Activated group 3 innate lymphoid cells promote T-cell–mediated immune responses Nicole von Burga,b,1, Stéphane Chappaza,c,d,1, Anne Baerenwaldta,b, Edit Horvatha,b, Somdeb Bose Dasguptae, Devika Ashokf, Jean Pieterse, Fabienne Tacchini-Cottierf,g, Antonius Rolinka, Hans Acha-Orbeaf, and Daniela Finkea,b,2 aDepartment of Biomedicine, University of Basel, 4058 Basel, Switzerland; bUniversity Children’s Hospital of Basel, 4056 Basel, Switzerland; cAustralian Cancer Research Foundation Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; dDepartment of Medical Biology, University of Melbourne, Parkville, VIC 3010, Australia; eBiozentrum, University of Basel, 4056 Basel, Switzerland; and fDepartment of Biochemistry and gWorld Health Organization Immunology Research and Training Centre, University of Lausanne, 1066 Epalinges, Switzerland Edited by Ruslan Medzhitov, Yale University School of Medicine, New Haven, CT, and approved July 25, 2014 (received for review April 15, 2014) Group 3 innate lymphoid cells (ILC3s) have emerged as important suggesting an unexpected crosstalk between these two cell types. cellular players in tissue repair and innate immunity. Whether Altogether, we show that peripheral ILC3s can mature upon these cells meaningfully regulate adaptive immune responses activation into bona fide APCs shaping T-cell–mediated immune upon activation has yet to be explored. Here we show that responses. upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express Results costimulatory molecules. ILC3s can take up latex beads, process ILC3s Elicit Ag-Specific T-Cell Proliferation and TD B-Cell Responses in + + protein antigen, and consequently prime CD4 T-cell responses Vivo. To investigate whether ILC3s can initiate CD4 T-cell in vitro. The cognate interaction of ILC3s and CD4+ T cells leads responses in vivo, we generated mice with a deletion of Iab ex- to T-cell proliferation both in vitro and in vivo, whereas its disrup- clusively in ILC3s by crossing mice expressing Cre recombinase tion impairs specific T-cell and T-dependent B-cell responses in under the control of the RORc promoter [RORc(γt)-Cretg] (12) vivo. In addition, the ILC3–CD4+ T-cell interaction is bidirectional to mice with a floxed H2-Ab1 allele (I-abneo) (13). Mice homo- and leads to the activation of ILC3s. Taken together, our data re- zygous for the floxed H2-Ab1 allele and carrying one copy of the Δ veal a novel activation-dependent function of peripheral ILC3s in Cre transgene, here referred to as I-ab ILC3 mice, were healthy, + eliciting cognate CD4 T-cell immune responses. did not show signs of spontaneous inflammation, and had a normal distribution of lymphocytes, macrophages (MΦ), and T-cell activation | antigen presentation dendritic cells (DCs) (Fig. 1A). Numbers of ILC3s were also Δ similar in WT and I-ab ILC3 mice (Fig. 1A). MHC class II ex- pression was normal on splenic B cells, DCs, and MΦ, whereas nnate lymphoid cells (ILCs) are a group of lymphocytes that − NCR ILC3s completely lacked MHC class II (Fig. 1B). To ex- Iplay a critical role in immediate immune host defense as well + amine Ag-specific CD4 T-cell proliferation in vivo, 3 × 106 as mucosal and lymphoid tissue homeostasis. Although they lack + carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4 T INFLAMMATION somatically rearranged antigen (Ag) receptors, they exhibit IMMUNOLOGY AND cells from OT-II (H-2b) T-cell receptor transgenic mice (OT-IItg) a transcription factor and cytokine profile similar to T helper ΔILC3 −/− (Th) cells. Therefore, ILCs are classified into three major fam- were adoptively transferred into WT, I-ab ,orI-ab mice. ilies (1). Reminiscent of Th1 cells, ILC1s are characterized by Following immunization with ovalbumin (OVA) peptide323–339 γ and OVA protein plus CpG, labeled OT-IItg T cells proliferated Interferon (IFN) production and developmental regulation by −/− ΔILC3 tg T-bet. ILC2s secrete interleukin (IL)-5, IL-9, and IL-13 and, in WT, but not in I-ab mice (Fig. 1C). In I-ab mice, OT-II analogous to Th2 cells, depend on Gata3. ILC3s produce IL-22 and IL-17A and together with Th17 cells express the retinoic acid Significance receptor-related orphan receptor RORγt (2, 3). ILC3s can be frac- + − tioned into NKp46 and NKp46 subsets including lymphoid tissue Group 3 innate lymphoid cells (ILC3s) play decisive roles in mam- − − + − + inducer (LTi) cells. Here, lin NKp46 CD4 / RORγt ILCs are re- malian physiology including tissue repair, lymphoid tissue de- − ferred to as natural cytotoxicity receptor (NCR) ILC3s. ILCs express velopment, and immune regulation. So far, the functions of ILC3s Toll-like receptors (TLRs) (4) and IL-1R, indicating that they can in the adult immune system have been mainly linked to their directly sense microbial products and inflammatory signals (5–7). The capacity to release cytokines in response to microbial or in- ability to rapidly release cytokines upon microbial challenge fostered flammatory signals. The results presented here show that acti- vated ILC3s can alter the outcome of adaptive immune responses the idea that ILCs may bias the outcome of T-cell responses. In ad- + + by directly stimulating CD4 T cells. Indeed, IL-1β–activated dition, both ILC2 and ILC3s were recently shown to modulate CD4 + T-cell responses through Ag-peptide presentation by MHC class II (8, ILC3s express costimulatory molecules and induce cognate CD4 9). In line with this, it was proposed a decade ago that ILC3s interact T-cell responses. More importantly, antigen-driven T- and B-cell + with T cells in secondary lymphoid organs and thereby promote CD4 responses are impaired in vivo when this cellular interaction is T-cell memory responses (10, 11). Whether peripheral ILC3s can disrupted. Overall, our data show that peripheral ILC3s play a yet – mature into Ag-presenting cells (APCs) providing costimulation for unappreciated role in T-cell mediated immunity. T-cell–mediated immunity has never been explored. In the present Author contributions: N.v.B., S.C., and D.F. designed research; N.v.B., S.C., E.H., D.A., H.A.-O., and study, we show that in mice where MHC class II is specifically deleted D.F. performed research; A.B., S.B.D., J.P., F.T.-C., A.R., and H.A.-O. contributed new reagents/ in ILC3s, Ag-specific T-cell and T-dependent (TD) B-cell responses analytic tools; A.R. performed cell sorting; N.v.B. and D.F. analyzed data; and N.v.B., S.C., and + are impaired, demonstrating that ILC3s present Ag to CD4 T cells in D.F. wrote the paper. vivo. IL-1β strongly activates fetal liver (FL)-derived and splenic The authors declare no conflict of interest. − NCR ILC3s and induces both the expression of costimulatory This article is a PNAS Direct Submission. molecules and the up-regulation of MHC class II molecules, 1N.v.B. and S.C. contributed equally to this work. thereby enhancing their T-cell priming potential. Finally, we show 2To whom correspondence should be addressed. Email: [email protected]. + that in the presence of Ag, the cognate interaction between CD4 T − This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cells and NCR ILC3s leads to the activation of the latter, 1073/pnas.1406908111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1406908111 PNAS | September 2, 2014 | vol. 111 | no. 35 | 12835–12840 Downloaded by guest on September 30, 2021 T-cell proliferation was significantly reduced, demonstrating that ILC3s were able to present Ag and to meaningfully alter OVA-specific T-cell responses in vivo. To study the role of ILC3s Δ in TD B-cell responses, we immunized WT, I-ab ILC3, and − − RORγ / mice intraperitoneally (i.p.) with a single dose of Alum- precipitated nitrophenylated-OVA (100 μg) and adoptively + transferred 2 × 106 OT-IItg CD4 T cells plus CpG. The loss of Δ MHC class II on ILC3s (I-ab ILC3) resulted in a significant re- duction of 4-hydroxy-3-nitrophenyl-acetyl (NP)-OVA–specific IgG 14 d after immunization (Fig. 1D). Additionally, a more detailed analysis of IgG subtypes revealed that specific IgG1, IgG2a, IgG2b, and IgG3 levels were significantly reduced (Fig. 1 − − E–H). In RORγ / mice, where ILC3s, Th17 cells, lymph nodes, and Peyer’s patches were completely absent, NP-OVA–specific IgG titers were even more reduced. Collectively, these data un- ambiguously show that Ag presentation by ILC3s contributes to + T-cell priming in vivo and that CD4 T-cell proliferation and TD B-cell responses were impaired when Ag presentation was abolished in ILC3s. − NCR ILC3s Can Internalize Latex Beads. Based on the in vivo finding that the lack of MHC class II molecules by ILC3s impaired T-cell–mediated immune responses, we tested the capacity of − + NCR ILC3s generated from α4β7 FL progenitor cells in vitro (14) or ex vivo isolated from the spleen of adult mice to take up red fluorescent latex beads. We and others have previously shown that FL-derived and adult ILC3s share phenotypic and functional properties such as lymphotoxin β-dependent induction of lymphoid tissue formation (15, 16). Both in vitro-generated − and ex vivo-isolated NCR ILC3s were capable of internalizing red fluorescent latex beads, although with slower kinetics than bone marrow (BM)-derived MΦ (BMMΦ) (Fig. 2 A and B and Fig. S1). Bead uptake was severely inhibited at 4 °C or in the presence of 0.5 μM Cytochalasin D (CytD), an inhibitor of actin polymerization, showing the specificity of internalization (Fig. − 2C). In vitro-generated NCR ILC3s could be subdivided into + − − CD4 and CD4 NCR ILC3 subsets (Fig. S2 A and B).