Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: T cells predominantly express the IL-32β isoform. (A) Expression distribution of total IL32 and the IL32 isoforms α (A) , β (B) , η (C) , D, γ (E) , ϵ, θ, and ζ in the functional transcriptome clusters of Early activated Tconv, Suppressive Treg, Uncommitted Teff, Th1 Teff and Protumorigenic Treg among intratumoral CD4 + T cells (n=4) or in circulating mammaglobulin (MAMI)-reactive IFNγ + CD4 + Teff (n=3) of breast cancer patients depicted as average Transcripts Per Million (TPM) of all single-cells included in each cluster and based on previously published single-cell transcriptome sequencing data . (B–D) Relative expression of the IL32 isoforms α (A) , β (B) , γ (E) , η (C) and D among (B) T cells isolated from peripheral blood of healthy donors (HDs, n=4) either after stimulation with anti-CD3/CD28 antibodies (+) or after resting (-) for 24 h, (C) myeloid infiltrating lymphocytes (MILs) isolated from a multiple myeloma patient and (D) Survivin-specific T cells measured by RT-qPCR analyses. (B) The proportion (%) of each isoform among all detected transcripts of the tested IL32 isoforms. (C, D) Ct value of each tested IL32 isoform normalized to the Ct value of the housekeeping gene (C) PPIB and (B, D) ACTB (2^-ΔCt). Mean+SD. MILs: n=2, Survivin T cells n=2, n corresponds to independent experiments. (E) WB analysis of IL-32 expression in cell lysates (27 µg) of Scr and IL-32KO Survivin T cells next to rIL-32β (4 ng, MW: 23.1 kDa) and rIL-32γ (4 ng, MW: 28.1 kDa). As a loading control, the housekeeping gene GAPDH (37 kDa) was detected on the same blot. Representative blot of n=6 independent experiments. MW, Molecular Weight.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Expressing, Functional Assay, Sequencing, Isolation, Quantitative RT-PCR, Control, Molecular Weight

Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: HD T cells upregulate IL-32 after TCR stimulation. (A) IL-32 expression in T cells isolated from peripheral blood of Healthy Donors (HDs) based on ICFC analysis. (Top) Representative gating strategy applied to identify live single CD3 + T cells in cultures that received 24 h of CD3/CD28 stimulation. Representative (bottom, left) dot plots of IL-32 + cells among CD3 + T cells after 24 h of resting (no stimulation, green) or CD3/CD28 stimulation (orange) and (bottom, right) histograms of IL-32 expression on CD3 + T cells normalized to modal based on the respective fluorescence minus one (FMO) control supplemented with the isotype control corresponding to the anti-IL-32 antibody (grey). (B) Representative dot plots of IL-32 expression of HD T cells after CD3 versus CD3/CD28 stimulation (n=2). (C) Representative WB analysis of IL-32 expression in cell lysates from unstimulated HD T cells compared to stimulation with anti-CD3 or anti-CD3/CD28 antibodies. As a loading control, the housekeeping gene GAPDH was detected on the same blot (n=2). (D, E) Cumulative data of (D) the frequency of IL-32 + cells or (E) the Mean Fluorescence Intensity (MFI) of IL-32 among live single CD3 + T cells without or with CD3/CD28 stimulation, n=9, mean+SD, data from individual experiments are represented as dots, paired Student’s t-test, ****p<0.0001.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Expressing, Isolation, Fluorescence, Control

Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: CD4 + and CD8 + T cells upregulate IL-32 after TCR activation similarly. ICFC analyses of IL-32 expression among CD4 + and CD8 + CD3 + T cells from blood of HDs (n=9) after 24 h of resting or CD3/CD28 stimulation. (A) Representative gating strategy used to identify (top) CD4 + and CD8 + (CD4 - ) cells among live single CD3 + T cells after 24 h of CD3/CD28 stimulation. Further identification (bottom) of IL-32 + cells among CD4 + and CD8 + CD3 + T cells according to the respective FMO + Isotype control. (B, C) Cumulative data of (B) the frequency of IL-32 + cells (%) and (C) the expression level of IL-32 per single-cell displayed by the Mean Fluorescence Intensity (MFI) among CD8 + and CD4 + CD3 + T cells after 24 h without or with CD3/CD28 stimulation, data from individual experiments are represented as dots, mean+SD, Student’s paired t-test, ****p<0.0001. (D) Frequency of CD4 + T cells (%) among live single CD3 + T cells after 24 h of CD3/CD28 stimulation, median with individual values. (E) Frequency of CD4 + IL-32 + or CD8 + IL-32 + T cells (%) among all live single CD3 + T cells after 24 h without or with CD3/CD28 stimulation. Mean+SD, dots depict data from individual experiments, Student’s paired t-test, *p<0.05, ****p<0.0001.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Activation Assay, Expressing, Control, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: Co-expression of IL-32 and CD25 by HD T cells. IL-32 expression in CD25 + compared to CD25 - CD3 + T cells from healthy donors (HDs) after 24 h of resting (no stimulation) or CD3/CD28 stimulation determined by ICFC analysis. (A) Representative gating of CD25 + and CD25 - cells among CD3/CD28 stimulated live single CD3 + T cells followed by further subgating of IL-32 + cells among CD25 + or CD25 - cells. Gates were set according to the respective FMO + Isotype controls for CD25 and IL-32. (B, C) Frequency of IL-32 + cells (%) in CD25 + and CD25 - cells among CD3 + , CD4 + , and CD8 + T cells after 24 h (B) without stimulation or (C) with CD3/CD28 stimulation. Cumulative data of n=9, Mean+SD, dots depict data from individual experiments, Student’s paired t-test, **p<0.01, ****p<0.0001.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: HD CD3 + T cells upregulate and secrete IL-32β after TCR stimulation. (A) IL-32 secretion by CD3 + T cells from healthy donors (HDs, n=9) after 24 h of culture without or with CD3/CD28 stimulation measured by ELISA analysis of culture supernatants. Mean+SD, dots depict data from individual experiments, Student’s paired t-test, **p<0.01. (B) Kinetics of the frequency of IL-32 + cells among all live T cells (%, black) in connection with the secretion of IL-32 (pg/ml, gray) in culture supernatants, as measured by ICFC and ELISA, respectively, over a time course of 0.5 – 72 h after CD3/CD28 stimulation of purified T cells from 3 different HDs. (C) WB analysis of IL-32 intracellular expression in total cell lysates (14 µg) and IL-32 secretion into the cell culture supernatant (SN; 14 µg, TCA/Ac enriched) by HD CD3 + T cells after 72 h of resting or CD3/CD28 stimulation next to human recombinant IL-32β (rIL-32β, 4 ng, MW: 23.1 kDa) and rIL-32γ (6 ng, MW: 28.1 kDa). As a loading control, the housekeeping gene GAPDH (MW: 37 kDa) was detected on the same blot. Representative data from n=2 HDs.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Enzyme-linked Immunosorbent Assay, Purification, Expressing, Cell Culture, Recombinant, Control

Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: IL-2 induces IL-32 but TCR activation is required for IL-32 secretion. (A) IL-32 expression in CD3 + T cells from healthy donors (HDs) after 24 h of resting (unstimulated) either alone or in the presence of IFN-γ and low (100 IU/ml) or high (3,000 IU/ml) amounts of IL-2 and after 24 h of either CD3 or CD3/CD28 stimulation determined by ICFC analysis. Representative data from n=2 independent experiments. (B–D) WB analyses of IL-32 expression in cell lysates and IL-32 secretion into the cell culture supernatants (enriched via Amicon Ultracentrifugal filters) by HD CD3 + T cells after treatment for 48 h with 500 or 3,000 IU/ml IL-2 alone (low or high IL-2, respectively) or treatment with high IL-2 for 48 h with subsequent CD3/CD28 stimulation for 72 (h) (B) Representative blot of lysates and supernatants (15 µg) from n=2 HDs (#1, #2) next to rIL-32β (4 ng, MW: 23.1 kDa) and rIL-32γ (4 ng, MW: 28.1 kDa). As a loading control, the housekeeping gene GAPDH (MW: 37 kDa) was detected on the same blot. (C) Cumulative data of quantified IL-32β expression in cell lysates normalized to GAPDH for n=2–5 HDs, mean+SD, dots depict data from individual experiments, Student’s paired t-test, *p<0.05. (D) Cumulative data of quantified IL-32β secretion into culture supernatants displayed by the Area Under the Curve (AUC) for n=2–5 HDs, mean+SD. (E, F) IL-32 expression in HD T cells after resting (Unstim) or CD3/CD28 stimulation alone (Stim) or together with either an αIL-2 neutralizing antibody (Stim + αIL-2) or the respective isotype control (Stim + Isotype) for 24 (h) (E) Representative dot plots depicting IL32 + CD25 + cells among live single T cells stimulated in the presence of the isotype or the αIL-2 antibody. (F) Cumulative data showing the frequency of IL-32 + CD25 + cells among live single T cells, n=2 HDs, Student’s unpaired t-test, *p<0.05.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Activation Assay, Expressing, Cell Culture, Control

Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: T cells actively secrete IL-32β mainly through unconventional secretion pathways bypassing the Golgi network. (A, C, D) IL-32 and (B, C) TNF-α secretion of Survivin T cells after 4 h of CD3/CD28 stimulation alone or in the presence of inhibitors that target (A, B) conventional or (C, D) unconventional protein secretion pathways analyzed by ELISA. (A, B) Treatment with Brefeldin A (BFA) or Monensin. (C) Application of ammonium chloride (NH 4 Cl), 2.5 µM Punicalagin or the respective volume of methanol (MeOH), the dissolution medium of Punicalagin, alone. (D) Combined treatment with NH 4 Cl and 2.5 µM Punicalagin. Related data within individual experiments are represented as dots, whereas each dot represents a mean of n=3 technical replicates. Individual experiments are connected by a line. Cumulative data of n=2–5 independent experiments, mean, Student’s paired t-test, *p<0.05, **p<0.01.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Enzyme-linked Immunosorbent Assay, Dissolution

Journal: Frontiers in Immunology

Article Title: IL-2 and TCR stimulation induce expression and secretion of IL-32β by human T cells

doi: 10.3389/fimmu.2024.1437224

Figure Lengend Snippet: IL-32 is secreted by T cells predominantly as a free protein. (A) Representative WB analyses of IL-32β and the microvesicle/exosome markers GRP94 and HSP70 in lysates of cells, cell-debris, microvesicles and exosomes, into the respective cell solution and cell-free, cell debris-free, microvesicle-free and exosome-free supernatant (SN), the PBS-wash SN, which was used to wash the exosome pellet, and in the EV-free medium used to culture Survivin-specific T cells for 4 h with anti-CD3/CD28 antibodies. The loaded quantity of each lysate and SN sample corresponds to 0.16x10 6 (1X), 1.6x10 6 (10X) and 16x10 6 (100X) Survivin-specific T cells and the amount of PBS-wash SN derived from this cell number or the volume of EV-free medium used to culture this cell number. MW in kDa (IL-32β: 23.1, GRP94: 98, HSP70: 70). IL-32β, GRP94, HSP70: n=4. n represents independent experiments. (B) Cumulative data of quantified IL-32β expression in lysates and supernatants displayed by the Area Under the Curve (AUC) for n=4 independent experiments normalized to 0.16x10 6 Survivin-specific T cells. Dots depict data from individual experiments, Student’s paired t-test, *p ≤ 0.05.

Article Snippet: T cells were activated using non-tissue treated FB culture plates (12-well, Falcon, Cat#351143; 24-well, Falcon, Cat#351147; 96-well, Falcon, Cat#351172; 48-well, Thermo Fisher Scientific, Cat#152640), which had been coated overnight at 4°C with 1X PBS (Sigma, Cat#D1408) containing 4 μg/ml anti-CD3 antibodies (eBioscience, Cat#14–0037-82, RRID: AB_467057).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The cleavage motifs derived from PIAS1 (LTYD*G and NGVD*G) were used to virtually screen the entire human proteome for proteins sharing the same sequences. The human proteome dataset containing approximately 20,000 human protein-coding genes represented by the canonical protein sequence was downloaded from UniProtKB/Swiss-Prot. (B) 16 additional proteins were extracted from the screen. 8 proteins carry the LTYD*G motif (left) and 8 proteins carry the NGVD*G motif (right). 6 proteins (underlined) were selected for further validation. (C) Protein downregulation during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot showing the downregulation of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes the cleaved fragment for EHMT2. (D) Caspase inhibition blocks the degradation of YTHDF2, MAGEA10, SORT1 MTA1 and EHMT2. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot showing the protein levels of 6 selected proteins using antibodies as indicated. SAMHD1 and β-actin were included as controls. Arrowhead denotes cleaved EHMT2 fragment.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Derivative Assay, Sequencing, Western Blot, Inhibition

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Schematic representation showing the relative positions of Cas9 target sites for small guide RNAs sg-1 to sg-3. (B) Akata (EBV+) cells were used to establish stable cell lines using 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated cross-linking of BCR. YTHDF2 and viral protein (ZTA and RTA) expression levels were monitored by Western Blot using antibodies as indicated. (C) RNAs from YTHDF2-depleted and control Akata cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (D) P3HR-1 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with TPA and sodium butyrate (NaBu) to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (E) RNAs from YTHDF2-depleted and control P3HR-1 cells were extracted and analyzed by RT-qPCR. The values of control were set as 1. Error bars indicate ±SD. IE, immediate early gene; Early, early gene; Late, late gene. (F) SUN-719 cells were used to establish stable cell lines as indicated. The cells were either untreated or treated with Gemcitabine to induce lytic reactivation. YTHDF2 and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (G) Akata (EBV+) cells were used to establish control and YTHDF2 overexpression cell line as indicated. The cells were untreated or lytically induced by anti-IgG treatment. The expression of YTHDF2 as monitored by anti-YTHDF2 and anti-Myc antibodies. Viral protein expression levels were monitored by Western Blot using antibodies as indicated. (H) Extracellular virion-associated DNA from cells treated in panel G was extracted and the relative EBV viral copy numbers were calculated by q-PCR analysis using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p<0.001. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2 or 3 different sgRNA constructs and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG treatment for 24 or 48 hrs as indicated. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. (A) EIF4H depletion promotes the expression of EBV ZTA and RTA. (B) MAGEA10 depletion does not affect EBV protein expression. (C) SORT1 depletion does not significantly affect EBV protein expression. (D) EHMT2 depletion does not affect EBV protein expression. Arrowhead denotes cleaved fragments. (E) MTA1 depletion does not uniformly affect EBV protein expression but slightly enhances the expression of its homolog MTA2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) YTHDF2-depleted and control Akata (EBV+) cells were lytically induced with anti-IgG for 0 to 48 hrs. (B) YTHDF2-depleted and control P3HR1 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. (C) YTHDF2-depleted and control SNU-719 cells were lytically induced with TPA and NaBu for 0 to 48 hrs. Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Western Blot showing YTHDF2 downregulation by IgG cross-linking induced BCR activation. Akata (EBV+) and Akata-4E3 (EBV-) cells were treated with anti-IgG antibody as indicated. YTHDF2 and viral protein expression levels were monitored by Western Blot. Arrowheads denote cleaved YTHDF2 in the longer exposure blot. (B) Caspase inhibition blocks YTHDF2 degradation. The cells were either untreated or pretreated with a pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Arrowheads denote cleaved YTHDF2. (C) Functional domains and putative cleavage sites in YTHDF2. CaspDB was used to predict the potential cleavage sites in YTHDF2. The locations of the putative cleavage sites D166 and D367 were labeled as indicated. CNOT1 binding domain: responsible for the degradation of associated RNA; P/Q/N rich region: aggregation-prone region; YTH domain: responsible for binding to m 6 A-modified RNA. (D) Schematic representation of V5-tagged YTHDF2 with two putative cleavage sites. Red oval, anti-YTHDF2 monoclonal antibody recognition site. (E-F). Wild-type V5-YTHDF2 was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using either anti-YTHDF2 (E) or anti-V5 (F) antibodies. The relative position of predicted cleavage fragments was labeled as indicated. (G-H) YTHDF2 (D166A/D367A) mutant protein was incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. (I) Motif analysis showing the conservation of the two cleavage sites and the surrounding amino acids. Amino acid sequences were extracted from 97 (D166) and 80 (D367) vertebrate species and motif logos were generated using WebLogo. (J) Structure modeling of full-length YTHDF2 by I-TASSER. The two cleavage sites D166 and D367 are labeled as indicated. N and C denote N-terminus and C-terminus, respectively. (K) Triple depletion of caspase-3, -8 and -6 reduces YTHDF2 and PIAS1 degradation and blocks viral protein accumulation. The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. The expression of caspases, cleaved caspases, YTHDF2, PIAS1 and viral proteins (ZTA and RTA) was monitored by Western Blot using antibodies as indicated. Arrowheads denote cleaved fragments. See also - and Table S2.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot, Activation Assay, Expressing, Inhibition, Functional Assay, Labeling, Binding Assay, Modification, Incubation, Recombinant, Mutagenesis, Generated

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-C) Akata (EBV+) cells were lytically induced with anti-IgG for 0, 6, 12, 24 and 48 hrs (A). P3HR1 (B) and SNU-719 (C) cells were lytically induced with TPA and NaBu for 0 6, 12, 24 and 48 hrs. YTHDF2, EBV ZTA and RTA, cleaved caspase substrate (CASP sub.), cleaved PARP, cleaved CASP3 and cleaved CASP8 were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (D-F) Apoptotic induction by an intrinsic trigger promotes EBV reactivation. Akata (EBV+) (D), P3HR1 (E) and SNU-719 (F) cells were untreated or treated with increasing amount of Taxol for 48 hrs. YTHDF2, EBV ZTA and RTA, and cleaved caspase substrate (CASP sub.) were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata EBV(+) cells were transduced with lenti-vector control or Myc-CASP8 to establish stable cell lines. The cells were treated with anti-IgG for 0, 24 and 48 hrs. (A) CASP8, EBV ZTA and RTA were monitored by Western Blot using antibodies as indicated. β-actin blots were included for loading controls. (B) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of vector control at 0 hr was set as 1. (C) Total RNA was extracted and then EBV lytic (ZTA and RTA) and latent (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1 and LMP2) genes were analyzed by RT-qPCR. The value of vector control at 0 hr was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The CASP3/CASP8/CASP6-triply-depleted Akata (EBV+) cells were lytically induced by anti-IgG treatment. (A-B) Total RNA was extracted and then EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. (C) Extracellular virion DNA from the medium were extracted and then analyzed by qPCR using primers specific to BALF5. The value of control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic Akata (EBV+) cells upon lytic induction. Akata (EBV+) cells were either untreated (control) or treated with anti-IgG antibody for 24 and 48 hrs as indicated. (A) The cells were stained with Propidium Iodide (PI) and then permeabilized for immnunostaining with anti-YTHDF2 antibody. (B) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. (C-D) Immunofluorescence assay showing YTHDF2 downregulation and EBV EAD upregulation in apoptotic SNU-719 cells upon lytic induction. SNU-719 cells were either untreated (control) or treated with TPA and NaBu for 24 and 48 hrs as indicated. (C) Cells were stained with Propidium Iodide (PI) and then permeabilized for immunostaining with anti-YTHDF2 antibody. (D) Cells were permeabilized and co-immunostained with anti-YTHDF2 and anti-EBV EAD antibodies as indicated. Scale bar, 20 μm

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Immunofluorescence, Staining, Immunostaining

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: The Akata (EBV+) (A), P3HR-1 (B) and SNU-719 (C) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then lytically induced with anti-IgG antibody or TPA/NaBu as indicated for 48 hrs. Western Blot showing the protein levels of RIP, phospho-RIP (p-RIP) and phospho-RIP3 (p-RIP3) using antibodies as indicated. β-actin blots were included as controls.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) The design of CRISPR/Cas9-resistant YTHDF2 variant was based on the sg-2 protospacer adjacent motif (PAM). D166A/D367A mutations were introduced into the PAM-mutated YTHDF2. Both constructs were cloned into a lentiviral vector with a C-terminal Myc-tag. (B-C) WT and cleavage-resistant YTHDF2 suppresses EBV replication. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing YTHDF2 and EBV protein expression levels in these cell lines upon IgG cross-linking as indicated (B). Arrowheads denote cleaved fragments. Extracellular and intracellular viral DNA was measured by qPCR using primers specific to BALF5 (C). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001. (D) Schematic representation of 5 YTHDF2 cleavage-mimicking fragments. These fragments were cloned into a lentiviral vector with a C-terminal Myc-tag. (E) SNU-719 cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. (F) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual fragment to establish stable cell lines. Western Blot analysis showing YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (G) Caspase-mediated cleavage impairs YTHDF2 binding to CNOT1. Halo-V5-tagged WT YTHDF2 and the individual fragments were co-transfected with HA-tagged CNOT1 SH domain into 293T cells as indicated. Co-immunoprecipitation (Co-IP) experiments were performed using anti-V5 antibody-conjugated magnetic beads. The immunoprecipitated samples and total cell lysates (Input) were analyzed by Western Blot with antibodies as indicated. (H) Model showing the functional consequences of YTHDF2 cleavage in CNOT1 binding and the targeting of m 6 A-modified RNA. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: CRISPR, Variant Assay, Construct, Clone Assay, Plasmid Preparation, Western Blot, Expressing, Transduction, Stable Transfection, Binding Assay, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Magnetic Beads, Functional Assay, Modification

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) SNU-719 cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines (see ). RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by adding TPA (20 ng/ml) for 24 hrs. The value of vector control was set as 1. (B) Akata (EBV+) cells were transduced with lentiviruses carrying vector control or individual YTHDF2 fragment to establish stable cell lines . RT-qPCR analysis showing EBV ZTA and RTA mRNA levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. The value of vector control at 24 hrs was set as 1. (C) Akata (EBV+) cells were transduced with lentiviruses carrying vector control, Halo-tag, WT YTHDF2 or individual YTHDF2 fragment to establish stable cell lines. Western Blot analysis showing Halo, YTHDF2 fragments and EBV protein expression levels in these cell lines upon lytic induction by anti-IgG treatment for 24 and 48 hrs. Shorter and longer exposures were included to show the differences in protein levels. (D) Total mRNA was extracted from cells treated in panel (C). EBV ZTA and RTA mRNA levels were analyzed by RT-qPCR. The value of vector control at 24 hrs was set as 1 Results from three biological replicates are presented. Error bars indicate ±SD. N.S., not significant; ***, p< 0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Transduction, Plasmid Preparation, Stable Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were lytically induced by IgG-cross linking for 24 hrs. (A) Total RNA was subjected to m 6 A RIP, followed by RT-qPCR using indicated primers. Values are displayed as fold change over 10% input. GAPDH and Dicer are cellular negative and positive controls, respectively. (B) Cell lysate was collected to detect YTHDF2 binding of viral RNAs by RIP-qPCR. Values are displayed as fold change over 10% input. MALAT1 and SON are cellular negative and positive controls, respectively. Results from three biological replicates are presented. Error bars indicate ±SD. **, p< 0.01.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Quantitative RT-PCR, Binding Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) A group of genes in the category of “ activation of cysteine-type endopeptidase activity involved in apoptotic process ” (also called “ caspase activation ”) were extracted from YTHDF2 target genes derived from YTHDF2 RIP-seq and PAR-CLIP-seq datasets ( , , ) (B-C) YTHDF2 reconstitution suppresses caspase-8 expression and subsequent caspase activation. Akata (EBV+) YTHDF2-sg2 cells were reconstituted with WT or cleavage-resistant YTHDF2 (D166A/D367A) using lentiviral constructs. Western Blot analysis showing the levels for caspase-8 (CASP8), cleaved caspase-8, and cleaved caspase substrates (CASP sub.) in these cell lines upon IgG cross-linking as indicated (B). CASP8 mRNA levels were analyzed by RT-qPCR using CASP8 primers (C). The value of vector control at 0 hr was set as 1. (D-E) Caspase-8 inhibition suppress EBV replication in YTHDF2-depleted cells. Control and YTHDF2-depleted Akata (EBV+) cells were either untreated or pretreated with caspase-8 inhibitor (Z-IETD-FMK, 50 μM) for 1 hr and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (D). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (E). The value of vector control at 0 hr was set as 1. (F-G) Caspase-8 depletion suppresses EBV replication in YTHDF2-depleted cells. YTHDF2-depleted Akata (EBV+) cells were transduced with lentivirus carrying control sgRNA or CASP8-sg1 to establish cell lines and then anti-IgG antibody was added for 0 to 48 hrs as indicated. Western Blot showing the protein levels of EBV ZTA and RTA as indicated (F). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (G). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Activation Assay, Activity Assay, Derivative Assay, Expressing, Construct, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Inhibition, Transduction

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-B) YTHDF2 depletion promotes CASP8 mRNA expression. Akata (EBV+) cells and P3HR-1 cells carrying different sgRNA targeting YTHDF2 or control (sg-NC) were used to extract total RNA and qPCR analyses were performed a group of YTHDF2-targeted cellular genes involved in caspase activation. The values were normalized with a non YTHDF2 target HPRT1 . The values of sg-NC were set as 1. (C-D) CASP8 is modified by m 6 A and YTHDF2 binding to CASP8 . Akata (EBV+) cells were used to perform m6A RIP-qPCR (C) and YTHDF2 RIP-qPCR (D), respectively. Values are displayed as fold change over 10% input. (E-G) YTHDF2 depletion promotes caspase-8 protein expression and PIAS1 cleavage upon lytic induction. Akata (EBV+) cells (E), P3HR-1 cells (F) and SNU-719 cells (G) carrying different sgRNA targeting YTHDF2 or control (sg-NC) were lytically induced by anti-IgG, TPA and sodium butyrate (NaBu) and gemcitabine treatment for 24 hrs. Protein expression was monitored by Western Blot using antibodies as indicated. (H) CASP8 m 6 A peaks were extracted from MeT-DB V2.0 database. YTHDF2-PAR-CLIP data were retrieved from Wang et al.. The Exon-7 of CASP8 with highest m 6 A peaks were analyzed for conservation among sequences derived from 100 vertebrate species. 15 potential m 6 A motifs (M1-M15) were extracted based on m 6 A motif DRACH. (I) Motif logos were generated for 15 individual sites. Red cycles denote highly conserved motifs (M2, M3, M5, M8 and M12) across 100 vertebrate species. (J-K) WT and mutant CASP8 -Exon-7 were cloned into the m 6 A-null Renilla luciferase (RLuc) reporter (3’UTR region) that also express Firefly luciferase (FLuc) from a separate promoter (J). These three reporter plasmids were transfected into parental or YTHDF2-depleted (YTHDF2 KD) SNU719 cells. Relative Renilla to Filefly luciferase activity (RLuc/FLuc) was calculated (K). The value of WT in parental cells was set as 1. (L) Model illustrating YTHDF2 regulation of CASP8 mRNA and caspase-8 regulation of YTHDF2 and PIAS1 in EBV reactivation. Results from three biological replicates are presented. Error bars indicate ±SD. *, p< 0.05; **, p< 0.01; ***, p< 0.001. See also , and Table S3.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Expressing, Activation Assay, Modification, Binding Assay, Western Blot, Derivative Assay, Generated, Mutagenesis, Clone Assay, Luciferase, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) Diagram summarizing the major writers, readers and erasers involved in the m 6 A RNA modification pathway. (B) The downregulation of m 6 A RNA modification pathway proteins during EBV reactivation. Akata (EBV+) cells was treated with anti-IgG antibody to induce EBV reactivation for 0, 24 and 48 hrs. Western Blot was performed using antibodies as indicated. N6AMT1 and β-actin blots were included as controls. (C) Caspase inhibition blocks the degradation of m 6 A RNA modification pathway proteins. The Akata (EBV+) cells were either untreated or pretreated with a caspase-3/-7 inhibitor (Z-DEVD-FMK, 50 μM) or pan-caspase inhibitor (Z-VAD-FMK, 50 μM) for 1 hr, and then anti-IgG antibody was added for 48 hrs. Western Blot was performed using antibodies as indicated. (D and E) V5-METTL14 (D) and V5-WTAP (E) were incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL14, anti-V5 and anti-WTAP antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. Arrowheads denote cleaved fragments. Star denotes non-specific bands. See also - .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Modification, Western Blot, Inhibition, Incubation

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A) V5-METTL3 was incubated with individual caspase for 2 hrs at 37°C. Western Blot was performed using anti-METTL3 and anti-V5 antibodies as indicated. The locations of antibody recognition epitopes were labelled as indicated. The positions of weakly cleaved fragments were labelled by arrowhead. Star denotes non-specific bands. (B) V5-tagged WTAP D301A/D302A and D301A mutants were incubated with individual recombinant caspase for 2 hrs. Western Blot was performed using antibodies as indicated. Arrowheads denote cleaved fragments. (C) Sequence alignment of WTAP sequences from 10 representative species using the Constraint-based Multiple Alignment Tool (COBALT). The cleavage motifs were highlighted by yellow color. (D) Motif analysis showing the conservation of the WTAP D302 and the surrounding amino acids. Amino acid sequences were extracted from 97 vertebrate species and motif logos were generated using WebLogo. (E-F) Akata (EBV+) cells were used to establish stable cell lines using 2 different guide RNA constructs targeting YTHDF1 (D) and ALKBH5 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Incubation, Western Blot, Recombinant, Sequencing, Generated, Stable Transfection, Construct, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: (A-E) Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A), METTL14 (B), WTAP (C), VIRMA (D) and YTHDF3 (E) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation. Cellular and viral protein expression levels were monitored by Western Blot using antibodies as indicated. See also and .

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Western Blot

Journal: bioRxiv

Article Title: Caspases switch off m 6 A RNA modification pathway to reactivate a ubiquitous human tumor virus

doi: 10.1101/2020.11.12.377127

Figure Lengend Snippet: Akata (EBV+) cells were used to establish stable cell lines using 2-3 different guide RNA constructs targeting METTL3 (A and B), METTL14 (C and D), WTAP (E and F), VIRMA (G and H) and YTHDF3 (I and J) and a non-targeting control (sg-NC). The cells were untreated or lytically induced with anti-IgG-mediated BCR activation for 24 or 48 hrs. EBV ZTA and RTA mRNA expression levels were monitored by RT-qPCR (A, C, E, G and I). Extracellular viral DNA was measured by qPCR using primers specific to BALF5 (B, D, F, H and J). The value of vector control at 0 hr was set as 1. Results from three biological replicates are presented. Error bars indicate ±SD. **, p<0.01; ***, p<0.001.

Article Snippet: For lytic induction in Akata (EBV+) cell lines, the cells were treated with IgG (1:200, Cat# 55087, MP Biomedicals) for 0 to 48 hrs.

Techniques: Stable Transfection, Construct, Activation Assay, Expressing, Quantitative RT-PCR, Plasmid Preparation

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) In-silico analysis of CAR-T cell-treated patients (n=4,219) revealed a high relapse rate, with 42.11% (n=216 of n=513 overall relapse patients) experiencing CD19-negative recurrence after monospecific CAR-Therapy (n=2,916). (B) Schematic overview of the CAR design strategy showing mono, bi, and trispecific constructs targeting CD19, CD20, and CD22. (C) Experimental workflow illustrating CAR screening: 1,452 CARs were transduced into primary T cells and analyzed for signal-1 (activation), signal-2 (exhaustion), and signal-3 (cell death). (D) Categorization of CARs into low (L), medium (M3), and high (H) levels based on fluorescence intensity cutoffs determined by monospecific CD19 CARs. (E) Distribution of 1,452 screened CARs across L-, M-, and H-CARMSeD categories using the CAR-Mediated Self-Destruction (CARMSeD) scoring system. (F) AI model development pipeline for CAR dysfunction risk prediction, based on 1,452 CAR constructs with an 80:20 split for training and testing. (G–J) Performance metrics of AI model predicting CAR-Mediated Self-Destruction (CAR-MSED) scores using 1,452 CAR constructs (G) Model accuracy over 50 epochs, achieving a training accuracy of 0.98 and validation accuracy of 0.95. (H) Scatter plot comparing measured versus predicted CAR-MSED scores for training (R 2 = 0.87) and validation (R 2 = 0.83) sets. (I) Predicted versus measured CAR-MSED scores on the validation set, categorized into low (L-CARMSED, blue), medium (M-CARMSED, orange), and high (H-CARMSED, green) groups. (J) Box plot of predicted signal scores for 9,372 unknown sequences, classified as L-CARMSED (2,749 sequences), M-CARMSED (1,468 sequences), and H-CARMSED (5,155 sequences). (K) Molecular dynamics simulation of CAR constructs with varying linker lengths, assessing CAR-CAR interaction. Structural conformations at 0 ns, 50 ns and 200 ns for different CAR scFv arrangements highlighting CDR regions (surface transparency 30%), Root Mean Square Deviation (RMSD) plots over 200 ns for the both constructs, respectively, indicating structural stability and conformational changes. (L) In vitro receptor binding affinity validation for top humanized scFvs of CD19, CD20, and CD22 CARs (n=6).

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Cat No. 130-129-550) and CD22 (Miltenyi Biotec).

Techniques: In Silico, Construct, Activation Assay, Fluorescence, Biomarker Discovery, In Vitro, Binding Assay

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Schematic illustration of the K562 cell line model expressing individual or triple combinations of CD19 (purple), CD20 (yellow), and CD22 (red) antigens. (B) Bar chart depicting the percentage expression of each antigen in K562 cell lines, both individually and in combination. (B) Cytotoxicity assays showing potent and antigen-specific killing of K562 target cells. All tested constructs surpassed the performance of second-generation monospecific CD19 (m19) CAR-T cells (n=5). (C) Comparison of proliferation rates for bispecific; b20/19 or b22/19, and trispecific; t20/19/22 CAR-T cells. Trispecific constructs showed reduced proliferation, consistent with increased structural rigidity predicted by CARMSeD scoring. (D) Schematic of the Raji WT cell line platform expressing CD19 (purple), CD20 (yellow), and CD22 (red) antigens, edited using CRISPR-Cas9 to generate knockout variants. (E) Cytotoxicity assays demonstrating the superior efficacy of b20/19 CAR-T cells in eliminating antigen-negative Raji variants, compared to ineffective m19 CARs (n=5). (F) Schematic representation of the tumor rechallenge (TR) model using the Raji WT cell line (Raji WT ). Gray circles represent initial engraftment and monitoring phases, while purple circles indicate the timing of the Raji CD19-/- rechallenge. (G) Heatmap representation of TR model showing IFN-γ secretion (pg/mL), percentage of tumor lysis, and the number of CAR-T cells detected on days 7, 9, 11, 15, and 17 post-rechallenge (n=5). (H) Schematic timeline of in vivo lymphoma model for evaluation of monospecific and bispecific CAR-T cells. Mice were xenografted with Raji WT cells (expressing CD19, CD20, and CD22) (day 0), followed by administration of m19 or b20/19 CAR-T cells on day 5 and subsequent Raji CD19-/- TR on day 12, 19 and 26. (I–J) Bioluminescent imaging and tumor burden quantification show effective tumor control by b20/19 CAR-T cells versus m19 CARs. (K) CAR-T cell persistence over time. (L) Kaplan-Meier survival curves showing survival outcomes over 70 days (n=5). (M) Analysis of residual tumor CD19 or CD20 tumor cells over time. (N, O) Bar plot showing Granzyme B and IFN-γ secretion from CD8 + CAR-T cells isolated post-treatment with m19 and b20/19 confirm functional cytotoxicity of b20/19 against CD19⁻ targets (n=5). (P–Q) Repeated TR induced upregulation of exhaustion markers PD-1 and LAG-3 (n=5). (R) Immunophenotyping of CAR-T cells post-TR shows loss of central memory (T cm ) populations and increased PD-1 expression, consistent with functional exhaustion and limited persistence (n=5). Data represents mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Cat No. 130-129-550) and CD22 (Miltenyi Biotec).

Techniques: Expressing, Construct, Comparison, CRISPR, Knock-Out, Lysis, In Vivo, Imaging, Control, Isolation, Functional Assay

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Pathway analysis of proteins involved in AKT3 interaction, modifications or regulation of its expression with emphasis on FOXO4. (B) Relative mRNA expression levels (normalized to beta actin) of key genes show upregulation of FOXO4 mRNA in b20/19-AKT3 PROTAC CAR-T. (C1) Flow cytometry histograms of total FOXO4 and phosphorylated FOXO4 (p-FOXO4) in CAR-T cells after TR with Raji CD19-/- cells (C2) Histogram analysis of the flow cytometry plots (n=10). (D) Bar graph shows the percentage of CD8 + CAR-T cells expressing different phenotypes. Pie charts illustrate the proportional distribution of these subsets across conditions. (E) Persistence of CAR-T cells over 15 days under various conditions (n=4). (F) Violin plots show the percentage of mTOR activity (% mTOR activity) in various conditions, with shRNA based FOXO4 knockdown significantly elevated mTOR activity (n=6). (G) Bar plots show the percentage of MFI of autophagy from autophagic flux assay (n=8). (H) ECAR in NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 conditions, with FOXO4 knockdown increasing shift from oxidative phosphorylation (OXPHOS) to glycolysis (n=12 data points). (I) Similarly, OCR with FOXO4 knockdown decreasing mitochondrial respiration. Individual data points are shown for each condition (n=12 data points). (J1) Percentage of expression (% expression) of CD19 (yellow), CD20 (blue), and CD22 (purple) across 129 ALL patient samples, with varying expression levels for each marker. (J2) Bar graph displays the number of patient samples categorized as Negative/Dim, Moderate, or Bright for CD19, CD20, and CD22 expression. (K) Schematic illustration of K562 WT cells based on CD20 expression levels, resulting in three populations: CD20 L (low), CD20 M (medium), and CD20 H (high). (L) Violin plots show the percentage of CD20 expression (% CD20 expression) in the sorted K562 WT cell populations, confirming distinct expression levels (n=10). (M) Representative super-resolution microscopy images of differential CD20 surface expression in K562 cells. Images show DAPI (blue, nuclear staining) and CD20 (red) in K562-C20 L (low), K562-C20 M (medium), and K562-C20 H (high) cell. Scale bar indicates 10 μm. (N) Survival of K562 cells expressing varying CD20 expression levels under CAR-T cell treatments. Panels N1 (K562-CD20 L ), N2 (K562-CD20 M ), and N3 (K562-CD20 H ) show the percentage of CD20 + cell survival when treated with Rituximab-based monospecific CAR (Rtx-m20, dark green), in-house humanized anti-CD20 CAR (AB21-m20, green) (N=4). (O) Persistence of CAR-T cells with varying CD20-targeting CAR constructs over 15 days (N=5). Data represents mean ± SEM. ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups. Scale bar indicates 10 μm.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Cat No. 130-129-550) and CD22 (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Activity Assay, shRNA, Knockdown, Flux Assay, Phospho-proteomics, Marker, Super-Resolution Microscopy, Staining, Construct

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Schematic timeline of the experiment showing Raji WT cell injection, CAR-T cell administration, and Raji CD19-/- TR. (B1) In vivo bioluminescence imaging of mice treated with NTP PROTAC+Scram , NTP PROTAC+shFOXO4 , AKT3 PROTAC+Scram , and AKT3 PROTAC+shFOXO4 , showing tumor burden (red indicates high tumor load, blue indicates low) over 84 days, with ‘X’ marking deceased mice. (B2) Pie charts depict survival outcomes on days 56 and 84 (n=5). (C) Tumor radiance over 84 days, demonstrating reduced tumor burden in the AKT3 PROTAC+scram group. (D) Kaplan-Meier survival curves, with the AKT3 PROTAC+scram group exhibiting the highest survival rate. (E1) Line graph shows the percentage of CAR-T cells in blood (% CAR-T cells in blood) over 84 days. (E2) Bar graph displays the percentage of CAR-T cells in blood at day 84, with the AKT3 PROTAC+scram group showing detectable levels (∼3%), while other groups show non-detectable (ND) levels (n=5). (F) Tumor burden assessment till day 56. The line graph shows the number of Raji cells over time. The AKT3 PROTAC+scram group exhibits no detectable Raji cell burden by day 56, while other groups show some detectable cells (n=5). (G) Bar graph shows the percentage of CD8 CAR-T cells (% CD8 T cells) expressing different phenotypes on day 28 post-infusion with corresponding pie charts illustrating the proportional distribution (n=5). (H-I) ECAR and OCR under various conditions on day 28 (n=12 data points). (J) Schematic of CLDN ScFv and EGFR ScFv CAR constructs with ICOS, 4-1BB, CD3ζ, and AKT3 PROTAC domains. (K-L) Cytotoxicity of CLDN-CAR and EGFR-CAR-T cells with NTP3 PROTAC or AKT3 PROTAC against AGS (K) and A549 (L) cells at varying T:E ratios (n=5). (M) CAR-T cell proliferation over 15 days, showing enhanced expansion with AKT3 PROTAC . (N) Bar graph and pie charts analysis of EGFR-CAR and CLDN-CAR-T cells with NTP PROTAC or AKT3 PROTAC , showing the percentage of various T cell subsets (n=5). Data represents mean ± SEM. ***p < 0.005; ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Cat No. 130-129-550) and CD22 (Miltenyi Biotec).

Techniques: Injection, In Vivo, Imaging, Expressing, Construct

Journal: bioRxiv

Article Title: AI-Guided CAR Designs and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

doi: 10.1101/2025.06.12.658477

Figure Lengend Snippet: (A) Schematic of the engineering strategy for trispecific CAR-T cells, integrating b20/19-AKT3 PROTAC with a secretory BiTE module consisting of nanobodies targeting CD3 and CD22 (nbCD3/22). (B) Correlation of expression of nbCD3, nb22, CD19 CAR, and CD20 CAR at various MOIs. The cells were treated with Brefeldin and data was obtained using intracellular flow cytometry. (C) Experimental setup for T cell activation, using Jurkat-GFP cells and Dynabeads (db) coated with CD3 to assess secreted nbCD3/22 functionality via flow cytometry. (D) Dose-dependent T cell activation (CD69 expression) in response to culture supernatants with nbCD3/22, using db coated with CD3 for validation. (E) HEK-293T synNotch reporter assay shows dose-dependent inhibition of CD22-CAR signaling by nbCD22 in CAR-T cell supernatants, confirming BiTE functionality under two conditions. (F) Experimental timeline for in vivo CAR-T cell therapy study in Raji WT or NALM6 WT model followed by CAR-T cell administration and TR with Raji CD19/CD20-/- or NALM6 CD19/CD20-/- cells (G) Bioluminescence imaging of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 CAR-T cells, monitored from Day 7 to Day 84. (H) Quantified tumor radiance over time, showing sustained tumor control in Raji and NALM6 models with b20/19-AKT3 PROTAC+nbCD3/22 . (I1) Percentage of CAR-T cells in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC or b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days (I2) Bar graph of CAR-T cell populations in blood at various time points. (J) Levels of nbCD3/22 (pg/mL) in the blood of Raji and NALM6 tumor-bearing mice treated with b20/19-AKT3 PROTAC+nbCD3/22 , measured over 56 days, showing sustained secretion. (K) Kaplan-Meier survival curves demonstrating improved survival with nbCD3/22-modified CAR-T cells. (L) Bar graph and pie charts compare b20/19-AKT3 PROTAC and b20/19-AKT3 PROTAC+nbCD3/22 , showing various memory T cell subsets over time (n=5) in all conditions. Data represents mean ± SEM. ****p < 0.001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Following transduction, cells were fixed and stained using anti-CAR antibodies that recognize the extracellular domain (ECD) of the respective receptors: CD19 (Cat No. 130-129-550) and CD22 (Miltenyi Biotec).

Techniques: Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery, Reporter Assay, Inhibition, In Vivo, Imaging, Control, Modification