Rôle des thymocytes CD4+ dans la formation de la médulla thymique

Dans ce travail, nous avons étudié le rôle respectif des thymocytes SP CD4+ et CD8+ dans la formation de la médulla thymique. Nous avons aussi étudié la régulation d'une molécule pressentie pour jouer un rôle important dans le développement des mTEC : la lymphotoxine α. Nous avons pu montrer grâce à plusieurs modèles de souris transgéniques, que les thymocytes SP CD4+ sont plus compétents que les thymocytes SP CD8+ pour induire l'expansion de la médulla. De plus, nous avons montré que seuls les thymocytes SP CD4+ autoréactifs, capables d'engager avec les mTEC des interactions physiques stables antigène-spécifiques, induisent l'expansion de la médulla. Lors de ces interactions, des signaux sont échangés, permettant ainsi le développement des mTEC. Dans un deuxième temps, nous avons montré que cette interaction antigène-spécifique entre les thymocytes SP CD4+ autoréactifs et les mTEC régule l'expression de la lymphotoxine α dans ce type de thymocytes

Three-dimensional visualization of the mouse thymus organization in health and immunodeficiency

Lymphoid organs exhibit complex structures tightly related to their function. Surprisingly, although the thymic medulla constitutes a specialized microenvironment dedicated to the induction of T cell tolerance, its three-dimensional topology remains largely elusive because it has been studied mainly in two dimensions using thymic sections. To overcome this limitation, we have developed an automated method for full organ reconstruction in three dimensions, allowing visualization of intact mouse lymphoid organs from a collection of immunolabeled slices. We validated full organ reconstruction in three dimensions by reconstructing the well-characterized structure of skin-draining lymph nodes, before revisiting the complex and poorly described corticomedullary organization of the thymus. Wild-type thymi contain ~200 small medullae that are connected to or separated from a major medullary compartment. In contrast, thymi of immunodeficient Rag2(-/-) mice exhibit only ~20 small, unconnected medullary islets. Upon total body irradiation, medullary complexity was partially reduced and then recovered upon bone marrow transplantation. This intricate topology presents fractal properties, resulting in a considerable corticomedullary area. This feature ensures short distances between cortex and medulla, hence efficient thymocyte migration, as assessed by mathematical models. Remarkably, this junction is enriched, particularly in neonates, in medullary thymic epithelial cells expressing the autoimmune regulator. The emergence of a major medullary compartment is induced by CD4(+) thymocytes via CD80/86 and lymphotoxin-α signals. This comprehensive three-dimensional view of the medulla emphasizes a complex topology favoring efficient interactions between developing T cells and autoimmune regulator-positive medullary thymic epithelial cells, a key process for central tolerance induction

Medulla formation is controlled by autoreactive CD4⁺ thymocytes.

<p>(A) Thymic sections from <i>Rag2</i>^−/− mice and <i>Marilyn:Rag2</i>^−/− 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 <i>Marilyn:Rag2</i>^−/− 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).</p

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

<p>(A) Sections from <i>Rag2</i>^−/−, <i>OTII:Rag2</i>^−/− and <i>Rip-mOVA:OTII:Rag2</i>^−/− 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 <i>OTII:Rag2</i>^−/− 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-Ab^hiCD80^hi mTECs: data from 4 experiments, each with 3–5 RTOCs per condition.</p

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

<p>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-Ab^hiCD80^hi mTECs (right) for the indicated conditions: data from 3 experiments; lines represent medians.</p

Ag-injection restores medulla formation in TCR transgenic mice.

<p>(A) Thymic sections from <i>OTII:Rag2</i>^−/− 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 <i>OTII:Rag2</i>^−/− mice injected with PBS or OVAp. (C) The graph shows percentages of Ki67⁺ mTECs in <i>OTII:Rag2</i>^−/− mice injected with PBS or OVAp: means and SD from 3 experiments, each with 3 mice per genotype. (D) Thymic sections from <i>Marilyn:Rag2</i>^−/− 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.</p

Medulla formation is defective in mice lacking CD4⁺ thymocytes.

<p>(A) Sections from WT, <i>β2m</i>^−/−, <i>H2-Aα</i>^−/− and <i>TCRα</i>^−/− 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, <i>β2m</i>^−/−, <i>H2-Aα</i>^−/− and <i>CIIta</i>^IV-/IV- mice: 3 mice per genotype; number of sections is 87 for WT, 90 for <i>β2m</i>^−/−, 91 for <i>H2-Aα</i>^−/− and 78 for <i>CIIta</i>^IV-/IV-; significance relative to WT. (C) Representative FACS profiles are shown (top) for Ly51 expression by CD45⁻EpCAM⁺ TECs from WT, <i>β2m</i>^−/−, <i>H2-A</i>α^−/− and CIIta^IV-/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.</p

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

<p>(A) RANKL, CD40L, LTα and LTβ mRNAs were quantified in DP and CD4⁺ thymocytes from <i>OTII:Rag2</i>^−/− and <i>Rip-mOVA:OTII:Rag2</i>^−/− 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 <i>OTII:Rag2</i>^−/− or <i>Marilyn:Rag2</i>^−/− mice: data representative of 3 experiments. (C) LTα mRNA was quantified in CD4⁺ thymocytes from <i>OTII:Rag2</i>^−/− mice co-cultured with unloaded (none) or OVAp-loaded mTECs: data representative of 2 experiments. (D) LTα mRNA was quantified in CD4⁺ thymocytes from <i>OTII:Rag2</i>^−/− 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 <i>OTII:Rag2</i>^−/− 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.</p