Journal: bioRxiv

Article Title: A novel antagonist of the CCL5/CCR5 axis suppresses the tumor growth and metastasis of triple-negative breast cancer by CCR5-YAP1 regulation

doi: 10.1101/2023.11.15.567291

Figure Lengend Snippet: (A) The cell viability was measured in the human ER + breast cancer cell line MCF-7, the human TNBC cell lines of MDA-MB-231 and BT549, and murine TNBC cells of 4T1, as well as human colon cancer cells of HCT-116 by MTS assay with maraviroc (MVC) or verteporfin (VP) treatment, respectively (n = 3). (B) The cell viability was measured in cisplatin (DDP)-resistant MDA-MB-231 cells by MTS assay with 0, 1.56, 3.13, 6.25, 12.5 and 25 μM of DDP or VP treatment, respectively (n = 3). (C-E) The quantification of apoptosis analyzed by flow cytometry in TNBC cells of MDA-MB-231 (C), BT549 (D) and 4T1 (E) treated with 25 μM MVC or indicated concentrations of VP, respectively (n = 3). (F) The tumor size and the body weight of mice were measured daily in BALB/c mice injected with 2×10 5 4T1 cells into the mammary fat pad and treated with vehicle, 8 mg/kg/day of MVC or VP, respectively. The representative tumor images were documented and the tumor weight was measured when the tumor-bearing mice were sacrificed on day 28 post injection (n = 6). (G) Kaplan-Meier analysis of the overall survival of the 4T1 tumor-bearing mice treated with vehicle, MVC or VP, respectively, as described in (F). One point denotes one mouse (n = 10). (H) Representative images and quantification of Ki67 and TUNEL staining of the tumor sections analyzed by IHC on day 28 post injection (n = 4). Scale bar: 10 μm. (I-K) The CD4 + T cells (I), CD8 + T cells (J) and NK cells (K) in spleens (SP) and tumors (T) of 4T1 tumor-bearing mice treated with vehicle, MVC or VP as described in (F) were analyzed by flow cytometry on day 14 post injection (n = 4). (L-N) The Tregs in T (L), macrophages in SP (M) and TAMs in SP and T (N) of 4T1 tumor-bearing mice treated with vehicle, MVC or VP as described in (F) were analyzed by flow cytometry on day 14 post injection (n = 4). (O- Q) Total MDSCs (O), G-MDSCs (P), and Ly6G + /Ly6C + MDSCs (Q) in bone marrow (BM), SP and T of 4T1 tumor-bearing mice treated with vehicle, MVC or VP as described in (F) were analyzed by flow cytometry on day 28 post injection (n = 3). (R) Representative images and quantification of CD4 + , CD8 + , CD49b + and Foxp3 + cells for the tumor sections analyzed by IHC on day 14, as well as Ly6G + cells on day 28 post injection (n = 3). Scale bar: 10 μm. (S-T) Serum anti-tumor cytokines (S) and pro-tumor cytokines (T) of 4T1 tumor-bearing mice treated with vehicle or VP as described in (F) were analyzed by ELISA on day 14 post injection (n = 4). (U) The tumor size was measured daily in immune competent BALB/c (n = 4) or immunodeficient NOD-SCID (n = 5) mice treated with vehicle or 8 mg/kg/day of verteporfin (VP), respectively. The representative tumor images were documented and the tumor weight was measured when the tumor-bearing mice were sacrificed on day 28 post tumor inoculation. (V) The tumor size was measured daily in 4T1 tumor-bearing BALB/c mice treated with or without anti-mouse/rat asialo GM1 (anti-NK) antibody, or combined with vehicle or 8 mg/kg/day of VP, respectively. The representative tumor images were documented and the tumor weight was measured when the tumor-bearing mice were sacrificed on day 14 post tumor inoculation (n = 4). (W) The tumor size was measured daily in 4T1 tumor-bearing BALB/c mice treated with or without anti-CD8α (anti-CD8) antibody, or combined with vehicle or 8 mg/kg/day of VP, respectively. The representative tumor images were documented and the tumor weight was measured when the tumor-bearing mice were sacrificed on day 22 post tumor inoculation (n = 4). The square in the tumor image is 1cm × 1cm. Data in (A, B, and R-T) were analyzed by Unpaired two-tailed Student’s t test, data in (C-F, H-Q, and U-W) were analyzed by one-way ANOVA, and data in (G) was analyzed by Kaplan– Meier method with the log-rank test. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: not significant.

Article Snippet: To deplete the CD8 + T cells in a mouse, 50 μg of anti-mouse CD8α (clone 2.43) antibody (BioXCell, BP0061) was intraperitoneally injected to the mouse on the days of -1 st , 2 nd , 7 th , 12 th , and 19 th post tumor inoculation [ ] and the depletion efficiency of CD8 + T cells was verified by the fluorescence-activated cell sorting (FACS) analysis on day 18 post treatment.

Techniques: MTS Assay, Flow Cytometry, Injection, TUNEL Assay, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: A novel antagonist of the CCL5/CCR5 axis suppresses the tumor growth and metastasis of triple-negative breast cancer by CCR5-YAP1 regulation

doi: 10.1101/2023.11.15.567291

Figure Lengend Snippet: In TNBC cells, the verteporfin (VP) treatment without PDT disrupted the binding of CCL5 to CCR5 and reduced the expression of CCR5 (1). The decreased CCR5 further led to the reduced HIF-1α (2), and decreased the expression of YAP1(3). The decreased YAP1 could up-regulate the expression of CXCL16 to further increase the recruitment of CD4 + T, CD8 + T and NK cells, as well as the anti-tumor cytokines like IFN-γ and TNF-α in TME (4). The decreased YAP1 could also down-regulate the expression of CXCL8 to further decrease the recruitment of immunosuppressive cells like MDSCs, TAMs and Tregs, as well as the pro-tumor cytokines like IL-10 and TGF-β1 in TME (5). With the regulation of both (4) and (5), VP, as well as CCR5, regulated the TNBC tumor growth mainly by immune regulation in TME. Moreover, the decreased YAP1 also inhibited the metastasis of TNBC to lung by down-regulating the expression of EMT markers β-catenin, ZEB1 and ZEB2 (6). Thus, VP targets CCL5/CCR5 axis to inhibit the TNBC growth by immune effects and metastasis by cell-intrinsic way via YAP1.

Article Snippet: To deplete the CD8 + T cells in a mouse, 50 μg of anti-mouse CD8α (clone 2.43) antibody (BioXCell, BP0061) was intraperitoneally injected to the mouse on the days of -1 st , 2 nd , 7 th , 12 th , and 19 th post tumor inoculation [ ] and the depletion efficiency of CD8 + T cells was verified by the fluorescence-activated cell sorting (FACS) analysis on day 18 post treatment.

Techniques: Binding Assay, Expressing

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: (A) Cluster of expanded Vβ1 + T cells within multiple sclerosis (MS) lesions. Immunohistochemistry for the expanded and persisting Vβ1 clone (green) in clusters of CD8 + T cells (red) in parenchymal MS lesions. Several such clusters were observed. Only very few scattered CD8 − Vβ1 + T cells could be identified in the brain lesion. Nuclei are visualized with 4',6-diamidino-2-phenylindole (white). Scale bar 20 µm. (B) Sequences of paired T-cell receptor (TCR) α and β chains. Single sorted or laser microdissected Vβ1 + CD8 + T cells from peripheral blood or brain sections were submitted to single-cell TCR PCR to identify Vβ1 chains and all possible matching α chains. The V, n(D)n, and J regions are indicated. Amino acids encoded by n(D)n nucleotides are printed in red. The expanded Vβ1-Jβ2.3 β chain (upper line) was found to pair with 4 different α chains. Three α chains were identified from brain lesions, and 1 α chain was found in blood. The α chains expressing the Jα33 (second line) and Jα16 (fifth line) elements were identified in 7 and 6 independent cells, respectively. All α chains share the Vα7.2 element, and even though they do not share the same Jα element, they all show homologous complementarity determining region 3α regions with a conserved valine (V) followed by a positively charged arginine (R) (with only one clone showing a glutamine [Q]), a negatively charged amino acid (D/E), and a relatively large hydrophilic amino acid. One of the α chains (highlighted in red) is the mucosal-associated invariant T (MAIT) cell canonical TCR Vα7.2-CAXXDSNYQLIW-Jα33 chain with 2 N nucleotide–encoded amino acids between Vα7.2 and Jα33 (here VR). The other clones with Jα16, Jα24.1, and Jα58 chains are atypical for MAIT cells, which usually carry Jα33, Jα20, or Jα12. PBMC = peripheral blood mononuclear cell.

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Immunohistochemistry, Expressing, Clone Assay

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Immunofluorescence staining of brain-infiltrating immune cells. All nuclei are stained with 4′,6-diamidino-2-phenylindole (white). Green and red dyes were used. Double-positive cells are therefore shown in yellow. Scale bars 50 µm. (A) Double staining for CD3 (green) and CD8α (red). Most CD3 + T cells coexpress CD8α. (B) CD8 + (red) T cells outnumber CD4 + (green) T cells. (C) Low numbers of CD45RA + (green) CD8 + (red) double-positive T cells in multiple sclerosis (MS) brain tissue. (D) Many CD8 + T cells (red) coexpress CD45RO (green). (E) Naive T cells double-positive for CCR7 (red) and CD45RA (green) are mostly found within blood vessels and are barely detectable in the parenchyma of MS CNS. (F) Effector memory (CD45RO + CCR7 − ) (green arrow) and central memory (CD45RO + CCR + ) (yellow arrow) T cells in MS lesions.

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Immunofluorescence, Staining, Double Staining

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Double fluorescence immunohistochemistry identifies brain-infiltrating mucosal-associated invariant T (MAIT) cells in multiple sclerosis (MS) lesions of patient A. Nuclei are stained with 4',6-diamidino-2-phenylindole (white). Green and red dyes were used. Double-positive cells are therefore shown in yellow. Scale bars 20 µm. (A) T cells expressing the T-cell receptor Vα7.2 (red) and Vβ1 chains (green). This combination (see ) was identified by single-cell PCR. (B) Most Vα7.2 + (green) T cells belong to the CD8 + (red) T-cell subset. (C) MAIT cells expressing Vα7.2 (red) and CD161 (green) in the parenchyma of MS brain. (D) The vast majority of CD161 + (green) cells in MS CNS coexpress CD8α (red).

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Fluorescence, Immunohistochemistry, Staining, Expressing

Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: αβ T-cell receptors from multiple sclerosis brain lesions show MAIT cell–related features

doi: 10.1212/NXI.0000000000000107

Figure Lengend Snippet: Analysis of the T-cell receptor (TCR) Vα7.2 repertoire of patient A by pyrosequencing shows oligoclonal T-cell expansions in different samples. Each pie chart represents the total number of nucleotide sequences found in the Vα7.2 repertoire of a certain compartment and each sector represents one distinct T-cell clone. We compared (A) samples from CNS tissue from the biopsy taken in 1996, (B) Vα7.2 + CD161 + mucosal-associated invariant T (MAIT) cells, (C) Vα7.2 + CD161 − cells, (D) CD8 + cells, and (E) CD4 + T cells from peripheral blood taken in 2013 and 2014. For each population we list the designation of the Jα elements and their relative percentage. Clones carrying the MAIT canonical TCR α chain (CAXXDSNYQLIW) are marked in bright blue, and clones containing the noncanonical α chains characterized by Jα12 (CAXXDSSYKLIF) and Jα20 (CAVXXDYKLSF) are marked in dark blue and light blue, respectively. (F) Percentages of identical complementarity determining region 3α amino acid sequences within the TCR Vα7.2 + repertoire between the different samples defined above (A–E). The numbers are percentages indicating how often a particular sequence detected in one sample was also found in another sample. The greatest overlap was between Vα7.2 + CD161 + and CD8 + T cells from peripheral blood, but there was also significant overlap between the CNS sample and the Vα7.2 + CD161 + sample from 2013 to 2014, as highlighted by the red and yellow colors. PBMC = peripheral blood mononuclear cell.

Article Snippet: To characterize T-cell infiltrates in sections of MS brain, the following antibodies against cell surface molecules were used: mouse anti-human CD161 (1:5, 191B8, Miltenyi Biotec, Bergisch Gladbach, Germany), mouse anti-human Vα7.2 (1:5, 3C10, BioLegend, San Diego, CA), mouse anti-human CD8α (1:50, LT8, AbD Serotec, Kidlington, UK; labeled with the Cy3 MAb labeling kit, GE Healthcare, Freiburg, Germany), rabbit anti-human CCR7 (1:800, Y59, Abcam, Cambridge, UK), mouse anti-human CD45RA (1:250, HI100, BioLegend), mouse anti-human CD45RO (1:250, UCHL1, BioLegend), fluorescein isothiocyanate (FITC)-labeled mouse anti-human Vβ1 (1:100, BL37.2, Beckman Coulter, Brea, CA), rabbit anti-human CD3 (1:500, Dako, Glostrup, Denmark), and Alexa Fluor 488–conjugated mouse anti-human CD4 (1:50, RPA-T4, eBioscience, San Diego, CA).

Techniques: Clone Assay, Sequencing

Journal: PLoS Pathogens

Article Title: Activated Brain Endothelial Cells Cross-Present Malaria Antigen

doi: 10.1371/journal.ppat.1004963

Figure Lengend Snippet: (A) IFNγ-stimulated MBECs were incubated with (or without) 3 × 10 6 thawed PbA mature iRBCs for 24 h. After washing, we added 10 6 CD8 + T cells from either a naïve mouse or one infected with PbA 6 days previously. The wells were washed gently and photographed (DIC) after 20 h. Images are representative of triplicate wells. (B) An olfactory bulb smear from a mouse with ECM was fixed and stained with antibodies against von Willebrand factor (green) and CD8 (red). CD8 + T cells were present within blood vessels (arrows), on the abluminal face of endothelial cells (arrow head) and in the parenchyma (asterisks), 40× objective. Inset: a perivascular CD8 + T cell in close contact with the endothelium (100× objective).

Article Snippet: Unconjugated rat antibodies against CD8α (clone YTS169.4) and CD8β (clone 53–5.8) from Bio X Cell were used at 5 μg/ml for immunofluorescence.

Techniques: Incubation, Infection, Staining

Journal: Nature biotechnology

Article Title: In vivo molecular imaging for immunotherapy using ultra-bright near-infrared-IIb rare-earth nanoparticles

doi: 10.1038/s41587-019-0262-4

Figure Lengend Snippet: a, Schematic of the CD8α blocking experiment. A CT-26 tumor mouse was first intravenously injected with mixed ErNPs-aPDL1 and free anti-CD8α mAb (left wide-field image, 48 hrs post first injection), followed by a second intravenous injection of PbS-aCD8 (right wide-field image, 24 hrs post second injection). b, The signal of tumor and spleen to background ratios were plotted to reveal the bio-distribution of ErNPs-aPDL1 (left) and PbS-aCD8 (right) with CD8 and no blocking (mice n = 3 for each group). c-e, Wide-field images of mice from different directions (left-arm, belly, and right-arm) revealed the in vivo bio-distribution of ErNPs-aPDL1 and PbS-aCD8 in ( c ) a CT-26 tumor mouse intravenously injected with mixed ErNPs-aPDL1 and PbS-aCD8, ( d ) a CT-26 tumor mouse intravenously injected with only PbS-aCD8, and ( e ) a 4T1 tumor mouse intravenously injected with mixed ErNPs-aPDL1 and PbS-aCD8, at 24 hrs post injection. f, Corresponding (T/spleen)_CD8 ratios in these mice (n = 3 for each group). Green color: ErNPs-aPDL1 and red color: PbS-aCD8 for all these images. All data are presented as means ± s.d. All the scale bar is 5 mm. Data in b , f is presented as box plots (center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, outliers). (****P = 0.0001, t = 14.4971, df = 4); (*****P = 0.0001, t = 15.0393, df = 4).

Article Snippet: Anti-CD8α monoclonal antibody (Clone 2.43) was purchased from Bio X Cell.

Techniques: Blocking Assay, Injection, In Vivo