CXCR6+ST2+ memory T helper 2 cells induced the expression of major basic in to reduce the fecundity of helminth

Kazushige Obata-Ninomiyaa, Kenji Ishiwatab, Hisanobu Nakanoc, Yusuke Endoc, Tomomi Ichikawac, Atsushi Onoderac,d, Kiyoshi Hiraharac, Yoshitaka Okamotoe, Hirotaka Kanukab, and Toshinori Nakayamac,1

aDepartment of Advanced Allergology of the Airway, Graduate School of Medicine, Chiba University, 260-8670 Chiba, Japan; bDepartment of Tropical Medicine, The Jikei University School of Medicine, 105-8461 Tokyo, Japan; cDepartment of Immunology, Graduate School of Medicine, Chiba University, 260-8670 Chiba, Japan; dInstitute for Global Prominent Research, Chiba University, 263-8522 Chiba, Japan; and eDepartment of Otorhinolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 260-8670 Chiba, Japan

Edited by Kenneth M. Murphy, Washington University, St. Louis, MO, and approved August 7, 2018 (received for review August 20, 2017) Memory T helper (mTh) cells play important roles in the reinfection important in ILC2 cells for triggering production of IL-5 and IL- of pathogens and drive the pathogenesis of diseases. While recent 13 and also, in Tpath2 cells for chromatin remodeling of the Il5 studies have characterized the pathogenic mTh2 cell subpopulations and up-regulation of ST2 expression (8). driving allergic , those that induce immune responses It has been reported that a subset of Treg cells expresses ST2 against helminth infection remain unknown. We found that IL-5– (14). Treg cells suppress immune-mediated inflammation (15, 16). + + + + producing CXCR6 ST2 CD44 mTh2 cells play a crucial role in the ST2 Treg cells are generated by TCR stimulation in the presence IL-33–dependent inhibition of the fecundity of helminth, whereas γ − of IL-33 in a process controlled by IRF4, BATF, and PPAR (17). other ST2 mTh2 cells do not. Although both cell types induced Helminth infection is known to induce the generation of the infiltration of granulocytes, especially eosinophils, into the lungs + Th2 cells and Treg cells (18, 19). In this study, we used the in response to helminth infection, the ST2 mTh2 cell-induced eosin- nematode helminth Nippostrongylus brasiliensis (Nb). Nb passes ophils expressed higher levels of major basic protein (MBP), which is through the lungs before reaching the gut and is expelled within important for reducing the fecundity of Nippostrongylus brasiliensis 10 d in mice. Mice acquire and maintain immunity against Nb for (Nb), than ST2− mTh2 cell-induced ones. Notably, we also found that + − + + over 1 y. Nb induces accumulation of Th2 cells in the lungs that ST2 Treg cells but not ST2 Treg cells suppressed CXCR6 ST2 peaked 10 d after infection (18). Helminth-induced Th2 cells mTh2 cell-mediated immune responses. Taken together, these find- ings show that we identified a mechanism against helminth elicited produce IL-4, IL-5, and IL-13, which results in elevated serum by a subpopulation of IL-5–producing mTh2 cells through the accu- IgE, eosinophilia, goblet cell hyperplasia, and ultimately, helminth mulation of eosinophils strongly expressing MBP in the lungs. expulsion (20). The type 2 inflammatory immune response in- duced by helminth infections is similar to that observed in allergic IL-33/ST2 | memory Th2 cell subset | Treg | helminth | allergic inflammation asthma (21). Helminth infection increased IL-33 levels in the lungs (22), such as occurs during asthma pathogenesis, and IL- ntigen recognition by the T-cell receptor (TCR) drives naïve 33 deficiency impairs the expulsion and inhibition of maturation + ACD4 T cells to differentiate into effector T helper (Th) cell subsets, such as Th1, Th2, and Th17 cells, that later become Significance memory T helper type 1 (mTh1), mTh2, and mTh17 cells that orchestrate long-term antigen-specific immune responses (1–3). Helminth infection elicits T helper type 2 (Th2) and Treg cells.

Recently, based on disparate production patterns, several However, the critical cell subpopulations of Th2 and Treg cells in INFLAMMATION antihelminth immunity remain unknown. We identified two IMMUNOLOGY AND functionally distinct mTh2 subpopulations have been identified; + + − Th2 + 1cells,IL-17–producing Th2 cells, and high IL-5–producing Th2 cell subpopulations: CXCR6 ST2 memory Th2 cells and ST2 pathogenic T helper type 2 (Tpath2) cells (4–7). Th2 + 1 cells memory Th2 cells. Although both subpopulations induced the ac- γ – cumulation of eosinophils into the lungs during helminth infection, produce IFN- in addition to Th2 , IL-17 producing + + Th2 cells produce IL-17 and Th2 cytokines, and the high IL-5– those induced by CXCR6 ST2 memory Th2 cells expressed high producing memory-type Tpath2 cells express ST2, a component levels of major basic protein (MBP) and contributed to the re- duction of fecundity of helminth. This response was suppressed by of the IL-33 receptor. Tpath2 cells produce large amounts of + − ST2 but not ST2 Treg cells, both of which are induced during IL-5 after TCR stimulation (7, 8). Several of these Th cell sub- + + populations possess effector functions that play crucial roles in helminth infection. We, therefore, identified CXCR6 ST2 memory the pathogenesis of Th1, Th2, and Th17 cell-mediated in- Th2 cells as a critical subpopulation to induce accumulation of flammatory diseases (3). In comparison with models where the eosinophils strongly expressing MBP in the lungs. balance of conventional Th cell subsets (Th1, Th2, and Th17) de- “ Author contributions: K.O.-N., Y.O., H.K., and T.N. designed research; K.O.-N., K.I., and termines certain disease states, we have proposed a pathogenic Th H.N. performed research; K.O.-N. contributed new reagents/analytic tools; K.O.-N. and population disease induction model,” in which the minority pres- A.O. analyzed data; and K.O.-N., K.I., H.N., Y.E., T.I., A.O., K.H., and T.N. wrote the paper. ence of unconventional Th cell subsets determines disease (3). The authors declare no conflict of interest. IL-33, a member of the IL-1 family, is released from various This article is a PNAS Direct Submission. cells, including epithelial cells, in response to cellular damage or Published under the PNAS license. inflammation (9, 10). Il33 and Il1rl1 are well-known to be Data deposition: The RNA sequence datasets reported in this paper have been deposited associated with the severity of asthma symptoms (11). IL- in the Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo 33 stimulation exacerbates allergic airway inflammation and is (accession no. GSE120173). associated with infiltration of eosinophils into the mucosa (12). 1To whom correspondence should be addressed. Email: [email protected]. The IL-33 receptor consisting of ST2 and IL-1 receptor accessory This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. protein is expressed on various inflammatory cells, including type 1073/pnas.1714731115/-/DCSupplemental. 2 innate lymphoid cells (ILC2s) and Tpath2 cells (8, 13). IL-33 is Published online October 1, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1714731115 PNAS | vol. 115 | no. 42 | E9849–E9858 Downloaded by guest on September 29, 2021 − + of worms (23). However, the functionally critical subpopulation of ST2 and ST2 mTh cells isolated on day 40 after Nb infection mTh2 cells that induces immune responses against helminth (Fig. 2A). To investigate population-specific gene expression, we remains unknown. selected genes with a difference in expression of over fivefold in + + In this study, we identified CXCR6 ST2 mTh2 cells that help one population compared with the other two populations and reduce the fecundity of the helminth, Nb, via the accumulation displayed these gene sets as heat maps. This analysis revealed that − + of eosinophils expressing high levels of major basic protein ST2 and ST2 mTh cells possess distinct transcriptional profiles (MBP) in the lung (24). Notably, the reduction of fecundity in- (Fig. 2B). MA plot analysis of transcription factors, cytokines, + + + duced by CXCR6 ST2 mTh2 cells was suppressed by the ST2 , and cell surface molecules (Fig. 2 C, D,andF) − + but not the ST2 Treg cells elicited in Nb infection. We showed a revealed that ST2 mTh cells expressed lower levels of central cellular and molecular mechanism underlying the immune re- mTh cell markers, such as Tcf7, Klf2, Ccr7,andSell, and higher sponse against helminth. levels of resident mTh cell markers, such as Pparg (26, 27). The expression of Th2 cell-specific genes (Il4, Il5, Il13, Epas1,and + Results Gata3) was higher in ST2 mTh cells, while Il2, Ifng, Tnf,andCcl5 + Helminth Infection Induces IL-5–Producing ST2 mTh Cells. We re- were lower. Real-time qPCR analysis confirmed the differential cently identified a crucial subpopulation of mTh2 cells that pro- expression of these genes (Fig. 2E). Analysis of cell surface mote allergen-induced eosinophilic airway inflammation, named markers revealed CCR7, CD62L, CXCR3, and IL-18Rα to be − memory-type Tpath2 cells (3). Tpath2 cells selectively express ST2 higher on ST2 mTh cells and CD69, CCR2, and CXCR6 to be + (a component of the IL-33 receptor). Although it is well-known higher on ST2 mTh cells (Fig. 2G). CD69 is a marker of activated that Th2 cells play critical roles in helminth infections, whether cells and also resident memory T cells, while CCR2 and an analogous ST2+ effector Th2 cell subpopulation also mediates CXCR6 are known to be involved in migration to the lung (28–30). the immune response to helminth remains unknown. To address + this question, we analyzed mice infected with the helminth Nb. In Nb-Induced ST2 mTh Cells Are Required for the Reduction of the Fecundity of Nb Infection. We next sought to investigate the this model, the number of Th2 cells in the lungs of wild-type mice + − ∼ physiological relevance of ST2 and ST2 mTh cells in the expul- reached a peak 10 d after infection, correlating with the timing of + + Nb expulsion from the gut (18, 25). We assessed ST2 expression in sion, maturation, and fecundity of Nb. Naïve CD4 T cells or ST2 + − − Th cells (CD4 CD44hiFoxP3 )fromthelungsandspleensofmice or ST2 mTh cells purified from BALB/c mice infected 40 d earlier infected with Nb for 0 (naïve time point), 10 (effector time point), with Nb were adoptively transferred to syngeneic recipients. Four and 40 d (memory time point), as depicted in Fig. 1A.Toavoid weeks later, adoptively transferred mice were infected with Nb (SI false-positive staining, we included the staining of ST2 expression Appendix,Fig.S3). Compared with recipients of naïve Th cells, re- − − + − on Th cells of Il1rl1 / mice as negative control (Fig. 1B). In naïve cipients of ST2 or ST2 mTh cells up-regulated mRNA level of lungs, less than 5% of Th cells expressed ST2. At the effector time Il33 in the lungs 2 d after Nb infection when Nb reached the lungs, point, expression of ST2 was up-regulated on Th cells in the lungs and we also noted moderate up-regulation of mRNA of Tslp in the + + − (Fig. 1B). At the memory time point, the number of ST2 Th cells lungs of recipients of ST2 or ST2 mTh cells (Fig. 3A and SI in the lungs had declined but remained at least two- to threefold Appendix,Fig.S4A). We observed an increase in the number of lung higher than in naïve mice (Fig. 1 C and D). eosinophils, , and neutrophils after Nb infection (Fig. 3B, orange bars vs white bars). The numbers of eosinophils and Next, we examined the cytokine production profile of Th cells + − macrophages were elevated in ST2 or ST2 mTh cell-transferred at the effector and memory time points. After Nb infection, the + production of Th2 cytokines (IL-4, IL-5, and IL-13) by Th cells in mice compared with naïve CD4 T cell-transferred mice 4 d after + Nb infection (Fig. 3B, white bars vs. gray or black bars). However, the lungs dramatically increased, particularly among ST2 cells + − at the effector and memory time points (Fig. 1E and SI Appendix, no marked difference was observed between the ST2 or ST2 mTh − Fig. S1 A and B). Although a substantial proportion of ST2 Th recipients (Fig. 3B, gray bars vs. black bars). We next assessed the number of eggs in the feces and the number and size of worms cells produced IL-4 and IL-13, IL-5 production was almost ex- + − + isolated from the small intestines of ST2 or ST2 mTh recipients clusively detected within the ST2 population. Low levels of + γ − + 6 d after Nb infection. Compared with recipients of naïve CD4 IFN- and IL-17A were detected from both ST2 and ST2 Th − + cells. These results indicate that Nb infection induced substantial T cells and ST2 mTh cells, the recipients of ST2 mTh cells – + exhibited dramatically fewer eggs and significantly smaller worms, numbers of IL-5 producing ST2 Th cells detectable at least + until day 40 postinfection. Although less dramatic, a similar indicating that the ST2 mTh cell-mediated immune responses − pattern of Th2 cytokine production was detected in splenic ST2 inhibited Nb maturation (Fig. 3C, Left and Fig. 3D). No detectable + and ST2 Th cells (SI Appendix, Fig. S1 C and D). Moreover, the worms were noted in the skin (days 2 and 6), the lungs (day 6), and + majority of ST2 Th cells expressed GATA3 at the effector and small intestine (day 2), and no marked difference was noted in the memory time points (Fig. 1F and SI Appendix, Fig. S1E). number of worms isolated from the lungs or small intestine among the three experimental groups (Fig. 3C, Right and SI Appendix,Fig. IL-33 Treatment Enhances ST2 Expression and IL-5 Production in ST2+ S4D). A kinetic study of the number of eggs was performed, and an − + mTh Cells. Next, we investigated the role of IL-33 in ST2 and inhibitory effect of ST2 mTh cells was observed at least on days 5– + + − − + + ST2 mTh cells (CD4 CD44hiCD25 ). Sorted ST2 and ST2 8(Fig.3E). The transfer of ST2 Th cells isolated from the mice Th cells recovered at the memory time point (SI Appendix, Fig. 10 d after Nb infection (effector time point) into naïve mice did not S2A) were cultured with or without IL-33 and IL-7 (SI Appendix, reduce the number of eggs in feces, while those from mice at 40 d Fig. S2B). Culture with IL-33 together with low levels of IL-7 led after Nb infection (memory time point) did reduce the number of + to significant elevation of IL-5 and IL-13 mRNA expression in eggs, indicating that ST2 mTh cells isolated from the mice at 40 d + ST2 mTh cells (SI Appendix, Fig. S2C). IL-33 + IL-7 treatment after Nb infection have a memory function against Nb (SI Appendix, + + enhanced ST2, IL-5, and IL-13 expression in ST2 mTh cells (SI Fig. S4 E and F). Collectively, these findings indicate that ST2 but − Appendix, Fig. S2D). These results indicate that IL-33 + IL- not ST2 mTh cells reduced the fecundity of Nb. + 7 treatment can enhance the ability of ST2 mTh cells to ex- + press ST2, IL-5, and IL-13. The ST2 mTh Cell-Mediated Reduction of Fecundity of Nb Depends on IL-33, IL-5, and Eosinophils. Next, to explore the cellular mech- − + + Nb-Induced ST2 and ST2 mTh Cells Exhibit Distinct Gene Expression anisms underlying ST2 mTh cell-dependent immunity against Profiles. Next, we used RNA sequence analysis to compare gene Nb, we assessed the role of IL-33 (SI Appendix, Figs. S5A and S6 + + expression profiles of Nb-induced lung naïve CD4 T cells and A and B). As described above, the adoptive transfer of ST2

E9850 | www.pnas.org/cgi/doi/10.1073/pnas.1714731115 Obata-Ninomiya et al. Downloaded by guest on September 29, 2021 − − + + mTh cells into BALB/c mice conferred the ability to suppress the mTh cells from Il1rl1 / mice into normal Il1rl1 / BALB/c mice number of fecal Nb eggs 6 d after Nb infection. Strikingly, the failed to confer this suppressive effect on the number of eggs + transfer of ST2 mTh cells into IL-33–deficient mice or whole recovered from the feces (Fig. 4A and SI Appendix, Fig. S6 A and

A Nb infection E Lung CD4+CD44hiFoxp3- gated ## # naive effector memory 010 40 (Day 0) (Day 10) (Day 40) (naïve) (effector) (memory) Il1rl1+/+ 105 24.5 0.9 105 29.2 32.0 105 21.2 18.2 # Assay: Cell surface, ICS Il1rl1-/- 104 104 104 B Lung CD4+Foxp3- gated 103 103 103 naive effector memory 102 102 102 (Day 0) (Day 10) (Day 40) 0 73.2 1.4 0 30.0 8.8 0 53.1 7.5 105 9.5 2.8 105 14.6 17.5 105 ST2 10.0 6.3 IL-4 00102103104105 102103104105 0102103104105 Il1rl1+/+ 4 4 4 10 10 10 105 22.9 1.4 105 19.6 31.0 105 35.8 4.0 3 3 3 10 10 10 104 104 104 102 102 102 103 103 103 0 0 0

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4 $!!" 2 2 $#!" 100 10 10 102 0 74.2 1.3 0 38.0 4.1 0 $!!"100 ST2 55.5 2.1 $!!"100 IL-10 00102103104105 102103104105 0102103104105 #!"50 F #!" #!" Lung CD4+CD44hiFoxp3- gated cell no. (x 10 !" naive effector memory !"0 0 !"0 (Day 0) (Day 10) (Day 40) naïve (Day 0) 105 14.2 13.5 105 4.8 54.2 105 13.7 25.7 effector (Day 10) 104 104 104 memory (Day 40) 3 *P <0.05 10 103 103 **P <0.01 102 102 102 ***P <0.001 0 59.5 12.8 0 22.3 18.7 0 51.6 9.0 n.s ST2 2 3 4 5 2 3 4 5 2 3 4 5 GATA3 0010 10 10 10 10 10 10 10 01010 10 10

Fig. 1. Helminth infection induces IL-5–producing ST2+ mTh cells. (A) Experimental protocols for the infection of Il1rl1+/+ and Il1rl1−/− mice with Nb. #Assay: cell surface, + + − intracellular staining (ICS). (B)CD4 T cells were isolated from the lungs before and after infection with Nb. Staining profiles of CD44 and ST2 in CD4 Foxp3 Tcellsare + − − shown with the percentages of cells in each area. (C and D) The percentages (C) and the absolute cell numbers (D)ofmemoryCD4 T cells (total), ST2 mTh cells (ST2 + + mTh), and ST2 mTh cells (ST2 mTh) in the lungs are shown. Mean values (five mice per group) are shown with SEM. n.s., not significant. *P < 0.05; **P < 0.01; ***P < + 0.001. (E and F)MemoryCD4 T cells shown in B were stimulated with phorbol 12-myristrate 13-acetate (PMA) and ionomycin for 6 h. Intracellular staining profiles of ST2, IL-4, IL-13, IL-5, IFN-γ, IL-17, and IL-10 (E) and intracellular staining profile of GATA3 (F) are shown with the percentages of cells in each area. We set positive/negative gates using staining of Il1rl1−/− cells (B) or with isotype controls (E and F). Five independent similar experiments were performed, and similar results were obtained.

Obata-Ninomiya et al. PNAS | vol. 115 | no. 42 | E9851 Downloaded by guest on September 29, 2021 AB

CD

E

FG

Fig. 2. Nb-induced ST2− and ST2+ mTh cells exhibit distinct gene expression profiles. (A) Experimental protocols for the infection of wild-type mice with Nb. #Assay: RNA sequencing. (B) The expression of the genes with a difference in expression of more than fivefold in ST2+ mTh cells compared with the other two groups obtained + by RNA sequence analysis. (C and D) MA plots comparing the different expression of transcription factors (C) as well as cytokines and chemokines (D)betweenST2 − + − − + and ST2 mTh cells. Horizontal line denotes log2[(ST2 mTh)/(ST2 mTh)], while vertical lines denote (1/2) × log2[(ST2 mTh) × (ST2 mTh)]. (E) A qRT-PCR analysis of + − the indicated genes in ST2 and ST2 mTh cells. The minimum data values were set as one. The mean values (four data per group) are shown with the SEM. n.s., not + − significant. **P < 0.01; ***P < 0.001. (F) MA plots comparing the differential expression of surface markers between ST2 and ST2 mTh cells. (G) Staining profiles of indicated markers on ST2+ and ST2− mTh cells are shown with the mean fluorescence intensity of cells in histograms. At least two independent experiments were performed and showed similar results. The mean of FPKM was used for analyses in B–D and F. We set positive/negative gates using staining with isotype controls in G.

E9852 | www.pnas.org/cgi/doi/10.1073/pnas.1714731115 Obata-Ninomiya et al. Downloaded by guest on September 29, 2021 40 ** A Lung Il33 B Lung ** ** )

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1 200 10 (/HPRT) ** * Cell number (x 10 ** * Cell number (x 10

Relative expression 0 0 0 Eos Mac Neu MC Baso ILC2 Lym. Total no transfer no infection *P <0.05 naive CD4+ T cells **P <0.01 ST2- mTh cells Nb infection ***P <0.001 ST2+ mTh cells

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(x 10 200 100 10 Number of worm 100 Length of worm ( 0 0 0 1 2 3 Number of egg per gram naive CD4+ T cells *P <0.05 ST2- mTh cells **P <0.01 ST2+ mTh cells ***P <0.001 E n.s 5 10 naive CD4+ T cells ST2- mTh cells 104 ST2+mTh cells 3 * P <0.05 (naïve CD4+ T cells vs ST2+ mTh cells) 10 * - + †††* †† †† P <0.01 (ST2 mTh cells vs ST2 mTh cells) * ††† P <0.001 (ST2- mTh cells vs ST2+ mTh cells) 102 n.s. n.s + + - + 1 (naïve CD4 T cells vs ST2 mTh cells, or ST2 mTh cells vs ST2 mTh cells) 10 n.s.

Number of egg per gram n.s. 100 59876 01 Days after infection

Fig. 3. Nb-induced ST2+ mTh cells are sufficient for the reduction of fecundity of Nb. (A–D) Mice were killed 2 (A), 4 (B), or 6 d (C and D) after infection with Nb as described in SI Appendix, Fig. S3.(A) A qRT-PCR analysis of the indicated genes in the lungs of indicated mice. The minimum data values were set as one. The mean values (four data points per group) are shown with the SEM. (B) The absolute cell numbers of eosinophils (Eos), macrophages (Mac), neutrophils (Neu), mast cells (MC), basophils (Baso), and lymphocytes (Lym) in the lungs are shown. The mean values (four mice per group) are shown with the SEM. (C) The absolute numbers of parasite eggs in feces per gram and the number of worms are shown. The mean values (three or four mice per group) are shown with the SEM. (D) The lengths of worms collected from the small intestine of the indicated mice are shown. The mean values (at least 15 worms from four mice per group) are shown with the SEM. n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001. (E) The feces were collected from the mice from 5 d after Nb infection as described in SI Appendix, Fig. S3. The absolute numbers of parasite eggs in feces per gram are shown. The mean values (four mice per group) are †† ††† shown with the SEM. At least two independent experiments were performed with similar results. n.s., not significant. *P < 0.05; P < 0.01; P < 0.001. INFLAMMATION IMMUNOLOGY AND

+ B). These results suggest that the IL-33–ST2 axis was required mice (32). ΔdblGATA recipients of ST2 mTh cells did not show + for ST2 mTh cells to induce immune responses against Nb. a suppressed number of fecal eggs compared with recipients of − Furthermore, IL-33 increased the production of IL-13 and IL- ST2 mTh cells (Fig. 4D and SI Appendix, Fig. S5D). Although + 5 from ST2 mTh cells (SI Appendix, Fig. S2). We, therefore, ILC2 cells are known to induce the accumulation of eosinophils + next examined the contribution of IL-13 and IL-5 to the ST2 via the production of IL-5, the depletion of ILC2 cells did not + mTh2 cell-dependent reduction of fecundity of Nb using Il4ra- significantly affect this ST2 mTh cell-mediated reduction of deficient mice lacking the expression of types I and II receptors fecundity of Nb (SI Appendix, Fig. S6 C and D) (13). These re- for IL-4 and IL-13 and neutralizing antibody against IL-5. Sur- sults indicate that IL-33, IL-5, and eosinophils are required for + + prisingly, the transfer of ST2 mTh cells into Il4ra-deficient mice ST2 mTh cell-mediated reduction of fecundity of Nb. Thus, the + reduced the number of eggs recovered from the feces compared IL-33–induced IL-5 production from ST2 mTh cells and the − with ST2 mTh cell-transferred mice, suggesting that IL-4 and resulting eosinophilic activation play important roles in the re- + IL-13 are dispensable for the ST2 mTh cell-dependent reaction duction of the fecundity of Nb. (Fig. 4B and SI Appendix, Fig. S5B). We next analyzed the role of + + − IL-5 in the ST2 mTh cell-mediated reduction of fecundity of Nb Eosinophils Induced by ST2 and by ST2 mTh Cells in Nb Infection (SI Appendix, Fig. S5C). Treatment with an IL-5–neutralizing Exhibit Distinct Gene Expression Profiles. We found that eosinophils + + antibody 1 d before Nb infection of ST2 recipients led to a play an important role in the ST2 mTh cell-dependent immu- dramatic increase in the number of fecal eggs compared with nity against Nb. However, the number of eosinophils accumu- + control antibody-injected mice (Fig. 4C). IL-5 is known to be an lated in the lungs by ST2 mTh cells was almost the same as that − important factor for proliferation, activation, and accumulated by ST2 mTh cells (Fig. 3). We, therefore, hy- + chemoattraction (31). We, therefore, assessed whether eosino- pothesized that the function of eosinophils induced by ST2 mTh + − phils are involved in the ST2 mTh cell-dependent reduction of cells was distinct from that of eosinophils induced by ST2 fecundity of Nb using eosinophil-deficient mice: ΔdblGATA mTh cells.

Obata-Ninomiya et al. PNAS | vol. 115 | no. 42 | E9853 Downloaded by guest on September 29, 2021 A ** n.s. n.s. *** n.s. n.s. n.s. 300 ST2- mTh cells Il33+/+ mice 20 n.s. ST2+ mTh cells

) 200 3 ST2- mTh cells Il33-/- mice ST2+ mTh cells

(x 10 10 100 **P <0.01

Number of worm ***P <0.001 0 0 n.s

B C D n.s. n.s. *** n.s. n.s. n.s. 300 n.s. 300 n.s. n.s. 12 * 60 300 30 9 200 ) ) ) 200 4 3 40 200 4 20 6 (x 10 (x 10 100 (x 10 20 100 10 100 3 Number of worm Number of worm Number of worm 0 0 0 0 0 0 ST2- mTh cells control Ab Il4ra-/- mice ST2+ mTh cells Ab ST2- mTh cells dblGATA ***P <0.001 *P ST2+ mTh cells n.s n.s n.s

+ Fig. 4. ST2 mTh cell-mediated reduction of fecundity of Nb depends on IL-33, IL-5, and eosinophils but not IL-4 and IL-13. (A–D) Mice were killed 6 d after infection with Nb. Experimental protocol is shown in SI Appendix, Fig. S5. The absolute numbers of parasite eggs in feces per gram and the numbers of worms are shown. The mean values (three or four mice per group) are shown with the SEM. At least two independent experiments were performed and showed similar results. n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

+ To explore the molecular mechanisms underlying ST2 mTh creased after Nb infection at the effector time point (day 10) cell-dependent immunity against Nb, we compared the gene (Fig. 6 B–D). At the memory time point (day 40), the number of + + expression profiles of lung eosinophils in naïve CD4 T cell- or ST2 Treg cells was elevated in the lungs compared with that at − + ST2 or ST2 mTh cell-transferred BALB/c mice on Nb in- the naïve time point (day 0). FACS analysis revealed that lung + fection (SI Appendix, Fig. S7) (8). We used heat maps to depict ST2 Treg cells expressed preferentially IL-10 and Latency- the genes that exhibited differential expression at least twofold associated protein (LAP), a surrogate marker of TGF-β pro- − higher in a given population than in the other two (Fig. 5A). The duction, compared with ST2 Treg cells (Fig. 6E). + + − ST2 mTh cell-induced eosinophils selectively expressed To analyze whether ST2 or ST2 Treg cells influenced the + 17 genes, including antihelminthic enzyme Prg2 coding MBP immunity conferred by adoptively transferred ST2 mTh2 cells, + + − (24). This result was confirmed by qRT-PCR (Fig. 5B, Left). We ST2 mTh2 cells were cotransferred with ST2 or ST2 Treg included the expression of Epx as a control (Fig. 5B, Right). In- cells compared with naïve CD4 T-cell cotransferred mice. In- + − deed, 10 ng/mL of IL-5 up-regulated the expression of mRNA of terestingly, the cotransfer of ST2 Treg cells but not ST2 Treg Prg2 during eosinophil development (SI Appendix, Fig. S8 A and cells prepared using CD25 expression (SI Appendix, Fig. S9A)or B). We next examined the functional consequence of MBP in the Foxp3-YFP expression (SI Appendix, Fig. S9B) increased the eosinophils during Nb infection. The expression level of mRNA number of fecal eggs compared with naïve CD4-transferred + − of Prg2 in eosinophils was almost depleted by the treatment of mice, indicating that ST2 but not ST2 Treg cells selectively + siRNA for Prg2 (SI Appendix, Fig. S8 C and D). The number of constrained the ST2 mTh2 cell-mediated immune responses fecal eggs from mice receiving intranasally transferred control against Nb (Fig. 6 F and G). In addition, on investigating the role + siRNA-treated eosinophils was significantly reduced compared of ST2 Treg cells in NSG mice lacking of T cells, B cells, and + with no transfer mice, and the number recovered if MBP- ILC2 cells (SI Appendix, Fig. S9C), we noticed that ST2 depleted eosinophils was introduced (Fig. 5 C and D). The mTh2 cells harvested from Nb-infected mice reduced the num- number of worms was not changed in these experimental set- ber of worms in the intestine and that the expulsion of Nb me- + + + tings. These results indicated that eosinophils induced by ST2 diated by ST2 mTh2 cells was significantly inhibited by ST2 − − mTh cells and ST2 mTh cells possess distinct gene expression but not ST2 Treg cells (SI Appendix, Fig. S9D). These results + + profiles and that ST2 mTh cells induced eosinophils with high indicate that ST2 mTh2 cells are sufficient for reducing the expression of Prg2 mRNA, which is sufficient to reduce the fe- number of worms in the intestine under lymphodeficient condi- + cundity of Nb in the lungs. tions and also support a role for ST2 Treg cells in suppressing + the function of ST2 mTh2 cells to reduce the fecundity. ST2+ Treg Cells Suppress ST2+ mTh Cell-Dependent Immunity Against Nb. In the colon and adipose tissues, some Treg cells express ST2 Discussion + + + (14, 17). Whether these ST2 Treg cells play a role in helminth We identified ST2 mTh2 cells and ST2 Treg cells as promoters + infection is not known. We sought to assess whether ST2 Treg and repressors, respectively, of immune responses in Nb in- + cells occurred in the lungs before and after Nb infection. The fection. Nb-induced ST2 mTh cells produced large amounts of + + frequency of CD4 FoxP3 Treg cells in the lungs increased after IL-5 and exhibited a transcriptional profile distinct from that of + − Nb infection (Fig. 6A). We detected a small number of ST2 ST2 mTh cells. This study revealed the cellular and molecular + Treg cells in the lungs of naïve mice, and these numbers in- mechanisms underlying the ST2 Th2 cell-mediated immune

E9854 | www.pnas.org/cgi/doi/10.1073/pnas.1714731115 Obata-Ninomiya et al. Downloaded by guest on September 29, 2021 A B Prg2 Epx Lung-eosinophil Nb *** n.s. naïve mTh *** 10 2 n.s. n.s. Th ST2- ST2+ 10 naive CD4+ T cells H2-DMb2 ST2- mTh cells H2-Eb1 55 n.s. 1 +

(/HPRT) ST2 mTh cells Olfr1046 ***P <0.001 Fkbp3

Relative expression n.s Prg2 00 0 Olfr470 C Nb infection (500) Fabp6 Transfer

Olfr145 -10 6 Olfr700 BALB/c Eosinophils Treated with Defb48-ps Assay: count number of egg and worm siRNA for control or Prg2 Grap n.s. Olfr1085 D n.s. Pdlim4 ** ** 300 n.s. n.s. Olfr160 1515

Izumo4 ) 200 3 Olfr1384 1010

Olfr1113 (x 10 100 55 Number of worm 0 -1.0 0 1.0 Number of egg per gram 0 0 Row Z-score no-transfer **P <0.01 Control siRNA n.s eosinophil siRNA for Prg2

+ Fig. 5. Helminth-induced ST2 mTh2 cells accumulated MBP-expressing eosinophils in the lungs, reducing the fecundity of Nb. (A) The expressions of the + genes with a difference in expression of more than fivefold in the sorted eosinophils obtained from the lungs of ST2 mTh cell-transferred mice compared with the other two groups according to an RNA sequence analysis were selected as depicted in SI Appendix, Fig. S7. The genes listed in two independent + − + experiments are shown. (B) A qRT-PCR analysis of Prg2 and Epx in the sorted eosinophils obtained from the lung of naïve CD4 T cell-, ST2 mTh cell-, or ST2 mTh cell-transferred mice. The minimum data values were set as one. The mean values (six data per group) are shown with the SEM. (C) Experimental protocols for the infection of the mice with Nb. #Assay: count numbers of eggs and worms. (D) Mice were killed 6 d after infection with Nb, as described in C. The absolute numbers of parasite eggs in feces per gram and the numbers of worms are shown. The mean values (six mice per group) are shown with the SEM. At least two independent experiments were performed and showed similar results. n.s., not significant. **P < 0.01; ***P < 0.001.

+ − + responses in Nb infection: Nb-induced ST2 mTh cells caused the promotes eosinophil (38). Although ST2 and ST2 mTh inhibition of worm maturation and a reduction of fecundity via a cells both induced eosinophil accumulation in the lungs, only + mechanism that was dependent on IL-5 and IL-33 and through ST2 mTh cells induced the inhibition of the maturation of Nb. the accumulation of eosinophils expressing high levels of MBP, an Thus, we thought that eosinophils in the lungs may have distinct + − antihelminthic enzyme (24). The reduction of the fecundity of Nb properties depending on the adoptive transfer of ST2 vs. ST2 mTh + − was suppressed by ST2 but not ST2 Treg cells. Thus, Nb in- cells. The role of eosinophils against helminth infections has +

fection leads to the induction of ST2 mTh cells capable of in- been poorly defined and remains controversial. With regard to INFLAMMATION ducing immune responses in Nb infection in the lungs. Nb infection, eosinophils have been suggested to be associated IMMUNOLOGY AND Recently, it has been reported that Nb infection elicited IL- with a reduction in the fecundity of Nb in primary infection (39). 5 production from Th2 cells resident in peripheral tissues but not We further revealed that eosinophils contributed to the inhibi- in lymphoid organs due to peripheral tissue checkpoint cytokines, tion of maturation of Nb in secondary infection (SI Appendix, + including IL-33, IL-25, and TSLP (21). In this study, ST2 mTh Fig. S10). IL-5 transgenic mice that exhibit eosinophilia are re- cell-dependent immune responses against Nb were found to re- sistant to primary Nb infection (37, 40). These observations quire IL-33. TSLP might be involved in this reaction. Nb-elicited suggest that IL-5 is a key factor in eosinophil-dependent immune + ST2 mTh cells expressed higher levels of Pparg and CD69 and responses in Nb infection. − lower levels of Klf2, Tcf7, Ccr7,andSell compared with ST2 mTh Regarding the role of IL-13 in the immune responses against cells. PPARγ modulates cellular metabolism to sustain cell via- Nb infection, it has been reported that IL-13 is required for the bility and ST2 expression (17). It has been reported that resident expulsion of adult worms from the small intestine (20, 41). memory T cells express higher levels of PPARγ and CD69 com- Furthermore, IL-13 produced by T cells was shown to play a pared with central memory T cells (33, 34). Lower Klf2 expression minor role in the expulsion of adult Nb worms from the small may correlate with lower expression of central memory markers intestine in primary infection (42). In our experimental settings, + + CCR7 and CD62L (26, 35, 36). Nb-induced ST2 mTh cells the ST2 mTh2 cells produced both IL-5 and IL-13 and showed exhibited characteristic markers of resident memory T cells; thus, a reduction in the fecundity but not the expulsion of the worms they might respond to IL-33 in peripheral tissues and thereby, in normal mice. Interestingly, however, we detected a decreased acquire their unique transcriptional profile, supporting their number of worms in the intestine of a lymphodeficient NSG ability to produce high levels of IL-5. mouse model, as shown in SI Appendix, Fig. S9, indicating that + + Nb-induced ST2 mTh cells produced higher levels of IL- ST2 mTh2 cells have the potential to promote the expulsion of − + 5 than ST2 mTh cells. IL-5 is reported to be important for Nb under certain conditions. ST2 mTh cell-derived IL-13 may the survival, proliferation, migration, and inflammatory response play a role in the process of expulsion of Nb. of eosinophils to Toll-like receptor (TLR) ligands (37). In con- The number of larvae in the lung 2 d after secondary Nb − + trast, ST2 mTh cells produced CCL5, a CCR3 ligand that exposure was slightly decreased when ST2 mTh cells were

Obata-Ninomiya et al. PNAS | vol. 115 | no. 42 | E9855 Downloaded by guest on September 29, 2021 A Lung CD4+ gated C Lung CD4+Foxp3+ cell naive effector memory total ST2- cells ST2+ cells (Day 0) (Day 10) (Day 40) n.s. n.s. #)"15 10 )"5 100 8.5% 100 12.9% 100 8.8% #!" ** 80 80 80 *** *** *** *** &"4 *** 60 60 60 ***

#!" cells 10 40 40 40 + %"3 5 20 20 20 )" $"2

percent 0 0 0 )"5 2 3 4 5 2 3 4 5 2 3 4 5 Foxp3 010 10 10 10 010 10 10 10 010 10 10 10 % in CD4 #"1 B CD4+Foxp3+ gated Lung naive effector memory 0 !"0 !"0 (Day 0) (Day 10) (Day 40) D Lung + + 5 5 5 CD4 Foxp3 cell 10 89.4 4.3 10 64.1 24.0 10 84.5 9.2 total ST2- cells ST2+ cells Il1rl1+/+ 104 104 104 ** ** ** 3 3 3 #$!"120 (!"80 10 10 10 ** n.s. ** n.s. ** ** %!"30 2 2 2 10 10 10 '!"60 cells)

0 0 0 (!"4 80 CD44 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 $!"20 ST2 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 &!"40 105 105 105 93.2 1.6 92.6 1.0 95.2 0.5 &!"40 -/- #!"10 Il1rl1 104 104 104 $!"20

103 103 103 cell no. (x 10 !"0 !"0 !"0 102 102 102 naïve (Day 0) *P <0.05 CD44 0 0 0 **P <0.01 ST2 effector (Day 10) 101102103104105 101102103104105 101102103104105 ***P <0.001 memory (Day 40) n.s E F n.s. Lung Treg (CD4+Foxp3+) n.s. 40 ** naive effector memory n.s. n.s. (Day 0) (Day 10) (Day 40) 300 100 100 100 59.2 89.3 82.9 ) ** 80 27.8 80 45.0 80 34.6 3 200 2000020 n.s. ** 60 60 60 (x 10 40 40 40 100

20 20 20 Number of worm Number of egg per gram percent 0 0 0 00 0 0102103104105 0102103104105 0102103104105 IL-10 no transfer **P <0.01 100 100 100 251.0 374.0 330.0 ST2+ mTh cells + naive CD4+ T cells n.s 80 92.8 80 149.0 80 119.0 ST2+ mTh cells + ST2- Treg 60 60 60 ST2+ mTh cells + ST2+ Treg 40 40 40 20 20 20 n.s. G n.s. percent 0 0 0 2 3 4 5 2 3 4 5 2 3 4 5 * n.s. LAP 010 10 10 10 010 10 10 10 010 10 10 10 n.s. * *** * n.s. n.s. n.s. n.s. 100 114.0 100 488.0 100 130.0 4040 200 200 80 33.3 80 91.8 80 30.9 * 60 150 60 60 ) 40 3 40 40 2020 100100

20 20 20 (x 10 n.s. percent 0 0 0 50 2 3 4 5 2 3 4 5 GATA3 010 10 10 10 010 10 10 10 0102103104105 Number of worm ST2+ Number of egg per gram 00 00 ST2- no transfer *P <0.05 control ST2- mTh cells ***P <0.001 ST2+ mTh cells + naive CD4+T cells n.s ST2+ mTh cells + ST2- Treg (Foxp3-YFP+) ST2+ mTh cells + ST2+ Treg (Foxp3-YFP+)

Fig. 6. ST2+ Treg cells suppress the immune response against helminth induced by ST2+ mTh cells. (A and B) CD4+ T cells were isolated from the lungs of Il1rl1+/+ (A and B) and Il1rl1−/− (B) before and after infection with Nb in Fig. 1A. Staining profiles of Foxp3, CD44, and ST2 expression in lung CD4+ T cells before and after infection with Nb. The percentages of cells in each area are depicted. (C and D) The percentages (C) and numbers (D) of lung Treg cells − + (total), ST2 Treg cells, and ST2 Treg cells are shown. Mean values (five mice per group) are shown with the SEM. (E) Treg cells depicted in B were stimulated with phorbol 12-myristrate 13-acetate (PMA) and ionomycin for 6 h and then stained for ST2, IL-10, LAP, and GATA3. The mean fluorescence intensity of cells is shown in histograms. (F and G) Mice were killed 6 d after infection with Nb. The number of parasite eggs per gram of feces and the number of worms are shown. Mean values (three mice per group) are shown with the SEM. At least three independent experiments were performed and showed similar results. We set positive/negative gates using staining of Il1rl1−/− cells (B) or with isotype controls (A and E). n.s., not significant. *P < 0.05; **P < 0.01; ***P < 0.001.

E9856 | www.pnas.org/cgi/doi/10.1073/pnas.1714731115 Obata-Ninomiya et al. Downloaded by guest on September 29, 2021 transferred as shown in SI Appendix, Fig. S4D. Indeed, similar Antibodies. Alexa488-conjugated anti-Foxp3 and control rat IgG2b; results were reported by Knott et al. (43). Those authors studied allophycocyanin (APC)-conjugated anti-CD25, anti-CD69, anti–IL-4 (11B11), primary and secondary infections using IL-5–deficient and anti–IL-5 (TRFK5), anti–IL-10, anti-GATA3, control rat IgG1, IgG2a, and IgG2b; eosinophil-deficient ΔdblGATA mice. During primary infection, BV421-conjugated anti-ST2 and control rat IgG2a; BV510-conjugated anti- Thy1.1, anti-CD4 (RM4-5), control mouse IgG1 and rat IgG2a; phycoerythrin they found enhanced numbers of eggs but no marked differences (PE)-conjugated anti-LAP, anti–IL-17, control mouse IgG1 and rat IgG1; and in the number of larvae and worms in the absence of eosinophils. PE-Cy7–conjugated anti-CD44 and control rat IgG2b were purchased from The resistance to secondary infections was impaired in IL-5– Biolegend. PE-conjugated anti-CXCR3, APC-conjugated anti-CCR2 and anti- deficient and ΔdblGATA mice, with significantly increased CXCR6, and Alexa Fluor 647-conjugated anti–IL-18Rα were purchased from R numbers of larvae in the lungs 2 d after infection. and D Systems. PE-conjugated anti–IFN-γ and anti–IL-13 were purchased + We further revealed that eosinophils induced by ST2 mTh from BD. APC-conjugated anti-CD62L was purchased from Thermo. cells in the lungs possess distinct gene profiles from those of eo- − sinophils induced by ST2 mTh cells, including a high expression Helminth Infection and ex Vivo Experiments. Mice were infected with Nb by of MBP. MBP is known to be expressed in eosinophils, and it the s.c. injection of 500 third-stage larvae. To quantity the egg production, damages parasites. MBP was highly expressed in the eosinophils in feces from individual Nb-infected mice were collected. The eggs were then counted using a phase-contrast microscope. For the isolation of larvae from culture with IL-5 in vitro, and the fecundity of Nb was not the skin, the flank skin around the inoculation site was excised. The isolated inhibited by MBP-depleted eosinophils. Our study highlights the skin was finely minced with scissors and incubated with simulated gastric juice fact that the induction of MBP in eosinophils is dependent on + (0.24% hydrochloric acid and 0.32% pepsin) under constant agitation at 37 °C IL-33–stimulated IL-5 high-producing ST2 mTh2 cells. for 2 h. For the isolation of larvae from the lung, the whole lung was sur- Helminth-secreted products increase the differentiation of gically excised. The isolated lung was finely minced with scissors followed by Treg cells to prolong residence within the gut, thereby increasing incubation in PBS with a Baermann apparatus at 37 °C for 2 h. For the iso- the number of progeny that can be produced (44). Recently, lation of adult worms, the small intestine was excised and opened longitu- + ST2 Treg cells were differentiated after TCR stimulation in the dinally followed by incubation under the same conditions as those of the presence of IL-33 (14). In this study, we observed an increase in lung samples. For the evaluation of worm length, photomicrographs of + + worms recovered from the small intestine of infected mice were traced and ST2 Treg cells in the lungs after Nb infection. Thus, ST2 mTh measured using the ImageJ software program (23). For the in vivo neutral- cells provide inhibition of maturation and reduction of fecundity ization of IL-5, mice were treated once with an i.p. injection of 200 μg anti– of helminth, whereas helminths reduce the antihelminth immune IL-5 (TRFK5) or control rat IgG 1 d before the inoculation. + response by inducing ST2 Treg cells. These results suggest that + adjuvants that could promote the number of ST2 mTh2 cells Real-Time qPCR and RNA Sequence Analyses. Total RNA was isolated with the + and reduce the number of ST2 Treg cells would be beneficial in TRIzol reagent (Thermo). cDNA was synthesized with an oligo (dT) primer and the development of helminth vaccines. Tissue Treg cells not only SuperScript II RT (Thermo). Real-time qPCR was performed with the ABI PRISM suppress Th2 responses that expel helminths but also, modulate 7500 Sequence Detection System as described previously (52). Primers and inflammation to promote host tissue repair (45). Although the Roche Universal proves were purchased from Sigma and Roche, respectively. + molecular mechanisms underlying the suppression of ST2 Gene expression was normalized with the Hprt mRNA signal. For cDNA li- + + brary construction, we used SMARTer Stranded Total RNA-Seq Kit-Pico Input mTh2 cell responses by ST2 Treg cells remain unknown, ST2 Mammalian (634412; Clontech) according to the manufacturer’s protocol. Treg cells and their products are attractive targets for helminth Sequencing the library fragments was performed on the HiSeq 1500 System vaccine development. (53). For data analysis, read sequences (50 bp) were aligned to the + Comparative RNA sequence analysis of Nb-induced ST2 mm10 mouse reference genome (University of California, Santa Cruz; De- − mTh cells and ST2 mTh cells revealed that the former selec- cember 2011) using Bowtie 2 (version 2.0.0) and TopHat (version 1.3.2). tively expressed two Th2-related transcription factors: Epas1 and Fragments per kilobase of exon per million mapped reads (FPKMs) for each Pparg. GATA3 binds to the Epas1 locus (46), and Epas1 ex- gene were calculated using Cufflinks (version 2.0.2). pression by effector memory T cells leads to elevated glycolysis (47). PPARγ may contribute to the ST2 expression and functions Flow Cytometric Analyses. For the flow cytometric analyses, lung samples + in ST2 mTh cells elicited by Nb infection as well as by allergic were treated with reagents of the lung dissociation (Miltenyi Biotec). Lung

cells and splenocytes were preincubated with anti-CD16/32 on ice for 15 min INFLAMMATION

inflammation (48, 49). Although the respective roles of these Nb to prevent the nonspecific binding of irrelevant antibodies. Cells were then IMMUNOLOGY AND infection-specific transcription factors remain unknown, they + stained with the indicated combination of antibodies and analyzed with FACS may contribute to the ability of ST2 mTh cells to induce the Verse (BD). inhibition of maturation and reduction of fecundity of Nb and/or + − + − + to promote their survival in vivo. Transfer of mTh Cells. ST2 and ST2 CD4 CD44hiCD25 mTh cells, ST2 and + In summary, we have identified a helminth-induced ST2 ST2− CD4+CD44hiFoxp3-YFP− mTh cells, ST2+ and ST2− CD4+CD25+ Treg cells, + − + + + + ST2 and ST2 CD4 Foxp3-YFP Treg cells, and CD4 CD44lo naïve CD4 T cells mTh2 cell subset that is crucial for the inhibition of worm mat- + uration involving a mechanism dependent on IL-5 and IL-33 that in spleen and the lungs were purified by the CD4 T-cell isolation kit (Mil- tenyi Biotec), AutoMACS (Miltenyi Biotec), and cell sorting (BD Aria III). works via the accumulation of eosinophils expressing high level + + − Those mTh cells (1 × 105 per mouse) or naïve CD4 T cells (1 × 105 per mouse) of MBP. This response was suppressed by ST2 but not ST2 + − 5 with or without ST2 or ST2 Treg cells (1 × 10 per mouse) were transferred Treg cells. Therefore, this study reveals the immune responses + – into indicated mice according to the method described (54, 55). Whole CD4 mediated by a subpopulation of IL-5 producing mTh2 cells T cells (5 × 106 per mouse) from spleens of naïve mice were injected i.p. into during helminth infection. the NSG mice and nude mice before transfer of mTh cells.

Methods Eosinophils. Mouse bone marrow was collected, and cells were cultured to Mice. The animals used in this study were backcrossed to BALB/c mice 10 times. differentiate into eosinophils as described previously (56). In brief, bone − − − − Il33 / mice, Il1rl1 / mice, and Foxp3-YFP-IRES-Cre mice were provided by marrow cells were cultured with 20% FCS (HyClone), 100 IU/mL penicillin Susumu Nakae, The Institute of Medical Science, The University of Tokyo, (Thermo), 10 μg/mL streptomycin (Thermo), and 2 mM glutamine (Thermo) Tokyo, Japan; Andrew N. J. McKenzie, Medical Research Council, Cambridge, and supplemented with 100 ng/mL SCF (R and D Systems) and 100 ng/mL United Kingdom; and Alexander Y. Rudensky, Memorial Sloan Kettering FLT3-L (R and D Systems). On day 4, the cells were resuspended with basic Cancer Center, New York, respectively (10, 50, 51). BALB/c mice were pur- medium containing the indicated concentration of IL-5 (Biolegend) only. + chased from CLEA Japan. ΔdblGATA mice and Il4ra-deficient mice were Over 90% of cultured cells were identified as Siglec-F eosinophils by flow purchased from Jakson Laboratory. NSG mice were purchased from Charles cytometric analysis from day 13 after cultivation. Eosinophils were electro- River Japan. Animal care was conducted in accordance with the guidelines of porated using a Neon Transfection System (Thermo) with silencer siRNA for a Chiba University (#30-63, #30-64) and The Jikei University School of Medicine control or Prg2 (Thermo) and then intranasally transferred (1 × 106 per (2015-102C2). mouse) into naïve BALB/c mice intranasally.

Obata-Ninomiya et al. PNAS | vol. 115 | no. 42 | E9857 Downloaded by guest on September 29, 2021 Statistical Analyses. Data were analyzed with the Excel software program. for valuable discussion. This work was supported by Ministry of Education, Comparisons of two groups were calculated with Student’s t tests. Differ- Culture, Sports, Science and Technology Grants-in-Aid for Scientific Research ences with P values <0.05 were considered significant. (S) 26221305, Young Scientists (B) 2687072 and 17K15675, and grants from the Practical Research Project for Allergic Diseases and Immunology (Re- ACKNOWLEDGMENTS. We thank Dr. Mark Bix for his helpful discussion and search on Allergic Diseases and Immunology JP18ek0410030) from Japan suggestions during the preparation of the manuscript. We also thank Kaoru Agency for Medical Research and Development, Japan Agency for Medical Sugaya, Miki Kato, Toshihiro Ito, and Masshardt Yuka for their excellent Research and Development (AMED), Takeda Science Foundation, and the technical assistance; Yoko Ozawa for animal care; and Naohiro Watanabe Naito Foundation.

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