Supplementary figures

A number of % of mitochondrial genes % of ERCC

60 100 7500

) ) ) 75 10 10 10 40 5000 50

20 2500 Expression (log Expression (log Expression (log 25

0 0 0

lck lck cd4 lck cd4 rag1 cd4 rag1 rag1 mhc2dab mhc2dab mhc2dab

B 100 7500 7500 75 Bulk 5000 5000 50 Empty

ERCC Single Cell 2500 2500 25 Number of Number of Content (%) Detected Genes 0 Detected Genes 0 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 Mitochondrial Content (%) ERCC Content (%) Mitochondrial Content (%)

60 30 150

40 20 100 Count Count 20 Count 10 50

0 0 0 0 2500 5000 7500 10000 0 25 50 75 100 0 25 50 75 100 Number of Detected Genes ERCC Content (%) Mitochondrial Content (%)

100 7500 7500 75 5000 Fail 5000 50 Pass

2500 2500 ERCC 25 Number of Number of Content (%) Detected Genes 0 Detected Genes 0 0 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 Mitochondrial Content (%) ERCC Content (%) Mitochondrial Content (%)

Supp. Figure 1. Quality control of SmartSeq2 data. A. Number of detected genes, mitochondrial and ERCC content in single cells from each of the transgenic lines. B. Plots for the number of detected genes, % ERCC and % mitochondria (MT) genes. Cells that passed QC (ERCC < 60 % MT < 45% and at least 900 detected genes) are coloured in blue. The bulk and empty wells were removed from the final plots.

Supp. Figure 2. Clustering of Smart-seq2 data from Tg(lck:EGFP), Tg(cd4-1:mcherry) and Tg(mhc2dab:GFP, cd45:dsRed) zebrafish transgenic lines. A. Brennecke method was used for identification of highly variable genes. The orange solid line shows the curve of the technical noise fit and the pink dashed line shows the position of genes with 20% biological coefficient of variance (CV). Blue dots indicate ERCCs, pink dots indicate highly variable genes, black dots represent the rest of genes expressed in the data set. B. Diffusion map showing origin of cells in each of the clusters.

LCK RORC GATA3 TBX21

3.0 2.0 3.0 ) ) ) )

1.02 2 2 2 1.5 2.0 2.0

1.0 0.5 1.0 1.0 Expression (log Expression (log Expression (log Expression (log 0.5

0.0 0.0 0.0 0.0 ILC1 ILC2 ILC3 ILC1 ILC2 ILC3 ILC1 ILC2 ILC3 ILC1 ILC2 ILC3 NCR1 NCR2 IL22 ID2 5.0 4.0 4.0 ) ) ) ) 2 2 2

1.5 2 2.0 3.0 3.0 1.0 2.0 2.0 1.0 Expression (log Expression (log Expression (log 0.5 Expression (log 1.0 1.0

0.0 0.0 0.0 0.0 ILC1 ILC2 ILC3 ILC1 ILC2 ILC3 ILC1 ILC2 ILC3 ILC1 ILC2 ILC3

Supp. Figure 3. Expression of marker genes in human ILCs. Violin plots showing the expression of marker genes in individual human ILCs. No expression of IL13 was detected in this dataset. The expression values were calculated from publicly available dataset (Björklund et al., 2016). rag1-/- PBS-injected rag1-/- V. anguillarum-injected rag1-/- A. simplex-injected 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 si:ch211-266a5.12 Cluster si:ch73-31d8.2 Cluster nitr9 Cluster si:busm1-194e12.12 tac4 1 rorcBX927125.1 1 CABZ01034691.1 1 rorc il22 CABZ01073795.1 2 2 si:dkey-78l4.8 2 si:dkey-79f11.10 mcl1b nitr4a 3 3 cxcr4a 3 tnfa zgc:194101 4 4 4 ccl20a.3 5 5 il17a/f3 5 ccr9a 6 6 6 7 7 7 ccr9a 8 tnf 8 8 hmgn3 9 9 9 nitr2b txn si:ch211-165b19.8 10 ifng1-2 10 10 sh2d1ab si:ch211-213a13.1 si:dkey-33i11.9 ddit4 11 cxcr3.1 11 ccr9a gzm3 12 si:dkey-79f11.10 12 Expression Expression nitr6b Expression 3 3 apoa1a 3 nitr10a nitr7b 2 nitr9 2 si:busm1-194e12.12 2 BX927125.1 ccl20a.3 zgc:103700 rbp4 nitr7b nitr9 nitr4a ctage5 1 1 BX927125.1 1 cxcr4b ccl20a.3 si:ch211-67e16.3 tnfa tagapa nitr10a 0 nitr2b 0 smarca2 0 nrros nitr2b klf2a -1 nitr6b -1 gzm3 jdp2b -1 BX936298.1 ifng1-1 ms4a17c.1 tnfrsf18 apoa1a nitr10a nitr6b -2 rbp2a -2 -2 tox apoa1a nitr1o

ccl38a.4 lygl1 nitr13 grn1 nkl.2 cxcr4a ponzr6 psmd10 grn2 si:dkey-78l4.4 si:ch211-256a21.4 si:dkey-21e2.12 si:dkey-31e10.5 ca2 si:ch211-284e13.12 anxa3b

hmgn2 nkl.2 si:ch211-188c18.1 CR352226.2 si:ch211-256a21.4 foxp3a si:dkey-21e2.12 egr2b si:ch211-214p13.9 si:ch211-277c7.6 zgc:194101 mmp13b tnfsf13b ifng1-2 igfp1a nitr1j sdc4

ca2 ifnphi2 si:dkey-78l4.4 foxp3a stmn1a nkl.2 gig2d si:ch211-277c7.6 zgc:123068 rrm2 nkl.4 si:ch211-214p13.9 CABZ01074745.1 tnfrsf18 pcna

hmgn2 s1pr4 klf2a si:ch211-66e2.3 flna mibp gzmk rrm2 rorc mcl1b pcna cxcr4a hmgb2b grn2 hmgb2a tac4 grn1 c1qb lygl1 lgals3bpb grn2 tppp zgc:112234 cxcl11.6 grn1 soul5 mxc notchl si:dkey-23a13.22 abcg4b si:ch211-207n23.2 BX539307.1 dapp1 sox13 atg101 si:ch73-31d8.2 nitr12 ccdc88c si:ch73-226l13.2 si:ch211-212d10.1 rrp15 cysltr1 il17a/f3 si:ch211-147a11.3 il13 zgc:112234 si:dkey-23a13.22 ENSDARG00000017314 sycp1 tuba7l si:dkey-78l4.10 h1f0 nkiras2 ENSDARG00000041723 nitr6a si:ch1073-456m8.1 napaa si:ch73-111k22.3 nudt8 scyl3 snrka ENSDARG00000042409 si:dkeyp-68b7.7 tbc1d14 si:ch73-113g13.3 txndc17 si:dkey-88e18.2 papd4 trnt1 CU550716.2 BX511215.2 si:ch73-269m14.2 cndp2

Supp. Figure 4. Expression of marker genes in lck+ cells collected from rag1-/- gut. Heatmaps showing expression of marker genes in each of the clusters for lck+ cells collected from rag1-/- gut, PBS- injected (left), V. anguillarum-injected (middle), A. simplex-injected (right). A rag1-/- PBS-injected Cluster 1 20 20 2 20 3 10 10 4 5 ILC-like 0 6 0 nitr-rorc+ 0 nitr+rorc+ 7 8 tSNE 2 tSNE 3 tSNE 3 naïve ILC-like naïve ILC-like 9 -10 -10 nitr+rorc+ -20 nitr-rorc+ nitr+rorc+ 10 11 ILC1-like ILC1-like 12 ifng1-1+ -20 ifng1-1+ -20 ILC1-like naïve ILC-like -40 ifng1-1+ nitr+rorc+ -30 -30 -40 -20 0 20 -40 -20 0 20 -40 -20 0 20 tSNE 1 tSNE 1 tSNE 2 -/- ILC3-like rag1 V. anguillarum-injected nitr+rorc+il22+ ILC3-like Cluster 40 nitr+rorc+il22+ 40 1 ILC3-like nitr-rorc+ 2 ILC1-like ILC1-like nitr+rorc+il22+ 20 + 3 ifng1-1+ ifng1-1 4 20 20 5 6 7 0 nitr-rorc+ nitr-rorc+ 8 tSNE 2 tSNE 3 0 tSNE 3 0 9 ILC1-like 10 ifng1-1+ 11 -20 -20 12 -20

-40 -20 0 20 -40 -20 0 20 -20 0 20 tSNE 1 tSNE 1 tSNE 2 rag1-/- A. simplex-injected Cluster ILC1-like 1 + 25 20 20 ifng1-1 2 3 4 5 6 0 ILC1-like 0 + 0 7 ifng1-1 8 tSNE 2 nitr-rorc+ tSNE 3 tSNE 3 9 10 -25 ILC2-like ILC2-like nitr+rorc+il13+ nitr+rorc+il13+ -20 -20 ILC2-like ILC1-like nitr+rorc+il13+ -50 ifng1-1+ nitr-rorc+ nitr-rorc+ -20 0 20 -20 0 20 -50 -25 0 25 tSNE 1 tSNE 1 tSNE 2

B Domain Biological Process Cellular Component Molecular Functon

nitr-rorc+ (Cluster 1) nitr+rorc+ (Cluster 3) rag1-/- PBS-injected rag1-/- PBS-injected

immune system process 7.2 cellular response to TNF 6.43 3.43 intracellular response to TNF 6.43 neutrophil chemotaxis 6.01 response to IFNG 6.01 intracellular CCR chemokine receptor binding 6.01 2.78 organelle ribosome 5.86 chemokine actvity 5.72 positve regulaton of ERK1 and ERK2 cascade 5.59 response 2.29 structural consttuent of ribosome 5.5 to stress cytosolic ribosome 5.06 response to biotc stmulus 4.17 response to other organism 3.58 folding 2.29 cytochrome complex 3.55 electron transfer actvity 3.32 ATP metabolic process 3.21 translaton 3.06 mitochondrial 2.28 mitochondrial ATP synthesis 2.97 respiratory chain coupled electron transport hydrogen ion transmembrane 2.72 transporter actvity hydrogen ion transmembrane transport 2.71 respiratory chain complex 2.57 cell 2.06 response to lipopolysaccharide 2.51 positve regulaton of actn nucleaton 2.49 cytochrome-c oxidase actvity 2.36 threonine-type 2.36 2.06 ubiquinol-cytochrome-c reductase actvity peptdase actvity regulaton of lymphocyte differentaton 2.06 regulaton of hemopoiesis 2.06 0.0 0.5 1.0 1.5 2.0 0.0 1.0 2.0 3.0 0.0 0.5 1.0 1.5 2.0 0.0 2.0 4.0 6.0 0.0 2.0 4.0 6.0 0.0 2.0 4.0 6.0 -log10(q-value) -log10(q-value)

Supp. Figure 5. Identification of nitr-rorc+ and nitr+rorc+ population of cells. A. tSNE plots representing distribution of clusters of lck+ cells from gut of rag1-/- fish in different dimensions. B. GO terms characteristic for nitr-rorc+ (Cluster 1) and nitr+rorc+ (Cluster 3) cells in PBS-injected rag1-/- fish. A rag1+/+ PBS-injected nitr-rorc+ Cluster 1 2 3 foxp3a+ 20 4 20 20 5 naïve ILC-like 6 nitr+rorc+ 7 8 9 tSNE 3 0 tSNE 3 0 tSNE 2 0 10

-20 -20 -20 nkl.2+ gzmk+ cd8a+ -20 0 20 -20 0 20 40 -20 0 20 40 tSNE 2 tSNE 1 tSNE 1

B avg.exp. 10 2.0

9 1.0 0.0 8 % cells 0.05 0.25 7 0.50 0.75 1.00 6 Clusters 5

4

3

2

1

rorc il13 il22 tnfa tnf ccr9a mcl1b gata3 cd4-1 cd8a nitr2b nitr7b nitr9 nitr4a gzm3 gzmk nkl.2 ifng1-2ifng1-1 foxp3a

Supp. Figure 6 Analysis of the lck+ cells collected from the gut of wild-type fish. A. 2D projection of tSNE analysis of 10x RNAseq data showing heterogeneity of innate and adaptive lymphocytes’ pool. B. Dotplot shows the level of expression of signature genes and percentage of cells per cluster that express the of interest. A rag1 -/- PBS - V. anguillarum - A. simplex-injected aligned datasets Cluster 1 2 25 25 20 3 4 nitr-rorc+ 5 6 nitr-rorc+ 0 0 0 7 ifng1-1+ 8 tSNE 3 tSNE 3 tSNE 2 ifng1-1+ ifng1-1+ 9 nitr-rorc+ 10 -25 -25 11 -20 12 nitr+rorc+ nitr+rorc+ 13 nitr+rorc+ -50 -50 -50 -25 0 25 -50 -25 0 25 -20 0 20 tSNE 1 tSNE 1 tSNE 2

B nitr+rorc+ (Cluster 7) nitr+rorc+ (Cluster 7) aligned data set aligned data set DE genes PBS-injected ccl38a.4 DE genes PBS-injected DE genes V. anguillarum-injected DE genes A. simplex-injected

4 BX936298.1 4 zgc:194101 si:ch211-266a5.12 si:dkey-78l4.8 mafa ENSDARG000000880mhc2dab40

A. simplex il22 faslg ccl38a.4

V. anguillarum si:ch211-222l21.1 zgc:152863 ccl36.1 tnfrsf9a m17 plk3 ccl38.1 2 banf1 2 si:dkey-78l4.10 ptmaa si:ch211-212d10.1 tnfrsf18gbp1 ENSDARG00000068995 zgc:158343 si:dkey-31e10.5 scinlb si:dkey-78l4.8 vmp1 isg15si:ch211-226m16.2 il17a/f3 junbb il13 si:dkey-78l4.10 ldhba hist2h2l si:ch211-231m23.4 smarca2cldni si:dkey-12f6.5 si:ch73-1a9.3 dap1b isg15 0 sepp1a hmgn6dap1b ms4a17c.1 0 si:busm1-194e12.12 0 2 4 0 2 4 PBS PBS nitr-rorc+ (Cluster 13) nitr-rorc+ (Cluster 13) aligned data set aligned data set DE genes PBS-injected DE genes PBS-injected DE genes V. anguillarum-injected DE genes A. simplex-injected

4 4

BX927125.1 si:dkey-53k12.2 calm1a

arl6ip5b psme2 A. simplex vma21 dapp1 V. anguillarum ENSDARG00000088040 higd1a cpa5 2 cmip m17 arpc5a 2 rnd1b rnaset2 myg1 akap12a hnrnpa0b ufm1 zgc:91910 chd3 uqcrb pa2g4b snrpe sbf1 prelid1a crf4 pa2g4a ccl38.6 cpa5 anp32b ccl38.6 anp32b med31 puf60a kmt2bb zgc:194101 phb dnajb1b zgc:162730 grhl2b zgc:198329 sla2 nduf10 sla2 arl1 ubl3b mphosph10 zgc:162730 syk her9 ccl38a.5 vat1 tp53inp1 hsp70.2 mt2 arl5c hsp70.2 si:dkeyp-85e10.3 dnaja2 hist2h2l nduf10 si:dkey-188i13.10 gucy1a3 lta 0 cabz01093075.1 dctn2 isg15 il12bb CABZ01013771.1 0 hsp70l tars bcl2l10 mt2 gucy1a3 spock3 si:dkey-188i13.10 si:dkeyp-85e10.3 si:ch211-24o10.6 isg15 0 2 4 0 2 4 PBS PBS

Supp. Figure 7. PBS - V. anguillarum - A. simplex-injected rag1-/- mutant fish aligned data sets. A. tSNE plots representing distribution of aligned clusters in different dimensions. B. Regression plots showing top 20 DE genes between: V. anguillarum and PBS-injected fish (left) and A. simplex and PBS- injected fish (right) in nitr+rorc+ cells (Cluster 7) and nitr-rorc+ (Cluster 13).