Journal: Nanomaterials

Article Title: Natural Killer Cell-Derived Extracellular Vesicles Exhibit Cytotoxicity Against Bulk Tumor Cells and Cancer Stem Cells in Triple-Negative Breast Cancer

doi: 10.3390/nano16090525

Figure Lengend Snippet: NK-EV treatment disrupts TNBC cancer stem cell-associated phenotypes. The tumorsphere formation assays were conducted on ( A ) SUM149PT (2000 cells) following 7 days of treatment and ( B ) E-cadherin + MDA-MB-231 (1500 cells) following 4 days of treatment with a single dosage of NK-EVs (orange) or 10 nm paclitaxel (PTX; blue). Results are presented as (i) RFU and (ii) EC 50 curve analysis for NK-EV treatment with 95% confidence interval and prediction bands (red dashed line represents 50% response). ( C ) RT-qPCR analysis of CTGF , CYR61 , and CD44 gene expression in MDA-MB-231 cells after 72 h of NK-EV treatment. Treatment groups include untreated control (gray), 1.0 × 10 9 NK-EVs/mL (orange), 10 nM paclitaxel (blue), a combination of 1.0 × 10 9 EVs/mL NK-EVs and paclitaxel (green). Data are shown as the mean ± SEM from three independent experiments, each with technical duplicates. ns: non-significant, *: p < 0.05, **: p < 0.01.

Article Snippet: Fluorescence-activated cell sorting (FACS) into the CD44 high /CD24 low and non-CD44 high /CD24 low cells was conducted using the SH800 Cell Sorter (Sony Biotechnology, San Jose, CA, USA).

Techniques: Quantitative RT-PCR, Gene Expression, Control

Journal: Nanomaterials

Article Title: Natural Killer Cell-Derived Extracellular Vesicles Exhibit Cytotoxicity Against Bulk Tumor Cells and Cancer Stem Cells in Triple-Negative Breast Cancer

doi: 10.3390/nano16090525

Figure Lengend Snippet: NK-EV treatment rapidly induces cytotoxicity in MDA-MB-231 TNBC cancer stem cells (CSC). RFP + MDA-MB-231 cells were FACS sorted as CD44 high /CD24 low CSCs and non-CSCs. Cells were incubated with green Annexin-V in the Sartorius S3 for live-cell imaging over a 24 h period. Treatment groups included a low (1 × 10 8 EVs/mL), medium (1 × 10 9 EVs/mL), or high (1 × 10 10 EVs/mL) dose of NK-EVs or 293F-EVs (control), along with an untreated control. Green Annexin-V fluorescence intensity was quantified for ( A ) non-CSCs and ( B ) CD44 high /CD24 low CSCs. Representative bright field and green fluorescence images illustrate Annexin-V expression of ( C ) non-CSCs and ( D ) CSCs following 20 h of treatment. Data are shown as the mean ± SEM from four independent experiments, each with technical triplicates. ns: non-significant, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Article Snippet: Fluorescence-activated cell sorting (FACS) into the CD44 high /CD24 low and non-CD44 high /CD24 low cells was conducted using the SH800 Cell Sorter (Sony Biotechnology, San Jose, CA, USA).

Techniques: Incubation, Live Cell Imaging, Control, Fluorescence, Expressing

Journal: PLOS Pathogens

Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1371/journal.ppat.1014150

Figure Lengend Snippet: (A) Experimental setup for (B-J) : (B, C) 2x10 7 purified T cells obtained from adult eGFP transgenic mice (eGFP) were transferred (Tx) into neonatal mice and analysed after 2 days. (D-G) 10 7 eGFP + T cells were stained with cell proliferation dye eF450 and adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received cells but were not infected. Animals were analysed at 7 days after cell transfer. (H,I) Various numbers of eGFP + T cells were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received no T cells. (J) 8x10 6 purified eGFP + CD44 + T cells obtained from adult non-infected mice were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV or buffer control. Animals were analysed at 8 days post infection (dpi). (B) Representative flow cytometry plots of spleen T cells. (C) Absolute numbers of T cells calculated to animal body weight 2 days after adoptive transfer. Cell numbers of non-treated PND 28 animals are depicted in for comparison. (D) Representative flow cytometry plots of eGFP + T cells isolated from lungs, indicating eF450 signal loss with each proliferation cycle. (E) Quantitative analysis of proliferated eGFP + T cells isolated from lungs. (F) Representative flow cytometry plots of eGFP + CD8 + T cells isolated from lungs simultaneously stained with M38-, M45-, and m139-pMHC tetramers. (G) Quantitative analysis of MCMV-specific tetramer + eGFP + CD8 + T cells isolated from lungs. (H) Immunohistology of lungs indicating localisation of transferred eGFP + T cells in nodular inflammatory foci (NIF). (I) Lung viral loads after adoptive transfer of various numbers of naïve T cells. RLU, relative light units. (J) Lung viral loads after adoptive transfer of CD44 + T cells from adult non-infected mice. (K) Experimental setup for (L-O) : MCMV-2DR contains no additional inserted sequences besides the fluorophore and luciferase reporters, whereas MCMV-4DR encodes for sequences of chicken ovalbumin. Neonatal wildtype or adult Rag2-/-Il2rg-/- mice were infected (104 or 2x105 PFU, respectively) with either MCMV-2DR or MCMV-4DR and received adoptive transfers of 5x10 6 OT-I eCFP and 5x10 6 OT-II eGFP proliferation dye-labelled cells; control animals did not receive T cells. Animals were analysed at 8 dpi. (L) Representative flow cytometry plots of T cells isolated from lung draining lymph nodes indicating eF450 signal loss with each proliferation cycle. (M) Quantitative analysis of proliferated T cells. (N) Immunohistology of MCMV-4DR -infected neonatal lungs, indicating localisation of transferred OT-I eCFP and OT-II eGFP T cells next to MCMV-infected cells in NIF. (O) Lung viral loads after infection with MCMV-4DR (upper panel) or MCMV-2DR (lower panel) and adoptive transfer of OT-I and OT-II cells. Data were acquired from two or more experiments (B and C, n = 5-6; D-G, n = 5; H and I, n = 5-13, J, n = 5-6 L-O, n = 7-11 animals per group). Scale bars, 100 µm. Statistical differences between groups were calculated with Mann-Whitney U (C, E, G, L, and N) or Kruskal-Wallis (I) tests. The p values are provided above each graph. GLuc, Gaussia luciferase; hMIEP, human major immediate early promotor; OVA, chicken ovalbumin; TfR, transferrin receptor sequence.

Article Snippet: Naïve CD44 - T cells were isolated by magnetically depleting CD44 + cells using CD44 Microbeads (Miltenyi Biotec).

Techniques: Purification, Transgenic Assay, Staining, Infection, Control, Flow Cytometry, Adoptive Transfer Assay, Comparison, Isolation, Luciferase, MANN-WHITNEY, Sequencing

Journal: PLOS Pathogens

Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1371/journal.ppat.1014150

Figure Lengend Snippet: (A) Experimental setup for (B-J) : (B, C) 2x10 7 purified T cells obtained from adult eGFP transgenic mice (eGFP) were transferred (Tx) into neonatal mice and analysed after 2 days. (D-G) 10 7 eGFP + T cells were stained with cell proliferation dye eF450 and adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received cells but were not infected. Animals were analysed at 7 days after cell transfer. (H,I) Various numbers of eGFP + T cells were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received no T cells. (J) 8x10 6 purified eGFP + CD44 + T cells obtained from adult non-infected mice were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV or buffer control. Animals were analysed at 8 days post infection (dpi). (B) Representative flow cytometry plots of spleen T cells. (C) Absolute numbers of T cells calculated to animal body weight 2 days after adoptive transfer. Cell numbers of non-treated PND 28 animals are depicted in for comparison. (D) Representative flow cytometry plots of eGFP + T cells isolated from lungs, indicating eF450 signal loss with each proliferation cycle. (E) Quantitative analysis of proliferated eGFP + T cells isolated from lungs. (F) Representative flow cytometry plots of eGFP + CD8 + T cells isolated from lungs simultaneously stained with M38-, M45-, and m139-pMHC tetramers. (G) Quantitative analysis of MCMV-specific tetramer + eGFP + CD8 + T cells isolated from lungs. (H) Immunohistology of lungs indicating localisation of transferred eGFP + T cells in nodular inflammatory foci (NIF). (I) Lung viral loads after adoptive transfer of various numbers of naïve T cells. RLU, relative light units. (J) Lung viral loads after adoptive transfer of CD44 + T cells from adult non-infected mice. (K) Experimental setup for (L-O) : MCMV-2DR contains no additional inserted sequences besides the fluorophore and luciferase reporters, whereas MCMV-4DR encodes for sequences of chicken ovalbumin. Neonatal wildtype or adult Rag2-/-Il2rg-/- mice were infected (104 or 2x105 PFU, respectively) with either MCMV-2DR or MCMV-4DR and received adoptive transfers of 5x10 6 OT-I eCFP and 5x10 6 OT-II eGFP proliferation dye-labelled cells; control animals did not receive T cells. Animals were analysed at 8 dpi. (L) Representative flow cytometry plots of T cells isolated from lung draining lymph nodes indicating eF450 signal loss with each proliferation cycle. (M) Quantitative analysis of proliferated T cells. (N) Immunohistology of MCMV-4DR -infected neonatal lungs, indicating localisation of transferred OT-I eCFP and OT-II eGFP T cells next to MCMV-infected cells in NIF. (O) Lung viral loads after infection with MCMV-4DR (upper panel) or MCMV-2DR (lower panel) and adoptive transfer of OT-I and OT-II cells. Data were acquired from two or more experiments (B and C, n = 5-6; D-G, n = 5; H and I, n = 5-13, J, n = 5-6 L-O, n = 7-11 animals per group). Scale bars, 100 µm. Statistical differences between groups were calculated with Mann-Whitney U (C, E, G, L, and N) or Kruskal-Wallis (I) tests. The p values are provided above each graph. GLuc, Gaussia luciferase; hMIEP, human major immediate early promotor; OVA, chicken ovalbumin; TfR, transferrin receptor sequence.

Article Snippet: CD44 + T cells were isolated from lungs of MCMV-infected Wt adult mice 7 dpi by positive selection of CD45 + cells with CD45 Microbeads (Miltenyi Biotec) followed by FACS cell sorting for CD3 + CD44 + T cells.

Techniques: Purification, Transgenic Assay, Staining, Infection, Control, Flow Cytometry, Adoptive Transfer Assay, Comparison, Isolation, Luciferase, MANN-WHITNEY, Sequencing

Journal: PLOS Pathogens

Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1371/journal.ppat.1014150

Figure Lengend Snippet: (A) Experimental setup for Figs 3 and 4: Neonatal (n = 3) and adult (n = 3) mice were infected with 10 4 or 2x10 5 PFU MCMV, respectively. 10 7 purified CD3 + CD44 - naïve T cells obtained from adult eGFP mice were transferred into neonatal mice. Control animals (n = 3 neonates and n = 3 adults) were not infected with MCMV. After 7 days, adoptively transferred eGFP + lung T cells in neonates and endogenous lung T cells from adults were isolated, and pooled for single-cell transcriptome profiling (scRNAseq) combined with TCR sequencing. (B) Unsupervised RNAseq analysis and UMAP dimensionality reduction of all cells, colours indicate infection status. (C) Cell expression of genes as indicated. (D) Relative distribution of T cells with identical TCRs indicating clonal enrichment, the filled area represents clonal cells. (E) Principal component analysis for the four different groups. (F) Subanalysis and UMAP dimensionality reduction of Cd4 + Cd8a - T cells. (G) Classification of CD4 T cells into subpopulations by expression of characteristic genes as illustrated. (H) Frequency of the four groups assigned to the different CD4 T cell subpopulations. (I and J) (I) Differential gene expression and (J) cytotoxicity module score of Th1 cells obtained from MCMV-infected adults and neonates. (K) Relative distribution of the clonal (filled section of bars) and non-clonal (open bars) CD4 T cell subpopulations of each group, with numbers indicating the absolute cell counts. (L) Relative distribution of clonally expanded CD4 T cells into subpopulations. Data acquired by one experiment with n = 3 animals per group. Statistical difference in (J) was calculated with Mann-Whitney U test, and the p-value is provided above the graph.

Article Snippet: CD44 + T cells were isolated from lungs of MCMV-infected Wt adult mice 7 dpi by positive selection of CD45 + cells with CD45 Microbeads (Miltenyi Biotec) followed by FACS cell sorting for CD3 + CD44 + T cells.

Techniques: Infection, Purification, Control, Isolation, Single Cell, Sequencing, RNA sequencing, Expressing, Gene Expression, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1371/journal.ppat.1014150

Figure Lengend Snippet: (A) Experimental setup for (B-J) : CD3 + CD44 + effector T cells were purified from adult lungs 7 dpi, and 8x10 6 cells per animal were transferred into MCMV-infected neonatal mice, which were then analysed 8 dpi. Controls did not receive T cells. (B and C) (B) Representative flow cytometry plots and (C) quantitative analysis of CD45 + cells obtained from neonatal lungs at 8 dpi. (D and E) (D) Representative flow cytometry histograms and (E) quantitative analysis of CD44 expression in isolated lung T cells. (F) Immunohistology of lungs in general view (left panels) and high resolution (right panels) illustrating inflammation and presence of NIFs with MCMV-infected cells. Dotted squares indicate the zoomed areas shown in the right panels. (G-H) Quantitative analysis of immunohistology with (G) number of NIFs per lung slice, (H) NIF area, and (I) number of MCMV-infected cells per NIF. (J) Quantitative analysis of lung viral loads as indicated. Data acquired from four independent experiments. Statistical differences were calculated in C , E , and G-J with Mann-Whitney U test, and the p-values are provided above the graphs.

Article Snippet: CD44 + T cells were isolated from lungs of MCMV-infected Wt adult mice 7 dpi by positive selection of CD45 + cells with CD45 Microbeads (Miltenyi Biotec) followed by FACS cell sorting for CD3 + CD44 + T cells.

Techniques: Purification, Infection, Flow Cytometry, Expressing, Isolation, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1371/journal.ppat.1014150

Figure Lengend Snippet: (A) Experimental setup for (B-J) : (B, C) 2x10 7 purified T cells obtained from adult eGFP transgenic mice (eGFP) were transferred (Tx) into neonatal mice and analysed after 2 days. (D-G) 10 7 eGFP + T cells were stained with cell proliferation dye eF450 and adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received cells but were not infected. Animals were analysed at 7 days after cell transfer. (H,I) Various numbers of eGFP + T cells were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV. Control mice received no T cells. (J) 8x10 6 purified eGFP + CD44 + T cells obtained from adult non-infected mice were adoptively transferred into neonatal mice, which were simultaneously infected with MCMV or buffer control. Animals were analysed at 8 days post infection (dpi). (B) Representative flow cytometry plots of spleen T cells. (C) Absolute numbers of T cells calculated to animal body weight 2 days after adoptive transfer. Cell numbers of non-treated PND 28 animals are depicted in for comparison. (D) Representative flow cytometry plots of eGFP + T cells isolated from lungs, indicating eF450 signal loss with each proliferation cycle. (E) Quantitative analysis of proliferated eGFP + T cells isolated from lungs. (F) Representative flow cytometry plots of eGFP + CD8 + T cells isolated from lungs simultaneously stained with M38-, M45-, and m139-pMHC tetramers. (G) Quantitative analysis of MCMV-specific tetramer + eGFP + CD8 + T cells isolated from lungs. (H) Immunohistology of lungs indicating localisation of transferred eGFP + T cells in nodular inflammatory foci (NIF). (I) Lung viral loads after adoptive transfer of various numbers of naïve T cells. RLU, relative light units. (J) Lung viral loads after adoptive transfer of CD44 + T cells from adult non-infected mice. (K) Experimental setup for (L-O) : MCMV-2DR contains no additional inserted sequences besides the fluorophore and luciferase reporters, whereas MCMV-4DR encodes for sequences of chicken ovalbumin. Neonatal wildtype or adult Rag2-/-Il2rg-/- mice were infected (104 or 2x105 PFU, respectively) with either MCMV-2DR or MCMV-4DR and received adoptive transfers of 5x10 6 OT-I eCFP and 5x10 6 OT-II eGFP proliferation dye-labelled cells; control animals did not receive T cells. Animals were analysed at 8 dpi. (L) Representative flow cytometry plots of T cells isolated from lung draining lymph nodes indicating eF450 signal loss with each proliferation cycle. (M) Quantitative analysis of proliferated T cells. (N) Immunohistology of MCMV-4DR -infected neonatal lungs, indicating localisation of transferred OT-I eCFP and OT-II eGFP T cells next to MCMV-infected cells in NIF. (O) Lung viral loads after infection with MCMV-4DR (upper panel) or MCMV-2DR (lower panel) and adoptive transfer of OT-I and OT-II cells. Data were acquired from two or more experiments (B and C, n = 5-6; D-G, n = 5; H and I, n = 5-13, J, n = 5-6 L-O, n = 7-11 animals per group). Scale bars, 100 µm. Statistical differences between groups were calculated with Mann-Whitney U (C, E, G, L, and N) or Kruskal-Wallis (I) tests. The p values are provided above each graph. GLuc, Gaussia luciferase; hMIEP, human major immediate early promotor; OVA, chicken ovalbumin; TfR, transferrin receptor sequence.

Article Snippet: Naïve CD44 - T cells were isolated by magnetically depleting CD44 + cells using CD44 Microbeads (Miltenyi Biotec).

Techniques: Purification, Transgenic Assay, Staining, Infection, Control, Flow Cytometry, Adoptive Transfer Assay, Comparison, Isolation, Luciferase, MANN-WHITNEY, Sequencing

Journal: PLOS Pathogens

Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1371/journal.ppat.1014150

Figure Lengend Snippet: (A) Experimental setup for Figs 3 and 4: Neonatal (n = 3) and adult (n = 3) mice were infected with 10 4 or 2x10 5 PFU MCMV, respectively. 10 7 purified CD3 + CD44 - naïve T cells obtained from adult eGFP mice were transferred into neonatal mice. Control animals (n = 3 neonates and n = 3 adults) were not infected with MCMV. After 7 days, adoptively transferred eGFP + lung T cells in neonates and endogenous lung T cells from adults were isolated, and pooled for single-cell transcriptome profiling (scRNAseq) combined with TCR sequencing. (B) Unsupervised RNAseq analysis and UMAP dimensionality reduction of all cells, colours indicate infection status. (C) Cell expression of genes as indicated. (D) Relative distribution of T cells with identical TCRs indicating clonal enrichment, the filled area represents clonal cells. (E) Principal component analysis for the four different groups. (F) Subanalysis and UMAP dimensionality reduction of Cd4 + Cd8a - T cells. (G) Classification of CD4 T cells into subpopulations by expression of characteristic genes as illustrated. (H) Frequency of the four groups assigned to the different CD4 T cell subpopulations. (I and J) (I) Differential gene expression and (J) cytotoxicity module score of Th1 cells obtained from MCMV-infected adults and neonates. (K) Relative distribution of the clonal (filled section of bars) and non-clonal (open bars) CD4 T cell subpopulations of each group, with numbers indicating the absolute cell counts. (L) Relative distribution of clonally expanded CD4 T cells into subpopulations. Data acquired by one experiment with n = 3 animals per group. Statistical difference in (J) was calculated with Mann-Whitney U test, and the p-value is provided above the graph.

Article Snippet: Naïve CD44 - T cells were isolated by magnetically depleting CD44 + cells using CD44 Microbeads (Miltenyi Biotec).

Techniques: Infection, Purification, Control, Isolation, Single Cell, Sequencing, RNA sequencing, Expressing, Gene Expression, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Limited protection against early-life lung murine cytomegalovirus infection results from deficiency of cytotoxic CD8 T cells

doi: 10.1371/journal.ppat.1014150

Figure Lengend Snippet: (A) Experimental setup for (B-J) : CD3 + CD44 + effector T cells were purified from adult lungs 7 dpi, and 8x10 6 cells per animal were transferred into MCMV-infected neonatal mice, which were then analysed 8 dpi. Controls did not receive T cells. (B and C) (B) Representative flow cytometry plots and (C) quantitative analysis of CD45 + cells obtained from neonatal lungs at 8 dpi. (D and E) (D) Representative flow cytometry histograms and (E) quantitative analysis of CD44 expression in isolated lung T cells. (F) Immunohistology of lungs in general view (left panels) and high resolution (right panels) illustrating inflammation and presence of NIFs with MCMV-infected cells. Dotted squares indicate the zoomed areas shown in the right panels. (G-H) Quantitative analysis of immunohistology with (G) number of NIFs per lung slice, (H) NIF area, and (I) number of MCMV-infected cells per NIF. (J) Quantitative analysis of lung viral loads as indicated. Data acquired from four independent experiments. Statistical differences were calculated in C , E , and G-J with Mann-Whitney U test, and the p-values are provided above the graphs.

Article Snippet: Naïve CD44 - T cells were isolated by magnetically depleting CD44 + cells using CD44 Microbeads (Miltenyi Biotec).

Techniques: Purification, Infection, Flow Cytometry, Expressing, Isolation, MANN-WHITNEY