Figure 1.
Immunofluorescence analysis of immune cell distribution and white pulp area.
Frozen sections of spleen and LN from p110δWT/WT, p110δD910A/D910A, and reconstituted mice were immunofluorescence-stained to detect T cells (CD3+, Thy1.2+), B cells (B220+), MMM (MOMA+) and DC (CD11c+). Representative images of spleen (A) and LN (B) sections for all conditions are shown (n = 6 mice/condition). Bar = 200 µm. (C) Measurement of white pulp area in hematoxylin/eosin-stained frozen spleen sections (3 sections/mouse, 6 mice/condition), quantified with ImageJ software. Mean ± SD; Kolmogorov-Smirnov test, ***p<0.001.
Figure 2.
Absolute numbers of spleen and LN total cells, CD4+ and CD8+ T cells before and after antigen stimulation.
Spleens and LN were extracted from p110δWT/WT, p110δD910A/D910A, and reconstituted mice in homeostatic conditions (t = 0) and after antigen stimulation (five days post-injection of inactivated C. albicans, t = 5 d). Whole organ cell suspensions were counted to determine total cell number (A, D) and stained to determine CD4+ T (B, E) and CD8+ (C, F) cell numbers by flow cytometry (n = 6 mice/condition). Mean ± SD.
Figure 3.
Absolute numbers of spleen B cells and DC before and after antigen stimulation.
Spleens were extracted from p110δWT/WT, p110δD910A/D910A, and reconstituted mice in homeostatic conditions (t = 0) and after antigen stimulation (five days post-injection of inactivated C. albicans, t = 5 d). B cell (A) and DC (B) were stained and cell numbers determined by flow cytometry (n = 6 mice/condition). Mean ± SD.
Figure 4.
FACS analysis of stromal cell populations in spleen from p110δWT/WT and p110δD910A/D910A mice.
Spleens from p110δWT/WT and p110δD910A/D910A mice were processed and stained with anti-CD45, -TER119, -CD31, and -gp38 mAb. A) Representative gating strategy for the analysis of stromal cell populations. Stromal cells were gated via the exclusion of dead, CD45-, and TER119-positive cells. B) Quantification of the percentage and absolute number of stromal cell populations in spleens of p110δWT/WT and p110δD910A/D910A mice (n = 3 experiments/spleen, 6 mice/group). Student's t-test, *p<0.05.
Figure 5.
p110δ mRNA expression in spleen stromal cell populations from p110δWT/WT and p110δD910A/D910A mice.
Total RNA was extracted from sorted p110δWT/WT and p110δD910A/D910A spleen stromal cell subsets (n = 5 mice/genotype). Lymphoid cells (CD45+) were sorted as control. Expression of p110δ mRNA was analyzed by qRT-PCR. Normalized quantities (mean 2−ΔCt) of p110δ mRNA are shown.
Figure 6.
qRT-PCR analysis of homeostatic chemokines and TNF family members in spleen, LN and spleen stromal cell subsets from p110δWT/WT and p110δD910A/D910A mice.
Total RNA was extracted from p110δWT/WT and p110δD910A/D910A spleen, LN, and sorted spleen stromal cell subsets (n = 5 mice/genotype). Expression of CCL19, CCL21, LTα, LTβ and LTβR was analyzed by qRT-PCR in spleen (A), LN (B), and stromal cell subsets (C). Normalized quantities (mean 2−ΔCt) of mRNA are depicted. Student's t-test, *p<0.05, **p<0.01, ***p<0.001.