Mice
SPF C57BL/6J (B6) mice (Jax, catalogue no. 000664, both sexes) were obtained from The Jackson Laboratories. CD45.1 congenic mice (B6.SJL-Ptprca Pepcb/BoyJ, JAX, catalogue no. 002014), Rosa-CAG-LSL-tdTomato reporter mice (B6;129S6-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, JAX, catalogue no. 007908), Il17a-Cre mice (STOCK Il17atm1.1(icre)Stck/J, JAX, catalogue no. 016879) and ROSA26-eGFP-DTA (B6.129S6(Cg)-Gt(ROSA)26Sortm1(DTA)Jpmb/J, JAX, catalogue no. 032078) were purchased from The Jackson Laboratory. TCR7B8 (C57BL/6-Tg(Tcra,Tcrb)2Litt/J) and TCRHh7-2 (C57BL/6-Tg(Tcra,Tcrb)5Litt/J) mice were generated in house as described previously. All transgenic lines were bred and maintained under SPF conditions at the Alexandria Center for Life Sciences animal facility, New York University School of Medicine.
For IL-17A fate mapping experiments, sex-matched littermates (both male and female) were used. Experimental cohorts were 6–8 weeks old at treatment onset. Sample sizes were determined by power analysis (power = 0.9, α = 0.05) using mean and s.d. estimates from previous and pilot studies (four to five animals per group). All animal procedures were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of New York University School of Medicine.
Antibodies, intracellular staining and flow cytometry
Monoclonal antibodies were obtained from eBioscience, BD Pharmingen, BioLegend, Thermo Fisher, Tonbo Bioscience and Invitrogen. The following fluorochrome-conjugated antibodies were used: CD4 BUV395 (GK1.5, BD, catalogue no. 563790, 1:400), CD25 APC (PC61, Thermo, catalogue no. 17-0251-82, 1:400), CD69 PE-Cy7 (H1.2F3, BioLegend, catalogue no. 104512, 1:200), CD44 AF700 (IM7, BD, catalogue no. 560567, 1:200) or BV510 (IM7, BD, catalogue no. 563114, 1:200), CD45.1 BV650 (A20, BD, catalogue no. 563754, 1:400), CD45.2 FITC (104, eBioscience, catalogue no. 11-0454-85, 1:400), CD19 PerCP-Cy5.5 (1D3, Tonbo, catalogue no. 65-0193-U100, 1:400), B220 PerCP-Cy5.5 (RA3-6B2, Invitrogen, catalogue no. 45-0452-82, 1:400), CD11c PerCP-Cy5.5 (N418, Invitrogen, catalogue no. 45-0114-82, 1:400) or PE-Cy7 (N418, BioLegend, catalogue no. 117318, 1:400), CD11b PerCP-Cy5.5 (M1/70, Invitrogen, catalogue no. 45-0112-82, 1:400) or BUV395 (BD, catalogue no. 563553, 1:400), MHCII I-A/I-E PerCP-Cy5.5 (M5/114.15.2, BioLegend, catalogue no. 107626, 1:400), NK1.1 PerCP-Cy5.5 (PK136, Invitrogen, catalogue no. 45-5941-82, 1:200), TCRβ BV711 (H57-597, BD, catalogue no. 563135, 1:200), TCRγδ PerCP-Cy5.5 (GL3, BioLegend, catalogue no. 118117, 1:400), FOXP3 FITC (FJK-16s, eBioscience, catalogue no. 11-5773-82, 1:200), RORγt BV421 (Q31-378, BD, catalogue no. 562894, 1:200), T-bet PE-CF594 (O4–46, BD, catalogue no. 562467, 1:70), IL-17A AF700 (TC11-18H10.1, BioLegend, catalogue no. 506914, 1:200), IFN-γ PE-Cy7 (XMG1.2, BioLegend, catalogue no. 505826, 1:200), Granzyme B AF700 (QA16A02, BioLegend, catalogue no. 372222, 1:200), TNF BV650 (MP6-XT22, BioLegend, catalogue no. 506333, 1:200), CXCR6 PE/Dazzle594 (SA051D1, BioLegend, catalogue no. 151117, 1:200), CD62L PE (MEL-14, BD Pharmingen, catalogue no. 553151, 1:400), and TCR Vβ14 FITC (14-2, BD Pharmingen, catalogue no. 553258, 1:400). Dead cells were excluded using 4′,6-diamidino-2-phenylindole (Sigma) or LIVE/DEAD Fixable Blue dye (Thermo Fisher).
For scTCR-seq coupled with scRNA-seq, cells were labelled with TotalSeq-C hashtag antibodies (BioLegend): Hashtag 1 (M1/42; 30-F11, catalogue no. 155861, 1:100), Hashtag 2 (catalogue no. 155863, 1:100), Hashtag 3 (catalogue no. 155865, 1:100), and Hashtag 4 (catalogue no. 155867, 1:100).
For transcription factor staining, cells were first stained for surface markers, then fixed and permeabilized using the FOXP3 staining buffer set (eBioscience), followed by nuclear staining. For intracellular cytokine analysis, cells were stimulated for 3 h in RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS), plus phorbol 12-myristate 13-acetate (50 ng ml−1, Sigma), ionomycin (500 ng ml−1, Sigma) and GolgiStop (BD Biosciences), then stained for surface markers, fixed, permeabilized and subjected to intracellular/nuclear staining with eBioscience buffers.
Flow cytometry was performed using BD LSR II or Aria II instruments (BD Biosciences), data acquisition was carried out using BD FACSDiva software (v.8.0.1; BD Biosciences) and data were analysed with FlowJo software (v.10.10.0) (Tree Star).
Design of SFB-3340 antigen construct and generation of cancer cell lines expressing SFB-3340
To establish a synthetic neoantigen mimicry model, we designed a construct encoding a small, independently folded domain containing a well-characterized immunogenic CD4+ T cell epitope (hereafter referred to as SFB-3340) derived from a large membrane protein of SFB (SFBNYU_003340, GenBank: EGX28318.1)36. A mammalian codon-optimized gene fragment encoding SFB-3340 antigen, fused via a flexible linker to a c-Myc tag, was synthesized chemically (GenScript) and cloned into the pEF1α-IRES-Neo vector (Addgene, catalogue no. 28019) between NheI and SalI restriction sites. Expression was driven by the constitutive EF1α promoter.
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