# paper.pdf ## Metadata - PDFFormatVersion=1.6 - Language=en-US - IsLinearized=true - IsAcroFormPresent=false - IsXFAPresent=false - IsCollectionPresent=false - IsSignaturesPresent=false - CreationDate=D:20260424143913+05'30' - Creator=Adobe InDesign 17.4 (Windows) - ModDate=D:20260424145517+05'30' - Producer=Adobe PDF Library 16.0.7 - Trapped=False - xmpmm:instanceid=uuid:f28a79bd-fa4d-466e-a369-1e0090a5b245 - xmpmm:originaldocumentid=xmp.did:A33916180F2068118083ACD1B377E157 - xmpmm:documentid=xmp.id:2e7bea8f-e58d-984e-a33a-6e2a7a724887 - xmpmm:renditionclass=proof:pdf - xmpmm:derivedfrom=xmp.iid:7e6425bb-8e13-3c40-8278-e42cdec9e9bdxmp.did:7e6425bb-8e13-3c40-8278-e42cdec9e9bdxmp.did:A33916180F2068118083ACD1B377E157default - xmpmm:history=convertedfrom application/x-indesign to application/pdfAdobe InDesign 17.4 (Windows)/2026-04-24T14:39:13+05:30 - xmp:createdate=2026-04-24T14:39:13+05:30 - xmp:modifydate=2026-04-24T14:55:17+05:30 - xmp:metadatadate=2026-04-24T14:55:17+05:30 - xmp:creatortool=Adobe InDesign 17.4 (Windows) - xmprights:marked=False - photoshop:modifydate=2019-01-31T16:16:25 - photoshop:createdate=2019-01-24T11:43:46 - dc:format=application/pdf - pdf:producer=Adobe PDF Library 16.0.7 - pdf:trapped=False - extensisfontsense:slug=3023672065GraphikOpenType - PS3023672065--0Graphik-Bold1.0011904271029GraphikOpenType - PS1904271029--0Graphik-Regular1.0012428528031News Gothic MT OTOpenType - PS2428528031Monotype Typography0NewsGothicMTOT-Regular1.000543760319News Gothic MT OTOpenType - PS543760319Monotype Typography0NewsGothicMTOT-Bold1.0002785939051KnockoutOpenType - PS2785939051--0Knockout-68FullFeatherwt1.200423856041Graphik X CondOpenType - PS423856041--0GraphikXCond-Super1.0001526035435Graphik X CondOpenType - PS1526035435--0GraphikXCond-Bold1.0003093728306Graphik CondOpenType - PS3093728306--0GraphikCond-Black1.0011341118539Graphik CondOpenType - PS1341118539--0GraphikCond-Regular1.0011385893531GraphikOpenType - PS1385893531--0Graphik-Semibold1.0012986479693Graphik CompactOpenType - PS2986479693--0GraphikCompact-Regular1.0024223138361Graphik CompactOpenType - PS4223138361--0GraphikCompact-Semibold1.0023673047546Harding SevenOpenType - PS3673047546--0HardingSeven-Extrabold0.000103712566Harding SevenOpenType - PS103712566--0HardingSeven-Roman0.0002232452979Harding SevenOpenType - PS2232452979--0HardingSeven-Medium0.000631916388Harding SevenOpenType - PS631916388--0HardingSeven-Bold0.000157629484Publico BannerOpenType - PS157629484--0PublicoBanner-Black1.005 ## Contents ### Page 1 nature immunologyhttps://doi.org/10.1038/s41590-026-02506-6ArticleThe long noncoding RNA lnc13 restrains inflammatory responses to maintain oral tolerance to glutenIn the format provided by the authors and uneditedSupplementary information ### Page 2 Supplementary Fig 1. lnc13 knockout does not alter Il18rap expression but enhancesinflammatory gene induction in macrophages.(A) Relative expression of lnc13 in WT and lnc13⁻/⁻ bone marrow–derived macrophages(BMDMs), either unstimulated (–) or stimulated with LPS for 8 hours (+). Lnc13 levels werenormalized to Rpl32 housekeeping gene, and DDCt was calculated by normalizing to the averageof unstimulated WT cells. lnc13 is inducibly degraded upon LPS exposure in WT BMDMs (n=4biological replicates across 4 independent experiments). (B) Il18rap expression is unchanged ### Page 3 between WT and lnc13⁻/⁻ BMDMs under both resting and LPS-stimulated conditions, indicatingthat lnc13 deletion does not affect the adjacent Il18rap gene. Il18rap levels are first normalized toRpl32 housekeeping gene and DDCt was calculated by normalizing to the average WT cells (n=4biological replicates across 4 independent experiments). (C) RT-qPCR of inflammatory transcriptsin BMDMs. Expression of genes were first normalized to Rpl32 housekeeping gene and DDCt wascalculated by normalizing to the average WT LPS stimulated samples (n=4 biological replicatesacross 4 independent experiments). lnc13⁻/⁻ cells show enhanced induction of Il6, Il1b, and Nos2following LPS stimulation, indicating a heightened proinflammatory state in the absence of lnc13.In all plots, red dots denote KO cells while blue dots denote WT cells. Data represent mean ±s.e.m.; unpaired, two-tailed, Student’s t-test. N=4. * p <0.05,** p<0.01, *** p<0.001, ****p<0.0001. ### Page 4 Supplementary Fig 2. HLA-DQ8 transgene validation. ### Page 5 (A) Schematic of the HLA-DQ8 transgenic mouse model. The human HLA-DQ8 allele isexpressed under its endogenous promoter, and the mouse MHC class II locus is knocked out.(B) Representative flow cytometry in mouse peripheral blood confirming that HLA-DQ8 miceexpress human HLA-DQ8 but lack endogenous mouse MHC-II. ### Page 6 ### Page 7 Supplementary Fig 3. Gating of LPLs and elevated IL-12 in myeloid cells in lnc13⁻/⁻-DQ8mice.(A) Representative flow cytometry gating strategy of LPL cells. Note, while we are showing acomprehensive gating strategy here, the flow panel for HLA-DQ8+ CD11c+ DCs were doneseparately from CD4/CD8 given the constraints on fluorophores. (B) Expression of Il12b,encoding IL-12, of CD11b+ lamina propria cells (live, CD45+, TCRb-, B220-, CD11b+) in WT-DQ8 and lnc13-/--DQ8 mice post gluten challenge by RT-qPCR. DDCt was calculated by firstnormalizing to Rpl32 housekeeping gene, then normalizing to the average of WT-DQ8 samples.N=4 across 2 independent experiments. (C) Normalized expression of IL12A and IL12B in humanceliac duodenal biopsy RNA-seq. For IL12A and IL12B, DESeq2 normalized read counts werenormalized to average of read counts from healthy controls to allow representation of both genesin one plot (GSE134900). Error bars represent mean ± s.e.m.; stastistical comparison done withDESeq2. * p <0.05,** p<0.01, *** p<0.001, **** p<0.0001. (D) Representative histogram plot(left), quantifications of % IL-12+ cells (middle) and IL-12 MFI (right) of lamina propria liveCD45+ TCRb- B220- HLA-DQ8+ CD11c+ dendritic cells. Dotted line in histogram representsisotype control. Error bars represent mean ± s.e.m.; statistics for scatter plots are done usingunpaired, two-tailed Student’s t-test. * p <0.05,** p<0.01, *** p<0.001, **** p<0.0001. ### Page 8 Supplementary Fig 4. Gating strategy for IELs.The representative gating strategy to identify populations in the IEL fraction is shown. Note, forGZMB expression, only NKG2D was used to gate NK cells as NK1.1-FITC overlapped withGZMB-FITC. ### Page 9 ### Page 10 Supplementary Fig 5. Individual villus and crypt measurements from Swiss roll histology.Representative images and quantitative examples illustrating measurement of: villus height, cryptdepth, and villous-to-crypt (V/C) ratio from H&E-stained Swiss roll sections of mouse smallintestine. These measurements were used to generate the pooled analyses shown in Figure 4G–H.Sections were assessed in a blinded fashion to avoid measurement bias. Black scale bar represents300μm. yellow scale bar represents 200μm ### Page 11 Supplementary Fig 6. Conceptual model: lnc13 restrains inflammatory signaling to preserveoral tolerance to gluten.Schematic illustration of how lnc13 regulates immune responsiveness and contributes to CeDsusceptibility. Left: In individuals without HLA-DQ2/8 alleles and carrying functional lnc13,gliadin presentation is limited and lnc13 suppresses inflammatory signaling. These individualsmaintain immune tolerance and do not develop CeD (analogous to WT mice). Middle: Inindividuals with HLA-DQ8 (or DQ2) but intact lnc13, gliadin is presented to T cells, but lnc13dampens immune activation, acting as a safeguard. These individuals remain tolerant (analogousto WT-DQ8 mice). Right: In individuals with both HLA-DQ8/2 and a lnc13 loss-of-function allele(e.g., rs917997[T]) immune cells become hyperresponsive to gliadin and inflammatory cues such ### Page 12 as IL-15. This leads to heightened immune activation, predisposing to CeD pathology (modeledby lnc13⁻/⁻-DQ8 mice). Model illustrates that lnc13 functions downstream of antigen presentation,desensitizing immune cells to inflammatory cascades and limiting pathogenic differentiation.Figure generated with BioRender. ### Page 13 Supplementary Table 2. Oligonucleotide sequences. For gRNAs, “^” denotes cut site.Oligo name Sequence 5’>3’Human lnc13 siRNAgeneration FTAATACGACTCACTATAGGGTACCACATGCCTGTGAAAAACTCTCHuman lnc13 siRNAgeneration RTAATACGACTCACTATAGGGAGACAGGATGGTGTTGTTCACTCLnc13 RAP probes 1. GGAGACCATATAGGGTCCATAAGGAAAGGTCTGCAGTGTGTCCTAGAAGGGTGCCATCCATACTCAAATGGTGATGCTTTCTTATACCGAAATAAAGCAT2. TGCTTTCATTCTGCTGAGCCTAGCTGCTCAGCCAAAGTCTTGCAAACACAGGAATTTTGTCTTGATTATTCCTCTTGTTTTAGTTGATAT3. TACAAGTCTGAGGCACTGTAAAGCCAAATGCTTGGGAGCTCAGTTATCTTGGAACAAGATAAACCTGGCTGCACTGGCCAGAAGAGGTTTCTGAGTCTATGATCTCTGAGAGAGGCTAAC4. CTCAGGGGATGTGACTCATGATGTGGGAGCCAGTTCATGAGGTGTTGCCTTCACTGCTCACATGATGAGCCACCCTGTTCCATCAGGAAAG5. ACCCAGCCCTTAAACACATCATCTTGTTACTAAGGAAGACTTCCCCTAAGACCACTCGGCCAATCAGTGTCCATACAGGATACCACTCTCAGCAGGCAAA6. TTGTAGACAACTACAAACCCATTCTTTGTCTTAATTGGACTAATGATCTGGGACAAATTAGTCTCTACTTGCAGAGACTCGGGTAAGGTCTCTAGCCAAGGAAACAAGGAGATATCGTAA7. ATATTTTCTGCCAACAACAGATACCATGCATGTTGTTCACCTGCAGGTCTAGAAACATATACCTAGTTCATGCCTTCAAGTGGGTCCTGTAGGGACCCA8. AAAATAAAATCCTATTTATCTGAATGCATATACACACACAAACGTATGATAAATTCATATATATAGTGCAAAACAGGCATGCGTGCATGAATGTTGGACAGCCCATGGAGCTATTTCTGT9. ACCAGTAGTCAGGACTGTGGGCATTTATTTTATATTACTTGAGGTATTGACCTTGGAATAACATGAATGTTGAGAACTTTCTCTCTGAGAACTGTCTG10. GGTTACCATTGTATTTGTTCAATGTATGTGAAGCAATAGACAAGATACATGGCCTCCTCATCTAGATGGTGCAGCCAGAAAACGTGGCTT11. GCAAGGCATAAATATTAATAACAGTTTGTCCCAAATGCATACCTGTCATCTCACTGGACCTAGCTATGGAAAAGAAGACATTCTATAAACATGGACTGGCTATTTCTGCTTTCATATTTG12. AAGAAACCCAGGAAGCCTGGCCAGAGCCAGGGCACAGTGGTTCAACAAGTGTGGGTTCCTCCCCACTGC ### Page 14 GATCCCACAGTGGTCCTTCCCACAAAAACTTCCTCCAGGGATAATTTATGC13. GTGGTGTTCAAAGGGCATAAACAGCCACATCAACTCAAACCCTGTTTCAGCTATAGACCAAGTCATGAGCATCAAATTCACTTTGTTAATCAAAACGACG14. TGACATGCAAAGCTATGGCATCTGTTGTATTCTTGCTGCTTGTCAACAAATGGGTGCCCTTATGCCCCACACTCTGCTTCATAATTGCTGCCTCTCTAGG15. TTAGTTCTATCCCATTCCTAAATCTGAATCCTGTAAAGAGTGTTCCAGCCAAGAGGAGGGCTCTGTCCTCTCTGTTATATACAATTTGGAAGAACTTGACTCAAATTTTTGACCCTCTGAGT16. GGAGATCATATGTTCTGTCTCACCTTCTATATTTCTCCTCTATAGAATTGATGAATAAACATGCAGCAAAATTTCTTTACAGAAGGGCTTTAGATGGAGTTTCAATCACTCGTATCGAACLnc13mm_gRNA-1 CCTAGTTCATGCCTTCA^AGTLnc13mm_gRNA-2 ATATTGGACCTTTCGAA^GCTLnc13mm_gRNA-3 ATGATCTCCTGGGTCCC^TACLnc13mm_gRNA-4 TTGTCCAGGGAGTACAA^GCCLnc13mm_gRNA-5 TGGTGACCTACTACAAT^TTCLnc13mm_gRNA-6 CTCATCAGAGCTATACC^ATCLnc13 WT genotypeprimer FAACACCACCTTTCCTGATGGLnc13 WT genotypeprimer RCAGGGCACAGTGGTTCAALnc13 KO genotypeprimer FATGGGCTGTCCAACATTCATLnc13 KO genotypeprimer RTTATCATTGTCCTGTGAATGGATGGHuman RPLP0 F GCAGCATCTACAACCCTGAAGHuman RPLP0 R CACTGGCAACATTGCGGACMouse Rpl32 F GACCGATATGTGAAAATTAAGCGAAMouse Rpl32 R CATCAGGATCTGGCCCTTGAHuman Phlda1 F GGAGATCGACTTTCGGTGCCHuman Phlda1 R GGCCTGACGATTCTTGTACTGHuman Ccr7 F AAGCGATGCGATGCTCTCTCHuman Ccr7 R TTGCGCTCAAAGTTGCGTGHuman Tnfsf11 F CAACATATCGTTGGATCACAGCAHuman Tnfsf11 R GACAGACTCACTTTATGGGAACCHuman Tnfrsf4 F GCAATAGCTCGGACGCAATCTHuman Tnfrsf4 R GAGGGTCCCTGTGAGGTTCTHuman S1pr3 F GTGATCCTCTACGCACGCATCHuman S1pr3 F CGCTCCGAGTTGTTGTGGTHuman Gzmb F TACCATTGAGTTGTGCGTGGG ### Page 15 Human Gzmb R GCCATTGTTTCGTCCATAGGAGAHuman Brca2 F TGCCTGAAAACCAGATGACTATCHuman Brca2 R AGGCCAGCAAACTTCCGTTTAHuman Tiam1 F ATGACGCTACATATTTGGCTGAGHuman Tiam1 R ACCCAAGATTTCTTCGTTGCTTHuman Junb F ACAAACTCCTGAAACCGAGCCHuman Junb R CGAGCCCTGACCAGAAAAGTAHuman Mapk7 F CTCGGGAGCGCATTAAGGAGHuman Mapk7 R GTCAGATCAATGGTGTCAGGTGHuman Il2rb F CTGCTTCACCAACCAGGGTTAHuman Il2rb R GGGGTCGTAAGTAAAGTACACCTHuman Il15ra F ACAACAGCAGCTATTGTCCCGHuman Il15ra R GTGGCCCTGTGGATACACACHuman Ikbke F GAGAAGTTCGTCTCGGTCTATGGHuman Ikbke R TGCATGGTACAAGGTCACTCCMouse Hprt F GTTAAGCAGTACAGCCCCAAAMouse Hprt R AGGGCATATCCAACAACAAACTTMouse Cd138 F CTTTGTCACGGCAGACACCTTMouse Cd138 R GACAGAGGTAAAAGCAGTCTCGMouse Blimp1 F AAGAGGTTATTGGCGTGGTAAGMouse Blimp1 R TAGACTTCACCGATGAGGGGT