP (green), mCherry (red), or both (blue). Circles represent individual donors; bars represent median values with range ( n = 4). This experiment was performed three times. *, P < 0.05; **, P < 0.01 in RM one-way ANOVA with Geisser–Greenhouse correction.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: Gene Knock-In, Plasmid Preparation, Knock-In, Construct, Expressing, Electroporation, Cell Recovery, Transfection

Journal: The Journal of Experimental Medicine

Article Title: High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA

doi: 10.1084/jem.20211530

Figure Lengend Snippet: Nonviral CRISPR gene editing with large payloads. (A) Diagram of nanoplasmid knock-in constructs TRAC _NotchICD_mNG, TRAC_ NotchICD_1G4, and TRAC _THEMIS_1G4. (B, D, and F) Representative contour plots showing the frequency of CD8 + T cells expressing mNG (B) or 1G4 TCR (D and F) 5 d after electroporation of a NotchICD_mNG (B), NotchICD_1G4 (D), or THEMIS_1G4 (F) nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. (C, E, and G) Bar graphs showing the frequency of CD8 + T cells expressing mNG (C) or 1G4 TCR (E and G) and cell viability 5 d after electroporation of a NotchICD_mNG (C), NotchICD_1G4 (E), or THEMIS_1G4 (G) nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors, and bars represent median values with range ( n = 3). This experiment was performed three times. *, P < 0.05; **, P < 0.01 in paired t test.

Article Snippet: Primary human CD8 + and CD4 + T cells were isolated by positive selection from buffy coats using the StraightFrom Buffy Coat CD8 MicroBead Kit or CD4 MicroBead Kit, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).

Techniques: CRISPR, Knock-In, Construct, Expressing, Electroporation

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: Total CD8 + T cells from individuals without HIV were treated with medium, vehicle (Veh.) control, or the GSK3 inhibitor (inh), followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( A and B ) Analysis of CD8 + T cell subpopulations in unstimulated cells ( n = 4). ( C ) Fold change of CD8 + T cell subpopulations upon vehicle control or GSK3 inhibitor treatment, relative to the medium alone condition ( n = 4). ( D ) Expression of TCF-1 in CD8 + T cell subsets ( n = 4). ( E ) Fold change in the expression of the indicated markers induced by anti-CD3/anti-CD28 antibody stimulation relative to unstimulated cells ( n = 5). ( F ) Analysis of dead cells by Aqua LIVE/DEAD + staining (Aqua L/D) among total and T-bet + CD8 + T cells, and fold change in dead CD8 + T cells induced by anti-CD3/anti-CD28 stimulation relative to the unstimulated (Unstim.) condition ( n = 5). ( G ) Frequencies of granzyme B + (GZMB + ), IL-2 + , IFN-γ + , and TNF-α + CD8 + T cells after anti-CD3/anti-CD28 stimulation. ( H ) Expression of 1 to 4 functions in CD8 + T cells ( n = 5). * P < 0.05, by Dunn’s test ( B ) and Wilcoxon test ( C , D , and F – H ). Data obtained from 2 ( A – F ) or 3 ( G and H ) independent experiments are shown.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Incubation, Expressing, Staining

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: ( A and B ) Total CD8 + T cells from individuals without HIV were treated with vehicle control or the GSK3 inhibitor, followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( A ) Analysis of the expression of 2-NBDG, BODIPY, MitoTracker Green, and CellROX among CD8 + T cell subsets. ( B ) Frequencies of 2-NBDG + , BODIPY + , MitoTracker Green + , and CellROX + cells among CD8 + T cell subpopulations after stimulation ( n = 5). ( C and D ) Total CD8 + T cells from individuals without HIV were treated with vehicle control or the GSK3 inhibitor, followed by incubation under basal conditions or with anti-CD3/anti-CD28 stimulation for 48 hours. ( C ) Flow cytometric analysis of the expression of p-S6 and p-AKT in total CD8 + T cells, and frequencies of p-S6 + p-AKT + , p-S6 – p-AKT + , and p-S6 – p-AKT – subsets in total CD8 + T cells ( n = 7). ( D ) Flow cytometric analysis of IL-2 and IFN-γ expression among TNF-α + CD8 + T cells, after anti-CD3/anti-CD28 stimulation. Histograms show the expression of p-S6 in the indicated cell subsets, in reprogrammed and nonreprogrammed cells. Frequency of p-S6 + cells among TNF-α + IFN-γ + IL-2 + or TNF-α + IFN-γ – IL-2 – subsets ( n = 6 individuals without HIV). * P < 0.05 and ** P < 0.01, by Wilcoxon test ( B and C ) and Šidák’s multiple-comparison test ( D ). Data are from 2 independent experiments.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Incubation, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: After treatment with the GSK3 inhibitor, CD8 + T cells from people with HIV were stained with HLA-matched HIV dextramers for analysis of the phenotype of HIV dextramer + cells. ( A ) UMAP plots generated from HIV dextramer + CD8 + T cells after data concatenation ( n = 4). Vehicle control and GSK3 inhibitor treatments as well as CD8 + T cell subpopulations were identified by manual gating and projected into the UMAP space. ( B ) Analysis of the expression of CCR7, CD27, and TCF-1 expression in HIV dextramer + CD8 + T cells ( n = 7). ( C ) Frequencies of CD8 + T cell subpopulations among HIV dextramer + CD8 + T cells ( n = 7). ( D – G ) After reprogramming, cells from people with HIV were stimulated for 6 hours with Gag peptides for analysis of the total frequency (IFN-γ + or CD107a + or IL-2 + or TNF-α + ) of antigen-specific CD8 + T cells ( n = 5) ( D ), the proportion of memory T cell subpopulations ( n = 5) ( E ), the expression of CD107a, IFN-γ, granzyme B, IL-2, and TNF-α on a per-cell basis ( n = 5–11) ( F ), and the frequency of polyfunctional cells ( n = 6) ( G ). ( H and I ) Cells from people with HIV were stimulated for 6 days with Gag peptides and restimulated with the same peptides for another 12 hours, followed by analysis of the viability of proliferating HIV Gag–specific CD8 + T cells ( n = 6) ( H ) and frequencies of the total (live IFN-γ + , IL-2 + , or TNF-α + ) HIV Gag–specific response ( n = 8) ( I ). ( J and K ) After reprogramming, cells from people with HIV ( n = 6) were stimulated for 6 hours with Gag peptides, followed by analysis of p-S6 + p-AKT – , p-S6 + p-AKT + , p-S6 – p-AKT + , and p-S6 – p-AKT – cell subsets ( J ) and the intensity of expression of p-S6 and p-AKT ( K ) in HIV Gag–specific (IFN-γ + and/or IL-2 + ) CD8 + T cells. * P < 0.05 and *** P < 0.001, by Wilcoxon test. Data are from 3 independent experiments.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Staining, Generated, Control, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: HIV-1 BaL–superinfected CD4 + T cells from people with HIV were cultured alone or in the presence of autologous nonreprogrammed or reprogrammed CD8 + T cells. After 7 days, the levels of infection were measured by flow cytometry (KC57 anti-Gag antibody) or ELISA (p24 in culture supernatant). ( A ) Representative flow cytometric analysis of the frequency of infected CD4 + T cells (from a total of 4 donors). ( B ) HIV-suppressive capacity of nonreprogrammed and reprogrammed CD8 + T cells (log 10 decrease of p24 levels in culture supernatant; n = 5 individuals, with the median of triplicates for each experiment). ( C and D ) The frequency of IFN-γ + HIV-specific CD8 + T cells ( C ) and expression of CCR7, PD-1, and LAG-3 in HIV-specific CD8 + T cells ( D ) were measured after 7 days of coculturing. * P < 0.05 and ** P < 0.01, by Wilcoxon test.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Cell Culture, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Reprogramming dysfunctional CD8 + T cells to promote properties associated with natural HIV control

doi: 10.1172/JCI157549

Figure Lengend Snippet: ( A ) Total CD8 + T cells from people without HIV were treated with vehicle control or the GSK3 inhibitor, followed by evaluation of Eomes and CD122 expression in CD8 + T cell subsets ( n = 5). ( B ) After vehicle control or GSK3 inhibitor treatment, CD8 + T cells from people without HIV were left unstimulated or stimulated with IL-7 or IL-15 for 6 days, followed by analysis of cell proliferation ( n = 5; data are from 2 independent experiments). ( C and D ) CD8 + T cells from people with HIV were treated with vehicle control or the GSK3 inhibitor, followed by stimulation with IL-15 for 6 days, for analysis of the proliferation of HIV dextramer + CD8 + T cells ( n = 4; data from 3 independent experiments). * P < 0.05, by Wilcoxon test.

Article Snippet: For reprogramming of CD8 + T cells followed by antigen-specific stimulation, we used PBMCs and magnetically separated CD8 + T cells and non-CD8 + T cells (REAlease CD8 MicroBead Kit; Miltenyi Biotec).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy

doi: 10.1101/2025.10.11.681829

Figure Lengend Snippet: A) Experimental layout describing FACS-based human NSCLC TIL screen for regulators of proliferation in CD8+ and CD8+ DP TILs. B) Z-scores of DP screen versus z-scores of CD8+ TILs from DESeq2 analysis. To ensure robust coverage of each TF, TF barcodes with less than 500 mean counts were filtered out. ORF enrichment was defined using a paired two-tailed DESeq2 test with Benjamini–Hochberg correction and using a p. value cutoff of < 0.01. C) Proliferation validation in four distinct NSCLC preREP TIL patient samples for RELB ORF overexpression versus Thy1.1 ORF control overexpression. D) Flow cytometry analysis at the end of proliferation experiment for T cell lineage (CD8), tissue residency (CD103), and exhaustion (CD39, LAG3, PD-1, TIGIT) markers.

Article Snippet: CD8+ TILs were isolated using a TIL CD8+ magnetic isolation kit (REAlease® CD8 (TIL) MicroBead Kit, human, Miltenyi) and CD8+ CD103+ CD39+ TILs were FACS sorted (n = 2 patient samples), activated with Transact (Miltenyi) according to manufacturer’s instructions, and transduced (n = 2 transduction replicates) with the MORF library at 8% v/v.

Techniques: Two Tailed Test, Biomarker Discovery, Over Expression, Control, Flow Cytometry

Journal: bioRxiv

Article Title: RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy

doi: 10.1101/2025.10.11.681829

Figure Lengend Snippet: A) Experimental layout describing transcriptomic and T cell receptor diversity profiling via bulk RNA-seq and TCR-seq on CD4+, CD8+, and CD8+ DP NSCLC TIL subsets. B) Effective TCR clone number in initial (day 0) and expanded (day 19). Expanded conditions were either not transduced with lentivirus or transduced with corresponding GFP+ORF (Thy1.1 or RELB) lentivirus 24 hours post-activation. P values were calculated using paired t-tests. (C) Principal component analysis (PCA) plots for all RNA-seq samples analyzed with DESeq2. (D) Significance (p adj ) versus fold change between RELB and Thy1.1 samples in different TIL subtypes. Gene enrichment was defined using a paired two-tailed DESeq2 test with Benjamini–Hochberg correction and a p adj cutoff of <0.05. (E) Heatmap of relevant genes related to T cell memory, exhaustion, and costimulation. Each column represents a different patient sample within a specified perturbation. Genes asterisked in red are differentially enriched in DP TILs overexpressing RELB when compared to Thy1.1, while genes asterisked in blue are differentially depleted in DP TILs overexpressing RELB when compared to Thy1.1. (F) GO Biological pathway enrichment analysis on differentially enriched pathways (p < 0.05) in DP TILs. (G) Relevant GSEA enriched and depleted gene sets in CD4+, CD8+, and DP TILs for RELB vs Thy1.1 comparison (p adj < 0.05). Positive Normalized Enrichment Score (NES) corresponds to enrichment of pathway in RELB-overexpressing conditions while a negative NES corresponds to depletion.

Article Snippet: CD8+ TILs were isolated using a TIL CD8+ magnetic isolation kit (REAlease® CD8 (TIL) MicroBead Kit, human, Miltenyi) and CD8+ CD103+ CD39+ TILs were FACS sorted (n = 2 patient samples), activated with Transact (Miltenyi) according to manufacturer’s instructions, and transduced (n = 2 transduction replicates) with the MORF library at 8% v/v.

Techniques: RNA Sequencing, Transduction, Activation Assay, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy

doi: 10.1101/2025.10.11.681829

Figure Lengend Snippet: (A) Experimental layout describing in vitro tumor rechallenge killing assay using a HER2-targetting CAR. (B) GFP+ count of SKBR3-GFP cells via live cell imaging over tumor multi-challenge assay (n = 3 distinct TIL patient samples, error bars represent standard error of the mean). * = p < 0.05 using two-way ANOVA analysis with Dunnett’s multiple comparisons test. (C) Flow cytometry analysis of side-scatter versus forward-scatter area of final rechallenge assay supernatant. (D) Total events count within side-scatter and forward-scatter gate. (E) CD8+ TIL events gated using fluorescence minus-one (FMO) controls. (F) Percent positive events for CD8+, CD8+ PD1+, CD8+ LAG3+, and CD8+ TIGIT+ based on flow gating on FMO controls. P value calculated for % CD8+ events with paired T-test.

Article Snippet: CD8+ TILs were isolated using a TIL CD8+ magnetic isolation kit (REAlease® CD8 (TIL) MicroBead Kit, human, Miltenyi) and CD8+ CD103+ CD39+ TILs were FACS sorted (n = 2 patient samples), activated with Transact (Miltenyi) according to manufacturer’s instructions, and transduced (n = 2 transduction replicates) with the MORF library at 8% v/v.

Techniques: In Vitro, Live Cell Imaging, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy

doi: 10.1101/2025.10.11.681829

Figure Lengend Snippet: (A) Experimental layout describing in vivo solid tumor challenge killing assay using a HER2-targetting CAR and flow cytometry analysis timeline pre-injection and post-tumor challenge (day 44 post CAR-TIL injection). (B) Flow cytometry phenotyping of pre-injection product for memory (IL7R, TCF1, CCR7, TCF1), exhaustion (PD-1, CD69), costimulation (ICOS), tissue-residency (TGF-β), and proliferation (Ki-67) markers. P values calculated using paired T-tests. * = p < 0.05 and ** = < 0.01. (C) Merged donor results of tumor volume (mm 3 ) of untreated (n=4) and treated mice with 5×10^5 (n = 2 patient TIL samples, 4 mice per treatment). * = p < 0.05 between CAR-TIL + RELB and CAR-TIL conditions using a two-way ANOVA analysis with Dunnett’s multiple comparisons test (top panel). CAR-TIL control conditions were significantly different (p < 0.05) from untreated sham control at final three timepoints (top panel). Bottom panel shows unmerged results for both patient samples tested. (D) Percent positive CD3, CD45, and CD45+ CD4-CD8-CAR-TILs gated using FMO controls from final tumor harvest samples for one patient sample. P values calculated using Welch’s unpaired t test. * = < 0.05 p. value. (E) Percent positive CD45+ TCF1hi+, CD45+ IL7R+, CD45+ PD-1+, CD45+ TIGIT+, CD45+ ICOS+, CD45+ OX40+ CAR-TILs gated using FMO controls from final tumor harvest samples for one patient sample. P values calculated using Welch’s unpaired t test. * = p < 0.05, ** = p < 0.01, and *** = p < 0.001.

Article Snippet: CD8+ TILs were isolated using a TIL CD8+ magnetic isolation kit (REAlease® CD8 (TIL) MicroBead Kit, human, Miltenyi) and CD8+ CD103+ CD39+ TILs were FACS sorted (n = 2 patient samples), activated with Transact (Miltenyi) according to manufacturer’s instructions, and transduced (n = 2 transduction replicates) with the MORF library at 8% v/v.

Techniques: In Vivo, Flow Cytometry, Injection, Control

Journal: bioRxiv

Article Title: RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy

doi: 10.1101/2025.10.11.681829

Figure Lengend Snippet: (A) Experimental layout describing in vitro TIL + matched tumor organoid co-culture killing assay conducted at 1:1 effector to target cell ratio. (B) Representative flow cytometry gating of viable CD3+ CD8+ TILs for T cell activation (4-1BB), and exhaustion (TIGIT, PD-1) markers. Gates were drawn based on FMO controls. (C) Summary data for all patient samples of CD3+ CD8+ TILs that were positive for 4-1BB, TIGIT, and PD-1. P values calculated using Welch’s unpaired t test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. (D) Representative flow cytometry gating of tumor cells (based on either cell size – patient 1 or EPCAM+ signal – patients 2 and 3) for T-cell mediated cytotoxicity (Granzyme B) or viability (LIVE/DEAD). Gates were drawn based on FMO controls. (E) Summary data for all patient samples of tumor cells (gated on cell size or EPCAM+ signal) that were positive for Granzyme B or LIVE/DEAD signal. P values calculated using Welch’s unpaired t test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: CD8+ TILs were isolated using a TIL CD8+ magnetic isolation kit (REAlease® CD8 (TIL) MicroBead Kit, human, Miltenyi) and CD8+ CD103+ CD39+ TILs were FACS sorted (n = 2 patient samples), activated with Transact (Miltenyi) according to manufacturer’s instructions, and transduced (n = 2 transduction replicates) with the MORF library at 8% v/v.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Activation Assay