Journal: Scientific Reports

Article Title: Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses

doi: 10.1038/s41598-023-44316-y

Figure Lengend Snippet: Influence of viral E3/49K and pUL11 on human PBMC proliferation. ( A ) Assessment of PBMC proliferation by tritiated thymidine incorporation. Human PBMC (1 × 10 5 /well) were stimulated with increasing numbers of irradiated L23-control, L23-E3/49K or L23-UL11 cells. The cells were cultured for 5 days, pulsed with tritiated thymidine and harvested after 16 h. One representative experiment out of a series of eight and four is shown comparing the stimulatory capacity of L23-E3/49K and L23-UL11 cells, respectively, with L23-control cells. Results are expressed as mean cpm of triplicate cultures. Statistical analysis was performed by applying the paired Student’s t -test. ( B ) Proliferative responses of PBMC from eight different human blood donors to stimulation with L23-control and L23-E3/49K cells. 1 × 10 5 human PBMC were stimulated with 25 × 10 3 irradiated L23 cells. Proliferation was determined after 6 days. Results are expressed as percent proliferation relative to L23-control cells (100%). Statistical significance was determined by ANOVA. ( C ) Studies using soluble E3/49K. Human PBMC (1 × 10 5 /well) were stimulated with irradiated L23-control cells (25 × 10 3 /well). Culture was performed without soluble E3/49K (unstimulated) or in the presence of purified soluble E3/49K (sec49K-His; n = 2) or supernatant from L23-E3/49K cells (sec49K; n = 4). The cells were cultured for 5 days, pulsed with tritiated thymidine and harvested after 16 h. Results are expressed as percent proliferation relative to cultures without E3/49K (100%). ( D ) Assessment of T cell proliferation by CFSE dilution. Human PBMC (1 × 10 5 /well) were stained with CFSE and stimulated with irradiated L23-control or L23-E3/49K cells (25 × 10 3 /well). The cells were cultured for 7 days, stained with anti-CD4-mAb and analyzed by flow cytometry. The numbers represent % positive cells in each quadrant of the representative histograms.

Article Snippet: Human CD8 + T cells were isolated by depletion of HLA-DR + CD14 + CD56 + CD4 + cells using an antibody cocktail and MACS (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and positively isolated after staining with CD8 by electronic cell sorting (Cell Sorting Core Facility, Hannover Medical School).

Techniques: Irradiation, Control, Cell Culture, Purification, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses

doi: 10.1038/s41598-023-44316-y

Figure Lengend Snippet: Phosphorylation of p56lck(p505) and Zap70(p319) in PBMC stimulated with L23-transfectants. ( A ) PBMC that had been stimulated for 48 h with L23-control, L23-UL11 or L23-E3/49K cells were intracellularly stained with p56lck(p505) or Zap70(p319) in combination with anti-CD4 and analyzed by flow cytometry. Dot plots show reactivity of p56lck(p505) and ZAP70(p319) in gated CD4 + cells and were obtained in a representative experiment. The numbers represent percent positive cells. ( B ) The bar graph summarizes mean percentages of CD4 + p56lck(p505) + and CD4 + Zap70(p319) + cells from 2 (2 h), 2 (24 h) or 6 (48 h) independent experiments. Statistical significance for the p56lck(p505) data at 48 h was determined by ANOVA.

Article Snippet: Human CD8 + T cells were isolated by depletion of HLA-DR + CD14 + CD56 + CD4 + cells using an antibody cocktail and MACS (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and positively isolated after staining with CD8 by electronic cell sorting (Cell Sorting Core Facility, Hannover Medical School).

Techniques: Phospho-proteomics, Control, Staining, Flow Cytometry

Journal: Scientific Reports

Article Title: Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses

doi: 10.1038/s41598-023-44316-y

Figure Lengend Snippet: Phenotypic characterization of human PBMC after chronic stimulation with L23 cells. Human PBMC were analyzed by flow cytometry on day 0 (unstimulated) and on day 28 after repetitive re-stimulation (every week plus fresh medium) using irradiated L23-control, L23-UL11, and L23-E3/49K as stimulator cells. ( A ) Activation induced alterations of CD45RA and CD45R0 isoforms. The cells were stained by CD45RA in combination with CD45R0 mAb. Forward-/side-scatter gating was performed to exclude porcine L23 cells from the analysis. Data presented as dot plots of CD45RA/CD45R0 co-expression were obtained in a representative experiment. The numbers represent the percentage of CD45RA + CD45R0 - (lower right quadrant) and CD45R0 + CD45RA - (upper left quadrant) cells. The bar graph summarizes percentages from 5 independent experiments. Statistical significance was determined by ANOVA. ( B ) Activation induced upregulation of CD25. The cells were stained by CD4 in combination with CD25 mAb. Forward-/side-scatter gating was performed to exclude porcine L23 cells from the analysis. The dot plots show CD4/CD25 co-expression patterns obtained in a representative experiment. CD4 + T cells expressing intermediate levels of CD25 are indicated by dashed ellipses, CD4 + CD25 high cells by solid ellipses. The numbers represent the percentage of cells in the ellipses. The bar graph summarizes percentages from 5 independent experiments. Statistical significance was determined by ANOVA. ( C ) Characterization of CD4 + CD25 high cells. Chronically L23-E3/49K-stimulated cells were stained by CD4 and CD25 in combination with either FoxP3, CD127 or CD28 mAbs. Dashed lines represent isotype controls (FoxP3) or secondary antibodies alone. The histograms were obtained in a single experiment and were confirmed in a second experiment using cells from another blood donor.

Article Snippet: Human CD8 + T cells were isolated by depletion of HLA-DR + CD14 + CD56 + CD4 + cells using an antibody cocktail and MACS (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and positively isolated after staining with CD8 by electronic cell sorting (Cell Sorting Core Facility, Hannover Medical School).

Techniques: Flow Cytometry, Irradiation, Control, Activation Assay, Staining, Expressing

Journal: Scientific Reports

Article Title: Expression of viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses

doi: 10.1038/s41598-023-44316-y

Figure Lengend Snippet: Susceptibility of porcine target cells to cell death induced by human cytotoxic effector cells. A total of 1 × 10 5 porcine target cells (L23-control and L23-E3/49K) were co-cultured with 1 × 10 5 human cytotoxic effector cells. After 2 h the cells were washed and stained with a combination of monoclonal antibody W6/32 (anti-human HLA class-I) and Annexin V. Analysis of Annexin V staining was performed in “gated” porcine L23 cells as defined by absence of W6/32 staining. ( A ) Representative histograms of Annexin V staining in L23-control and L23-E3/49K cells alone (dashed lines) or after co-cultures with human anti-L23 sensitized T cell lines (CD8 + or CD4 + ), CD56 + NK and NKT cells or porcine CTL generated in vitro by allogeneic mixed lymphocyte reaction (pTeff) (solid lines). The dotted line discriminates viable (Annexin V low ) and apoptotic (Annexin V high ) cells. Numbers represent the proportion of apoptotic/dead L23 cells. ( B ) The bar graph summarizes percentages of apoptotic cells from 5 different experiments using cells from 2 blood donors (CD8 + ), 12 experiments with cells from 2 donors (CD4 + ), 4 experiments using cells from 3 different blood donors (CD56 + ), and 5 experiments with cells from 2 donors (pTeff). Statistical analysis was performed by applying the paired Student’s t -test.

Article Snippet: Human CD8 + T cells were isolated by depletion of HLA-DR + CD14 + CD56 + CD4 + cells using an antibody cocktail and MACS (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and positively isolated after staining with CD8 by electronic cell sorting (Cell Sorting Core Facility, Hannover Medical School).

Techniques: Control, Cell Culture, Staining, Generated, In Vitro