Fig 1.
Plasmacytoid dendritic cells in hukman kidney.
(a) Gating strategy with the markers used to differentiate between the pDC in yellow, cDC1 in purple and cDC2 in green. (b) CD303+ pDCs are less then 2% of the total CD11b+ cells (upper panels). Plasmacytoid DCs are small and round cells positive for HLA-DR (lower panels). In here the plots of the kidney n° 4 are shown, similar results were obtained with the kidney n° 5. (c) In here the plots of the kidney n°2 characterized by high numbers of infiltrating pDC are shown, similar results were obtained with the kidneys n° 1 and 3.
Table 1.
Antibody panels.
Table 2.
Characterization of nephrectomy specimen used for isolation of infiltrating leukocytes and histological analysis.
Table 3.
Relative percentage of Dendritic cell subtype.
Fig 2.
CD141 positive cells in human kidney.
Two distinct populations of CD11c+ CD11b- (upper panels) and CD11c- CD11b+ (lower panels) express the cDC1 marker CD141. CD11c+ CD141+ cells have the typical dendritic cell shape and high expression of HLA-DR but are negative for DC-SIGN. These characteristics identify them as cDC1 cells. The CD11b+ CD141+ have similar granularity to macrophages and express DC-SIGN together with HLA-DR. In here the plots of the kidney n° 5 are shown, similar results were obtained with the kidney n° 4. (b) In here the CD141 positive cells, plots of the kidney n°2 characterized by high numbers of infiltrating pDC are shown similar results were obtained with the kidneys n° 1 and 3.
Fig 3.
cDC2 dendritic cells in human kidney.
The marker CD1c is expressed by CD11b+ cells (left panel) but not by CD11c+ cells. The cDC2 subtype has low granularity and size, all express HLA-DR and 73% express high levels of DC-SIGN. In here the plots of the kidney n° 5 are shown, similar results were obtained with the kidney n°4. (b) In here the CD1c positive cells, plots of the kidney n°2 characterized by high numbers of infiltrating pDC are shown, similar results were obtained with the kidneys n° 1 and 3.
Fig 4.
(a) Gating strategy with the markers used to identify monocytes and macrophages. The boxes are color-coded with classical monocytes in blue, non-classical in green and intermediate in red. (b) Dot-plot of the CD45 gate followed by CD14 versus CD16 gate showing the relative percentage of classical, non-classical and intermediate monocytes (lower left). The monocytes were homogeneous in size and granularity (upper right). Differential expression of HLA-DR (lower central) can be use instead of CD16 to differentiate between the three subtypes which are distinguished by the same color coding as in 1a (lower right). In here the plots of the kidney n°5 are shown, similar results were obtained with kidney n°4.
Fig 5.
Monocytes derived macrophages in inflamed kidney.
The proportion of CD14 positive cells that express CD16 cells is greater in kidneys with low numbers of pDC than in those with high numbers of pDC, and includes both CD14int CD16+ and CD14+CD16+(central panel). The addition of 25F9, a marker for monocyte derived infiltrating macrophages and the active phagocytic marker CD68 reveals that the expanded population includes CD14 positive macrophages in addition to non-classical and intermediate monocytes, as it shown also by the scatter plot property. In here the plots of the kidney n° 2 are shown, similar results were obtained with the kidneys n° 1 and 3.
Fig 6.
Gating out macrophages reconstitutes a monocyte percentage similar to the less inflamed kidney.
Gating out the CD14+ CD68+25F9+ macrophages reveals similar proportions of the three monocyte subtypes as in the less inflamed kidney. The CD68+25F9+ have the shape of macrophages and they share similar levels of CD14 and CD16 in respect to intermediate and non-classical macrophages as indicated by the merge plot were blue dots are macrophages and red dots are monocytes (lower right). In here the plots of the kidney n° 2 are shown, similar results were obtained with the kidneys n° 1 and 3.
Table 4.
Percentage monocytes subtype (Gated on 25F9- CD68-) and Macrophages.
Fig 7.
The correlation among the renal MPh suggests a co-ordinate response to inflammation.
The relative percentage of the DC and monocytes subtypes and macrophages of the five kidneys clustered into two groups based on the level of pDC.
Fig 8.
Immunohistological characterization.
Sections from two RTN and CDN specimen adjacent to those used for FACS were stained for morphological evaluation with PAS and an antibody to CD45.
Fig 9.
Localization of the DC subtypes in kidney cortex.
Histological sections from CDN (case 5) and RTN (case 1) adjacent to those used for FACS were stained with CD303, CD141 and CD1c in order to localize the DC subtype and count their absolute number. CD303 expressing pDC (double staining with CD68 in brown) are rare in the renal cortex of the donor nephrectomy specimens (CDN) whereas CD141+ cells (arrows) were more common and CD1c+ cells (arrows) were easily detectable. There were three-to seven-fold more pDC (arrows) in the tumour nephrectomies (RTN) compared to the donor kidneys, but rarely any CD141 expressing cells (arrows).
Table 5.
Absolute count and relative percentage of Dendritic cell subtype in histological sections.