Figure 1.
Recruitment of skin-derived DC in mice developing Amela-melanomas is impaired, but their expression of CCR7 is unaltered.
(A) Ratio of MigDC versus ResDC in Amela-TDLN or corresponding LN from control mice. (B) % TRITC-labeled cells among MigDC in Amela-TDLN or corresponding LN from control mice 20 hrs after TRITC-skin painting (see Methods). (C-F) Analysis of DC subpopulations in LNs from control (C, E) or Amela-bearing (D, F) mice after TRITC-painting. (C-D) After gating on CD11c+NK1.1−CD25−CD45R− cells, TRITC-staining is shown versus SSC. TRITC– and TRITC+ cells were analyzed for expression of CD11c versus MHCII. ResDC and MigDC were found within TRITC− cells; only MigDC were found within TRITC+ cells. Only MigDC expressed CD207 corresponding to Langerhans cells and CD207+ Dermal DC. (E–F) Histograms of CCR7 expression for the different DC subpopulations indicated in C and D, respectively, show low, intermediate and high fluorescence values (mean), respectively, for ResDC, CD207−MigDC and CD207+MigDC.
Figure 2.
TL localization in TDLN of mice developing Amela-melanomas is impaired.
(A) CFSE-labeled CD8 TL from B10.D2 mice (white) were adoptively transferred into control, Mela- and Amela-bearing recipient mice. 15 hrs later their LN (A) were stained with anti-B220-PE-Texas red (red) and anti-CD3-APC (blue) and spleens (B) were stained with anti-B220-APC (blue) (upper pictures) or anti-MOMA-1-biotyl/streptavidin-Alexa647 (blue) (lower pictures) and analyzed by confocal microscopy (see Methods). The graph in (A) represents the number of CFSE+ cells per section of LN. The graph in (B) represents the number of CFSE+ cells per splenic white pulp. Number of white pulps analyzed per mouse was > or = 2. Data are from 8 (control), 3 (Mela-bearing) and 4 (Amela-bearing) mice. Statistics are as described in Methods.
Figure 3.
Lymphoid organization in developing Amela-melanomas is disrupted.
Analysis of LN (A) and spleen (B) from control, Mela- and Amela-bearing mice. (A) In LN, B- and T-zones were identified with anti-B220-PE-Texas red (red) and anti-CD3-FITC (green), respectively. Graphs represent the density of B (left) and T (right) cells, determined by pixel quantification. For tumor-bearing mice, TDLN are shown. Data are from 7 (control), 4 (Mela-bearing) and 4 (Amela-bearing) mice. (B) Splenic B-zones, T-zones and marginal zones were identified with anti-B220-APC (blue), anti-CD3-FITC (green), and anti-MOMA-1/goat anti-rat-Alexa647 (red), respectively. The white pulp areas are delineated by MOMA-1 expression. One graph (left) shows the size of white pulp areas in the different mice (each symbol represents a white pulp area). Density of B (middle graph) and T (right graph) cells were determined by pixel quantification. Data are from 6 (control), 4 (Mela-bearing) and 9 (Amela-bearing) mice.
Figure 4.
Decrease in splenic lymphoid chemokines in mice developing Amela-melanomas.
Spleen sections from control, Mela- and Amela-bearing mice stained in (A) with anti-B220-APC (blue) to detect the B-zone, ER-TR7/chicken anti-rat-Alexa488 (green) to detect the FRC network and anti-CCL21/donkey anti-goat-Alexa546 (red). (B) Expression of ccl21a encoding Ccl21-Ser, ccl19 and cxcl13 relative to tbp transcripts measured by QRT-PCR in total spleens of control, Mela- or Amela- bearing mice. Means (+/− s.e.m.) for independent samples (N = 10; 8; 14 for ccl21a; N = 5; 4; 8 for ccl19; N = 3; 3; 9 for cxcl13) from spleens of control, Mela- or Amela- bearing mice, respectively, A is representative of 5 mice per group.
Figure 5.
Loss of gp38+ FRC-associated CCL21 in spleens and TDLN of mice developing Amela-melanomas.
In (A) spleen sections were stained with ER-TR7/chicken anti-rat-Alexa647 (blue), anti-gp38/donkey anti-goat-Alexa488 (green) and anti-CCL21-biotyl/SA-Alexa546 (red). In (B) LN sections were stained with anti-B220-Alexa647 (blue), anti-gp38/donkey anti-goat-Alexa546 (red) and anti-CCL21-biotyl/SA-Alexa488 (green). A and B are representative of 3 mice per group. Lower images show magnifications of the fields highlighted in the upper images and show stainings with each of the antibodies separately and merged, as indicated.
Figure 6.
Splenic architecture is disrupted, with loss of contractile fibroblasts, in mice developing Amela-melanomas.
Spleens from control, Mela-, and Amela-bearing mice stained in (A) with ER-TR7/chicken anti-ratAlexa488 (left), anti-laminin/donkey anti-rabbit-Alexa488 (middle), and anti-collagen IV/donkey anti-rabbit-Alexa488 (rigth) to detect stromal networks, and in (B) with anti-B220-FITC (green), anti-Desmin/donkey anti-rabbit-Alexa555 (red). Representative of 5 mice per group.
Figure 7.
Gr1+CD11b+ cells recruited in SLO interact with the stroma in mice developing Amela-melanomas.
LN (A) or spleen (B) sections from control, Mela- and Amela-bearing mice were stained with anti-Gr1-PE (red) and anti-Lyve-1-biotyl/streptavidin-FITC (green) to detect LN medullas or for MOMA-1 (green) to delineate splenic white pulp areas, respectively. LN (C) or spleen (D) sections from Amela-bearing mice were stained with anti-Gr1-APC (green) and anti-ER-TR7/chicken anti-rat-Alexa647 (blue). Magnification showing a cluster of Gr1+ cells on the stromal network is shown.