A pSS human transcriptional signature (1255 probes; 933 )

100

10 1 (human parotid glands)

1 GSE406 1 Fold change pSS versus non-pSS

-10 -10 1 10 100 Fold change pSS versus non-pSS GSE23117 (human minor salivary glands)

B human pSS signature (1740 probes)

[1013 probes with FC>1;p-value(FDR)<0.05, i.e. 58%]

0 118 1013

-3

-6

-9

-12

log10(p-value) -15

-18

-21

-24

-27 -100 -10 1 10 100 Fold change 16-week versus 8-week old NOD mice GSE32681 (mouse lacrimal glands) Supplementary Figure 1. 58% of the human pSS transcriptional signature is upregulated in inflamed NOD lacrimal glands. (A) Publically-available microarray datasets were used to generate a FC-FC plot comparing expression in minor salivary glands (GSE23117, x-axis) and parotid glands (GSE40611, y-axis) from pSS and non-pSS subjects. Filtered on genes with p-value(FDR)<0.05 in at least one of the two datasets and mean expression >6 in at least one group of subjects. 933 genes (1255 probes) showed significant FC>2 in the two datasets and were defined as the pSS transcriptional signature (black square). Blue lines, FC=2. See complete list of genes included in the human pSS signature in Supplementary Table 1A. (B) Volcano plot comparing whole lacrimal gland tissue transcriptomes from 16-week old (i.e., at the peak of immune infiltration) and 8-week old NOD mice (i.e., at the initiation of immune infiltration) (GSE32681). The human pSS signature defined in panel A (933 genes corresponding to 1740 mouse probes) is highlighted in red on the mouse dataset with the corresponding number of gene probes significantly upregulated and downregulated in inflamed mouse lacrimal glands, i.e., in 16-week old NOD mice (see complete list of genes in Supplementary Table 1B).

1 CD73high CD200high CD73low CD200low - - Hspa4l Kif1b PD1 ICOS Tnfrsf9 Bambi-ps1 Lbh T cells Adora2a Psap Casp1 Fmnl2 Lclat1 Pltp Casp3 Gbp2 Pdcd1 S100a6 Dusp10 Endod1 Csf2rb2 Zdhhc2 Trim13 Mcm6 Mdfic Csf2rb Hivep3 Cd244 Casp4 Gimap6 Acot11 Ahr Gpr183 Klra17 Fam129a Rell1 Il2rb Trim36 Tbx21 Cxcr3 Tubb2a Pear1 Dnajb4 Cdca8 Rasl11b Cst7 Uhrf1 Slc41a2 Anxa2 Ctsl Akap12 Dirc2 S100a4 Gramd3 Acot7 Gng2 Mgat4a Comt Plac8 Nfatc1 Pde3b Prkca Cd55 Dusp16 Nt5e Ctsh Tmem2 Gramd4 Cd200 Gpr18 Plscr1 Mrps6 Ampd1 Tjp2 Sgk3 Spock2 Ly86 Gfod1 Rapgef4 Ptprv Myo1e Sema4a Tox H2-Aa Bcl2a1a Bc H2-Eb1 Ppap2c Zan Filip1l Serpina3g Pdlim1 Lag3 Pik3ip1 Kdm2b Als2cl Slc5a3 Axl Klhdc1 Rgs16 Rreb1 Twsg1 Sell Ccr8 Cyfip1 Eef2k Irak1bp1 Acss2 Syt11 Ndrg3 Rbpj Klhdc2 Penk Wisp1 Aqr Gdpd5 Commd8 Spry1 Atp1b1 Ifng Zc3h12d Slpi Tpi1 Dnah8 Hnrpll Klf2 Maf Treml2 Tnfsf11 Farp1 Hs3st3b1 Raph1 Lair1 Prnp Tyrobp Sh3rf1 Bmpr2 Basp1 H1f0 Acvr1b Abi2 Ugcg Ubash3b Zbtb18 Ptgfrn Arhgap31 Cnn3 Tox2 Zdhhc14 Nrn1 Fam69a Gpm6b Tiam1 Ccr9 Dsel Klrd1 Socs2 Dapl1 Dst Amigo2 Cyfip1 Tnfrsf4 Actn1 Itgb8 Igfbp4 Galm S1pr1 Slc22a15 Spib Angptl2 Cd44 Mctp2 Ptger2 Scml4 Eea1 Dtx1 Stx11 Trps1 Xkrx Tnfrsf1b Rras2 Icos BC018473 Cish Trat1 Folr4 Ikzf2 Itga6 Rora Satb1 Ehd1 Emb Rhbdl3 Dab2ip Rasgrp2 Atxn1 Pik3r5 Ahi1 Prkd3 Gna13 Arhgap29 Msi2 Alcam Pdlim4 Impa2 Itgb3 Bhlhe40 St8sia6 Dusp4 Ypel2 Tbc1d8 Nfil3 Adh1 Cxcl10 Myo10 Gem Klf3 Nr4a2 Ctla4 Mpeg1 Ramp1 Tcf4 Mapre2 Plaur Rrm2 Lyrm2 Bach2 Gpr68 Spice1 Il18rap Grcc10 Nkg7 Supplementary Figure 2. Transcriptional signatures from PD1highICOShigh and PD1−ICOS− LGT-infiltrating T cell subsets. Expression heatmaps displaying transcriptional signatures for PD1highICOShigh (left) and PD1−ICOS− (right) LGT-infiltrating T cell subsets. The PD1highICOShigh signature corresponds to the genes which are similarly regulated in CD73lowCD200low and CD73highCD200high subsets relative to the PD1−ICOS− subset. Signatures were generated by filtering genes displayed in Figure 4 panel A on genes showing mean expression >6 and at least 3-fold significant upregulation (p-value(FDR)<0.05) in the subset(s) of interest relative to the other LGT-infiltrating CD4+ T cell subset(s). Samples are color-coded and hierarchically-clustered according to (see complete list of genes in Supplementary Table 1E).

2 CD73high CD200high CD73low CD200low PD1- ICOS- Ikzf3 Spleen T cells Prdm1 Foxp3 Mxi1 Cebpb Maff Jun Crem Nfkbia Rel Nfil3 Pawr Nr4a2 Mxd4 Maf Tbx21 Pbx3 Tox Nfatc1 Rbpj Hif1a Jarid2 Hivep3 Elk3 E2f3 Ahr Trps1 Gfi1 Id2 Ikzf4 Ikzf2 Rora Nfat5 Plagl1 Gata3 Pou2af1 Ets1 Bptf Smad7 Irf8 Stat1 Irf7 Tsc22d3 Mafk Tsc22d1 Arid5a Trim13 Mef2a Ncoa4 Runx3 Bach1 Pbx2 Rreb1 Klf2 Klf7 Smad1 Klf3 Foxp1 Tcf4 Spib Rere Gtf2i Bach2 Tcf7 Nfe2l2 Supplementary Figure 3. profiles expressed by LGT-infiltrating CD4+ T cell subsets. Expression heatmap of the genes displayed in Figure 4 panel A that encode with transcription factor activities (based on annotations) and that show at least 2-fold significant differential expression (p-value(FDR)<0.05) in one of the two PD1highICOShigh T cell subsets relative to the PD1−ICOS− subset. Samples are color-coded (see key) and hierarchically-clustered according to gene expression.

3