Journal: Nature

Article Title: Clonally expanded CD8 T cells characterize Amyotrophic Lateral Sclerosis 4

doi: 10.1038/s41586-022-04844-5

Figure Lengend Snippet: a, b, Peripheral blood cells from 8 month old WT and KI mice were analyzed by mass cytometry. a, t-SNE plots after gating on live CD45high (top) and CD19−NK1.1−CD11b−CD11c−TCRβ+ (bottom). b, Frequency of PD-1+ expressing cells after gating on CD44+ CD8+ T cells. Three mice/group were pooled for mass cytometry analysis. c, Gating strategy for flow cytometry analysis of CD8 T cells in the CNS. d, Frequency of PD-1+ CD8 T cells in the spleen and lungs of WT and KI mice at the indicated ages (2 independent experiments, 4 mice/group; mean +/− SEM). d, Frequency of PD-1+ (left) and Foxp3+ (right) CD4 T cells in the peripheral blood of WT and KI mice at the indicated ages (3 independent experiments, n=6 (left) and 2 independent experiments, n=4 (8 months) and n=6 (right) individual mice/group. SB=super bright.

Article Snippet: The following isotope conjugated anti-mouse antibodies were used at 1:100 dilution: 113 In CD45.1 (clone A20, eBioscience, Catalog # 14–0453-85), 115 In CD45.2 (clone 104, eBioscience, Catalog # 14–0454-85), 141 Pr Ly6G (clone 1A8, Fluidigm, Catalog # 3141008B), 142 Nd CD11c (clone N418, Biolegend, Catalog # 117302), 143 Nd TCRbeta (clone H57–597, Fluidigm, Catalog # 3143010B), 144 Nd CD24 (clone M1/69, Biolegend, Catalog # 101802),145 Nd CD69 (clone H1.2F3, Fluidigm, Catalog # 3145005B), 146 Nd F4/80 (clone BM8, Fluidigm), 147 Sm CD357/GITR (clone DTA-1, Biolegend), 148 Nd CD11b (clone M1/70, Biolegend, Catalog #), 149 Sm CD19 (clone 6D5, Fluidigm, Catalog #), 150 Nd IgD (clone 11–26c.2a, Biolegend, Catalog #), 151 Eu CD25 (clone 3C7, Fluidigm, Catalog # 3146008B), 152 Sm SiglecF (clone S17007L, Biolegend, Catalog # 155502), 153 Eu NKp46 (clone 29A1.4, Fluidigm, Catalog # 3153006B), 155 Gd CD73 (clone TY/11.8, Biolegend, Catalog # 127202), 156 Gd CD64 (clone X54–5/7.1, Biolegend, Catalog # 139302), 158 Gd CD117 (clone 2B8, Biolegend, Catalog # 105802), 159 Tb PD-1 (clone RMP1, Biolegend, Catalog #), 160 Gd CD62L (clone MEL-14, Fluidigm, Catalog # 109113),162 Dy Ly6C (clone HK1.4, Biolegend, Catalog # 128002),163 Dy anti-APC (Biolegend, Catalog # 408002),164 Dy Sca-1 (clone D7, Fluidigm, Catalog # 3164005B), 165 Ho anti-PE (Biolegend, Catalog # 408102), 166 Er KLRG1 (clone 2F1/KLRG1, Biolegend, Catalog # 93135),167 Er CXCR3 (clone CXCR3–173, Biolegend, Catalog # 126526), 168 Er CD8alpha (clone 53–6.7, Biolegend, Catalog # 100702), 169 Tm CD206 (clone C068C2, Fluidigm, Catalog # 3169021B), 170 Er NK1.1 (clone PK136, Fluidigm, Catalog # 3170002B), 171 Yb CD44 (clone IM7, Biolegend, Catalog # 103002), 172 Yb CD4 (clone RM4–5, Fluidigm, Catalog # 3172003B), 174 Yb MHCII (I-A/I-E) (clone M5/114.15.2, Biolegend, Catalog # 107602), 175 Lu CD127 (clone A7R34, Biolegend, Catalog # 135002), 176 Yb B220 (RA3–6B2, clone Biolegend, Catalog # 103202).

Techniques: Mass Cytometry, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells

doi: 10.1038/s41598-020-72181-6

Figure Lengend Snippet: Percentages of CD45 + immune cells in control and noise-exposed cochleae.

Article Snippet: The whole-mount preparations and sections were stained using the following antibodies at 1:50 to 1:100 dilution: goat anti-mouse CD45 (AF114, R&D Systems; a kind gift from Dr. Tejbeer Kaur), rat anti-mouse CD11b (MCA711, Bio-Rad), rat anti-mouse F4/80 (CI-A3-1: NB600-404SS, Novus Biologicals), rabbit monoclonal CD3e (SP7:SAB5500058, Sigma-Aldrich), rat anti-mouse CD45R/B220 (RA3- 6B2; 553083, BD Pharmingen), mouse monoclonal neutrophil elastase (NP57; sc-53388, Santa Cruz Biotechnology, Inc.), CD161/ NK1.1 Antibody (PK136; NB100-77528SS, Novus Biologicals), and rabbit polyclonal myosin VI (25-6791, Proteus BioSciences).

Techniques: Control

Journal: Scientific Reports

Article Title: The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells

doi: 10.1038/s41598-020-72181-6

Figure Lengend Snippet: Immune cell phenotypic markers.

Article Snippet: The whole-mount preparations and sections were stained using the following antibodies at 1:50 to 1:100 dilution: goat anti-mouse CD45 (AF114, R&D Systems; a kind gift from Dr. Tejbeer Kaur), rat anti-mouse CD11b (MCA711, Bio-Rad), rat anti-mouse F4/80 (CI-A3-1: NB600-404SS, Novus Biologicals), rabbit monoclonal CD3e (SP7:SAB5500058, Sigma-Aldrich), rat anti-mouse CD45R/B220 (RA3- 6B2; 553083, BD Pharmingen), mouse monoclonal neutrophil elastase (NP57; sc-53388, Santa Cruz Biotechnology, Inc.), CD161/ NK1.1 Antibody (PK136; NB100-77528SS, Novus Biologicals), and rabbit polyclonal myosin VI (25-6791, Proteus BioSciences).

Techniques:

Journal: STAR Protocols

Article Title: Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes

doi: 10.1016/j.xpro.2021.100537

Figure Lengend Snippet:

Article Snippet: CyTOF: anti-NK1.1-165Ho (PK136) , Fluidigm , Cat#3165018B RRID: N/A.

Techniques: Recombinant, Staining, Blocking Assay, Saline, Multiplex Assay, Software

Journal: STAR Protocols

Article Title: Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes

doi: 10.1016/j.xpro.2021.100537

Figure Lengend Snippet: CyTOF Antibody Volumes (per 50 μL Maxpar Cell Staining Buffer/sample)

Article Snippet: CyTOF: anti-NK1.1-165Ho (PK136) , Fluidigm , Cat#3165018B RRID: N/A.

Techniques: Staining

Journal: STAR Protocols

Article Title: Isolation of mouse brain-infiltrating leukocytes for single cell profiling of epitopes and transcriptomes

doi: 10.1016/j.xpro.2021.100537

Figure Lengend Snippet: CITE-seq Antibody Volumes (per 50 μL CITE-seq Staining Buffer/sample)

Article Snippet: CyTOF: anti-NK1.1-165Ho (PK136) , Fluidigm , Cat#3165018B RRID: N/A.

Techniques: Staining

Journal: bioRxiv

Article Title: Rapid establishment of a tumor-retained state curtails the contribution of conventional NK cells to anti-tumor immunity in solid cancers

doi: 10.1101/2023.08.10.552797

Figure Lengend Snippet: (A) Expression of CD49a vs CD49b by NK cells (CD3- NK1.1+) isolated from MC38 tumors grafted subcutaneously on the flank. (B) Expression of CD49a vs CD11b by NK cells isolated from MC38 tumors, alongside blood. (C) Expression of CD49a vs CD11b by NK cells across multiple tumor models. NK cells identified as CD3- NK1.1+ in C57BL/6 mice, CD3- NKp46+ in BALB/c mice. (D) Proportion of NK cells within the CD11b+ CD49a-, CD11b- CD49a- (double negative, DN) and CD11b- CD49a+ gates across the different tumor models. Data collect from B16F10-OVA and EO771 tumors was from 1 independent repeat, and MC38, CT26, and MMTV-PyMT tumors pooled from 2 independent repeats, where MC38 (24 hr n=12, 72hr n=12), CT26 (24 hr n=8, 72hr n=8), EO771 (24 hr n=4, 72hr n=4), B16F10-OVA (24 hr n=4, 72hr n=4), and MMTV-PyMT (24 hr n=7, 72hr n=12). (E) Number of Kaede Green+ and Kaede Red+ NK cells at 5, 24 and 72 hrs post photoconversion of MC38 tumors. (F) Expression of CD49a versus CD11b by Kaede Green+ and Kaede Red+ NK cells at 5, 24 and 72 hrs post photoconversion. (G) The proportion of Kaede Green/Red for each NK cell subset at each time point post photoconversion. Data at 5 (n=5) hrs is representative of 1 independent repeat, whereas 24 (n=8) and 72 (n=11) hrs pooled from 2 independent repeats. (H) UMAPs showing protein expression of CD11b and CD49a alongside Kaede Green/Red expression by NK cells isolated from MC38 tumors at 24 and 72 hrs post photoconversion. (I) UMAPS showing expression of CD11b, CD49a, NKG2A, LAG-3 and CD69. (J) Enumeration of the proportion of cells expressing NKG2A, LAG-3 and CD69 across the NK cell subsets. Data in UMAPs 24hr (n=5), and 72hr(n=8) are representative of two independent repeats. Statistical significance was determined by two-way ANOVA with Šidák’s multiple comparisons test (J). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Anti- NK1.1, anti-CD8α, and anti-OX40 antibodies were provided by AstraZeneca.

Techniques: Expressing, Isolation

Journal: bioRxiv

Article Title: Rapid establishment of a tumor-retained state curtails the contribution of conventional NK cells to anti-tumor immunity in solid cancers

doi: 10.1101/2023.08.10.552797

Figure Lengend Snippet: Evidence for dysfunctional NK cells within human CRC was sought through bioinformatics analysis of publicly available data sets alongside flowcytometric analysis of primary human CRC samples. (A) UMAP of 3521 NK cells from scRNA-seq of 62 human CRC samples, from GSE178341. (B) Expression of selected marker genes for clusters shown in ‘A’. (C) Expression of ITGAM and ITGA1 in NK1 subsets. (D) Granzyme and perforin gene expression in NK1 subsets, in tumor (T) versus normal adjacent (N) tissue. (E) Gene set enrichment for KEGG Natural killer cell-mediated cytotoxicity between NK1 clusters. (F) Gaussian kernel density embedding of cells in CRC tumors by tumor staging, and (G) lymph node (LN) metastases. (H) Representation flow plots depicting the frequency of CD49a+ and CD11b+ NK cells across PBMC, unaffected colon tissue and CRC primary tumor for the same donor (n=10). (I) Bar plot showing the frequency of CD11b+CD49a-, CD11b-CD49a- and CD11b-CD49a+ NK cells across compartments. Significance was determined using Wilcoxon test to show specific comparisons. (J) Ratio of CD11b+CD49a- to CD11b-CD49a+ NK cells across tissue compartments. Significance was determined using Kruskal-Wallis. (K) Representative histograms showing expression of selected markers on tumor infiltrating NK cell subsets. (L) Bar plots depicting geoMFI values for specific markers in tumor infiltrating NK cells. Significance was determined using the Kruskal-Wallis test.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Anti- NK1.1, anti-CD8α, and anti-OX40 antibodies were provided by AstraZeneca.

Techniques: Expressing, Marker