Antigen recognition by autoreactive CD4⁺ thymocytes drives homeostasis of the thymic medulla - PubMed (original) (raw)

Antigen recognition by autoreactive CD4⁺ thymocytes drives homeostasis of the thymic medulla

Magali Irla et al. PLoS One. 2012.

Abstract

The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4⁺ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4⁺ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4⁺ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Medulla formation is defective in mice lacking CD4+ thymocytes.

(A) Sections from WT, β2m −/−, H2-Aα −/− and TCRα −/− thymi were stained with antibodies against K14 and K8; m, medulla. The graph shows medullary areas obtained from 3 experiments: symbols represent individual confocal images; lines represent medians. (B) The distribution of medullary areas (mm2) counterstained with HE is shown for WT, β2m −/−, H2-Aα −/− and CIIta IV-/IV- mice: 3 mice per genotype; number of sections is 87 for WT, 90 for β2m −/−, 91 for H2-Aα −/− and 78 for CIIta IV-/IV-; significance relative to WT. (C) Representative FACS profiles are shown (top) for Ly51 expression by CD45−EpCAM+ TECs from WT, β2m −/−, _H2-A_α−/− and CIItaIV-/IV- mice: percentages of cells are indicated. Percentages (bottom left) and numbers per thymus (bottom right) of CD45−EpCAM+Ly51−/lo mTECs and CD45−EpCAM+Ly51+ cTECs are shown: means and SD from 3 measurements; significance relative to WT.

Figure 2

Figure 2. Medulla formation is controlled by autoreactive CD4+ thymocytes.

(A) Thymic sections from Rag2 −/− mice and Marilyn:Rag2 −/− females or males were stained with antibodies against K8 and K14: m, medulla. The graph shows quantifications of medullary areas: symbols represent individual confocal images; lines represent medians; data from 3 experiments, each with 2–3 mice per group. (B) The graph shows numbers per thymus of CD45−EpCAM+Ly51−/lo mTECs and CD45−EpCAM+Ly51+ cTECs in Marilyn:Rag2 −/− females and males: means and SD from 3 experiments. (C) Representative FACS profiles are shown for Ly51 expression by CD45−EpCAM+ TECs from WT→WT and mixed H-Y+WT→WT (1∶1 ratio) chimeras. Graphs show percentages of CD45−EpCAM+Ly51−/lo mTECs and numbers per thymus of CD45−EpCAM+Ly51−/lo mTECs and CD45−EpCAM+Ly51+ cTECs for chimeras prepared with the indicated H-Y∶WT BM ratio: means and SD derived from 3 measurements; significance relative to WT→WT chimeras (0∶1 ratio). (D) Representative FACS profiles are shown for Ki67 expression by CD45−EpCAM+Ly51−/lo mTECs from WT→WT and mixed H-Y+WT→WT (1∶1 ratio) chimeras. Graphs show percentages and numbers per thymus of Ki67+ mTECs for chimeras prepared with the indicated H-Y∶WT BM ratio: means and SD from 3 measurements; significance relative to WT→WT chimeras (0∶1 ratio).

Figure 3

Figure 3. mTEC cellularity is controlled by Ag-specific interactions with CD4+ thymocytes.

(A) Sections from Rag2 −/−, OTII:Rag2 −/− and Rip-mOVA:OTII:Rag2 −/− thymi were stained with antibodies against K8 and K14: m, medulla. The graph shows quantifications of medullary areas: symbols represent individual confocal images; lines represent medians; data from 3 experiments, each with 2–3 mice per group. (B) The graph shows numbers per thymus of CD45−EpCAM+Ly51−/lo mTECs and CD45−EpCAM+Ly51+ cTECs: means and SD from 3 measurements; significance relative to WT. (C) RTOCs using OTII:Rag2 −/− DP thymocytes were cultured for 5 days with (+) or without (−) OVAp. Control cultures contained no thymocytes (none) or WT DP thymocytes (DP-B6). Representative FACS profiles are shown for Ly51 expression by CD45−EpCAM+ TECs: percentages of cells are indicated. Graphs show frequencies of EpCAM+Ly51+ cTECs and EpCAM+Ly51−/lo mTECs (left) or numbers of EpCAM+Ly51−/lo mTECs (right). (D) Representative FACS profiles are shown for the expression of I-Ab and CD80 by mTECs in RTOCs cultured with (+) or without (−) OVAp: percentage of cells are indicated. Graphs show frequencies and numbers of mature I-AbhiCD80hi mTECs: data from 4 experiments, each with 3–5 RTOCs per condition.

Figure 4

Figure 4. Ag-injection restores medulla formation in TCR transgenic mice.

(A) Thymic sections from OTII:Rag2 −/− mice injected with PBS or OVAp were stained with antibodies against K14: m, medulla; c, cortex. The graph shows medullary areas: data from 3 experiments, each with 3 mice per group; symbols represent individual confocal images; lines represent medians. (B) The graph shows numbers per thymus of CD45−EpCAM+Ly51−/lo mTECs in OTII:Rag2 −/− mice injected with PBS or OVAp. (C) The graph shows percentages of Ki67+ mTECs in OTII:Rag2 −/− mice injected with PBS or OVAp: means and SD from 3 experiments, each with 3 mice per genotype. (D) Thymic sections from Marilyn:Rag2 −/− females injected with PBS or H-Yp were stained with antibodies against K14: m, medulla; c, cortex. The graph shows medullary areas: data from 3 experiments, each with 3 mice per group; symbols represent individual confocal images; lines represent medians.

Figure 5

Figure 5. Roles of LTβR, RANK and CD40 signaling in mTEC expansion and maturation.

2-dGUO-treated WT embryonic thymic lobes were cultured for 4 days in medium containing agonistic anti-LTβR antibodies, CD40L and/or RANKL. Control cultures were un-supplemented (medium) or not treated with 2-dGUO. (A) Representative FACS profiles are shown for Ly51 expression by CD45−EpCAM+ TECs (top) and I-Ab and CD80 expression by CD45−EpCAM+Ly51−/lo mTECs (bottom) for the indicated cultures: percentages of cells are indicated. (B) Graphs show mTEC numbers (left) and frequencies of mature I-AbhiCD80hi mTECs (right) for the indicated conditions: data from 3 experiments; lines represent medians.

Figure 6

Figure 6. LT expression is induced by Ag-specific activation of CD4+ thymocytes.

(A) RANKL, CD40L, LTα and LTβ mRNAs were quantified in DP and CD4+ thymocytes from OTII:Rag2 −/− and Rip-mOVA:OTII:Rag2 −/− mice: means and SEM are from 3 experiments, each with 2 mice per group. (B) LTα mRNA and cell surface LT were assessed for unstimulated and anti-CD3/CD28-activated CD4+ thymocytes from OTII:Rag2 −/− or Marilyn:Rag2 −/− mice: data representative of 3 experiments. (C) LTα mRNA was quantified in CD4+ thymocytes from OTII:Rag2 −/− mice co-cultured with unloaded (none) or OVAp-loaded mTECs: data representative of 2 experiments. (D) LTα mRNA was quantified in CD4+ thymocytes from OTII:Rag2 −/− mice isolated 1.5 days after injection of PBS or OVAp: data representative of 3 experiments. (E) β-casein, CRP and RANK mRNAs were quantified in mTECs from WT, LTα−/− mice and OTII:Rag2 −/− mice 5 days after injection of PBS or OVAp. (F) LTα mRNA was quantified in DP and CD4+ thymocytes from CD80/86−/− mice: means and SEM are derived from 2 experiments, each with 2 mice per group. (G) Graphs show distributions of medullary areas (mm2) in WT, CD80/86−/− and CD28−/− thymi (left), and thymi from DT-treated WT and Foxp3-DTR mice (right): significance relative to WT. (H) Positive selection induces CD40L and RANKL expression in thymocytes. After migrating into the medulla, CD4+ thymocytes scan the surface of mTECs for the presence of auto-Ag–MHCII complexes. Ag-specific and CD28-CD80/86 dependent interactions between CD4+ thymocytes and mTECs induce the expression of LT in CD4+ thymocytes and RANK in mTECs, thereby completing the signaling axes required for promoting mTEC expansion and maturation.

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