KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Alexa Fluor® 700 Anti-Human CD45 (Clone HI30) | BioLegend | Cat #: 304023; RRID: AB_493760 |
| V500 Mouse Anti-Human CD14 (Clone M5E2) | BD Biosciences | Cat #: 561391; RRID: AB_10611856 |
| V500 Mouse Anti-Human CD15 (Clone HI98) | BD Biosciences | Cat #: 561585; RRID: AB_10896278 |
| V500 Mouse Anti-Human CD19 (Clone HIB19) | BD Biosciences | Cat #: 561125; RRID: AB_10563208 |
| PerCP/Cyanine5.5 Anti-Human CD3 (Clone UCHT1) | BioLegend | Cat #: 300430; RRID: AB_893299 |
| BB515 Mouse Anti-Human CD14 (Clone RPA-T4) | BD Biosciences | Cat #: 564419; RRID: AB_2744419 |
| Brilliant Violet 605™ Anti-Human CD8a (Clone RPA-T8) | BioLegend | Cat #: 301040; RRID: AB_2563185 |
| PE Anti-Human TCR Va7.2 (Clone 3C10) | BioLegend | Cat #: 351706; RRID: AB_10899577 |
| PE/Cyanine7 Anti-Human CD161 (Clone HP-3G10) | BioLegend | Cat #: 339918; RRID: AB_11126745 |
| APC/Fire™ 750 Anti-Human CD56 (NCAM) Recombinant (Clone QA17A16) | BioLegend | Cat #: 392408; RRID: AB_2728404 |
| PE-CD594 Mouse Anti-Human CD69 (Clone FN50) | BD Biosciences | Cat #: 562645; RRID: AB_2737699 |
| APC Anti-Human CD25 (Clone BC96) | BioLegend | Cat #: 302610; RRID: AB_314280 |
| Brilliant Violet 650™ Anti-Human CD38 (Clone HB-7) | BioLegend | Cat #: 356620; RRID: AB_2566233 |
| Brilliant Violent 421™ Anti-Human CD279 (PD-1) (Clone: EH12.2H7) | BioLegend | Cat #: 329920; RRID: AB_10960742 |
| BV650 Mouse Anti-Human CD274 (Clone: MIH1) | BD Biosciences | Cat #: 563740; RRID: AB_2738398 |
| Brilliant Violent 785™ Anti-Human HLA-DR (Clone: CL243) | BioLegend | Cat #: 307641; RRID: AB_2561360 |
| PE anti-human CD206 (MMR) Antibody | BioLegend | Cat #: 321106; RRID: AB_571911 |
| APC Mouse Anti-Human CD14 (CLONE M5E2) | BD Biosciences | Cat #: 561383; RRID: AB_10612009 |
| PerCP/Cy5.5 Anti-Human CD11b (Clone: ICRF44) | BioLegend | Cat #: 301327; RRID: AB_10900072 |
| FITC Mouse Anti-Human CD163 (Clone GHI/61) | BD Biosciences | Cat #: 563697; RRID: AB_2738379 |
| BV421 Mouse Anti-Human CD3 (Clone UCHT1) | BD Biosciences | Cat #: 562426; RRID: AB_11152082 |
| Brilliant Violet 421™ Anti-Human CD19 (Clone HIB19) | BioLegend | Cat #: 302234; RRID: AB_11142678 |
| PE/Cy™7 Mouse Anti-Human CD68 (Clone Y1/82A) | BD Biosciences | Cat #: 565595; RRID: AB_2739298 |
| BV510 Mouse Anti-Human CD11c (Clone: B-ly6) | BD Biosciences | Cat #: 563026; RRID: AB_2737960 |
| BD Pharmingen™ APC Mouse Anti-Human IFN-γ | BD Biosciences | Cat#: 554702 RRID: AB_398580 |
| Human BD Fc Block™ | BD Biosciences | Cat #: 564220; RRID: AB_2728082 |
| Purified anti-mouse CD16/32 Antibody | BioLegend | Cat #: 101302; RRID: AB_312800 |
| PerCP/Cyanine5.5 anti-mouse CD62L Antibody (Clone: MEL-14) | BioLegend | Cat #: 104432, RRID: AB_2285839 |
| PE/Cyanine7 anti-mouse/human CD44 Antibody (Clone: IM-7) | BioLegend | Cat #: 103030, RRID: AB_830787. |
| Brilliant Violet 650™ anti-mouse CD69 Antibody (Clone: H1.2F3) | BioLegend | Cat #: 104541, RRID: AB_2616934 |
| APC/Fire™ 750 anti-mouse TCR β chain Antibody (Clone: H57–597) | (BioLegend | Cat #: 109246, RRID: AB_2629697 |
| PE anti-mouse NK-1.1 Antibody (Clone: PK136) | BioLegend | Cat #: 108708, RRID: AB_313395 |
| Alexa Fluor® 700 anti-mouse F4/80 Antibody (Clone: BM8) | BioLegend | Cat #: 123130, RRID: AB_2293450 |
| Alexa Fluor® 700 anti-mouse/human CD45R/B220 Antibody (Clone: RA3–6B2) | BioLegend | Cat #: 103232, RRID: AB_493717 |
| Alexa Fluor® 700 anti-mouse/human CD11b Antibody (Clone: M1-70) | BioLegend | Cat #: 101222, RRID: AB_493705 |
| Alexa Fluor® 594 anti-mouse CD3 Antibody (Clone: 17A2) | BioLegend | Cat #: 100240, RRID: AB_2563427 |
| Brilliant Violet 605™ anti-mouse CD4 Antibody (Clone: GK1.5) | BioLegend | Cat #: 100451, RRID: AB_2564591 |
| Alexa Fluor® 700 Rat Anti-Mouse CD4 (Clone: RM4–5) | BD Biosciences | Cat #: 557956, RRID: AB_396956 |
| BV786 Rat Anti-Mouse CD8a (Clone: 53–6.7) | BD Biosciences | Cat #: 563332, RRID: AB_2721167 |
| Pacific Blue™ anti-mouse/human CD11b Antibody (Clone: M1/70) | BioLegend | Cat #: 101224, RRID: AB_755986 |
| Alexa Fluor® 700 anti-mouse Ly-6G Antibody (Clone: 1A8) | BioLegend | Cat #: 127622, RRID: AB_10643269 |
| APC/Cyanine7 anti-mouse Ly-6C Antibody (Clone: HK1.4) | BioLegend | Cat #: 128026, RRID: AB_10640120 |
| Brilliant Violet 421™ anti-mouse Ki-67 Antibody | BioLegend | Cat# 652411, RRID: AB_2562663 |
| ROR gamma (t) Monoclonal Antibody (B2D), PE, eBioscience™ | Thermo Fisher Scientific | Cat# 12–6981–82, RRID: AB_10807092 |
| PE/Cyanine7 anti-T-bet Antibody | BioLegend | Cat# 644824, RRID: AB_256176 |
| FOXP3 Monoclonal Antibody, APC, eBioscience™ (Clone: FJK-16s) | Thermo Fisher Scientific | Cat #: 17–5773–82, RRID: AB_469457) |
| APC anti-mouse IFN-γ Antibody (Clone: XMG1.2) | BioLegend | Cat #: 505810, RRID: AB_315404 |
| APC anti-mouse Perforin Antibody (Clone: S16009A) | BioLegend | Cat #: 154304, RRID: AB_2721463 |
| FITC anti-human/mouse Granzyme B Antibody (Clone: GB11) | BioLegend | Cat #: 515403, RRID: AB_2114575 |
| FITC anti-mouse IL-17A Antibody (Clone: TC11–18H10.1) | BioLegend | Cat #: 506908, RRID: AB_536010 |
| CD45-BX001 (HI30)—ALEXA FLUOR™ 488-RX001 Kit (Clone: HI30) | Akoya Biosciences | Cat #: 4150003; RRID: AB_2895052 |
| CD8-BX004 (SK1)—ALEXA FLUOR™ 488-RX004 Kit (Clone: SK1) | Akoya Biosciences | Cat #: 4150004; RRID: AB_2895049 |
| CD38 comm (Clone: HB-7) | Akoya Biosciences | Cat #: 4150007; RRID: AB_2909414 |
| Purified anti-human CD45RO (Maxpar(R) Ready) antibody (Clone: UCHL1) | Biolegend | Cat #: 304239; RRID: AB_2563752 |
| pancytokeratin comm (Clone: AE-1/AE-3) | Akoya Biosciences | Cat #: 4150020; RRID: AB_2909509 (TEMPORARY) |
| Purified anti-human CD39 (Maxpar(R) Ready) antibody (Clone: A1) | Biolegend | Cat #: 328221; RRID: AB_2563747 |
| Purified anti-human CD15 (SSEA-1) antibody (Clone: HI98) | Biolegend | Cat #: 301902; RRID: AB_314194 |
| Purified anti-human CD40 antibody (Clone: HB14) | Biolegend | Cat #: 313002; RRID: AB_314965 |
| Purified anti-CD68 antibody (Clone: KP1) | Biolegend | Cat #: 916104; RRID: AB_2616797 |
| FOXP3 Monoclonal Antibody (PCH101), eBioscience (Clone: PCH101) | ThermoFisher | Cat #: 14–4776–82; RRID: AB_467554 |
| Purified anti-human CD163 antibody (Clone: GHI/61) | Biolegend | Cat #: 333602; RRID: AB_1088991 |
| Anti-CD56 Monoclonal Antibody, Unconjugated (Clone: 123.C3.5D) | Cell Marque | Cat #: 156M-86; RRID: AB_1158184 |
| Purified anti-human CD326 (Ep-CAM) antibody (Clone: 9C4) | Biolegend | Cat #: 324202; RRID: AB_756076 |
| Purified anti-human CD66b antibody (Clone: G10F5) | Biolegend | Cat #: 305102; RRID: AB_314494 |
| Purified anti-human CD25 antibody (Clone: M-A251) | Biolegend | Cat #: 356102; RRID: AB_2561752 |
| Anti-Human CD62L - Purified (CODEX® Ready) antibody (Clone: DREG56) | Leinco | Cat #: C512–50 μg; RRID: AB_2893569 |
| CD279 comm (Clone: EH12.2H7) | Akoya Biosciences | Cat #: 4250010; RRID: AB_2909510 |
| ICOS comm (Clone: C398.4A) | Akoya Biosciences | Cat #: 4250013; RRID: AB_2909511 |
| LEAF™ Purified anti-human CD11b antibody (Clone: ICRF44) | Biolegend | Cat #: 301312; RRID: AB_314164 |
| HNF-4alpha (H-1) antibody (Clone: H-1) | Santa Cruz | Cat #: sc-374229; RRID: AB_10989766 |
| HLA-DR-BX026 (L243)—ATTO 550-RX026 Kit (Clone: L243) | Akoya Biosciences | Cat #: 4250006; RRID: AB_2895054 |
| Purified anti-human CD161 antibody (Clone: HP-3G10) | Biolegend | Cat #: 339902; RRID: AB_1501090 |
| Goat Anti-Human Il-18 r alpha / il-1 r5 Polyclonal antibody, Unconjugated (Clone: goat polyclonal) | R&D Systems | Cat #: AF840; RRID: AB_355650 |
| CD34 comm (Clone: 561) | Akoya Biosciences | Cat #: 4250020; RRID: AB_2909512 (TEMPORARY) |
| CD69 comm (Clone: FN50) | Akoya Biosciences | Cat #: 4250022; RRID: AB_2909513 (TEMPORARY) |
| KI67-BX047 (B56)—ATTO 550-RX047 Kit (Clone: B56) | Akoya Biosciences | Cat #: 4250019; RRID: AB_2895046 |
| CD19-BX003 —CY5-RX003 (Clone: HIB19) | Akoya Biosciences | Cat #: 4350003 |
| Anti-Human CD19 - Purified In vivo GOLD™ Functional Grade antibody (Clone: SJ25-C1) | Leinco | Cat #: C2854–1.0 mg; RRID: AB_2829610 |
| PD-L1 (E1L3N) XP Rabbit Antibody (Clone: E1L3N) | Cell Signalling | Cat #: 13684S; RRID: AB_2687655 |
| CD3-BX015 (UCHT1)—CY5-RX015 Kit (Clone: UCHT1) | Akoya Biosciences | Cat #: 4350008; RRID: AB_2895047 |
| CD4-BX021—CY5-RX021 Kit (Clone: SK3) | Akoya Biosciences | Cat #: 4350010 RRID: AB_2895048 |
| LEAF™ Purified anti-human CD4 antibody (Clone: OKT4) | Biolegend | Cat #: 317404; RRID: AB_571961 |
| Purified anti-human CD57 antibody (Clone: HNK-1) | Biolegend | Cat #: 359602; RRID: AB_2562403 |
| CD11C-BX027 —CY5-RX027 Kit (Clone: S-HCL-3) | Akoya Biosciences | Cat #: 4350012; RRID: AB_2895050 |
| Anti-Human CD16 - FITC antibody (Clone: 3G8) | Leinco | Cat #: C6854; RRID: AB_2893584 |
| Purified anti-human TCR Valpha7.2 antibody (Clone: 3C10) | Biolegend | Cat #: 351702; RRID: AB_10900258 |
| Alpha-Smooth Muscle Actin Monoclonal Antibody, eBioscience (Clone: 1A4) | ThermoFisher | Cat #: 14–9760–82; RRID: AB_2572996 |
| LYVE1 antibody (Clone: polyclonal) | Abcam | Cat #: ab36993; RRID: AB_2138663 |
| Mouse Anti-CD45RA Monoclonal Antibody, Unconjugated (Clone: HI100) | BD Biosciences | Cat #: 555486; RRID: AB_395877 |
| Biological samples | ||
| Patient HCC core samples | This paper | N/A |
| Patient HCC rim samples | This paper | N/A |
| Patient HCC adjacent normal liver tissue | This paper | N/A |
| Control liver tissue from patients undergoing liver transplantation | This paper | N/A |
| Chemicals, peptides, and recombinant proteins | ||
| 2-methylbutane | FisherScientific | Cat #: AC167840010; CAS #: 78–78–4 |
| Optimal cutting temperature (O.C.T.) compound | FisherScientific | Cat #: 23–730–571 |
| MACS tissue storage solution | Miltenyi Biotec | Cat #: 130–100008 |
| Serum-free RPMI 1640 | Thermofisher | Cat #: 61870127 |
| Collagenase IV | STEMCELL technologies | N/A |
| DNAse I | STEMCELL Technologies | Cat #: 07900 |
| Percoll PLUS solution (in PBS) | GE Healthcare | Cat #: GE17–544501 |
| RBC lysis buffer | ThermoFisher | Cat #: 50–1129751 |
| Recovery™ Cell Culture Freezing Medium | Gibco | Cat #: 12648010 |
| FACS buffer | BD Biosciences | Cat #: 554656 |
| Stain Buffer (FBS) | BD Biosciences | Cat #: 564220; RRID: AB_2869006 |
| Fixable Viability dye ZOMBIE-UV | BioLegend | Cat #: 423108 |
| Acridine Orange / Propidium Iodide Stain | Logos Biosystems | F23001 |
| RPMI 1640 Medium, GlutaMAX™ Supplement | ThermoFisher | Cat #: 61870127 |
| Corning™ Regular Fetal Bovine Serum | Corning | Cat #: MT35010CV |
| Sodium Pyruvate 100 mM | ThermoFisher | Cat #: 11360070 |
| HEPES (1M) | ThermoFisher | Cat #: 15630080 |
| MEM Non-Essential Amino Acids Solution (100X) | ThermoFisher | Cat #: 11140050 |
| Puromycin | InvivoGen | Cat #: ant-pr-1; CAS #: 58–58–2 |
| Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, 10 mL | Coning | Cat #: 354230 |
| Percoll | Cytiva | Cat #: 17089109 |
| Lympholyte Cell Separation Media | Cedarlane Laboratories | Cat. No. CL5035 |
| ACK-lysis buffer | Quality Biologicals | Cat #: 118–156–101 |
| Murine and human MR1-tetramers loaded with MAIT ligand 5-OP-RU | NIH Tetramer Core | NA |
| Leukocyte Activation Cocktail, with BD GolgiPlug™ | BD Biosciences | Cat #: 550583, RRID: AB_2868893 |
| eBioscience™ Intracellular Fixation & Permeabilization Buffer Set | ThermoFisher | Cat #: 88–8824–00 |
| Critical commercial assays | ||
| N/A | ||
| Deposited data | ||
| CRISPR/Cas9 lentiviral vector targeting murine Cd274 | This paper | Addgene ID: 199279 |
| GRCh38 reference | refdata-gex-mm10-2020-A | |
| GRCh38 VDJ reference | refdata-cellranger-vdj-GRCh38-alts-ensembl-5.0.0 | |
| Transcriptomic data of 371 patients diagnosed with HCC | The Cancer Genome Atlas Project- LIHC cohort | https://cancergenome.nih.gov/ |
| scRNA-seq Data from human HCC patients | This paper | The database of Genotypes and Phenotypes (dbGaP); Accession number: phs003279.v1.p1 |
| scRNA-seq Data from murine tumor-bearing vs tumor-free livers | This paper | GEO: GSE231712 |
| CODEX images | This paper | The Canccer imaging Archives (TCIA) under https://doi.org/10.7937/bh0r-y074 |
| Sc-RNA-seq data HCC patients + ICB | Ma et al., 2021 | GEO: GSE12544 |
| Experimental models: Cell lines | ||
| Luciferase-expressing RIL-175 | Kapanadze et al., 2013 | N/A |
| Syngeneic Hep55.1c | CLS Cell Lines Service GmbH | Cat #: 400201 |
| B16F10 (GFP and luciferase-expressing) | Kreiter et al., 2015 | N/A |
| MC38 | Dr, Jay Berzofsky (NCI) | RRID:CVCL_B288 |
| Experimental models: Organisms/strains | ||
| NCI C57BL/6NCr Mice | Charles River Laboratories | Code: 556 |
| Mr1−/− mice | Treiner et al., 2013 | N/A |
| Lyz2Cre mice | Jackson Laboratory | Strain#: strain #004781 |
| Csf1rLsL-DTRmice | Jackson Laboratory | Strain#: 024046 |
| Csf1rCre mice | Jackson Laboratory | Strain#: 029206 |
| Cd274fl/fl | Jackson Laboratory | Strain #: 036255 |
| Oligonucleotides | ||
| Primer: MR1 5 8763–8783: AGC TGA AGT CTT TCC AGA TCG, | Ruf et al., 2021 | N/A |
| Primer: MR1 9188–9168 rev: ACA GTC ACA CCT GAG TGG TTG | Ruf et al., 2021 | N/A |
| Primer: MR1 10451–1043: GAT TCT GTG AAC CCT TGC TTC | Ruf et al., 2021 | N/A |
| BX001—Alexa Fluor™ 488-RX001 | Akoya Biosciences | Cat #: 5450013 |
| BX004—Alexa Fluor™ 488-RX004 | Akoya Biosciences | Cat #: 5450014 |
| BX007—Alexa Fluor™ 488-RX007 | Akoya Biosciences | Cat #: 5450015 |
| BX010—Alexa Fluor™ 488-RX010 | Akoya Biosciences | Cat #: 5450016 |
| BX013—Alexa Fluor™ 488-RX013 | Akoya Biosciences | Cat #: 5450017 |
| BX016—Alexa Fluor™ 488-RX016 | Akoya Biosciences | Cat #: 5150001 |
| BX019—Alexa Fluor™ 488-RX019 | Akoya Biosciences | Cat #: 5150002 |
| BX022—Alexa Fluor™ 488-RX022 | Akoya Biosciences | Cat #: 5150003 |
| BX025—Alexa Fluor™ 488-RX025 | Akoya Biosciences | Cat #: 5150004 |
| BX028—Alexa Fluor™ 488-RX028 | Akoya Biosciences | Cat #: 5150005 |
| BX031—Alexa Fluor™ 488-RX031 | Akoya Biosciences | Cat #: 5150006 |
| BX034—Alexa Fluor™ 488-RX034 | Akoya Biosciences | Cat #: 5150007 |
| BX037—Alexa Fluor™ 488-RX037 | Akoya Biosciences | Cat #: 5150008 |
| BX040—Alexa Fluor™ 488-RX040 | Akoya Biosciences | Cat #: 5150009 |
| BX043—Alexa Fluor™ 488-RX043 | Akoya Biosciences | Cat #: 5150010 |
| BX046—Alexa Fluor™ 488-RX046 | Akoya Biosciences | Cat #: 5150011 |
| BX049—Alexa Fluor™ 488-RX049 | Akoya Biosciences | Cat #: 5150012 |
| BX002—Atto 550-RX002 | Akoya Biosciences | Cat #: 5450023 |
| BX005—Atto 550-RX005 | Akoya Biosciences | Cat #: 5450024 |
| BX014—Atto 550-RX014 | Akoya Biosciences | Cat #: 5450025 |
| BX017—Atto 550-RX017 | Akoya Biosciences | Cat #: 5250001 |
| BX020—Atto 550-RX020 | Akoya Biosciences | Cat #: 5250002 |
| BX023—Atto 550-RX023 | Akoya Biosciences | Cat #: 5250003 |
| BX026—Atto 550-RX026 | Akoya Biosciences | Cat #: 5250004 |
| BX029—Atto 550-RX029 | Akoya Biosciences | Cat #: 5250005 |
| BX032—Atto 550-RX032 | Akoya Biosciences | Cat #: 5250006 |
| BX035—Atto 550-RX035 | Akoya Biosciences | Cat #: 5250007 |
| BX041—Atto 550-RX041 | Akoya Biosciences | Cat #: 5250008 |
| BX047—Atto 550-RX047 | Akoya Biosciences | Cat #: 5250009 |
| BX003—Cy5-RX003 | Akoya Biosciences | Cat #: 5450026 |
| BX006—Cy5-RX006 | Akoya Biosciences | Cat #: 5450027 |
| BX015—Cy5-RX015 | Akoya Biosciences | Cat #: 5350001 |
| BX021—Cy5-RX021 | Akoya Biosciences | Cat #: 5350002 |
| BX024—Cy5-RX024 | Akoya Biosciences | Cat #: 5350003 |
| BX027—Cy5-RX027 | Akoya Biosciences | Cat #: 5350004 |
| BX030—Cy5-RX030 | Akoya Biosciences | Cat #: 5350005 |
| BX033—Cy5-RX033 | Akoya Biosciences | Cat #: 5350006 |
| BX036—Cy5-RX036 | Akoya Biosciences | Cat #: 5350007 |
| BX042—Cy5-RX042 | Akoya Biosciences | Cat #: 5350008 |
| BX045—Cy5-RX045 | Akoya Biosciences | Cat #: 5350009 |
| Software and algorithms | ||
| 10x Genomics Cell Ranger pipeline (v5.0 or v6.0) | 10x Genomics | N/A |
| S3-CIMA | Babaei et al.2023 | https://github.com/claassenlab/S3-CIMA |
| Visreg R package | Breheny and Burchett, 2013 | N/A |
| HALO image analysis platform V3.2 | Indica Labs | N/A |
| ImageJ (Fiji version 2.8.5) | NIH | N/A |
| FlowJo | FlowJo | RRID: SCR_008520 |
| GraphPad Prism 9 | GraphPad Prism | RRID: SCR_002798 |
| Other | ||
| LUNA-FL fluorescent cell counter | Logos Biosystems | N/A |
| 10x Genomics Chromium platform, 5’ v1.1 immune profiling chemistry | 10x Genomics | PN-1000165 |
| Illumina NextSeq 550 instrument | Illumina | N/A |
| Illumina NextSeq 2000 instrument | Illumina | N/A |
| gentleMACS Octo Dissociator Tissue Dissociator | Miltenyi | RRID: SCR_020272 |
| CytoFLEX LX flow cytometer | Beckman Coulter | RRID: SCR_019627 |