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: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Semiquantitative evaluation of protein expression using proteome profiler arrays. Representative protein profiler arrays of defects treated with hADM, hADM + BMC, or hADM + BMC-CD8 after three days are shown in ( A ). The differentially expressed proteins are marked by a red square. Protein expression was analyzed denitometrically and spot density was normalized to control spots located in the upper left, upper right, and lower left corners. Heatmap generated on basis of protein expression is shown in ( B ). Heatmap contains data from differentially pretreated hADM recovered on D3 and 7. Proteins newly secreted on D7 are marked in red. Color code indicates fold change. The pie charts ( C ) show the number of secreted proteins from membranes that covered the bone defect for 3 (upper pie chart) and 7 days (lower pie chart). The categories inflammation (red), anti-inflammation (blue) and regeneration (orange) are shown. Bi- or multifunctional proteins were assigned to the respective categories, so that the sum of the fractions is higher than the total number of proteins detected. There was a general increase in the number of expressed proteins from D3 to D7. Detected proteins and their main biological functions is listed in .

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Expressing, Control, Generated

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Protein profiler analysis of conditioned media from defects treated with hADM, hADM + BMC, or hADM + BMC-CD8 after three days. Protein levels are normalized to the intensity of the control spots (=100%). Only proteins with significant differences ( p < 0.05) or trends ( p < 0.1) are shown. Data are presented as boxplots (median, minimum, maximum; n = 3 per group). * indicates explorative significance ( p < 0.05) versus the respective comparison group.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Control, Comparison

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Protein profiler analysis of conditioned media from defects treated with hADM, hADM + BMC, or hADM + BMC-CD8 after 7 days . Protein levels are normalized to the intensity of the control spots (=100%). Only proteins with significant differences ( p < 0.05) or trends ( p < 0.1) are shown and presented in alphabetical order. Data are presented as boxplots (median, minimum, maximum; n = 3 per group). * indicates explorative significance ( p < 0.05) versus the respective comparison groups.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Control, Comparison

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Direct comparison of protein profiler data obtained after 3 and 7 days in defects treated with hADM (white), hADM + BMC (light gray), or hADM + BMC-CD8 (dark gray). Values are normalized to the density of the control spots (=100%). Only proteins with significant differences ( p < 0.05) or trends ( p < 0.1) are shown and presented in alphabetical order. Data are presented as boxplots (median, minimum, maximum; n = 3 per group). * indicates explorative significance ( p < 0.05) versus the respective comparison group.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Comparison, Control

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Osteogenic gene expression (A–D) and ALP protein expression (E) in MSC after cultivation in osteogenic differentiation medium (ODM) supplemented with CM. CM was generated from hADM (white), hADM seeded with BMC (light gray), or hADM seeded with CD8-depleted BMC (BMC-CD8, dark gray) wrapped around the bone defect for 3 or 7 days, then explanted and incubated in medium for 24 h. MSC were cultured for 3 weeks in ODM, with 10% CM added during the first 3 days, followed by analysis of gene expression relative to the Gapdh housekeeping gene ( A – D ) or ALP measurement ( E ). Control MSC (salmon) were cultured in expansion medium (M, only in E ) or in ODM, both without CM. # = p < 0.05 vs. ODM; * = p < 0.05 vs. indicated group. All significances are exploratory.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Gene Expression, Expressing, Generated, Incubation, Cell Culture, Control

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Influence of CM on expression of M1 ( Cd80, Nos2 ) and M2 ( Arg1, Socs1 ) marker genes in macrophages differentiated into M0 (A–D), M1 (E,F), or M2 (G,H). CM was generated from hADM (white), hADM seeded with BMC (light gray), or hADM seeded with CD8-depleted BMC (BMC-CD8, dark gray) wrapped around the bone defect for 3 or 7 days, then explanted and incubated in medium for 24 h. Primary rat monocytes from bone marrow aspirates were cultured for 7 days, with 10% CM added during the first 3 days. Monocytes cultured in differentiation media without CM served as controls (salmon). # = p < 0.05 vs. control; * = p < 0.05 vs. indicated group. All significances are exploratory.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Expressing, Marker, Generated, Incubation, Cell Culture, Control

Journal: Cells

Article Title: Cell Supported Single Membrane Technique for the Treatment of Large Bone Defects: Depletion of CD8 + Cells Enhances Bone Healing Mechanisms During the Early Bone Healing Phase

doi: 10.3390/cells15030215

Figure Lengend Snippet: Influence of CM on expression of CCL2 and IL-1RA genes in macrophages differentiated into M0 (A,B), M1 (C,D), or M2 (E,F). CM was generated from hADM (white), hADM seeded with BMC (light gray), or hADM seeded with CD8-depleted BMC (BMC-CD8, dark gray) wrapped around the bone defect for 3 or 7 days, then explanted and incubated in medium for 24 h. Primary rat monocytes from bone marrow aspirates were cultured for 7 days, with 10% CM added during the first 3 days. Monocytes cultured in differentiation media without CM served as controls (salmon). # = p < 0.05 vs. control; * = p < 0.05 vs. indicated group. All significances are exploratory.

Article Snippet: CD8 + cells from the BMC fraction (10 cells) were depleted by positive magnetic selection applying anti-rat CD8α MicroBeads (130-090-318, Miltenyi Biotec, Bergisch Gladbach, Germany) and LD columns (Miltenyi Biotec) thereby following the instructions of the manufacturer and described in [ ].

Techniques: Expressing, Generated, Incubation, Cell Culture, Control

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: 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

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 2 The anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations suppress melanoma brain metastases in the SM1 mouse melanoma model. (A) The ICI doublets were more effective than single agent ICI in the brain tumors of SM1 mouse melanoma model with 2/10 mice relapsing in each combination group. Mice received 200 µg/100 µL i.p. doses of single agents and doublets every 5 days for 80 days. (B) MRI T2 sequence of brain of mice with SM1 MBM tumors demonstrating a lack of tumor regrowth after holding the therapy for 28 days. MRIs were generated using standard T2-weighted Turbo Spring Echo. Brain tumors were manually contoured on all MRI slices using ImageJ. A custom program was used to extract voxels within the manually drawn contours and compute total volume burden, in mm3. (C) Monitoring melanoma brain metastases by MRI. Time course of MRI T2 sequences of brain of mouse melanoma brain metastases following treatment with IgG, anti-PD- 1+LAG-3 or anti-PD-1+CTLA-4. MRIs were taken twice a week and were generated using standard T2-weighted Turbo Spring Echo. Brain tumors were manually contoured on all MRI slices using ImageJ. A custom MATLAB program was used to extract voxels within the manually drawn contours and compute total volume burden, in mm3. (D–F) Kinetics of CD4+ and CD8+ T cell, Myeloid-derived suppressor cells (MDSCs) and macrophage infiltrate following treatment with each ICI doublet over a period of 28 days in SM1 brain tumors. Tumors were collected at days 7 and 14 for IgG and at days 7, 14, and 28 after treatment initiation for the doublets. The results were represented as average ±SEM with 10 mice per group for all the growth curve experiments. The results were represented as average±SEM of 3 mice per group for panels D–F. Statistical significance was assessed with one-way ANOVA test (*p<0.05, **p<0.01, ***p<0.001). ANOVA, analysis of variance; ICI, immune checkpoint inhibitor.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Sequencing, Generated, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 3 Immune landscape of flank and brain tumors in SM1 mouse model following the treatment with single agent and combination ICI therapy. A) t-SNE plots showing major cell types identified in relapsed brain tumors and responding flank SM1 tumors following treatment with single agent and combination ICI. (B) Proportion of each cell type in the responding flank tumors from the indicted treatment groups. (C) Proportion of different T-cell clusters identified in the responding flank tumors from the indicated treatment groups. (D) Heatmap showing expression of activation/exhaustion markers across the identified T-cell clusters. (E) Violin plots showing expression of T cell activation markers and immune checkpoints in each T-cell cluster. (F) IHC analysis of tumors treated with anti-PD-1+LAG-3 or anti-PD-1+CTLA-4 demonstrates increased CD4+and CD8+ T cell infiltration in responding flank tumors. The samples were stained with anti-CD4 and anti-CD8 by IHC. Tumors were harvested at day 9 for IgG and single agents and at day 19 for anti-PD-1+LAG-3 and day 25 for anti-PD-1+CTLA-4 following the initiation of treatment. ICI, immune checkpoint inhibitor.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Expressing, Activation Assay, Staining

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 4 Anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 differentially polarize CD4+T cells in the SM1 mouse melanoma model. (A) Proportion of T cell clusters and three major subsets of CD4+T cells identified in the responding flank tumors from the indicated treatment groups. (B) Expression of immune checkpoints and markers for T helper subsets and T regulatory cells in the three major subsets of CD4+T cells identified by violin plots. (C) Percentage of CD4+T cells and activated CD69+CD4+ T cells in the responding flank tumors treated with combination of anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 identified by flow cytometry. Tumors were harvested at the endpoint following the treatment initiation. (D) Flow cytometry histogram plot showing activated CD4+T cells in tumors treated with IgG, anti-PD-1+LAG-3 or anti-PD-1+CTLA-4 as evidenced by cell surface CD69 and CD4 staining. (E) Treatment with anti-PD-1+CTLA-4 increased Tregs infiltration in the responding flank tumors and relapsing brain tumors. The samples were stained with anti-FOXP3 by IHC. Tumors were harvested at day 9 for IgG and single agents and at day 19 for anti-PD-1+LAG-3 and day 25 for anti-PD-1+CTLA-4 following the initiation of treatment for sc-RNAseq and IHC. The results were represented as average±SEM of 3 mice per group for panels C and D. Statistical significance was assessed with one-way ANOVA test (***p<0.001). ANOVA, analysis of variance ; IHC, immunohistochemistry.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Expressing, Flow Cytometry, Staining, Immunohistochemistry

Journal: Journal for immunotherapy of cancer

Article Title: Differential requirements for CD4+ T cells in the efficacy of the anti-PD-1+LAG-3 and anti-PD-1+CTLA-4 combinations in melanoma flank and brain metastasis models.

doi: 10.1136/jitc-2023-007239

Figure Lengend Snippet: Figure 5 Antitumor responses to anti-PD-1+LAG-3 are dependent on CD4+T helper function. (A, B) Depletion of CD4+T cells demonstrates that responses to anti-PD-1+LAG-3 but not anti-PD-1+CTLA-4 are dependent on CD4+T cells in both SM1 flank and brain tumor models. The results were represented as an average±SEM of 5 mice per group. (C) Decreased percentage of tumor-infiltrating CD69+CD8+ T cells and CD8+T cells in SM1 flank tumors, respectively, following CD4+T cell depletion and anti-PD-1+LAG-3 treatment. Increased percentages of CD69+CD8+ T cells and total CD8+T cells were seen in SM1 flank tumors following CD4+T cell depletion and anti-PD-1+CTLA-4 treatment. Tumors were collected at day 10 after the treatment initiation. (D) Antitumor responses of the PD-1+LAG-3 but not the PD-1+CTLA-4 combination are dependent on CD4+T cells in B16 flank tumors. Results show the average±SEM of 5 mice per group. (E) Decreased percentage of tumor-infiltrating CD69+CD8+ T cells and CD8+T cells in B flank tumors, respectively, following CD4+T cell depletion and anti-PD-1+LAG-3 treatment. Increased percentages of CD69+CD8+ T cells and total CD8+T cells were seen in B16 flank tumors following CD4+T cell depletion and anti-PD-1+CTLA-4 treatment. Tumors were harvested for flow cytometry analysis at day 5 following the start of the treatment. (F) Responses to Anti-PD-1+LAG-3 but not Anti-PD-1+CTLA-4 are dependent on CD4+T cells in the flank tumors of D4M- UV2 mouse melanoma model. The results were represented as an average±SEM of 5 mice per group. (G) G) Percentage of CD69+CD8+ and CD8+ T cells decrease in the D4M-UV2 flank tumors significantly, following CD4+T cell depletion and Anti-PD- 1+LAG-3 treatment. Tumors were collected for flow cytometry analysis at day 16 of treatment for IgG and day 19 of treatment for the doublets. (H, I) ELISPOT assays showing decreased levels of IFNγ production following CD4+T cell depletion and treatment with the PD-1+LAG-3 combination in both the SM1 and B16 mouse melanoma models. The results were represented as average±SEM of 3 mice per group for panels D-G. Statistical significance was assessed with one-way ANOVA test (*p<0.05, **p<0.01, ***p<0.001). ANOVA, analysis of variance.

Article Snippet: CD4+ and CD8+ T cell depletion CD4- specific antibody (clone YTS191; cat. #BE0003- 1; Bio X Cell) and CD8a- specific antibody (clone YTS169.4; cat. #BE0117; Bio X Cell) from Bio X Cell were used to deplete CD4+ T cells and CD8+ T cells, respectively.

Techniques: Flow Cytometry, Enzyme-linked Immunospot