Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glucocorticoids regulate TCR-induced elevation of CD4: functional implications.

doi: 10.4049/jimmunol.164.12.6213

Figure Lengend Snippet: FIGURE 1. CD4 expression increases after T cell activation indepen- dently of the stimulus used to activate CD41 T cells. A, Cells were acti- vated with either plate-bound anti-TCR or anti-CD3 mAb (1 mg/well) or the soluble stimulants SEA (1 mg/ml) and Con A (1.5 mg/ml). Cells were cultured for 24–120 h and analyzed by double-immunofluorescence stain- ing with PE-conjugated anti-CD25 mAb and FITC-conjugated anti-CD4 mAb. Unrelated PE- or FITC-conjugated IgG1 mAb were used as controls. Results (representative of four to six independent experiments) are ex- pressed as the mean fluorescence intensity (MFI). Mean CD4 fluorescence of freshly isolated cells was 580 vs 9 for mAb control. Similar results were obtained after staining with OX35 (data not shown). B, FACS plot of SEA-activated CD41 T cells (as shown in A) after 24 h (solid lines) or 96 h (dashed lines) days of incubation. The histograms on the left show control staining with unrelated FITC-conjugated IgG1 mAb. C, CD4 expression by CD251 vs CD252, SEA-treated CD41 T cells (as described in A) after 24–72 h of culture.

Article Snippet: For flow cytometry, FITC-conjugated mouse anti-rat TCRab IgG1 mAb, FITC-conjugated mouse anti-rat CD4 IgG1 mAb (clone W3/25, which recognizes an epitope of domain 1 of CD4), FITCconjugated mouse anti-rat CD8a IgG1 mAb (clone OX8), PE-conjugated anti-CD4 mAb (W3/25), PE-conjugated anti-CD8a mAb (OX8), and PEconjugated mouse anti-rat CD25 IgG1 mAb (clone OX39) were purchased from Serotec.

Techniques: Expressing, Activation Assay, Cell Culture, Staining, Isolation, Control, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Glucocorticoids regulate TCR-induced elevation of CD4: functional implications.

doi: 10.4049/jimmunol.164.12.6213

Figure Lengend Snippet: FIGURE 6. Activation-induced TCR down-regulation is not affected by CORT. A, Cells were activated as described in Fig. 1 and cultured for 24–120 h. TCR expression was analyzed by double-immunofluorescence staining with PE-conjugated anti-CD4 mAb and FITC-conjugated anti- TCR mAb. Unrelated PE- or FITC-conjugated IgG1 mAb were used as controls. Results (representative of four independent experiments) are ex- pressed as the percent MFI of TCR expression by fresh cells (5100%; MFI 5 389.8; MFI for mAb control, 8). B, Cells were incubated in the absence or the presence of CORT (1026 M) and were simultaneously ac- tivated with plate-bound anti-TCR mAb (1 mg/well). Cell culture and TCR analysis were performed as described in A.

Article Snippet: For flow cytometry, FITC-conjugated mouse anti-rat TCRab IgG1 mAb, FITC-conjugated mouse anti-rat CD4 IgG1 mAb (clone W3/25, which recognizes an epitope of domain 1 of CD4), FITCconjugated mouse anti-rat CD8a IgG1 mAb (clone OX8), PE-conjugated anti-CD4 mAb (W3/25), PE-conjugated anti-CD8a mAb (OX8), and PEconjugated mouse anti-rat CD25 IgG1 mAb (clone OX39) were purchased from Serotec.

Techniques: Activation Assay, Cell Culture, Expressing, Staining, Control, Incubation

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: 21h10 improves antitumor efficacy in in vivo B16F10 murine melanoma and ex vivo human PDOTs models, with non-neutralizing anti-drug antibodies, and toxicity alleviated with TNFα blockade. ( A ) Naïve CD8 + TRP1 high/low melanoma-specific T cells were adoptively transferred to mice prior to B16F10 inoculation. Cytokine therapy began on day 5 and continued every day until the stop of treatment (dashed line). ( B ) Mice received no prior T cell adoptive transfer before cytokine therapy. ( C ) Same as ( B ), but with adoptive transfer of naïve CD8 + TRP1 high/low T cells. ( D ) Scheme of PDOTS preparation. ( E ) Viability assessment of ex vivo human melanoma PDOTS following treatment of ICB (anti-PD-1, pembrolizumab, or anti-PD-1/anti-LAG-3), 21h10, or untreated control. ( F ) Representative images of PDOTS viability assessment shown in ( E ). PI-dead cells in red and Hoechst-nuclei in blue. ( G ) Mice died due to toxicity or after 40 days of treatment were bled and their serum was analyzed by ELISA for anti-drug antibodies against 21h10. Positivity (dotted line) was determined as two standard deviations above the mean of samples from mice treated only with PBS. Fractions indicate the number of mice classified as “positive” for anti-drug antibodies out of the total mice in each treatment group. ( H ) Mice with various titer levels of anti-21h10 antibodies were inoculated with MC38 tumors. Starting on day 6, mice were treated with 21h10 or PBS daily for 17 days. ( I ) Wild-type (WT), Rag2-/- , or Rag2-/- Il2rg-/- mice were treated with PBS or 21h10 daily and isotype or anti-NK1.1 depleting antibodies every three days. Mice were sacrificed when weight loss exceeded 20% of the initial starting weight. ( J ) Serum TNFα level was measured in C57BL/6 and Rag2-/- mice with PBS or 21h10 treatments. ( K ) Antitumor activity comparison on B16F10 melanoma between 21h10 and 21h10+aTNFα treatment groups.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: In Vivo, Ex Vivo, Adoptive Transfer Assay, Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Mice were treated as in , A and C, but on day 15 of tumor growth, tumors were processed for single-cell RNA-sequencing. UMAP of all samples are combined. ( B ) scRNAseq cluster composition average across treatment groups. ( C ) T cells in each scRNAseq sample from ( B ). Different colors for each bar indicate individual mice from each group. ( D ) Flow cytometry quantification of CD8 + T cell infiltration. ( E ) TRP1 high and TRP1 low melanoma antigen-specific cells captured by sequencing. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS groups. ( F ) T cells were sub-clustered from all samples from ( A ). The average T cell sub-cluster composition across treatment groups is shown on the right. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( G ) IFN-𝛾-response score total UMAP from ( A ) based on scaled and summed Hallmark IFN-𝛾-response genes for each cell. ( H ) Heatmap from pseudo-bulk differential gene expression analysis across T cell sub-clusters. Significant differences compared to PBS samples. ( I ) Similar to ( H ), but for TRP1 cells only, values from individual mice are shown. P-values displayed vs. PBS samples. ( J ) Flow cytometry quantification of CD8 + IFN-𝛾/granzyme B levels in endogenous or transferred CD8 + TRP1 high/low T cells. ( K ) CD4 + T cell sub-clustering UMAP and cluster composition. Two-way ANOVA with Dunnett’s multiple comparisons test vs. PBS samples. ( L ) Flow cytometry of IFN-𝛾 + CD4 + T cells. ( M ) Quantification and representative flow cytometry plots of Foxp3-GFP Treg cells. One-way ANOVA with Dunnett’s multiple comparisons for flow quantification and scRNA-seq total T cell comparison. For all dot plots with error bars: dots indicate individual mice, bars are S.E.M. *p ≤ 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001, or p-values displayed.

Article Snippet: Two days before and on the day of tumor inoculations, mice were treated with anti-CD4 (150 µg per mouse; Bioxcell Cat# BE0003-1) and/or anti-CD8 depleting antibodies (150 µg per mouse; Bioxcell (valid) # BE0061) or rat IgG2b isotype (150 µg per mouse; clone LTF-2, BioXCell Cat# BE0090) diluted in sterile endotoxin-free PBS.

Techniques: RNA Sequencing Assay, Flow Cytometry, Sequencing, Expressing, Comparison

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: Cross-reactivity of anti-bovine PD-L1 and TIM-3 monoclonal antibodies in ovine PBMCs. ( A ) Cross-reactivity of anti-bovine PD-L1 monoclonal antibody (mAb) with fresh ovine PBMCs. The gray histogram shows fluorescence intensity of the rat IgG2a isotype control. The pink histogram shows staining with anti-bovine PD-L1 mAb. All data were gated on live IgM + cells within PBMCs. ( B – D ) Cross-reactivity of anti-bovine TIM-3 mAb with fresh ovine PBMCs. The gray histograms show fluorescence intensity of the mouse IgG1 isotype control. The red histograms show staining with anti-bovine TIM-3 mAb. Histograms were gated on CD4 + ( B ), CD8 + ( C ), and γδTCR + T cells ( D ). Gating strategies for the flow cytometric assays were shown in .

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 °C for 20 min.

Techniques: Bioprocessing, Fluorescence, Control, Staining

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: Blockade effects of anti-bovine PD-L1 and TIM-3 monoclonal antibodies in PBMCs of healthy sheep. ( A – C ) T-cell functional analysis in PBMCs from BLV-uninfected healthy sheep ( n = 10) with blockade by anti-PD-L1 and anti-TIM-3 mAbs under ConA stimulation. ( A ) Frequency of activated T cells (CD25 + CD69 + ) within CD4 + and CD8 + T-cell subsets. ( B , C ) Frequency of cytokine-producing activated cells (CD69 + IFN-γ + and CD69 + TNF-α + ) within CD4 + and CD8 + T-cell subsets. Gating strategies for the flow cytometric assays are shown in . Each symbol represents data from an individual animal. Red lines indicate median values. Significant differences compared between treatment groups: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 °C for 20 min.

Techniques: Bioprocessing, Functional Assay

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: Comparison of immunoinhibitory receptor expression between healthy and experimentally BLV-infected sheep. ( A ) Percentage of PD-L1 + cells among IgM + B cells in BLV-uninfected ( n = 4) and BLV-infected sheep ( n = 7). ( B – D ) Comparison of TIM-3 + cell frequencies in CD4 + , CD8 + , and γδTCR + T-cell subsets between BLV-uninfected ( n = 4) and BLV-infected sheep ( n = 7). Gating strategies for the flow cytometric assays are shown in . Each dot represents data from an individual animal. Red lines indicate group median values. * p < 0.05.

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 °C for 20 min.

Techniques: Comparison, Expressing, Infection

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: T-cell activation and cytokine responses in BLV-infected sheep following immune checkpoint blockade. ( A , B ) Frequencies of activated CD25 + CD69 + cells in CD4 + and CD8 + T-cell subsets in PBMCs from BLV-infected sheep ( n = 7) following ConA stimulation with blockade by anti-PD-L1 and anti-TIM-3 mAbs. ( C – F ) Frequencies of cytokine-producing CD69 + cells expressing IFN-γ ( C , D ) or TNF-α ( E , F ) under the same blockade conditions. ( G ) IFN-γ concentration of PBMCs from BLV-infected sheep ( n = 7) following ConA stimulation with blockade by anti-PD-L1 and anti-TIM-3 mAbs. Gating strategies for the flow cytometric assays are shown in . Each dot represents data from an individual animal. Red lines indicate median values. Significant differences compared between treatment groups: * p < 0.05, ** p < 0.01.

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 °C for 20 min.

Techniques: Activation Assay, Infection, Expressing, Concentration Assay

Journal: Cancers

Article Title: Implication of COPB2 Expression on Cutaneous Squamous Cell Carcinoma Pathogenesis

doi: 10.3390/cancers14082038

Figure Lengend Snippet: Expression of tumor immune microenvironment (TIME) indicators in cSCC: ( A ) COPB2 expression showed a positive correlation with the expression of both (i) MHC I and (ii) MHC II transcripts in cSCC cell lines in the GSE98767 dataset; ( B ) Representative expression patterns of MHC I, MHC II, and hot spots of CD4+/CD8+ tumor-infiltrating lymphocytes (TILs) in cSCC tissues (Original magnification, 100×; scale bar, 200 μm; magnification for inset, 400×; scale bar for inset, 50 μm).

Article Snippet: COPB2 (rabbit polyclonal IgG, working dilution 1/50, Abnova, PAB23074, Walnut, CA, USA), MHC I (rabbit monoclonal IgG, working dilution 1/1000, Abcam, Cambridge, UK, ab134189), MHC II (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab55152), CD4 (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab133616), and CD8 (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab108343) antibodies were used as primary antibodies herein.

Techniques: Expressing

Journal: Cancers

Article Title: Implication of COPB2 Expression on Cutaneous Squamous Cell Carcinoma Pathogenesis

doi: 10.3390/cancers14082038

Figure Lengend Snippet: Clinicopathologic significance of COPB2 expression in 95 cSCC patients.

Article Snippet: COPB2 (rabbit polyclonal IgG, working dilution 1/50, Abnova, PAB23074, Walnut, CA, USA), MHC I (rabbit monoclonal IgG, working dilution 1/1000, Abcam, Cambridge, UK, ab134189), MHC II (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab55152), CD4 (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab133616), and CD8 (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab108343) antibodies were used as primary antibodies herein.

Techniques: Expressing

Journal: Cancers

Article Title: Implication of COPB2 Expression on Cutaneous Squamous Cell Carcinoma Pathogenesis

doi: 10.3390/cancers14082038

Figure Lengend Snippet: Significance of clinical factors and molecular markers by Cox regression analysis.

Article Snippet: COPB2 (rabbit polyclonal IgG, working dilution 1/50, Abnova, PAB23074, Walnut, CA, USA), MHC I (rabbit monoclonal IgG, working dilution 1/1000, Abcam, Cambridge, UK, ab134189), MHC II (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab55152), CD4 (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab133616), and CD8 (rabbit monoclonal IgG, working dilution 1/1000, Abcam, ab108343) antibodies were used as primary antibodies herein.

Techniques:

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) Schematic for tumor dissociation, isolation of tumor infiltrated leukocytes (TILs) and Drop-seq based single cell RNA sequencing. Ctrl, vehicle treated control; Ab, anti-Her2/Neu antibody; Pal, CDK4/6 inhibitor Palbociclib; TILs, tumor infiltrated leukocytes. ( B ) Clustering of 1444 TILs derived from Ctrl, APP and APR tumors and t-SNE plot colored by clusters (left) and annotation of TIL-clusters on t-SNE plot colored by treatment groups (right). Each point represents a single cell. ( C ) Abundance of each cluster [as clustered and annotated in (B)] in TILs with indicated treatment. ( D ) Analysis of immune cell populations among CD45+ TILs by mass cytometry. Immune cell populations were identified by overlaying the expression of markers on viSNE plots as shown in . ( E ) Representative images and quantification of CD3 and CD8 immunofluorescence staining for Ctrl, APP and APR tumors. Arrows indicate CD3 and CD8 double positive cells. Scale bar, 50 μm. ( F ) Single cell transcriptional analysis of tumor-produced cytokines and chemokines. The size of each circle reflects the percentage of cells in a treatment group where the gene is detected, and the color intensity reflects the average expression level within each treatment group.( G ) Schematic for tumor dissociation, isolation of CD45+ TILs and Gr1+ cells and Drop-seq based single cell RNA sequencing. ( H ) t-SNE plot of single cell RNA sequencing data from CD45+ TILs and Gr1+ cells. Each point represents a single cell.( I ) Overlay of marker genes for Gr1+ cell population identified from experiment (F) on t-SNE plots derived from experiment (A) and (F), respectively. ( J ) Violin plot showing distribution of ssGSEA enrichment score among TIL-clusters identified from experiment (A). Experiment (F) generated MDSCs signature (top 300 differential expressed genes for MDSC population) was used for enrichment analysis. Each point represents a single cell. ( K ) Growth of Ab+Pal resistant tumors by adding Gr1 antibody and ICB. Ab+Pal resistant tumors were transplanted to recipient MMTV-Neu mice and first treated with Ab+Pal to acquire the resistance phenotype (left) and then adding IgG (n=6), Anti-Gr1 (n=5) or Anti-Gr1+ICB (n=7) treatments were followed (right). P- value in D,E and K by one-way ANOVA with Tukey’s test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Isolation, RNA Sequencing Assay, Derivative Assay, Mass Cytometry, Expressing, Immunofluorescence, Staining, Produced, Marker, Generated

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) Gr1+ MDSCs isolated from Ab+Pal treatment resistant tumors inhibited CD3/CD28 stimulated proliferation of CD4+ and CD8+ T cells in vitro. Left, representative flow cytometry histograms measuring carboxyfluorescein succinimidyl ester (CFSE); right, quantification of T-cell proliferation. P -value by Student’s t-test. ( B ) t-SNE plot of single cell RNA sequencing data from CD45+ TILs, Gr1high Ly6G+cells and Gr1dim Ly6G-cells. Each point represents a single cell. ( C ) Expression of key marker genes used for immune cell-type identification and annotation was overlaid on t-SNE plots as shown in (B). ( D ) mRNA levels of Lcn2 and Mgst1 in sorted T cells (CD3+), macrophages (F4/80+) and MDSCs/IMCs (Gr1+) from APR tumors were quantified by RT-PCR. P -value by one-way ANOVA with Tukey’s test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Isolation, In Vitro, Flow Cytometry, RNA Sequencing Assay, Expressing, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Single-cell profiling guided combinatorial immunotherapy for fast-evolving CDK4/6 inhibitor resistant HER2-positive breast cancer

doi: 10.1101/671198

Figure Lengend Snippet: ( A ) t-SNE plots of 3168 TILs derived from resistant tumors with continuous Ab+Pal, Ab+Cabo or Ab+Cabo+ICB treatment [merged (left) or subdivided into individual group (right)] were shown]. Each point represents a single cell. ( B ) Expression of key marker genes used for immune cell-type identification and annotation was overlaid on t-SNE plots. ( C ) Representative images and quantification of CD3 and CD8 immunofluorescence staining. Arrows indicate CD3 and CD8 double positive cells. Scale bar, 50 μm. P- value by one-way ANOVA with Tukey’s test. ( D ) Enrichment scores of cabozantinib target genes among tumor infiltrated IMCs after indicated treatment. The P values showed were determined by Student’s t-test. ( E ) Distribution of Kit and/or Met expression cells on t-SNE plot of single cell RNA sequencing data from (A), and abundance of Kit or Met expressing IMCs within CD45+ TILs after indicated treatment. P -value by three-sample Chi-square test. ( F ) Representative images and quantification of CD8 and Granzyme B immunofluorescence staining. Arrows indicate CD8 and Granzyme B double positive cells. Scale bar, 50 μm. P- value by one-way ANOVA with Tukey’s test.

Article Snippet: The following pre-conjugated antibodies purchased from Fluidigm were used in this study: CD45-089Y (3089005B, 30-F11); Ly-6G-141Pr (3141008B, 1A8,); CD11c-142Nd (3142003B, N418); CD45R-144Nd (3144011B, RA3-6B2); CD4-145Nd (3145002B, RM4-5); CD11b-148Nd (3148003B, M1/70); CD44-150Nd (3150018B, IM7); CD25-151Eu (3151007B, 3C7); CD3e-152Sm (3152004B, 145-2C11); PD-L1-153Eu (3153016B, 10F.9G2); CTLA-4-154Sm (3154008B, UC10-4B9); PD-1-159Tb (3159024B, 29F.1A12); Ly-6C-162Dy (3162014B, HK1.4); CX3CR1-164Dy (3164023B, SA011F11); NK1.1-165Ho (3165018B, PK136); c-Kit-166Er (3166004B, 2B8); CD8a-168Er (3168003B, 53-6.7); CD86-172Yb (3172016B, GL1); I-A/I-E-209Bi (3209006B, M5/114.15.2).

Techniques: Derivative Assay, Expressing, Marker, Immunofluorescence, Staining, RNA Sequencing Assay