Tissue residency of innate lymphoid cells in lymphoid and nonlymphoid organs

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Science  20 Nov 2015:
Vol. 350, Issue 6263, pp. 981-985
DOI: 10.1126/science.aac9593

Establishing a longtime residency

Innate lymphoid cells (ILCs) are a subset of immune cells that promote barrier immunity in tissues such as the gut and lungs and help to maintain immune homeostasis. Gasteiger et al. investigated how the body maintains its pools of ILCs in such peripheral tissues, as well as in immune tissues such as the lymph nodes and the spleen. In mice surgically joined to share their bloodstreams, unlike lymphocytes, most ILCs did not circulate through the blood. Instead, ILCs resided long term in tissues, even in the face of inflammation or infection.

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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.

Tissue-resident leukocytes can be categorized by their cellular origin and means of maintenance as either self-renewing cells that seed nonlymphoid organs during ontogeny or cells that are replenished hematogenously from precursors in the bone marrow (BM) or secondary lymphoid organs (SLOs), such as the spleen and lymph nodes (LNs). Innate lymphoid cells (ILCs) have been identified in embryonic tissues, BM, SLOs, peripheral blood, and many nonlymphoid tissues, including mucosal sites such as the lung and small intestine, where they contribute to tissue immunosurveillance, immunoregulation, repair, and homeostasis (1, 2). How ILC populations in lymphoid and nonlymphoid organs are maintained, and whether ILCs recirculate from lymphoid to nonlymphoid tissues, have been controversial questions. Based on the identification of fetal ILC progenitors that seed the mouse intestine (3) and the proposed development of ILCs in human tonsils (4), it is reasonable to expect that ILCs may self-renew locally or be generated from immature precursors in these tissues. However, recent studies have identified progenitors to all currently known ILC subsets in adult BM, raising the possibility that ILCs in lymphoid and nonlymphoid organs are continuously replenished through medullary lymphopoiesis and subsequent recruitment to peripheral tissues (5, 6).

To directly test whether hematogenous precursors continuously replenish the pool of peripheral tissue ILCs in adult mice, we generated parabiotic mice, which establish blood chimerism through joint circulation (7, 8). Congenically marked CD45.1+ and CD45.2+ mice were surgically connected for 30 to 40 days until complete chimerism (~50:50 ratio of CD45.1+ to CD45.2+) of major lymphocyte populations was established in the peripheral blood and spleens of the parabionts (fig. S1). We analyzed the percentages of cells that derived from the donor or host parabiont for currently known populations of “helper-like” ILCs, including LinRORγtEomesNK1.1+ ILC1, LinRORγtGATA-3+ ILC2, LinRORγt+CD4 ILC3, and LinRORγt+CD4+ lymphoid tissue inducer (LTi) cells (fig. S2). We found that >95% of ILC1, ILC2, ILC3, and LTi cells residing in the small intestine lamina propria, salivary gland, lung, and liver (9) were of host origin, indicating that these ILC types are bona fide tissue-resident cells (Fig. 1, A to C, and fig. S3). One exception to the overwhelming tissue residency of ILCs were EomesNK1.1+ ILC1s in the lung and peripheral blood, which originated from both parabionts (fig. S3). Because cells isolated from enzymatically dissociated lung tissue include cells from both the vasculature and the parenchyma (10), we injected parabiotic mice intravenously with fluorescently labeled antibody to CD45 5 min before mice were euthanized, in order to identify intravascular leukocytes. This revealed that virtually all ILC2s, but only ~20% of EomesNK1.1+ ILC1s, localized to the lung parenchyma (Fig. 1, D and E, and fig. S3). In contrast to intravascular ILC1s, which derived evenly from both parabionts (Fig. 1E and fig. S3), extravascular ILC1s were >90% host-derived. This observation suggests that these cells are lung parenchymal ILC1s that do not continuously exchange with and are distinguishable from their intravascular counterparts, in agreement with the observation that EomesNK1.1+ ILC1s in the small intestine, salivary gland, and liver were also overwhelmingly of host origin. Together, these experiments identified all analyzed ILC types as tissue-resident cells in all examined nonlymphoid organs.

Fig. 1 Innate lymphoid cells are tissue-resident cells.

Congenically marked CD45.1+ and CD45.2+ mice underwent parabiosis surgery and were analyzed on days (d) 30 to 40 [(A to E) and (J)] or day 30 versus days 104 to 130 of parabiosis [(F to I) and (K)]. The percentage of cells derived from the host parabiont was determined for all “helper-like” ILCs, including LinRORγtEomesNK1.1+ ILC1, LinRORγtGATA-3+ ILC2, LinRORγt+CD4 ILC3, and LinRORγt+CD4+ LTi cells; LinRORγtEomes+NK1.1+ conventional NK cells; and CD4+ and CD8+ T cells in the small intestine lamina propria [(A), (F), (G)], salivary gland [(B) and (H)], lung [(C) and (I)], and mesenteric LN [(J) and (K)]. Intravascular leukocytes were stained with fluorescently labeled antibody to CD45, administered intravenously 5 min before the isolation and analysis of lung leukocytes on day 40 of parabiosis [(D) and (E)]. In the bar graphs, each circle represents an individual parabiont. The red dotted line at 50% marks complete chimerism (equal contribution from host and donor parabionts). The data are shown as mean ± SD and represent two or three independent experiments (n = 4 to 8 parabionts; na, not analyzed; *P < 0.05). In (D), (E), and (J), numbers in the plot indicate the percentage of cells in the respective gates.

Next, we explored whether the long-term maintenance of tissue-resident ILCs was accomplished through self-renewal or through progressive replacement by hematogenous precursors. We analyzed mice that had been in parabiosis for >3 months and found that all analyzed ILC subsets remained >95% host-derived in all nonlymphoid organs tested (Fig. 1, F to I, and fig. S4). In contrast, conventional natural killer (NK) and T lymphocytes, which appeared relatively tissue-resident in the small intestine in our analysis at day 30 to 40 of parabiosis, had equilibrated with donor-derived cells at 3 months (Fig. 1, F and G). However, over time, we did detect a minor increase from <2 to ~5% donor-derived cells for ILC2s in the small intestine and ILC1s in the salivary gland, which we did not observe for the other ILC subsets (fig. S4). Therefore, although our data are consistent with a minor contribution of hematogenous precursors (5, 6) or circulating mature ILCs (11) to the physiologic renewal of ILC subsets, the majority of ILCs appear to be tissue-resident cells that are locally maintained in peripheral organs. This is in contrast to, for example, the progressive replacement of tissue-resident embryonic macrophages in some adult tissues, such as the intestine, which experience constant immune stimulation and tissue renewal (12).

Our observation of tissue-resident ILCs in nonlymphoid tissues raised the question of whether ILCs in SLOs are replenished from hematogenous sources or whether they also are locally renewing cells. Unexpectedly, our analysis of mesenteric LNs and spleens of parabiotic mice showed that all ILC subsets tested were >95% host-derived at both 1 and 3 months (Fig. 1, J and K, and fig. S5), demonstrating that ILCs establish tissue residency in both lymphoid and nonlymphoid organs. Although ILCs that leave peripheral tissues via afferent lymphatics can contribute to ILC populations in draining LNs (13), the identification of tissue-resident ILCs in the spleen, which does not recruit cells via afferent lymphatics, further supports the idea of ILC residency in SLOs. The regional maintenance of ILCs in SLOs thus distinguishes helper-like ILCs from developmentally related NK cells (“killer” ILCs), which, like αβ T cells, are continuously replaced by BM-derived precursors and recirculate systemically and through SLOs. Nevertheless, Eomes+ conventional NK cells can also establish tissue residency in some organs—e.g., the salivary gland and small intestine (Fig. 1) (9, 14).

To test whether the tissue residency of ILCs is maintained under inflammatory conditions, we generated parabiotic Foxp3DTR mice. In these mice, Foxp3+ regulatory T (Treg) cells express the human diphtheria toxin receptor (DTR) under the control of the endogenous Foxp3 locus (15). Upon administration of diphtheria toxin (DTX), the depletion of Treg cells initiates a systemic lymphoproliferative syndrome and fatal autoimmunity (15, 16). Unexpectedly, we failed to detect an increased contribution of hematogenous cells to the expanded ILC populations in the analyzed organs of parabiotic mice depleted of Treg cells (Fig. 2 and fig. S6). This suggests that the mobilization of ILCs or ILC precursors into peripheral tissues is negligible even during systemic autoimmunity, and that the associated increase in ILC populations in lymphoid and nonlymphoid organs probably results from local expansion, despite the influx of hematogenously derived myeloid cells and adaptive lymphocytes (15, 16).

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

Congenically marked pairs of Foxp3DTR or wild-type (WT) control mice underwent parabiosis surgery. After 40 days, both mice within Foxp3DTR 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 LN (B) on day 9 of DTX. Each circle represents an individual parabiont. The data are shown as mean ± SD and were pooled from two independent experiments (n = 6 parabionts).

Next, we examined whether local ILC expansion is observed during infection with a helminth, Nippostrongylus brasiliensis (Nb) (17), that induces strong proliferation and activation of ILC2s in the lung and small intestine. Recent work suggests that Nb infection triggers the appearance of “inflammatory” ILC2s that differentiate further to “natural” ILC2s to sustain the expansion of ILC2s necessary for worm expulsion (18). To determine whether the ILC response in this setting is due to the mobilization of inflammatory ILCs from the BM or elsewhere, or to local differentiation and expansion, we infected parabiotic mice with Nb and analyzed ILC2s during the acute phase of helminth infection (day 7 after infection). At this time point, ILC2s in the lung, small intestine, and mesenteric LN remained host-derived, despite their robust proliferation and expansion (Fig. 3, A to D). In contrast, we observed a small but statistically significant increase in donor-derived ILC2s during the chronic inflammation phase on day 15 after infection (Fig. 3, C and D) (19, 20). At this time point, ILC2s continued to exhibit increased proliferation (Fig. 3C) and have been shown to serve critical functions in tissue repair (21). Thus, the local expansion of resident ILCs during the acute phase of Nb infection is followed by moderately increased hematogenous recruitment or redistribution of ILCs during the chronic inflammation and repair phase. The increase in donor-derived cells was specific to ILC2s among innate immune cell types; we could not detect any increase in donor-derived ILC3s or tissue-resident alveolar macrophages (Fig. 3E and fig. S7). However, even at this later stage, >90% of ILC2s originated from the host parabiont.

Fig. 3 Local expansion of tissue-resident ILC2s 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 (Nb) or mock-infected. The percentages of Ki-67+ (A and C) and donor-derived (B and D) ILC2s were analyzed in the lung, small intestine lamina propria, and mesenteric LN on days 7 and 15 after infection. (E) Analysis of ILC2s and ILC3s in the small intestine lamina propria on day 15 after infection. In the bar graphs, each symbol represents an individual parabiont. The data are shown as mean ± SD and represent two or three independent experiments (n = 6 to 12 parabionts; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001; ns, not significant).

The local proliferation of tissue-resident ILCs led us to ask whether these cells can access the cytokine interleukin (IL)–2, whose availability in lymphoid organs is controlled by Treg cells (22) but whose physiological relevance to ILC responses in nonlymphoid tissues has been controversial. For this purpose, we generated mixed bone marrow chimeras harboring both wild-type ILCs and ILCs deficient in CD25, the high-affinity α-chain of the IL-2 receptor (Fig. 4A), as previously described (23). We found that CD25-sufficient (wild-type) and CD25-deficient ILC2s proliferated to a similar extent and were found at equal ratios in Nb-infected and mock-infected chimeric mice, indicating that IL-2 did not directly influence the proliferation of ILC2s in response to Nb infection (Fig. 4, B and C). However, we observed that CD25-sufficient ILC2s produced more IL-13 (Fig. 4D), which is required for worm expulsion. These results demonstrate that ILC2s can indeed access physiological levels of IL-2 in nonlymphoid organs, supporting the notion that one function of locally produced IL-2 may be to modulate ILC effector function (22, 24, 25). We previously suggested competition for IL-2 as one mechanism by which Foxp3+ Treg cells could restrain the homeostasis of splenic ILC1s (23). It remained unclear in that study whether Treg cells are able to restrain IL-2–dependent ILC activation in nonlymphoid tissues, where they form co-clusters with ILC2s (26). We found that, transient depletion of Treg cells induced the IL-2– and CD25-dependent proliferation of ILC2s (Fig. 4, E to G), suggesting that ILC2s can directly access IL-2 in the lung and the small intestine and that Treg cells restrain the IL-2–dependent expansion of these tissue-resident ILCs.

Fig. 4 IL-2 acts directly on tissue-resident ILC2s to promote cytokine production.

(A to D) Mixed chimeras were generated by cotransfer of BM from CD45.2+ Il2ra/ mice (CD25−/−) and CD25-sufficient CD45.1+ Foxp3DTR mice (CD25+/+) into irradiated RAG-γcKO mice. In (B) to (D), mixed chimeras were infected with Nb, and lung and small intestine (SI) lamina propria were analyzed on day 7 post-infection. (B) shows the percentage of Ki-67+ ILC2 cells among CD25+/+ and CD25−/− cells; (C) shows ratios of CD25−/− to CD25+/+ ILC2s; and (D) shows the percentage of cells stimulated with phorbol 12-myristate 13-acetate and ionomycin that exhibited intracellular IL-13 staining. (E to F) Foxp3DTR mice were subjected to DTX or mock treatment and additionally received IL-2–neutralizing antibodies JES6-1A and S4B6-1 or isotype control immunoglobulin G, as indicated. On day 9 of treatment, the absolute numbers of ILC2s per lung (E) and the percentages of Ki-67+ lung ILC2s (F) were analyzed. (G) Mixed chimeras were generated as in (A). Ratios of CD25−/− to CD25+/+ lung and small intestine lamina propria lymphocytes were analyzed on day 9 of DTX or mock treatment. The data are shown as mean ± SD and represent two to three independent experiments (n = 4 to 8 mice).

Our experiments identified ILCs in both lymphoid and nonlymphoid organs as tissue-resident cells that are locally renewed and expanded in response to acute environmental challenges. These findings also suggest that the plasticity of ILCs—exemplified by the differentiation of “inflammatory” to “natural” ILC2s (18), the differentiation of ILC3s to RORγtEomesNK1.1+ “ex-ILC3” cells (27), the polarization of ILC1s toward ILC3s (28), and the differentiation of CD4+ LTi cells (29)—comprises local processes occurring within peripheral organs. Our study further demonstrates that the local pool of resident ILCs can be replenished and complemented, albeit only in part, through contributions of hematogenously derived precursors or mature cells in situations of extended inflammation and tissue repair. Consistent with our findings, it has been reported that ILC subsets are elevated in the peripheral blood of patients suffering from psoriasis (30, 31). Furthermore, peripheral blood ILC2s have been shown to dynamically modulate the expression of molecules that regulate tissue homing in mice and humans (20, 32). In addition, we have detected donor-derived lymphoid and ILC progenitors in parabiotic BM (fig. S8), raising the possibility that ILC progenitors can physiologically seed tissues not only during embryonic development (3) but also in adult mice. It remains to be determined whether this observation reflects the physiologic migration of ILC progenitors or the engraftment of donor-derived hematopoietic stem cells (33, 34) giving rise to ILCs. Independent of these considerations, our data support a model in which ILCs are locally maintained and expanded as tissue-resident cells during homeostasis and acute infection. This “sedentary” lifestyle of ILCs in broadly differing microenvironments is consistent with the proposed roles of ILCs as sentinels and local keepers of tissue function.

Supplementary Materials

Materials and Methods

Figs. S1 to S8


  1. ACKNOWLEDGMENTS: We thank R. Franklin, S. Dadi, M. Li, and A. Chaudhry for help with parabiosis or cell isolations; D. Artis, L. Monticelli, and B. Hoyos for providing and maintaining worms; K. Wu and A. Bravo for general laboratory support; and T. O’Sullivan, J. Sun, A. Diefenbach, W. Kastenmüller, and members of the Rudensky and Gasteiger laboratories for critical discussions. Data from this study are tabulated in the main paper or in the supplementary materials. This work was supported by an Irvington Fellowship from the Cancer Research Institute (G.G.), NIH Medical Scientist Training Program grant T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program (X.F.), Cancer Center Support Grant P30CA008748 from the NIH National Cancer Institute, NIH grant R37AI034206 (A.Y.R.), the Ludwig Center at Memorial Sloan Kettering Cancer Center, and the Hilton-Ludwig Cancer Prevention Initiative (Conrad N. Hilton Foundation and Ludwig Cancer Research) (A.Y.R.). G.G. is an investigator with the Deutsche Forschungsgemeinschaft Emmy Noether Programme, and A.Y.R is an investigator with the Howard Hughes Medical Institute.
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