cxcl12  (R&D Systems)

Journal: NPJ Biofilms and Microbiomes

Article Title: Natural killer cell effector function is critical for host defense against alcohol-associated bacterial pneumonia

doi: 10.1038/s41522-024-00558-w

Figure Lengend Snippet: Mouse primary NK cells were collected from alcohol-fed and treated mice and added to the apical side of a Transwell with migration assessed in response to ( A ) media alone, and ( B ) media with CCL2/CXCL12 following 5 h of incubation. C Primary NK cells were harvested from control and binge-on-chronic alcohol-fed mice with and without treatment. NK cells were then incubated with K. pneumoniae for 3 h. and bacterial viability was determined by serial dilution. p -values as determined by one‐way ANOVA with Sidak’s multiple comparisons are shown in the figure. N = 3 wells of primary NK cells/group (6 mice/group, NK cells per 2 mice were pooled).

Article Snippet: Cat. No. 45-SRP3215-10UG), 100 ng/mL of CXCL12 (R&D Systems, Cat. No. 460-SD-050), 100 ng/mL of CXCL9 (R&D Systems, Cat. No. 492-MM-010), 100 ng/mL CXCL10 (R&D Systems, Cat. No. 466-CR-010), 100 ng/mL CXCL11 (R&D Systems, Cat. No. 572-MC-025), 300 ng/mL CX3CL1 (R&D Systems, Cat. No. 472-FF-025) was added to the bottom of the of the Transwell.

Techniques: Migration, Incubation, Control, Serial Dilution

Journal: NPJ Biofilms and Microbiomes

Article Title: Natural killer cell effector function is critical for host defense against alcohol-associated bacterial pneumonia

doi: 10.1038/s41522-024-00558-w

Figure Lengend Snippet: Mouse primary splenic NK cells were collected from alcohol-fed and treated mice and added to the apical side of a Transwell and migration in response to different chemokines was assessed following 5 h of incubation. A Confirmation of NK cell purification. (a1) Percentage of NK cells pre-isolation, and (a2) percentage of NK cells post-isolation. NK cell migration response profiles to ( B ) medial alone, ( C ) CCL2, ( D ) CXCL12, ( E ) CX3CL1, and ( F ) CCL9-11. Percent migration was calculated as the total number of viable NK cells in the bottom well divided by the total number of viable NK cells added to the Transwell insert. p values are indicated in the figure by one‐way ANOVA with Sidak’s multiple comparison. N = 6–7 wells of primary NK cells/group (12–14 mice/group, NK cells per 2 mice were pooled) derived from 2 independent experiments.

Article Snippet: Cat. No. 45-SRP3215-10UG), 100 ng/mL of CXCL12 (R&D Systems, Cat. No. 460-SD-050), 100 ng/mL of CXCL9 (R&D Systems, Cat. No. 492-MM-010), 100 ng/mL CXCL10 (R&D Systems, Cat. No. 466-CR-010), 100 ng/mL CXCL11 (R&D Systems, Cat. No. 572-MC-025), 300 ng/mL CX3CL1 (R&D Systems, Cat. No. 472-FF-025) was added to the bottom of the of the Transwell.

Techniques: Migration, Incubation, Purification, Isolation, Comparison, Derivative Assay

Journal: NPJ Biofilms and Microbiomes

Article Title: Natural killer cell effector function is critical for host defense against alcohol-associated bacterial pneumonia

doi: 10.1038/s41522-024-00558-w

Figure Lengend Snippet: Human NK cells (NK-92 cell line) were treated with alcohol, indole, or recombinant TGF-β1, and NK cell migration in response to the CCL2/CXCL12 was assessed. p values are indicated in the figure by one‐way ANOVA with Sidak’s multiple comparison. N = 9 wells of NK-92 NK cells/group derived from 3 independent experiments.

Article Snippet: Cat. No. 45-SRP3215-10UG), 100 ng/mL of CXCL12 (R&D Systems, Cat. No. 460-SD-050), 100 ng/mL of CXCL9 (R&D Systems, Cat. No. 492-MM-010), 100 ng/mL CXCL10 (R&D Systems, Cat. No. 466-CR-010), 100 ng/mL CXCL11 (R&D Systems, Cat. No. 572-MC-025), 300 ng/mL CX3CL1 (R&D Systems, Cat. No. 472-FF-025) was added to the bottom of the of the Transwell.

Techniques: Recombinant, Migration, Comparison, Derivative Assay

Journal: Development (Cambridge, England)

Article Title: CXCL12 promotes the crossing of retinal ganglion cell axons at the optic chiasm

doi: 10.1242/dev.202446

Figure Lengend Snippet: Cxcl12 is expressed by ventral diencephalon meninges and Cxcr4 is expressed by RGCs. (A,B) In situ hybridisation for Cxcl12 and Cxcr4 on coronal vibratome sections through the ventral diencephalon (A) and retina (B) of E12.5, E14.5 and E17.5 mouse embryos. Asterisks in A indicate the position in the ventral diencephalon where the optic chiasm (oc) will form. In B, arrows indicate expression of Cxcr4 in the RGC layer of the retina and arrowhead indicates the hyaloid vasculature. (C) Cryosection through the E17.5 peripheral ventrotemporal (VT) retina stained after Cxcr4 in situ hybridisation by immunofluorescence with antibodies specific for BRN3A (labels contralaterally projecting RGCs) and ZIC2 (labels ipsilaterally projecting RGCs). Staining is shown as the combined and single channels. Dotted lines indicate the boundary between ZIC2-positive and -negative RGCs. Scale bars: 200 µm (A,B); 100 µm (C). D, dorsal; V, ventral; VT, ventrotemporal.

Article Snippet: The explants were treated with 0-250 ng ml −1 recombinant mouse CXCL12 (Bio-Techne, 460-SD-010), 0-100 ng ml −1 recombinant chicken CXCL12 (Cambridge Bioscience, RP1354CT) or positioned 100-400 µm from pieces of ventral diencephalon meninges.

Techniques: In Situ, Hybridization, Expressing, Staining, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: CXCL12 promotes the crossing of retinal ganglion cell axons at the optic chiasm

doi: 10.1242/dev.202446

Figure Lengend Snippet: CXCL12 and CXCR4 are required for contralateral RGC axon growth at the optic chiasm. (A) Ventral views of RGC axons labelled from one eye in E14.5 Cxcl12 and Cxcr4 wild-type and mutant littermates captured using a stereo microscope. The optic nerve (on), optic chiasm (oc), contralateral optic tract (ot c ) and ipsilateral optic tract (ot i ) are indicated. Boxed region is shown at higher magnification in the lower panels; brackets indicate the width of the ipsilateral projection. Number analysed of each genotype was: Cxcl12 +/+ n =8, Cxcl12 +/− n =12, Cxcl12 −/− n =6; Cxcr4 +/+ n =5, Cxcr4 +/− n =10, Cxcr4 −/− n =5. (B) Ipsilateral (Ipsi) and contralateral (Contra) flatmounted retinas from retrogradely labelled E15.5 Cxcl12 and Cxcr4 wild-type and mutant littermates. Dotted line demarcates the ventrotemporal crescent (VTC). d, dorsal; n, nasal; t, temporal; v, ventral. Number analysed of each genotype was: Cxcl12 +/+ n =12, Cxcl12 +/− n =11, Cxcl12 −/− n =4; Cxcr4 +/+ n =10, Cxcr4 +/− n =19, Cxcr4 −/− n =4. Graphs display quantification of the Ipsilateral Index (A) and proportion of ipsilaterally projecting RGCs relative to the total number of labelled cells in both eyes (B), with each dot representing the value for one embryo and horizontal bars the mean values. Statistical analyses were performed using one-way ANOVA with Tukey post-hoc comparison. Scale bars: 200 µm.

Article Snippet: The explants were treated with 0-250 ng ml −1 recombinant mouse CXCL12 (Bio-Techne, 460-SD-010), 0-100 ng ml −1 recombinant chicken CXCL12 (Cambridge Bioscience, RP1354CT) or positioned 100-400 µm from pieces of ventral diencephalon meninges.

Techniques: Mutagenesis, Microscopy, Comparison

Journal: Development (Cambridge, England)

Article Title: CXCL12 promotes the crossing of retinal ganglion cell axons at the optic chiasm

doi: 10.1242/dev.202446

Figure Lengend Snippet: CXCL12 and ventral diencephalon meninges promote RGC axon outgrowth. (A-C) Representative examples and quantification of E14.5 mouse (A,B) or E6 chicken (C) retinal explants cultured in the presence and absence of CXCL12 (A,C) or ventral diencephalon meninges (B). Mouse explants from peripheral ventrotemporal (VT) retina contain predominately ipsilaterally projecting RGCs; peripheral dorsotemporal (DT), ventronasal (VN) and dorsonasal (DN) explants contain contralaterally projecting RGCs. All RGCs in chicken project contralaterally. Dotted lines in B indicate the edge of the meningeal tissue. Quantitative data are shown as boxplots with the median values (middle bars) and first to third interquartile ranges (boxes); whiskers indicate 1.5× the interquartile ranges; each dot represents the value from one explant. A minimum of 22 explants from three independent experiments (A), 13 explants from three independent experiments (B) or 18 explants from four independent experiments (C) were analysed per condition. Statistical analyses were performed using Kruskal–Wallis rank sum test with TUKEY-Kramer post-hoc comparison. Scale bars: 200 µm.

Article Snippet: The explants were treated with 0-250 ng ml −1 recombinant mouse CXCL12 (Bio-Techne, 460-SD-010), 0-100 ng ml −1 recombinant chicken CXCL12 (Cambridge Bioscience, RP1354CT) or positioned 100-400 µm from pieces of ventral diencephalon meninges.

Techniques: Cell Culture, Comparison

Journal: Nature immunology

Article Title: T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control

doi: 10.1038/s41590-023-01443-y

Figure Lengend Snippet: (A) Heatmap of select genes differentially expressed by sorted CD45−CD31+gp38+ lymphatic endothelial cells (LEC) from naïve skin (N; n= 3 mice) or BPC mouse melanomas (BPC; n= 4 mice). (B) Normalized CXCL12 transcript counts from naïve (n=3 mice) or BPC tumor-associated LECs (n=4 mice). (C) Frequency of CXCL12-dsRed+ cells of CD45−CD31+gp38+ LECs from normal adjacent skin or YUMMER.17 tumors in CXCL12-dsRed reporter mice. (D) Frequency of CXCL12-dsRed+ cells of gp38+LYVE-1lo or gp38+LYVE-1hi LECs. For C-D, n=8. (E) Representative images of CXCL12 (green) in YUMMER1.7 tumors implanted in Prox1-TdTomato (red) reporter mice. Nuclei, DAPI (blue). Arrows = CXCL12+ lymphatic vessels; scale bar = 100 μm. Inset, far right; scale bar = 50 μm. Immunofluorescence staining was repeated in 3 independent rounds with at least 3 independent samples per round. (F) Representative images of LYVE-1+ and LYVE-1− lymphatic vessels (LYVE-1, blue; Prox1, red; CXCL12, green; CD8, cyan; Scale bar = 50μm). (G) Quantification of CXCL12 pixel area of (left) and CD8+ T cell density proximal to (right) Prox1+LYVE-1+ and Prox1+LYVE-1− peritumoral lymphatic vessels. Each point is a vessel, sampled over three mice. (H) Representative peritumoral and intratumoral images of CD8+ T cells (green) and LYVE-1+ (red) lymphatic vessels in YUMMER1.7 tumors (day 14) of CXCL12WT or CXCL12ΔiProx1 mice. Nuclei, DAPI (blue). Scale bars = 50 μm. (I) Frequency of LYVE-1+ pixels per image in the peritumoral region of YUMMER1.7 tumors in CXCL12WT (n=9) or CXCL12ΔiProx1 (n=8) mice. (J) Frequency of CD8+ pixels per image in peritumoral and intratumoral regions of YUMMER1.7 tumors in CXCL12WT (n=11) or CXCL12ΔiProx1 (n=8) mice; results of 2 independent experiments. For all experiments, each symbol represents one mouse except in G. Two-sided, unpaired (B, G, and I-J) and paired (C-D) student’s t-tests.

Article Snippet: 100 ng/ml recombinant mouse CXCL12 (R&D Systems) was added when indicated.

Techniques: Immunofluorescence, Staining

Journal: Nature immunology

Article Title: T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control

doi: 10.1038/s41590-023-01443-y

Figure Lengend Snippet: (A) Frequency of CD8+ Tcells in the blood (left) or lymph (right) following acute treatment with FTY720 (FTY) or AMD3100 (AMD). n=3–5 miceper group. (B) Shannon diversity index for TCRb sequences detected in YUMMER1.7 tumors 7 days post treatment withAMD3100 (AMD; n=10) or vehicle control (Cont; n=8). Error bars = standard error; center = mean. (C) Frequency ofIFNg+TNFa+ producing CD44+CD8+ T cells following ex vivo CD3/CD28 restimulation in the presence or absence ofAMD3100. n=5 (D) Frequency of IFNg+TNFa+ producing endogenous CXCR4WT (n=3) or CXCR4ΔUBC (n=3) CD44+CD8+T cells following ex vivo CD3/CD28 restimulation. (E) Frequency of IFNg+TNFa+ producing CD44+CD8+ T cells followingex vivo CD3/CD28 restimulation in the presence (n=3) or absence (n=3) of recombinant murine 100 ng/ml CXCL12. ForD-E, error bars = standard deviation; center = mean. For all graphs, each symbol represents one mouse. One-way ANOVAadjusted for multiple comparisons (A and C), two-sided Mann-Whitney test (B), two-sided, unpaired student’s t-test (D andE). (F) YUMM1.7 tumor growth during treatment with vehicle control (Cont; black; n=5), AMD3100 (AMD; blue; n=5), aPD-L1 (PD-L1; red; n=5), or combination AMD3100+aPD-L1 (A+P; green n=5); one experiment. Fractions indicate rate of tumor control defined as daily growth rate <30mm3 post onset of therapy.

Article Snippet: 100 ng/ml recombinant mouse CXCL12 (R&D Systems) was added when indicated.

Techniques: Inhibition, Control, Ex Vivo, Recombinant, Standard Deviation, MANN-WHITNEY

Journal: Nature immunology

Article Title: T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control

doi: 10.1038/s41590-023-01443-y

Figure Lengend Snippet: (A) RNAScope of Cxcl12 transcripts in YUMMER1.7 tumors (purple); hematoxylin (blue). Repeated in 3 tumors. Leftimage: scale bar = 400 μm; middle and right images: scale bar = 200 μm (B) Frequency of Cxcl12-dsRed+ cells inYUMMER1.7 tumors (n=11 across 2 independent experiments). (C) Representative flow plots of CD45-CD31+gp38+dsRed+lymphatic endothelial cells (LEC) in Cxcl12-dsRed reporter mice or littermate controls (Cxcl12+/+). (D) Frequency ofCxcl12+ in LECs, blood endothelial cells (CD45-CD31+gp38-, BEC), epithelial cells (CD45-CD31-EpCAM+, Epi),fibroblasts (CD45-CD31-PDGFRa+, Fibro) in YUMMER1.7 tumors of Cxcl12-dsRed reporter mice (n=14; 3 independentexperiments). (E) Frequency of Cxcl12+ in B cells (CD3e-B220+), T cells (CD3e+B220-), neutrophils (CD3e-B220-CD11b+Ly6G+, Neutro), dendritic cells (CD3e-B220-CD11c+MHCII+, DCs), immature monocytes (CD3e-B220-CD11b+Ly6C+, Imm Mono), and macrophages (CD3e-B220-CD11b+Ly6C-F4/80+MHCII+/−, TAMs) in YUMMER1.7tumors of Cxcl12-dsRed reporter mice (n=3). (F) RNAScope of Cxcl12 transcripts in YUMM1.7 tumors (purple);hematoxylin (blue). Repeated in 3 tumors. Left image: scale bar = 400 μm; middle and right images: scale bar = 200 μm(G) Frequency of Cxcl12+ in CD45-stromal cells in YUMM1.7 tumors of Cxcl12-dsRed reporter mice (n=3). (H) Frequencyof Cxcl12+ CD45+ cells in YUMM1.7 tumors of Cxcl12-dsRed reporter mice (n=3). (I and J) Frequency of Cxcl12+ BECs(I) or fibroblasts (J) from normal adjacent skin or YUMMER1.7 tumors in Cxcl12-dsRed reporter mice (n=8; 2 independentexperiments). (K) qRT-PCR of CXCL12 transcript levels from hypoxic LN LECs isolated from CXCL12WT orCXCL12ΔiProx1 mice (n=3). Transcripts normalized to b-actin and represented as fold change from WT. (L) Lymphaticvessel dilation in tumors implanted in CXCL12WT or CXCL12ΔiProx1 mice (n=5). (M and N) Quantification of total LEC aspercent of live (M) and proportion of LYVE-1+ LECs (N); n=6–7 mice. (O and P) Quantification of regulatory T cells(TREG, CD3e+CD4+CD25+Foxp3+) as a percent of live (O) or of CD4+ T cells (P); n=4–5 mice. For all graphs, each symbolrepresents one mouse. Two-sided paired (B, I, and J) and unpaired (K-P) student’s t-test. n.s. = not significant.

Article Snippet: 100 ng/ml recombinant mouse CXCL12 (R&D Systems) was added when indicated.

Techniques: Expressing, RNAscope, Quantitative RT-PCR, Isolation

Journal: Nature immunology

Article Title: T cell egress via lymphatic vessels is tuned by antigen encounter and limits tumor control

doi: 10.1038/s41590-023-01443-y

Figure Lengend Snippet: (A) Representative plot of intratumoral CD44+CD8+ T cells used in figures B-D: PD-1−TIM3− (−/−); PD-1+TIM3− (+/−); PD-1+TIM3+ (+/+). (B-D) Quantification of CXCR4 (B), CXCR6 (C), and ACKR3 (D) on intratumoral PD-1 vs TIM3 CD44+CD8+ T cells from YUMMER1.7 tumors. Each symbol represents one mouse. (E) Number of wild type (WT) (n=8) or CXCR4-deficient (KO) (n=12) effector CD8+ OT-1 TCR-Tg T cells in B16.F10OVA tumor dLNs 20hrs post intratumoral transfer (per 105 transferred cells). For E, 2 independent experiments. (F) UMAP plot depicting Seurat clusters (1269 cells; pooled from 3 mice), (G) feature plot representation of Cxcr4 (blue) and Cxcr6 (red) expression in each cell, (H) and Monocle 3 pseudotime analysis on intratumoral CD8+ T cells from day 21 YUMMER1.7 tumors. (I and J) Scoring of cells with a Retained vs. Egress signature derived from bulk RNAseq (expression of Retained program – Egress program). Scores were visualized on individual cells (I) and compared across Monocle branches (J). Monocle Branch 1 (n=623 cells), Branch 2 (n=260 cells), Branch 3 (n=386 cells). (K) Skin residence (GSE47045) and (L) TCR signaling (Reactome: R-MMU-202424). Boxplots, center represents median, upper and lower box bounds represent upper and lower quartiles respectively, and whiskers represent observations within 1.5*IQR of the upper and lower quartiles. (M) Time course of CXCR4 (left) and ACKR3 (right) surface expression on OVA-specific CD44+CD8+ T cells following ex vivo stimulation in the absence (unstim) or presence of CD3ε/CD28 antibodies. (N) Frequency of OVA-specific CD44+CD8+ T cells migrated toward CXCL12 following stimulation with (+) or without (−) SIINFEKL (N4) peptide. (O and P) Representative histograms (O) and quantification (P) of CXCR4 and ACKR3 on OVA-specific CD44+CD8+ T cells following 24hr ex vivo stimulation in the absence (U) or presence of CD3ε/CD28 antibodies (Ab) or peptides: SIINFEKL (N4), SIITFEKL (T4), or SIIQFEKL (Q4). For M and P, n=4 mice; error bars = standard deviation; for N, n=3 mice; representative graphs from 3 independent experiments. MFI=mean fluorescence intensity. One-way ANOVA adjusted for multiple comparisons (B-D, M-N, P); two-sided, unpaired student’s t-test (E); two sample wilcoxon rank-sum with a two-sided test (J-L).

Article Snippet: 100 ng/ml recombinant mouse CXCL12 (R&D Systems) was added when indicated.

Techniques: Expressing, Derivative Assay, Ex Vivo, Standard Deviation, Fluorescence