Journal: Scientific Reports

Article Title: Next-generation Candida albicans vaccine VXV-01 containing recombinant Als3p and Hyr1p antigens for invasive Candida infections

doi: 10.1038/s41598-025-32488-8

Figure Lengend Snippet: Immunogenicity of Als3p/Hyr1p dual antigen vaccine formulated with alum, CAF01, or BDX100 adjuvants. (A). Combination of Als3p/Hyr1p doses by checkerboard method. (B). Schematic of the experimental design for determining vaccine-induced immunogenicity is shown. The ICR CD-1 mice ( N = 5/group) vaccinated SC (Alum, CAF01) or IN (BDX100) with different vaccine formulations (Als3p and Hyr1p antigens ratio on the x-axis) on days 0 and 21. Two weeks after the final vaccination, serum antigen-specific IgG titers and T cells were evaluated using ELISA and FluroSpot assay, respectively. Comparison of (C). Anti-Als3p and (D). anti-Hyr1p IgG endpoint titers in alum, CAF01 or BDX100 adjuvant-antigen formulations. Anti-Als3p and anti-Hyr1p IgG endpoint titers in (E). alum, (F). CAF01, (G). BDX100 adjuvant-antigen formulations. (H). Heat map showing the mean frequency ( n = 5 mice/cell/formulation) of Als3p or Hyr1p-specific Th1, Th2, and Th17 cells (IFN- γ, IL-4 or IL17 producing cells) in mice vaccinated with alum, CAF01 or BDX100 vaccine formulations. Each row represents data from each vaccine formulation. (I). Bar graph showing Als3p or Hyr1p-specific Th1, Th2, and Th17 cells.

Article Snippet: For CD4 + T cell depletion, 200 μg/mouse dose of rat anti-mouse CD4 IgG2b (clone GK1.5, BioXcell, Cat #BE0003-1)) or rat IgG2b isotype antibodies (Clone: LTF-2, BioXcell, Cat #BE0090) were administered intraperitoneally on day − 3 and 0 relative to infection.

Techniques: Immunopeptidomics, Enzyme-linked Immunosorbent Assay, Flurospot, Comparison, Adjuvant, Formulation

Journal: Scientific Reports

Article Title: Next-generation Candida albicans vaccine VXV-01 containing recombinant Als3p and Hyr1p antigens for invasive Candida infections

doi: 10.1038/s41598-025-32488-8

Figure Lengend Snippet: Mechanism of VXV-01-mediated protection. Naïve mice or vaccinated mice ( n = 10 mice/group) were infected with C. albicans or C. auris . For vaccination, the VXV-01 or placebo (adjuvant only) was administered on days 0, 21, and 35 (for C. albicans infection) or days 0 and 21 (for C. auris infection). Mice were immunosuppressed with cyclophosphamide and cortisone acetate for C. auris infection. Naïve mice ( n = 10 mice/group) infected with (A). C. albicans or (C). C. auris received two intraperitoneal anti-sera injections at 2 and 168 h relative to infection. Vaccinated mice ( n = 10 mice/group) received anti-CD4 or isotype control antibodies to deplete the CD T cells. The mice were infected with (B). C. albicans or (D). C. auris . Mice survivals were compared by Mantel-Cox test, and p < 0.5 was considered statistically significant.

Article Snippet: For CD4 + T cell depletion, 200 μg/mouse dose of rat anti-mouse CD4 IgG2b (clone GK1.5, BioXcell, Cat #BE0003-1)) or rat IgG2b isotype antibodies (Clone: LTF-2, BioXcell, Cat #BE0090) were administered intraperitoneally on day − 3 and 0 relative to infection.

Techniques: Infection, Adjuvant, Control

Journal: Scientific Reports

Article Title: Next-generation Candida albicans vaccine VXV-01 containing recombinant Als3p and Hyr1p antigens for invasive Candida infections

doi: 10.1038/s41598-025-32488-8

Figure Lengend Snippet: The durability of VXV-01 vaccine-induced immunity. The ICR (CD-1) mice were vaccinated sub-cutaneously with VXV-01 or placebo (adjuvant alone) on days: (A). 0, 21; or (B). 0, 21, 35. The serum anti-Als3p and anti-Hyr1p IgG endpoint titers and T cells were evaluated using ELISA and FluroSpot assay on days 14, 28, 90, 180, and 270 post-final vaccination. ( C ). Anti-Als3p, and ( D ). Hyr1p IgG endpoint titers and T-cell responses were compared between two and three vaccination schedules over the period of 270 days post-vaccination. Data presented as mean ± SE of N = 5 mice/group. ( E ). VXV-01 efficacy was tested after 15 weeks of final two doses vaccination regimen (day 0, 21) against C. auris infection in immunosuppressed mice ( N = 10 mice/group). Survival curves were compared by the Mantel-Cox test, and p < 0.5 was considered statistically significant.

Article Snippet: For CD4 + T cell depletion, 200 μg/mouse dose of rat anti-mouse CD4 IgG2b (clone GK1.5, BioXcell, Cat #BE0003-1)) or rat IgG2b isotype antibodies (Clone: LTF-2, BioXcell, Cat #BE0090) were administered intraperitoneally on day − 3 and 0 relative to infection.

Techniques: Adjuvant, Enzyme-linked Immunosorbent Assay, Flurospot, Infection

Journal: bioRxiv

Article Title: CD4 + T-cells sensitize quasi-mesenchymal breast tumors lacking CD73 to anti-CTLA4 immune checkpoint blockade therapy

doi: 10.1101/2025.05.12.653467

Figure Lengend Snippet: (A) UMAP plot of unbiased clustering of Snail HI qM and sgCD73 tumors, where each color-coded cluster represents a specific cell type or state. (B) Genes representing each cluster depicted in the UMAP plot in (A). See additional details in Supplementary Table 1 (C) Representation of each cluster in Snail HI qM and sgCD73 tumors. Expression levels represent log transformed values. (D) Immune-fluorescence analysis of primary tumor sections obtained from Snail HI qM or sgCD73 tumor-bearing mice receiving Control or anti-CTLA4 antibodies stained for CD4 (red), CD8 (white) and DAPI (blue) at 63X magnification. ( E ) Quantification of CD8 + T-cells and CD4 + T-cells in each high-power field (HPF) at 63X magnification from (D). Data represent three independent experiments, with n=3-5 mice in each group. 3-5 fields of view from the tumor interior were obtained for each group at 63X magnification. (F) Immune-fluorescence analysis of primary tumor sections obtained from Snail HI qM tumor-bearing mice receiving Control, anti-CD73, anti-CTLA4 or combinations of anti-CD73 and anti-CTLA4 antibodies stained for CD4 (red), CD8 (white) and DAPI (blue) at 63X magnification. ( G ) Quantification of CD8 + T-cells and CD4 + T-cells in each high-power field (HPF) at 63X magnification from ( F ). Data represent three independent experiments, with n=3-5 mice in each group. 3-5 fields of view from the tumor interior were obtained for each group at 63X magnification. ( E, G ) Data represent SEM, two-tailed unpaired t-test, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001 .

Article Snippet: Other depletion antibodies were administered as follows: 200 ug anti-CD4 (clone GK1.5, BioXCell), 200ug anti-CD8a (clone 53-6.7, BioXCell), 200 ug of anti-NK1.1 (clone PK136, BioXCell) and 200 ug of anti-CSF1R (clone AFS98, BioXCell) administered once a week after 1-3 days of implantation until control tumors reached approximately 2000mm 3 in size.

Techniques: Expressing, Transformation Assay, Fluorescence, Control, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: CD4 + T-cells sensitize quasi-mesenchymal breast tumors lacking CD73 to anti-CTLA4 immune checkpoint blockade therapy

doi: 10.1101/2025.05.12.653467

Figure Lengend Snippet: (A) Schema and tumor kinetics for sgCD73 tumor-bearing mice treated with the indicated antibodies. Data represent three independent experiments where n=3-4 for each group (B) Representative immune-fluorescence images of primary tumor sections obtained from (A) stained for CD4 (red) and DAPI (blue) at 63X magnification. Bar graph on the right represents quantification of CD4 + T-cells in each high-power field (HPF) at 63X magnification for the indicated treatment groups. 3-5 fields of view from the tumor interior were obtained for each tumor. Data represent three independent experiments, with n=3-5 mice in each group. (C) Schema and tumor kinetics for sgCD73 tumors propagated in Wild Type (WT) or CD8 knock-out (CD8-KO) mice treated with the indicated antibodies. Data represent three independent experiments where n=3-4 for each group (D) Representative immune-fluorescence images of primary tumor sections obtained from (C) stained for CD4 (red) and DAPI (blue) at 63X magnification. Bar graph on the right represents quantification of CD4 + T-cells in each high-power field (HPF) at 63X magnification for the indicated treatment groups. 3-5 fields of view from the tumor interior were obtained for each tumor. Data represent three independent experiments, with n=3-5 mice in each group. ( E ) Schema and tumor kinetics for sgCD73 tumors propagated in Wild Type (WT) or B2M knock-out (B2M-KO) mice treated with the indicated antibodies. Data represent three independent experiments where n=3-5 for each group (F) Representative immune-fluorescence images of primary tumor sections obtained from (E) stained for CD4 (red) and DAPI (blue) at 63X magnification. Bar graph on the right represents quantification of CD4 + T-cells in each high-power field (HPF) at 63X magnification for the indicated treatment groups. 3-5 fields of view from the tumor interior were obtained for each tumor. Data represent three independent experiments, with n=3-5 mice in each group. ( G ) Schema and tumor kinetics for Snail HI qM tumor-bearing mice treated with the indicated antibodies. Data represent two independent experiments where n=3-4 for each group. ( H ) Representative immune-fluorescence images of primary tumor sections obtained from (G) stained for CD4 (red) and DAPI (blue) at 63X magnification. Bar graph on the right represents quantification of CD4 + T-cells in each high-power field (HPF) at 63X magnification for the indicated treatment groups. 3-5 fields of view from the tumor interior were obtained for each tumor. Data represent three independent experiments, with n=3-5 mice in each group. ( A, C, E, G ) Data represent SEM, two-way ANOVA, **, p<0.01, ***, p<0.001, ****, p<0.0001. ( B, D, F, H ) Bar graph data represent SEM, two-tailed unpaired t-test, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Scale bars are 100um.

Article Snippet: Other depletion antibodies were administered as follows: 200 ug anti-CD4 (clone GK1.5, BioXCell), 200ug anti-CD8a (clone 53-6.7, BioXCell), 200 ug of anti-NK1.1 (clone PK136, BioXCell) and 200 ug of anti-CSF1R (clone AFS98, BioXCell) administered once a week after 1-3 days of implantation until control tumors reached approximately 2000mm 3 in size.

Techniques: Fluorescence, Staining, Knock-Out, Two Tailed Test

Journal: bioRxiv

Article Title: CD4 + T-cells sensitize quasi-mesenchymal breast tumors lacking CD73 to anti-CTLA4 immune checkpoint blockade therapy

doi: 10.1101/2025.05.12.653467

Figure Lengend Snippet: (A) Schema and tumor kinetics for sgCD73 tumor-bearing mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group (B) Schema and tumor kinetics for sgCD73 tumors propagated in Wild Type (WT) or CD4 knock-out (CD4-KO) mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group ( C ) Schema and tumor kinetics for sgCD73 tumors propagated in Wild Type (WT) or MHC-II knock-out (MHC-II-KO) mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group ( D ) Schema and tumor kinetics for Snail HI qM tumor-bearing mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group ( E ) Schema and tumor kinetics for Snail HI qM tumors propagated in Wild Type (WT) or CD4 knock-out (CD4-KO) mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group ( F, G ) Flow cytometry analysis for ( F ) IFN-gamma and ( G ) T-BET from Snail HI qM control or sgCD73 tumor-bearing mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group ( A-E ) Data represent SEM, two-way ANOVA, **, p<0.01, ***, p<0.001, ****, p<0.0001. ( F, G ) Bar graph data represent SEM, two-tailed unpaired t-test, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001 .

Article Snippet: Other depletion antibodies were administered as follows: 200 ug anti-CD4 (clone GK1.5, BioXCell), 200ug anti-CD8a (clone 53-6.7, BioXCell), 200 ug of anti-NK1.1 (clone PK136, BioXCell) and 200 ug of anti-CSF1R (clone AFS98, BioXCell) administered once a week after 1-3 days of implantation until control tumors reached approximately 2000mm 3 in size.

Techniques: Knock-Out, Flow Cytometry, Control, Two Tailed Test

Journal: bioRxiv

Article Title: CD4 + T-cells sensitize quasi-mesenchymal breast tumors lacking CD73 to anti-CTLA4 immune checkpoint blockade therapy

doi: 10.1101/2025.05.12.653467

Figure Lengend Snippet: (A) Schema and tumor kinetics for CD73 and B2M double knock-out (DKO) tumor-bearing mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group (B) Schema and tumor kinetics for CD73 and B2M double knock-out (DKO) tumor-bearing mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group. ( C ) Schema and tumor kinetics for CD73 and B2M double knock-out (DKO) tumors propagated in Wild Type (WT) or CD4 knock-out (CD4-KO) mice treated with the indicated antibodies. Data represent 3 independent experiments where n=3-7 for each group ( D ) Schema and tumor kinetics for CD73 and B2M double knock-out (DKO) tumor-bearing mice treated with the indicated antibodies. Responders were rechallenged with the same cell line as indicated with or without treatment with anti-CD4. Data represent 3 independent experiments where n=3-7 for each group. ( E, F ) Representative immune-fluorescence images of primary tumor sections obtained from (B) stained for CD4 (red), CD8 (white) and DAPI (blue) at 63X magnification. Bar graph on the right represents quantification of CD4 + T-cells in each high-power field (HPF) at 63X magnification for the indicated treatment groups. 3-5 fields of view from the tumor interior were obtained for each tumor. Data represent three independent experiments, with n=3-5 mice in each group. ( A-D ) Data represent SEM, two-way ANOVA, **, p<0.01, ***, p<0.001, ****, p<0.0001. ( F ) Data represent SEM, two-tailed unpaired t-test, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001. Scale bars are 100um.

Article Snippet: Other depletion antibodies were administered as follows: 200 ug anti-CD4 (clone GK1.5, BioXCell), 200ug anti-CD8a (clone 53-6.7, BioXCell), 200 ug of anti-NK1.1 (clone PK136, BioXCell) and 200 ug of anti-CSF1R (clone AFS98, BioXCell) administered once a week after 1-3 days of implantation until control tumors reached approximately 2000mm 3 in size.

Techniques: Knock-Out, Fluorescence, Staining, Two Tailed Test

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Design of the MICA/B vaccine. pAbs, polyclonal serum IgG. b, MICB-specific serum antibody (Ab) titres quantified by flow cytometry (n = 4 mice per group) in MICB-transgenic mice immunized with Ctrl-vax (blue) or MICB-vax (red). MFI, median fluorescence intensity. c, MICB-specific CD4+ T cell responses following immunization with MICB-vax or Ctrl-vax; CFSE dilution of splenocytes stimulated with MICB or control protein (ovalbumin (OVA)); shown are representative flow cytometry plots (left) and quantification for three mice per group (right). d, Cell-surface levels of MICB on B16F10 (MICB) tumours from mice immunized with MICB-vax or Ctrl-vax (n = 4 mice per group); staining of tumour cells with isotype-control monoclonal antibody (grey) or anti-MICA/B monoclonal antibody (specific for the α1–α2 domains, monoclonal antibody not blocked by vaccine-induced antibodies). e, Therapeutic efficacy of MICB-vax (red) or Ctrl-vax (blue) in mice with established B16F10 (MICB) tumours immunized at the indicated time points (n = 7 mice per group). Vax, vaccination. *P = 0.0137, ****P <0.0001; NS (not significant), P > 0.999. f, g, Vaccine efficacy in two models of spontaneous metastasis. Mice were immunized with Ctrl-vax (blue) or MICB-vax (red) following surgical removal of primary tumours using the B16-B6 melanoma (f; 10 mice per group) or 4T1 breast cancer (g; 13 mice per group) models. Shown are the size of primary tumours at the time of surgery (left), representative images of lung metastases (middle) and quantification of the total number of lung surface metastases (right). D, day; s.c., subcutaneous. h, i, Immunogenicity of the rhesus MICA/B α3 domain vaccine in the rhesus macaque model. h, Timeline of vaccination; blood was drawn 24 h before indicated immunization or boost. i, Serum titres of antibody to rhesus MICA/B for animal ID 9312. Representative data are shown from at least three (b) or two (c–g) independent experiments. Data from a single experiment with technical replicates for each time point are shown in i. Statistical significance was assessed by two-tailed unpaired Student’s t test (b), two-way ANOVA with Sidak’s multiple-comparison test (c), one-way ANOVA with Tukey’s multiple-comparison test (d), two-way ANOVA with Bonferroni’s post hoc test (left) and the log-rank (Mantel–Cox) test (right) (e), two-tailed Mann–Whitney test (f, g) and two-way ANOVA with Tukey’s multiple-comparison test (i). Data are depicted as the mean ± s.e.m. (b–g) or mean±s.d. (i).

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Flow Cytometry, Transgenic Assay, Fluorescence, Control, Staining, Drug discovery, Immunopeptidomics, Two Tailed Test, Comparison, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Characterization of the MICA/B α3 immunogen used in the primate study. The α3 domains of rhesus macaque MICA and MICB were expressed as a fusion protein with ferritin to generate nanoparticles that displayed both α3 domains on the surface. Nanoparticles formed by this fusion protein were conjugated using click chemistry to CpG ODN 2935 as the adjuvant and characterized by HPLC gel filtration chromatography. Shown are HPLC traces of the protein following conjugation to the CpG oligonucleotide (red: 280 nm trace for detection of protein; blue: 350 nm trace for detection of bis-aryl hydrazone bond). b, SDS-PAGE analysis of purified macaque MICA/B α3–ferritin protein under reducing (+) and non-reducing (−) conditions following CpG conjugation and final purification using a HPLC gel filtration column. c–e, Characterization of serum antibody responses to rhesus macaque MICA (left) and MICB (right) proteins (full-length extracellular domains without ferritin fusion partner) at different steps in the immunization process. Antibody responses were investigated in three animals (RBQ12, RVf10 and RQq15) at multiple timepoints (pre-immunization; three weeks following initial immunization and boosts 1-3, as illustrated in Fig. 1h) using a fluorescence-based ELISA (RFU, relative fluorescence units) at multiple serum dilutions (1:102 to 1:104). f–m, Binding of purified polyclonal serum IgG to cell surface MICA (left) and MICB (right) using HEK293T transfectants that displayed rhesus macaque MICA (Mamu-A*01) or MICB (Mamu-B*01) proteins. Preimmune sera were used as a control (grey) for sera obtained following immunization (red). Representative histograms (left in f, g) and graphical summaries of flow cytometry data (right in f, g, h–m) are shown for the four immunized macaques (9312, RBQ12, RVf10 and RQq15). Representative data from two experiments (a–b). Data from a single macaque immunization experiment with technical replicates for each macaque analyzed (c–m). Two-way ANOVA with Tukey’s multiple comparison test (c-e). Data depict mean+/− SD.

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Adjuvant, Filtration, Chromatography, Conjugation Assay, SDS Page, Purification, Fluorescence, Enzyme-linked Immunosorbent Assay, Binding Assay, Control, Flow Cytometry, Comparison

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a–b, Representative histograms (a) and quantification (b) of PD-1, CTLA-4, Tim-3, Tigit and Lag3 expression by tumor-infiltrating CD8 T-cells from Ctrl-vax (blue) or MICB-vax (red) mice (n = 9 mice/group). c–d, Comparison of T-cell and NK cell populations in B16F10 (MICB) tumors following treatment with a MICA/B mAb or the MICB vaccine. In the vaccine arm, mice received Ctrl-vax (C-vax) (n = 9 mice) or MICB-vax (M-vax) (n = 10 mice) on days 0 and 14, while mice in the mAb treatment group (n = 8 mice/ group) received two buffer injections. Mice were implanted with B16F10 (MICB-dox) tumor cells on day 21. MICB expression was induced on tumor cells by doxycycline treatment starting on day 28, and mice in the mAb treatment group received either mouse IgG2a isotype control mAb (iso) or MICA/B mAb (mAb) treatment every 48 h starting on day 28. Tumor-infiltrating immune cells were analyzed in all groups on day 35. Total numbers of tumor-infiltrating CD4+ T-cells, CD8+ T-cells and NK cells were quantified by flow cytometry (c), and intracellular staining was performed for IFNγ (d) in all four treatment groups. e–g, scRNA-seq analysis of changes in tumor-infiltrating immune cells induced by the vaccine. CD45+ immune cells in B16F10 (MICB-dox) tumors were investigated by scRNA-seq under four experimental conditions: the MICB-vax (+dox) experimental group and the three control groups, Ctrl-vax (−dox), Ctrl-vax (+dox) and MICB-vax (−dox). Doxycycline was administered to mice for seven days prior to scRNA-seq analysis to induce MICB expression on tumor cells in two of these groups (+dox). For each of the four groups, CD45+ immune cells were pooled from five mice to reduce variation from individual tumors. e, UMAP projection of CD45+ immune cells combined from all experimental groups. Major immune cell populations are annotated based on differentially expressed genes. f, Comparison of immune subpopulations across all clusters for the experimental MICB-vax (+dox) group (red) versus the three combined control groups (blue). g, Distribution of CD45+ cells across individual clusters (color-coded as in e) for the experimental MICB-vax (+dox) group (MICB) and the three combined control groups (Ctrl). Representative data of two experimental repeats (a–b). Data from a single experiment (c–d). ScRNA-seq data from a single experiment with sorted CD45+ cells pooled from 5 mice/group (e–g). Two-tailed Mann Whitney test (b); one-way ANOVA with Tukey’s multiple comparison test (c–d). Data depict mean+/− SEM.

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Expressing, Comparison, Control, Flow Cytometry, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, b, Comparison of vaccine efficacy against B16F10 (MICB) WT tumours and tumours with resistance mutations in the B2m (a) or Ifngr1 (b) gene. Mice received MICB-vax or Ctrl-vax and were then challenged with tumours of the indicated genotype (n = 7 mice per group). KO, knockout. c, Effect of CD4+ T cell and NK cell depletion on immunity to B2m-knockout tumours. Mice were immunized with MICB-vax or Ctrl-vax; treatment with depleting or isotype-control monoclonal antibody was started 2 d before injection of B2m-knockout B16F10 (MICB) tumour cells (n = 7 mice per group). d, Contribution of vaccine-induced anti-MICB antibodies to NK cell-mediated cytotoxicity against B2m-knockout B16F10 (MICB) tumour cells. CFSE-labelled B2m-knockout B16F10 (MICB) tumour cells were pre-incubated with 10 μg per well of purified serum IgG from mice immunized with MICB-vax or Ctrl-vax before the addition of NK cells at different effector to target (E:T) ratios as indicated. The percentage of dead target cells was assessed by flow cytometry. e, Effect of CD4+ T cell depletion on vaccine-induced NK cell infiltration into tumours. Flow cytometry quantification of total NK cell numbers is shown in WT (left) and B2m-knockout (right) tumours for the following treatment groups: Ctrl-vax+isotype-control monoclonal antibody (blue), Ctrl-vax + anti-CD4 (orange), MICB-vax+isotype-control monoclonal antibody (red) and MICB-vax + anti-CD4 (green) (n = 7 mice per group). Representative data from two independent experiments are shown in a–e. Statistical significance was assessed by two-way ANOVA with Bonferroni’s post hoc test (left) and log-rank (Mantel–Cox) test (right) (a–c), two-way ANOVA with Sidak’s multiple-comparison test (d) and one-way ANOVA with Tukey’s multiple-comparison test (e). Data are depicted as the mean±s.e.m.

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Comparison, Knock-Out, Control, Injection, Incubation, Purification, Flow Cytometry

Journal: Nature

Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack

doi: 10.1038/s41586-022-04772-4

Figure Lengend Snippet: a, Effect of CD4+ T cells on migratory DC populations in the tdLNs of mice immunized with MICB-vax versus Ctrl-vax. Total migratory DCs as well as cDC1 and cDC2 cells were quantified 2 d after induction of MICB expression in tumour cells by doxycycline treatment (n = 7 mice per group, except n = 6 for Ctrl-vax without anti-CD4). b, Migratory DC subsets within the tdLN of MICB-vax-immunized mice treated following immunization (days 28 + 30) with isotype-control, CD4-depleting or CD40L-blocking monoclonal antibody (n = 7 mice per group). c, Quantification of DC populations within the tumours of mice immunized with Ctrl-vax (blue) or MICB-vax (red) on day 7 following induction of MICB expression with doxycycline (n = 7 mice per group). d, Effect of cDC1 depletion on MICB vaccine-induced T cell and NK cell accumulation within tumours in Xcr1DTR mice. Mice were treated with DT or left untreated starting on day 26 following immunization with Ctrl-vax or MICB-vax (days 0 + 14) and B16F10 (MICB-dox) tumour implantation (day 21). Immune cells were analysed in tumours 7 d after induction of MICB expression on tumours with doxycycline (day 37) (n = 7 mice per group). e, Contribution of vaccine-induced anti-MICB antibodies to DC-mediated cross-presentation of tumour antigens to CD8+ T cells. Bone marrow-derived DCs (BMDCs) were pre-incubated with B2m-knockout B16F10 (MICB-OVA) tumour cells in the presence of affinity-purified serum IgG from mice immunized with Ctrl-vax or MICB-vax at the indicated concentrations. DCs were co-cultured with CFSE-labelled OT-1 CD8+ T cells with T cell proliferation as the readout. The role of activating Fc receptor (FcR) was assessed using BMDCs from Fcer1g−/− mice (orange) or pre-incubation of BMDCs with FcR-blocking antibody (yellow) before tumour cell addition. Representative data from two independent experiments are shown in a–e. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple-comparison test (a, b, d), two-tailed Mann–Whitney test (c) and two-way ANOVA with Tukey’s multiple-comparison test (e). Data are depicted as the mean ± s.e.m. (a–d) or mean ± s.d. (e).

Article Snippet: The following isotype-control antibodies were used: rat IgG2b (clone LTF-2, BioXcell; control for anti-CD4), rat IgG1 (clone TNP6A7, BioXcell; control for anti-CD8β), mouse IgG2a (clone C1.18.4, BioXcell; control for anti-NK1.1) and rat IgG2a (clone 2A3, BioXcell; control for anti-CSF1R).

Techniques: Expressing, Control, Blocking Assay, Derivative Assay, Incubation, Knock-Out, Affinity Purification, Cell Culture, Comparison, Two Tailed Test, MANN-WHITNEY

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Comparison of patient characteristics between CD8 + T-cell high and CD8 + T-cell low . The data is.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques: Comparison, Cell Differentiation, Mutagenesis, Adjuvant

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Assessment of CD8 + T cells in patient samples. ( a and b ) analysis of CD8 + T cells in stroma and epithelium of the primary tumor of 19 patients ( a ) and the PM lesions of 37 patients. The graphs illustrate the number of CD8 + T-cells normalized to the corresponding area of stroma or epithelium. ( c ).The bar graph shows the distribution of intraepithelial CD8 + T-cells normalized by area in PM-lesions among the 37 patients. The dotted line indicates the median and divide the cohort into CD8 + T-cell high and low. ( d ) The scanned histological slide of PM lesions. The upper left picture presents an example with high CD8 + T-cell infiltration and the corresponding HALO classified picture below. The left upper example is CD8 + T-cell low. The classified pictures show the different areas of the tumor (green: stroma, violet: necrosis, red: intraepithelial, yellow: white space). DFS ( e ) and OS ( f ) based on intraepithelial CD8 + T-cell counts of PM lesions. 18 patients belong to the CD8 + T-cell high group and 19 patients to the CD8 + T-cell low group. DFS ( g ) and OS ( h ) based on the stroma content and CD8 + T-cell distribution. 15 patients with a CD8 + T-cell high PM lesion were associated with low stromal content (continuous line), 9 patients with a CD8 + T-cell low PM-lesion had a rich stroma (fine dotted line),10 patients with a CD8 + T-cell low PMlesion had a poor stroma (bold dotted line) and 3 patients with a CD8 + T-cell high PM-lesion were stroma rich (dotted line). Error bars represent the median and the lines the interquartile range. Each dot represents a patient. **** = p ≤ 0.0001, *** = p ≤ 0.001, ** = = p ≤ 0.01, * = p ≤ 0.05, ns = p > 0.05.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques:

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Impact of HIPEC treatment in PM mouse model. ( a ) measurement of peritoneal tumor load as PCI. mice were treated with PBS ( n = 9) or heated PBS ( n = 7) or with M/D (n = 10) or with heated M/D (n = 11). ( b ) Staining of tumor tissues for the presence of CD8 + T cells, Granzyme B + cells and macrophages. ( c and d ) Quantification of CD8 + T cells and GZMB + cells. ( e ) PCI of the treated mice with and without CD8 + T-cells. Each dot represents one mouse. Error bars show the mean +/-SD. **** = p ≤ 0.0001, *** = p ≤ 0.001, ** = = p ≤ 0.01,* = p ≤ 0.05, ns = p > 0.05.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques: Staining

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Induction of antigen-specific CD8 + T cells via heated chemotherapy. ( a )Time-line of the experiment. ( b ) flow cytometry data on the maturation state of Mo-DC`s depending on the cancer-cell treatment. ( c )Time-line of the experiment with additional co-culture of CD8 + T-cells. ( d ) Shows flow cytometry data of CD8 + T-cells and their IFN-γ production depending on the cancer-cell treatment. ( e ) Shows the time-line of a similar experiment using splenocytes from OT-I mice, which have a specific TCR for the ovalbumin. ( f ) Presents the ratio of IFN-γ positive CD8 + T-cells after co-culturing with PBS, or M/D or heated M/D treated MC-38-Ova cancer cells with splenocytes from a OT-I mouse. Cancer cell killing by specific CD8 + T-cells. ( g ) time-line of the co-culture experiment. The % of cancer cell killing by the treatment with or without specific CD8 + T-cells is shown in ( h ).The experiments were performed in triplicates. Error bars show the mean +/-SD. **** = p ≤ 0.0001, *** = p ≤ 0.001, ** == p ≤ 0.01, * = p ≤ 0.05, ns = p > 0.05.

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques: Flow Cytometry, Co-Culture Assay

Journal: Scientific Reports

Article Title: CD8 + T-cells restrict the development of peritoneal metastasis and support the efficacy of hyperthermic intraperitoneal chemotherapy (HIPEC)

doi: 10.1038/s41598-024-72826-w

Figure Lengend Snippet: Intraepithelial CD8 + T-cell number after HIPEC in 2 patients. ( a ) Numbers of intraepithelial CD8 + T-cells before first HIPEC and after the first HIPEC treatment of 2 different patient and the DFS and OS of these two patients ( b ).

Article Snippet: CD-4 + and CD8+-T-cell depletion in mice was achieved by the intraperitoneal injection of 100ug CD4 or CD8a depletion antibody (BioXCell, USA; clone GK 1.5 for CD4 + T-cells and clone YTS 169.4 was used for CD8 + T-cells) 1 day prior to tumor cells injection and 1 day prior to the treatment.

Techniques:

Journal: Cancer cell

Article Title: Taxanes trigger cancer cell killing in vivo by inducing non-canonical T cell cytotoxicity.

doi: 10.1016/j.ccell.2023.05.009

Figure Lengend Snippet: Figure 5. Pre-clinical assessment of the therapeutic benefits of T cells pre-treated with docetaxel (A) Representative images and quantification of cleaved-caspase 3 (CC3) immunofluorescent staining in MMTV-PyMT organoids expressing OVA and co- cultured with T cells isolated from the spleen of OT I mice upon pre-treatment with a vehicle control or docetaxel. n = 3 biological repeats, scale bar = 100 mm. Data are presented as mean ± SEM, p values were determined using unpaired, nonparametric t-test with a Mann-Whitney U correction in GraphPad Prism. (B) Schematic representation of experiment design. (C and D) Tumor growth in mice bearing KB1P tumors (C) or MMTV-PyMT tumors (D) and transplanted with CD4+ T cells pre-treated in vitro with a vehicle control or with docetaxel. For Figure 5C, the p value was determined in R using a linear mixed-effects model, for Figure 5D, due to the lower number of animals, the p value was determined in Prism GraphPad using a mixed-effects analysis with a Geisser-Greenhouse correction. (E and F) Kaplan-Meier analysis of survival from time of T cell transfer in mice bearing KB1P tumors (E) or MMTV-PyMT tumors (F) transplanted with CD4+ T cells pre-treated in vitro with a vehicle control or with docetaxel. For E and F, p = values were determined using a log rank Mantel-Cox test in GraphPad Prism. See also Figure S5, Tables S1, and S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Cultrex PathClear Reduced Growth Factor Basement Membrane Extract Type 2 R&D Systems 3533-005-02 TryplE Thermo Fisher Scientific 12605–010 Thiazolyl Blue Tetrazolium Bromide Sigma Aldrich 298-93-1 Collagenase A Roche 10103586001 HEPES Thermo Fisher Scientific 15630106 Streptomycin/penicillin Thermo Fisher Scientific 15140122 DNAse I Roche 10104159001 DAPI Thermo Fisher Scientific D1306 Opti-MEM Thermo Fisher Scientific 31985070 Polybrene Sigma Aldrich TR-1003-G LCKi Merck Millipore 213743-31-8 FGF Thermo Fisher Scientific PHG0261 Uranylacetate Polysciences Inc 21447–25 Y-27632 Bio Connect S1049 Ploy-D-lysine Thermo Fisher Scientific A3890401 Lipofectamine 2000 Thermo Fisher Scientific 11668019 Rimadyl (carpofen) Zoetis N/A Temgesic (buprenorphine) Indivior Europe Limited N/A Duratears Alcon N/A InVivoMAb rat IgG2b isotype control, clone LTF-2 BioXCell BE0090 InVivoMAb anti-mouse CD4, clone GK1.5 BioXCell BE003-1 InVivoMAb anti-mouse CD8, clone YTS 169.4 BioXCell BP0117 Fc block CD16/CD32, clone 2.4G2 BD Biosciences 553141 Permeabilization kit eBioscience 00-5523-00 Golgi Plug BD Biosciences 555029 Ultraglutamine Lonza BE17-605E/U1 PMA Abcam Ab120297 Ionomycin MedChem Express HY-13434 DAPI-containing Vectashield mounting medium Vectorlab H-1200-10 Foxp3 fixation/permeabilization buffer eBioscience 00-5523-00 Sepharose CL-2B GE Healthcare 17-0140-01 Critical commercial assays MycoAlert PLUS Kit Lonza LT07-118 LegendPlexMulti-Analyte Flow Assay kit for mouse Th Cytokine panel VbP VO3 BioLegend 741044 D-Luciferin Promega E1605 Experimental models: Cell lines Cell lines MDA-MB-231, HEP1, A375, RKO, RPE, HEK293T, Jurkat T cells and A549 ATCC N/A EGI-1 cell line DSMZ DSMZ N/A HuH7 cell line CLS.

Techniques: Staining, Expressing, Cell Culture, Isolation, Control, MANN-WHITNEY, In Vitro