T cells regulate lymph node-resident ILC populations in a tissue and subset-specific way

doi:10.1016/j.isci.2021.102158

. 2021 Feb 7;24(3):102158.

doi: 10.1016/j.isci.2021.102158. eCollection 2021 Mar 19.

T cells regulate lymph node-resident ILC populations in a tissue and subset-specific way

Priscillia Bresler¹, Emmanuel Tejerina¹, Jean Marie Jacob^{2

3}, Agnès Legrand¹, Véronique Quellec¹, Sophie Ezine¹, Lucie Peduto², Marie Cherrier⁴

Affiliations

¹ Institut Necker Enfants Malades, Université Paris Descartes, INSERM U1151, CNRS UMR 8253, Faculté de Médecine Necker, 156 rue de Vaugirard, 75015 Paris, France.
² Stroma, Inflammation & Tissue Repair Unit, Institut Pasteur, Inserm U1224, Paris, France.
³ Université Paris Diderot, Sorbonne Paris Cité, Paris, France.
⁴ Institut Imagine, Université Paris Descartes, INSERM U1163, Laboratory of Intestinal Immunity, 24 Boulevard du Montparnasse, 75015 Paris, France.

PMID: 33665576
PMCID: PMC7907429
DOI: 10.1016/j.isci.2021.102158

T cells regulate lymph node-resident ILC populations in a tissue and subset-specific way

Priscillia Bresler et al. iScience. 2021.

. 2021 Feb 7;24(3):102158.

doi: 10.1016/j.isci.2021.102158. eCollection 2021 Mar 19.

Authors

Priscillia Bresler¹, Emmanuel Tejerina¹, Jean Marie Jacob^{2

3}, Agnès Legrand¹, Véronique Quellec¹, Sophie Ezine¹, Lucie Peduto², Marie Cherrier⁴

Affiliations

¹ Institut Necker Enfants Malades, Université Paris Descartes, INSERM U1151, CNRS UMR 8253, Faculté de Médecine Necker, 156 rue de Vaugirard, 75015 Paris, France.
² Stroma, Inflammation & Tissue Repair Unit, Institut Pasteur, Inserm U1224, Paris, France.
³ Université Paris Diderot, Sorbonne Paris Cité, Paris, France.
⁴ Institut Imagine, Université Paris Descartes, INSERM U1163, Laboratory of Intestinal Immunity, 24 Boulevard du Montparnasse, 75015 Paris, France.

PMID: 33665576
PMCID: PMC7907429
DOI: 10.1016/j.isci.2021.102158

Abstract

Innate lymphoid cells (ILCs) have been shown to be significantly affected in the small intestine lamina propria and secondary lymphoid organs (SLOs) of conventional lymphopenic mice. How ILCs are regulated by adaptive immunity in SLOs remains unclear. In T cell-deficient mice, ILC2s are significantly increased in the mesenteric lymph nodes (MLNs) at the expense of CCR6⁺ ILC3s, which are nonetheless increased in the peripheral lymph nodes (PLNs). Here, we show that T cells regulate lymph node-resident ILCs in a tissue- and subset-specific way. First, reducing microbial colonization from birth restored CCR6⁺ ILC3s in the MLNs of T cell-deficient mice. In contrast, T cell reconstitution resulted in the contraction of both MLNs ILC2s and PLNs ILC3s, whereas antagonizing microbial colonization from birth had no impact on these populations. Finally, the accumulation of MLNs ILC2s was partly regulated by T cells through stroma-derived IL-33.

Keywords: Components of the Immune System; Immunology; Microbiome.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

**Figure 1**
T cells regulate ILC homeostasis through distinct mechanisms depending on the ILC subtype and tissue microenvironment (A–C) (A) Flow cytometry analysis of ILCs from the mesenteric lymph nodes (MLNs) and peripheral lymph nodes (PLNs) of 8-week-old T cell-deficient mice (CD3ε⁻^/−) and control littermates (CD3ε⁺^/−). Histograms showing cell numbers in each ILC subset in the MLNs (B) and PLNs (C) of T cell-deficient mice and control littermates before (3 weeks old CD3ε⁻^/⁻ n = 8 and CD3ε^+/− n = 6) and after weaning (8-week-old CD3ε⁻^/− n = 13 and CD3ε^+/− n = 9). (D) Group 3 ILCs were subdivided into three subsets according to the expression of NKp46 and CCR6: NKp46⁺ (orange), CCR6⁺ (purple), and NKp46⁻ CCR6⁻ (DN) (black). (E and F) Histograms showing the number of cells in each ILC3 subset in MLNs and PLNs of T cell-deficient mice and control littermates before (3 weeks old CD3ε⁻^/− n = 8 and CD3ε^+/− n = 6) and after weaning (8-week-old CD3ε⁻^/− n = 14 and CD3ε^+/− n = 12). (G) Flow cytometry analysis of type 2 cytokine production by ILCs in the MLNs and PLNs of 8-week-old T cell-deficient mice CD3ε⁻^/− and control littermates CD3ε^+/−. (H and I) Histograms showing the number of ILCs expressing IL-13 alone or in combination with IL-5 in the MLNs (H) and PLNs (I) of 8-week-old T cell-deficient mice CD3ε⁻^/− (n = 16) and control littermates CD3ε^+/− (n = 15). Statistical analysis was performed using the two-way ANOVA method and Bonferroni's multiple comparison test, with alpha = 0.05. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗p < 0.05. Data are pooled from at least 3 independent experiments. Data are represented as mean ± SEM. See also Figures S1 and S2.

**Figure 2**
Commensal bacteria play a dominant role in the regulation of CCR6⁺ ILC3 homeostasis in the mesenteric lymph nodes of T cell-deficient mice (A–E) (A) Experimental procedure: T cell-deficient mice (CD3ε⁻^/−) and control littermates (CD3ε^+/−) were treated with a cocktail of broad-spectrum antibiotics (Abx) (ampicillin 0.5 g/L; streptomycin 1 g/L; metronidazole 0.5 g/L) in the drinking water from birth. Histograms showing the percentage of each ILC (B and C) and each ILC3 subset (D and E) among CD45⁺ cells in MLNs (B and D) and PLNs (C and E) of 8-week-old T cell-deficient mice and control littermates treated with a cocktail of broad-spectrum antibiotics (CD3ε⁻^/− Abx n = 9 and CD3ε^+/− Abx n = 7) with 2% glucose or with 2% glucose only in the drinking water (CD3ε⁻^/− SPF n = 8 and CD3ε^+/− SPF n = 5). (F and G) Histograms showing the percentage of ILCs expressing IL-13 alone or in combination with IL-5 among CD45⁺ cells in the MLNs (F) and PLNs (G) of 8-week-old T cell-deficient mice and control littermates treated with a cocktail of broad-spectrum antibiotics (CD3ε⁻^/− Abx n = 9 and CD3ε^+/− Abx n = 8) or with glucose in the drinking water (CD3ε⁻^/− SPF n = 8 and CD3ε^+/− SPF n = 6). (H and I) Percentage of Ki67⁺ cells among ILC3 subsets in the MLNs and PLNs of the mice described above. (J) Absolute numbers of each ILC3 subset in the MLNs of 8-week-old T cell-deficient mice and control littermates born from either SPF CD3ε⁻^/− or SPF CD3ε^+/− Dam (CD3ε⁻^{/− Dam}⁻^/− n = 8 and CD3ε^{+/− Dam}⁻^/− n = 6; CD3ε⁻^{/− Dam+/−} n = 3 and CD3ε^{+/− Dam+/−} n = 5). (K) Mating strategy used to generate T cell-deficient mice and control littermates born from either CD3ε⁻^/− or CD3ε^+/− Dam. Statistical analysis was performed using the two-way ANOVA method with alpha = 0.05. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. Data are pooled from at least 3 independent experiments. Data are represented as mean ± SEM. See also Figure S1.

**Figure 3**
T cell reconstitution suppresses the expansion of LN-resident ILCs T cell-deficient mice (CD45.1 CD3ε⁻^/−) received a mix of 1.5 × 10⁶ CD4⁺ and 1.5 × 10⁶ CD8⁺ T cells or 3 × 10⁶ CD4⁺ T cells or 1 × 10⁶ CD4⁺ Foxp3⁺ T cells isolated from the peripheral lymph nodes and spleen of CD45.2 B6 mice or CD45.2 Foxp3EGFP reporter mice. When OT-II T cells were injected, mice received 3 × 10⁶ CD4⁺ T cells isolated from the peripheral lymph nodes and the spleen of CD45.2 RAG2⁻^/⁻ OT-II TCR transgenic mice. When specified, ovalbumin was added at 1.5% in the drinking water throughout the procedure. Control mice were injected with PBS. Mice were sacrificed 12 weeks following adoptive transfer, and flow cytometry analysis was performed. (A–C) (A) Flow cytometry analysis of the fraction of Lin⁻ GATA-3^hi cells in the MLNs and of Lin⁻ RORγt⁺ cells in the PLNs among live recipient CD45⁺ cells in T cell-deficient mice 12 weeks after injection with PBS or 3 × 10⁶ CD4⁺ T cells or a combination of 1.5 × 10⁶ CD4⁺ and 1.5 × 10⁶ CD8⁺ T cells. Histograms showing cell numbers in each ILC subset in the MLNs and PLNs (B and C) of T cell-deficient mice injected with PBS (n = 9), CD4⁺ and CD8⁺ T cells (n = 6), CD4⁺ T cells (n = 7), Foxp3⁺ CD4⁺ T cells (n = 8), or OT-II T cells with (n = 3) or without (n = 6) oral administration of ovalbumin, 12 weeks after the injection. WT controls are shown as a reference. (D and E) Histograms showing the number of each ILC3 subset in the MLNs (D) and PLNs (E) of the same mice. (F and G) Histograms showing the number of ILCs expressing IL-13 alone or in combination with IL-5 in the MLNs (F) and PLNs (G) of T cell-deficient mice treated as described above. T cell-deficient mice (CD45.1 CD3ε⁻^/−) were treated with a cocktail of broad-spectrum antibiotics with 2% glucose (Abx) or 2% glucose only (SPF) in the drinking water and received 3 × 10⁶ CD4⁺ cells or PBS at 8 weeks of age. Antibiotics treatment was maintained for 12 more weeks, and flow cytometry analysis was performed. (H) Histogram showing the frequency of ILCs expressing IL-13 alone or in combination with IL-5 among CD45⁺ cells in the MLNs of SPF and antibiotic-treated T cell-deficient mice injected with PBS (SPF and Abx n = 8) or CD4⁺ T cells (SPF and Abx n = 7). (I) Histogram showing the frequency of each ILC3 subset among CD45⁺ cells in the PLNs of the same mice. Statistical analysis was performed using two-way ANOVA method with alpha = 0.05. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05. Data are pooled from at least 3 independent experiments. Data are represented as mean ± SEM. See also Figure S3.

**Figure 4**
IL-33 participates in the regulation of mesenteric lymph nodes-resident ILC2 by T cells (A) Histograms showing the relative expression of genes coding for *Il25*, *Il33,* and *Tslp* in the MLNs, PLNs, and small intestine (SI) of 8-week-old T cell-deficient mice (n = 4) and control littermates (n = 4). Statistical analysis was performed using the t test method and Mann-Whitney comparison test, with alpha = 0.05. ∗∗p < 0.01. Data are represented as mean ± SEM. (B) Experimental procedure: Age-matched CD3ε⁻^/− mice were treated with blocking antibodies directed against ST2 (n = 5), IL-17RB (n = 5), and TSLP (n = 5) or control isotype (Rat IgG2b, IgG1, and IgG2a respectively, n = 5 each) and sacrificed 24 h later for flow cytometry analysis. (C) Histogram showing the number of ILC2s in the MLNs and SILP in each experimental condition. (D and E) Histograms showing the number of ILCs producing IL-13 alone or in combination with IL-5 in the MLNs and SILP of the same mice. (F) Histogram showing the percentage of Ki67⁺ cells in each ILC subset from the MLNs of mice treated with anti-ST2 or control isotype. (G) Histogram showing the percentage of cells positive for Annexin V, DAPI, or both among MLNs ILC2. Statistical analysis was performed using the two-way ANOVA method with alpha = 0.05. ∗∗∗p < 0.001, ∗∗p < 0.01. Data are from one representative experiment out of two. Data are represented as mean ± SEM. (H) Experimental procedure: thymic lobes from CD45.2 C57BL/6 neonates were grafted under the kidney capsule of 8-week-old CD45.1 CD3ε⁻^/− mice, and recipients were sacrificed 16 weeks later. Flow cytometry analysis was performed on MLNs CD45⁺ cells (I), and CD45⁻ cells were isolated and sorted for qPCR analysis (J). (I) Histogram showing the number of ILC2s in the MLNs of T cell-deficient mice grafted with neonatal thymic lobes from CD45.2 B6 mice (n = 8), aged-matched T cell-deficient mice (n = 8), and control littermates (n = 8). Statistical analysis was performed using the two-way ANOVA method with alpha = 0.05. ∗∗∗∗p < 0.0001, ∗∗p < 0.01. Data are pooled from 3 independent experiments. Data are represented as mean ± SEM. (J) Relative *Il33* mRNA expression in CD45⁻ cells sorted from the MLNs of T cell-deficient mice grafted with neonatal thymic lobes (n = 8) and age-matched T cell-deficient mice (n = 8) and control littermates (n = 8). Statistical analysis was performed using the two-way ANOVA method with alpha = 0.05. ∗∗p < 0.01. Data are pooled from 3 independent experiments. Data are represented as mean ± SEM. See also Figures S4 and S5.

**Figure 5**
Stromal IL-33 is not required for the accumulation of bone marrow-derived ILC2s in the mesenteric lymph nodes and small intestine lamina propria of lymphopenic hosts (A–C) (A) Experimental procedure: 8-week-old IL-33^Gt/Gt-deficient mice or wild-type mice were lethally irradiated and injected with 2 × 10⁶ bone marrow cells isolated from CD3ε⁻^/− mice and depleted from Lineage⁺ cells. Histograms showing the percentage of each ILC subset in donor-derived cells 8 weeks after bone marrow transfer in the MLNs (B) and SILP (C) of IL-33^Gt/Gt-deficient (n = 8) and wild-type recipients (n = 8). (D and E) Histograms showing the fraction of ILCs producing IL-13 alone or in combination with IL-5 in MLNs and SILP donor-derived cells of the same mice. Statistical analysis was performed using the t test method and Mann-Whitney comparison test, with alpha = 0.05. Data are pooled from two independent experiments. Data are represented as mean ± SEM.

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[1] Bordon Y. Mucosal immunology: ILCs broker peace deals in the gut. Nat. Rev. Immunol. 2013;13:473. - PubMed

[2] Bordon Y. Mucosal immunology: ILCs broker peace deals in the gut. Nat. Rev. Immunol. 2013;13:473. - PubMed

[3] von Burg N., Turchinovich G., Finke D. Maintenance of immune homeostasis through ILC/T cell interactions. Front. Immunol. 2015;6:416. - PMC - PubMed

[4] von Burg N., Turchinovich G., Finke D. Maintenance of immune homeostasis through ILC/T cell interactions. Front. Immunol. 2015;6:416. - PMC - PubMed

[5] Cherrier M., Ramachandran G., Golub R. The interplay between innate lymphoid cells and T cells. Mucosal Immunol. 2020;13:732–742. - PubMed

[6] Cherrier M., Ramachandran G., Golub R. The interplay between innate lymphoid cells and T cells. Mucosal Immunol. 2020;13:732–742. - PubMed

[7] Cupedo T., Kraal G., Mebius R.E. The role of CD45+CD4+CD3- cells in lymphoid organ development. Immunol. Rev. 2002;189:41–50. - PubMed

[8] Cupedo T., Kraal G., Mebius R.E. The role of CD45+CD4+CD3- cells in lymphoid organ development. Immunol. Rev. 2002;189:41–50. - PubMed

[9] Ebbo M., Crinier A., Vély F., Vivier E. Innate lymphoid cells: major players in inflammatory diseases. Nat. Rev. Immunol. 2017;17:665–678. - PubMed

[10] Ebbo M., Crinier A., Vély F., Vivier E. Innate lymphoid cells: major players in inflammatory diseases. Nat. Rev. Immunol. 2017;17:665–678. - PubMed

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T cells regulate lymph node-resident ILC populations in a tissue and subset-specific way

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T cells regulate lymph node-resident ILC populations in a tissue and subset-specific way

Authors

Affiliations

Abstract

Conflict of interest statement

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