Journal: The Journal of Experimental Medicine

Article Title: STARTRAC analyses of scRNAseq data from tumor models reveal T cell dynamics and therapeutic targets

doi: 10.1084/jem.20201329

Figure Lengend Snippet: CCR8 expression in tumor T reg cells and characterization of anti-CCR8 antibodies . (A) Representative histogram plot depicting expression of CCR8 on 4-1BB + and 4-1BB − CD4 + Foxp3 − T EFF population in MC38 tumors. (B) Representative histogram plot depicting expression of CCR8 on CCR7 + and CCR7 − CD8 + T cells in MC38 tumors. (C) Flow cytometry analysis of tumor-infiltrating T reg cells across multiple mouse tumor models shown as representative FACS plots (left) and frequencies in tumors and peripheral lymphoid organs (right). *, P = 0.0250; ****, P < 0.0001; two-way ANOVA with Tukey’s post-hoc analysis. (D) Flow cytometry analysis of CCR8 expression on immune cell types (Foxp3 + T reg cells, Foxp3 − T effectors, CD8 + , NKp46 + NK, CD19 + B, and CD11b + ) in tumors and peripheral lymphoid tissues across tumor models. (E) In vitro chemotaxis assay evaluating the ability of CCR8-depleting and -nondepleting antibodies to block 100 pM CCL1-induced chemotaxis. (F) Bar graph comparing IC 50 values from the chemotaxis assay testing CCR8-depleting and -nondepleting antibodies ( n = 6). (G) Pharmacokinetic assessment of serum antibody concentration after single-dose administration of CCR8-depleting and -nondepleting antibodies (10 mg/kg i.p.). (H) Serum concentration at 72 h of animals treated as in G. DLN, draining LN; SPL, spleen; MFI, mean fluorescence intensity.

Article Snippet: Recombinant human CCL1 (R&D Systems) was prepared at suboptimal concentration of 100 pM and added to the bottom Transwell chambers at 100 μl per well.

Techniques: Expressing, Flow Cytometry, In Vitro, Chemotaxis Assay, Blocking Assay, Concentration Assay, Fluorescence

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Il1a , Ccl5 , and Ccl1 mRNA levels in the dorsal horn of female mice. n = 5 to 8. ( B ) Immunofluorescence image of IL-1α (green) in vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). ( C ) Double immunofluorescence staining of CCL1 (green) and vascular endothelial cells (red). Scale bars, 50 μm (low) or 20 μm (high). ( D and E ) Coimmunostaining image of F4/80 (green) and CD31 (red) in the spinal dorsal horn and the statistical graph. Scale bar, 200 μm. n = 5. ( F ) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. ( G ) Immunofluorescence image shows IL-1R1 (red) expression in macrophages (green). Scale bar, 10 μm. ( H and I ) Intrathecal IL-1R1 injection inhibited the up-regulation of F4/80 (green) and CD31 (red) in the near vascular of the spinal dorsal horn. Scale bar, 200 μm (H). The number of F4/80 high cells in the dorsal horn of the spinal cord [(I), left]. The percentage of F4/80 high area among CD31 + area [(I), right]. n = 4 to 5. ( J and K ) Immunostaining shows F4/80 (green) and CD31 (red) expression in the spinal dorsal horn. Scale bar, 200 μm (J). The number of F4/80 high cells in the dorsal horn of the spinal cord [(K), left]. The percentage of F4/80 high area among CD31 + area [(K), right]. n = 5. ( L ) Intrathecal injection of IL-1α siRNA attenuated the mechanical allodynia induced by BTZ. n = 5. Significance: * P < 0.05, # P < 0.05, ** P < 0.01, and ## P < 0.01.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Immunofluorescence, Double Immunofluorescence Staining, Injection, Expressing, Immunostaining

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Coimmunofluorescence of CCL1 (green) and macrophages (purple) on day 3 following BTZ treatment in female mice. Scale bar, 50 μm. ( B ) Quantification of CCL1 fluorescence intensity in macrophages. n = 5. ( C ) Coimmunofluorescence of CCR8 (red) and NeuN (marker of neuron, green). Scale bars, 20 μm. ( D ) Coimmunofluorescence of CCR8 (red) and glial fibrillary acidic protein (GFAP; a marker of astrocyte, green). Scale bars, 20 μm. ( E ) Representative traces (left) and statistical data (right) for the action potential firing recorded in spinal cord lamina II neurons. n = 4 mice. ( F ) Intrathecal injection of CCL1-neutralizing antibody attenuated the mechanical allodynia induced by BTZ. n = 5 to 7. ( G ) CCL1 increased the action potentials of neurons in female mouse spinal slices. n = 6. ( H ) Intraspinal CCL1 injection reduced the mechanical withdrawal threshold. n = 5. ( I ) R243 preincubation blocked the CCL1-induced action potential increase in female mouse spinal slices. n = 6. ( J ) Intrathecal injection of R243 attenuated the mechanical allodynia induced by BTZ. n = 5. ( K ) Intrathecal injection of R243 attenuated the mechanical allodynia induced by recombinant CCL1. n = 5. ( L ) Recombinant CCL1 increased the action potentials in astrocyte CCR8 knockdown mouse spinal slices. n = 5. ( M ) Intraspinal CCL1 injection reduced the mechanical withdrawal threshold in astrocyte CCR8 knockdown female mice. n = 5. Significance: not significant (n.s.), * P < 0.05 and ** P < 0.01.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Fluorescence, Marker, Injection, Recombinant, Knockdown

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Immunofluorescence imaging showed BTZ-activated astrocytes (turquoise) in close proximity to macrophages (green) and vascular endothelial cells (red). Scale bars, 20 μm. ( B ) Schematic of two-photon imaging configuration. ( C ) Representative image showed the increased GCaMP6s (green) in spinal cord astrocytes (SR101, red) following CCL1 incubation. Scale bar, 150 μm. ( D ) Example signal trace (left) and histogram of area under the curve (AUC) (right) of calcium signaling value from the spinal dorsal horn astrocyte in mice treated with CCL1. n = 3. ( E to G ) R243 incubation prevented CCL1-induced calcium signaling increase in GCaMP6s + astrocytes. Representative image (E) and signal trace (F) show R243 blocked CCL1-induced calcium up-regulation. Scale bar, 20 μm. AUC quantification (G). n = 3. ( H to J ) Response of astrocytes to recombinant CCL1 during bath application of TTX (1 μM). Representative image (H) and signal trace (I) showed that TTX did not inhibit the increase in intracellular calcium signaling induced by CCL1. Scale bar, 20 μm. AUC quantification (J). n = 3. ( K to M ) Application of CNO increased the astrocyte intracellular calcium signaling in the female with injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. Scale bar, 20 μm. ( N ) The action potential number in female mouse spinal cord slice was increased by CNO application. Representative trace (left) and statistical data (right) were shown. n = 3. ( O ) Application with CNO decreased the paw withdrawal threshold in female mice with intraspinal injection of AAV2/5-GfaABC1D-CRE and AAV-DIO-hM3Dq-mCherry. n = 5. Significance: not significant (n.s.) and * P < 0.05.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Immunofluorescence, Imaging, Incubation, Recombinant, Injection

Journal: Science Advances

Article Title: Earlier onset of chemotherapy-induced neuropathic pain in females by ICAM-1–mediated accumulation of perivascular macrophages

doi: 10.1126/sciadv.adu2159

Figure Lengend Snippet: ( A ) Increased ICAM-1 promotes monocyte adhesion to vascular endothelial cells following BTZ treatment. ( B ) IL-1α/ICAM-1 collaboratively promote monocyte transmigration across the vascular wall. ( C ) Infiltrated macrophage-released CCL1 enhances neuron excitability directly or indirectly via the activated astrocytes.

Article Snippet: For neutralizing antibodies CCL1 (20 μg/kg; R&D Systems, MAB845) and IL-1R1 (20 μg/kg; Bio X Cell, BE0256), mice were administered intrathecally 30 min before the injection of BTZ or the IL-1R1 ligand, IL-1α.

Techniques: Transmigration Assay

Journal: PLoS ONE

Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

doi: 10.1371/journal.pone.0065413

Figure Lengend Snippet: Expression of selected chemokine-related genes differentially expressed in CD69-activated B6 compared to CD69 −/− CD4 T cells analyzed by microarray.

Article Snippet: In one experiment mice were treated with chemokine antagonists (anti-CCL-1 Ab 148113 (cat. no. MAB845), anti-CXCL-10 Ab 134013 (cat. no. MAB466) and anti-CCL-19 Ab 87201 (cat no. MAB880) all purchased from R&D Systems, Wiesbaden, Germany).

Techniques: Expressing, Microarray

Journal: PLoS ONE

Article Title: The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

doi: 10.1371/journal.pone.0065413

Figure Lengend Snippet: CD4 T cells were enriched from the spleen of B6 or CD69 −/− mice and migration of these cells was analysed in vitro in transwell system. Medium alone or containing indicated concentrations of CCL-1, CXCL-10, CCL-5 and CCL-4 were loaded in the lower chamber of the transwell system, while CD4 T cells in the medium were loaded in the upper chamber. Cell number migrating from the upper chamber to the chemokine containing chamber trough polycarbonate membrane after 1 h at 37°C, 5% CO 2 was counted by flow cytometry. All the reactions were done in at least four repeats and average cell number per chemokine concentration per cell type was calculated. Results are presented as the number of cells migrating to the wells containing medium alone ( A ) or indicated concentration of CCL-1 ( B ), CXCL-10 ( C ), CCL-5 ( D ) or CCL-4 ( E ). Mean (± SEM) for at least four repeats per each chemokine concentration and cell type is presented. The lower graph set represents chemotactic indexes of B6 and CD69 −/− CD4 T cells for CCL-1 ( F) , CXCL-10 ( G) , CCL-5 ( H ) and CCL-4 ( I ). The chemotactic index was calculated as: the number of cells migrating to the media with chemokine divided by the average number of cells migrating to the media alone. *p≤0.05.

Article Snippet: In one experiment mice were treated with chemokine antagonists (anti-CCL-1 Ab 148113 (cat. no. MAB845), anti-CXCL-10 Ab 134013 (cat. no. MAB466) and anti-CCL-19 Ab 87201 (cat no. MAB880) all purchased from R&D Systems, Wiesbaden, Germany).

Techniques: Migration, In Vitro, Membrane, Flow Cytometry, Concentration Assay