Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: 21h10 improves antitumor efficacy in in vivo B16F10 murine melanoma and ex vivo human PDOTs models, with non-neutralizing anti-drug antibodies, and toxicity alleviated with TNFα blockade. ( A ) Naïve CD8 + TRP1 high/low melanoma-specific T cells were adoptively transferred to mice prior to B16F10 inoculation. Cytokine therapy began on day 5 and continued every day until the stop of treatment (dashed line). ( B ) Mice received no prior T cell adoptive transfer before cytokine therapy. ( C ) Same as ( B ), but with adoptive transfer of naïve CD8 + TRP1 high/low T cells. ( D ) Scheme of PDOTS preparation. ( E ) Viability assessment of ex vivo human melanoma PDOTS following treatment of ICB (anti-PD-1, pembrolizumab, or anti-PD-1/anti-LAG-3), 21h10, or untreated control. ( F ) Representative images of PDOTS viability assessment shown in ( E ). PI-dead cells in red and Hoechst-nuclei in blue. ( G ) Mice died due to toxicity or after 40 days of treatment were bled and their serum was analyzed by ELISA for anti-drug antibodies against 21h10. Positivity (dotted line) was determined as two standard deviations above the mean of samples from mice treated only with PBS. Fractions indicate the number of mice classified as “positive” for anti-drug antibodies out of the total mice in each treatment group. ( H ) Mice with various titer levels of anti-21h10 antibodies were inoculated with MC38 tumors. Starting on day 6, mice were treated with 21h10 or PBS daily for 17 days. ( I ) Wild-type (WT), Rag2-/- , or Rag2-/- Il2rg-/- mice were treated with PBS or 21h10 daily and isotype or anti-NK1.1 depleting antibodies every three days. Mice were sacrificed when weight loss exceeded 20% of the initial starting weight. ( J ) Serum TNFα level was measured in C57BL/6 and Rag2-/- mice with PBS or 21h10 treatments. ( K ) Antitumor activity comparison on B16F10 melanoma between 21h10 and 21h10+aTNFα treatment groups.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: In Vivo, Ex Vivo, Adoptive Transfer Assay, Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Mice were treated as in , A and C, but on day 15 of tumor growth, tumors were processed for single-cell RNA-sequencing. UMAP of all samples are combined. ( B ) scRNAseq cluster composition average across treatment groups. ( C ) T cells in each scRNAseq sample from ( B ). Different colors for each bar indicate individual mice from each group. ( D ) Flow cytometry quantification of CD8 + T cell infiltration. ( E ) TRP1 high and TRP1 low melanoma antigen-specific cells captured by sequencing. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS groups. ( F ) T cells were sub-clustered from all samples from ( A ). The average T cell sub-cluster composition across treatment groups is shown on the right. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( G ) IFN-𝛾-response score total UMAP from ( A ) based on scaled and summed Hallmark IFN-𝛾-response genes for each cell. ( H ) Heatmap from pseudo-bulk differential gene expression analysis across T cell sub-clusters. Significant differences compared to PBS samples. ( I ) Similar to ( H ), but for TRP1 cells only, values from individual mice are shown. P-values displayed vs. PBS samples. ( J ) Flow cytometry quantification of CD8 + IFN-𝛾/granzyme B levels in endogenous or transferred CD8 + TRP1 high/low T cells. ( K ) CD4 + T cell sub-clustering UMAP and cluster composition. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( L ) Flow cytometry of IFN-𝛾 + CD4 + T cells. ( M ) Quantification and representative flow cytometry plots of Foxp3-GFP Treg cells. One-way ANOVA with Dunnett’s multiple comparisons for flow quantification and scRNA-seq total T cell comparison. For all dot plots with error bars: dots indicate individual mice, bars are S.E.M. *p ≤ 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001, or p-values displayed.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: RNA Sequencing Assay, Flow Cytometry, Sequencing, Expressing, Comparison

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: Depletion of CD8+ T cells results in more severe thrombocytopenia and impairs responsiveness to steroid therapy in vivo. For the active model, WT mice were transplanted with immunized β3−/− splenocytes with or without depletion of CD8+ T cells. DEX treatment began at day 6. (A) Thrombocytopenia was more severe in mice given CD8+ T cell–depleted splenocytes compared with those transplanted with nondepleted splenocytes as indicated by &&&P < .001. Mice transplanted with CD8+ T cell–depleted immunized β3−/− splenocytes were less responsive to oral DEX compared with mice transplanted with nondepleted splenocytes as indicated by ###P < .001. ***P < .001, nondepleted splenocytes vs nondepleted splenocytes + DEX. N = 8. In our passive mouse model, CD8+ T cells were depleted from WT mice by injection of anti-CD8 mAb (400 μg intravenously) before inducing passive ITP with anti-β3 mAb (9D2, 1 μg intraperitoneally). Platelet counts were not significantly affected by anti-CD8 mAb depletion of CD8+ T cells. DEX was administered (10 mg/kg/day, intraperitoneally, daily) beginning at 4 hours after anti-β3 mAb injection. (B) Thrombocytopenia was more severe in CD8+ T cell–depleted mice compared with those given the anti-β3 integrin mAb alone. N = 6. (C) CD8+ T cell–depleted thrombocytopenic mice were less responsive to DEX compared with mice with normal levels of CD8+ T cells. N = 6. ***P < .001. Mean ± SD.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: In Vivo, Injection

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ T-cell transfusion therapeutically attenuated platelet clearance and enhanced response to DEX in passive murine model of ITP. In the passive model of ITP, mice were injected with anti-β3 integrin mAb (9D2, 1 μg, intraperitoneally) at day 0 and transfused with 106 CD8+ T cells from either WT, naïve β3−/−, or immunized β3−/− mice. Control cells are remaining splenocytes following CD8+ purification. (A) CD8+ T cells significantly increased platelet count in the absence of any DEX treatment. N = 6. *P < .05, **P < .01, ***P < .001 vs PBS. (B) DEX (10 mg/kg) was administered at 4 hours after mAb injection and CD8+ T-cell transfusion. N = 6. *P < .05, **P < .01, ***P < .001 vs PBS or indicated groups. Mean ± SD.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: Injection, Control, Purification

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: Steroid treatment selectively increased CD8+ Tregs in both the passive and active models of ITP. (A) In the passive model of ITP, mice were injected with anti-β3 mAb (9D2, 1 μg, intraperitoneally) at day 0. In some mice, 10 mg/kg DEX was administered via intraperitoneal injection 4 hours after antibody injection. On indicated days, mice were bled, and different CD8+ subsets were detected via flow cytometry. N = 8 to 10. (B) In the active model, mice were transfused with immunized β3−/− splenocytes on day 0. DEX treatment (oral, 10 mg/kg) or control water was initiated on day 6. Mice were bled on the indicated days, and CD8+ subsets were detected as above. N = 6, *P < .05, **P < .01, ***P < .001. Mean ± SD.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: Injection, Flow Cytometry, Control

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ T cells suppressed platelet apoptosis and clearance in vitro. (A-B) CD8+ T cells from immunized β3−/− mice were added to CD8+-depleted splenocytes and then cocultured with WT platelets. Control has no CD8+ T cells added. (A) After 72 hours, platelets remaining in culture were identified by forward and side scatter characteristics and gated on 7-AAD− and assessed for apoptosis with caspase 3/7 and Annexin V. N = 4. (B) CD8+ T cells from immunized β3−/− mice were cocultured with platelets for 72 hours, and then the remaining platelets in solution were counted, with flow cytometry as events per second. More platelets remained in the coculture system after incubation with CD8+ T cells from immunized β3−/− mice compared with control or naïve β3−/− CD8+ T cells. N = 4. (C) Platelets were labeled with 9D2 primary antibody and FITC anti-mouse IgG. Platelets were then cocultured with macrophages with or without CD8+ T cells. After 24 hours, FITC-positive macrophages were detected by flow cytometry. CD8+, CD8+CD25+, CD8+CD122+, and CD8+CD103+ T cells from immunized β3−/− and WT mice significantly suppressed phagocytosis of platelets by macrophages. Furthermore, the CD8+ T cells from immunized β3−/− mice suppressed phagocytosis more effectively (P < .05). This inhibitory function was also dose dependent (supplemental Figure 4) *P < .05, **P < .01. Mean ± SEM.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: In Vitro, Control, Flow Cytometry, Incubation, Labeling

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ Tregs from immunized β3−/− mice inhibit the adaptive antiplatelet immune response in vitro. For proliferation assays, purified splenic CD8+ T cells or CD8+ Tregs from WT, naïve β3−/−, or WT platelet immunized β3−/− mice were added to CD8+ T cell–depleted splenocytes in the presence of 1 × 107 WT platelets per well to induce T- or B-cell proliferation. Control cells were CD8+-depleted splenocytes with PBS or platelets (PLT) as indicated. Both immunized β3−/− CD8+ T cells and CD8+ Tregs inhibited (A,E) CD4+ T-cell proliferation, (B,F) CD19+ B-cell proliferation, and (C,G) platelet-associated IgG production. (D) Immunized β3−/− CD8+ T cells also inhibited IL-10 cytokine production dose dependently. N = 8. Imm, immunized. *P < .05, **P < .01. Mean ± SEM.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: In Vitro, Purification, Control

Journal: Blood

Article Title: CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

doi: 10.1182/blood-2015-03-635417

Figure Lengend Snippet: CD8+ Tregs from immunized β3−/− mice inhibit CD8+ cytotoxic activity against platelets. Platelets (5 × 106) were cultured with purified 1 × 105 cytotoxic CD8+ T cells (CD8+CD178+) with or without 5 × 105 CD8+ Treg cells (according to the original ratio) for 24 hours. Platelet cytotoxicity was measured with (A) remaining platelet number in supernatant, determined as number of platelets per second counted by flow cytometry69 and (B) caspase 3/7 expression. N = 4. PLT, platelet; CTL, cytotoxic CD8+ T cells. *P < .05, **P < .01. Mean ± SEM.

Article Snippet: In vivo CD8 + T-cell depletion Anti-CD8 mAb (400 μg; clone 2.43; ATCC) was injected intravenously to deplete CD8 + T cells from recipient mice before induction of thrombocytopenia.

Techniques: Activity Assay, Cell Culture, Purification, Expressing

Journal: Immunity

Article Title: Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK + CD8 + T Cells as Conserved Hallmark of Inflammaging.

doi: 10.1016/j.immuni.2020.11.005

Figure Lengend Snippet: Figure 3. Effects of Aging on CD8+ T Cell Populations in Mouse (A) scRNA-seq clusters of 4,299 CD8+ T cells. (B) Differential representation of CD8+ T cell clusters. (C) Scaled gene expression. (legend continued on next page)

Article Snippet: In some experiments, we depleted CD8+ T cells from aged mice using depleting anti-CD8a antibody (BioXcell, clone 2.43, cat# BE0061) injected IP 0.4 mg per mouse followed by injections with 0.25 mg per mouse of anti-CD8a antibody every 4 days for 3 weeks.

Techniques: Gene Expression

Journal: Immunity

Article Title: Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK + CD8 + T Cells as Conserved Hallmark of Inflammaging.

doi: 10.1016/j.immuni.2020.11.005

Figure Lengend Snippet: Figure 4. CD49d Plays a Role in Crosstalk between CD8+ Taa Cells and Other Cell Types in Tissues (A) scRNA-seq cell clusters (young and old mice, GSE132901) and scaled gene expression. (B) Predicted ligand-receptor interactions between a4b1 integrin in CD8+ Taa cells and its ligands in other cell types; EC, epithelial cells; LEC, lungs epithelial cells; CEC, ciliated epithelial cells; SMC, smooth muscle cells; CDC, collecting duct cells; GCEC, epithelium cells of glomerular capsule. (C–E) Membrane expression of CD49d protein by mouse CD8+ T cells (flow cytometry) (n = 3 mice in each group).

Article Snippet: In some experiments, we depleted CD8+ T cells from aged mice using depleting anti-CD8a antibody (BioXcell, clone 2.43, cat# BE0061) injected IP 0.4 mg per mouse followed by injections with 0.25 mg per mouse of anti-CD8a antibody every 4 days for 3 weeks.

Techniques: Gene Expression, Membrane, Expressing, Cytometry

Journal: Immunity

Article Title: Comprehensive Profiling of an Aging Immune System Reveals Clonal GZMK + CD8 + T Cells as Conserved Hallmark of Inflammaging.

doi: 10.1016/j.immuni.2020.11.005

Figure Lengend Snippet: Figure 7. Single-Cell Analysis of Age-Associated CD8+ T Cells in Human Blood (A) Overview illustrating single-cell analysis of PBMCs from non-obese healthy young and old men. (B–D) Flow cytometry analysis of expression of GZMK and GZMB in CD8+ T cells.

Article Snippet: In some experiments, we depleted CD8+ T cells from aged mice using depleting anti-CD8a antibody (BioXcell, clone 2.43, cat# BE0061) injected IP 0.4 mg per mouse followed by injections with 0.25 mg per mouse of anti-CD8a antibody every 4 days for 3 weeks.

Techniques: Single-cell Analysis, Flow Cytometry, Expressing

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload‐associated ZDHHC12 primarily participates in maintaining the activation of CD8+T cells in renal cell carcinoma. (a) UMAP plot showing the subpopulation classification of CD8+ T cells ( GSE121636 ). (b) UMAP visualization of ZDHHC12 expression across different CD8+ T cell subsets. (c) Violin plot depicting ZDHHC12 expression patterns in various CD8+ T cell subpopulations. (d) BALB/c mice fed either a high‐iron‐load diet or a normal diet were subcutaneously inoculated with Renca tumor cells on the dorsum, and starting from the day of inoculation, 200 µg of anti‐CD8 depletion antibody (αCD8) or an isotype control antibody was intraperitoneally administered every three days; tumor volume was measured from day 7 post‐inoculation. Data were presented as mean ± SD. e and f, Western blot (e) and RT‐qPCR (f) analyses were performed on CD8+ T cells sorted from renal cancer tissues of both the normal diet and high‐iron diet groups. (g–i), The figure illustrates schematic diagrams of different treatment groups (g). Specifically, tumor cells were isolated and sorted from the tumor tissues of the renal cancer model. Subsequently, the collected tumor cells were subcutaneously injected into the dorsum of either Zdhhc12 wild‑type or knockout mice. Tumor size was measured regularly, and growth curves were plotted (h). On day 19, the mice were sacrificed, and tumors were excised and weighed (i). (j–m), Portions of tumor tissues were processed for cell extraction, staining, and flow cytometry analysis to evaluate CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, the proportions of CD3+/CD45+ (j), CD8+/CD3+ (k), GZMB+/CD8+ (l), and IFN‐γ+/CD8+ (m) T cells within tumor masses were quantified. (n,o) Portions of tumor tissues were embedded, sectioned, stained, and subjected to immunofluorescence analysis to assess CD8+ T cell function in Zdhhc12 wild‐type and KO mice. Specifically, multiplex staining was performed for DAPI/CD3/CD8/GZMB, and the proportions of positive cells were quantified (n); similarly, multiplex staining for DAPI/CD3/CD8/IFN‐γ was conducted with subsequent quantification of positive cell ratios (o).

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Activation Assay, Expressing, Control, Western Blot, Quantitative RT-PCR, Isolation, Injection, Knock-Out, Extraction, Staining, Flow Cytometry, Cell Function Assay, Immunofluorescence, Multiplex Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Iron overload promotes the ubiquitin‐proteasome pathway degradation of ZDHHC12 by enhancing its binding with TRIM28. (a) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentrations for 48 h. (b) Cells were collected for Western blot analysis after treating sorted CD8+ T cells with FAC at the indicated concentration gradients for 48 h. (c) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentrations for 48 h. (d) Cells were collected for Western blot analysis after treating 293T cells with FAC at the indicated concentration gradients for 48 h. (e) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay after treatment with or without FAC (20 µg/mL) for 48 h. (f) HA‐ZDHHC12 was transfected into 293T cells, and co‐immunoprecipitation was performed using an anti‐HA‐ZDHHC12 antibody, followed by mass spectrometry to identify potential ZDHHC12‐interacting proteins. The peptide spectrum of TRIM28 is shown. (g) The degradation of ZDHHC12 in 293T cells was assessed by CHX chase assay with or without TRIM28 knockout. (h) Schematic diagrams depicting the protein domain architecture of TRIM28 and the TRIM28 △RING truncation mutant. (i) Sorted CD8+ T cells were stably transfected with the plasmids illustrated in the figure, followed by cell collection for Western blot analysis and quantitative assessment. (j) After transfection with the indicated plasmids, 293T cells were treated with FAC, followed by MG132 treatment, and then collected for Co‐IP and Western blot analysis. (k) After treatment with FAC (0, 20, 40 µg/mL), 293T cells were treated with MG132, collected, and subjected to Co‐IP and Western blot analysis. (l) After transfection with the indicated plasmids for 24 h, 293T cells were collected for Co‐IP and Western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Concentration Assay, Transfection, Immunoprecipitation, Mass Spectrometry, Knock-Out, Mutagenesis, Stable Transfection, Co-Immunoprecipitation Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12 inhibits cuproptosis in CD8+T cells through interaction with FDX1. (a) We reviewed the mass spectrometry results and found that FDX1 may interact with ZDHHC12, and the peptide spectrum of FDX1 is shown in the figure. (b) HA‐ZDHHC12 and Flag‐FDX1 were transfected into 293T cells, followed by cell lysis and co‐immunoprecipitation using Flag or HA antibodies, and then analyzed by western blot. (c) 293T cells were lysed and subjected to co‐immunoprecipitation with ZDHHC12 or FDX1 antibodies, followed by western blot detection. (d) Immunofluorescence staining was performed in 293T cells using ZDHHC12 and FDX1 antibodies, and images were acquired and analyzed by confocal microscopy. (e) GST pull‐down assay was performed using the recombinant protein of ZDHHC12. (f) GST pull‐down assay was performed using the recombinant protein of FDX1. (g) Protein structure prediction and molecular docking were conducted for ZDHHC12 and FDX1. (h,i) Western blot (h) and RT‐qPCR (i) were performed in 293T cells after transfection with the indicated plasmids. (j,k) Western blot (j) and RT‐qPCR (k) were performed in 293T cells after transfection with the indicated plasmids. (l) Western blot analysis was conducted in 293T cells 72 h after transfection with the indicated plasmids. (m) Western blot analysis was performed in 293T cells after transfection with the indicated plasmids. (n) CD8+ T cells were isolated from renal cancer tissues of Zdhhc12 WT and Zdhhc12 KO mice and subjected to western blot analysis. (o) After successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (p,q) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and immunostained with DLAT and Mito‐tracker, then imaged by confocal microscopy (p) to quantify (q) DLAT oligomerization. (r) After successful transfection with the indicated plasmids in 293T cells, cells were treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu for 48 h in the presence or absence of tetrathiomolybdate (TTM) (1 µ m ) as indicated, followed by CCK‐8 assay to measure and quantify cell viability. (s,t) Following successful transfection with the indicated plasmids in 293T cells, cells were collected after treatment with or without tetrathiomolybdate (TTM) (1 µ m ), then immunostained with DLAT and Mito‐tracker and imaged by confocal microscopy (s) to quantify (t) DLAT oligomerization. (u) After successful transfection with the indicated plasmids in CD8+ T cells, cells were treated with or without tetrathiomolybdate (TTM) (1 µ m ), then collected for western blot analysis. (v) Following successful transfection with the indicated plasmids in 293T cells, cells were collected and treated with different concentrations of elesclomol‐Cu or disulfiram‐Cu as shown in the figure for 48 h, followed by CCK‐8 assay to measure and quantify cell viability. (w) After successful transfection with the indicated plasmids in sorted CD8+ T cells, cells were collected for western blot analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Mass Spectrometry, Transfection, Lysis, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Confocal Microscopy, Pull Down Assay, Recombinant, Quantitative RT-PCR, Isolation, CCK-8 Assay

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: ZDHHC12‐mediated palmitoylation of FDX1 at Cys152/155 promotes its degradation. (a) In 293T and CD8+T cells, FDX1 was immunoprecipitated using an anti‐FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed either in the presence or absence of hydroxylamine (HAM) treatment, with subsequent streptavidin‐HRP pulldown of biotin‐conjugated proteins to specifically detect palmitoylated FDX1. (b) In 293T cells, FDX1 was immunoprecipitated using an anti‐ FDX1 antibody, followed by the acyl‐biotin exchange (ABE) assay performed with or without hydroxylamine (HAM) treatment, and subsequently enriched with streptavidin‐HRP to isolate biotin‐conjugated proteins, thereby enabling the detection of FDX1 palmitoylation levels in both 2‐BP (25 µ m , 24 h)‐treated and untreated conditions. (c) 293T cells treated with or without palmitic acid azide were collected for Click‐IT reaction and streptavidin pulldown. (d) 293T cells were treated with different concentrations of 2‐BP (+, 20 µ m ; ++, 40 µ m ) for 24 h, then collected for western blot analysis. (e) 293T cells treated with or without 2‐BP (25 µ m , 24 h) were exposed to CHX and collected at different time points for western blot analysis. (f) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (g) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (h) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (i) 293T cells transfected with the indicated plasmids were treated with or without 2‐BP (25 µ m , 24 h) after 24 h, then collected for western blot analysis. (j) The peptide spectrum for FDX1 palmitoylation site identification. (k) Conservation sequences of FDX1 C152 and FDX1 C155 sites across different species. (l) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment. m, AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to ABE assay and western blot analysis with or without HAM treatment. (n) AFG3L2‐knockout 293T cells transfected with the indicated plasmids were subjected to Click‐iT pull‐down assay and western blot analysis with or without palmitic acid azide treatment, followed by quantitative analysis of FDX1 palmitoylation levels. (o) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis. (p) CD8+ T cells were transfected with the indicated plasmids, then collected and stained with the indicated fluorescent dyes for flow cytometry analysis. (q) CD8+ T cells were transfected with the indicated plasmids, then treated with PBS or FAC (20 µg/mL), collected, and stained with the indicated fluorescent dyes for flow cytometry analysis.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Immunoprecipitation, Western Blot, Knock-Out, Transfection, Pull Down Assay, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Development of lipid nanoparticles encapsulating Zdhhc12 mRNA (Zdhhc12‐LNP). (a) Schematic diagram of the LNP synthesis process; (b) The average zeta potential and the average particle size; (c) The average PDI and the average encapsulation efficiency; (d) Representative cryo‐EM image showing the morphology of synthesized LNPs. (e) Spectrum showing the purity of the sample. (f) Representative chromatogram showing mRNA capping efficiency. (g) Western blot analysis of Zdhhc12 expression in CD8+ T cells isolated from mice and treated with control, blank LNP, or Zdhhc12‐encapsulated LNP, following a 24 h pre‐stimulation with 2 µg/mL anti‐CD3/CD28 and subsequent 24 h incubation with LNPs. (h) ABE assay and Western blot analysis were performed on CD8+ T cells isolated from mice to assess the palmitoylation level of Fdx1, where cells were first cultured for 24 h post‐infection without anti‐CD3/CD28, restimulated with 2 µg/mL anti‐CD3/CD28 for 24 h, incubated with LNPs for 24 h, and then harvested for analysis. (i) Renca cells were subcutaneously injected into the backs of BALB/c mice, LNPs were administered via intratumoral injection, and when tumors reached an appropriate size, tumors were harvested for sorting of CD8+ T cells, followed by Western blot analysis to assess Zdhhc12 expression level in CD8+ T cells. (j–m) Renca cells were subcutaneously injected into the dorsal region of BALB/c mice. After tumor establishment, mice received intratumoral injection of LNPs or no injection, and serum levels of ALT (j), AST (k), CRE (l), and BUN (m) were measured at appropriate time points. Ns, not significant. (n,o) BALB/c mice were subcutaneously injected with Renca cells, intratumorally administered Zdhhc12‐LNP or control, and intraperitoneally injected with 200 µg anti‐CD8 (αCD8) or control; tumor growth was recorded starting from day 7 post‐tumor inoculation (n), and flow cytometry analysis of CD8+ T cell content in the peripheral blood of mice (n = 5) was performed at the end of the experiment (o), with data presented as mean ± SD.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Zeta Potential Analyzer, Encapsulation, Cryo-EM Sample Prep, Synthesized, Western Blot, Expressing, Isolation, Control, Incubation, Cell Culture, Infection, Injection, Flow Cytometry

Journal: Advanced Science

Article Title: Materials‐Guided Gene‐Ionizable Lipid Nanoparticles to Reverse Iron‐Associated Immune Resistance in Renal Cancer

doi: 10.1002/advs.202600078

Figure Lengend Snippet: Under normal iron conditions, ZDHHC12‐mediated palmitoylation of FDX1 promotes FDX1 degradation by the mitochondrial matrix protease AFG3L2, ultimately suppressing cuproptosis in CD8+ T cells and maintaining their immune clearance function. Under iron overload conditions, increased binding between ZDHHC12 and TRIM28 leads to ZDHHC12 degradation, consequently inducing cuproptosis in CD8+ T cells and facilitating immune escape.

Article Snippet: For in vivo CD8+ T cell depletion experiments, 200 μg of anti‐CD8α antibody (MCE, HY‐ P99129 ) was intraperitoneally administered every three days, with an equivalent amount of IgG isotype antibody as the control.

Techniques: Binding Assay

Journal: Cancer Immunology Research

Article Title: Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054

doi: 10.1158/2326-6066.cir-21-0405

Figure Lengend Snippet: Figure 1. Characterization of REGN5054 and DFO-REGN5054 binding to primary cells and in vitro functionality. A, Binding to primary human (top) and cynomolgus monkey (bottom) CD8þ T cells shown by flow cytometry. Mean fluorescence intensities (MFI) are plotted. REGN5054 (blue), DFO-REGN5054 (red), and their isotype controls (IgG4P-PVA control, gray, open triangles, and DFO-IgG4P-PVA control, gray, closed triangles). Values indicated are the concentration of antibodies at half-maximum binding. B, Immobilized or soluble DFO-REGN5054 does not induce the proliferation of human PBMCs, as determined by a viability luciferase assay. DFO-REGN5054 (red), DFO-isotype control (DFO-IgG4P-PVA control, gray), TeGenero (TGN) superagonist anti-CD28 IgG4P (orange; contains S228P mutation), and IgG4P control (black). RLUs are plotted. Data represent average RLU values SD from 4 donors. Statistical significance was determined on Log10 transformed data using a 2-way ANOVA with Tukey post hoc test. Asterisks indicate statistically significant (P < 0.05) differences as compared with the respective isotype-matched control groups. C, A T-cell/antigen-presenting cell–based reporter assay was used to assess concentration-dependent inhibition of human CD8-dependent TCR signaling. These experiments were performed using JRT3.T3.5/AP1-Luc/1G4AB/hCD8AB T cells stimulated by parental 3T3 or 3T3/hb2M/HLA-A2/NYESO1p cells. Black closed squares and green open squares represent the commercial anti-CD8 antibody RPA-T8 and its isotype control (mIgG1). Other reagents used are as in A. RLUs are plotted. D, Mitomycin-treated human PBMCs were treated with purified allogeneic CD8þ T cells in an MLR. Various antibodies were titrated at a range of concentrations (0.8–100 nmol/L). Supernatant was collected after 120 hours and was tested in a human IFNg alphaLISA assay.

Article Snippet: For assessment of anti-CD8A with different functional properties on primary infection virus clearance, mice were treated as indicated with either a depleting anti-human CD8A (OKT8; BE0004-2, Bio X Cell) or with Regeneron CD8A antibodies with different blocking activities as defined previously by in vitro functional assay.

Techniques: Binding Assay, In Vitro, Cytometry, Control, Concentration Assay, Luciferase, Mutagenesis, Transformation Assay, Reporter Assay, Inhibition

Journal: Cancer Immunology Research

Article Title: Immuno-PET Monitoring of Lymphocytes Using the CD8-Specific Antibody REGN5054

doi: 10.1158/2326-6066.cir-21-0405

Figure Lengend Snippet: Figure 4. 89Zr-DFO-REGN5054 specifically localizes to lymphoid tissues in immunocompetent mice humanized for CD8. VelociT mice received a single dose of 89Zr-DFO- REGN5054, and PET/CT was acquired on days 0, 1, 4, and 6 after injection. Quantitative ex vivo biodistribution of the mice (n ¼ 4 per dose group) was conducted on day 6 after injection. A, Representative PET/CT images demonstrate the localization of 89Zr-DFO-REGN5054 over time when dosed at 0.1, 0.5, or 1.5 mg/kg. Blue, yellow, and green arrows highlight the spleen, axillary, and inguinal lymph nodes, respectively. B, Tissue biodistribution values presented as percent injected dose per gram (%ID/g). C, Tissue biodistribution values presented as tissue-to-blood ratios (T:B). n ¼ 3–4 mice per group, and data are representative of 4 experiments.

Article Snippet: For assessment of anti-CD8A with different functional properties on primary infection virus clearance, mice were treated as indicated with either a depleting anti-human CD8A (OKT8; BE0004-2, Bio X Cell) or with Regeneron CD8A antibodies with different blocking activities as defined previously by in vitro functional assay.

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Ex Vivo