Journal:

Article Title: T cell pathways involving CTLA4 contribute to a model of acute lung injury 1

doi: 10.4049/jimmunol.0903238

Figure Lengend Snippet: Lipopolysaccharide (LPS, 100 μg) or phosphate buffered saline (PBS) was administered to mice (Balb/c) intratracheally. Lungs were harvested at indicated days (D1 - D6). Lungs were digested as described in Methods. Expression levels of CTLA4 and Foxp3 in the CD3+CD4+ cell population in lung tissue were determined by flow cytometry: representative histograms of CTLA4 and Foxp3 expression (shaded areas represent isotype control) (A, C); mean fluorescence intensity (MFI) for CTLA4 and Foxp3 (B, D). Data are shown as geometrical mean ± SEM (n = 3 - 6 per group). *, LPS group vs respective PBS group (p < 0.05); #, vs LPS D6 (p < 0.05); †, p < 0.05.

Article Snippet: Mouse anti-CTLA4 antibody (αCTLA4, UC10-4F10-11) was purchased from Bio X Cell (West Lebanon, NH).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal:

Article Title: T cell pathways involving CTLA4 contribute to a model of acute lung injury 1

doi: 10.4049/jimmunol.0903238

Figure Lengend Snippet: Mice were injected intraperitoneally with anti-CTLA4 antibody (αCTLA4, 100 μg) or hamster IgG (IgG, 100 μg) one day prior to intratracheal administration of LPS (100 μg). D6 group was reinjected intraperitoneally with αCTLA4 (100 μg) on day 3. BAL fluid was collected at indicated days (D1 - D6) after the LPS exposure. Levels of albumin in BAL were determined by ELISA (A). Neutrophil and lymphocyte counts in BAL fluid were determined by differential staining of cells (B, C). Data are shown as geometric mean ± SEM (n = 3 - 6 per group). †, p < 0.05.

Article Snippet: Mouse anti-CTLA4 antibody (αCTLA4, UC10-4F10-11) was purchased from Bio X Cell (West Lebanon, NH).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal:

Article Title: T cell pathways involving CTLA4 contribute to a model of acute lung injury 1

doi: 10.4049/jimmunol.0903238

Figure Lengend Snippet: Mice were injected intraperitoneally with anti-CTLA4 antibody (αCTLA4, 100 μg) or hamster IgG (IgG, 100 μg) one day prior to intratracheal administration of LPS (100 μg). D6 group was reinjected intraperitoneally with αCTLA4 (100 μg) on day 3. BAL fluid and lungs were collected at indicated days (D1 - D6) after the LPS exposure. Lungs were digested as described in Methods. Levels of IL-17A in BAL fluid were determined by a multiplexed immunoassay (A). Expression of CD69 and Foxp3 in the CD3+CD4+ cell population in lung tissue was determined by flow cytometry: representative histograms of CD69 and Foxp3 expression at day 2 (shaded areas represent isotype control) (B); mean fluorescence intensity (MFI) for CD69 and Foxp3 (C, D). Data are shown as geometric mean ± SEM (n = 3 - 6 per group). †, p < 0.05.

Article Snippet: Mouse anti-CTLA4 antibody (αCTLA4, UC10-4F10-11) was purchased from Bio X Cell (West Lebanon, NH).

Techniques: Injection, Expressing, Flow Cytometry, Fluorescence

Journal:

Article Title: T cell pathways involving CTLA4 contribute to a model of acute lung injury 1

doi: 10.4049/jimmunol.0903238

Figure Lengend Snippet: Mice were injected intraperitoneally with anti-CTLA4 antibody (αCTLA4, 100 μg) or hamster IgG (IgG, 100 μg) one day prior to intratracheal administration of LPS (100 μg). Lungs were collected at day 2 after the LPS exposure and digested as described in Methods. Absolute cell counts and ratio of CD4+Foxp3+ (T regulatory) and CD4+Foxp3− (T effector) cells in the CD4+ cell population were determined by flow cytometry (A, B). Expression of Foxp3 in the CD4+Foxp3 cell population and CD69 in the CD4+CD25+ cell population in lung tissue was determined by flow cytometry: representative histograms of CD69 and Foxp3 expression (shaded areas represent isotype control) (C, E); mean fluorescence intensity (MFI) for Foxp3 and CD69 (D, F). Data are shown as geometric mean ± SEM (n = 3 - 6 per group). †, p < 0.05; ns, not significant.

Article Snippet: Mouse anti-CTLA4 antibody (αCTLA4, UC10-4F10-11) was purchased from Bio X Cell (West Lebanon, NH).

Techniques: Injection, Flow Cytometry, Expressing, Fluorescence

Journal: Nature

Article Title: MHC-II neoantigens shape tumor immunity and response to immunotherapy

doi: 10.1038/s41586-019-1671-8

Figure Lengend Snippet: (a) One million KP9025 sarcoma cells were injected subcutaneously into syngeneic 129S4 mice and animals were treated with either control mAb or the αPD-1+αCTLA4 combination on days 3, 6, and 9 post transplant. Representative data from two independent experiments are shown as average tumor diameter ± SEM (n=5 in all groups per experiment). (b) KP9025 sarcoma cells were injected as above and tumors were surgically resected followed by rechallenge with the same line. Representative data from one of two independent experiments are shown as average tumor diameter ± SEM (n=3 in all groups per experiment). (c) Cohorts of 5 mice were injected with 1x10 6 KP.mLAMA4, KP.mITGB1, KP.mLAMA4.mITGB1, or KP.mSB2.SIINFEKL and treated with either control mAb (top) or the αPD-1 + αCTLA4 combination (bottom) on days 3, 6, and 9 post transplant. Representative data from one of three independent experiments is shown as individual tumor diameters.

Article Snippet: For immune checkpoint therapy, rat IgG2a αPD1 (RMP1-14, Leinco) and murine IgG2b αCTLA4 (9D9, Leinco Technologies) were used.

Techniques: Injection, Control

Journal: Nature

Article Title: MHC-II neoantigens shape tumor immunity and response to immunotherapy

doi: 10.1038/s41586-019-1671-8

Figure Lengend Snippet: (a) Rag2 −/− or WT 129S4 mice were injected with 1x10 6 KP9025 or KP.mLAMA4 cells and treated with αPD-1, αCTLA or αPD-1 + αCTLA4 on days 3, 6 and 9. Tumors were harvested once the average diameter reached 20 mm and sarcoma cell lines were established ex vivo . Cell lines were stimulated with IFNγ to upregulate MHC-I and subsequently used to stimulate the mLAMA4-specific CD8 + 74.14 T cell clone. IFNγ secretion by T cells was measured by ELISA. Representative data from 2 independent experiments is represented as the average of 2 independent tumor samples in each group. (b) WT 129S4 mice were injected with 1x10 6 KP.mSB2.SIINFEKL cells and treated with αPD-1+αCTLA4 combination ICT on days 3, 6 and 9. Tumors were harvested as described in (a). Established ex vivo cell lines were cloned via limiting dilution and parental KP.mSB2.SIINFEKL cells or individual clones from outgrown tumors were used to stimulate the mSB2-specific C3 CD8 + T cell clone and IFNγ production quantified by ELISA. Representative data from four independent experiments is presented as average IFNγ concentration of 8 individual clones ± SEM. Significance was determine using an unpaired, two sided t test. (c) Cell surface staining of SIINFEKL-H-2-K b expressed by unstimulated or IFNγ-stimulated parental KP.mSB2.SIINFEKL or individual clones described in (b). A representative histogram is shown. (d) Quantification of average SIINFEKL-H-2-K b MFI from 8 individual clones described in (c) ± SEM. NS not significant. (e) Survival curves of WT 129S4 mice injected subcutaneously with 1x10 6 KP.mSB2.SIINFEKL.mITGB1. Mice were treated with control mAb or αPD-1+αCTLA4 combination ICT on days 3, 6 and 9. n=10 mice per group from two independent experiments. ****indicates p=1.5x10 −5 as calculated using Mantel-Cox test.

Article Snippet: For immune checkpoint therapy, rat IgG2a αPD1 (RMP1-14, Leinco) and murine IgG2b αCTLA4 (9D9, Leinco Technologies) were used.

Techniques: Injection, Ex Vivo, Enzyme-linked Immunosorbent Assay, Clone Assay, Concentration Assay, Staining, Control

Journal: Nature

Article Title: MHC-II neoantigens shape tumor immunity and response to immunotherapy

doi: 10.1038/s41586-019-1671-8

Figure Lengend Snippet: (a) Whole TIL from KP.mLAMA4.mITGB1 tumors 12 days post transplant were stained with mITGB1-I-A b tetramers. Populations were previously gated on viable CD11b − CD4 + cells. Representative data from one of two independent experiments of 5 pooled tumors each is shown. (b) mITGB1-I-A b tetramer-negative and tetramer-positive cells described in (a) were analyzed for expression of T-BET and FOXP3. Representative plots are shown. (c) Quantification of two independent experiments described in (b) is shown as average percent of tetramer-negative and tetramer-positive cells staining positive for the indicated protein. Tumor-bearing animals received control mAb or α-CTLA4 treatment on days 3, 6, and 9-post transplant where indicated. (d) mITGB1-I-A b tetramer-positive and tetramer-negative cells described in (a) were analyzed for expression of PD-1. Representative plots are shown. (e) Quantification of two independent experiments described in (d) is shown as average percent of tetramer-negative and tetramer-positive cells staining positive for PD-1. (f) mITGB1-I-A b tetramer-positive cells described in (a) were analyzed for expression of the indicated proteins. Representative histograms from one of two independent experiments using pools of 5 tumors each are shown.

Article Snippet: For immune checkpoint therapy, rat IgG2a αPD1 (RMP1-14, Leinco) and murine IgG2b αCTLA4 (9D9, Leinco Technologies) were used.

Techniques: Staining, Expressing, Control

Journal: Nature

Article Title: MHC-II neoantigens shape tumor immunity and response to immunotherapy

doi: 10.1038/s41586-019-1671-8

Figure Lengend Snippet: (a) Representative tetramer staining of mLAMA4-specific CD8 + T cells from the spleens of KP.mLAMA4 (left) or KP.mLAMA4.mITGB1 (right) tumor-bearing mice 12 days post transplant. Mice received the indicated ICT treatment on days 3, 6, and 9. Cells were gated from viable CD45 + CD11b − Thy1.2 + cells. (b) Quantification of three independent experiments described above is shown as average percent mLAMA4 tetramer-positive of CD8 + T cells ± SEM. *indicates p=.04, ***indicates p=.0007 and ****indicates p=.00003 (2-way ANOVA with multiple comparisons corrected with the Bonferroni method). (c) In vivo cytotoxic function of mLAMA4-specific CD8 + T cells. Naïve splenocytes were labeled with 0.5 μM CFSE and pulsed with 1 μM SIINFEKL peptide (white histograms) or 5 μM CFSE and pulsed with 1 μM mLAMA4 peptide (green histograms) and transferred into control naïve or tumor-bearing mice 11 days post tumor transplant. Tumor-bearing animals received the indicated ICT treatment on days 3, 6, and 9 post transplant. Representative data is shown. (d) Quantification of percent mLAMA4-specific lysis from independent in vivo cytotoxicity assays described above is shown as average ± SEM (n=6 in αCTLA4, n=8 in all other groups). p values calculated using a 2-way ANOVA with multiple comparisons corrected with the Bonferroni method.

Article Snippet: For immune checkpoint therapy, rat IgG2a αPD1 (RMP1-14, Leinco) and murine IgG2b αCTLA4 (9D9, Leinco Technologies) were used.

Techniques: Staining, In Vivo, Labeling, Control, Lysis

Journal: Nature

Article Title: MHC-II neoantigens shape tumor immunity and response to immunotherapy

doi: 10.1038/s41586-019-1671-8

Figure Lengend Snippet: (a) Rag2 −/− mice were simultaneously injected with 1x10 6 KP.mLAMA4 and KP.mLAMA4.mITGB1 cells on contralateral flanks. Representative data from one of two independent experiments is shown as individual tumor diameter (n=3 in each experiment). (b) WT 129S4 mice were injected with 1x10 6 KP.mITGB1 cells and were treated with αPD-1+αCTLA4 combination ICT on days 3, 6, and 9. Representative data from one of two individual experiments is shown as individual tumor diameters (n=5 in all experiments). (c) WT 129S4 mice were simultaneously injected with 1x10 6 KP.mLAMA4 and KP.mLAMA4.mITGB1 cells on contralateral flanks and treated as in (b). Representative data from one of two individual experiments is shown as individual tumor diameters (n=5 in all experiments). (d) WT 129S6 mice were injected subcutaneously with 2x10 6 T3 sarcoma cells and were treated with αPD-1+αCTLA4 combination ICT on days 3, 6, and 9. Following tumor rejection and a 30-day recovery period, tumor-experienced mice were rechallenged with 2x10 6 T3 cells in the presence of control mAb or CD4-depleting antibody, or with irrelevant sarcoma cells. Representative data from one of two independent experiments are shown as average tumor diameter ± SEM (n=5 in all groups per experiment). (e) WT 129S4 mice were injected subcutaneously with 1x10 6 KP.mLAMA4.mITGB1 cells followed by surgical resection 10 days post transplant. After a 30-day recovery period, tumor-experienced mice were rechallenged with 1x10 6 KP9025, KP.mLAMA4.mITGB1, or KP.mLAMA4. Representative data from one of two independent experiments are shown as average tumor diameter ± SEM (n=5 in all groups per experiment). ****indicates p=2x10 −6 as calculated using a 2-way ANOVA with multiple comparisons corrected with the Bonferroni method. (f) Quantification of data from three independent experiments described in is shown as average number of spots ± SEM (left) and average number of mITGB1-specific CD4 + cells ± SEM (right). **indicates p=.003, ****indicates p=7.2x10 −5 (unpaired, two sided t test). (g) CD45 + Ly6G − MHCII + CD64 + CD25 − CD11b + F4/80 + macrophages in TIL from animals bearing the indicated contralateral tumors were analyzed for expression of iNOS 11 days post tumor transplant. Representative data is shown. (h) Quantification of iNOS + macrophages from experiments described in (f) as a percent of total CD45 + cells. Data is shown as average ± SEM of four independent experiments. *indicates p=.03 as calculated using an unpaired, two sided t test. (i) CD45 + Ly6G − MHCII + CD64 + CD25 − CD11b + F4/80 + macrophages from the indicated contralateral tumors described were isolated 11-days post transplant and analyzed for expression of iNOS. Representative plots are shown. (j) Quantification of iNOS + macrophages from two independent experiments described in (h) is shown as average percent of total CD45 + cells.

Article Snippet: For immune checkpoint therapy, rat IgG2a αPD1 (RMP1-14, Leinco) and murine IgG2b αCTLA4 (9D9, Leinco Technologies) were used.

Techniques: Injection, Control, Expressing, Isolation

Journal: Nature

Article Title: MHC-II neoantigens shape tumor immunity and response to immunotherapy

doi: 10.1038/s41586-019-1671-8

Figure Lengend Snippet: (a) WT syngeneic 129S4 mice were injected with 1x10 6 KP.mLAMA4 cells followed by treatment with αPD-1+αCTLA4 on days 3, 6, and 9 post-transplant. Representative data from one of three individual experiments is shown as individual tumor diameters (n=5 per group per experiments) (b) Mice were injected contralaterally with 1x10 6 KP.mLAMA4 and 1x10 6 KP.mLAMA4.mITGB1 followed by treatment as described in (a). Representative data from one of three individual experiments is shown as individual tumor diameters (n=5 per group per experiments). (c) Mice were injected as described in (b) and IFNγ ELISPOT analysis of tumor infiltrating CD4 + T cells stimulated with naïve splenocytes pulsed with 2 μg/mL of the indicated peptides was performed 11 days post-transplant. Italicized numbers indicate the average number of spots in mITGB1-stimulated wells from three independent experiments. (d) Tetramer staining of mLAMA4-specific CD8 + TIL 11 days post transplant of mice described in (b). Representative data from one of four independent experiments is shown as percent of mLAMA4-specific cells within the CD8 + T cell population. (e) Quantification of tumor-infiltrating T cells from mice described in (b) 11 days post transplant. Data is shown as percent of total viable CD45 + cells ± SEM. *indicates p=.02, **indicates p=.009 (unpaired, two sided t tests).

Article Snippet: For immune checkpoint therapy, rat IgG2a αPD1 (RMP1-14, Leinco) and murine IgG2b αCTLA4 (9D9, Leinco Technologies) were used.

Techniques: Injection, Enzyme-linked Immunospot, Staining

Journal: bioRxiv

Article Title: Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment

doi: 10.1101/2021.05.21.445190

Figure Lengend Snippet: (A) UMAP of scRNA-seq analysis of 22,635 cells isolated from KPF CAF-HIF2 WT tumors (10,703 cells; n = 3 mice) and KPF CAF-HIF2 KO tumors (11,932 cells; n = 3 mice). Cell types were identified through graph-based clustering followed by manual annotation using marker genes. (B) Percentage of myeloid cells in each tumor. (C) M2-polarized TAMs were identified within the myeloid cell population via expression of Arg1 and Mrc1. (D) Immunosuppressive TAMs were identified within the myeloid cell population via expression of Cd274 ( Pdl1 ) and Cd86 ( B7-2 ). (E) Violin plots showing findings on scRNA-seq analysis of Ctla4 in KPF CAF-HIF2 WT and KO tumors in all identified cell types. (F) Left: Representative IHC images of CAF-HIF2 WT and KO tumors stained for FoxP3 (n = 5-6/group); scale bars, 50 µm. Right: Quantification of FoxP3+ Tregs per field. (G) Schematic for administration of PT2399 + αCTLA4 in a syngeneic flank KPC model. i.p., intraperitoneal; o.g., oral gavage; b.i.d., bid in die (twice a day). (H) Tumor growth curve from (A) (n = 10/group). Veh, vehicle; P , by Mann–Whitney U test. (I) Schematic for administration of PT2399 + αCTLA4/αPD1 in a syngeneic orthotopic KPC model. (J) Kaplan-Meier curves showing percentage survival for (C) (n = 10/group); P , by log-rank test. All error bars represent mean ± SEM; P , by Student’s t test unless otherwise noted. See also Supplementary Figure 6 and Supplementary Table 2.

Article Snippet: After 2 weeks of recovery, murine αCTLA4 (clone 9D9, Merck) and murine αPD1 (muDX400, Merck) or isotype control were administered IP every 4 days at 20 μg/mouse, 200 μg/mouse, and 220 μg/mouse, respectively for 2 weeks.

Techniques: Isolation, Marker, Expressing, Staining, MANN-WHITNEY