Journal: Respiratory Research

Article Title: Lung Inflammation in alpha-1-antitrypsin deficient individuals with normal lung function

doi: 10.1186/s12931-023-02343-3

Figure Lengend Snippet: Large airway inflammatory cells in normal and alpha1- antitrypsin deficient Individuals. A Bronchial biopsies from normal and alpha1- antitrypsin deficient individuals were incubated with primary antibodies against CD68, CD45, CD8, neutrophil elastase (NE), and AA1; HRP-conjugated secondary antibody and HRP-DAB developer as described in the methods. B The cells were counted and expressed as the number of cells per mm2 of basement membrane in the section. * P < 0.05, ** P < 0.005

Article Snippet: Cat # MA5-32166) (T-helper cells), anti-human CD8 (ThermoFischer Scientifics, Waltham, MA.

Techniques: Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

doi: 10.1186/s13046-023-02609-0

Figure Lengend Snippet: HERC2 participated in the immune evasion of HCC cells. A HERC2-deficient Huh7 cells and HERC2-overexpressing HCC-97 h cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. B and C Activated PBMCs were cocultured with Huh7 cells or HERC2-overexpressing HCC-97 h cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( B ). C IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. D The correlation between HERC2 expression and levels of PD-L1 based on TCGA datasets ( n = 369). E The correlation between HERC2 expression and liver immune cell recruitment based on TCGA datasets ( n = 369). ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

Article Snippet: Cells were then stained with anti-CD8 (17–0086-42, Thermo Fisher Scientific), anti-CD4 (12–0049-42, Thermo Fisher Scientific), or anti-CD56 (11–0566-42, Thermo Fisher Scientific) antibodies at 4 °C in the dark for 30 min.

Techniques: Western Blot, Flow Cytometry, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

doi: 10.1186/s13046-023-02609-0

Figure Lengend Snippet: HERC2 regulated JAK2/STAT3 signaling through direct interaction with PTP1B. A Huh7 cells were treated with 50 ng/ml IL-6 for 20 min. The extracted protein was precipitated with an antibody against HERC2 and separated by SDS-PAGE followed by silver staining. On the other hand, the band was analyzed with 4-D label-free quantitative proteomics. B Huh7 cells were treated with 50 ng/ml IL-6 for 20 min, and the interaction between HERC2 and PTP1B was validated by an immunoprecipitation assay. C-D HEK293T cells were co-transfected with HERC2-Flag and PTP1B-HA ( C ) or PTP1B-mcherry ( D ) plasmids. C Immunoprecipitation was performed using an anti-FLAG magnetic beads. The presence of coprecipitated HERC2 was determined by immunoblotting with the anti-HA antibody. D The colocalization of HERC2 and PTP1B in the cells was analyzed by immunofluorescence assay. E HERC2 and PTP1B double-deficient Huh7 cell lines were established. The cells were treated with 50 ng/ml IL-6 for 20 min, and the phosphorylation levels of JAK2 and STAT3 were detected by western blot analysis. F Cells were treated with 50 ng/ml IL-6 for 24 h. The RT-qPCR assay was used to detect the mRNA expression of CSC-related genes. G Cells were cultured with medium containing 100 × N2, 50 × B27, 20 ng/ml EGF, 10 nmol FGF, 5 μg/ml insulin, and 0.4% BSA for 7 days, scale bars = 100 μm. H The cells were treated with 20 μM sorafenib for 24 h. A flow cytometry assay was used to determine the percentage of apoptotic cells. I Cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. J and K Activated PBMCs were cocultured with HCC cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( J ). K IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

Article Snippet: Cells were then stained with anti-CD8 (17–0086-42, Thermo Fisher Scientific), anti-CD4 (12–0049-42, Thermo Fisher Scientific), or anti-CD56 (11–0566-42, Thermo Fisher Scientific) antibodies at 4 °C in the dark for 30 min.

Techniques: SDS Page, Silver Staining, Immunoprecipitation, Transfection, Magnetic Beads, Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing, Cell Culture, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

doi: 10.1186/s13046-023-02609-0

Figure Lengend Snippet: HERC2 promoted hepatic STAT3 activation and facilitated HCC tumorigenesis in vivo. A Diagram for mouse primary HCC induction. 15-day-old male mice were intraperitoneally injected with 25 μg/g DEN. Two weeks later, the mice were intraperitoneally injected with 0.5 μl/g CCl4 once a week for consecutive 22 weeks. B Tumor formation in liver tissues was observed, and the red arrow indicates typical tumor nodes. C The statistical diagram for tumor numbers is presented. D The statistical diagram for the liver index is presented. ( E ) H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. F The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 100 μm. G Western blot analysis was performed to detect JAK2/STAT3 signaling activation. H RT-qPCR analysis was conducted to evaluate the levels of cancer stem cell-related genes. I Western blot analysis was performed to detect PD-L1 expression in liver tissues. J PD-L1 expression was evaluated by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. K The expression of CD8 was determined by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. L-N A total of 2 × 10 6 or 2 × 10 5 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. L A diagram of mouse orthotopically implanted HCC is presented. M Tumor formation in liver tissues is presented. N A statistical diagram for liver tumor volume is presented. O-Q A total of 2 × 10 6 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. O H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. P The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 250 μm. Q CD133 expression in liver tissues was determined by immunohistochemical assay. Top scale bars = 100 μm, and bottom scale bars = 25 μm. * p < 0.05, ** p < 0.01. Data from one representative experiment of three independent experiments are presented

Article Snippet: Cells were then stained with anti-CD8 (17–0086-42, Thermo Fisher Scientific), anti-CD4 (12–0049-42, Thermo Fisher Scientific), or anti-CD56 (11–0566-42, Thermo Fisher Scientific) antibodies at 4 °C in the dark for 30 min.

Techniques: Activation Assay, In Vivo, Injection, Staining, Expressing, Immunohistochemical staining, Western Blot, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV

doi: 10.1038/s41467-023-36163-2

Figure Lengend Snippet: TCR-stimulated HTOC was infected with HIV. Control cultures were not infected (Uninfected). In some cultures, ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. A FOXP3 and CD25 expression (top) and PD-1 and IFN-γ expression (bottom) in CD4 + (CD3 + CD8 neg ) FOXP3 + cells, showing the frequency of T regs and T regDys . Expression of IL-17A and ROR-γt ( B ), and IL-17A and CCR6 ( C ) in CD4 + cells, showing the frequency of Th17 cells. Representative contour plots (left), statistical analyses of proportions of the cells from 5 independent experiments showing the frequency of these subsets ( A , B , right). D Statistical analysis of T regDys /Th17 ratio based on the results in A and B . Results derived from five independent experiments are presented as mean values +/− SEM. A – D **** P < 0.0001; *** P < 0.0002; * P < 0.02; Two-tailed; Unpaired t test. Circles within box plots represent each replicate.

Article Snippet: Flow cytometry antibodies for ROR-γt (AFKJS-9), IL-1β (CRM56), CCR6 (R6H1), IL-17A(eBio64DEC17), CD25 (M-A251), Ki-67 (SolA15), HIF-1α (Mgc3), CD4 (OKT4), CD45(HI30), CD8 (RPA-T8), IFN-γ (4S.B3), FOXP3(236A/E7), AREG (AREG559), HLADR, and phospho-caspase 1 (Ser376) were all purchased from Invitrogen/Thermofisher Scientific.

Techniques: Infection, Blocking Assay, Expressing, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV

doi: 10.1038/s41467-023-36163-2

Figure Lengend Snippet: A HTOC were TCR-activated and infected with HIV. ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) and ARI for 6 days as described in methods ( n = 5 independent experiments). Representative flow cytometric data showing ODC-1 expression in CD3 + CD8 negative cells (CD4+ T cells) (top) and statistical analyses of MFI from five independent experiments (bottom) are shown. B Key intermediate steps in polyamine synthesis. Cells were stimulated and processed for flow cytometry as in A for determining the expression of EIF5A ( C ) and hypusinated EIF5A in CD4 + T cells ( D ). Light and dark shaded gray histograms represent staining controls for EIF5A ( C ) and hypusinated EIF5A ( D ) respectively. Mean values +/− SEM; **** P < 0.0001; *** P < 0.0002; Two-tailed; Unpaired t test in A – D . E Fluorometric polyamine estimation in cellular lysates from cells stimulated as in A for 4 or 7 days ( n = 3 independent experiments). Polyamine concentrations were normalized to cell numbers. Mean values +/− SD; *** P = 0.0002; Two-tailed; Welch’s t test. Circles within box plots represent each replicate.

Article Snippet: Flow cytometry antibodies for ROR-γt (AFKJS-9), IL-1β (CRM56), CCR6 (R6H1), IL-17A(eBio64DEC17), CD25 (M-A251), Ki-67 (SolA15), HIF-1α (Mgc3), CD4 (OKT4), CD45(HI30), CD8 (RPA-T8), IFN-γ (4S.B3), FOXP3(236A/E7), AREG (AREG559), HLADR, and phospho-caspase 1 (Ser376) were all purchased from Invitrogen/Thermofisher Scientific.

Techniques: Infection, Blocking Assay, Expressing, Flow Cytometry, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

doi: 10.1038/s41467-023-36045-7

Figure Lengend Snippet: ICIE combines cryosurgery with intravenously injected (i.v. ) irinotecan (or camptothecin/CPT) and PD-L1 silencing siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting immunogenic cell death (ICD) and reducing the expression of programmed death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immuno-active). As a result, the CD8 + T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAAm-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and T EM : effective memory T cell.

Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

Techniques: Injection, Expressing, Modification

Journal: Nature Communications

Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

doi: 10.1038/s41467-023-36045-7

Figure Lengend Snippet: a Expression of HMGB1, CRT, HSP-70, and HSP-90 in EO771 cells after incubating them with various formulations at −20, −4, or 37 °C for 10 min ( n = 3 independent experiments). b A schematic illustration of ICD production following treatment of CPT&siR CRNPs in combination with cryosurgery to mature DCs, present the tumor-specific antigen to T cells by the matured DCs, and activate CD8 + cytotoxic T lymphocytes (CTLs) to attack cancer cells with reduced expression of PD-L1 via the release of granzyme B (GZMB). c , d Typical flow cytometry plots ( c ) and quantitative data ( d ) on maturing bone marrow dendritic cells (BMDCs) after co-culturing them with EO771-OVA cells for 24 h ( n = 3 independent experiments). The EO771-OVA cells were pretreated with the indicated various formulations. “+C” represents cold treatment at −20 °C for 10 min. e , f Quantitative data ( e ) and typical flow cytometry plots ( f ) of CD11c + Kb-SIINFEKL + BMDC percentage following co-culture with the aforementioned, pretreated EO771-OVA cells ( n = 3 independent experiments). g , h Typical flow cytometry histograms ( g ) and quantitative data ( h ) of CD8 + T cell proliferation after incubating them with the BMDCs matured by the EO771-OVA cells treated with aforementioned formulations ( n = 3 independent experiments). i, j Typical flow cytometry plots ( i ) and quantitative data ( j ) of activated CD8 + T cells following co-culture with the aforementioned BMDCs ( n = 3 independent experiments). k Representative images showing the process of activated CD8 + T cells attacking EO771-OVA cells labeled with CellTracker™ Green CMFDA Dye. The T cells were activated by the BMDCs co-cultured with EO771-OVA cells that received CPT&siR CRNPs+C treatment. The experiment was repeated three times independently with similar results. l , m Typical flow cytometry plots ( l ) and quantitative data ( m ) of dead EO771-OVA cells after T cell attacking. T cells were activated by BMDCs cocultured with EO771-OVA cells with different formulations ( n = 3 independent experiments). Statistical analyses were done using one-way ANOVA with Tukey’s multiple comparisons and correction. Data are presented as mean ± SD ( a , d , e , h , j , m ). Source data are provided as a Source Data file.

Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

Techniques: Expressing, Flow Cytometry, Co-Culture Assay, Labeling, Cell Culture

Journal: Nature Communications

Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

doi: 10.1038/s41467-023-36045-7

Figure Lengend Snippet: a – d Quantitative flow cytometry data of matured DCs (CD11c + CD86 + ) in inguinal lymph nodes (LNs, a ), and percentage of infiltrated CD8 + T cells ( b ), ratios of CD8 + /Treg cells ( c ), and percentage of cytotoxic T cells (CTLs, CD8 + GZMB + , d ) in primary (Prim) tumors of mice with cryosurgery and L (left side) tumors of mice with no cryosurgery ( n = 3 mice). e Representative immunofluorescence images of infiltrated CTLs in primary (Prim) tumors from mice with cryosurgery ( n = 3 mice). f Representative flow cytometry plots showing the percentage of effector memory T cell (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood (BL) collected from mice with cryosurgery and injection of one of the various formulations. g Quantitative flow cytometry data of T EM in blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). h CD8 + /Treg ratios in the blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). i Representative flow cytometry plots of Treg cells in blood collected from mice with cryosurgery and injection of one of the different formulations. j Quantitative flow cytometry data of CD8 + /Treg ratios in distant (Dist) tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. k Quantitative flow cytometry data of the percentage of infiltrated CTLs in Dist tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. l Representative immunofluorescence images of infiltrated CTLs in Dist tumors from mice with cryosurgery on Prim tumors and injection of one of the different formulations ( n = 3 mice). Statistical analyses were done using two-way ANOVA with Sidak’s post-test and correction for multiple comparisons. The experiments for e and I were repeated three times independently ( n = 3 mice) with similar results. Data are presented as mean ± SD ( a – d , g , h , j , k ). Source data are provided as a Source Data file.

Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

Techniques: Flow Cytometry, Immunofluorescence, Injection

Journal: Nature Communications

Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

doi: 10.1038/s41467-023-36045-7

Figure Lengend Snippet: a A schematic illustration of the in vivo experimental design. Mice received an intravenous injection of various formulations every 3 days. Cryosurgery was performed on the orthotopic tumor (OT) at 8 h after the first injection of the various formulations. The lung metastasis model was induced by intravenous injection of 4T1 cancer cells on day 10 after the first injection of the different formulations. b Weight of lungs collected from mice at the end of the study, suggesting inhibition of lung metastasis by ICIE ( n = 6 mice). c Representative photographs of lung tissues isolated at the end of the study, showing inhibition of lung metastasis by the ICIE treatment. Scale bar: 1 mm. d Quantification of lung metastasis nodes per mouse from mice at the end of the study ( n = 6 mice). e Representative H&E staining images of lung tissues collected at the end of the study, showing the decreased formation of metastasis in the lungs of mice with the ICIE treatment. Scale bar: 1 mm for low-magnification images and 200 µm for zoom-in images. The areas circled by blue dashed lines are metastases in the lungs. The experiments were repeated three times independently ( n = 3 mice) with similar results. f The percentage of effector memory T cells (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood collected from mice with the various treatments ( n = 3 mice). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test and correction. Data are presented as mean ± SD ( b , d , f ). Source data are provided as a Source Data file.

Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

Techniques: In Vivo, Injection, Inhibition, Isolation, Staining

Journal: Indian Journal of Pharmacology

Article Title: Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment

doi: 10.4103/ijp.ijp_695_21

Figure Lengend Snippet: Comparison of lymphocyte subsets by acne severity

Article Snippet: CD3/CD16/CD56 monoclonal antibody (M2AB, C5.9, J5511), fluorescein isothiocyanate, PE (catalog number: MA1–12207), MS anti-HU CD45 PERCP (catalog number: MHCD4531), anti-human CD19 APC SJ25C1 (catalog number: 17-0198-41), human CD3/CD4/CD8 mixture CD3/CD4/CD8 (catalog number: CD348A), anti-human CD38 APC-eFluor® 780 (catalog number: 47-0389-41), anti-human HLA-DR APC (LN3) (catalog number: 17-9956-41), anti-human CD45RA APC HI100 (catalog number: 17-0458-41), and anti-human CD45RO APC-eFluor® 780 (catalog number: 47-0457-41) were obtained from Thermo Fisher Scientific (Meridian Road Rockford, IL 61105 USA).

Techniques:

Journal: Indian Journal of Pharmacology

Article Title: Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment

doi: 10.4103/ijp.ijp_695_21

Figure Lengend Snippet: Comparison of lymphocyte subsets in patients with acne vulgaris before and after systemic isotretinoin treatment

Article Snippet: CD3/CD16/CD56 monoclonal antibody (M2AB, C5.9, J5511), fluorescein isothiocyanate, PE (catalog number: MA1–12207), MS anti-HU CD45 PERCP (catalog number: MHCD4531), anti-human CD19 APC SJ25C1 (catalog number: 17-0198-41), human CD3/CD4/CD8 mixture CD3/CD4/CD8 (catalog number: CD348A), anti-human CD38 APC-eFluor® 780 (catalog number: 47-0389-41), anti-human HLA-DR APC (LN3) (catalog number: 17-9956-41), anti-human CD45RA APC HI100 (catalog number: 17-0458-41), and anti-human CD45RO APC-eFluor® 780 (catalog number: 47-0457-41) were obtained from Thermo Fisher Scientific (Meridian Road Rockford, IL 61105 USA).

Techniques:

Journal: bioRxiv

Article Title: FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies

doi: 10.1101/2023.01.19.522856

Figure Lengend Snippet: A) Cartoon illustration of humanized CTLA-4 mice crossed to hFcyR mice to generate a hCTLA-4/hFcyR colony for the in vivo evaluation of fully human anti-CTLA-4 antibody variants. B) Flow cytometry evaluation of endogenous murine CTLA-4 cell surface expression in WT C57BL/6 mice compared to hCTLA-4/hFcyR mice, expressed as mean fluorescence intensity (MFI). C) Ipilimumab is a human IgG1 antibody whereas tremelimumab is a human IgG2 subclass. D) Surface plasmon resonance characterization of binding to human CTLA-4 by recombinant variants of ipilimumab and tremelimumab and their respective KDs. E) Evaluation of the ability of 10D1 and 1121 Fabs on IgG2 backbone to block interaction of hCTLA-4 with B7.1 as measured by ELISA. F) In vivo evaluation of the MC38 colon carcinoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). G) Evaluation of the in vivo activity of anti-CTLA-4 subclass variant on MC38 tumor growth. Ipilimumab and tremelimumab were tested as both IgG1 and IgG2 Fc subclass variants. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0, 3 and 6. H) Ipilimumab and tremelimumab were both placed on an IgG2 Fc backbone and tested for antitumor activity in the MC38 tumor model. I) In vivo evaluation of the B16 melanoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). J) Ipilimumab in either and IgG1 or IgG2 format was tested for antitumor activity in the B16 tumor model. n = 4-8 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

Article Snippet: For cell surface staining, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Biolegend CD4-FITC, clone GK1.5, CD8a-BV786, clone 53-6.7; CD3e-Percp-Cy5.5, clone 17A2; and ThermoFisher CD45-AlexaFluor 700, clone 30-F11.

Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, SPR Assay, Binding Assay, Recombinant, Blocking Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Variant Assay

Journal: bioRxiv

Article Title: FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies

doi: 10.1101/2023.01.19.522856

Figure Lengend Snippet: A) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 (200 ug alone or in combination with 50 ug intratumoral 2B6 blockade prior on days 0 and 3) comapred to 2B6 only control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). Blockade of the inhibitory FcyR (FcyRIIB) leads to significantly less Tregs when coadministered with 10D1-IgG1 compared to 10D1-IgG2. B) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants (e.g. IgG1 or IgG2) were given alone or in combination with 2B6 antibody blockade (50 ug given intratumoral) on days 0, 3, and 6. The combination of the IgG1-subclass variant led to significantly improved tumor control when compared to anti-CTLA-4 antibody alone or the anti-CTLA-4 IgG2 subclass antibody with 2B6. C) Table of the 10D1 IgG subclass variants generated for these studies, along with their relative binding affinities for activating vs inhibitory FcyRs. D) ELISA-based quantification of the relative affinity of the various subclass variants tested (in E and F). E) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of anti-CTLA-4 antibody variants (200 ug on days 0 and 3) comapred to to IgG control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). F) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to control, Fc-null (N297A), or IgG1. G) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1 or 1121) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced GAALIE variants led to significantly improved tumor control, even when placed on the 1121 (tremelimumab) backbone. n = 4-7 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

Article Snippet: For cell surface staining, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Biolegend CD4-FITC, clone GK1.5, CD8a-BV786, clone 53-6.7; CD3e-Percp-Cy5.5, clone 17A2; and ThermoFisher CD45-AlexaFluor 700, clone 30-F11.

Techniques: In Vivo, Blocking Assay, Flow Cytometry, Variant Assay, Generated, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies

doi: 10.1101/2023.01.19.522856

Figure Lengend Snippet: A) In vivo evaluation of the Fc-optimized anti-CCR8 antibody (GAALIE variant with decreased FcyRIIB binding) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma and MB49 bladder tumor microenvironment following systemic (i.p.) administration of 200 ug of CCR8-IgG1, CCR8-GRLR, or CCR8-GAALIE on days 0 and 3 following randomization. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). B) In vivo evaluation of the effect of Fc-enhanced anti-CCR8 antibodies on tumor control in the MC38, MB49, and B16 tumor models. Here, 200 ug of anti-CCR8 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variant GAALIE led to significantly improved tumor control when compared to Fc-null (GRLR), or IgG1. C) In vivo comparison of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) versus anti-CCR8 antibodies on tumor control in the MC38 tumor model. Here, 200 ug of each subclass variant was given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to IgG1 or afucosylated versions, with anti-CCR8 GAALIE demonstrating improved activity over anti-CTLA-4 GAALIE. n = 5-10 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

Article Snippet: For cell surface staining, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Biolegend CD4-FITC, clone GK1.5, CD8a-BV786, clone 53-6.7; CD3e-Percp-Cy5.5, clone 17A2; and ThermoFisher CD45-AlexaFluor 700, clone 30-F11.

Techniques: In Vivo, Variant Assay, Binding Assay, Flow Cytometry, Activity Assay