Lymphotoxin β Receptor Controls T Cell Progenitor Entry to the Thymus

Abstract

The recruitment of lymphoid progenitors to the thymus is essential to sustain T cell production throughout life. Importantly, it also limits T lineage regeneration following bone marrow transplantation, and so contributes to the secondary immunodeficiency that is caused by delayed immune reconstitution. Despite this significance, the mechanisms that control thymus colonization are poorly understood. In this study, we show that in both the steady-state and after bone marrow transplant, lymphotoxin β receptor (LTβR) controls entry of T cell progenitors to the thymus. We show that this requirement maps to thymic stroma, further underlining the key importance of this TNFR superfamily member in regulation of thymic microenvironments. Importantly, analysis of the requirement for LTβR in relationship to known regulators of thymus seeding suggests that it acts independently of its regulation of thymus-homing chemokines. Rather, we show that LTβR differentially regulates intrathymic expression of adhesion molecules known to play a role in T cell progenitor entry to the thymus. Finally, Ab-mediated in vivo LTβR stimulation following bone marrow transplant enhances initial thymus recovery and boosts donor-derived T cell numbers, which correlates with increased adhesion molecule expression by thymic stroma. Collectively, we reveal a novel link between LTβR and thymic stromal cells in thymus colonization, and highlight its potential as an immunotherapeutic target to boost T cell reconstitution after transplantation.

FIGURE 1.

Reduced early thymus progenitors in LTβR^−/− mice. CD4⁻CD8⁻ (DN) thymocytes (A) and ETP (B) in WT and Ltbr^−/− thymus, gated on lineage⁻ and lineage⁻CD25⁻ cells, respectively. Representative FACS plots are shown (n ≥ 23). (C) Representative FACS plots and proportions of BrdU⁺ ETP in WT and Ltbr^−/− thymus (n = 10). (D) Total cellularity and numbers of immature single-positive 8 (ISP8) and CD4⁺CD8⁺ double-positive (DP) thymocytes in WT and Ltbr^−/− thymus (n ≥ 8). (E) BrdU incorporation and proportions of BrdU⁺ DN3 thymocytes from WT and Ltbr^−/− thymus (n ≥ 8). All data are from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2.

LTβR expression by thymic stroma controls thymus entry. (A–D) Lethally irradiated WT/Ltbr^−/− mice were reconstituted with congenically marked T cell–depleted WT/Ltbr^−/− BM cells as indicated. Representative FACS plots are shown, after gating on congenically marked donor-derived thymocytes (n = 10 from three independent experiments). (E) Frequency and absolute number of ETP in WT and Ltbr^−/− dGuo thymus grafts following transplant into WT hosts for 6–8 wk (n ≥ 8 grafts from three independent experiments). Representative FACS plots are shown. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3.

LTβR differentially regulates known mediators of thymus seeding. (A) Purified stromal samples from WT and Ltbr^−/− mice were analyzed by qPCR for the indicated genes. mRNA levels were normalized to β-actin (mean ± SEM) and represent at least two independent biological experiments. (B) Frequency of DN thymocyte subsets and ETP in thymuses from adult WT and plt/plt mice (n ≥ 9 from three independent experiments). (C) Comparison of Selp mRNA expression in WT and Ltbr^−/− endothelium, and mean fluorescence intensity (MFI) analysis of VCAM-1 and ICAM-1 in the indicated stromal subsets of WT and Ltbr^−/− mice. *p < 0.05, **p < 0.01.

FIGURE 4.

Initial thymic reconstitution after BMT is controlled by LTβR. Lethally irradiated WT (A) and Ltbr^−/− (B) mice were reconstituted with T-depleted congenically marked WT BM cells and harvested after 13 d. Thymic reconstitution was determined by calculating the intrathymic frequency of CD45.1⁺ donor cells, and bar charts in (C) show numbers of total donor thymocytes and percentages of donor-derived double-positive and DN thymocytes. n ≥ 13 from five independent experiments; representative FACS plots are shown. (D) Analysis of BrdU incorporation in WT donor-derived CD45.1⁺ thymocytes from WT and Ltbr^−/− hosts (n = 6 from three independent experiments). ****p < 0.0001.

FIGURE 5.

LTβR stimulation enhances thymic reconstitution after BMT. (A) Lethally irradiated WT mice were reconstituted with T-depleted congenic WT BM cells, injected i.p. with 100 μg of agonistic anti-LTβR or isotype on days 1, 3, 5, 7, and 9, and harvested on day 10 or 28. (B and C) Thymic reconstitution was determined by calculating the intrathymic frequency of total CD45.1⁺ donor thymocytes and donor DN thymocyte subsets at day 10. Representative FACS plots are shown (n ≥ 8 from three independent experiments). (D) Frequencies of host- or donor-derived splenic T cells were determined at day 28. (E) Mean fluorescence intensity (MFI) expression of VCAM-1 and ICAM-1 on CD45⁻EpCAM1⁺ TEC, CD31⁺podoplanin⁻ endothelium, and CD31⁻podoplanin⁺ mesenchyme from mice treated with either anti-LTβR or control Ab control treated mice at day 10. n ≥ 8 from two independent experiments. *p < 0.05, **p < 0.001.