magnetic cell-sorting kit by negative selection easysep mouse cd4+cd25+ regulatory t cell isolation kit  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: Cryosections from murine HCC tumors treated with Reo/ uv -Reo alone (1×10 7 pfu) and in combination with sorafenib (10 mg/Kg), sampled during the “ Therapy-phase ”, were immunofluorescently labelled for ( A ) CTLs (CD3 + CD8 + ) and ( B ) T H -cells (CD3 + CD4 + ). ( C ) The abundance was of CTLs and T H -cells was quantified in random fields of view (F.O.V.) obtained by confocal microscopy and the ratio of the two cell types was determined ( D ). (Scale bar = 50 µm; * p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001; n = 5 mice per condition).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: ( A ) Cytokine array analysis of pooled protein extracts generated from murine HCC tumors treated with Reo/ uv -Reo alone (1×10 7 pfu) or in combination with sorafenib (10 mg/Kg), data are shown for IFNG and TNFA (samples pooled from 5 mice per treatment group). ( B ) Quantification of IFNG and TNFA in supernatants from unstimulated naive human CD4 + T-cells or following three days of activation in the presence of antibodies against CD3/CD28 and the cytokines IL2 (50 U/mL) and IL12 (10 ng/mL). ( C ) Cytokine array data generated as described in ‘A’ showing data for CCL5. ( D ) Chemotaxis assay comparing migration of naive or T H 1-activated human CD4 + T-cells towards recombinant human CCL5 (100 ng/mL) in the presence of the CCR5 inhibitor ‘Maraviroc’ (1 µM) or vehicle (DMSO). ( E ) Flow cytometric data showing expression of CCR5 on the surface of human CD4 + T H 1-cells ( left ) and CD45 + CD3 + CD4 + T H -cells in murine HCC tumors treated with uv -Reo/sorafenib therapy ( right ) (Sora. = Sorafenib, CCR5i = Maraviroc; * p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Generated, Activation Assay, Chemotaxis Assay, Migration, Recombinant, Expressing

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: ( A ) Co-culture assay between human HCC cell line ‘HLE’ in the presence of either naive or T H 1-activated human CD4 + T-cells. HLE target cell viability determined by flow cytometry following incubation overnight. ( B ) Co-culture assay between HLEs and human CD4 + T-cells as described in ‘A’, but with one additional group in which T-cells and HLE target cells are separated by a tissue culture insert with 0.4 µm pore size. ( C ) Co-culture assay setup as described in (A), but in the presence of increasing concentrations of anti-human TNFA neutralizing antibody or isotype-matched control. ( D ) Human HLE HCC cells were treated with increasing concentrations of recombinant human TNFA overnight and tumor cell viability was then determined by flow cytometry. ( E ) Human HLE HCC cells were pre-treated with rhIFNG (100 U/mL) or sorafenib (7 µM) followed by increasing concentrations of TNFA overnight then tumor cell viability determined by flow cytometry. ( F ) Murine HCC cells ‘1MEA’ were treated overnight with increasing concentrations of rmTNFA alone or following pre-treatment with rmIFNG (100 U/mL) or sorafenib (7 µM) then tumor cell viability was determined by flow cytometry (* p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001; n = 4 – 8 per condition).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Co-culture Assay, Flow Cytometry, Incubation, Pore Size, Control, Recombinant

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: Human and murine HCC cells were treated with increasing concentration of rIFNG overnight and tumor cell viability determined by flow cytometry. ( B ) Co-culture assay between human HLE HCC cells pre-treated with or without rhIFNG (100 U/mL) and T H 1-activated CD4 + T-cells. Target HCC cell viability determined by flow cytometry following overnight incubation ( C ) Co-culture assay between human HCC cells pre-treated with or without sorafenib (7 µM) and T H 1-activated CD4 + T-cells. Target HCC cell viability was determined by flow cytometry following overnight incubation. ( D ) Human and murine HCC cells pre-treated with sorafenib (7 µM) prior to exposure to increasing concentrations of rIFNG overnight. HCC viability was determined by flow cytometry. Co-culture assays were performed overnight between human HLE HCC cells and either naive or T H 1-activated CD4 + T-cells in the presence of increasing concentrations of neutralizing antibodies against either TRAIL ( E ) or LTA ( F ) and target HCC cell viability was determined by flow cytometry (* p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001; n = 4 – 8 per condition).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Concentration Assay, Flow Cytometry, Co-culture Assay, Incubation, Co-Culture Assay

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: Human ( A ) and murine ( B ) HCC cells were treated with Reo/ uv -Reo (2 pfu/cell) alone or in combination with sorafenib (7 µM) overnight and IFNB was quantified in supernatants by ELISA (n = 3). ( C ) Total tumor lysates were generated from murine HCC tumors, grown in vivo, treated with either live- or uv -reo alone (1×10 7 pfu) and in combination with sorafenib (10 mg/kg) or vehicle then IFNB was quantified by ELISA (n = 5). ( D ) Co-culture assay between human HLE HCC cells and either naive or T H 1-activated CD4 + T-cells with or without rhIFNB (500 ng/mL). Target HCC cell viability was determined by flow cytometry following overnight incubation. ( E ) Co-culture assay using human ( left ) and murine ( right ) cells setup as described in (D), but with one additional group in which T-cells and target HCC cells were separated by a tissue culture insert with a 0.4 µm pore size. ( F ) Co-culture assay between human HLE HCC cells and either naive or T H 1-activated CD4 + T-cells with or without rhIFNB (500 ng/mL) added concurrently with T-cell seeding or added to HCC cells prior T-cell seeding (Sora. = Sorafenib; * p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001; n = 4 – 8 per condition).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Enzyme-linked Immunosorbent Assay, Generated, In Vivo, Co-culture Assay, Flow Cytometry, Incubation, Pore Size

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: ( A ) T H 1-activated CD4 + T-cells were treated with increasing concentrations of rhIFNB overnight and the concentration of soluble TRAIL was measured in supernatants by ELISA (n = 9). ( B - C ) T H 1-activated CD4 + T-cells were treated overnight as described in ‘A’ and cell surface expression of TRAIL was quantified by flow cytometry (n = 3). ( D ) Co-culture assay between human HLE HCC cells and either naive of T H 1-activated CD4 + T-cells with or without rhIFNB in the presence of increasing concentrations of anti-human TRAIL neutralizing antibody. Following incubation overnight, target HCC cell killing was determined by flow cytometry (n = 8). ( E ) Human HLE HCC cells were treated with increasing concentrations of rhIFNB overnight and cell surface expression of FasL was quantified by flow cytometry (n = 3). ( F ) Expression of FasL receptor – Fas – on human HLE HCC cells was confirmed by flow cytometry (n = 3). ( G ) Co-culture assay between human HLE HCC cells and either naive or T H 1-activated CD4 + T-cells, with or without IFNB, in the presence of increasing concentrations of anti-human Fas neutralizing antibody or isotype matched control (csTRAIL = cell surface TRAIL; * p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001; n = 8).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Co-culture Assay, Incubation, Control

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: ( A ) Flow cytometric analysis of cell surface MHCII proteins on human (HLE) and murine (1MEA) HCC cell lines. ( B ) Co-culture assay between human HLE HCC cells and either naive or T H 1-activated CD4 + T-cells in the presence of rhIFNB with or without anti-MHCII neutralizing antibodies or isotype matched control. ( C ) Co-culture assay between human HLE HCC cells and either naive or T H 1-activated CD4 + T-cells alone, with rhIFNB and in the presence of increasing concentrations of degranulation inhibitor ‘EGTA’. Flow cytometric analysis of cell surface degranulation marker CD107a and intracellular expression of GZMB using human ( D ) and murine ( E ) naive or T H 1-activated CD4 + T-cells treated overnight with rIFNB (mouse 50 ng/mL; human 500 ng/mL). ( F ) The presence of GZMB was quantified in supernatants from human naive or T H 1-activated CD4 + T-cells treated with or without rhIFNB overnight. ( G ) Co-culture assay between human HLE HCC cells and either naive or T H 1-activated CD4 + T-cells with or without rhIFNB (500 ng/mL) overnight in the presence of increasing concentrations of a GZMB inhibitor, pan-caspase inhibitor or vehicle. (GZMBi = GZMB inhibitor; * p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001; n = 3 – 8 per condition).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Co-culture Assay, Control, Marker, Expressing

Journal: bioRxiv

Article Title: TKI-conditioned immunotherapy using uv -inactivated reovirus promotes survival in hepatocellular carcinoma, mediated by cytotoxic CD4 + T-cells

doi: 10.1101/2024.02.23.581738

Figure Lengend Snippet: The proportion of perforin-expressing human ( top ) and murine ( bottom ) CD4 + T-cells was quantified by intracellular flow cytometry using either naïve, T H 1-activated, or T H 1-activated T-cells in the presence of rIFNB (n = 3). ( B ) Co-culture assay between human HLE HCC cells and either naive or activated T-cells in the presence of absence of rhIFNB (500 ng/mL). (C) Co-culture assay between human HLE HCC cells and naive or activated CD8 + T-cells in the presence of anti-human TNFA neutralizing antibody (10 µg/mL) or isotype matched control. ( D ) Co-culture assay between human HLE HCC cells and either naive or activated CD8 + T-cells with or without rhIFNB (500 ng/mL) in the presence of the degranulation inhibitor ‘EGTA’ (2 mM). The degree of target cell killing was determined in all co-culture assay by flow cytometry following incubation overnight. ( D ) Comparison of CD3 + CD4 + and CD3 + CD8 + T-cell ratio in cryosections taken from murine 1MEA HCC tumors grown in syngeneic hosts and treated for two weeks with either Reo/ uv -Reo alone (1×10 7 pfu) or in combination with sorafenib (7 µM) or vehicle (Act. = activated, Sora. = sorafenib; * p<0.05, ≠ p<0.01, + <0.001, ^ <0.0001; n = 8 per condition).

Article Snippet: Murine CD4 + T-cells were isolated from BALB/c mice using a CD4 negative magnetic selection kit (Miltenyi Biotechnology, Cologne, Germany) on single cell suspensions generated from mouse spleens and lymph nodes.

Techniques: Expressing, Flow Cytometry, Co-culture Assay, Control, Incubation, Comparison