Tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs

Abstract

Innate lymphoid cells (ILCs) contribute to barrier immunity, tissue homeostasis, and immune regulation at various anatomical sites throughout the body. How ILCs maintain their presence in lymphoid and peripheral tissues thus far has been unclear. We found that in the lymphoid and nonlymphoid organs of adult mice, ILCs are tissue-resident cells that were maintained and expanded locally under physiologic conditions, upon systemic perturbation of immune homeostasis and during acute helminth infection. However, at later time points after infection, cells from hematogenous sources helped to partially replenish the pool of resident ILCs. Thus, ILCs are maintained by self-renewal in broadly different microenvironments and physiological settings. Such an extreme "sedentary" lifestyle is consistent with the proposed roles of ILCs as sentinels and local keepers of tissue function.

Figure 1. Innate lymphoid cells are tissue-resident cells

Congenically marked CD45.1⁺ and CD45.2⁺ mice underwent parabiosis surgery and were then analyzed on d30–40 (A–E, J) or d104–130 of parabiosis (F–I, K). The percentage of cells derived from the host parabiont was determined for all “helper-like” ILCs including Lin⁻RORγt⁻Eomes⁻NK1.1⁺ ILC1, Lin⁻RORγt⁻GATA-3⁺ ILC2, Lin⁻RORγt⁺CD4⁻ ILC3, and Lin⁻RORγt⁺CD4⁺ LTi cells; Lin⁻RORγt⁻Eomes⁺NK1.1⁺ conventional NK cells (cNK); as well as CD4⁺ and CD8⁺ T cells in the small intestine lamina propria (A, F, G), salivary gland (B, H) lung (C, I) and mesenteric LN (J, K). Intravascular leukocytes were stained by anti-CD45-PE administered intravenously 5 min prior to the isolation and analysis of lung leukocytes on d40 of parabiosis (D, E). Each symbol represents an individual parabiont. The red dotted line at 50% marks complete chimerism (equal contribution from host and donor parabionts). na = not analyzed. The data are shown as mean ± SD and represent 2 or 3 independent experiments (n = 4–8).

Figure 2. Tissue-residency of ILCs is maintained upon systemic immune activation

Congenically marked pairs of Foxp3^DTR or wild-type (WT) control mice underwent parabiosis surgery. After 40 days, both mice within Foxp3^DTR or WT pairs were subjected to diphtheria toxin (DTX) treatment. For the indicated populations of lymphocytes, the percentage of host-derived cells was analyzed in the small intestine lamina propria (A) and mesenteric lymph node (B) on d9 DTX. Each symbol represents an individual parabiont. The data are shown as mean ± SD and were pooled from 2 independent experiments (n = 6).

Figure 3. Local expansion of tissue-resident ILC2 upon helminth infection

Congenically marked CD45.1⁺ and CD45.2⁺ mice underwent parabiosis surgery. After 40 days, both mice within each parabiotic pair were infected with Nippostrongylus brasiliensis (N.b.) or mock-infected. The percentages of Ki-67⁺ (A, C) and donor-derived (B, D) ILC2 were analyzed in the lung, small intestine lamina propria, and mesenteric lymph node on d7 and d15 post-infection (pi). (E) Analysis of ILC2 and ILC3 in the small intestine lamina propria on d15 pi. Each symbol represents an individual parabiont. The data are shown as mean ± SD and represent 2–3 independent experiments (n = 6–12).

Figure 4. Interleukin-2 acts directly on tissue-resident ILC2 to promote cytokine production

(A–D, G) Mixed chimeras were generated by co-transfer of bone marrow from CD45.2⁺ Il2ra^−/− mice (CD25^−/−) and CD25-sufficient CD45.1⁺ Foxp3^DTR mice (CD25^+/+) into irradiated RAG-γc^KO mice. (B–D) Mixed chimeras were infected with Nippostrongylus brasiliensis, and lung and small intestine lamina propria were analyzed on d7 post-infection. (B) Percentage of Ki-67⁺ ILC2 among CD25^+/+ and CD25^−/− cells. (C) Ratios of CD25^−/− to CD25^+/+ ILC2. (D) Intracellular IL-13 staining of cells stimulated with PMA/ionomycin. (E–G) Foxp3^DTR mice were subjected to diphtheria toxin (DTX) or mock treatment, and additionally received IL-2 neutralizing antibodies JES6-1A and S4B6-1, or isotype control IgG, as indicated. On d9 of DTX, the absolute numbers of ILC2 per lung (E) and the percentages of Ki-67⁺ lung ILC2 (F) were analyzed. (G) Ratios of CD25^−/− to CD25^+/+ lung and small intestine lamina propria lymphocytes were analyzed in mixed chimeras on d9 of DTX. The data are shown as mean ± SD and represent 2–3 independent experiments (n = 4–8).