pe-cy7 anti-mouse tim-3 (rmt3-23, 12-5870-82)  (Thermo Fisher)

Journal: bioRxiv

Article Title: Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion

doi: 10.1101/2024.05.09.593421

Figure Lengend Snippet: (A,B) Quantification of intracellular amino acids 10 min (a), SAM and SAH up to 60 min (b) in OT-I T cells activated with 10 ng/ml SIINFEKL (n=3). (C) Quantification of essential amino acids (left) and peak area of Met (right) in LN CD8 + T cells and CD8 + TIL, isolated at day 12 post tumor implantation (n=3). (D) Quantification of intracellular Met in CD8 + PBMC and CD8 + TIL isolated from patient primary colorectal tumors (n= 8). (E) Tumor growth (left) and survival (right) of B16-OVA tumors in Rag1 -/- mice after transfer of 24 hr activated OT-I T cells initially activated in 0.1 or 0.03 mM Met with 2.5ng/ml SIINFEKL for 30 min, followed by restoration of 0.03 mM to 0.1 mM Met (n=5). (F) Schematic of experimental design. OT-I CD8 + T cells with different congenic markers were initially activated in 0.1 mM or 0.03 mM Met for 30 min with replenishment of Met in 0.03 mM to 0.1 mM Met for 24 hrs, transferred into B16-OVA tumor bearing Rag1 -/- mouse at a 1:1 ratio and analyzed day 12 post T cell transfer. (G) Frequency of PD1 + and PD1 + Tim-3 + cells of OT-I CD8 + TIL, initially activated in 0.1 mM and 0.03 mM Met and assessed at D12 post injection (n=7). (H-I) Contour plot and frequency of TOX + cells (h) and frequency of IFNψ + TNFα + (i) of OT-I CD8 + TIL as in G (n=7). (J-L) Frequency of (j) TCF1 + Tim-3 - TEXprog, CX3CR1 + Ly108 + TEXprogenitor-like (k) and, TCF1 - Tim-3 + TEXterm (l) cells in OT-I CD8 + T cells initially activated as in E (n=7, n=4). (M) T cell exhaustion gene sets in GSEA analysis of RNA-Seq of OT-I CD8 + TIL as in E n=4. Data are mean±s.e.m. Unpaired two-tailed Student’s t-test (A-D), two-way ANOVA (E-left), Mantel-Cox log rank test (E--right) and paired two-tailed Student’s t-test (G-L) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: BUV395 anti-mouse CD44 (IM7, 363-0441-82), PerCP-Cy 5.5 anti-mouse IL-2 (JES6-5H4, 45-7021-82), PerCP-eF710 anti-mouse CD27 (O323, 46-0279-42), and PE-Cy7 anti-mouse Tim-3 (RMT3-23, 12-5870-82, eBioscience) were obtained from Thermo Fisher.

Techniques: Isolation, Tumor Implantation, Injection, RNA Sequencing Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion

doi: 10.1101/2024.05.09.593421

Figure Lengend Snippet: (A) Frequencies of T cells isolated from B16-OVA tumors at D12 post transfer of OT-I T cells activated as in (n=7). (B) Expression of TCF1 + on OT-I CD8 + T cells at D12 post transfer of OT-I T cells, activated as in (n=7). (C, D) Representative contour plot (c) and quantification (d) of TCF1 and Tim-3 expressing cells from TOX + CD8 + TIL from OT-I CD8 + T cells isolated at D12 post transfer of OT-I T cells activated as in into B16-OVA tumor-bearing Rag1 -/- mice (n=7). (E-G) Frequency of Tim-3 - Ly108 + TEXprog (e), Tim-3 + Ly108 - , CXCR3 - Tim-3 + TEXterm (f), CD62L + T memory and CD62L + CD44 + T central memory cells (g) in OT-I TIL as in (A) (n=7 (f), n=4 (g)). (H-J) Representative contour plot and quantification of CD127 + CD27 + memory cells (h, i) and KLRG1 + CD127 - terminal effector cells (j) in OT-I CD8 + TIL isolated at D12 post of cells activated as in into B16-OVA tumor-bearing Rag1 -/- mice (n=4). Data are mean±s.e.m with statistical analyses performed using paired two-tailed Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: BUV395 anti-mouse CD44 (IM7, 363-0441-82), PerCP-Cy 5.5 anti-mouse IL-2 (JES6-5H4, 45-7021-82), PerCP-eF710 anti-mouse CD27 (O323, 46-0279-42), and PE-Cy7 anti-mouse Tim-3 (RMT3-23, 12-5870-82, eBioscience) were obtained from Thermo Fisher.

Techniques: Isolation, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion

doi: 10.1101/2024.05.09.593421

Figure Lengend Snippet: (A) Fluo-8 AM analysis of Ca 2+ flux in CD8 + T cells activated with anti-CD3 and anti-CD28 by anti-hamster IgG crosslinking in either 0.1 mM or 0.03 mM Met containing Ca 2+ Ringer solution with addition of 2 mM Ca 2+ to measure Ca 2+ influx. Representative of 2 experiments. (B, C) Representative images of T cells, cultured with or without anti-CD3/28 dynabeads (dark grey masked), in 0.1 mM and 0.03 mM Met, with or without cyclosporin-A treatment for 30 min, stained with NFAT1 (red, marked), Hoechst (blue) and Phalloidin (green) (b). Quantification of NFAT1 intensity as nuclear to cell ratio (c) (See for unmasked figure). Scale bar=10μm. (Each circle represents one cell, n=40 cells). (D, E) Histogram of NFAT1 binding signals (d), read count per million reads normalized to background) and quantification of known NFAT1-binding regions of Lag3, Pdcd1, Havcr2, Ctla4 and Tnfrsf9 and Tox (e, normalized read count (see differential binding analysis)) in T cells initially activated in 0.1 or 0.03 mM Met for 30 min and assessed 24 hrs post activation (n=2). Data are mean±s.e.m. Unpaired two-tailed Student’s t-test (C, D). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: BUV395 anti-mouse CD44 (IM7, 363-0441-82), PerCP-Cy 5.5 anti-mouse IL-2 (JES6-5H4, 45-7021-82), PerCP-eF710 anti-mouse CD27 (O323, 46-0279-42), and PE-Cy7 anti-mouse Tim-3 (RMT3-23, 12-5870-82, eBioscience) were obtained from Thermo Fisher.

Techniques: Cell Culture, Staining, Binding Assay, Activation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion

doi: 10.1101/2024.05.09.593421

Figure Lengend Snippet: (A) Un-edited image of showing the unmasked, Red CD3/38 dynabeads which are masked grey in the main figure to highlight NFAT1 staining. (B) Quantification of nuclear NFAT1 from the experiment shown in . (C) NFAT1 intensity quantification (nuclear to cell ratio) of CD8 + T cells activated in either 0.1 mM, 0.00 mM or 0.03 mM Met for 30 min with anti CD3/28 dynabeads and stained for NFAT1 (each circle represents one cell, n=40 cells). (D) Fluo-8 AM analysis of Ca 2+ flux in CD8 + T cells activated with anti-CD3 and anti-CD28 and anti-hamster IgG crosslinking in either 0.1 mM or 0.03 mM Met containing Ca 2+ -free Ringer solution treated with DMSO or 5μM YM58483. (E) NFAT1 intensity quantification (nuclear to cell ratio) of CD8 + T cells activated for 30 min by CD3/28 dynabeads in either 0.1 mM or 0.03 mM Met in the presence of DMSO or 5μM YM58483 (n=40 cells). (F) NFAT1 quantification (nuclear to cell ratio) of previously activated OT-I T cells, treated with anti-CD3/28 dynabeads for 30 min. T cells were initially activated with 2.5ng/ml SIINFEKL for 24 hrs in control medium and rested with 10 ng/ml mIL-7 for 48 hrs (each circle represents one cell, n=40). (G) NFAT2 quantification (nuclear to cell ratio) of CD8 + T cells, activated in 0.1 mM or 0.03 mM Met for 30 min with anti-CD3/28 dynabeads (each circle represents one cell, n=40 cells). (H) NFAT1 Cut&Run tracks at Pdcd1, Havcr2, Lag3, and Tnfrsf9 locus. The gray highlights the peaks showing increased NFAT1 binding (n=2). (I) Normalized RNA expression quantified by quantitative-PCR performed 24 hrs post activation of OT-I CD8 + T cells activated with 2.5 ng/ml SIINFEKL in 0.1 mM or 0.03 mM Met for 30 min followed by 0.1 mM (n=6). Data are mean±s.e.m. Unpaired two-tailed Student’s t-test (B, C, E, F, G) and one-tailed Student’s t-test (I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: BUV395 anti-mouse CD44 (IM7, 363-0441-82), PerCP-Cy 5.5 anti-mouse IL-2 (JES6-5H4, 45-7021-82), PerCP-eF710 anti-mouse CD27 (O323, 46-0279-42), and PE-Cy7 anti-mouse Tim-3 (RMT3-23, 12-5870-82, eBioscience) were obtained from Thermo Fisher.

Techniques: Staining, Binding Assay, RNA Expression, Real-time Polymerase Chain Reaction, Activation Assay, Two Tailed Test, One-tailed Test

Journal: bioRxiv

Article Title: Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion

doi: 10.1101/2024.05.09.593421

Figure Lengend Snippet: (A) Quantification of methyl-arginine in CD8 + T cells activated with anti-CD3/28 dynabeads in 0.1, 0.0, or 0.03 mM Met for 30 min (each circle represents one cell. n= 25 cells). (B) Fluo-8 AM analysis of Ca 2+ flux in CD8 + T cells activated with anti-CD3 and anti-CD28 by anti-hamster IgG crosslinking in either 0.1 mM or 0.03 mM Met containing Ca 2+ -free Ringer solution treated either with DMSO or 1μM TRAM-34. (C) Tumor growth of B16-OVA tumors in Rag1 -/- mouse after transfer of OT-I CD8 + T cells activated in 0.1 mM or 0.03 mM Met with either DMSO or 1 μM TRAM-34 for 30 min and cultured for 24 hrs (n=5). (D) ICR-1 AM analysis of Ca 2+ flux in KCa3.1 WT and KCa3.1 R350A T cells activated with anti-CD3 and anti-CD28 by anti-hamster IgG crosslinking in Ca 2+ -free Ringer solution. (E, F) Representative image (e) and quantification of NFAT1intensity as nuclear to cell ratio (f) in KCa3.1 WT and KCa3.1 R350A OT-I T cells activated for 30 min with anti-CD3/28 dynabeads (dark grey masked) (See for unmasked figure), scale bar=10 μm (each circle represents one cell, n=40 cells). (G, H) Tumor growth (g) and survival (h) of B16-OVA tumor-bearing RAG -/- mice after transfer of KCa3.1 WT and KCa3.1 R350A OT-I T cells (n=5). (I) B16-OVA tumors harvested at D12 post transfer of KCa3.1 WT and KCa3.1 R350A OT-I T cells (n=4). (J) Schematic of experimental design to assess congenically distinct KCa3.1 WT and KCa3.1 R350A OT-I T cells, mixed at a 1:1 ratio and transferred into B16-OVA tumor bearing Rag1 -/- mice and analyzed day 12 post T cell transfer. (K-L) Frequency of Tim-3 + PD1 + (k), histogram and quantification of TOX MFI, and frequency of IFNψ + TNFα + (l) in KCa3.1 WT and KCa3.1 R350A OT-I TIL at D12 post T-cell transfer as in J (n=7). (M) Frequency of TCF1 + Tim-3 - TEXprog and TCF1 - Tim-3 + TEXterm in KCa3.1 WT and KCa3.1 R350A OT-I TIL at D12 post T-cell transfer as in J (n=7). (N) Schematic of experimental design to assess congenically distinct KCa3.1 WT and KCa3.1 R350A Thy1.1 P14 T cells, mixed at a 1:1 ratio, transferred into WT mice, and infected with either LCMV-Armstrong or LCMV-Clone-13. Spleens were analyzed at D9 post infection. (O, P) Frequency of Tox + and PD1 + Tim-3 + in KCa3.1 WT and KCa3.1 R350A P14 CD8 + T cells from spleens at D9 post infection with either LCMV-Armstrong (o) or LCMV-Clone-13 (p) (n=4). (Q-S) Frequency of CX3CR1 + PD1 + TEXprog (q), Tim-3 + of CX3CR1 + PD1 + TEXeffector-like (r) and TCF1 - Tim-3 + TEXterm (s) in KCa3.1 WT and KCa3.1 R350A P14 CD8 + T cells from spleens at D9 post infection as in N (n=4). Data are mean±s.e.m. Unpaired two-tailed Student’s t-test (A, F), 2-way ANOVA (C, G), Mantel-Cox test (H) and paired two-tailed Student’s t-test (K-M, O-S). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: BUV395 anti-mouse CD44 (IM7, 363-0441-82), PerCP-Cy 5.5 anti-mouse IL-2 (JES6-5H4, 45-7021-82), PerCP-eF710 anti-mouse CD27 (O323, 46-0279-42), and PE-Cy7 anti-mouse Tim-3 (RMT3-23, 12-5870-82, eBioscience) were obtained from Thermo Fisher.

Techniques: Cell Culture, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion

doi: 10.1101/2024.05.09.593421

Figure Lengend Snippet: (A) Tumor weight of B16-OVA tumors isolated at D12 post transfer of OT-I T cells expressing KCa3.1 WT or KCa3.1 R350A (n=4). (B) Surface expression of PD1 + , Lag3 + and PD1 + Tim-3 + in CD8 + TIL isolated at D12 post transfer into B16-OVA tumor-bearing mice of OT-I T cells expressing KCa3.1 WT or KCa3.1 R350A (n=4). (C) Representative histogram of TOX expression and quantification in CD8 + TIL isolated at D12 post transfer of OT-I T cell expressing KCa3.1 WT or KCa3.1 R350A as (b) (n=4). (D) Representative contour plot (left) and quantification of IFNψ + TNFα + (right) in OT-I T cells expressing KCa3.1 WT or KCa3.1 R350A at D12 post transfer as (b) (n=4). (F, G) Expression of EOMES + PD1 + (f) and representative contour plot of expression of TCF1 and Tim-3 (g) on congenically marked OT-I T cells expressing KCa3.1 WT or KCa3.1 R350A , mixed and transferred at a ratio of 1:1. TIL were isolated from B16-OVA tumors at D12 post T cell transfer (n=7). (H, I) Expression of memory markers CD62L and CD127 + CD27 + (h), representative contour plot and quantification of KLRG1 + CD127 - terminal effector cells (i) on congenically marked OT-I T cells expressing KCa3.1 WT or KCa3.1 R350A , as in (F), isolated at D12 post transfer into B16-OVA tumor-bearing mice (n=7). (J) Frequency of KCa3.1 WT - and KCa3.1 R350A -expressing P14 T cells in spleens, D9 post infection with either LCMV-Armstrong or LCMV-Clone-13 (n=4). (K-M) Representative contour plot (k) and quantification of KLRG1 + CD127 - terminal effector cells (l) and KLRG1 - CD127 + memory precursor cells (m) in spleens of animals injected with congenically marked P14 T cells expressing KCa3.1 WT or KCa3.1 R350A , mixed 1:1, and infected with either LCMV-Armstrong or LCMV-Clone-13. Analysis on d9 post infection (n=4). Data are mean±s.e.m. Unpaired two-tailed Student’s t-test (A-D) and paired two-tailed Student’s t-test (F-M). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: BUV395 anti-mouse CD44 (IM7, 363-0441-82), PerCP-Cy 5.5 anti-mouse IL-2 (JES6-5H4, 45-7021-82), PerCP-eF710 anti-mouse CD27 (O323, 46-0279-42), and PE-Cy7 anti-mouse Tim-3 (RMT3-23, 12-5870-82, eBioscience) were obtained from Thermo Fisher.

Techniques: Isolation, Expressing, Infection, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: Rapid metabolic regulation of a novel arginine methylation of KCa3.1 attenuates T cell exhaustion

doi: 10.1101/2024.05.09.593421

Figure Lengend Snippet: (A) Schematic of experimental design of acute HBSS/Met treatment in sub-cutaneous tumors. (B) Nuclear NFAT1 quantification of CD44 + CD8 + T cells isolated from B16 subcutaneous tumors and dLN, 5 days post daily peri-tumoral supplementation of 50 μl 61μM Met (n=4 mice, each circle represents one cell, n=40 cells). (C-E) Tumor growth (left) and survival (right) of WT mice with sub-cutaneous MC38 colorectal tumors (c, n=10), Lewis-lung carcinoma (LLC) tumors (d, (representative of two experiments, n=5) and F420 osteosarcoma tumors (e, n=5), treated with either HBSS or 50 μl 61μM Met peri-tumorally daily for 5 days (n=10). (F, G) Expression of PD1, Tim-3, CD69 + (f), Tcm (CD62L hi Cd44 hi ) and Tem (CD62L lo CD44 hi ) (g) on CD8 + TIL isolated from B16 tumor bearing mice 2 days after 5 daily peri-tumoral injection with HBSS or Met (n=4). (H) Tumor growth of B16f10 and MC38 tumors in NSG mice treated peri-tumorally with 50 μl HBSS or 50 μl 61μM Met daily for 5 days (n=5). (I) Representative flow cytometry plot of blood showing CD8 + T cell depletion in anti-CD8 antibody treated mice compared to IgG-Isotype treated mice (n=4). (J) Schematic of experimental design of contralateral tumor experiment. (K) Growth of B16f10 tumors implanted on left shoulder (dashed lines) and right flank (solid lines) following peri-tumoral injection of flank tumor with 50 μl HBSS or 61μM Met daily for 5 days (n=5, representative of two experiments). (L) B16 tumor growth in WT mice fed with either 1 % Met chow or 1.5 % Met chow, 7 days post tumor implantation (n=5). Data are mean±s.e.m. Two-way ANOVA (C-F, J and K), Mantel-Cox log rank test (C, D and E), unpaired two-tailed Student’s t-test (B, G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: BUV395 anti-mouse CD44 (IM7, 363-0441-82), PerCP-Cy 5.5 anti-mouse IL-2 (JES6-5H4, 45-7021-82), PerCP-eF710 anti-mouse CD27 (O323, 46-0279-42), and PE-Cy7 anti-mouse Tim-3 (RMT3-23, 12-5870-82, eBioscience) were obtained from Thermo Fisher.

Techniques: Isolation, Expressing, Injection, Flow Cytometry, Tumor Implantation, Two Tailed Test