www.sciencemag.org/content/352/6284/459/suppl/DC1
Supplementary Materials for
Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes
Laura K. Mackay,* Martina Minnich, Natasja A. M. Kragten, Yang Liao, Benjamin Nota,
Cyril Seillet, Ali Zaid, Kevin Man, Simon Preston, David Freestone, Asolina Braun, Erica Wynne-Jones, Felix M. Behr, Regina Stark, Daniel G. Pellicci, Dale I. Godfrey, Gabrielle T. Belz, Marc Pellegrini, Thomas Gebhardt, Meinrad Busslinger, Wei Shi,
Francis R. Carbone, Rene A. W. van Lier, Axel Kallies,* Klaas P. J. M. van Gisbergen*
*Corresponding author. E-mail: [email protected] (L.K.M.); [email protected] (A.K.); [email protected] (K.P.J.M.v.G.)
Published 22 April 2016, Science 352, 459 (2016) DOI: 10.1126/science.aad2035
This PDF file includes
Materials and Methods Figs. S1 to S17 References
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Materials and Methods:
Mice
Zfp683-‐/-‐ (Hobit KO; (2)), Prdm1 GFP/+ (Blimp1-‐GFP; (31)), Prdm1flox/flox x Lck Cre (Blimp1 KO;
(3)), Prdm1-‐Bio/Bio x Rosa26-‐BirA/BirA (Blimp1-‐Bio; (32)), CAGs-‐rtTA3 mice (33), Tbx21-‐/-‐ (T-‐bet
KO; (34)) and Il15-‐/-‐ (Il15 KO; (35)) mice were maintained on a C57Bl/6 background. Blimp1
KO were crossed onto Hobit KO mice to generate Blimp1 × Hobit DKO mice. gBT-‐I mice are
CD8+ TCR transgenic mice that recognize the H-‐2Kb-‐restricted HSV-‐1 gB epitope of amino
acids 498-‐505 (gB498-‐505). For the generation of mixed bone marrow (BM) chimeric mice,
B6.SJL-‐PtprcaPep3b/BoyJ (Ly5.1) or B6.SJL-‐PtprcaPep3b/BoyJ × C57Bl/6 (Ly5.1 × Ly5.2) were
used as recipients. Following irradiation (2 × 550 Rads), recipient mice were reconstituted by
intravenous transfer of 2 × 107 BM cells. Recipients were used in experiments 8-‐12 weeks
after reconstitution. Chimerism was analyzed in the blood prior to experiments using flow
cytometry using the congenic markers Ly5.1 and Ly5.2 to establish the relative size of host
and donor compartments. The ratio between donor compartments within the blood prior to
infection was used to calibrate the ratio of the indicated lymphocyte populations within
experiments. Mice were maintained under SPF conditions and animal experiments were
performed according to national and institutional guidelines.
Viral infections
Mice were infected with 1 × 106 plaque forming units (PFU) of HSV type 1 by epicutaneous
application after scarification, as described (28) or with 30 PFU of LCMV (strain WE) by
intravenous injection. Infected mice were sacrificed at the indicated time points after
infection and organs were harvested for analysis by flow cytometry.
Cell preparation
Single cell preparations of thymus, spleen, kidney and liver were obtained by passing organs
over cell strainers (70 µM, BD Biosciences). Kidney and liver lymphocytes were isolated after
resuspension of cell preparations in 44% Percoll (GE Healthcare) and density gradient
centrifugation. Contaminating erythrocytes were removed using red blood cell lysis buffer
(155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA). Skin tissue (1.5 × 1 cm2) was removed from
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the flank following hair removal using Veet depilation cream (Reckitt Bencksier). To separate
epidermis from dermis, skin pieces were digested in dispase (2.5 mg/ml) at 37°C for 1.5 h.
To obtain single cell suspensions, the epidermis was digested in trypsin (0.25%) and EDTA
(0.1%) at 37°C for 30 min and dermis in collagenase type III (3 mg/ml) and DNAse I (5 ug/ml)
at 37°C for 30 min. Single cell suspensions of epidermis and dermis were pooled and
sequentially passed over 70 µm and 30 µm nylon mesh. Intraepithelial lymphocytes (IELs)
and lamina propria lymphocytes (LPLs) were isolated from small intestine. After removal of
associated fat tissue, Peyers patches and fecal content, 1 cm2 pieces of small intestine were
incubated at 37°C for 30 min in Ca2+ and Mg2+ Free Hanks buffer containing 5 mM EDTA and
1mM DTT. IELs were isolated from the released epithelial fraction after resuspension in 44%
Percoll, layering on top of 70% Percoll and density gradient centrifugation. LPLs were
obtained after digestion of the remaining gut tissue in 1 mg/ml collagenase type III, 0.4 U/ml
dispase and 100 µg/mL DNase at 37°C for 45 minutes.
Antibodies and Flow cytometry
Single cell suspensions were labeled with the indicated fluorescently conjugated antibodies
at 4°C for 30 min in PBS containing 0.5% FCS. Tetramer labeling was performed at room
temperature. Antibody and tetramer binding was analyzed on Canto or Fortessa (BD
Biosciences) flow cytometers. The following antibodies were purchased from eBioscience,
BD Biosciences or BioLegend: CD3 (145-‐2C11), CD4 (GK1.5), CD8 (53-‐6.7), CD24 (M1/69),
CD49a (Ha31/8), CD49b (DX5), CD62L (MEL-‐14), CD69 (H1.2F3), CD103 (2E7), CD122 (TM-‐
BETA1), CD127 (A7R34), CCR7 (4B12), CXCR6 (221002), Eomes (DAN11MAG), Granzyme B
(GB11), IFN-‐γ (XMG1.2), KLRG1 (2F1), Ly5.1 (A20), Ly5.2 (104), NK1.1 (PK136), NKp46
(29A1.4), T-‐bet (4B10), TCRβ (H57-‐597), TNF-‐α (MP6-‐XT22), and TRAIL (N2B2). PBS57-‐loaded
and vehicle-‐loaded CD1d tetramers were obtained from the tetramer core facility of the
NIH; gB-‐loaded tetramers were generated in the Dept of Microbiology and Immunology,
University of Melbourne, and gp33-‐ and np396-‐loaded tetramers were obtained from BD
Biosciences.
CD8 T cell cultures
CD8 T cells were isolated from spleen and lymph nodes using CD8 microbeads (Miltenyi) and
magnetic sorting on LS columns (Miltenyi). Isolated CD8 T cells were stimulated with plate-‐
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bound anti-‐CD3 antibodies (5 µg/ml) in the presence of anti-‐CD28 (2 µg/ml) and IL-‐2 (100
U/ml). CD8 T cells were expanded further in medium containing only IL-‐15 (20 ng/ml), IL-‐2
(100 U/ml) or IL-‐2 and IL-‐12 (5 ng/ml), as indicated. For analysis of Hobit and Blimp1
expression in CD8 T cells, anti-‐CD3 and anti-‐CD28 stimulated T cells were cultured for 7 days
in the presence of IL-‐2, and then cultured in medium containing the following cytokines for
a further 24 hr: IL-‐15/IL-‐15-‐antibody complexes (50 ng/ml IL-‐15 and 250 ng/ml IL-‐15Rα) or
TGFβ (5 ng/ml). For retroviral transduction experiments, activated CD8 T cells were
transduced with retroviruses overexpressing T-‐bet or Eomes (or control empty retrovirus),
as previously described (17).
NKT cell cultures
Single cell preparations of thymus were enriched for NKT cells using magnetic depletion of
CD8+ and CD24+ thymocytes with anti-‐CD8 and anti-‐CD24 antibodies and goat anti-‐rat
beads (Qiagen). NKT cells were isolated using cell sorting with anti-‐TCRβ antibodies and
PBS57-‐loaded CD1d tetramers. Isolated NKT cells were stimulated with plate-‐bound anti-‐
CD3 antibodies (5 µg/ml) in medium containing anti-‐CD28 (2 µg/ml) and IL-‐2 (100 U/ml).
Expanded NKT cells were re-‐plated after 3 days in medium containing IL-‐15 (20 ng/ml) and
cultured for an additional 7 days.
T cell transfers
For experiments using transgenic gBT-‐I cells, adoptive transfers of naïve gBT-‐I cells were
carried out intravenously with lymph node suspensions (5 × 104 cells) prior to HSV infection.
To study the effect of IL-‐15, WT and Il15 KO mice were infected with HSV and 4 days later
received effector gBT-‐I cells (5-‐12 × 104 cells) enriched from the spleens of WT mice 6 days
after HSV infection. For experiments where CD8 T cells were transferred by intradermal
injection, in vitro expanded CD8 T cells (1 x 106 cells) were injected into the skin (five 20-‐μl
injections over an area of skin 1 × 1.5 cm2) using a 30-‐gauge needle, as described (9). For
adoptive transfer experiments with NKT cells, in vitro expanded NKT cells (1 x 106 cells) were
transferred by intravenous injection. Recipient mice were sacrificed at the indicated time
points.
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Quantitative PCR and Taqman
To perform qPCR analysis, RNA was isolated using RNeasy Mini Kit according to the
manufacturer’s instructions (Qiagen). RNA was synthesized into cDNA using iScript cDNA
Synthesis Kit (BioRad). qPCR was run on a C1000 Thermal Cycler (BioRad) using Sensimix
SYBR No ROX Kit (Bioline). The following primer pairs were used: Hobit (forward: 5’-‐
CTCAGCCACTTGCAGACTCA-‐3’, reverse: 5’-‐CTGTCGGTGGAGGCTTTGTA-‐3’), Blimp1 (forward:
5’-‐TTCTCTTGGAAAAACGTGTGGG-‐3’, reverse: 5’-‐GGAGCCGGAGCTAGACTTG-‐3’), S1pr1
(forward: 5’-‐GTGTAGACCCAGAGTCCTGCG-‐3’, reverse: 5’-‐AGCTTTTCCTTGGCTGGAGAG-‐3’),
Klf2 (forward: 5’-‐CTCAGCGAGCCTATCTTGCC-‐3’, reverse: 5’-‐CACGTTGTTTAGGTCCTCATCC-‐3’),
Ccr7 (forward: 5’-‐GGGTTCCTAGTGCCTATGCTGGCTATG-‐3’, reverse: 5’-‐
GGCAATGTTGAGCTGCTTGCTGGTT-‐3’), and Hprt (forward: 5’-‐GGGGGCTATAAGTTCTTTGC-‐3’,
reverse: 5’-‐TCCAACACTTCGAGAGGTCC-‐3’). Expression was normalized using Hprt and the
expression was quantified relative to expression in total or naïve WT CD8 T cells set to 1.
The expression of Hobit and Blimp1 in gBT-‐I cells after HSV infection was analysed using
Taqman assay. RNA extraction, cDNA synthesis, preamplification and quantitative RT-‐PCR
were performed using Taqman Gene Expression Cells-‐To-‐Ct Kit, Taqman Fast Preamp master
mix and assorted commercially available Taqman assays (Mm00437762_m1,
Mm00446973_m1, Mm00446968_m1, Mm00476128_m1) with Taqman Fast Advanced
Mastermix on a StepOnePlus Real-‐Time PCR cycler (Life Technologies). To perform Taqman
on Hobit the following probe was used in combination with the qPCR primers of Hobit: 6-‐
FAM-‐5’-‐TCATGACTTAGCCTGGAGCGAGAGGATGT-‐3’-‐TAMRA. The threshold cycle of
respective genes for each cell population was normalized to the arithmetic mean of Hprt,
B2m and Tbp housekeeping genes (DCt). Normalized gene expression of each cell type was
compared to the gene expression of naïve gBT-‐I cells (set to 1) according to the 2(-‐DDCT)
method.
RNA-‐sequencing
LCMV-‐specific CD8 T cell populations were isolated by flow cytometric sorting from LCMV-‐
infected mice at day 40+ post-‐infection using gp33 and np396 tetramers. Following HSV
infection (day 40+) gBT-‐I cells were isolated by sorting on Vα2+CD45.1+ cells. Tcm
(CD62L+CD69-‐) and Tem (CD62L-‐CD69-‐) populations were obtained from the spleen. LCMV-‐
specific Tem were also obtained from the liver. CD103+ Trm populations (CD69+CD103+)
6
were obtained from skin after HSV infection and the intraepithelial fraction of the small
intestine after LCMV infection. LCMV-‐specific CD103-‐ Trm (CD62L-‐CD69+) populations were
isolated from liver. Splenic naïve CD8 T cells (CD8+CD44-‐CD62L+) and liver-‐derived NKT cells
(CD3+CD1d-‐PBS57 tetramer+), trNK cells (CD3-‐NK1.1+NKp46+CD49a+CD49b-‐) and cNK cells
(CD3-‐NK1.1+NKp46+CD49a-‐CD49b+) were isolated from WT mice under homeostatic
conditions. To establish the role of Hobit and Blimp-‐1, NKT cells (TCRb+CD1d-‐PBS57
tetramer+) were sorted to purity from NKT cell cultures, established as described above.
RNA purification was performed following the manufacturer's protocol using the RNAeasy
Plus Mini Kit (Qiagen). RNA samples were sequenced on an Illumina HiSeq analyzer,
producing between 13 and 33 million 100-‐bp single-‐end reads per sample. Two or more
biological replicates were generated and sequenced for each sample. Sequence reads were
aligned to the GRCm38/mm10 build of the Mus musculus genome using the Subread aligner
(36). Only uniquely mapped reads were retained. Genewise counts were obtained using
featureCounts (37). Reads overlapping exons in annotation build 38.1 of NCBI RefSeq
database were included. Genes were filtered from downstream analysis if they failed to
achieve a CPM (counts per million mapped reads) value of at least 1 in at least two libraries.
Counts were converted to log2 counts per million, quantile normalized and precision
weighted with the ‘voom’ function of the limma package (38, 39). A linear model was fitted
to each gene, and empirical Bayes moderated t-‐statistics were used to assess differences in
expression (40). Genes were called differentially expressed if they achieved a false discovery
rate (FDR) of 0.05 or less and had an expression change of >1.5 fold or >2 fold, as indicated.
The called differentially expressed genes must also have at least 4 or 8 RPKMs (reads per
kilobase of exon length per million mapped reads) in one or both of the two cell types being
compared, as indicated. The gene set enrichment plots were generated with the
‘barcodeplot’ function in limma. Gene set enrichment analysis was carried out using the
‘roast’ method in limma with 999 rotations (41). One-‐sided P values are reported.
Hobit ChIP
For retroviral transduction experiments, we cloned full-‐length mouse Hobit cDNA with a N-‐
terminal V5-‐tag into a pSIN retroviral vector (pSIN-‐TRE3G-‐V5-‐mHobit-‐P2A-‐GFP). Tet-‐
inducible V5-‐tagged Hobit expression was driven by the TRE3G promoter and followed by
P2A-‐GFP. The retrovirus was produced using retroviral helper plasmid (pCMV-‐Gag-‐Pol, Cell
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Biolabs) into Platinum-‐E (Cell Biolabs) packaging cells using calcium phosphate transfection
in the presence of chloroquine (25 µM). Viral supernatant was collected at 24-‐36 hr after
transfection and passed through a 0.45-‐µm filter. Tet-‐on competent CD8 T cells were
isolated from the spleen and lymph nodes of CAGs-‐rtTA3 mice and stimulated with anti-‐
CD3, anti-‐CD28 and IL-‐2 (100 U/ml) for 3 days. To establish transduction, the activated CD8
T cells were centrifuged 3 times at 8 hr-‐intervals in the presence of viral supernatant and
polybrene (4 µg/ml) at 2,000 rpm for 30 min. The transduced CD8 T cells were further
stimulated with IL-‐2 and IL-‐12 (5 ng/ml) for another 2 days together with doxycycline (1
µg/ml) to induce Hobit expression. Transduced cells were sorted based on GFP expression
with a FACS Aria machine (Becton Dickinson) and subjected to chromatin precipitation with
anti-‐V5 agarose (clone V5-‐10, Sigma). Briefly, 70 x 107 cells were fixed with 1%
formaldehyde for 10 min followed by quenching with 0.125 M glycine for 5 min. The cells
were lysed in 0.25% SDS buffer for 1 hr. The released chromatin was sheared to an average
size of 500 bp using a BiorupterTM
sonicator (Diagenode). After removal of cell debris, the
chromatin (400 µg) was diluted in buffer containing 1% Triton and pre-‐cleared using Protein
G Plus agarose (Santa Cruz Biotechnology). The pre-‐cleared chromatin was incubated
overnight at 4°C with anti-‐V5 agarose. After washing, the Hobit-‐V5 binding protein-‐DNA
complexes were eluted using buffer containing 1% SDS and 100 mM NaHCO3. Reverse
cross-‐linking was performed on the eluates with buffer containing 10 mM Tris-‐HCl (pH 8.0),
1 mM EDTA and 200 mM NaCl in the presence of proteinase K (500 g/ml). Genomic DNA
was isolated from the precipitated material by phenol extraction and ethanol precipitation.
The precipitated genomic DNA was quantified by real-‐time PCR.
Blimp1 ChIP
Splenic CD8 T cells from Blimp1-‐Bio mice were stimulated first with anti-‐CD3, anti-‐CD28 and
IL-‐2 (100 U/ml) for 3 days and then with IL-‐2 and IL-‐12 (5 ng/ml) for another 2 days. Blimp1-‐
Bio mice carry a biotin acceptor sequence at the carboxyl terminus of Blimp1, which was
biotinylated in vivo by coexpression of the Escherichia coli biotin ligase BirA from the
Rosa26BirA allele. Chromatin from ~3 x 108 CD8 T cells was prepared using a lysis buffer
containing 0.25% SDS prior to chromatin precipitation by streptavidin pulldown (Bio-‐ChIP),
as described (42). The precipitated genomic DNA was quantified by real-‐time PCR.
8
ChIP-‐sequencing
Approximately 1-‐5 ng of ChIP-‐precipitated DNA was used as starting material for the
generation of sequencing libraries with the NEBNext Ultra Ligation Module and NEBNext
End Repair/dA-‐Tailing module. DNA fragments of 200–500 bp were selected with AMPure
XP beads (Beckman Coulter). PCR amplification was performed with the KAPA Real Time
Amplification kit (KAPA Biosystems). Completed libraries were quantified with the Agilent
Bioanalyzer dsDNA 1000 assay kit and Agilent QPCR NGS Library Quantification kit. Cluster
generation and sequencing was carried out by using the Illumina HiSeq 2000 system with a
read length of 50 nucleotides according to the manufacturer’s guidelines. Sequence reads
that passed the Illumina quality filtering were considered for alignment. Reads were aligned
to the mouse genome assembly version of July 2007 (NCBI37/mm9) using bowtie version
1.0.0. For peak calling of ChIP-‐seq data we used the MACS program version 1.3.6.1 with
default parameters, a genome size of 2,654,911,517 bp (mm9) and a mixture of pro-‐B and
mature B cell input control sample. Peaks were filtered for P values of < 10-‐7.5. Peaks were
assigned to target genes, as described (43).
Peak-‐overlap and motif analysis
The Multovl program (44) was used for the peak overlap analysis. The minimal overlap
length was set to one. Motif analyses were performed with the Gimme-‐Motifs program (45)
with default settings for de novo Blimp1 and Hobit motif prediction from the top 250 peaks
of each data set.
Migration assays
Migration assays were performed with cultured thymic NKT cells using 24-‐well plates with
transwell inserts (5 µm pores, Costar). The chemo-‐attractants CCL21 (R & D Systems) or S1P
(Sigma) were added to the lower chamber. NKT cells were allowed to migrate for 3 h at
37°C. Migrated NKT cells were enumerated using counting beads on a flow cytometer. NKT
cell migration was calculated as the ratio of migrated cells relative to control conditions
without chemo-‐attractants.
Immunofluorescence staining and confocal microscopy
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Spleens were cut in small pieces and incubated in 40 µg/mL CD1d-‐PBS57 or vehicle-‐loaded
PE-‐labelled tetramer in PBS 2% FCS for 18 h at 4°C. Spleens were washed in PBS and fixed in
4% PFA for 1 h on ice, then washed in PBS, dehydrated in 30% sucrose and frozen in OCT
(Sakura, Finetek). Spleen sections were cut on a cryostat at 10 µm, and stained using B220-‐
Pacific Blue and CD3-‐eFluor660 (eBiosciences). PE-‐labelled tetramer was detected using a
polyclonal rabbit-‐anti-‐PE antibody (Novus Biologicals), followed by an anti-‐rabbit Alexa Fluor
555 antibody (Life Technologies). Sections were mounted using Prolong Gold Antifade (Life
Technologies) and imaged on a Zeiss LSM710 confocal microscope. Image files were
processed using Imaris 7.5 (Bitplane).
Statistical analysis
Values are expressed as mean ± S.E.M. Differences between two groups were assessed by
Student’s t test and differences between more than two groups were assessed using one-‐
way ANOVA followed by a Bonferroni post-‐hoc test. After log2 transformation ratios were
compared to 0 by one-‐sample Student’s t test. A p-‐value of less than 0.05 was considered
statistically significant. * denotes P < 0.05, ** denotes P < 0.01 and *** denotes P < 0.001.
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Supplementary Figure Legends:
Figure S1: Hobit and Blimp1 are co-‐expressed in LCMV-‐specific Trm. (A) Hobit and (B)
Blimp1 expression was determined by qPCR in LCMV-‐specific central memory (Tcm),
effector memory (Tem) and tissue-‐resident memory CD8 T cells (Trm) from the
intraepithelial lymphocyte fraction of the small intestine (SI IEL) at day 40+ after LCMV
infection. Data in (A,B) represent pooled results of 3 mice per group. Bars denote mean ±
S.E.M.
Figure S2: Hobit and Blimp1 are required for development of CD8 T cells with a Trm
phenotype in skin. Activated congenically marked CD8 T cells from WT mice (Ly5.1/2+) were
intradermally injected at a 1:1 ratio together with CD8 T cells from Hobit KO, Blimp1 KO or
Blimp1 x Hobit DKO mice (Ly5.2+) into recipient mice (Ly5.1+). (A) Representative dot plots
display WT and Blimp1 x Hobit DKO donor CD8 T cell populations as detected by Ly5.1 and
Ly5.2 expression in the spleen (top row) and skin (bottom row) at the indicated time-‐points
after transfer. (B) Bar graph shows the ratio (log2) of transferred WT and Blimp1 x Hobit
DKO CD8 T cells in spleen, draining lymph node and skin at day 6 after transfer. (C)
Representative histograms display CD69 (left) and CD103 (right) expression on donor WT
(black line) and Hobit KO or Blimp1 KO (grey filled) CD8 T cells 14-‐15 days post intradermal
injection. Data in (A-‐C) are representative of 5-‐8 mice per group. *** P < 0.001 as
determined by one-‐way ANOVA (B). Bars denote mean ± S.E.M.
Figure S3: Hobit and Blimp1 regulate effector CD8 T cell differentiation during HSV
infection. (A,B) Mixed BM chimeric mice containing WT (Ly5.1+) hematopoietic cells
together with WT control or Blimp1 x Hobit DKO (Ly5.2+) cells were analyzed for virus-‐
specific effector CD8 T cells in spleen at day 10-‐11 after infection with HSV. (A) Plots display
expression of KLRG1 and CD127 on gB tetramer+ CD8 T cells in the WT and Blimp1 x Hobit
DKO compartments of mixed BM chimeric mice. (B) Bar graph displays the percentage of
KLRG1+CD127-‐ short-‐lived effector cells (SLEC) and KLRG1-‐CD127+ memory precursor
effector cells (MPEC) within the gB tetramer+ CD8 T cell population. Data in (A,B) are
11
representative of two independent experiments with 3-‐4 mice per group. * P < 0.05 and ***
P < 0.001 as determined by two-‐tailed Student’s t test (B). Bars denote mean ± S.E.M.
Figure S4: Blimp1, but not Hobit, regulates effector CD8 T cell differentiation during LCMV
infection. Mixed BM chimeric mice containing WT (Ly5.1+) hematopoietic cells together
with WT control, Hobit KO, Blimp1 KO or Blimp1 x Hobit DKO (Ly5.2+) cells were analyzed for
virus-‐specific effector CD8 T cells in spleen at day 8-‐11 after infection with LCMV. (A) Plots
display KLRG1 and CD127 expression on gp33 tetramer+ CD8 T cells of the indicated
genotypes. (B) Bar graph displays the ratio (log2) between WT (Ly5.1+) and WT or mutant
(Ly5.2+) compartments within SLEC and MPEC gp33 tetramer+ CD8 T cells. (C)
Representative plots display the expression of granzyme B within WT (Ly5.2-‐) and the
indicated WT or mutant (Ly5.2+) compartments of virus-‐specific CD8 T cells. (D) Expression
of intracellular IFN-‐γ and TNF-‐α was determined in CD8 T cells of the indicated genotype
following brief restimulation with gp33 peptide. (E-‐G) The percentage of CD8 T cells of the
indicated genotype that displayed expression of (E) granzyme B, (F) IFN-‐γ, and (G) TNF-‐α was
quantified. Data in (A,B) are the pooled results of two independent experiments with 3-‐7
mice per group. Data in (C-‐G) are representative of two independent experiments with 3 to
4 mice per group. ** P < 0.01 and *** P < 0.001 as determined by one-‐sample Student’s t
test (B) or by one-‐way ANOVA (E). Bars denote mean ± S.E.M.
Figure S5: Hobit and Blimp-‐1 regulate the maintenance of gut-‐resident memory T cells
after LCMV infection. Mixed BM chimeric mice with WT (Ly5.1+) and WT control, Hobit KO,
Blimp1 KO, or Blimp1 x Hobit DKO (Ly5.2+) compartments were infected with LCMV to
analyze the LCMV-‐specific CD8 T cell response within spleen and gut. (A) Representative
flow cytometry plots display Ly5.1 and Ly5.2 expression of gp33 tetramer+ CD8 T cells from
spleen, and intraepithelial and lamina propria fractions of the small intestine (SI IELs and SI
LPLs, respectively) in mixed BM chimeric mice containing a WT (Ly5.1+) and Blimp1 x Hobit
DKO (Ly5.2+) compartment. (B) Bar graphs display the ratio (log2) of gp33+ CD8 T cells from
Ly5.1+ WT and Ly5.2+ WT control or mutant compartments in the indicated tissues at day
10 (top row) and day 50+ (bottom row) after LCMV infection. Data are representative of two
independent experiments with 3-‐4 mice per group. * P < 0.05, ** P < 0.01 and *** P < 0.001
as determined by one-‐sample Student’s t test (B). Bars denote mean ± S.E.M.
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Figure S6: Hobit maintains tissue-‐resident NK cells in the liver. (A) Expression of Blimp1
was analyzed on tissue-‐resident NK cells (trNK) and conventional NK cells (cNK) in liver using
GFP expression in heterozygous Blimp1-‐GFP reporter mice (black line) and WT controls
(filled grey). (B) Representative plots display expression of CD49a and Eomes, CD69 and
CD62L, TRAIL and CD49b, and CXCR6 and CD49b of total liver NK cells of WT (left panels) and
Hobit KO mice (right panels). Data are representative of (A) three or (B) two independent
experiments.
Figure S7: Hobit and Blimp1 regulate the Trm-‐phenotype of NKT cells. (A) Dot plots display
expression of CD49a and CD69 on splenic NKT cells of the indicated genotypes. (B) The
number of CD69-‐ and CD69+ NKT cells was determined in spleens of WT, Hobit KO, Blimp1
KO and Blimp1 x Hobit DKO mice. (C) Expression of Blimp1 was analyzed on NKT cells in
thymus, spleen and liver using GFP expression in heterozygous Blimp1-‐GFP reporter mice
(black line) and WT controls (filled grey). Data in (A,B) are representative of three
independent experiments with 6-‐11 mice per group. Data in (C) are representative of at
least three independent experiments. * P < 0.05 and *** P < 0.001 as determined by one-‐
way ANOVA (B). Bars denote mean ± S.E.M.
Figure S8: The maintenance of NKT cells in the liver but not the spleen depends on Hobit
and Blimp1. (A) Representative flow cytometry plots display the binding of anti-‐TCRβ
antibodies and PBS57-‐loaded CD1d tetramers to total splenocytes (top panel) and total liver
lymphocytes (bottom panel) from WT, Hobit KO, Blimp1 KO and Blimp1 x Hobit DKO mice.
(B) Plots display the binding of anti-‐Ly5.1 antibodies and PBS57-‐loaded CD1d tetramers to
total donor splenocytes (top panel) and total donor liver lymphocytes (bottom panel) in
mixed BM chimeric mice containing control WT (Ly5.1+) and WT, Hobit KO, Blimp1 KO or
Blimp1 x Hobit DKO compartments (Ly5.2+). Results represent pooled data of three (A) or
four (B) different experiments with 6-‐11 mice per group.
Figure S9: Hobit and Blimp1 co-‐regulate maintenance of liver but not splenic NKT cells. (A)
Congenically marked NKT cells of WT or Blimp1 x Hobit DKO mice (Ly5.2+) were co-‐
transferred together with competitor WT NKT cells (Ly5.1+) into WT recipients (Ly5.1/2+) in
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a 1:1 ratio. Representative dot plots display WT and Blimp1 x Hobit DKO donor NKT cell
populations as detected by Ly5.1 and Ly5.2 expression in the liver at indicated time points
after transfer. (B) Bar graph displays the ratio (log2) between Ly5.1+ and Ly5.2+ donor NKT
cells. (C) The ratio (log2) between WT (Ly5.1+) and WT, Hobit KO, Blimp1 KO and Blimp1 x
Hobit DKO mice (Ly5.2+) donor NKT cells was determined in spleen (left) and liver (right) at
day 15+ post transfer. (D) Histograms display CTV dilution of donor NKT cells of WT and
Blimp1 x Hobit DKO mice. (E) The division index of donor NKT cells of WT and Blimp1 x Hobit
DKO mice was calculated. Results are representative of two independent experiments with
3 mice per group. * P < 0.05, ** P < 0.01 and *** P < 0.001 as determined by two-‐tailed
Student’s t test (B) or by one-‐sample Student’s t test (C). Bars denote mean ± S.E.M.
Figure S10: LCMV-‐specific CD69+ CD8 T cells in liver and kidney display a Trm phenotype
and require Hobit and Blimp1. Antigen-‐specific CD8 T cells were analyzed in WT mice using
gp33 tetramer at day 40+ after infection. (A) The percentage of CD62L-‐CD69+ Trm within
the LCMV-‐specific CD8 T cell population was quantified in blood and liver. (B-‐D) Expression
of (B) S1pr1, (C) Klf2 and (D) Ccr7 as determined by qPCR in the indicated populations of
LCMV-‐specific CD8 T cells in spleen and liver. (E,F) Expression of (E) Hobit and (F) Blimp1 as
determined in the indicated populations of naïve CD8 T cells and LCMV-‐specific gp33+ CD8 T
cells using qPCR. Short-‐lived effector cells (KLRG1+CD127-‐, SLEC) and memory-‐precursor
effector cells (KLRG1-‐CD127+, MPEC) were analyzed at day 8, and Tcm (CD62L+CD69-‐), Tem
(CD62L-‐CD69-‐), and Trm (CD62L-‐CD69+) were analyzed at day 30+ after infection with
LCMV. (G,H) LCMV-‐specific CD8 T cells were analyzed within the kidney of mixed BM
chimeric mice containing control WT (Ly5.1+) and WT, Hobit KO, Blimp1 KO, or Blimp1 x
Hobit DKO (Ly5.2+) compartments at day 50+ following infection with LCMV. (G) Expression
of CD69 and CD103 was determined on gp33 tetramer+ CD8 T cells of the indicated
genotype. (H) Bar graph displays the ratio of CD69+ LCMV-‐specific CD8 T cells from WT
(Ly5.1+) and WT or mutant (Ly5.2+) compartments within kidney. Results represent 3-‐6
samples of two independent experiments. * P < 0.05 and ** P < 0.01 as determined by two-‐
tailed Student’s t test (A) or by one-‐way ANOVA (H). Bars denote mean ± S.E.M.
Figure S11: Hobit expression is regulated by IL-‐15 and T-‐bet. (A) Hobit (left) and Blimp1
(right) expression in adoptively transferred WT gBT-‐I cells sorted from skin of WT or Il15 KO
14
mice at day 14 after HSV infection, as determined by Taqman qPCR. (B) Activated WT and T-‐
bet KO CD8 T cells were incubated with IL-‐15/IL-‐15-‐antibody complexes, TGFβ or without
stimuli (control) for 24 hr. Hobit expression in the cultured CD8 T cells was examined using
qPCR. (C) Similarly, Hobit expression was determined in activated CD8 T cells transduced
with overexpression vectors for T-‐bet and Eomes or with empty vector (control). Results in
(A-‐C) are presented as mean values relative to expression in WT CD8 T cells. Data in (A)
represents 10 mice per group and data in (B,C) is representative of 2-‐4 independent
experiments. Bars denote mean ± S.E.M.
Figure S12: NKT1 in contrast to NKT2 display characteristics of tissue-‐resident
lymphocytes. (A) Thymic NKT cells of WT Balb/c mice were identified through binding of
CD1d tetramers and anti-‐CD3 antibodies. Surface expression of CD122 was used to separate
the NKT1 and NKT2 populations. (A) Representative histograms show expression of T-‐bet
(left) and CD69 (right). (B) The geometric mean fluorescence intensity (geo MFI) of CD69
expression was quantified. (C) Thymic NKT1 and NKT2 cells were sorted from Balb/c mice
using CD1d tetramers, anti-‐CD3 antibodies and anti-‐CD122 antibodies. Expression of Hobit,
Blimp1, S1pr1 and Ccr7 was determined using qPCR. Data in (A-‐C) are representative of two
independent experiments with 3 to 4 mice per group. * P < 0.05 and *** P < 0.001 as
determined by two-‐tailed Student’s t test (B,C). Bars denote mean ± S.E.M.
Figure S13: Liver Trm are distinct from splenic and liver Tem and share characteristics with
epithelial Trm. HSV-‐specific and LCMV-‐specific Tcm, Tem and Trm populations of spleen,
liver, skin and the intraepithelial fraction of the small intestine (SI IEL) were isolated and
analyzed using RNA-‐sequencing. (A) Heatmap displays the expression profile of genes
associated with HSV-‐specific skin Trm and LCMV-‐specific gut Trm in the indicated
populations of virus-‐specific CD8 T cells. Relative expression levels (Z-‐scores) of genes are
shown, color-‐coded according to the legend. Rows are scaled to have a mean of 0 and an
s.d. of 1. (B) Differentially expressed (DE) genes (FDR < 0.05, fold change >2, and RPKM >8)
in pairwise comparisons of LCMV-‐specific Tem from spleen and liver, and liver Trm were
enumerated. Number of upregulated (black bars) and downregulated genes (white bars) are
displayed separately. (C) DE genes were determined between HSV-‐specific Trm (skin) or
LCMV-‐specific Trm (liver and gut) and corresponding virus-‐specific Tcm and Tem subsets
15
from spleen and liver in pairwise comparisons. Numbers in overlapping parts of the Venn
diagram indicate gene transcripts that overlap between Trm populations and numbers in
unique parts of the Venn diagram display tissue-‐specific gene transcripts. (D) Enrichment of
the set of 192 genes associated with epithelial Trm was analyzed in LCMV-‐specific liver Trm
compared to liver Tem. The horizontal axis shows empirical Bayes moderated t-‐statistics for
all genes comparing liver Trm to liver Tem and the vertical bars show the ranks of the up-‐
and downregulated epithelial Trm signature genes. Up-‐ and downregulated genes are
shown in red and blue, respectively. Red and blue worm lines show enrichment of up-‐ and
down-‐regulated signature genes relative to random ordering. Roast P values for enrichment
of up-‐ and downregulated signature genes are < 0.001. RNA-‐sequencing data are pooled
from two independent experiments.
Figure S14: The gene signature of epithelial Trm is enriched in innate tissue-‐resident
lymphocytes. (A) The 192 epithelial Trm signature genes associated with virus-‐specific Trm
from skin and gut after HSV and LCMV infection, respectively, were overlaid with
differentially regulated gene transcripts in pairwise comparative analysis of liver-‐derived
cNK with trNK and of liver-‐derived Tem with NKT cells. Numbers in overlapping parts of the
Venn diagram indicate gene transcripts that overlap between adaptive and innate tissue-‐
resident populations and numbers in unique parts of the Venn diagram display specific gene
transcripts of Trm, trNK and NKT cells. (B,C) Gene set enrichment tests show that epithelial
Trm signature genes are significantly enriched in (B) trNK compared to cNK cells and in (C)
NKT cells compared to liver-‐derived Tem. Universally upregulated and downregulated genes
in epithelial Trm are shown in red and blue vertical bars, respectively. Roast P values for
enrichment of up-‐ and downregulated genes in (B,C) are < 0.001. RNA-‐sequencing data are
pooled from two independent experiments.
Figure S15: Cultured NKT cells maintain expression of the tissue-‐residency-‐associated
molecule CD69 in a Hobit and Blimp1-‐dependent manner. Thymic NKT and splenic CD8 T
cells were stimulated with anti-‐CD3, anti-‐CD28 and IL-‐2 for 3 days and further expanded in
IL-‐15 for another 7 days. (A) Expression of Hobit and Blimp1 was analyzed in cultured NKT
cells and CD8 T cells of WT mice using qPCR. (B) Expression of CD69 was analyzed on
cultured NKT cells of WT, Hobit KO, Blimp1 KO or Blimp1 x Hobit DKO mice using flow
16
cytometry. Histograms display expression of CD69 on NKT cells of WT mice (black line)
overlaid with that of the indicated KO mice (filled grey). (C) Gene set enrichment graph
shows that the 30 genes associated with tissue-‐resident lymphocytes are significantly
enriched in WT compared to Blimp1 x Hobit DKO NKT cells. Vertical bars display the ranks of
the tissue-‐residency signature genes. Upregulated and downregulated genes in tissue-‐
resident lymphocytes are shown in red and blue vertical bars, respectively. Worm line shows
enrichment score of upregulated (red lines) and downregulated tissue-‐residency signature
genes (blue lines) relative to random ordering. Roast P values for enrichment of up-‐ and
downregulated genes are < 0.001. Data are representative of three independent
experiments. Bars denote mean ± S.E.M.
Figure S16: Hobit and Blimp1 suppress pathways of tissue-‐egress at the transcriptional
level and promote localization of NKT cells in the red pulp of the spleen. (A-‐C) The
expression of (A) Klf2, (B) S1pr1 and (C) Ccr7 was quantified by qPCR in splenic NKT cells of
WT, Hobit KO, Blimp1 KO and Blimp1 x Hobit DKO mice. (D-‐F) NKT cells in the red pulp but
not in the white pulp of the spleen were labeled by intravenous (i.v.) injection of (D,E) anti-‐
CD44 and (F) anti-‐CD45 antibodies. (D) Histograms display labeling of splenic NKT cells with
i.v. injected anti-‐CD44 within the Ly5.1+ WT compartment (black line) and the indicated
Ly5.2+ WT or mutant compartments (filled grey) in mixed BM chimeric mice. (E) The ratio
between the indicated Ly5.2+ and Ly5.1+ compartments of mixed BM chimeric mice was
quantified for red pulp (i.v. CD44+) and white pulp (i.v. CD44-‐) NKT cells in spleen. (F)
Numbers of splenic red pulp (i.v. CD45+) and white pulp (i.v. CD45-‐) NKT cells were
determined in WT and Blimp1 x Hobit DKO mice. (G) Spleen sections of WT (left panel) and
Blimp1 x Hobit DKO mice (right panel) were stained with anti-‐CD3 to label T cells (white),
with anti-‐B220 to label B cells (blue) and with PBS57-‐loaded tetramers to label NKT cells
(red). Data in (A-‐E) are pooled from two independent experiments with 3-‐6 mice per group.
Data in (F) are representative of two independent experiments with three mice per group.
Data in (G) represent two independent experiments. * P < 0.05, ** P < 0.01 and *** P <
0.001 as determined by one-‐way ANOVA (A-‐C,E) or by two-‐tailed Student’s t test (F). Bars
denote mean ± S.E.M.
17
Figure S17: Hobit and Blimp1 share genome-‐wide binding sites. Venn diagram displays the
overlap between DNA binding sites of Hobit and Blimp1 in CD8 T cells, as identified by ChIP-‐
sequencing. Data is based on one Hobit and Blimp1 ChIP-‐sequencing experiment.
Mackay et al. Figure S2
17.5
31.3
4.8
6.2
1.4
1.8
1.6
9.0
1.1
7.0
1.1
1.0
0 10 3
10 4
10 5
0
10 3
10 4
10 5
Ly5.2
Ly5.
1 Day 4 Day 14 Day 45
Spl
een
Ski
n
WT
Blimp1 x Hobit KO
A
C
B
WT Hobit KO
0 10 3
10 4
10 5
0
20
40
60
80
100 WT Blimp1 KO
CD69 CD103
Cel
ls (%
of m
ax)
Spleen dLN Total
Skin CD103+ Epi
Don
or C
D8
T ce
lls (l
og2
KO
/ W
T)
Blimp1 x Hobit DKO Day 6
-4
-2
0
2
4
*** ***
***
***
Mackay et al. Figure S3
0 10 2 10 3 10 4 10 5
0
10 3
10 4
10 5 50.8 16.8
19.0 13.3
10.3 37.8
45.7 6.1
CD127
KLR
G1
WT Blimp1 x Hobit DKO
SLEC MPEC 0
20
40
60
80
Effe
ctor
cel
ls (%
of g
p33+
cel
ls)
Blimp1 x Hobit DKO WT
*** **
A B
Mackay et al. Figure S4
0 10 2
10 3
10 4
10 5
0 10 2
10 3
10 4
10 5 36.3 14.4
31.8 17.4
39.4 11.3
28.1 21.2
19.7 14.3
50.2 15.8
13.6 7.3
51.2 28.0
CD127
KLR
G1
WT Hobit KO Blimp1 KO Blimp1 x Hobit DKO
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
-4
-2
0
2
4 SLEC MPEC
gp33
+ C
D8
T ce
lls (l
og2
KO
/ W
T)
** ***
** **
A
B
0 10 2
10 3
10 4
10 5
0 10 2
10 3
10 4
10 5
TNF-a
15.3 10.2
68.8 5.7
14.1 8.7
73.0 4.2
14.7 10.0
68.8 6.6
10.8 12.9
73.2 3.0
0 10 2 10 3 10 4 10 5
0 10 2
10 3
10 4
10 5 35.4 23.8
23.9 17.8
6.8 47.7
17.1 28.3
IFN
-g
Ly5.2
gran
zym
e B
D
E F
0
5
10
15
TNF-
a ex
pres
sion
(%)
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit KO
G
0 5
10 15 20
IFN
-g e
xpre
ssio
n (%
)
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit KO
0 20 40 60 80
gran
zym
e B
exp
ress
ion
(%)
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit KO
*** ***
WT 2.7 54.0
24.6 28.3
Hobit KO 26.7 24.3
21.7 27.3
Blimp1 KO Blimp1 x Hobit DKO
WT Hobit KO Blimp1 KO Blimp1 x Hobit DKO
C
Mackay et al. Figure S5
Ly5.2
42.8
37.3
78.2
1.2
75.1
3.4
0 10 3 10 4 10 5 0
10 3 10 4 10 5 60.1
35.6
91.9
4.3
94.8
1.4 Ly5.
1 Spleen SI IELs SI LPLs
Day
10
Day
50+
WT
Blimp1 x Hobit DKO
A
B
-6
-4
-2
0
2
4
6
gp33
+ C
D8
T ce
lls (l
og2
KO
/ W
T)
-6
-4
-2
0
2
4
6
gp33
+ C
D8
T ce
lls (l
og2
KO
/ W
T)
-4
-2
0
2
4
-4
-2
0
2
4
-4
-2
0
2
4
-6
-4
-2
0
2
4
6
gp33
+ C
D8
T ce
lls (l
og2
KO
/ W
T)
Day 10 Spleen
Day 10 SI IELs
Day 10 SI LPLs
Day 50+ Spleen
Day 50+ SI IELs
Day 50+ SI LPLs
* **
*** * * *
* **
*
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
Mackay et al. Figure S6 A
0 10 3 10 4 10 5 0
20
40
60
80
100
Blimp1 GFP
trNK cNK
Cel
ls (%
of m
ax)
63 330
49 616
B
WT Blimp1 GFP
TRAI L
5.0 5.8
81.4 7.9
TRAI L
28.6 3.9
60.9 6.5 TRA
IL
5.2 5.0
81.3 8.6
19.4 7.9
58.0 14.7
CD62L
CD
69
8.8 1.3
73.5 16.4
1.8 2.2
89.0 7.0
0 10 2
10 3
10 4
10 5
0
10 3
10 4
10 5
CD49b
CX
CR
6
49.8
1.3 40.3
8.7 85.0
1.5
9.9
3.6
Eomes
CD
49a
WT Hobit KO
Mackay et al. Figure S7
b
0 10 3 10 4 10 5 0
20
40
60
80
100
Blimp1 GFP
Cel
ls (%
of m
ax)
Thymus Spleen Liver 152 376
113 496
134 455
NKT:
WT Blimp1 GFP
B
A
C
CD49a
WT Hobit KO Blimp1 KO Blimp1 x Hobit DKO C
D69
0 10 3 10 4 10 5
0
10 3
10 4
10 5 40.7 37.7
4.6 17.0
41.2 25.3
7.5 25.9
37.5 43.8
4.3 14.4
35.4 10.7
12.0 41.9
0.0
0.4
0.8
1.2 CD69- NKT cells CD69+ NKT cells *
***
NK
T ce
lls (x
106
cel
ls)
***
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit KO
9.5 26.1 25.9 22.7
1.6 2.0 2.0 1.8
PBS57-loaded CD1d tetramer
WT Hobit KO
0
10 3
10 4
0 10 3
10 4
Blimp1 KO Blimp1 x Hobit DKO
10 5
10 5
Spl
een
Live
r TC
Rb
Mackay et al. Figure S8
A
35.1 1.3
2.2 61.5
30.7 10.4
16.2 42.8
47.1 1.7
1.5 49.7
51.7 2.2
1.9 44.3
51.5 1.8
1.1 45.6
0 10 3 10 4 10 5
0
10 3
10 4
10 5 36.0 18.4
12.4 33.2
45.7 19.5
3.7 31.2
65.4 10.6
0.9 23.2
PBS57-loaded CD1d tetramer
Ly5.
1
WT Hobit KO Blimp1 KO Blimp1 x Hobit DKO
Spl
een
Live
r
B
Mackay et al. Figure S9
E
C
D
Spleen Liver
Blimp1 x Hobit DKO WT
NK
T ce
ll pr
olife
ratio
n (D
ivis
ion
Inde
x)
Day 15+
0
20
40
60
80
100
0 10 3 10 4 10 5
NKT cell proliferation (CTV)
WT Blimp1 x Hobit DKO
Cel
ls (%
of m
ax)
Spl
een
Live
r
-4
-2
0
2
4
Don
or N
KT
cells
(lo
g 2
KO
/ W
T)
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
Day 15+ Spleen
-4
-2
0
2
4
Don
or N
KT
cells
(lo
g 2
KO
/ W
T)
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
Day 15+ Liver
**
* *
0.0
0.2
0.4
0.6
Day 1 Day 8 Day 15+ -4
-2
0
2
4
Don
or N
KT
cells
(lo
g 2
Ly5.
2 / L
y5.1
)
*** ***
WT Blimp1 x Hobit DKO
Liver
0 10 3
10 4
10 5
0
10 3
10 4
10 5
Ly5.2
Ly5.
1 Day 1 Day 8 Day 15+
3.8
5.8
3.7
5.7
6.1
4.3
6.4
0.8
3.0
2.9
0.2
1.1
WT
Blimp1 x Hobit DKO
WT
WT
A B
Mackay et al. Figure S10 A
B C
naive
Spleen
CM EM
Liver
EM Trm 0.001
0.01
0.1
1
10
S1p
r1 m
RN
A ex
pres
sion
(AU
)
Ccr
7 m
RN
A ex
pres
sion
(AU
)
naive
Spleen
CM EM
Liver
EM Trm
D
E F
naive
Spleen
CM EM
Liver
SLEC MPEC EM Trm
20
40
60
80
Hob
it m
RN
A ex
pres
sion
(AU
)
0
50
100
150
Blim
p1 m
RN
A ex
pres
sion
(AU
)
naive
Spleen
CM EM
Liver
SLEC MPEC EM Trm
Blood Liver 0
5
10
15
20
25 C
D69
exp
ress
ion
(% o
f gp3
3+ c
ells
) **
CD103
CD
69
0 10 3 10 4 10 5
0
10 3
10 4
10 5
WT Hobit KO
Blimp1 KO Blimp1 x Hobit DKO 4.9 1.8
10.4 82.9
11.1 3.9
5.6 79.3
27.8 3.6
3.2 65.4
7.1 1.3
7.1 84.5
-8
-4
0
4
8
gp33
+ C
D8
T ce
lls (l
og2
KO
/ W
T)
Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
* **
WT
Kidney
Klf2
mR
NA
expr
essi
on (A
U)
naive
Spleen
CM EM
Liver
EM Trm 0.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
G H
0
Mackay et al. Figure S11
A
B WT Il15 KO
0.0
0.5
1.0
1.5
Blim
p1 m
RN
A e
xpre
ssio
n (A
U)
Blimp1
WT Il15 KO 0.0
0.5
1.0
1.5
Hob
it m
RN
A ex
pres
sion
(AU
) Hobit
C
0
1
2
3
4
5
control IL-15 TGFβ control IL-15 TGFβ
WT T-bet KO
Hobit
Hob
it m
RN
A ex
pres
sion
(AU
)
control T-bet Eomes 0
2
4
6
8
10
12
Hob
it m
RN
A ex
pres
sion
(AU
) Hobit
Mackay et al. Figure S12
A
C
NKT1 NKT2 0.0
0.5
1.0
1.5
S1p
r1 m
RN
A ex
pres
sion
(AU
)
NKT1 NKT2 0
2
4
6
8
10
Hob
it m
RN
A ex
pres
sion
(AU
)
NKT1 NKT2 0
2
4
6
8
10
Blim
p1 m
RN
A ex
pres
sion
(AU
)
Blimp1 Hobit S1pr1 Ccr7
NKT1 NKT2
CD69 T-bet
Cel
ls (%
of m
ax)
0
20
40
60
80
100
0 10 3 10 4 10 5
NKT1 NKT2 0.0
0.5
1.0
1.5
Ccr
7 m
RN
A ex
pres
sion
(AU
)
NKT1 NKT2 0
500
1000
1500
CD
69 e
xpre
ssio
n (g
eo M
FI)
CD69 B ***
* * * *
Mackay et al. Figure S13 A
Insig1 Gsg2 Ddx3x Dhcr24 Ppp1r16b Klf6 Btg2 Cxcr6 F osb J un Hspa5 Nedd4 Plk3 Stard4 Tn f aip3 B4galnt4 Cd244 Hobit Irf4 Cish Mapkapk3 Sik1 Pygl Ctnna1 Odc1 P er1 Dusp1 Atf3 Ldl r ad4 Sc4mol J unb Gpr56 Nfkbid F osl2 Rgs2 Nr4a2 Dgat1 Arrdc3 F r md4b Nr4a1 Gpr171 Smim3 E y a2 Gpr55 Amica1 L y6g5b Cs r np1 T r af4 Zfp36 Glrx Dusp5 Litaf Gadd45b Ifng Osgin1 Abi3 8430419L09Rik Egr1 Hilpda Skil Rnf149 Hmgcs1 P4hb Pnrc1 Gpr34 Ppp1r15a Itgae P2 r y10 Ehd1 Dusp6 Xcl1 Spsb1 Isg20 Inpp4b Neu r l3 Hpgds Rhob Vdac1 Lad1 F os Cdh1 Cd69 Qpct Hspd1 Hba-a2 Fgf13 Cmah AB124611 Snx10 Haao P ogk Sbk1 Klf2 Bcl9l L y6c2 Thap7 Asrgl1 Elmo1 H e xb Racgap1 Cxcr4 Cdc25b Lfng S1pr5 Arhgap26 Mpnd Kcnab2 Atp1b3 S1pr4 Gm11346 Tmem71 Kbtbd11 Emb Ms4a4c G r amd4 Ehd3 Kcnn4 Tcf7 Aaed1 Ms4a4b P aqr7 F am89b L yst Glipr2 Eml3 S1pr1 Pik3r5 Setx Txndc5 Ncln Stk38 2010016I18Rik BE692007 Itga4 Ccl5 Pyhin1 Gm8369 Gm1966 Cd97 Cd84 Klf3 Abtb2 Tbxa2r L y r m2 Lcn4 Acp5 BC147527 P odnl1 Lef1 BC094916 AI413582 Sh2d1a Phf11b Tsr3 Gmfg Gm9835 Eomes Gnpda2 Pde2a Sidt1 Gab3 Txk A v en Icam2 Klhl6 Samhd1 Smpdl3b Itgb2 Gm20140 Ttc7b D1E r td622e Atp10d St3gal1 V opp1 Pced1b Dock2 Itgb1 Acpl2 F am117a Il10 r a Lpin1 F am65b Rbm43 A430078G23Rik Arhgef18 Rasa3 Abhd8 F am49a Rasgrp2 B3gat3 P r kcq X r n2
LCM
V S
I IE
L
HS
V s
kin
LCM
V s
plee
n
HS
V s
plee
n
LCM
V s
plee
n
HS
V s
plee
n
CM EM Trm
-2 -1 0 1 2
Row Z-Score
Down Up
200 100 0 100 200 DE genes (adj p value < 0.05)
Spleen Tem vs Liver Tem
Spleen Tem vs Liver Trm
Liver Tem vs Liver Trm
374 141
808
51 9
40
84
Gut Trm Skin Trm
Liver Trm
t-statistic
0
4.0
5.7
0
Up in Liver Trm
Down in Liver Trm
Enr
ichm
ent
10 2 0.9 0.5 0.2 0.0 -0.3 -0.5 -0.9 -2 -20
Enr
ichm
ent
B
C
D
Mackay et al. Figure S14
t-statistic
0
2.3
4.6
0
Up in NKT
Down in NKT
Enr
ichm
ent
15.9 3.8 2.3 1.5 0.8 0.1 -0.4 -1.2 -2.3 -4.1 -26.6
Enr
ichm
ent
t-statistic
0
3.2
5.0
0
Up in trNK
Down in trNK
Enr
ichm
ent
16.3 2.3 1.0 0.3 -0.1 -0.4 -0.7 -1.0 -1.4 -2.1 -20.2
Enr
ichm
ent
Epithelial Trm associated trNK vs cNK
NKT vs Liver Tem
98 17
337
42 152
1123
35
A
B
C
Mackay et al. Figure S15
CD8 T cells
NKT 0
10
20
30
40
Hob
it m
RN
A ex
pres
sion
(AU
)
CD8 T cells
NKT 0
1
2
3
4
Blim
p1 m
RN
A ex
pres
sion
(AU
)
0 10 3 10 4 10 5 0
20
40
60
80
100
CD69
Cel
ls (%
of m
ax)
WT KO
Blimp1 KO Blimp1 x Hobit KO Hobit KO
A
B
Hobit Blimp1
t-statistic
0
5.2
4.9
0
Up in Blimp1 x Hobit DKO
NKT cells
Enr
ichm
ent
20.5 2.6 1.7 1.1 0.6 0.1 -0.5 -1.0 -1.6 -2.5 -14.8
Enr
ichm
ent
Down in Blimp1 x Hobit DKO
NKT cells
C
Mackay et al. Figure S16
-4
-2
0
2
4 white pulp red pulp
NK
T ce
lls (l
og2
KO
/ W
T)
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
** ***
WT Blimp1 x Hobit DKO
white pulp red pulp
0
0.2
0.4
0.6
0.8
NK
T ce
lls (x
106
cel
ls)
***
0 10 3 10 4 10 5 0
20
40
60
80
100
iv CD44
D
E F
G
WT Blimp1 x
Hobit DKO
WT WT
WT Hobit KO
WT Blimp1 KO
WT Blimp1 x Hobit DKO
CD3
PBS57-CD1d tetramer
B220
100 mm 100 mm
A B
0.0
0.2
0.4
0.6
0.8
1.0
Ccr
7 m
RN
A ex
pres
sion
(AU
)
Ccr7
***
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
0.0
0.2
0.4
0.6
Klf2
mR
NA
expr
essi
on (A
U)
Klf2 *
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
C
0.0
0.1
0.2
0.3
0.4
S1p
r1 m
RN
A ex
pres
sion
(AU
)
S1pr1
*** ***
WT Hobit KO
Blimp1 KO
Blimp1 x Hobit DKO
Mackay et al. Figure S17 Direct Blimp1 binding sites
4306 200
Direct Hobit binding sites
204
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