Overexpression of lymphotoxin in T cells induces fulminant thymic involution

Abstract

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

Figure 1

Generation and characterization of tg mice expressing T cell derived LT. A: Schematic representation of tgLTα and tgLTβ constructs used for pronuclear microinjection on the basis of the 5′HG expression cassette. Arrows 1 to 6 indicate various primers used for PCR and RT-PCR analysis (see Material and Methods). B: Genomic Southern blot analysis identified founder mice positive for tgLtα, tgLtβ or DP for tgLtαβ. LTα or LTβ, endogenous (end.) and transgenic (tg) LTα or LTβ EcoRI fragments detected by a radioactively labeled probe. kbp: kilobase pairs C: RPA showing increased LTα and LTβ mRNA expression in spleen, thymus, MLN, and Peyer’s patches of double and single tg mice (lines: tg(Ltαβ)856 and tg(Ltα)54). TNF, IL6, IFNγ, IFNβ TGFβ1, TGFβ2, and MIF mRNA expression levels remained unchanged. tgLtαβ thymi mice displayed a three- to fivefold up-regulation of TGFβ3 mRNA. D: Histogram of FACS analysis for LTα₁β₂ expression on T (CD4⁺ and CD8⁺) and B cells (B220⁺/CD19⁺) in spleen, thymus, and MLN. Green lines depict tg(Ltαβ)856, purple fill Ltα^−/− and pink line wt mice. B220⁺/CD19⁺ cells positive for LTα₁β₂ were detected in tg thymi. E: Analysis of LTα₂β₁, LTα₁β₂, and LTα₃ in organ homogenates (thymus, spleen, MLN) of various transgenic lines, wt mice, and Ltα^−/− mice by ELISA, reveals the presence of LTα₃ in tgLtαβ and tgLtα lines but not in Ltα^−/− mice.

Figure 2

Macroscopic and cellular characterization of thymic involution in tgLtαβ mice. A: RNA expression of LT and various cytokines using RPA during postnatal development (P1 to P42). One representative experiment out of three for each time point is shown. LTα and LTβ were up-regulated in tg thymi during early postnatal development and in adult mice. B: Macroscopic analysis of postnatal thymus development in tgLtαβ mice and negative littermates. Thymic involution of tg mice can be detected between P7 and 42 (scale bar: 1 mm). C: Time course of alterations in relative thymus weight (ie, thymus weight with respect to body weight). Each time point is the mean of at least 3 mice analyzed. Onset of thymic involution in male C57BL/6 mice is earlier than in C57BL/6 female mice. tg thymi start involuting between postnatal day 7 and 14. D: FACS analysis of tg and wt thymi at P42. Numbers indicate the percentage of the respective cell populations. Pooled tgLtαβ thymi (n = 3 to 4) (lower row) show a reduction in DP T cells and an inverted CD4:CD8 ratio. A representative experiment from four is shown. E: FACS analysis of Lin⁻ DN thymocytes of tgLtαβ and wt thymi (n = 4).

Figure 3

Analysis of thymic microenvironment in tg mice. Immunohistochemical analysis of tg and wt thymi at P1. Even though no obvious macroscopic differences were observed at P1, tg thymic cortex was already reduced and thymic medulla enlarged (asterisks). Scale bar: 200 μm.

Figure 4

Increase in MTS24⁺ staining in tgLtαβ mice. A: H&E staining of wt, tgLtαβ, or tgLtα mice showing the relative enlargement of the thymic medullary compartment in tg mice when compared to wt mice. Scale bar: 400 μm. B: Confocal microscopy of thymi cryosections stained for CK5, CK18, and MTS24 were performed to investigate medullary and cortical micro-architecture by confocal microscopy. No apparent differences were observed by CK5 and CK18 staining. However, an increase in MTS24⁺ staining was observed in tg mice. Scale bar: 50 μm. C: Confocal microscopy of tg and wt thymi stained for UEA-1 and CK18 were performed. No differences in the distribution pattern or UEA-1/CK18 double positivity were observed between wt and tgLtαβ or tgLtα mice. Scale bar: 50 μm.

Figure 5

T cell triggered thymic involution is induced via TNFR1 and LTβR signaling. A: Macroscopic analysis of thymi from various knockout mice crossed to tgLtαβ mice (6 to 8 weeks old). Backcrossing to Tnfr1^−/− and Ltβr^−/− mice rescued the thymic phenotype due to LTαβ overexpression on T cells. Scale bar: 2 mm. B and C: Quantitative analysis of relative thymic weight and absolute number of thymocytes from these crossing experiments confirmed this observation. D: FACS analysis of tgLtαβ x Tnfr1^−/− or tgLtαβ x Ltβr^−/− mice reveals an amount of SP CD4⁺ and CD8⁺, DP CD4⁺CD8⁺ and DN CD4⁻CD8⁻ T cell populations in thymus comparable to wt mice. A representative of three independent experiments is shown.

Figure 6

Stromal TNFR1 and LTβR signaling triggered by tg T cells is responsible for thymic involution in tgLtαβ mice. A: Flow cytometric analysis of total thymus cells for Annexin V and 7-AAD was performed to investigate the magnitude of apoptosis in pooled tg (n = 3) and wt thymi (n = 3). A representative of three independent experiments is shown. B: Macroscopic analysis of thymi from mice reconstituted by BM transplantation from several genotypes: wt BM into wt, tg BM into wt, Tnfr1^−/−, or Ltβr^−/− mice. Transfer of tgLtαβ BM cells into wt mice induced thymic involution. In contrast, tgLtαβ BM failed to induce thymic involution in Tnfr1^−/− and Ltβr^−/− mice. C and D: Quantitative analysis of relative thymic weight and absolute number of thymocytes from the respective crossing experiments revealed prevention of the thymic, hypoplastic phenotype, particularly in Tnfr1^−/− mice. E: Analysis of thymic micro-architecture confirmed normal distribution of CD4⁺ and CD8⁺ T cells in Tnfr1^−/− or Ltβr^−/− mice crossed to tgLtαβ mice. Scale bars: 50 μm (left 2 columns) and 200 μm (right 2 columns).

Figure 7

Viral infection induces thymic involution and LT expression on T cells that is partially rescued in Tnfr1^−/− mice. A and B: Reduction in relative thymic weight and absolute number of thymocytes in wt mice infected LCMV-WE, detected to greater extent in wt mice infected with LCMV-DOCILE at day 9 post-infection. C: Immunohistochemistry analysis of thymi from LCMV-DOCILE infected wt mice and controls revealed a reduction of the thymic cortex. Scale bar = 200 μm. D: Increased levels of TNFα in serum and thymus protein homogenate of LCMV-DOCILE infected wt and Tnfr1^−/− mice. E and F: Partial rescue of thymic involution in Tnfr1^−/− female mice and to a lesser degree in Tnfr1^−/− male mice, as compared with uninfected wt mice or wt mice infected with LCMV-DOCILE. G: FACS analysis of thymi of LCMV-WE infected and non-infected wt mice. Thymus DP cells are reduced in LCMV-WE infected wt mice, whereas SP CD4⁺ and CD8⁺ T cells are increased. Histograms gated on CD8⁺ T cells indicate a strong up-regulation of LTα₁β₂ in 2 out 3 of thymi from LCMV-WE infected wt mice, and weak up-regulation in the third one. Interestingly, a similar but reduced effect was also detected on DN CD4⁻CD8⁻ thymocytes.