Journal: Clinical and Experimental Immunology

Article Title: Laryngeal transplantation in minipigs: early immunological outcomes

doi: 10.1111/j.1365-2249.2011.04531.x

Figure Lengend Snippet: Antibodies, isotypes and combinations used in multiple colour immunofluorescence studies of the laryngeal mucosa. Five combinations, each of three antibodies, were used

Article Snippet: Percentage areas of positive pixel staining were calculated for each fluorochrome and fluorochrome combination. table ft1 table-wrap mode="anchored" t5 caption a7 Primary antibody † to Clone Isotype Secondary antibody ‡ False colour Combination Myeloid cells CD172 742215 IgG2B IgG2b-FITC a Green 1 CD163 2A/10/11 IgG1 IgG1 - TRITC a Red 1 MHC-II MSA3 EPICS IgG2A IgG2a-AF633 b Blue 1 and 2 CD14 MIL2 IgG2B IgG2b-FITC a Green 2 CD16 c G7 IgG1 IgG1-TRITC a Red 2 Lymphoid cells CD45RC MIL15 IgM IgM-FITC a Green 3 and 4 CD25 K231.3B2 IgG1 IgG1-TRITC a Red 3 and 4 CD4 MIL17 H-M IgG2B IgG2b-AF633 b Blue 3 CD8 MIL12 IgG2A IgG2a-AF633 b Blue 4 and 5 γδ PPT 27 IgG2B IgG2b-FITC a Green 5 CD4 STH293 IgG1 IgG1-TRITC a Red 5 Open in a separate window † Mouse anti-pig; ‡ goat anti-mouse. a Southern Biotech, Birmingham, Alabama 35260, USA. b Molecular Probes, Oregon, USA. c AbD Serotec, Oxford, UK.

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Membrane localisation and checkpoint blockade enhance xenoantigen delivery to redirect pre-existing immunity against tumours

doi: 10.64898/2026.03.01.708859

Figure Lengend Snippet: A) Experimental timeline of the immunisation (i.e., vaccination) against OVA and subsequent tumour inoculation of the OVA-expressing B16F10 cell lines in mice. B) Titers of anti-OVA IgG per IgG subtypes in the plasma of the mice at d18. C) Percentage of OVA-specific CD8 + T cells against the immunogenic epitope OVA 257-264 (SIINFEKL) in the blood at d18 in pre-immunised mice (vax) or naïve mice (no vax) (n ≥ 3, mean ± SD, unpaired t-test). D, E) B16F10 melanoma cells, either wild-type (WT) or genetically modified to express high ( HI ) or low ( LO ) doses of membrane-bound OVA (B16mOVA) or soluble OVA (B16-OVA), were injected intradermally in C57BL/6 mice. Tumour growth (D) and associated survival (E) of the different OVA-expressing B16 cell lines in pre-immunised mice (n ≥ 5, mean ± SEM, Kruskal-Wallis with Dunn’s post-test on d10, d14 or d26 for tumour growth, log-rank tests for survival). F) Potential anti-tumour mechanisms engaged by soluble (left) or membrane-bound (right) xenoantigens when expressed by cancer cells.

Article Snippet: Samples were applied to the antigen coated plate and incubated at RT for 2 h. The plates were washed three times with PBST and HRP-conjugated antibodies were used to detect antigen-specific antibodies: anti-mouse IgG1 (#1070-05), anti-mouse IgG2a (#1080-05), anti-mouse IgG2b (#1090-05) and anti-mouse IgG3 (#1100-05) from Southern Biotech (Birmingham, AL, USA).

Techniques: Expressing, Clinical Proteomics, Genetically Modified, Membrane, Injection

Journal: bioRxiv

Article Title: Membrane localisation and checkpoint blockade enhance xenoantigen delivery to redirect pre-existing immunity against tumours

doi: 10.64898/2026.03.01.708859

Figure Lengend Snippet: A) Immunofluorescent staining of B16F10 WT melanoma with the TRP1-targeting TA99 antibody (scale bar = 100 μm). B) B16F10 WT tumour growth upon treatment with TA99 or IgG2a isotype control (n ≥ 4, mean ± SEM, Mann-Whitney test on d11). C) Design of FabTRP-OVA fusion protein and analysis of its production by SDS-PAGE (expected size = 92,3 kDa). D) Illustration of the FabTRP-OVA fusion protein approach for targeting the xenoantigen to the cancer cell membrane. E) Representative flow cytometry plot of FabTRP-OVA, OVA and no OVA (i.e., secondary antibody only) binding to the surface of B16F10 WT cells. F) B16F10 WT tumour growth when treated with FabTRP-OVA in pre-immunised or naïve mice (n ≥ 4, mean ± SEM, Kruskal-Wallis with Dunn’s post-test on d12). G) B16F10 WT tumour growth when treated with FabTRP-OVA in combination with anti-PD-1 checkpoint blocade therapy (n ≥ 4, mean ± SEM, Kruskal-Wallis with Dunn’s post-test on d12). H) Experimental treatment timeline of xenoantigen delivery (i.e., FabTRP-OVA, OVA or PBS (vehicle only)) and anti-PD-1 checkpoint blockade therapy in B16F10 WT in mice pre-immunised against OVA (Vax) or naïve (No vax). I, J) B16F10 WT tumour growth (I) and associated mouse survival (J) upon treatment with FabTRP-OVA, OVA or PBS in combination with anti-PD-1 checkpoint blockade therapy (n ≥ 5, mean ± SEM, Kruskal-Wallis with Dunn’s post-test on d16, log-rank tests for survival).

Article Snippet: Samples were applied to the antigen coated plate and incubated at RT for 2 h. The plates were washed three times with PBST and HRP-conjugated antibodies were used to detect antigen-specific antibodies: anti-mouse IgG1 (#1070-05), anti-mouse IgG2a (#1080-05), anti-mouse IgG2b (#1090-05) and anti-mouse IgG3 (#1100-05) from Southern Biotech (Birmingham, AL, USA).

Techniques: Staining, Control, MANN-WHITNEY, SDS Page, Membrane, Flow Cytometry, Binding Assay

Journal: bioRxiv

Article Title: Membrane localisation and checkpoint blockade enhance xenoantigen delivery to redirect pre-existing immunity against tumours

doi: 10.64898/2026.03.01.708859

Figure Lengend Snippet: A) Experimental timeline of the immunisation (i.e., vaccination) against varicella VZVO using Varivax or a combination of gE/CpG. B) IFΝγ quantification in the supernatant of gE restimulated (+) or non-restimulated (-) splenocytes, collected from mice immunized with Varivax, gE/CpG or PBS (i.e., naïve). Ionomycin+PMA was used as a positive control. C) Titers of anti-gE total IgG and per IgG subtypes in the plasma of the pre-immunised mice at d55 (before tumour implantation). D) Experimental treatment timeline of xenoantigen delivery (i.e., Varivax, gE or PBS (vehicle only)) and anti-PD-1 checkpoint blockade treatment in B16F10 WT in mice pre-immunised with Varivax against varicella VZVO . E, F) B16F10 WT tumour growth (E) and associated mouse survival (F) upon treatment with Varivax, gE or PBS in combination with anti-PD-1 checkpoint blockade (n ≥ 7, mean ± SEM, Kruskal-Wallis with Dunn’s post-test on d16, log-rank tests for survival).

Article Snippet: Samples were applied to the antigen coated plate and incubated at RT for 2 h. The plates were washed three times with PBST and HRP-conjugated antibodies were used to detect antigen-specific antibodies: anti-mouse IgG1 (#1070-05), anti-mouse IgG2a (#1080-05), anti-mouse IgG2b (#1090-05) and anti-mouse IgG3 (#1100-05) from Southern Biotech (Birmingham, AL, USA).

Techniques: Positive Control, Clinical Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: Tuning of CD73 blockade is required to improve the response of low CD73-expressing MC38 tumors to IR. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 day post-IR. (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–12, *p<0.05 ****p<0.0001 (two-way ANOVA). (D, right panel) Representative histograms of CD73 expression in MC38 and TS/A cells 24-hour post-IR at 6 and 12 Gy compared with non-irradiated (NIR) cells. (D, left panel) cultured cells were analyzed by flow cytometry for their CD73 expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, **p<0.01, ***p<0.001, ****p<0.0001 (two-way ANOVA). (E) TS/A tumor cells were injected subcutaneously in BALB/c mice and when tumors reached 60–70 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 days post-IR. (F) Tumor growth was monitored in treated mice. (G) The Kaplan-Meier survival curves for the treated mice are shown. (I) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=12–13, **p<0.01 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Injection, Irradiation, Cell Culture, Flow Cytometry, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 expression level controls the MC38 tumor response to CD73 blockade treatment. (A) Representative histograms of transfected and non-transfected MC38 cell with CD73 gene analyzed by flow cytometry. (B) Cultured MC38 control (Ctrl) and CD73 high MC38 cells were analyzed by flow cytometry 24-hour post-IR at 12 Gy for their CD73 membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, ***p<0.001, ****p<0.0001 (one-way ANOVA). (C) CD73 high MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 starting 1 day before IR then 2, 6 and 9 days post-IR. (D, F) Tumor growth was monitored in treated mice. (E, G) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=13–14, *p<0.05, ***p<0.001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation; NIR, non-irradiated; ssc-a, side scatter-a.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Control, Membrane, Fluorescence, Staining, Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 blockade treatment regimen affects the expression level of iCOS in tumor infiltrating CD4 + T lymphocytes. C57BL/6 mice with the subcutaneous MC38 tumors and BALB/c mice with subcutaneous TS/A tumors were irradiated and treated with either one dose or four doses of anti-CD73 starting 1 day before IR, then 2, 6 and 9 days post-IR. At day 10 post-IR, tumors were harvested and analyzed for immune infiltrating tumor cells by flow cytometry. CD4 + T lymphocytes infiltrating MC38 tumor were analyzed by flow cytometry for iCOS (A, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (A, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 tumors. (B, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. (B, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=8–10, *p<0.05 (two-way ANOVA). CD4 + T lymphocytes infiltrating MC38 CD73 high tumors were analyzed by flow cytometry for iCOS (C, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (C, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 CD73 high tumors. (D) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 CD73 high tumors are presented for each treatment group. CD4 + T lymphocytes infiltrating TS/A tumor were analyzed by flow cytometry for their iCOS (E, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (E, right panel) Representative histograms of iCOS expression in CD4 + T lymphocytes infiltrating TS/A tumor. (F, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating TS/A tumor are presented for each treatment group. (F, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=4–8, *p<0.05 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Irradiation, Flow Cytometry, Membrane, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: iCOS signaling is involved in CD73 blockade-mediated antitumor effect in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 (starting 1 day before IR then 2, 6 and 9 days post-IR) and anti-iCOS (starting 2 days post IR, then 6 and 9 days post-IR). (B and D) Tumor growth was monitored in treated mice. (C and E) The Kaplan-Meier survival curves for the treated mice are shown. (F) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=6–7, *p<0.05, ****p<0.001, ***p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: One dose of aCD73 improves the antitumor effect of aPD-L1 and IR treatment in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with one dose of anti-CD73 (starting 1 day before IR) and anti-PD-L1 (starting the same day as IR, then 3, 6 and 9 days post-IR). (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. (D) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–14, *p<0.05, **p<0.01, ****p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation