Journal: Cell reports. Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects.

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet: Figure 1. Anti-TGF-b CAR T cells reduced tumor growth and promoted T cell expansion in vivo (A) Anti-TGF-b CAR vector (T28zT2), anti-GPC3 CAR vector (G28zT2), and anti-CD19 CAR vector (1928zT2) based on an anti-TGF-b scFv (US20140127230A1), anti-GPC3 scFv (GC33), or anti-CD19 scFv (FMC63), respectively. All contained expression cassettes encoding a human CD8 leader signal peptide, CD28, CD3z, and TLR2 signaling domain along with eGFP using 2A self-cleaving peptide (2A). The eGFP expression was used to monitor CAR-transduced cells. (B) The percentage of Huh7 cells with 1928zT2, G28zT2, or T28zT2 T cell-induced lysis after 72 h; data are the mean percentage of tumor cell-specific lysis ± SEM values; n = 3 independent experiments; two-way ANOVA with Tukey’s multiple comparisons test; ****p % 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb Cell Signaling Technology Cat# 4494 RRID: AB_11178659 OPA1 (D7C1A) Rabbit mAb Cell Signaling Technology Cat# 67589 RRID: AB_2799728 MFF (E5W4M) XP Rabbit mAb Cell Signaling Technology Cat# 84580 RRID: AB_2799819 SMAD4 (D3R4N) XP Rabbit mAb Cell Signaling Technology Cat# 46535 RRID: AB_2736998 SMAD4 Polyclonal antibody Proteintech Cat# 10231-1-AP RRID: AB_2193323 a-Smooth Muscle Actin (D4K9N) XP Rabbit mAb Cell Signaling Technology Cat# 19245 RRID: AB_2734735 Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signaling Technology Cat# 9664 RRID: 10828837 CD8a (C8/144B) Mouse mAb Cell Signaling Technology Cat# 70306 RRID: AB_2799781 Anti-CD4 antibody Abcam Cat# ab133616 RRID: AB_2750883 Goat Anti-Rabbit IgG H&L (Alexa Fluor 568) Abcam Cat# ab175471 RRID: AB_2576207 Goat Anti-Mouse IgG H&L (Alexa Fluor 647) Abcam Cat# ab150115 RRID: AB_2687948 Biological samples Patient-derived xenografts (PDX) Jiang. et al.,26 Qin. et al.46 N/A Heathy PBMCs Guangzhou Tianhe Noah Biological Engineering Co., LTD NA Chemicals, peptides, and recombinant proteins TGF beta 1 Protein, Human, Rhesus, Cynomolgus, Canine, Recombinant SinoBiological CAS number: 10804-HNAC TMRM MedChemExpress Cat# HY-D0984 MitoTracker Green Yeasen Cat# 40742ES50 MitoTracker Deep Red Yeasen Cat# 40743ES50 GenCRISPRTM Cas9 v1.2 GenScript Cat# Z03702-1 Seahorse XF Cell Mito Stress Test Kit Agilent Cat# 103010-100 Critical commercial assays Human Granzyme B Precoated ELISA Kit DAKEWE Cat# 1118503 Human IFN-g Precoated ELISA Kit DAKEWE Cat# 1110003 Human IL-2 Precoated ELISA Kit DAKEWE Cat# 1110203 Human TGFb1 Precoated ELISA Kit DAKEWE Cat# 1117102 Human PRF1 Precoated ELISA Kit Bioswamp Cat# HM10300 MACS Pan T cell Isolation Kit Miltenyi Biotec Cat# 130-096-535 MACS GMP T cell TransAct Miltenyi Biotec Cat# 170-076-156 Deposited data Original data of the bulk RNAseq This paper GSA-human: HRA001397 Original western blot images This paper https://doi.org/10.17632/n7htzcxmw4.1 Links: https://data.mendeley.com/drafts/ n7htzcxmw4/1 Experimental models: Cell lines Huh7 Procell Cat# CL-0120 HepG2 Procell Cat# CL-0103 (Continued on next page) Cell Reports Medicine 6, 102020, March 18, 2025 e3

Techniques: In Vivo, Plasmid Preparation, Expressing, Lysis

Journal: Cell reports. Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects.

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet: Figure 3. CD4+ but not CD8+ T28zT2 T cells are effective for tumor growth inhibition (A) CD4+ and CD8+ T cell compartments were sorted, and CD8+ T28zT2 T cells and CD4+ T28zT2 cells were generated. We also mixed the CD4+ and CD8+ T28zT2 T cells at a ratio of 1:1 to obtain CD3+ T28zT2 T cells. We then infused a total of 5 3 106 of the three types of T28zT2 T cells, CD3+ 1928zT2 T cells, or CD3+ M28zT2 T cells peritumorally into subcutaneous xenografts of NSI mice inoculated with 1 3 106 AsPc-1 cells (day 0). Tumor volumes were monitored on the indicated days

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb Cell Signaling Technology Cat# 4494 RRID: AB_11178659 OPA1 (D7C1A) Rabbit mAb Cell Signaling Technology Cat# 67589 RRID: AB_2799728 MFF (E5W4M) XP Rabbit mAb Cell Signaling Technology Cat# 84580 RRID: AB_2799819 SMAD4 (D3R4N) XP Rabbit mAb Cell Signaling Technology Cat# 46535 RRID: AB_2736998 SMAD4 Polyclonal antibody Proteintech Cat# 10231-1-AP RRID: AB_2193323 a-Smooth Muscle Actin (D4K9N) XP Rabbit mAb Cell Signaling Technology Cat# 19245 RRID: AB_2734735 Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signaling Technology Cat# 9664 RRID: 10828837 CD8a (C8/144B) Mouse mAb Cell Signaling Technology Cat# 70306 RRID: AB_2799781 Anti-CD4 antibody Abcam Cat# ab133616 RRID: AB_2750883 Goat Anti-Rabbit IgG H&L (Alexa Fluor 568) Abcam Cat# ab175471 RRID: AB_2576207 Goat Anti-Mouse IgG H&L (Alexa Fluor 647) Abcam Cat# ab150115 RRID: AB_2687948 Biological samples Patient-derived xenografts (PDX) Jiang. et al.,26 Qin. et al.46 N/A Heathy PBMCs Guangzhou Tianhe Noah Biological Engineering Co., LTD NA Chemicals, peptides, and recombinant proteins TGF beta 1 Protein, Human, Rhesus, Cynomolgus, Canine, Recombinant SinoBiological CAS number: 10804-HNAC TMRM MedChemExpress Cat# HY-D0984 MitoTracker Green Yeasen Cat# 40742ES50 MitoTracker Deep Red Yeasen Cat# 40743ES50 GenCRISPRTM Cas9 v1.2 GenScript Cat# Z03702-1 Seahorse XF Cell Mito Stress Test Kit Agilent Cat# 103010-100 Critical commercial assays Human Granzyme B Precoated ELISA Kit DAKEWE Cat# 1118503 Human IFN-g Precoated ELISA Kit DAKEWE Cat# 1110003 Human IL-2 Precoated ELISA Kit DAKEWE Cat# 1110203 Human TGFb1 Precoated ELISA Kit DAKEWE Cat# 1117102 Human PRF1 Precoated ELISA Kit Bioswamp Cat# HM10300 MACS Pan T cell Isolation Kit Miltenyi Biotec Cat# 130-096-535 MACS GMP T cell TransAct Miltenyi Biotec Cat# 170-076-156 Deposited data Original data of the bulk RNAseq This paper GSA-human: HRA001397 Original western blot images This paper https://doi.org/10.17632/n7htzcxmw4.1 Links: https://data.mendeley.com/drafts/ n7htzcxmw4/1 Experimental models: Cell lines Huh7 Procell Cat# CL-0120 HepG2 Procell Cat# CL-0103 (Continued on next page) Cell Reports Medicine 6, 102020, March 18, 2025 e3

Techniques: Inhibition, Generated

Journal: Cell reports. Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects.

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet: Figure 5. A combination of CD4+ anti-TGF-b CAR T cells and CD8+ anti-MSLN CAR T cells exhibits augmented antitumor effects in NSCLC PDX (A) NSCLC PDX tumors were diced into 30 mm3 pieces, and tissue were inoculated subcutaneously into the right flanks of 8-week-old male NSI mice. 5 3 106

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb Cell Signaling Technology Cat# 4494 RRID: AB_11178659 OPA1 (D7C1A) Rabbit mAb Cell Signaling Technology Cat# 67589 RRID: AB_2799728 MFF (E5W4M) XP Rabbit mAb Cell Signaling Technology Cat# 84580 RRID: AB_2799819 SMAD4 (D3R4N) XP Rabbit mAb Cell Signaling Technology Cat# 46535 RRID: AB_2736998 SMAD4 Polyclonal antibody Proteintech Cat# 10231-1-AP RRID: AB_2193323 a-Smooth Muscle Actin (D4K9N) XP Rabbit mAb Cell Signaling Technology Cat# 19245 RRID: AB_2734735 Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signaling Technology Cat# 9664 RRID: 10828837 CD8a (C8/144B) Mouse mAb Cell Signaling Technology Cat# 70306 RRID: AB_2799781 Anti-CD4 antibody Abcam Cat# ab133616 RRID: AB_2750883 Goat Anti-Rabbit IgG H&L (Alexa Fluor 568) Abcam Cat# ab175471 RRID: AB_2576207 Goat Anti-Mouse IgG H&L (Alexa Fluor 647) Abcam Cat# ab150115 RRID: AB_2687948 Biological samples Patient-derived xenografts (PDX) Jiang. et al.,26 Qin. et al.46 N/A Heathy PBMCs Guangzhou Tianhe Noah Biological Engineering Co., LTD NA Chemicals, peptides, and recombinant proteins TGF beta 1 Protein, Human, Rhesus, Cynomolgus, Canine, Recombinant SinoBiological CAS number: 10804-HNAC TMRM MedChemExpress Cat# HY-D0984 MitoTracker Green Yeasen Cat# 40742ES50 MitoTracker Deep Red Yeasen Cat# 40743ES50 GenCRISPRTM Cas9 v1.2 GenScript Cat# Z03702-1 Seahorse XF Cell Mito Stress Test Kit Agilent Cat# 103010-100 Critical commercial assays Human Granzyme B Precoated ELISA Kit DAKEWE Cat# 1118503 Human IFN-g Precoated ELISA Kit DAKEWE Cat# 1110003 Human IL-2 Precoated ELISA Kit DAKEWE Cat# 1110203 Human TGFb1 Precoated ELISA Kit DAKEWE Cat# 1117102 Human PRF1 Precoated ELISA Kit Bioswamp Cat# HM10300 MACS Pan T cell Isolation Kit Miltenyi Biotec Cat# 130-096-535 MACS GMP T cell TransAct Miltenyi Biotec Cat# 170-076-156 Deposited data Original data of the bulk RNAseq This paper GSA-human: HRA001397 Original western blot images This paper https://doi.org/10.17632/n7htzcxmw4.1 Links: https://data.mendeley.com/drafts/ n7htzcxmw4/1 Experimental models: Cell lines Huh7 Procell Cat# CL-0120 HepG2 Procell Cat# CL-0103 (Continued on next page) Cell Reports Medicine 6, 102020, March 18, 2025 e3

Techniques:

Journal: Cell reports. Medicine

Article Title: CD4 + anti-TGF-β CAR T cells and CD8 + conventional CAR T cells exhibit synergistic antitumor effects.

doi: 10.1016/j.xcrm.2025.102020

Figure Lengend Snippet: Figure 6. TGF-b1 suppressed OXPHOS activity in CD4+ human T cells (A–D) CD4+ and CD8+ T cells (1 3 106) were activated with CD3/CD28 mAbs for 24 h, followed by PBS or TGF-b1 (10 ng/mL) treatment for 16 h. (A) Mitochondrial morphology of CD4+ and CD8+ T cells upon PBS or TGF-b1 (10 ng/mL) treatment, as determined by spinning disk confocal microscopy. Mitochondria are green (MitoTracker Green), and nuclei are blue (DAPI). Scale bar, 5 mm. (B) Relative lengths of mitochondria, as analyzed by ImageJ software, in CD4+ and CD8+ T cells (2 independent experiments). Each dot represents the mean relative length of the mitochondria in a sample. Data are shown as the mean ± SEM values; paired two-tailed t test; ****p % 0.0001. (C) Immunoblot analysis of cellular protein extracts probed with antibodies against pSMAD2S465/467 (top)/pSMAD3S423/425

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Phospho-DRP1 (Ser616) (D9A1) Rabbit mAb Cell Signaling Technology Cat# 4494 RRID: AB_11178659 OPA1 (D7C1A) Rabbit mAb Cell Signaling Technology Cat# 67589 RRID: AB_2799728 MFF (E5W4M) XP Rabbit mAb Cell Signaling Technology Cat# 84580 RRID: AB_2799819 SMAD4 (D3R4N) XP Rabbit mAb Cell Signaling Technology Cat# 46535 RRID: AB_2736998 SMAD4 Polyclonal antibody Proteintech Cat# 10231-1-AP RRID: AB_2193323 a-Smooth Muscle Actin (D4K9N) XP Rabbit mAb Cell Signaling Technology Cat# 19245 RRID: AB_2734735 Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb Cell Signaling Technology Cat# 9664 RRID: 10828837 CD8a (C8/144B) Mouse mAb Cell Signaling Technology Cat# 70306 RRID: AB_2799781 Anti-CD4 antibody Abcam Cat# ab133616 RRID: AB_2750883 Goat Anti-Rabbit IgG H&L (Alexa Fluor 568) Abcam Cat# ab175471 RRID: AB_2576207 Goat Anti-Mouse IgG H&L (Alexa Fluor 647) Abcam Cat# ab150115 RRID: AB_2687948 Biological samples Patient-derived xenografts (PDX) Jiang. et al.,26 Qin. et al.46 N/A Heathy PBMCs Guangzhou Tianhe Noah Biological Engineering Co., LTD NA Chemicals, peptides, and recombinant proteins TGF beta 1 Protein, Human, Rhesus, Cynomolgus, Canine, Recombinant SinoBiological CAS number: 10804-HNAC TMRM MedChemExpress Cat# HY-D0984 MitoTracker Green Yeasen Cat# 40742ES50 MitoTracker Deep Red Yeasen Cat# 40743ES50 GenCRISPRTM Cas9 v1.2 GenScript Cat# Z03702-1 Seahorse XF Cell Mito Stress Test Kit Agilent Cat# 103010-100 Critical commercial assays Human Granzyme B Precoated ELISA Kit DAKEWE Cat# 1118503 Human IFN-g Precoated ELISA Kit DAKEWE Cat# 1110003 Human IL-2 Precoated ELISA Kit DAKEWE Cat# 1110203 Human TGFb1 Precoated ELISA Kit DAKEWE Cat# 1117102 Human PRF1 Precoated ELISA Kit Bioswamp Cat# HM10300 MACS Pan T cell Isolation Kit Miltenyi Biotec Cat# 130-096-535 MACS GMP T cell TransAct Miltenyi Biotec Cat# 170-076-156 Deposited data Original data of the bulk RNAseq This paper GSA-human: HRA001397 Original western blot images This paper https://doi.org/10.17632/n7htzcxmw4.1 Links: https://data.mendeley.com/drafts/ n7htzcxmw4/1 Experimental models: Cell lines Huh7 Procell Cat# CL-0120 HepG2 Procell Cat# CL-0103 (Continued on next page) Cell Reports Medicine 6, 102020, March 18, 2025 e3

Techniques: Activity Assay, Confocal Microscopy, Software, Two Tailed Test, Western Blot

Journal: eBioMedicine

Article Title: Enhancing the efficacy of near-infrared photoimmunotherapy through intratumoural delivery of CD44–targeting antibody–photoabsorber conjugates

doi: 10.1016/j.ebiom.2025.105566

Figure Lengend Snippet: Intratumoural delivery of CD44-IR700 enhanced efficacy of photoimmunotherapy in vivo . (a) Schematic of experimental schedule. Mice bearing subcutaneous Lewis lung carcinoma tumours were treated with either no CD44-IR700 (Ctrl), CD44-IR700 via IV injection (CD44-IR700_IV), or CD44-IR700 via IT injection (CD44-IR700_IT), followed by NIR light irradiation on subsequent days. (b) The ratio of tumour-infiltrating CD8-positive T cells in live cells, as determined by flow cytometry on day 7 post-treatment, from the CD44-IR700_IV and CD44-IR700_IT groups (n = 6 per group). (c) Immunohistochemical staining of tumours 7 days after photoimmunotherapy. CD8-positive T cells were stained. The scale bar represents 50 μm. The numbers of CD8-positive T cells per view field were counted (n = 5 mice for each group). (d) Cytokine expression in tumours, as determined by quantitative PCR analysis on day 3 post-treatment with photoimmunotherapy, from the CD44-IR700_IV and CD44-IR700_IT groups (n = 5 per group). (e) Tumour volume monitoring. Tumour size was measured starting from the day of CD44-IR700 administration (day 0), and the change in size was expressed as a ratio relative to the initial volume on day 0 (n = 12 per group). (f) Tumour volume ratio on day 7. The graph shows the fold increase in tumour volume on day 7 compared with that on day 0 (n = 12 per group). (g) Representative images displaying tumours from each treatment group on day 7 post-administration. (h) Representative images of tumours excised on day 7 post-treatment from each therapeutic group. Data are presented as mean + standard error of the mean. Statistical difference was assessed using Mann–Whitney U tests, with significance denoted by asterisks (∗ p < 0.05 and ∗∗∗∗ p < 0.0001). CD44-IR700, IR700-conjugated anti-CD44 monoclonal antibody; CD, cluster of differentiation; NIR, near-infrared; Ctrl, control; IV, intravenous; IT, intratumoural; Ifnγ, interferon-γ; and Tnf, tumour necrosis factor-alpha.

Article Snippet: For immunohistochemistry, the primary antibody was anti-mouse CD8a antibody (98941S, Cell Signalling Technology, RRID: AB_2756376 ).

Techniques: In Vivo, IV Injection, Injection, Irradiation, Flow Cytometry, Immunohistochemical staining, Staining, Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Control

Journal: European Journal of Immunology

Article Title: The Microbiome Modifies Manifestations of Hemophagocytic Lymphohistiocytosis in Perforin‐Deficient Mice

doi: 10.1002/eji.202451061

Figure Lengend Snippet: Transfer of a diverse microbiome into PKO mice does not provoke spontaneous HLH. (A) Experimental setup for generating PKO‐Wildlings. (B) Microbiome analysis of fecal samples from Wildlings and SPF mice. Principal coordinate analysis (PCoA) with distance calculation using Bray‐Curtis dissimilarity (left). Relative abundance at the rank of phylum (right). (C) Mean litter size and wean‐to‐born ratio for PKO‐Wildling, PKO‐SPF, and C57BL/6J mice . (D) Kaplan–Meier curve of cumulative survival probability for PKO‐Wildling and PKO‐SPF mice at indicated time points after birth ( n = number of mice). Ticks indicate censored animals. (E) Representative contour plots of indicated antigens on isolated splenocytes (left). Relative and absolute abundance of CD8 + T cells, CD44 + , and KLRG1 + antigen‐experienced CD8 + T cells in the spleen and liver (right). (F) Contour plots depicting FoxP3, CD25, and RORγt expression (left). Frequency and total counts of FoxP3 + CD25 + CD4 + T regs and RORγt + T cells. (G) Abundance of indicated populations in the spleen and liver. (E–G) Data represent mean ± SD, pooled from at least two independent experiments with 3 mice/group, aged 9–16 weeks of age. Statistical differences were determined using unpaired Student's t ‐test, Mann–Whitney test, or multiple unpaired t ‐tests with correction for multiple comparisons using the Holm–Šidák method.

Article Snippet: For CD8 + staining, sections were pretreated with Dako Target Retrieval Solution (pH 9) for 20 min, blocked with 10% goat serum and Avidin/Biotin solution, and incubated with 1:200 rabbit anti‐mouse CD8a (Cell Signaling, #98941S) and biotinylated anti‐rabbit IgG overnight.

Techniques: Isolation, Expressing, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: The Microbiome Modifies Manifestations of Hemophagocytic Lymphohistiocytosis in Perforin‐Deficient Mice

doi: 10.1002/eji.202451061

Figure Lengend Snippet: Presence of Wildling microbiota in PKO mice promotes liver pathology during LCMV‐induced HLH. (A) Glutamate‐pyruvate transaminase (GPT) and lactate‐dehydrogenase (LDH) in serum of LCMV‐infected mice analyzed on day 11/12 after infection. Noninfected (n.i.) B6‐SPF mice are shown as a reference. (B) Viral titers in the liver, spleen, lung, and kidney. Shown is the median with an interquartile range. (C) The contour plot depicts the staining of F4/80 and Ly‐6G on density‐isolated immune cells from the liver of LCMV‐infected mice (left). The graph shows absolute numbers of NK cells (NK1.1 + CD3 − ), F4/80 + CD11b + macrophages, dendritic cells (DC, CD11c + SiglecH + /CD11c + MHCII + ), and Neutrophils (CD11b + Ly‐6G + ) in the liver (right). (D) Representative flow cytometry plots showing expression of CD8, CD4, FoxP3, and CD25 on liver immune cells (left), and percentages and total numbers of indicated cell subsets (right). (E) Representative images of CD8 and F4/80 immunohistochemistry staining of liver sections with H&E counterstaining (scale bar = 50 µm). Insert: Examples of hemophagocytosis. Quantification of hemophagocytic macrophages per 10 high‐power fields (HPF) assessed based on F4/80 staining and number of CD8 + cells per mm 2 liver tissue. (A, C–E) Shown is mean ± SD. Symbols represent individual mice pooled from 2–4 independent experiments with 2–4 mice/group. Statistical testing was performed using unpaired Student‘s t ‐test (A), Mann–Whitney test (A, B, D, E), and multiple t ‐tests with correction for multiple comparisons by Holm‐Šidák (C).

Article Snippet: For CD8 + staining, sections were pretreated with Dako Target Retrieval Solution (pH 9) for 20 min, blocked with 10% goat serum and Avidin/Biotin solution, and incubated with 1:200 rabbit anti‐mouse CD8a (Cell Signaling, #98941S) and biotinylated anti‐rabbit IgG overnight.

Techniques: Infection, Staining, Isolation, Flow Cytometry, Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: European Journal of Immunology

Article Title: The Microbiome Modifies Manifestations of Hemophagocytic Lymphohistiocytosis in Perforin‐Deficient Mice

doi: 10.1002/eji.202451061

Figure Lengend Snippet: Wildling microbiota modulates the cytokine pattern in LCMV‐triggered HLH. (A) PCA Biplot of 32 cytokines analyzed by multiplex assay in serum of mice on day 12 after LCMV‐WE infection. Points represent individual mice and colored arrows indicate the contribution of selected cytokines. (B) Serum concentration of selected HLH‐related cytokines. Horizontal line and error bars indicate the median with interquartile range. (C) IFN‐γ expression of CD8 + T cells after ex vivo restimulation with GP 33‐41 peptide (left). The abundance of IFN‐γ‐producing CD8 + T cells in spleens upon ex vivo restimulation with GP 33‐41 peptide or PMA/Ionomycin (right). (D) RORγt expression in TCRβ + T cells isolated from the liver (left). Frequency and absolute number of RORγt + T cells in the liver (right). (C, D) Shown is mean ± SD. (A–D) Results shown are representative of two experiments performed with similar outcomes (A, B) or pooled from two independent experiments with 2–4 mice/group (C, D). Statistical testing was done by unpaired Student's t ‐test (B‐D).

Article Snippet: For CD8 + staining, sections were pretreated with Dako Target Retrieval Solution (pH 9) for 20 min, blocked with 10% goat serum and Avidin/Biotin solution, and incubated with 1:200 rabbit anti‐mouse CD8a (Cell Signaling, #98941S) and biotinylated anti‐rabbit IgG overnight.

Techniques: Multiplex Assay, Infection, Concentration Assay, Expressing, Ex Vivo, Isolation

Journal: European journal of immunology

Article Title: The Microbiome Modifies Manifestations of Hemophagocytic Lymphohistiocytosis in Perforin-Deficient Mice.

doi: 10.1002/eji.202451061

Figure Lengend Snippet: FIGURE 1 Transfer of a diverse microbiome into PKO mice does not provoke spontaneous HLH. (A) Experimental setup for generating PKO- Wildlings. (B) Microbiome analysis of fecal samples from Wildlings and SPF mice. Principal coordinate analysis (PCoA) with distance calculation using Bray-Curtis dissimilarity (left). Relative abundance at the rank of phylum (right). (C) Mean litter size and wean-to-born ratio for PKO-Wildling, PKO- SPF, and C57BL/6J mice [31]. (D) Kaplan–Meier curve of cumulative survival probability for PKO-Wildling and PKO-SPF mice at indicated time points after birth (n = number of mice). Ticks indicate censored animals. (E) Representative contour plots of indicated antigens on isolated splenocytes (left). Relative and absolute abundance of CD8+ T cells, CD44+, and KLRG1+ antigen-experienced CD8+ T cells in the spleen and liver (right). (F) Contour plots depicting FoxP3, CD25, and RORγt expression (left). Frequency and total counts of FoxP3+ CD25+ CD4+ Tregs and RORγt+ T cells. (G) Abundance of indicated populations in the spleen and liver. (E–G) Data represent mean ± SD, pooled from at least two independent experiments with 3 mice/group, aged 9–16 weeks of age. Statistical differences were determined using unpaired Student’s t-test, Mann–Whitney test, or multiple unpaired t-tests with correction for multiple comparisons using the Holm–Šidák method.

Article Snippet: For CD8+ staining, sections were pretreated with Dako Target Retrieval Solution (pH 9) for 20 min, blocked with 10% goat serum and Avidin/Biotin solution, and incubated with 1:200 rabbit anti-mouse CD8a (Cell Signaling, #98941S) and biotinylated anti-rabbit IgG overnight.

Techniques: Isolation, Expressing, MANN-WHITNEY

Journal: European journal of immunology

Article Title: The Microbiome Modifies Manifestations of Hemophagocytic Lymphohistiocytosis in Perforin-Deficient Mice.

doi: 10.1002/eji.202451061

Figure Lengend Snippet: FIGURE 4 Presence of Wildling microbiota in PKO mice promotes liver pathology during LCMV-induced HLH. (A) Glutamate-pyruvate transaminase (GPT) and lactate-dehydrogenase (LDH) in serum of LCMV-infected mice analyzed on day 11/12 after infection. Noninfected (n.i.) B6- SPF mice are shown as a reference. (B) Viral titers in the liver, spleen, lung, and kidney. Shown is the median with an interquartile range. (C) The contour plot depicts the staining of F4/80 and Ly-6G on density-isolated immune cells from the liver of LCMV-infected mice (left). The graph shows absolute numbers of NK cells (NK1.1+ CD3−), F4/80+ CD11b+ macrophages, dendritic cells (DC, CD11c+ SiglecH+/CD11c+ MHCII+), and Neutrophils (CD11b+ Ly-6G+) in the liver (right). (D) Representative flow cytometry plots showing expression of CD8, CD4, FoxP3, and CD25 on liver immune cells (left), and percentages and total numbers of indicated cell subsets (right). (E) Representative images of CD8 and F4/80 immunohistochemistry staining of liver sections with H&E counterstaining (scale bar = 50 µm). Insert: Examples of hemophagocytosis. Quantification of hemophagocytic macrophages per 10 high-power fields (HPF) assessed based on F4/80 staining and number of CD8+ cells per mm2 liver tissue. (A, C–E) Shown is mean ± SD. Symbols represent individual mice pooled from 2–4 independent experiments with 2–4 mice/group. Statistical testing was performed using unpaired Student‘s t-test (A), Mann–Whitney test (A, B, D, E), and multiple t-tests with correction for multiple comparisons by Holm-Šidák (C).

Article Snippet: For CD8+ staining, sections were pretreated with Dako Target Retrieval Solution (pH 9) for 20 min, blocked with 10% goat serum and Avidin/Biotin solution, and incubated with 1:200 rabbit anti-mouse CD8a (Cell Signaling, #98941S) and biotinylated anti-rabbit IgG overnight.

Techniques: Infection, Staining, Isolation, Flow Cytometry, Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: European journal of immunology

Article Title: The Microbiome Modifies Manifestations of Hemophagocytic Lymphohistiocytosis in Perforin-Deficient Mice.

doi: 10.1002/eji.202451061

Figure Lengend Snippet: FIGURE 5 Wildling microbiota modulates the cytokine pattern in LCMV-triggered HLH. (A) PCA Biplot of 32 cytokines analyzed by multiplex assay in serum of mice on day 12 after LCMV-WE infection. Points represent individual mice and colored arrows indicate the contribution of selected cytokines. (B) Serum concentration of selected HLH-related cytokines. Horizontal line and error bars indicate the median with interquartile range. (C) IFN-γ expression of CD8+ T cells after ex vivo restimulation with GP33-41 peptide (left). The abundance of IFN-γ-producing CD8+ T cells in spleens upon ex vivo restimulation with GP33-41 peptide or PMA/Ionomycin (right). (D) RORγt expression in TCRβ+ T cells isolated from the liver (left). Frequency and absolute number of RORγt+ T cells in the liver (right). (C, D) Shown is mean ± SD. (A–D) Results shown are representative of two experiments performed with similar outcomes (A, B) or pooled from two independent experiments with 2–4 mice/group (C, D). Statistical testing was done by unpaired Student’s t-test (B-D).

Article Snippet: For CD8+ staining, sections were pretreated with Dako Target Retrieval Solution (pH 9) for 20 min, blocked with 10% goat serum and Avidin/Biotin solution, and incubated with 1:200 rabbit anti-mouse CD8a (Cell Signaling, #98941S) and biotinylated anti-rabbit IgG overnight.

Techniques: Multiplex Assay, Infection, Concentration Assay, Expressing, Ex Vivo, Isolation