A type 1 immunity-restricted promoter of the IL−33 receptor gene directs antiviral T-cell responses

The pleiotropic alarmin interleukin-33 (IL-33) drives type 1, type 2 and regulatory T-cell responses via its receptor ST2. Subset-specific differences in ST2 expression intensity and dynamics suggest that transcriptional regulation is key in orchestrating the context-dependent activity of IL-33–ST2 signaling in T-cell immunity. Here, we identify a previously unrecognized alternative promoter in mice and humans that is located far upstream of the curated ST2-coding gene and drives ST2 expression in type 1 immunity. Mice lacking this promoter exhibit a selective loss of ST2 expression in type 1- but not type 2-biased T cells, resulting in impaired expansion of cytotoxic T cells (CTLs) and T-helper 1 cells upon viral infection. T-cell-intrinsic IL-33 signaling via type 1 promoter-driven ST2 is critical to generate a clonally diverse population of antiviral short-lived effector CTLs. Thus, lineage-specific alternative promoter usage directs alarmin responsiveness in T-cell subsets and offers opportunities for immune cell-specific targeting of the IL-33–ST2 axis in infections and inflammatory diseases.


Article
https://doi.org/10.1038/s41590-023-01697-6ST2 expression by CTLs and Th1 cells, but not by Th2 cells or T reg cells, relies on IL-12 and the transcription factors T-bet and STAT4 (Extended Data Fig. 1f-j) 24,27 .Hence, we analyzed T-bet-and STAT4 binding as well as activation-induced changes in chromatin accessibility at the Il1rl1 locus in type 1-polarized T cells using publicly available chromatin immunoprecipitation sequencing (ChIP-seq) [36][37][38] and assay for transposase-accessible chromatin sequencing (ATAC-seq) 39,40 data.Although GATA-3 binds predominantly upstream of the type 2 promoter, T-bet and STAT4 binding was detected in the vicinity of exons A and B, at sites that were inaccessible in naive T cells but accessible in LCMV-primed Th1 cells and CTLs (Fig. 1g).
Alternative promoters are abundant 41 .We were unaware, however, of other genes with comparably selective alternative promoter usage in type 1 and type 2 immune cells.We thus used RNA-seq data to conduct a genome-wide search to identify additional genes with highly type 1 or type 2 immunity-specific promoters 42 .Il1rl1 was reliably detected when comparing Th2 cells to CTLs and Th1 cells but no other gene with lineage-specific TSS usage was found (Fig. 1h-j).This suggested that such highly restrictive type 1 and type 2 T-cell lineage-specific utilization of alternative first exons is rare, with the Il1rl1 gene potentially representing a unique case.

Il1rl1 promoter usage is conserved between mice and humans
Next, we assessed DNA conservation at the type 1 promoter [43][44][45] and compared it to T-bet-and STAT4-ChIP-seq as well as ATAC-seq data (Fig. 2a).Thereby, we identified a 275-nt-spanning, well-conserved sequence located ~5 kb upstream of exon A (CNS-5), which is bound by T-bet and STAT4 in Th1 cells and is marked by a sharp ATAC-seq peak in CTLs (Fig. 2a).In contrast, the sequence surrounding prominent peaks ~1.5 kb downstream of exon A appears less conserved.Mapping of T-bet and STAT4 binding motifs within CNS-5 indicated that both transcription factors putatively bind in close proximity at sequences almost identical between mice and humans 46 (Fig. 2b).
To assess whether T-cell lineage-specific promoter usage at the Il1rl1 locus is conserved between mice and humans, we first examined cap analysis of gene expression (CAGE) and transcriptomic data from the FANTOM5 resource 41,47 , which provided evidence for a putative TSS upstream of the IL1RL1 gene.In human Th1 cells this site is preceded by T-bet binding sites (Fig. 2c) 48 .Of note, the exon structure closely resembles the exons A and B identified in mice (cf.Fig. 1d).To determine whether this TSS is utilized in primary human CTLs and Th1 cells, we isolated in vivo-differentiated T cells from peripheral blood to quantify IL1RL1 promoter usage (Extended Data Fig. 2a-f).Congruently with mouse T cells, IL1RL1 transcripts of human CTLs and Th1 cells contained exons A and B within their 5′ UTRs, whereas Th2 cells had incorporated exon 1a (Fig. 2d).In summary, we have identified a previously unrecognized type 1 immunity-restricted Il1rl1 promoter, which is instructed by the lineage-associated transcription factors T-bet and STAT4 and orchestrates ST2 expression in CTLs and Th1 cells of humans and mice.

Il1rl1 promoters allow lineage-specific targeting of ST2
Modulation of the IL-33-ST2 axis could represent a promising approach in treating inflammatory diseases 49 .For instance, blockade of IL-33 using therapeutic antibodies has shown encouraging efficacy in clinical trials of asthma and chronic obstructive pulmonary disease 50 .However, due to hard-to-predict effects on the balance between IL-33-mediated inflammation and tissue repair, fine-tuned targeting approaches may offer critical advantages.We thus asked whether lineage-specific promoters can be leveraged to target ST2 expression in a T-cell subset-specific manner.Hence, we retrovirally transduced T cells to express small-hairpin RNAs (shRNAs) targeting distinct Il1rl1 5′ UTRs (Fig. 3a).Selective downregulation of ST2 was achieved in either CTLs and Th1 cells or Th2 cells using shRNAs binding exons A and B or exon 1a, respectively (Fig. 3b,c).
IL-33 has also emerged as a key driver of type 1 immune responses.It is released by fibroblastic reticular cells in lymphoid organs and promotes clonal expansion and activation of antiviral CTLs and T-helper 1 (Th1) cells to confer protection against replicating viruses [23][24][25][26][27] .Moreover, IL-33-mediated amplification of type 1 immune cells was shown to exacerbate tissue damage during graft-versus-host disease (GVHD) 28,29 and contribute to immune dysregulation in systemic inflammatory diseases 30 .
Considering its multifaceted mode of action, IL-33 is now recognized to amplify pro-or anti-inflammatory T-cell subsets in a context-specific manner 31,32 .To accomplish this versatility, it was suggested that transcription of the ST2-coding gene interleukin-1 receptor-like 1 (Il1rl1) requires cell-type specific regulation, such that certain T-cell subsets become sensitive to IL-33 signals dependent on the inflammatory environment 31,32 .ST2 is absent from naive T cells but expressed constitutively at high levels by type 2-biased immune cells in which its transcription is controlled by the master-regulator transcription factor of type 2 immunity GATA- 3 (refs.6,21,33).In contrast, antiviral CTLs and Th1 cells express low levels of ST2 transiently upon infection, and this expression depends on STAT4 and T-bet, key transcription factors of type 1 immunity 24,27 .This dynamic expression pattern renders it difficult to study ST2 on type 1 immune cells.Consequently, the molecular mechanism allowing for T-cell lineage-specific ST2 expression patterns has remained enigmatic.

Identification of a type 1 immunity-restricted Il1rl1 promoter
Previous studies have shown that the protein-coding exons of the Il1rl1 gene are preceded by two non-coding exons (exon 1a and exon 1b) located in a distal and proximal promoter region, respectively 4,34 (Fig. 1a).The proximal promoter drives ST2 expression in fibroblasts, whereas the distal promoter mediates ST2 expression in Th2 cells and mast cells 33,35 .To assess which promoter is used by type 1-polarized T cells, we generated CTLs, Th1 or Th2 cells in vitro, which all express substantial levels of ST2 (Fig. 1b and Extended Data Fig. 1a-c).Of note, at the per-cell level, type 1 T cells express less ST2 than Th2 cells (Fig. 1b).Thus, to stain ST2, we utilized a multi-step amplification protocol, yielding a more sensitive detection compared to stainings with frequently used ST2 antibodies (Extended Data Fig. 1d,e).By analyzing leader exons of 5' untranslated regions (UTRs) of ST2-coding transcripts, we found that none of the described promoters could possibly account for the expression of Il1rl1 by type 1-polarized T cells (Fig. 1c).Thus, to map the origin of Il1rl1 transcripts in these cells, we next subjected ST2 + CTLs, Th1 and Th2 cells to RNA-sequencing (RNA-seq) analysis.Thereby, we discovered a transcriptional start site (TSS) located ~40 kb upstream of the annotated Il1rl1 gene, which was selectively used in CTLs and Th1 cells (type 1 promoter) (Fig. 1d).This TSS gave rise to two Il1rl1 transcript isoforms with distinct leader sequences but unaltered protein-coding sequences.We refer to the leader exons in these transcripts as exons A, B and D. Further, alternative splicing of exon B to exon C resulted in a transcript that did not contain ST2-coding exons.As expected, the 'distal' promoter (exon 1a) presented the primary origin of Il1rl1 transcripts in Th2 cells (type 2 promoter).To assess the usage of the type 1 promoter in vivo, we next transferred naive lymphocytic choriomeningitis virus (LCMV)-specific T-cell receptor (TCR)-transgenic CD4 + T cells (Smarta) or LCMV-specific TCR-transgenic CD8 + T cells (P14) into wild-type (WT) mice and infected the recipients with LCMV.At day 7 postinfection (d7 p.i.), we reisolated transferred cells and quantified Il1rl1 promoter usage.In contrast to Th2 cells, CTLs and Th1 cells had largely incorporated exons A and B but not exon 1a into 5' UTRs of ST2-coding transcripts (Fig. 1e,f).

Article
https://doi.org/10.1038/s41590-023-01697-6 Furthermore, to study the type 1 immunity-restricted promoter in vivo, we generated knockout mice with a deletion of exons A and B (Il1rl1-ExAB −/− ).A second mouse strain lacking exon C (Il1rl1-ExC −/− ) was generated to control for potential effects of exon C-containing transcripts (Fig. 3d and Extended Data Fig. 3a-c).In steady state, both strains harbored normal numbers of T cells at an activation state comparable to WT mice (Extended Data Fig. 3d-i).Likewise, introduced deletions did not affect the composition of splenic innate immune cells (Extended Data Fig. 3j-l).Importantly, deletion of the type 1 promoter severely impaired ST2 expression by in vitro activated CTLs and Th1 cells, whereas Il1rl1-ExAB −/− Th2 cells differentiated from the same pool of naive CD4 + T cells exhibited normal ST2 expression (Fig. 3e,f).

Exon 1a
Exon 1b Expression relative to Hprt
To investigate the requirement of the type 1 Il1rl1 promoter for ST2 expression by T cells responding to viral challenge, we infected WT, Il1rl1 −/− , Il1rl1-ExAB −/− and Il1rl1-ExC −/− mice with LCMV (Fig. 3h).ST2 surface expression was almost absent from CTLs in Il1rl1-ExAB −/− mice and was significantly reduced in Th1 cells at day 7 p.i., whereas T reg cells displayed unaltered ST2 surface levels (Fig. 3i-n).Conversely, Il1rl1-ExC −/− CTLs and Th1 cells exhibited slightly enhanced ST2 expression (Fig. 3i-l), suggesting that exon C may act as a transcriptional decoy (cf.Fig. 1d).Altogether, we found that targeting of individual Il1rl1 promoters allowed for a selective T-cell lineage-specific manipulation of ST2 expression.

The type 1 Il1rl1 promoter drives antiviral T-cell responses
Next, we studied whether CD8 + T-cell responses to LCMV required the type 1 Il1rl1 promoter.At the peak of the response (d7 p.i.), Il1rl1-ExAB −/− mice harbored substantially reduced numbers of CTLs in spleens and livers, resembling in its extent the impairment observed in Il1rl1 −/− mice (Fig. 4a-c and Extended Data Fig. 6a-c).Diminished CTL counts were largely accounted for by a reduction in CD44 + CD62L − effector T cells expressing the proliferation marker Ki67 (Fig. 4d-f).Accordingly, CTLs specific for the immunodominant GP 33-41 and NP 396-404 epitopes of LCMV were substantially reduced (Fig. 4g and Extended Data Fig. 6d-f).Ultimately, Il1rl1-ExAB −/− mice displayed significantly lower numbers of CTLs expressing effector cytokines or cytolytic molecules, and systemic IFN-γ levels were reduced by >70% (Fig. 4h,i and Extended Data Fig. 6g-i).In contrast and as expected, CTL responses of Il1rl1-ExC −/− mice were comparable to those of WT mice (Fig. 4b,c,e-i and Extended Data Fig. 6b-i).
To determine whether observed effects were due to a T-cellintrinsic impairment in ST2 expression, mixed bone marrow chimeras were generated by reconstituting irradiated WT mice with bone marrow from Il1rl1 −/− , Il1rl1-ExAB −/− or Il1rl1-ExC −/− mice, each of them mixed 1:1 with WT bone marrow.Following LCMV infection, WT:Il1rl1-ExC −/− chimeras mounted CTL responses that derived at approximately equal parts from both bone marrow compartments.In contrast, WT bone marrow-derived CTLs outnumbered the CTLs derived from Il1rl1 −/− or Il1rl1-ExAB −/− bone marrow in the respective chimeras (Fig. 4j-m and Extended Data Fig. 6j,k).Lastly, to study the impact of type 1 promoter-driven ST2 expression on T-cell responses in the absence of any potentially confounding irradiation effects, congenically marked naive Il1rl1-ExAB −/− P14 CTLs and WT P14 cells were cotransferred into recipients, which were infected with LCMV and analyzed at d10 p.i. Analogously to the data from mixed bone marrow chimeras, Il1rl1-ExAB −/− and Il1rl1 −/− P14 T cells expanded much less than their respective cotransferred WT P14 T-cell populations (Fig. 4n-r).Similarly, albeit less pronounced, Il1rl1-ExAB −/− Smarta T cells expanded less than cotransferred WT Smarta cells (Fig. 4s-w and Extended Data Fig. 6n-r).Interestingly, analysis of Smarta cells revealed that ExonAB-deficient, but not WT, Smarta cells used the proximal promoter Proximal promoter Exon 1b (exon 1b) (Extended Data Fig. 6s-v).In summary, optimal expansion of antiviral T cells critically depends on T-cell-intrinsic activity of the type 1 Il1rl1 promoter.

The type 1 Il1rl1 promoter drives short-lived effector formation
At the peak of the acute response, the antiviral CTL population is heterogeneous and comprises functionally distinct subsets 51 .To delineate the impact of type 1 promoter-driven ST2 expression on CTL differentiation, we sorted activated CD44 + CTLs from LCMV-infected WT or Il1rl1-ExAB −/− mice for combined single-cell gene expression and TCR repertoire analysis.T cells were clustered into six separate populations using nearest neighbor modularity optimization and annotated based on signature gene expression (Fig. 5a,b, Extended Data Fig. 7a,b and Supplementary Table 1).The two dominant clusters showed expression of genes associated with short-lived effector cells (SLECs; Klrg1, Gzma and Id2) or memory precursor effector cells (MPECs; Il7r, Sell and Ccr7) (Fig. 5c).The third cluster was enriched in CTLs expressing Pdcd1 (encoding PD-1) and Lag3, markers of exhausted CTLs 52 , whereas cells of the fourth cluster exhibited higher expression of Tcf7 (encoding TCF-1) and Id3, thus likely presenting stem-like precursors of effector CTLs 53,54 .Lastly, the two remaining clusters were enriched in CTLs expressing higher levels of mitochondrial genes or cell cycle-related markers (Mki67, Top2a).A cluster-wise comparison between genotypes revealed that type 1 Il1rl1 promoter disruption affected gene expression in all CTL subsets (Extended Data Fig. 8) but resulted in a particularly pronounced curtailment of SLECs (Fig. 5d).This translated into a 90-95% reduction of SLEC numbers in Il1rl1-ExAB −/− mice, mirroring the phenotype of Il1rl1 −/− mice (Fig. 5e,f).Further, fewer Il1rl1-ExAB −/− CTLs exhibited surface expression of the SLEC-associated molecule CXCR3 (Fig. 5g) 55 .Importantly, despite a relative increase in the frequency of MPECs, MPEC counts were slightly decreased (Fig. 5h).WT  Correspondingly, Il1rl1-ExAB −/− CTLs in mixed bone marrow chimeras featured a pronounced defect in SLEC generation and moderately lower MPEC numbers, similar to Il1rl1 −/− CTLs (Extended Data Fig. 6l,m).In contrast, Il1rl1-ExC −/− bone marrow-derived CTLs were as proficient as WT cells in populating the SLEC and MPEC compartments.Lastly, the proportion of Il1rl1-ExAB −/− P14 T cells differentiating into SLECs and/ or CXCR3 + cells was reduced as compared to adoptively cotransferred WT P14 cells, whereas MPEC counts were largely unaffected (Fig. 5i-l).
In line with these results, analysis of Il1rl1-ExAB −/− and Il1rl1 −/− P14 cells at d30 p.i. revealed a modest decrease in numbers of memory CTLs compared to WT P14 cells (Extended Data Fig. 9a-d).However, both Il1rl1-ExAB −/− and Il1rl1 −/− P14 cells were able to give rise to both effector memory (T em ) as well as central memory cells (T cm ) (Extended Data Fig. 9e-g) and formed tissue-resident memory cells (Extended Data Fig. 9h-k).Thus, a lack of ST2 signaling leads to a generalized impairment in CTL expansion.This was accentuated in the SLEC compartment during the acute antiviral response and extended in part to the population of circulating memory CTLs, whereas formation of tissue-resident memory cells appeared less ST2 dependent.
Next, we asked whether ST2 expression by the type 1 promoter drives the selective proliferation of SLEC-differentiated T-cell clones or whether it enforces the differentiation of precursors into SLECs.ST2 is found on both KLRG1 + and KLRG1 − CTLs of WT mice (Fig. 5m), suggesting IL-33 signaling can occur prior to SLEC differentiation.Further, we integrated single-cell gene expression data with a TCR repertoire analysis.Despite an eight-fold difference in CD44 + CTL counts per spleen (Fig. 5n), equivalent numbers of clonotypes were identified in Il1rl1-ExAB −/− and WT mice when equal numbers of CD44 + CTLs were compared (Fig. 5o).This finding suggested that during the acute phase of infection, IL-33 expands activated T cells in a clonotype-unselective manner, which does not substantially alter the TCR diversity amongst the most abundant clonotypes.Of note, the majority of clonotypes identified were represented less than three times per mouse and no clonotype was found more often than seven times (Fig. 5p).In line with previous reports, this indicated that TCR diversity within the CTL population was high during the acute phase of infection 56 .By consequence, the severe reduction in SLEC numbers in Il1rl1-ExAB −/− mice resulted in reduced SLEC clonotype numbers (Fig. 5q,r).Taken together, without type 1 promoter-driven ST2 expression, most CTL clones achieve basal activation, but fail to develop into fully differentiated SLECs.Thus, type 1 promoter-driven ST2 expression is vital to establish a numerically relevant and clonally diverse population of short-lived antiviral effector CTLs.

RNA profiling indicates a TCR-cooperative role of IL-33
To gain mechanistic insight on how IL-33 signaling modulates T-cell activation and differentiation, we performed a comprehensive analysis of early IL-33 target genes.To this end, naive T cells were differentiated into CTLs, Th1 or Th2 cells, followed by a resting period without antigenic stimulation.Because ST2 signaling is subject to negative feedback mechanisms and oxidation of IL-33 rapidly reduces its activity 57,58 , gene expression was analyzed before (0 h) and 2 h after treatment with or without IL-33 (Fig. 6a).Short-term stimulation with IL-33 had a profound effect on the transcriptome of all subsets, and it strongly induced or, in fewer cases, reduced expression of target genes rather than preventing a loss or gain of transcription (Fig. 6b).Whereas many differentially regulated genes were shared between subsets, others were regulated in a lineage-specific manner (Fig. 6c).Gene Ontology-enrichment analysis revealed a broad role of IL-33-responsive genes in T-cell activation, proliferation and differentiation (Fig. 6d).Importantly, IL-33 stimulation of CTLs amplified expression of Tbx21, Zeb2 and Prdm1, encoding transcription factors critical for SLEC differentiation [59][60][61] (Fig. 6b,e).Across the three T-cell subsets we observed a prominent upregulation of genes frequently used as indicators of recent TCR activation (Nr4a1, Cd69 and Batf) (Fig. 6b,e) [62][63][64] .Coherently, gene set enrichment analysis showed a significant overlap between IL-33-and TCR-downstream signaling in CTLs (Fig. 6f and Supplementary Table 2).This finding suggested that IL-33 may support TCR stimulation to promote potent antiviral T-cell responses.
To further investigate the interplay between ST2 and TCR signaling strength, we made use of a genetically engineered LCM virus that differs from the WT counterpart only by a GP-A39C mutation, rendering its GP33 epitope a weak P14 TCR agonist 65 .P14 and Il1rl1 −/− P14 cells were cotransferred into WT recipients, which were subsequently infected with LCMV expressing either the high-or the low-affinity GP33 variant (Fig. 6g).We found that ST2-sufficient and ST2-deficient P14 cells expanded less when primed with the low-affinity ligand (Fig. 6h).Further, P14 cells depended on ST2 for optimal expansion and effector differentiation, irrespective of the TCR stimulation strength (Fig. 6i).Interestingly, the response of WT P14 cells responding to low-affinity virus was comparable to or exceeded the one of high-affinity ligand-primed Il1rl1 −/− P14 cells in terms of total and effector cell progeny, respectively (Fig. 6h,i).This finding suggested that ST2 signals can help reaching effector T-cell responses of critical size even when confronted with low-affinity ligands.Lastly, in comparison to WT P14 cells, Il1rl1 −/− P14 cells yielded slightly fewer MPECs, irrespective of the TCR stimulation strength (Fig. 6j).Of note, the impairment in T-cell expansion and effector differentiation between mice infected with high-or low-affinity GP33-expressing LCMV were unlikely due to any potential differences in inflammation or IL-33 release, as the endogenous NP396-specific T-cell responses to the two LCMV variants were indistinguishable (Fig. 6k).
In summary, these data demonstrate that IL-33-ST2 signaling provides a strong costimulatory signal that can act cooperatively with TCR signaling to promote the expansion and effector cell differentiation of antiviral CTLs.

Discussion
IL-33 has long been recognized as a type 2 immunity-related cytok ine 2,3,6,7,12,31,32 .Over the past decade its important role in promoting type 1 immunity has become widely accepted yet remains mechanistically less well understood, particularly due to a lack of understanding how ST2 expression is regulated in these cells 31,32 .Here, we studied the transcriptional regulation of ST2 expression in antiviral T cells and discovered a dedicated type 1 immunity-restricted promoter located ~40 kb upstream of the curated Il1rl1 gene in mice and humans.This type 1 promoter drives ST2 expression by CTLs and Th1 cells in vitro and in viral infections in vivo.As opposed to the previously described type 2 promoter, which is regulated by GATA-3 (ref.33) and is utilized by type 2 immune cells and T reg cells 21 , the identified promoter is controlled by the type 1 immunity-associated transcription factors T-bet and STAT4 and is subject to epigenetic remodeling during type 1 T-cell differentiation.Thus, we provide evidence for a dedicated regulatory genetic element to control ST2 expression selectively in type 1-polarized T cells, as well as NKT and NK cells.Il1rl1-ExC -/-: n = 7).i-l, Frequencies and counts of KLRG1 + CD127 − SLECs (i), CXCR3 + P14 cells (j and k) and KLRG1 − CD127 + MPECs (l) in adoptive cotransfer experiments on d10 p.i. (n = 7).m, ST2 expression by GP 33-41 -specific SLECs and MPECs in WT mice at d7 p.i. (n = 9).n, counts of CD44 + CTLs per spleen.o, TCR clonotypes in sequenced CD44 + CTLs.p, graph displaying number of TCR clonotypes and their abundance among all analyzed CD44 + CTLs.q, TCR clonotypes in SLEC and MPEC clusters.r, TCR repertoire occupation in individual WT and Il1rl1-ExAB −/− mice.Data represent one (b-d and m-r) or two (e-l) independent experiments and are presented as mean ± standard deviation, with each dot or line representing one mouse (f-i, k, l and n-q).P was determined using two-tailed t-tests (n and q), one-way ANOVA with Tukey's post hoc test (f-h) or two-way repeated measures ANOVA with Šidák's post hoc test (i, k and l).were adoptively cotransferred with Il1rl1 −/− P14 T cells (CD45.1 + ) into WT mice (CD45.2+ ).Recipients were infected with high-or low-affinity GP33-expressing LCMV-Cl13 and analyzed at d10 p.i. (high-affinity group: n = 7, low-affinity group: n = 6).g, experimental outline.h, counts of recovered P14 and Il1rl1 −/− P14 T cells.i, frequencies and counts of KLRG1 + CD127 − P14 cells.j, frequencies and counts of KLRG1 − CD127 + P14 cells.k, counts of endogenous NP 396-404 -specific CTLs and KLRG1 + CD127 − or KLRG1 − CD127 + NP 396-404 -specific CTLs.Data represent one (a-f) or two (g-k) independent experiments.Data are presented as mean ± standard deviation, with each dot representing one mouse (h-k).P value was determined using one-way ANOVA with Tukey's post hoc test (h-j), two-tailed t-tests (k), two-sided Wald test with BH correction (b and e), one-sided hypergeometric test with BH correction (d) and two-sided permutation test with BH correction (f).

Article
https://doi.org/10.1038/s41590-023-01697-6 Although the Il1rl1 gene has been studied intensively 8,35,66 , the type 1 promoter has remained unrecognized, likely because it is only transiently active, often resulting in a low abundance of ST2-coding transcripts 24,27 .The latter renders it difficult to obtain adequate read coverage for a clear definition of exon structures by commonly used RNA-seq techniques 67 -a challenge we approached by analyzing T cells that express a high amount of Il1rl1 transcripts.Subsequently, we have validated the crucial role of this regulatory element in vivo, and by analyzing human T cells have extended the concept to our species.Above all, our finding was surprising, as to the best of our knowledge no other gene has been identified to date, for which type 1 and type 2 immune cells exhibit a similarly distinct lineage-specific promoter usage.Consistently, our own attempts at identifying genes with an analogous promoter usage were unsuccessful.We acknowledge that technical limitations might have prevented us from identifying such genes.Still, our results suggest that this is not a common feature but represents a fairly unique mechanism to spatiotemporally orchestrate ST2 expression.
IL-33 is an exceptionally potent alarmin, which can act as a proor anti-inflammatory cytokine, depending on the local composition of immune cells and their responsiveness to IL- 33 (ref.32).Likely due to its potential to cause severe inflammation, IL-33 responsiveness requires stringent regulation.Transcription from the type 1 immunity-restricted promoter enables transient ST2 expression by CTLs and Th1 cells in response to inflammatory stimuli 23,24,27 , which may serve to prevent continuous activation of cells with a high tissue-destructive potential.In contrast, constitutive type 2 promoter-driven ST2 expression on T reg cells and ILC2s allows for rapid anti-inflammatory responses to tissue damage [19][20][21]32 .
Importantly, this dual mode of action constitutes a major hurdle for the therapeutic modulation of IL-33-ST2 signaling 32,68 .We here demonstrate that the usage of distinct promoters offers opportunities for a T-cell subset-specific targeting of ST2 expression.Il1rl1-ExAB −/− mice exhibit a type 1 immunity-restricted impairment of ST2 expression and display curtailed CTL and Th1 responses against LCMV, whereas ST2 expression by T reg cells and type 2 immune cells was fully preserved.Of note, in CTLs ST2 expression was almost exclusively dependent on the type 1 promoter, whereas some Il1rl1-ExAB −/− Th1 cells could compensate for the defect by engaging the proximal Il1rl1 promoter.Nevertheless, the type 1 promoter was critical for optimal expansion of antiviral Th1 cells.T-cell subset-specific targeting approaches could be of interest to modulate IL-33 responses in inflammatory diseases.For instance, IL-33 administration was shown to drive T reg expansion in the context of GVHD, promoting tolerance induction and disease amelioration [69][70][71] .However, IL-33 also augments type 1 alloimmunity by acting as a costimulatory molecule for donor CTLs and Th1 cells 28,29 .A targeted disruption of ST2 selectively on type 1 immune cells might minimize the pathological response during GVHD, whereas the protective effects of IL-33 should remain preserved.
Our study provides insight into the role of type 1 promoter-driven ST2 expression and IL-33 signaling in CTL differentiation.scRNA-seq analysis of antiviral CTLs revealed that Il1rl1-ExAB −/− mice display a pronounced reduction in SLECs.Moreover, clonotype diversity in the SLEC population was high in WT mice and diminished proportionally to cell counts in Il1rl1-ExAB −/− mice.This suggests that IL-33 can foster the transition of an activated CTL into a cell with potent effector functions rather than selectively expanding a pool of predifferentiated SLECs.Of note, although the effect was strongly magnified in the SLEC compartment, Il1rl1 −/− as well as Il1rl1-ExAB −/− CTLs showed a generalized reduction in expansion that in most instances also negatively affected MPECs.Consequently, this impairment in primary expansion likely accounts for the lower numbers of circulating memory CTLs 1 month after infection.The formation of tissue-resident memory cells appeared less affected.This finding might suggest a particular importance of IL-33 signals for the generation of antiviral effector CTLs but only to a lower extent for tissue-resident memory CTLs.However, further work is needed to thoroughly test this hypothesis.
Terminal differentiation has been associated with STAT4 signaling and with high levels of T-bet, Blimp-1 and Zeb2 (refs.59-61).Likewise, the activity of the type 1 Il1rl1 promoter is positively regulated by STAT4 and T-bet.Interestingly, RNA-seq of IL-33 target genes in CTLs demonstrated an induction of Tbx21 (T-bet), Prdm1 (Blimp-1) and Zeb2 expression, thus inferring a positive feedback loop that further reinforces ST2 expression and effector differentiation via T-bet.Besides these cell-intrinsic factors, TCR signaling strength is linked to acquisition of effector properties 72 .Our data show that IL-33 stimulation of CTLs strongly induces the transcription of several TCR-dependent genes.Moreover, IL-33 signals can restore the otherwise suboptimal expansion and effector differentiation of CTLs in response to a low-affinity antigenic peptide.Recent studies demonstrated a loss of IL-33 in lymphoid organs early after LCMV infection, suggesting substantial release during T-cell priming 26 .Together, this implies that ST2 signaling might act in conjunction with TCR signaling to achieve above-threshold activation required for fully functional effector differentiation.
In summary, we here uncover lineage-specific promoter usage as molecular mechanism governing disparate expression patterns of ST2 in distinct T-cell subsets.Using newly generated knockout mice, we demonstrate that the type 1 immunity-restricted Il1rl1 promoter is essential for fully functional antiviral T-cell responses and critical for the formation of a clonally diverse population of effector CTLs.These findings open new avenues for the modulation and exploitation of IL-33 signaling in type 1 immunity-mediated inflammatory diseases and T-cell-based cancer immunotherapy, respectively.https://doi.org/10.1038/s41590-023-01697-6Generation of Il1rl1-ExAB −/− and Il1rl1-ExC −/− mice Il1rl1-ExAB −/− and Il1rl1-ExC −/− mice were generated in the Transgenics Core Facility of the Max Delbrück Centrum Berlin using established protocols 78 .In brief, gRNA sequences with minimal predicted off-target effects were identified using the web-based tool CRISPOR 79 .Zygotes were collected from C57BL/6 J mice (Charles River), microinjected with synthetic gRNAs (Integrated DNA Technologies) and recombinant Cas9 protein (Integrated DNA Technologies) and subsequently transferred into pseudo-pregnant C57BL/6 J mice.Resulting F0 offspring mice were screened for successful deletion by PCR amplification of WT or knockout alleles.gRNA and PCR primer sequences are listed in Supplementary Table 4.

Flow cytometry
Surface stainings of purified lymphocytes were performed using different combinations of antibodies diluted in PBS.A list of antibodies and dilutions used in this study is provided in Supplementary Table 3.
Unspecific staining was minimized by blocking with rat immunoglobulin G ( Jackson ImmunoResearch) and anti-mouse CD16/32 (2.4G2, DRFZ inhouse production) prior to staining.Dead cells were labeled using Zombie Aqua or Zombie NIR fixable live/dead staining reagents (BioLegend) or by adding propidium iodide (PI) prior to acquisition.For detection of ST2 on murine T cells, lymphocytes were first stained with digoxigenin-conjugated antibody against ST2 (DJ8), followed by a secondary staining with PE-or APC-conjugated anti-digoxigenin Fab fragments (Roche).Further, stainings were enhanced by two rounds of PE-or APC-FASER amplification (Miltenyi Biotec).To identify LCMV-specific T cells, lymphocytes were stained with LCMV GP 33-41  or NP 396-404 peptide-loaded MHC class I (H2-Db) tetramers (PE or APC conjugated, respectively) for 30 min at 37 °C.For detection of transcription factors or Ki67 expression, surface-stained cells were fixed and stained using the FoxP3 staining buffer set (Thermo Scientific).Briefly, cells were fixed with 1x fixation/permeabilization reagent for 30 min at 4 °C and washed with permeabilization buffer.Subsequently, cells were stained with antibodies diluted in permeabilization buffer for 30 min at 4 °C.

Viruses and LCMV infection
LCMV-WE and LCMV-Cl13 strains were propagated on L929 or BHK-21 cells, respectively.Viral titers in stock solutions were determined by immunofocus assay on MC57G cells as described before 80 .In brief, MC57G cells were plated with virus stock dilutions and overlaid with 2% methylcellulose.After 48 h at 37 °C, the confluent monolayer of cells was fixed with 4% formaldehyde, permeabilized with Triton X-100 (1%, v/v) and stained with antibodies against LCMV nucleoprotein.After a secondary staining step with peroxidase-conjugated anti-rat immunoglobulin G antibody, foci were developed by 20-min incubation with OPD substrate (Sigma-Aldrich).Mice were infected intravenously (i.v.) with either 200 plaque-forming units (PFU) of LCMV-WE (mixed bone marrow chimera experiments), 200 PFU LCMV-Cl13 (adoptive transfer experiments, LCMV-Cl13 WT or C6 variant where indicated) or 2 × 10 6 PFU of LCMV-WE in minimal essential medium (Thermo Scientific).

In vivo labeling of T cells
To distinguish between tissue-resident and intravascular T cells, mice were injected i.v. with 3 µg PE-conjugated CD90.2 antibody (30-H12, BioLegend) and sacrificed 3 min after injection.

Mixed bone marrow chimeras
To generate mixed bone marrow chimeras, CD45.1 +/+ WT recipients were lethally irradiated (two doses of 5.5 Gy given in a 6-h interval).One day later, recipients were reconstituted with a 1:1 mixture of CD45.1 +/-WT and CD45.2 +/+ knockout bone marrow cells and splenocytes.After 8 weeks of hematopoietic reconstitution, CTL frequencies of respective donor populations were determined in blood, and mice were infected with LCMV-WE (200 PFU i.v.).Data were analyzed on d10 p.i. and normalized to CTL frequencies before infection.

Legendplex cytometric bead assay
To assess cytokine production by T cells in response to IL-33, 5 × 10 5 T cells were stimulated in 48-well plates with IL-33 (R&D, 10 ng ml −1 ) for 24 h at 37 °C.Afterwards, individual wells were harvested and centrifuged for 5 min at 350 g.To obtain serum, blood of individual mice was collected using yellow microtainers (BD).Serum and cell-free supernatant were frozen at −80 °C until analysis.Cytokine content was measured using LEGENDplex bead-based immunoassays (BioLegend) according to manufacturer's instructions and acquired at a Canto II flow-cytometer (BD).Cytokine concentration was extrapolated from standard titrations.

Retroviral transduction of T cells
For cloning of shRNA expression vectors, sense-and antisense-shRNA sequences were ordered as phosphorylated oligos with a 5' SalI restriction overhang (Eurofins Genomics) and annealed by subjecting equimolar amounts of oligos diluted in oligo annealing buffer (100 mM Tris-HCl, 1 M NaCl and 10 mM EDTA, pH 7.5) to a decreasing temperature gradient (95 °C to 25 °C with 1 °C min −1 ).Oligo sequences are provided in Supplementary Table 4. PQCXIX-GFP target vector 81 was digested by SalI and HpaI restriction enzymes (Thermo Scientific) and dephosphorylated with FastAP alkaline phosphatase (Thermo Scientific).Annealed oligos were ligated using T4 Ligase according to standard protocols (NEB).Heat-inactivated ligation reactions were directly used for heat-shock transformation into Oneshot TOP10 chemically competent Escherichia coli (Thermo Scientific).Single transformed bacterial clones were selected on LB-agar plates (MP Biomedicals) containing ampicillin (100 µg ml −1 , Sigma-Aldrich), and plasmid DNA was prepared using QIAprep Spin Plasmid Maxi or Midi kits (Qiagen).Correct plasmid sequences were verified by Sanger-sequencing (Eurofins Genomics).Virus particles were generated by co-transfection of HEK293T cells with shRNA-containing vectors and packaging plasmids pCGP and pECO 82 using Transporter 5 transfection reagent (Polysciences).For retroviral transduction, mouse T cells were activated in the presence of irradiated APCs and antibodies against CD3ε and CD28 described above.36-48 h after plating, culture medium was temporarily replaced with virus-containing supernatant, polybrene (8 µg ml −1 , Sigma-Aldrich) was added and plates were centrifuged for 90 min at 450 g at room temperature.T cells were incubated at 37 °C for 6-8 h.Afterwards, viral supernatant was replaced with conditioned cell culture medium and cells were split in a 1:3 ratio with fresh IL-2-containing medium.Transduced T cells were analyzed between day 5 and day 7 of culture.

RNA isolation and qRT-PCR
To isolate RNA for qRT-PCR analysis or bulk RNA-seq, 10 5 -10 6 T cells were harvested and lysed in RA-1 buffer (Macherey & Nagel).Total RNA was purified using the Nucleospin RNA XS Micro kit (Macherey & Nagel) according to manufacturer's instructions, without addition of carrier RNA.For qRT-PCR analysis, RNA was transcribed into cDNA utilizing Taqman Reverse Transcription Reagents (Applied Biosystems).cDNA was then subjected to qRT-PCR analysis using PowerUp SYBR Green or Taqman Fast Advanced Mastermix reagents (Applied Biosystems).Primer sequences and Taqman probes are listed in Supplementary Table 4. Amplifications were performed in triplicates by using a QuantStudio 7 device (Applied Biosystems) and expression levels were quantified with the ΔΔCt-method by normalizing target gene expression to levels of Hprt (mouse) or GAPDH (human).

Single-cell RNA library preparation, sequencing and analysis
Single-cell suspensions of CD90 + CD8 + CD44 + T cells were obtained by flow-cytometrical sorting of CD19-depleted splenocytes from LCMV-infected WT and Il1rl1-ExAB −/− mice at day 7 p.i. with LCMV-WE (2 × 10 6 PFU).Sorted T cells of individual mice were barcoded using TotalSeq-C anti-mouse Hashtags (anti-mouse Hashtag 1, 2 and 3, all BioLegend).T cells of each genotype were then pooled and applied to the 10x Genomics workflow for cell capturing.For the preparation of scRNA gene expression (GEX), TCR and CiteSeq libraries, the Chromium Next GEM Single Cell 5' Library & Gel Bead Kit v1.1 as well as the Chromium Single Cell 5' Feature Barcode Library Kit were used in conjunction with Chromium Controller (10x Genomics).After cDNA amplification the CiteSeq libraries were prepared separately using the Single Index Kit N Set A (10x Genomics).TCR target enrichment was performed using the Chromium Single Cell V(D)J Enrichment Kit for mouse T cells (10x Genomics).Final GEX and TCR libraries were obtained after fragmentation, adapter ligation and final Index PCR using the Single Index Kit T Set A. The Qubit dsDNA HS assay kit (Life Technologies) and a Qubit 2.0 Fluorometer were used for library quantification.Fragment sizes were determined using a Fragment Analyzer device with the NGS Fragment Kit (1-6,000 bp) (Agilent).Sequencing was performed on a NextSeq2000 device (Illumina) using P2 Reagents v3 (200 cycles) with the recommended sequencing conditions for 5' GEX and barcode libraries (read 1: 26 nt, read 2: 98 nt, index1: 8 nt, index 2: n.a.) and on a NextSeq500 device (Illumina) using a Mid Output v2 Kit (300 cycles) for TCR libraries (read 1: 150 nt, read 2: 150 nt, index 1: 8 nt, index 2: n.a., 20% PhiX spike-in).Raw data were processed using cellranger-3.1.0with refdata-gex-mm10-2020-A and refdata-cellranger-vdj_GRCm38_alts_ensembl-mouse-2.2.0 as reference.Mkfastq, count and vdj were used in default parameter settings with 3,000 expected cells for demultiplexing, detection of intact cells, quantification of gene expression, antibody capture as well as assembly and quantification of T-cell receptor sequences.
The cellranger output was further analyzed in R using the Seurat package (version 4.0.0) 83.Hashtag sequences of three individual Il1rl1-ExAB −/− and WT mice were imported and combined.Centered log ratio transformation was used for normalization.Seurat's default method was used for scaling.Features with correlation coefficients >0.85 to Gm42418, Malat1, AY036118 and Lars2 were removed from the count matrix.Hashtag demultiplexing (representing the three biological replicates per genotype) was performed based on Seurat's HTODemux with the parameter 'positive-quantile' at 0.99.Doublets and untagged cells were filtered out.Cells with expression values for Cd8a or Cd8b1 and Cd3g, Cd3d or Cd3e, with >200 and <4,500 features, and <10% UMI for mitochondrial genes were kept for further analysis.
After ranking by residual variance, 3000 variable genes were determined.The genes encoding TCR variable regions (Trav, Trbv, Trdv and Trgv) were removed.30 principal components were computed and stored.UMAP and t-distributed stochastic neighbor embedding were run using the first 15 principal components.Transcriptionally similar clusters were identified using shared nearest neighbor modularity optimization, with a resolution of 0.35.For visualization, cells of the Il1rl1-ExAB −/− condition were down-sampled to match the number of cells in the WT condition.Signature genes were identified using the FindAllMarkers function in default parameter settings (only.pos= TRUE, min.pct= 0.25, logfc.threshold= 0.25).Heatmaps and dotplots for the single-cell data were plotted with Seurat's DoHeatmap and Dot-Plot function, respectively, using default settings.Identified clusters were annotated based on the expression of key markers for CD8 + T-cell subsets and cell functions.Two Klrg1 expressing clusters were merged to the SLEC cluster.Further, two clusters were merged to form the Mito hi cluster based on their expression of mitochondrial genes.After combining the stated clusters, signature genes were identified again using the same method as described above.Cluster size was defined as the number of cells in one cluster.Relative cluster sizes were calculated by analyzing the number of cells in one cluster per genotype divided by the total number of cells.For feature plots, the expression of single features was plotted on the UMAP by using Seurat's default function FeaturePlot with the option "keep.scale='all'".For differential expression analysis between clusters the FindMarkers function was used.
For the analysis of TCRs, immune profiles were integrated using identical cellular barcodes.For cells with more than one contig for the heavy or light TCR chain the most abundant, productive contig was chosen.Cell numbers were equalized by subsampling the larger condition.Cells without TCR annotation were excluded from the analysis.The R package immunarch (version 0.6.6) 84was used for clonality analysis after downsampling to the WT condition.

RNA-seq
Smarta and P14 T cells used for bulk RNA-seq experiments were differentiated as described above.To enrich for ST2-expressing CTLs and Th1 and Th2 cells, ST2 + T cells were flow-cytometrically sorted at day 10 of culture.For global analysis of IL-33-responsive genes, T cells were harvested at day 10 of culture and rested for 3 days in the presence of IL-2 (5 ng ml −1 ) and IL-7 (5 ng ml −1 , both Miltenyi Biotec), without irradiated splenocytes or cognate peptide.At day 13, IL-12 (5 ng ml −1 ) was added to CTLs and Th1 cells to trigger ST2 expression.At day 14 of culture, T cells were subjected to Histopaque density centrifugation (1.083 g ml −1 , Sigma-Aldrich) and stimulated in conditioned medium with IL-33 (10 ng ml −1 , R&D Systems) for 2 h.When used for RNA-seq, quality of the isolated RNA was assessed with a Fragment Analyzer System (Agilent).All processed samples showed high RNA integrity (RQN > 8).cDNA libraries were prepared using the Smart-seq v4 mRNA Ultra Low Input RNA Kit (Clontech) with up to 10 ng RNA (IL-33-stimulated T cells) or TrueSeq stranded total RNA library kit (Illumina) with up to 1 µg of RNA (ST2-enriched T cells) according to manufacturer's instructions.Paired-end sequencing (2 × 75 bp) of cDNA libraries was performed on an Illumina NextSeq500 device using the NextSeq 500/550 High output Kit v2.Obtained reads were mapped to the mm10 genome (annotation release GRCm38.p6)using Tophat2 (ref.85) and Bowtie2 (ref.86) with very sensitive settings.Read counts were determined with featureCounts 87 .DESeq2 (ref.88) was used in RStudio for differential gene expression analysis.The DESeq2 count matrix was pre-filtered https://doi.org/10.1038/s41590-023-01697-6 for genes with ≥100 summarized read counts across the analyzed samples.A gene was considered as differentially expressed when log 2 fold change > 1.0 and P adjusted < 0.01.AnnotationDbi 89 , Enhanced-Volcano 90 , ComplexHeatmap 91 , pheatmap 92 and ggplot2 (ref.93) were used in RStudio for data visualization.
For PCA and sample distance calculation, a blind variance stabilizing transformation was performed on the unnormalized counts across all samples.Sample distance plots are based on pairwise calculation of the Pearson correlation.

Gene set enrichment and overrepresentation analysis
For gene set enrichment analysis, the R package clusterProfiler 94 was used.A gene list ranked by log 2 fold change containing all expressed genes served as input and was tested for enrichment of biological process gene sets from the gene ontology resource 95,96 .
For overrepresentation analysis, differentially expressed genes were split into up-and downregulated genes.Overrepresentation analysis was performed against biological process gene sets from the gene ontology resource using a one-sided hypergeometric test with BH correction.All expressed genes in the respective conditions were used as a background gene list.The results were simplified to reduce overlaps between ontology terms by using the clusterProfiler::simplifyGO function with a cutoff of 0.7.

Analysis of alternative transcription start sites
Raw RNA-seq reads of indicated T-cell subsets were aligned using hisat2 (version 2.2.1) 97 and assembled using Cufflinks (version 2.2.1) 98 in RABT mode with EnsEMBL annotation release 67.Assemblies were then merged into a new reference annotation with the public reference using the cuffmerge function.The resulting annotation was used for an analysis with the R package ProActiv 42 .The getAlternativePromoters function was used with standard parameters except for minAbs = 5.Only results with false discovery rate < 0.01 were considered.

Processing of published RNA-seq, ChIP-seq and ATAC-seq data
Fastq files of published RNA-seq datasets were obtained from the NCBI Sequence read archive and aligned using hisat2 with default settings 97 .ChIP-and ATAC-seq data sets were downloaded from the NCBI GEO Database and if required crossmapped to the mm10 genome using CrossMap 99 .All NGS data tracks were visualized in the IGV browser 100 .

Quantification and statistical analysis
Statistical analysis on untransformed or log 2 -transformed values was performed using GraphPad Prism (v10.0.3).Normal distribution was tested using Shapiro-Wilk and Kolmogorov-Smirnov tests.Unpaired or paired two-tailed Student's t-tests were used when two groups were compared with respect to one parameter.More than two groups were analyzed by one-way ANOVA with Tukey's post hoc test for multiple comparison.For comparisons of more than one parameter between two or more groups, two-way ANOVA with Tukey's post hoc tests (unpaired samples) or two-way repeated measures ANOVA with Šidák's post hoc tests (paired samples) were performed as indicated in the figure legends.

Gating strategy
Lymphocytes were defined using FSC-A and SSC-A and singlets were further selected using FSC-W vs. FSC-H and SSC-W vs. SSC-H or FSC-A vs. FSC-H.For cell sorting, dead cells were labelled with propidium iodide.For analysis of surface markers and intracellular stainings, dead cells were labelled with Zombie Aqua or Zombie NIR fixable live/dead staining reagents (Biolegend).Further gatings were defined based on bimodal expression of surface markers.Gating strategies are indicated in Figure Legends and representative plots showing gating strategies are provided in the Supplementary Information.Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

Fig. 1 |
Fig. 1 | A previously unrecognized alternative promoter drives IL-33 receptor expression in antiviral T cells.a, Scheme depicting the curated Il1rl1 gene.b, ST2 surface expression by in vitro differentiated T-cell subsets.Percentages in black, mean fluorescence intensity of ST2 + T cells in red.c, Il1rl1 first exon usage by differentiated T cells (CTL: n = 4 with one sample less than the limit of quantification (LOQ) in exon 1a and 1b reactions, Th1: n = 3, Th2: n = 4, NIH3T3: n = 2).d, RNA-seq coverage and splice junction tracks of ST2 + CTLs and Th1 and Th2 cells (n = 3 per subset) at the Il1rl1 locus.chr1:40,377,000-40,465,500; GRCm38.p6/mm10 is shown.e,f, WT mice received LCMV-specific P14 or Smarta T cells and were infected with LCMV-WE.P14 CTLs and Smarta Th1 cells were isolated on d7 p.i., and Il1rl1 first exon usage was analyzed by qPCR (e; CTL: n = 4 with two samples <LOQ in exon 1a reaction, Th1: n = 4, Th2 control (ctrl) (in vitro): n = 2) and RT-PCR (f).g, ChIP-seq tracks indicating T-bet 36 , STAT4 (ref.38) and GATA-3 (ref.37) binding and ATAC-seq tracks showing chromatin accessibility in naive or activated LCMV-specific T cells39,40 .h, Computational pipeline to identify alternative TSSs between type 1-(CTL, Th1) and type 2-(Th2) polarized T cells.i, Manhattan plot showing identified hits (parameters minAbs = 0.25 and promoter fold change (FC) = 2, dashed line: P = 0.01).j, Heatmap of identified TSSs with P < 0.01 and their respective ProActiv-normalized expression across all replicates.In i and j, red texts are used to highlight the important transcriptional start sites detected.Data in panels c, e and f are representative of two independent experiments.Data are presented as mean ± standard deviation, with each dot representing T cells isolated from individual mice.P was determined using two-tailed t-tests with Benjamini-Hochberg (BH) correction (i and j).

Il2rb-Fig. 6 |
Fig. 6 | Transcriptional profiling indicates broad costimulatory and TCRcooperative functions of IL-33-ST2 signaling in T cells.a-f, Differentiated CTLs, Th1 cells and Th2 cells were stimulated with IL-33 or left untreated for 2 h and subjected to RNA-seq analysis (n = 3 independent cultures per subset).a, experimental outline.b, heatmaps depicting differentially expressed genes in each T-cell subset (log 2 fold change > 1.0; P adjusted < 0.01).c-f, comparison of IL-33-stimulated and untreated T cells (2 h timepoint).c, Venn diagram illustrating the overlap in differentially regulated genes between CTLs, Th1 cells and Th2 cells.d, gene ontology biological process overrepresentation analysis of IL-33-induced genes shared among all subsets.e, expression of selected transcription factors, cytokines and chemokines, and TCR-regulated genes in each T-cell subset.f, gene set enrichment analysis of TCR-downstream genes in IL-33-stimulated versus unstimulated CTLs.g-k, P14 T cells (CD45.1 + CD45.2 + )
Article https://doi.org/10.1038/s41590-023-01697-6Extended Data Fig. 2 | Flow-cytometric sorting and analysis of human T cells.a, Gating strategy for the sorting of human CXCR3 + CRTH2 -Th1 cells and CXCR3 − CRTH2 + Th2 cells.b, Gating strategy for the sorting of human CD8 + T cells.c,d, Representative histograms and quantification of T-bet (c) and GATA-3 (d) expression by activated T cells at day 5 of culture (n = 3).e,f, Representative histograms and quantification of IFN-γ (e) and IL-13 (f) expression by T cells stimulated with PMA/ionomycin at day 5 of culture (n = 3).Data represent two independent experiments and are presented as mean ± SD with each dot representing one culture performed with T cells from individual donors (c-f).P was determined using one-way ANOVA with Tukey's post hoc test (c-f).
software (v8.1),FlowJo (v10.7.1)Cell population abundanceFrequencies of analyzed cell populations are indicated in representative plots.Purity of sorts are shown in representative plots in the Supplementary Information.