Table 1.
Primers used for amplification cattle XCR1 and XCL1 genes.
Table 2.
Antibodies used to isolate cattle blood cell populations.
Table 3.
Primers used for SYBR green real-time RT-PCR.
Table 4.
Primary and secondary antibodies used for 6-color flow cytometric analysis of cattle DCs subsets.
Fig 1.
Alignment of cattle XCR1 and XCL1 deduced from the nucleotide sequence of the cloned cDNA.
(A)The cloned cattle XCR1 showed 89, 92, 77, 71 and 78% aa identity homology with that of horse, pig, monkey, mouse and human, respectively. The conserved regions were marked with black frame. The mutation site in signal motif and Cystine sites forming potential disulfide were tagged in black circle and diamond patterns, separately. (B)The cloned cattle XCL1 showed 67, 78, 61, 57 and 67% aa identity homology with that of horse, pig, monkey, mouse and human, respectively. The different signal peptide cleavage sites were denoted with arrow.
Fig 2.
Analysis of gene expression of cattle XCR1 and Clec9A from isolated blood subset cells by RT-PCR.
The relative expression of cattle XCR1 mRNA from isolated subset cells (A) and lin- subset cells (B). The relative expression of cattle Clec9A mRNA from isolated subset cells(C) and lin- subset cells (D). Data are representative of four independent experiments in three cattle. Error bars represent standard deviation.
Fig 3.
Phenotypic characterization of cattle blood DC subsets.
6-color flow cytometric was performed to determinate the distribution of the CD26, CADM1, CD205 and CD172a on subsets of cattle blood DCs. The pre-enriched DCs (A) obtained by depletion of lineage cells (anti-bovine CD3/CD11b/CD14/CD21/CD335) from PBMCs were subjected to flow cytometry. Gate 1 (B) was selected to exclude cells debris with lower values of SSC-A and FSC-A, and further analyzed to gate singlets based on diagonal streak of FSC-A and FSC-H plot. Both of dead cells and the remained lineage cells (C) were together excluded by staining with Fixable viability Stain 620 and anti-mouse IgG1 PE-CF594. lin- cells were then gated to identify the MHCII+ and CD4+cells (D) as well as the MHCII+ and CD4+ cells (E). CD11c+ cDCs were further gated to analysis the plot (F) of CD26+ and CD172a+ cells. Overlap histograms for surface expression of CD11c (G), CD26 (H), CD205 (I) and CADM1 (J) on lin- cells, CD172a (K) and CD26 (L) on CD11c-cDCs, CD172a (M) and CD26 (N) on CD11c+cDCs, CADM1 (P) and CD205 (Q) on CD26+ DCs, as well as CD26 (O) on pDCs, based on FMO control. Numbers in histograms represent average percentage of cells expressing the surface molecules in three cattle, and error bar indicate standard error.
Fig 4.
Analysis of gene expression of cattle XCL1 in isolated blood subset cells and CD8+ T cells after stimulation.
The relative expression of cattle XCL1 mRNA from isolated subset cells(A) as well as obtained CD8+ T cells after stimulation with the PMA plus calcium ionomycin, or the Concanavalin A, respectively for different time (B). All the data above was representative of four independent experiments in three cattle.
Fig 5.
Chemotactic migration of cattle XCR1+ DC by mouse XCL1 and human XCL1.
(A) The relative expression of cattle XCR1 mRNA from input cells (CD26+ DCs) in upper chamber and migrated cells (XCR1+ DC) in lower chamber. Difference in XCR1 expression between the two populations was calculated using one-tailed nonparametric t-test (Mann-Whitney test). A significant difference was defined as P ≤ 0.05. (B) Transwell assay on CD26+ DCs by mouse XCL1 and human XCL1 with concentration varied from 100 ng/ml to 500 ng/ml.