Epithelial LTβR signaling controls the population size of the progenitors of medullary thymic epithelial cells in neonatal mice - PubMed (original) (raw)

Epithelial LTβR signaling controls the population size of the progenitors of medullary thymic epithelial cells in neonatal mice

Weiwei Wu et al. Sci Rep. 2017.

Abstract

The establishment of T cell central tolerance critically relies on the development and maintenance of the medullary thymic epithelial cells (mTECs). Disrupted signaling of lymphotoxin beta receptor (LTβR) results in dramatically reduced mTEC population. However, whether LTβR directly or indirectly control mTECs remains undetermined; how LTβR controls this process also remain unclear. In this study, by utilizing K14-Cre × Ltbrfl/fl conditional knockout (cKO) mice, we show that epithelial intrinsic LTβR was essential for the mTEC development postnatally. Mechanistically, LTβR did not directly impact the proliferation or survival of mTECs; the maturation of mTECs from MHC-IIlo to MHC-IIhi stage was also unaltered in the absence of LTβR; interestingly, the number of mTEC progenitors (Cld3,4hiSSEA-1+) was found significantly reduced in LTβR cKO mice at the neonatal stage, but not at E18.5. Consequently, epithelial deficiency of LTβR resulted in significant defect of thymic negative selection as demonstrated using OT-I and RIP-OVA transgenic mouse system. In summary, our study clarifies the epithelial intrinsic role of LTβR on mTEC development and function; more importantly, it reveals a previously unrecognized function of LTβR on the control of the size of mTEC progenitor population.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. Epithelial LTβR is required for postnatal mTEC development.

(a) The distribution of mTECs and cTECs was assessed by immunofluorescence staining of UEA-1 (green) and Ly51 (red) on the thymi from _Ltbr_fl/flK14Cre and _Ltbr_fl/+K14Cre mice of 4 weeks old. The scale bar represents 1000 μm. (b) Quantitative analysis of thymic medulla area as determined by UEA-1 staining. Values indicate mean ± SD from at least 3 thymus sections from mice of 4 weeks old. Representative of at least 3 independent experiments. (c) Thymic stromal cells from _Ltbr_fl/flK14Cre and control mice of different ages as indicated were prepared and then stained with anti-CD45, anti-EpCAM, UEA-1 and anti-Ly51. Within TEC population (CD45−EpCAM+), mTECs and cTECs were identified as UEA-1+Ly51− and UEA-1−Ly51+, respectively. (d,e) The frequency and absolute number of mTECs are shown. (f) The absolute number of cTECs is shown. One representative data was shown as mean ± SD for more than 3 mice each group. All the experiments were repeated more than three times. An unpaired two-tailed Student’s _t_-test is used: *P < 0.05; **P < 0.01, ***P < 0.001, ****P < 0.0001. cKO, conditional knockout; cTEC, cortical thymic epithelial cell; mTEC, medullary thymic epithelial cell; mTECp, medullary thymic epithelial cell progenitor; PN, postnatal.

Figure 2

Figure 2. Epithelial LTβR is not required for MHC-II and Aire expression on mTECs.

(a) After gating mTECs (CD45−EpCAM+UEA-1+Ly51−) from TECs, the maturation of mTECs in adult mice was assayed by MHC-II and Aire expression. (b) The frequency of each mTEC subset (Aire−MHC-II+,Aire−MHC-II++,Aire+MHC-II++) of mTECs and their absolutely numbers are shown. (c) MFI of Aire expression on Aire+MHCII++ mTEC subpopulation was determined by flow cytometry. (d) The relative mRNA expression of Aire, Ins2, Ins1, Col II and Crp in total thyme of indicated mice were determined by RT-PCR. All the experiments shown have been repeated for more than 3 times with at least 3 mice each group. An unpaired two-tailed Student’s _t_-test is used: *P < 0.05, **P < 0.01. Aire, autoimmune regulator.

Figure 3

Figure 3. Epithelial LTβR is not required for mTEC proliferation and apoptosis.

(a,b) After the surface staining, mTECs (CD45−EpCAM+UEA-1+Ly51−) from each group were intracellularly stained with Ki-67 for comparing their proliferation ability. Representative plots from mice at various time points are shown (a). The percentages of Ki-67 positive cells in mTECs of each group are plotted (b). (c and d) Intracellular staining of active caspase 3 in mTECs was used to distinguish the apoptosis cells in _Ltbr_fl/flK14Cre and _Ltbr_fl/+K14Cre control mice. Representative plots from mice at various time points are shown (c). The percentages of active caspase 3 positive cells in mTECs of each group are plotted (d). All experiments have been repeated for more than 3 times with at least 3 mice per group each time. An unpaired two-tailed Student’s _t_-test is used. No significant difference was found between comparing groups.

Figure 4

Figure 4. Epithelial derived LTβR controls mTECp development.

(a) Thymic stromal cells were prepared from neonatal _Ltbr_fl/flK14Cre and _Ltbr_fl/+K14Cre control mice. mTECp is identified as Cld3,4hiSSEA-1+ within CD45−EpCAM+ TEC population. (b) Statistic data are shown as mean ± SD of more than 3 mice each group. One representative FACS plot and statistic result from more than 3 repeats are shown. An unpaired two-tailed Student’s _t_-test is used: *P < 0.05. Cld3,4, Claudin3,4.

Figure 5

Figure 5. Deficiency of epithelial LTβR results in impaired negative selection.

(a) OT-I BM cells were depleted of mature T cells and transferred into lethally irradiated recipients mice as indicated. 6 wk later, OT-I thymocyte development was analyzed by flow cytometry. Total thymocytes were first analyzed with CD4 and CD8 (up row) and then OT-I thymocyte gated with Vα2 and Vβ5 from CD8 single positive T cells (middle row). After gating OT-I thymocyte (CD4−CD8+Vα2+Vβ5+), T cell maturation was determined by CD24 (bottom row). Representative profiles from mice of indicated genotypes are shown. (b) Statistical analysis shows the pooled data from three independent experiments with 6–7 mice each group. (c,d) The proportion of splenic OT-I (CD4−CD8+Vα2+Vβ5+) was plotted (c) and the statistical analysis shows the pooled data from three independent experiments with 6–7 mice each group (d). The statistic results are shown as mean ± SD. An unpaired two-tailed Student’s _t_-test is used: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. RIP, rat insulin promoter.

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