Journal: Cancer Research

Article Title: Base Editing of TIGIT Reprograms CD155 Signaling in Natural Killer Cells to Enhance Cancer Immunotherapy Efficacy

doi: 10.1158/0008-5472.CAN-25-0733

Figure Lengend Snippet: Cytotoxicity activities of TIGIT BE-NK cells in vitro . A–E, A variety of different cancer cell lines (GFP expressing or red dye labeled) were inoculated into 96-well plates in quantities of 10 4 cells/well and left for 6 hours to allow cells to attach, then 10 4 PB-NK or BE-NK cells were added, and images of the same field of view were recorded at different times. A, NSCLC cell line H1299 (GFP expressing). B , NSCLC cell line A549 (GFP expressing). C , Fibrosarcoma cell line HT-1080 (GFP expressing). D , Hepatocellular cell line Huh7 (red dye labeled). E , Hepatoma cell line PLC/PRF/5 (red dye labeled). Images recorded and data analyzed by IncuCyte S3, and data are shown as mean ± SD.

Article Snippet: Plates were then transferred into the Incucyte S3 (Sartorius, RRID: SCR_023147), and images were captured by the Incucyte S3 system with a × 10 objective lens at 6-hour time intervals.

Techniques: In Vitro, Expressing, Labeling

Journal: Cancer Research

Article Title: Base Editing of TIGIT Reprograms CD155 Signaling in Natural Killer Cells to Enhance Cancer Immunotherapy Efficacy

doi: 10.1158/0008-5472.CAN-25-0733

Figure Lengend Snippet: TIGIT BE-NK cells specifically recognized target cells in a CD155-/CD226-dependent manner. A, The mean fluorescence intensity (MFI) of CD155 on H1299, H1975, A549, and H460 (NSCLC cell line), K652 (chronic myelogenous leukemia cell line), and CEM (acute lymphoblastic leukemia cell line) was assessed by flow cytometry. B, Tumor cells and PB-NK or TIGIT BE-NK cells were inoculated into 48-well plates at a 1:1 ratio, and CD107a expression on NK cells was detected by flow cytometry 4 hours later. The increase in CD107a-positive cells in the TIGIT BE-NK group was normalized to the PB-NK group. C, Tumor cells were coincubated with PB-NK or TIGIT BE-NK cells for 24 hours at an E:T ratio of 1:1, and then NK cell–mediated lysis was evaluated by luminescent cell viability assay. E:T corresponds to E:T cells. D, The mean fluorescence intensity of CD155 on U251-MG (glioblastoma cell line), Huh7 (hepatocellular cell line), HT-1080 (fibrosarcoma cell line), PLC/PRF/5 (hepatoma cell line), BxPC-3 (pancreatic adenocarcinoma cell line), T24 (urinary bladder cancer cell line), U2OS (osteosarcoma cell line), MCF7 (breast cancer cell line), HeLa (cervical carcinoma cell line), SKOV3 (ovarian cancer cell line), HCT116 (colorectal carcinoma cell line), HGC-27 (gastric cancer cell line), KG-1A, OCI-AML3 (acute myeloid leukemia cell line), and MM.1S (multiple myeloma) was assessed by flow cytometry. E, Tumor cells and PB-NK or TIGIT BE-NK cells were inoculated into 48-well plates at a 1:1 ratio, and CD107a expression on NK cells was detected by flow cytometry 4 hours later. The increase in CD107a-positive cells in the TIGIT BE-NK group was normalized to the PB-NK group. F, The correlation analysis of the result is shown in D and E . Simple linear regression analysis. G and H, The NK cell–mediated cytotoxicity was evaluated using a luminescent cell viability assay following coincubation of A549 ( G ) or Huh7 ( H ) cells with NK cells at a 1:1 ratio for 24 hours. I and J, Cytotoxic effects of individual and combined TIGIT, CD96, and PVRIG KO in NK cells. Data recorded and analyzed by IncuCyte S3; data are shown as mean ± SD. K and L, PB-NK, TIGIT BE-NK, or TIGIT/CD226 double–KO NK cells were cocultured with A549 or Huh7 tumor cells. NK cells were harvested at 0, 2, 15, and 30 minutes after stimulation, and p44/42 MAPK (ERK1/2) and phospho-p44/42 MAPK (ERK1/2; Thr202/Tyr204) activity was assessed by Western blotting. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: Plates were then transferred into the Incucyte S3 (Sartorius, RRID: SCR_023147), and images were captured by the Incucyte S3 system with a × 10 objective lens at 6-hour time intervals.

Techniques: Fluorescence, Flow Cytometry, Expressing, Lysis, Cell Viability Assay, Activity Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: The influence of GVHD on T cell development in allogeneic chimeras. Irradiated euthymic (A and C) or thymectomized (B and D) A.BY (Thy1.2, Ly5.2) recipients received a graft containing 10 7 T cell–depleted B6.PL (Thy1.1, Ly5.2) bone marrow cells (as a source of hematopoietic progenitors) with or without mature T cells (0.4 or 1.6 × 10 6 ) harvested from the LNs of B6.SJL donors (Thy1.2, Ly5.1). Chimeras' spleen cells were analyzed by three-color staining on day 100 ± 5 posttransplant. The origin of recipient T cells was determined according to their Thy1/Ly5 phenotype. Results are presented as the mean ± SD (three to four mice per group). Note that the ordinate scale is different in A and B versus C and D. nd, not determined.

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Irradiation, Staining

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: Skewing of Vβ usage by CD4 + and CD8 + T cells in athymic GVHD + chimeras. Three-color staining was performed using Abs against CD4 or CD8, Thy1.1 or Thy1.2, and specific Vβ elements. Chimeras were studied on day 100 ± 5. Results are presented as the mean ± SD (three to four mice per group).

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: Expansion of T cells from GVHD + mice after adoptive transfer into normal hosts. Three-color staining was performed on spleen cell suspensions using Abs specific for CD4 or CD8, Ly5.1 or Ly5.2, and Thy1.1 or Thy1.2. (A) Numbers of CD4 + and CD8 + T cells of B6.PL origin (Thy1.1, Ly5.2) found in the spleen of day 100 nonthymectomized GVHD + recipients (negative controls). (B) Numbers of CD4 + and CD8 + T cells of B6.PL origin (Thy1.1, Ly5.2) found in the spleen of irradiated/thymectomized B6.SJL or A.BY secondary hosts, on day 30 after injection of 10 7 T cell–depleted C57BL/6 bone marrow cells + 2 × 10 6 T cells harvested from the spleen of day 70 GVHD + mice (test group). (C) Numbers of Thy1.2 + Ly5.2 + T cells found in the spleen of irradiated/thymectomized B6.SJL or A.BY hosts, on day 30 after injection of 10 7 T cell–depleted B6.PL bone marrow cells + 2 × 10 6 T cells harvested from the spleen of normal syngeneic donors (A.BY for A.BY secondary recipients, C57BL/6 for B6.SJL secondary recipients) (positive controls). GVHD + mice were prepared as in Fig. C (irradiation followed by transplantation of 10 7 B6.PL bone marrow cells + 0.4 × 10 6 B6.SJL LN T cells). Results are presented as the mean ± SD (three to four mice per group). * P < 0.05, ** P < 0.005 relative with numbers of CD4 + or CD8 + T cells in GVHD + mice (Student's t test). No significant difference was found between T cell numbers presented in B and C.

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Adoptive Transfer Assay, Staining, Irradiation, Injection, Transplantation Assay

Journal: The Journal of Experimental Medicine

Article Title: The Effect of Graft-versus-Host Disease on T Cell Production and Homeostasis

doi:

Figure Lengend Snippet: Adoptive transfer of normal host (A.BY)-tolerant T cells into nonthymectomized GVHD + mice and thymectomized GVHD − recipients. (A) Number of T cells in the spleen of day 100 GVHD + mice. GVHD + mice were prepared as in Fig. C (irradiation followed by transplantation of 10 7 B6.PL bone marrow cells + 0.4 × 10 6 B6.SJL T cells). (B) Number of T cells in the spleen of day 100 GVHD + mice previously injected, on day 60, with 5 × 10 6 host-tolerant T cells of C57BL/6 (Thy1.2,Ly5.2) origin. (C) Number of T cells in the spleen of thymectomized A.BY mice 40 d after irradiation and injection of 10 7 T cell–depleted B6.SJL bone marrow cells + 5 × 10 6 host-tolerant B6.PL T cells. Host (A.BY)-tolerant T cells used in B and C experiments were obtained from the spleen of nonthymectomized A.BY hosts, 60 d after irradiation and injection of T cell–depleted C57BL/6 or B6.PL bone marrow cells. Results are presented as the mean ± SD (three to four mice per group).

Article Snippet: Bone marrow cells were obtained from the tibias and femurs of donor mice and T cell depleted with a specific anti-Thy1.2 mAb (5a-8; mouse IgG) or with a rabbit anti–mouse T cells (Thy1) antiserum, both obtained from Cedarlane Labs., and rabbit serum (Low-Tox-M rabbit complement; Cedarlane Labs.) as a source of complement.

Techniques: Adoptive Transfer Assay, Irradiation, Transplantation Assay, Injection