gzmb cre  (Jackson Laboratory)

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 deficiency restricts tumor progression by promoting T PEX cell transition. (A) Schematic diagram of the adoptive transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 CD45.2 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. (B) Tumor growth in each group of the mice in A. n = 8 per group. (C) Survival curve in each group of the mice in A. n = 8 per group. (D) The absolute number of tumor infiltrating OT-I cells from the mice in A. n = 8 per group. (E and F) Representative flow cytometry plots and cumulative data show the frequency and absolute number of Tcf1 + Tim3 − T PEX , Tim3 + Tcf1 − T EX (E), and CX3CR1 + Tcf1 − Int-T EX cells (F) gated on tumor-infiltrating OT-I cells. n = 6–8 per group. (G) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of GzmB + , IFN-γ + , and perforin + cells gated on tumor-infiltrating OT-I cells. n = 7 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and MFI of GzmB, IFN-γ, and perforin gated on tumor-infiltrating Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX, and CX3CR1 − Tcf1 − T EX subsets. n = 5–6 per group. Data are representative of three independent experiments. P value was calculated by two-way ANOVA (B), Log-rank test (C), and two-tailed Student’s t test (D−H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The C57BL/6J (B6, CD45.2), B6.SJL (CD45.1), Cd4 cre , Gzmb cre , P14, and OT-I mice were from the Jackson Laboratory.

Techniques: Injection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 promotes T PEX differentiation without affecting their proliferation and apoptosis. (A and B) Representative flow cytometry plots and cumulative data show the frequency of CD44 (A) and PD-1 (B) OT-I cells infiltrating in tumors. (C) Representative flow cytometry plots and cumulative data show the frequency of caspase and Ki67 in tumor-infiltrating OT-I cells. (D) Cumulative data show the frequency of caspase and Ki67 in Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX , and CX3CR1 − Tcf1 − T EX subsets. (E) Cumulative data show the frequency of GzmB, IFN-γ, and perforin in tumor-infiltrating OT-I subsets mentioned above. (F) Schematic diagram of the classic CRC liver metastases model: Mettl8 fl/fl Cd4 cre and Mettl8 fl/fl mice were intrasplenically injected with 2 × 10 5 MC38 cells (left), and imaging of livers on day 21 after injection (right). (G) Representative flow cytometry plots and cumulative data show the ratio of Tcf1 + Tim3 − T PEX to Tim3 + Tcf1 − T EX cells gated on CD44 hi CD62L lo CD8 + T cells of the livers from mice in F. (H) Representative flow cytometry plots and cumulative data show the frequency of GzmB, IFN-γ, and TNF-α gated on CD44 hi CD62L lo CD8 + T cells from the livers of mice in F. (I) Schematic diagram of the classic melanoma lung metastases model: Mettl8 fl/fl Gzmb cre and Mettl8 fl/fl mice were i.v. injected with 2 × 10 5 B16F10 cells (top) and the survival curve (bottom). (J) Schematic diagram of adoptive transfer model: CD45.1.2 + Mettl8 −/− or WT P14 CD8 + T cells were adoptively transferred into CD45.2 + WT recipients, followed by LCMV-clone 13 (LCMV-C13) infection 24 h later and then analyzed on 30 dpi. (K) Statistical analysis show the absolute number of P14 cells from the spleens of mice in J. (L) Representative flow cytometry plots and cumulative data show Tcf1 + Tim3 − T PEX , Tim3 + Tcf1 − T EX , and CX3CR1 + Tcf1 − Int-T EX cells gated on P14 cells from the spleens of mice in J. n = 4–8 mice per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test (A–H, K, and L) or Log-rank test (I); *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The C57BL/6J (B6, CD45.2), B6.SJL (CD45.1), Cd4 cre , Gzmb cre , P14, and OT-I mice were from the Jackson Laboratory.

Techniques: Flow Cytometry, Injection, Imaging, Adoptive Transfer Assay, Infection, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Reconstitution of Mettl8 expression in Mettl8 −/− OT-I cells restored their phenotype to that of WT OT-I cells. (A) Schematic diagram of the rescue experiment: CD45.2 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells. Mettl8 overexpression (OE) or empty vector (EV) retrovirus were transduced to CD45.1.2 WT or Mettl8 −/− OT-I cells. 5 × 10 5 GFP + cells were sorted 48 h after transduction and adoptively transferred into the tumor-bearing mice at 9 dpi. Mice were harvested at 19 dpi. (B) Tumor growth in each group of the mice in A. n = 6 per group. (C) Tumor growth in each group displayed in each replicate. n = 6 per group. (D) Tumor weight (left) and the absolute number of tumor infiltrating OT-I cells (right) from the mice in A. n = 6 per group. (E) Representative flow cytometry plots and cumulative data show the frequency of GFP in OT-I cells. n = 6 per group. (F) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of Tcf1 + Tim3 − T PEX and Tim3 + Tcf1 − T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (G) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of GzmB + , IFN-γ + , and perforin + cells gated on tumor-infiltrating OT-I cells. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-way ANOVA (B) and two-tailed Student’s t test (D to H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The C57BL/6J (B6, CD45.2), B6.SJL (CD45.1), Cd4 cre , Gzmb cre , P14, and OT-I mice were from the Jackson Laboratory.

Techniques: Expressing, Injection, Over Expression, Plasmid Preparation, Transduction, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Intraepithelial lymphocytes exhibit selective immunity to intestinal pathogens

doi: 10.1101/2025.05.14.654011

Figure Lengend Snippet: A ) Flow cytometry gating strategy used to define IEL subsets from small intestinal epithelial preparations. B) Sorting strategy for scATAC-seq analysis. Live CD45⁺CD3⁺CD8α⁺ IEL were isolated for chromatin profiling. C–D) Bar graphs quantifying the proportion of IELs with accessible chromatin at (C) Itgae (encoding CD103) and (D) Ifng , canonical markers of IEL identity and effector function. E) Area-proportional Venn diagram illustrating the overlap in accessible chromatin regions between TCRγδ⁺ and TCRαβ⁺ IEL based on previously published bulk ATAC-seq data (Semenkovich et al., 2016), indicating extensive epigenetic convergence. F) Normalized RNA expression of Gzma and Gzmb across T cell developmental stages, mature T cell subsets, and NK cells, based on publicly available ImmGen datasets ( https://www.immgen.org/ ). G) Representative histogram showing inefficient IEL labeling in previously reported GzmbCre transgenic mice crossed to Rosa26 lsl-EYFP . H) Representative histogram of tdTomato⁺ IEL in Gzmb Cre/+ × Rosa26 lsl-tdTomato mice, demonstrating robust and specific labeling across IEL subsets. I) Flow cytometry quantification of intracellular GzmB protein expression across major IEL subsets from Gzmb Cre/+ mice.

Article Snippet: Rosa26 lsl-tdTomato (B6.Cg- Gt(ROSA)26Sor tm (CAG-tdTomato)Hze /J), Rosa26 lsl - iDTR (CBy.B6- Gt(ROSA)26Sor tm1(HBEGF)Awai /J) and transgenic Gzmb-Cre (B6;FVB-Tg(GZMB-cre) 1Jcb/J ) mice strains were purchased from Jackson Laboratories, USA.

Techniques: Flow Cytometry, Isolation, RNA Expression, Labeling, Transgenic Assay, Expressing

Journal: bioRxiv

Article Title: Intraepithelial lymphocytes exhibit selective immunity to intestinal pathogens

doi: 10.1101/2025.05.14.654011

Figure Lengend Snippet: A) UMAP projection of CD45⁺CD103⁺ IEL isolated from the small intestinal epithelium, revealing phenotypic heterogeneity across subsets. Accompanying pie chart depicts relative frequencies of each subset. Representative of three mice. B) Area-proportional Venn diagram illustrating shared proteomic profiles among three major IEL subsets: TCRαβ⁺CD8αα⁺, TCRγδ⁺CD8αα⁺, and TCRαβ⁺CD8αβ⁺. C) UMAP embedding of CD45⁺CD3⁺CD8α⁺ IEL profiled by single-cell ATAC-seq, demonstrating convergence in chromatin accessibility landscapes. D–E) Representative intracellular flow cytometry histograms of GzmB protein expression in (D) IEL and (E) peripheral immune populations, including NK cells, thymocytes, and splenic T cells. F) Confocal images of jejunal tissue sections stained for GzmB (green), CD3 (red), E-cadherin (gray), and nuclei (DAPI, blue), showing GzmB⁺CD3⁺ cells localized to the epithelial compartment. Scale bar, 100 μm. Representative of five mice. G) Schematic of the Gzmb -Cre knock-in allele, in which a T2A-Cre cassette is inserted downstream of the endogenous Gzmb stop codon, enabling bicistronic expression. H) Pie chart depicting the relative frequencies of each IEL subset in Gzmb Cre/+ mice. Representative of three mice. I) Representative immunofluorescence image of jejunal sections from Gzmb Cre/+ × Rosa26 LsL-tdTomato mice, showing tdTomato⁺ IELs (red) within the epithelium. Actin (phalloidin, green); nuclei (DAPI, blue). Representative of three mice. J) Flow cytometry plots and pooled quantification of tdTomato⁺ frequencies across IEL subsets, with individual bar labels denoting mean percentage of tdTomato⁺ cells within each population. Data represents mean ± SEM from 3 mice/group.

Article Snippet: Gzmb KI-Cre was generated by Ingenious Targeting Laboratory for Dr. Mahima Swamy.

Techniques: Isolation, Flow Cytometry, Expressing, Staining, Knock-In, Immunofluorescence

Journal: bioRxiv

Article Title: Intraepithelial lymphocytes exhibit selective immunity to intestinal pathogens

doi: 10.1101/2025.05.14.654011

Figure Lengend Snippet: A ) Flow cytometry gating strategy used to define IEL subsets from small intestinal epithelial preparations. B) Sorting strategy for scATAC-seq analysis. Live CD45⁺CD3⁺CD8α⁺ IEL were isolated for chromatin profiling. C–D) Bar graphs quantifying the proportion of IELs with accessible chromatin at (C) Itgae (encoding CD103) and (D) Ifng , canonical markers of IEL identity and effector function. E) Area-proportional Venn diagram illustrating the overlap in accessible chromatin regions between TCRγδ⁺ and TCRαβ⁺ IEL based on previously published bulk ATAC-seq data (Semenkovich et al., 2016), indicating extensive epigenetic convergence. F) Normalized RNA expression of Gzma and Gzmb across T cell developmental stages, mature T cell subsets, and NK cells, based on publicly available ImmGen datasets ( https://www.immgen.org/ ). G) Representative histogram showing inefficient IEL labeling in previously reported GzmbCre transgenic mice crossed to Rosa26 lsl-EYFP . H) Representative histogram of tdTomato⁺ IEL in Gzmb Cre/+ × Rosa26 lsl-tdTomato mice, demonstrating robust and specific labeling across IEL subsets. I) Flow cytometry quantification of intracellular GzmB protein expression across major IEL subsets from Gzmb Cre/+ mice.

Article Snippet: Gzmb KI-Cre was generated by Ingenious Targeting Laboratory for Dr. Mahima Swamy.

Techniques: Flow Cytometry, Isolation, RNA Expression, Labeling, Transgenic Assay, Expressing

Journal: bioRxiv

Article Title: Intraepithelial lymphocytes exhibit selective immunity to intestinal pathogens

doi: 10.1101/2025.05.14.654011

Figure Lengend Snippet: A) Representative immunofluorescence image of jejunal tissue from IEL iDTR ( G zmb Cre/+ × Rosa26 LsL-iDTR ) and control IEL WT (Gzmb Cre/+ ) mice 24 hours following a single intraperitoneal injection of diphtheria toxin (DT, 50 μg/kg), showing loss of CD3⁺ IELs in the epithelium. Scale bar, 100 μm. Representative of 5 mice. B) Flow cytometric quantification of CD45⁺CD103⁺ IELs and CD45⁺CD103⁻ cells in the epithelial fraction 24 h post-DT. Data represents mean ± SEM from 3 mice/group. C) Quantification of TCRγδ⁺ and TCRβ⁺ cells by flow cytometry in both IEL and lamina propria (gated on CD45+CD103-) (LP) compartments of the small intestine 24 h post-DT. Data represents mean ± SEM from 3 mice/group. D) Percent decrease across individual IEL subsets in IEL iDTR mice 24 h after DT, compared to IEL WT with individual bar labels denoting mean percentage of cells deleted within each population. Data represents mean ± SEM from 3 mice/group. E) Flow cytometric analysis of TCRγδ⁺ and TCRβ⁺ T cell populations in the spleen 24 h post-DT administration. Data represents mean ± SEM from 3 mice/group. F–G) Time-course quantification of total IELs (CD45⁺CD103⁺), TCRγδ⁺, and TCRαβ⁺ subsets at multiple time points post-DT (days 1, 4, 7, 10, 15), showing near-complete repopulation by day 15. Data represents mean ± SEM from 3 mice/group. H) Distribution of IEL subsets 15 days following DT treatment, demonstrating restoration of pre-depletion composition. Representation of 3 mice/group. I–J) Quantification of IEL and spleen T cells recovery at day 7 post-DT in mice treated with FTY720 (to block thymic egress) versus vehicle, demonstrating thymus-independent regeneration. Data represents mean ± SEM from 5 mice/group. K) EdU incorporation in CD45⁺CD103⁺ IEL on day 4 post-DT, indicating robust local proliferation during repopulation. Data represents mean ± SEM from 5 mice/group. L–M) Ki67 expression in IEL across time points post-depletion, showing a transient spike in proliferation that returns to baseline by day 15. Data represents mean ± SEM from 4-6 mice/group. Statistical analyses: ANOVA with Sidak’s post hoc correction (B,C,E, F, G, L, M); unpaired t-test (I, J, K). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = non-significant.

Article Snippet: Gzmb KI-Cre was generated by Ingenious Targeting Laboratory for Dr. Mahima Swamy.

Techniques: Immunofluorescence, Control, Injection, Flow Cytometry, Blocking Assay, Expressing

Journal: bioRxiv

Article Title: Intraepithelial lymphocytes exhibit selective immunity to intestinal pathogens

doi: 10.1101/2025.05.14.654011

Figure Lengend Snippet: A ) Flow cytometric quantification of CD45⁺, CD45⁺CD103⁺ (IEL), and CD45⁺CD103⁻ (non-IEL) subsets in the small intestine of Gzmb Cre/+ mice 24 hours after diphtheria toxin (DT) or PBS administration. Data represent mean ± SEM from 3 mice per group. B) Quantification of individual IEL subsets in the small intestine of IEL WT and IEL iDTR mice at day 1 post-DT, confirming broad depletion across γδ and αβ IEL lineages. Data represent mean ± SEM from 3 mice per group. C) Flow cytometric analysis of total CD45⁺ immune cells in multiple peripheral and mucosal compartments, including Peyer’s patches, colon IEL, colon lamina propria (LP), mesenteric lymph nodes (mLN), liver, and thymus of IEL WT and IEL iDTR mice, 24 hours after DT treatment. Data represent mean ± SEM from 3 mice per group. D–G) Quantification of TCRγδ⁺ and TCRβ⁺ lymphocyte subsets across mucosal and systemic tissues—Peyer’s patches, colon IEL, colon LP, mLN, and liver of IEL WT and IEL iDTR mice —24 hours post-DT administration. Data represent mean ± SEM from 3 mice per group. Statistical comparisons: ANOVA with Sidak’s post hoc correction (A, D, E, F, G) and unpaired t-test (B, C). * p < 0.05, ** p < 0.01, *** p < 0.001, ns = non-significant.

Article Snippet: Gzmb KI-Cre was generated by Ingenious Targeting Laboratory for Dr. Mahima Swamy.

Techniques:

Journal: bioRxiv

Article Title: Intraepithelial lymphocytes exhibit selective immunity to intestinal pathogens

doi: 10.1101/2025.05.14.654011

Figure Lengend Snippet: A–B ) Time-course quantification of (A) TCRγδ⁺ and (B) TCRβ⁺ IEL subsets following a single DT injection in IEL WT and IEL iDTR mice, demonstrating progressive repopulation over 15 days. Data represent mean ± SEM from 3-5 mice per group. C–D) Representative histograms of tdTomato expression in (D) CD45⁺CD103⁺ and (E) TCRγδ⁺ and TCRβ⁺ IEL from Gzmb CreERT2 × Rosa26 lsl-tdTomato mice, assessed 7 days after final tamoxifen or oil-only control administration. E) Quantification of tdTomato⁺ IEL across indicated time points, demonstrating the longevity of labelled IEL subsets. Data represent mean ± SEM from 4 mice per group. Statistical comparisons: ANOVA with Sidak’s post hoc correction (A, B). * p < 0.05, ** p < 0.01.

Article Snippet: Gzmb KI-Cre was generated by Ingenious Targeting Laboratory for Dr. Mahima Swamy.

Techniques: Injection, Expressing, Control

Journal: Cell Reports Medicine

Article Title: Ex vivo activation of the GCN2 pathway metabolically reprograms T cells, leading to enhanced adoptive cell therapy

doi: 10.1016/j.xcrm.2024.101465

Figure Lengend Snippet:

Article Snippet: Mouse: GzmB Cre , The Jackson Laboratory , RRID: IMSR_JAX:003734.

Techniques: In Vivo, Recombinant, Cell Stimulation, RNA Sequencing, Software