anti ifnγ receptor antibody  (Bio X Cell)

Journal: bioRxiv

Article Title: Acute IFN-γ responses drive sensory neuron injury and chronic pain after chikungunya virus infection

doi: 10.64898/2026.02.02.702518

Figure Lengend Snippet: (A–E) qPCR analysis of pro-inflammatory mediators in ipsilateral knee joints after intra-articular CHIKV infection. Tnf (A) and Il6 (B) were transiently induced, peaking at 7 and 3 dpi, respectively. Type I interferons ( Ifna , C; Ifnb , D) peaked at 1 dpi and returned to baseline thereafter. In contrast, Ifng (E) was strongly upregulated, peaking at 7 dpi and remaining elevated through at least 28 dpi. One-way ANOVA followed by Tukey’s post hoc test in (A) P = 0.032, (B) P = 0.0186, (C) # P = 0.065, (D) P = 0.026 and (E) P = 0.022; n = 3-6 per group. (F–H) Flow cytometry analysis of synovial fluid shows marked T cell accumulation after CHIKV infection. (F) Representative plots of CD4⁺ and CD8⁺ T cells in Mock, 7 dpi, and 28 dpi joints. Quantification revealed significant increases in CD4⁺ (G) and CD8⁺ (H) T cells at 7 dpi, which declined by 28 dpi but remained above Mock levels. One-way ANOVA followed by Dunnett’s post hoc test in (G) P < 0.0001 and (H) P = 0.005; n = 3–4 animals per group. (I–K) Flow cytometry analysis of CD11b⁺CD45⁺ myeloid cells. (I) Representative plots of synovial fluid from Mock, 7 dpi, and 28 dpi animals. (J) Quantification showed a strong increase in CD11b⁺CD45⁺ cells in joint fluid at 7 dpi, which declined toward baseline by 28 dpi. (K) In parallel, the frequency of circulating CD11b⁺CD45⁺ cells in blood was significantly reduced at 7 dpi, consistent with migration into the joint. One-way ANOVA followed by Dunnett’s post hoc test in (J) P = 0.027 and (K) P = 0.018; n = 4 animals per group. (L–O) Flow cytometry analysis of IFN-γ–producing T cells in the synovial fluid after CHIKV infection. (L, N) Representative plots of IFN-γ expression in CD8⁺ (L) and CD4⁺ (N) T cells from Mock, 7 dpi, and 28 dpi joints. (M) Quantification revealed a marked increase in the frequency of IFN-γ⁺ CD8⁺ T cells at 7 dpi, which was markedly reduced by 28 dpi. (O) A similar but less pronounced increase was observed in IFN-γ⁺ CD4⁺ T cells at 7 dpi. One-way ANOVA followed by Dunnett’s post hoc test in (M) P < 0.0001 and (O) P = 0.004; n = 3–4 animals per group.

Article Snippet: Pharmacological inhibition of IFN-γ signalling was achieved via intra-articular administration of a monoclonal anti-IFN-γ receptor antibody (anti-CD119; clone GR-20, Bio X Cell, Cat# BE0245).

Techniques: Infection, Flow Cytometry, Migration, Expressing

Journal: bioRxiv

Article Title: Acute IFN-γ responses drive sensory neuron injury and chronic pain after chikungunya virus infection

doi: 10.64898/2026.02.02.702518

Figure Lengend Snippet: (A–B) Intra-articular (i.a.) injections of IFN-γ (300 U or 600U/site; days 0, 2 and 4) induced a rapid and persistent reduction in paw withdrawal thresholds compared with saline, as shown by the time course (A) and AUC analysis (B) , demonstrating robust mechanical allodynia. Two-way ANOVA followed by Tukey’s post hoc test in (A) P < 0.05 and One-way ANOVA followed by Tukey’s post hoc test in (B) P < 0.001; n = 9–14 animals per group. (C–E) IFN-γ did not alter Adamts4 (C) or Mmp13 (D) expression in the joint but markedly increased Atf3 mRNA levels in dorsal root ganglia (DRG; E), consistent with a neuronal stress response independent of joint catabolism. Two-tailed unpaired t test in (C) n.s., (D) n.s., (E) P = 0.047; n = 7-9 animals per group. (F–H) IFN-γ directly activates cultured DRG neurons following 1 h exposure (1 ng/mL). (F) Representative double immunostaining for NeuN (red), p-ERK (green), and DAPI (blue). (G) Quantification of p-ERK mean fluorescence intensity in NeuN⁺ neurons, showing enhanced neuronal activation following IFN-γ treatment. (H) Capsaicin (0.5 µg, 5 min), used as a positive control, similarly increased p-ERK immunoreactivity. Quantifications were performed in three independent cultures, with two coverslips per culture; fluorescence intensity was measured at the single-cell level in NeuN⁺ neurons. Each data point represents one cell. Statistical analysis was performed using two-tailed unpaired t test; (G) P < 0.0001; (H) P = 0.0056. (I–K) IFN-γ also induced ATF3 expression in cultured DRG neurons. (I) Representative double immunostaining for NeuN (red), ATF3 (green), and DAPI (blue). (J) Quantification of ATF3 mean fluorescence intensity in NeuN⁺ neurons. (K) qPCR confirmed upregulation of Atf3 mRNA following IFN-γ exposure. Quantifications were performed in three independent cultures, with two coverslips per culture; fluorescence intensity was measured at the single-cell level in NeuN⁺ neurons. Each data point represents one cell. Two-tailed unpaired t test in (J) P = 0.0011 and (K) P = 0.0033. (L–U) Pharmacological or genetic blockade of IFN-γ signaling prevents CHIKV-induced pain and neuronal stress without altering viral load. (L) Anti-CD119 (IFN-γR1) monoclonal antibody prevented CHIKV-induced mechanical allodynia, as shown by time course (L) and AUC (M) analyses. Two-way ANOVA followed by Tukey’s multiple-comparisons test in (L) P < 0.0001; One-way ANOVA followed by Tukey’s post hoc test in (M) P = 0.0017; n = 8 animals per group. (N) Anti-CD119 treatment did not alter viral RNA levels in the joint, indicating that pharmacological IFN-γ blockade alleviates pain without impacting local viral burden. Two-tailed unpaired t test, n.s.; n = 7-8 animals per group (O, P) Anti-CD119 treatment attenuated CHIKV-induced upregulation of Adamts4 (O) and Mmp13 (P) in the joint, indicating that while IFN-γ alone is insufficient to drive catabolic gene expression, its signaling contributes to maintaining joint-destructive responses in the context of CHIKV infection. One-way ANOVA followed by Tukey’s post hoc test in (O) P = 0.002 and (P) P =0.001; n = 5-7 animals per group. (Q–T) Similarly, IFN-γ receptor-deficient (Ifngr1⁻/⁻) mice were protected from CHIKV-induced mechanical allodynia (Q, R) and exhibited abolished Adamts4 (S) and Mmp13 (T) induction, with no change in joint viral RNA burden. (U) Atf3 mRNA upregulation in DRG observed in WT animals was absent in Ifngr1⁻/⁻ mice, demonstrating that IFN-γ signaling is essential for CHIKV-induced neuronal stress and pain persistence. Two-way ANOVA followed by Tukey’s multiple-comparisons test in (Q) P < 0.05; (R-U) One-way ANOVA followed by Tukey’s post hoc test in (R) P = 0.003, (S) P = 0.043, (T) P = 0.003 and (U) P = 0.015. n = 8 animals per group.

Article Snippet: Pharmacological inhibition of IFN-γ signalling was achieved via intra-articular administration of a monoclonal anti-IFN-γ receptor antibody (anti-CD119; clone GR-20, Bio X Cell, Cat# BE0245).

Techniques: Saline, Expressing, Two Tailed Test, Cell Culture, Double Immunostaining, Fluorescence, Activation Assay, Positive Control, Single Cell, Gene Expression, Infection

Journal: bioRxiv

Article Title: Acute IFN-γ responses drive sensory neuron injury and chronic pain after chikungunya virus infection

doi: 10.64898/2026.02.02.702518

Figure Lengend Snippet: (A) Schematic representation of intra-articular (i.a.) CHIKV (10⁶ PFU) inoculation into the mouse knee joint. (B) Time course of paw withdrawal thresholds (PWT, g) to von Frey stimulation in Mock- and CHIKV-inoculated mice (0–77 dpi). CHIKV lowered PWT compared with Mock. Two-way ANOVA followed by Tukey’s post hoc test, P < 0.05. n = 9-24 animals per group. (C) Area under the curve (AUC) of PWT from panel B. AUC was significantly lower in CHIKV mice. Two-tailed unpaired t test, P < 0.0001. n = 9-24 animals per group. (D) CHIKV-infected mice were stratified at 77 dpi by mechanical sensitivity: ≥30 % reduction in PWT defined persistent (CHIKV-Persis., orange circles) and <30 % defined recovered (CHIKV-Recov., blue circles) groups. This classification revealed two distinct pain phenotypes in CHIKV-infected mice. (E) Time course of PWT (% baseline) in CHIKV-infected mice stratified at 77 dpi as demonstrated in D. CHIKV-Persis. mice maintained significantly lower PWT during the late phase (42-77 dpi) when compared with CHIKV-Recov. group. Two-way ANOVA with Tukey’s multiple-comparisons test, P < 0.05. n = 9-13 animals per group. (F) AUC (42-77 dpi) of PWT from panel E. AUC was significantly lower in CHIKV-Persis. group. One-way ANOVA with Tukey’s post hoc test, P = 0.043 (CHIKV-Recov. vs. CHIKV-Persis.); n = 9-13 animals per group. (G, H) Quantification of CHIKV RNA (Eq. PFU/µg RNA) in knee joints (G) and DRG (H) from CHIKV-Persis., CHIKV-Recov., and Mock groups at 77 dpi. Joint viral loads did not differ between groups, and viral RNA was undetectable in DRG, suggesting that persistent pain is independent of viral persistence. Two-tailed unpaired t tests in (G) and (H); n.s. n = 5-9 animals per group. (I-K) Levels of catabolic genes in knee joints of CHIKV-Recov. and CHIKV-Persis. mice at 77 dpi. Mmp13 (I) and Adamts4 (J) levels, and the Rankl/Opg mRNA ratio ( K) did not differ between groups. Two-tailed unpaired t tests in (I) , (J) and (K) , n.s. n = 7-11 animals per group. (L, M) Serum IFN-γ concentrations were elevated in CHIKV-Persis. mice during the acute phase, but not during the chronic phase of infection. (L) At 7 dpi (acute phase), IFN-γ levels were significantly higher in CHIKV-Persis. animals compared with Mock and CHIKV-Recov. groups. One-way ANOVA followed with Tukey’s post hoc test in (L) P = 0.023 (Mock vs. CHIKV-Persis.); in (M) n.s.. n = 6-7 animals per group.

Article Snippet: Pharmacological inhibition of IFN-γ signalling was achieved via intra-articular administration of a monoclonal anti-IFN-γ receptor antibody (anti-CD119; clone GR-20, Bio X Cell, Cat# BE0245).

Techniques: Two Tailed Test, Infection

Journal: bioRxiv

Article Title: Acute IFN-γ responses drive sensory neuron injury and chronic pain after chikungunya virus infection

doi: 10.64898/2026.02.02.702518

Figure Lengend Snippet: (A) Cohort of patients with confirmed CHIKV infection, with blood collected during the acute phase and clinical classification 3 months later as recovered (CHIKV-Recov., n = 20) or with persistent arthralgia (CHIKV-Chronic, n = 22). (B) Forest plot showing odds ratios (ORs) and 95% confidence intervals (CIs) for acute-phase clinical symptoms comparing patients who developed chronic arthralgia versus those who recovered 3 months after CHIKV infection. No symptom showed a significant association with chronic outcome. (C, E, G, I) Serum TNF-α (C), IL-6 (E), IL-1β (G), and IFN-γ (I) levels measured during the acute phase in CHIKV-Recov. and CHIKV-Chronic patients. IFN-γ levels were significantly higher in CHIKV-Chronic patients. Two-tailed unpaired t tests: TNF-α, IL-6, and IL-1β, n.s.; IFN-γ, P = 0.011. n = 14–22 per group. (D, F, H, J) Receiver operating characteristic (ROC) analyses corresponding to TNF-α (D), IL-6 (F), IL-1β (H), and IFN-γ (J). IFN-γ showed the highest discriminative performance for chronic arthralgia (area under the curve, AUC = 0,73), whereas TNF-α, IL-6, and IL-1β displayed limited predictive value (AUCs = 0.52–0.66). Sample size per group ranged from n = 14 to 22.

Article Snippet: Pharmacological inhibition of IFN-γ signalling was achieved via intra-articular administration of a monoclonal anti-IFN-γ receptor antibody (anti-CD119; clone GR-20, Bio X Cell, Cat# BE0245).

Techniques: Infection, Two Tailed Test

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet: (A) Representative immunoblot showing expression of TMEM11 in effector T cells cultured under non-polarizing (ThN) and Th1-, Th2-, non-pathogenic (np-) Th17-, pathogenic (p-) Th17-, and inducible Treg (Treg)-polarizing conditions. β-actin, loading control. (B) Representative flow plots and line graphs showing cell trace violet (CTV) dilution rate in WT and Tmem11 −/− T cells cultured under Th1-polarizing conditions for 4 days. Unstimulated WT naive T cells served as control (light gray histogram). (C) Representative flow plots and bar graphs showing IFN-γ-expressing populations and mean fluorescence intensity (MFI) of IFN-γ in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions. Cells were restimulated on day 4 with anti-CD3 and anti-CD28 antibodies for 5 h. (D) Representative flow plots (left) and bar graphs showing MFI of TBET expression (right) in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions. WT T cells cultured under Th2-polarizing conditions were used as negative controls (gray, flow plot). (E) Representative flow plots and bar graphs showing IFN-γ and TBET expression in CD4 + cells from the draining lymph nodes of control or Tmem11 −/− mice injected with MOG peptide for EAE induction and cultured under Th1-expansion conditions for 72 h. (F) Time course of the mean clinical score (left) and body weight measurement (right) of EAE in Rag2 −/− recipients of control or Tmem11 −/− draining lymph node cells cultured under Th1-expansion conditions. The line graphs show mean ± SEM from the indicated number of animals from one representative experiment of a total of three experiments. (G) Scatterplots showing the numbers of total mononuclear (left) or CD4 + T cells (right) isolated from the CNS of recipients of control or Tmem11 −/− cells at the peak of EAE. (H) Representative flow plots showing the cytokine profile of CD4 + T cells from the CNS of recipients of control or Tmem11 −/− cells at the peak of the disease. Bar graphs show average (±SEM) of normalized frequency of IFN-γ + and GM-CSF + cells. Individual points in bar graphs in (C), (D), (E), (G), and (H) show data from independent animals. Representative immunoblots and graphs summarize results from at least three independent experiments except where stated otherwise. Data represent means ± SEM; significance was determined by unpaired two-tailed t test (B, C, D, G, and H) and Mann-Whitney U test (F). ### p < 0.0001, * p < 0.05, and ** p < 0.005. N.S., not significant.

Article Snippet: CD4 + CD25T − cells were cultured without any polarizing cytokines or antibodies for non-polarizing (ThN) conditions, with 10 mg/ml anti-IL-4 antibody (Peprotech) and 2 ng/ml IL-12 for Th1 differentiation; 20 μg/ml anti-IFN-γ antibody (Bio X Cell), 2.5 μg/ml anti-IL-12 antibody and 10 ng/ml IL-4 for Th2 differentiation; 10 μg/ml anti-IL-4 antibody, 10 μg/ml anti-IFN-γ antibody, 30 ng/ml IL-6 (Peprotech) and 1 ng/ml TGF-β (Peprotech) for non-pathogenic Th17 differentiation; 10 μg/ml anti-IL-4 antibody, 10 μg/ml anti-IFN-γ antibody, 30 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23 (R&D Systems) and 10 ng/ml IL-1β (R&D Systems) for pathogenic Th17 differentiation and cultured in T cell medium as described above.

Techniques: Western Blot, Expressing, Cell Culture, Control, Fluorescence, Injection, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet: (A) Measurements of basal and oligomycin-induced maximal mitochondrial inner membrane potential in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions and restimulated with anti-CD3 and anti-CD28 antibodies for indicated times. Bar graphs on the right show ΔMFI for tetramethylrhodamine-ethyl ester (TMRE) as calculated by subtracting the MFI values after carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP) treatment. Light gray flow plots show T cells with FCCP treatment. (B) Intracellular ATP levels in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions. (C) Measurement of ROS levels in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions after restimulation with anti-CD3 and anti-CD28 antibodies for the indicated times. (D) Cellular oxidative stress levels in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions as determined by measuring the GSH/GSSH ratio after 2 h of restimulation with anti-CD3 and anti-CD28 antibodies. (E) Representative flow plots (left) and bar graphs (right) showing measurement of mtROS levels in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions and treated overnight with 250 μM NAC and restimulated for indicated times in the presence of NAC. (F) Intracellular IFN-γ measurement in WT and Tmem11 −/− cells cultured under Th1-polarizing conditions, treated overnight with 250 μM NAC, and restimulated with anti-CD3 and anti-CD28 antibodies for 5 h in the presence of NAC. Bar graphs in (A)–(F) show means ± SEM from three independent experiments; significance was determined by unpaired two-tailed t test. * p < 0.05, ** p < 0.005, and *** p < 0.0005.

Article Snippet: CD4 + CD25T − cells were cultured without any polarizing cytokines or antibodies for non-polarizing (ThN) conditions, with 10 mg/ml anti-IL-4 antibody (Peprotech) and 2 ng/ml IL-12 for Th1 differentiation; 20 μg/ml anti-IFN-γ antibody (Bio X Cell), 2.5 μg/ml anti-IL-12 antibody and 10 ng/ml IL-4 for Th2 differentiation; 10 μg/ml anti-IL-4 antibody, 10 μg/ml anti-IFN-γ antibody, 30 ng/ml IL-6 (Peprotech) and 1 ng/ml TGF-β (Peprotech) for non-pathogenic Th17 differentiation; 10 μg/ml anti-IL-4 antibody, 10 μg/ml anti-IFN-γ antibody, 30 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23 (R&D Systems) and 10 ng/ml IL-1β (R&D Systems) for pathogenic Th17 differentiation and cultured in T cell medium as described above.

Techniques: Membrane, Cell Culture, Two Tailed Test

Journal: Cell reports

Article Title: Mitochondrial reactive oxygen species regulate acetyl-CoA flux between cytokine production and fatty acid synthesis in effector T cells

doi: 10.1016/j.celrep.2025.115430

Figure Lengend Snippet:

Article Snippet: CD4 + CD25T − cells were cultured without any polarizing cytokines or antibodies for non-polarizing (ThN) conditions, with 10 mg/ml anti-IL-4 antibody (Peprotech) and 2 ng/ml IL-12 for Th1 differentiation; 20 μg/ml anti-IFN-γ antibody (Bio X Cell), 2.5 μg/ml anti-IL-12 antibody and 10 ng/ml IL-4 for Th2 differentiation; 10 μg/ml anti-IL-4 antibody, 10 μg/ml anti-IFN-γ antibody, 30 ng/ml IL-6 (Peprotech) and 1 ng/ml TGF-β (Peprotech) for non-pathogenic Th17 differentiation; 10 μg/ml anti-IL-4 antibody, 10 μg/ml anti-IFN-γ antibody, 30 ng/ml IL-6 (Peprotech), 10 ng/ml IL-23 (R&D Systems) and 10 ng/ml IL-1β (R&D Systems) for pathogenic Th17 differentiation and cultured in T cell medium as described above.

Techniques: FLAG-tag, Virus, Recombinant, Protease Inhibitor, Adjuvant, Staining, Isolation, Citrate Assay, Luminescence Assay, Chromatin Immunoprecipitation, Sample Prep, Plasmid Preparation, Software, Imaging, Microscopy, Transmission Assay, Gene Expression, Flow Cytometry, Mass Spectrometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enhancement of mycobacterial pathogenesis by host interferon-γ

doi: 10.1007/s00018-024-05425-7

Figure Lengend Snippet: Interferon-γ promotes the intracellular growth of M. bovis BCG and M. tuberculosis in macrophages. ( A ) Exposure of internalized mycobacteria to IFNγ inside phagosome. Control or IFNγ-expressing macrophages were treated with 0.8 μm latex bead for 4 h infected with M. bovis BCG for 1 h followed by a 24 h chase and subjected to subcellular fractionation followed by immunoblotting with antibodies against IFNγ, Hsp65, and actin. Hsp65 and actin were used as control markers for fraction purity. N.P., non-phagosome fraction; P, phagosomal fraction. ( B ) Interferon-γ promotes intracellular growth of mycobacteria in macrophages. Control, IFNγ-expressing macrophages were infected with M. bovis BCG for 1 h, followed by chase for the indicated times before lysis and determination of CFU. ( C ) Blocking host IFNγ signaling enhances IFNγ-dependent mycobacterial growth in macrophages. Control or IFNγ-expressing macrophages were infected with M. bovis BCG for 1 h followed by chase for the indicated times. Antibodies against IFNγ receptor 1 (CD119) or control isotype were concurrently treated with infection. Intracellular growth of mycobacteria was evaluated by CFU assay. ( D ) Chimera assay demonstrated that IFNγ promotes intracellular growth of mycobacteria in macrophages. Control and IFNγ-expressing macrophages were mixed at a 1:1 ratio and infected with M. bovis BCG-mCherry for 1 h. At 2 and 72 h post-infection, cells were fixed, blocked, immunostained with FITC anti-IFNγ antibody, and analyzed by flow cytometry. ( E ) Interferon-γ production by interferon-γ-expressing macrophages. Control or interferon-γ-expressing macrophages were infected with M. bovis BCG for 1 h followed by a 48 h chase. Interferon-γ released to culture supernatant was quantified by sandwich ELISA. ( F ) Intracellular mycobacteria are more susceptible to IFNγ than cultured mycobacteria. Cultured or isolated intracellular M. bovis BCG was cultured in 7H9 broth without or with 10 µg/ml IFNγ supplementation. At the indicated times, the inoculum was serially diluted in PBS, plated on 7H10 agar plates, and incubated at 37 o C for 3–4 weeks. The normalized growth rates were calculated as CFU values at indicated time points divided by their initial CFU values. ( G ) Host immune responses upregulate mycobacterial mmpL10 expression. M. bovis BCG was isolated from macrophages at 24 h post-infection or collected from cultures without or supplemented with 10 µg/ml IFNγ for 24 and 72 h, and subjected to RNA extraction. Expression of the mmpL10 gene was analyzed by qRT-PCR assay and the housekeeping rpsO transcript was used for normalization. ( H and I ) Interferon-γ promotes intracellular growth of M. tuberculosis but not M. smegmatis in macrophages. Control or IFNγ-rexpressing macrophages were infected with M. tuberculosis ( H ) or M. smegmatis ( I ) for 1 h, followed by chase for the indicated times before lysis and determination of CFU after 3 weeks ( H ) or 4 days ( I ) of incubation at 37 o C. Data information: Statistical analyses in Fig. 3B, C, F and H, and 3I were performed with two-way ANOVA followed by multiple comparisons among groups, whereas statistical analysis in Fig. 3E and G was performed with one-way ANOVA followed by multiple comparisons among groups. The results are the mean values ± standard deviations of three biological replicates each with three (Fig. 3B, C, E, F and H, and 3I) or two (Fig. 3G) technical replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns , not significant. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant

Article Snippet: Ten days after the final injection, serum samples were obtained, and ELISA was performed to determine the antibody titer; recombinant mouse IFNγ protein (#ab259378), recombinant mouse IL12 protein (#ab259419), recombinant mouse IL1β protein (#ab259421), recombinant mouse TNFα protein (#ab259411), and TMB ELISA substrate (#ab171523) from Abcam; mouse monoclonal anti-β-actin antibody (#3700S) from Cell Signaling; recombinant mouse IFNγ protein (#IF005) and glass bead (#G8772) from Sigma; recombinant mouse IL1β protein (#BMS332), recombinant mouse IL18 protein (#PMC0184), IFNγ rabbit monoclonal antibody (#701121), IFNγ monoclonal antibody (clone XMG1.2), eBioscience (#14-7311-81), rat anti-mouse IL12 p70 (clone 9A5) (#ENMM120), rabbit anti-mouse IL1β (#500-P51), rabbit anti-mouse IL18 (#210-401-323 S), rat IgG1 kappa isotype control (eBRG1), eBioscience (#14-4301-82), goat anti-rabbit IgG-HRP (#31460), goat anti-rat IgG-HRP (#31470), geneticin selective antibiotic (G418 Sulfate) (#10131035), and fluoromount-G mounting medium (#00-4958-02) from Thermo Fisher Scientific; FITC anti-mouse IFNγ antibody (#505806), FITC goat anti-rat IgG antibody (#405404), and Alexa fluor 647 anti-mouse IgG1 antibody (#406618) from BioLegend; rat anti-mouse IFNγ receptor 1 (CD119) (clone GR-20) and rat IgG2a isotype control (clone 2A3) from BioXCell; and heat shock protein 65 (mycobacterial) monoclonal antibody (clone 4H11) (#ADI-SPA-882-E) from Enzo Life Science.

Techniques: Control, Expressing, Infection, Fractionation, Western Blot, Lysis, Blocking Assay, Colony-forming Unit Assay, Flow Cytometry, Sandwich ELISA, Cell Culture, Isolation, Incubation, RNA Extraction, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Memory CD8 T cells are vulnerable to chronic IFN-γ signals but not to CD4 T cell deficiency in MHCII-deficient mice

doi: 10.1038/s41467-024-48704-4

Figure Lengend Snippet: a , b Thy1.1 memory P14 T cells were generated and transferred into Thy1.2 WT or MHCII −/− secondary host mice, as described in Fig. . Two weeks after the transfer, Thy1.1 + CD8α + Vα2 + P14 T cells were sorted from the spleen and subjected to RNA-Seq analysis. a Gene expression of P14 T cells from MHCII −/− mice compared with those from WT mice. DEGs (log 2 [Fold Change] < −0.5 or > 0.5 and FDR < 0.05) are highlighted in red (up in MHCII −/− ) or blue (down in MHCII −/− ). b GO enrichment analysis of DEGs. Pathways and GO terms significantly enriched in memory P14 T cells from MHCII −/− (red) or WT host mice (blue) are shown ( p < 0.05). c Plasma concentrations of IFN-γ (mean ± SD; WT: n = 13; MHCII −/− : n = 12; CD4 −/− : n = 15). d – h Thy1.1 memory P14 T cells were generated as in Fig. and transferred into Thy1.2 WT or MHCII −/− mice treated with anti-IFN-γ mAb or PBS. Spleen cells were subjected to flow cytometric analysis 40–50 ( d – f ) or 14–15 days ( g , h ) after the transfer. d Representative flow cytometric profiles of splenocytes. The number of Thy1.1 + CD8α + Vα2 + P14 T cells in the spleen is summarized (mean ± SD; n = 4 or 5 per group). e Representative flow cytometric profiles of Thy1.1 + CD8α + Vα2 + P14 T cells from spleens of MHCII −/− mice treated with anti-IFN-γ or PBS. The number of indicated subsets is summarized (mean ± SD; n = 7 per group). f Representative histograms of CD127, CXCR3 and CD27 expression on KLRG1 − and KLRG1 + Thy1.1 + CD8α + Vα2 + P14 T cells from spleens of MHCII −/− mice treated with anti-IFN-γ (black) or PBS (red). Geometric MFI values of each marker were normalized by the average value of KLRG1 − cells from PBS-treated mice and summarized (mean ± SD; n = 6 or 7 per group). g , h Representative histograms of Ki67 expression in Thy1.1 + CD8α + Vα2 + P14 T cells from spleens of MHCII −/− (red) or WT mice (black) ( g ), or of MHCII −/− mice treated with anti-IFN-γ (black) or PBS (red) ( h ). Percentages of Ki67 + cells among total Thy1.1 + CD8α + Vα2 + P14 T cells (upper right panel of g , h ) or in the indicated subsets of Thy1.1 + CD8α + Vα2 + P14 T cells (lower panels of g ) are summarized (mean ± SD; n = 4 or 5 for g , n = 6 for h ). Each symbol represents one mouse. Results are pooled from two independent experiments. Data were analyzed using the Wald test with the Benjamini-Hochberg procedure ( a ), one-way ANOVA with Tukey’s multiple comparison test ( c , d ) or two-tailed unpaired t test ( e – h ). Source data are provided as a Source Data file.

Article Snippet: For neutralization of IFN-γ, WT and MHCII −/− mice were injected intravenously with 200 μg of anti-IFN-γ mAb (XMG1.2) (Bio X Cell) on days 7 and 3 before the transfer of memory P14 T cells, and then injected with 100 μg of anti-IFN-γ mAb on day 3 and every seven days thereafter.

Techniques: Generated, RNA Sequencing, Gene Expression, Clinical Proteomics, Expressing, Marker, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Memory CD8 T cells are vulnerable to chronic IFN-γ signals but not to CD4 T cell deficiency in MHCII-deficient mice

doi: 10.1038/s41467-024-48704-4

Figure Lengend Snippet: Thy1.1 naïve P14 T cells were transferred into Thy1.2 WT mice and infected with Vac-GP33, as in Fig. . More than 30 days later, rmIFN-γ or PBS was administered to host mice 3 times per week. Splenocytes were subjected to flow cytometric analysis 24–25 days after the initial IFN-γ administration. a Time-course of the percentage of Thy1.1 + CD8α + Vα2 + P14 T cells in PBLs following the first rmIFN-γ administration (mean ± SD). b Representative flow cytometric profiles of splenocytes. The number of Thy1.1 + CD8α + Vα2 + P14 T cells in spleens is summarized (mean ± SD). c Representative histograms of Ki67 expression in Thy1.1 + CD8α + Vα2 + P14 T cells from spleens. The percentage of Ki67 + cells in P14 T cells is summarized (mean ± SD). d Representative flow cytometric profiles of Thy1.1 + CD8α + Vα2 + P14 T cells from spleens. The number of indicated subsets is summarized (mean ± SD). e Representative histograms of CD127, CXCR3 and CD27 expression on KLRG1 − and KLRG1 + Thy1.1 + CD8α + Vα2 + P14 T cells from spleens of WT mice treated with IFN-γ (red) or PBS (black). Geometric MFI values of each marker were normalized by the average value of KLRG1 − cells from PBS-treated mice and summarized (mean ± SD). Each symbol indicates one mouse ( n = 6 per group). Results are pooled from two independent experiments. Data were analyzed using two-way ANOVA with Sidak’s multiple comparisons test ( a ) or two-tailed unpaired t test ( b – e ). Source data are provided as a Source Data file.

Article Snippet: For neutralization of IFN-γ, WT and MHCII −/− mice were injected intravenously with 200 μg of anti-IFN-γ mAb (XMG1.2) (Bio X Cell) on days 7 and 3 before the transfer of memory P14 T cells, and then injected with 100 μg of anti-IFN-γ mAb on day 3 and every seven days thereafter.

Techniques: Infection, Expressing, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Memory CD8 T cells are vulnerable to chronic IFN-γ signals but not to CD4 T cell deficiency in MHCII-deficient mice

doi: 10.1038/s41467-024-48704-4

Figure Lengend Snippet: a IFN-γ levels in different tissues of WT, MHCII −/− , and CD4 −/− mice ( n = 5–9 per group). IFN-γ levels were normalized by total protein levels. SI; small intestine. b Flow cytometric analysis of colonic and splenic lymphocytes from IFN-γ Venus MHCII −/− and MHCII +/− (WT) mice. Representative flow cytometric profiles of IFN-γ Venus expression levels in the indicated subsets are shown. c Frequency and number of IFN-γ Venus + cells in the indicated subsets are shown (mean ± SD; MHCII −/− : n = 11; WT: n = 10). Each symbol represents one mouse. Data were analyzed using multiple two-tailed unpaired t tests ( a ) or two-way ANOVA with Sidak’s multiple comparisons test ( c ). Source data are provided as a Source Data file.

Article Snippet: For neutralization of IFN-γ, WT and MHCII −/− mice were injected intravenously with 200 μg of anti-IFN-γ mAb (XMG1.2) (Bio X Cell) on days 7 and 3 before the transfer of memory P14 T cells, and then injected with 100 μg of anti-IFN-γ mAb on day 3 and every seven days thereafter.

Techniques: Expressing, Two Tailed Test