Figures and data in Discovery of surrogate agonists for visceral fat Treg cells that modulate metabolic indices in vivo

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7 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements

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Development of a peptide-A^b yeast library.

(A) A degenerate codon with maximum diversity at TCR-facing positions (blue) and optimal residues at anchor positions (red) was used to generate a peptide-MHC yeast library containing up to 1 × 10⁹ peptides. (B) To validate the library a proxy TCR of known specificity, YAe, was used to perform four consecutive rounds of selection. (C) Enriched yeast clones were isolated at all steps and deep-sequenced following appropriate plasmid purification and PCR amplification in order to quantify the peptide frequency. The top eight enriched peptides found in the fourth round of YAe selection are shown. (D) Positional frequency matrix based on the deep sequence of the fourth round of selection.

Figure 1—figure supplement 1

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Evolution of peptide-Ab yeast display.

(A) Cartoon representation and protein sequence used for the peptide-A^b yeast display. (B) TCR tetramer staining of the naïve library and the four consecutive rounds of yeast selection using the YAe TCR. The peptide-A^b library was generated following the random mutagenesis by error-prone PCR of the construct shown in (A). (C) Size exclusion chromatography of the vTreg53 TCR following purification by Ni-NTA column and overnight biotinylation using BirA enzyme. (D) Reducing and non-reducing SDS-PAGE of the vTreg53 TCR fractions collected in (C).

Figure 1—figure supplement 2

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Validation of the peptide-Ab yeast display platform.

(A) 2W TCR tetramer staining together with c-myc staining is shown for the naïve library and four consecutive rounds of selection of the peptide-Ab yeast library. TCR tetramer and c-myc staining of enriched peptide sequences (single clones) isolated from the YAe (B) or 2W (C) TCR selections. Naïve library, 3 k peptide and single point mutations (red) in central positions of the enriched peptides were used as controls to confirm the specificity of the peptide-Ab/TCR SA interaction.

Figure 2 with 1 supplement

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Identification of surrogate peptides recognized by the vTreg53 TCR.

(A) The vTreg53 TCR was used to screen and isolate the peptide-MHC yeast display. vTreg53 TCR tetramer staining (500 nM of TCR-SA) together with c-Myc staining is shown for the naïve library and four consecutive rounds of selection. (B) Deep sequencing of the fourth round of selection reveals a small subset of peptides which share key residues at TCR facing positions (shown in green). (C) Positional frequency representation from the deep sequencing of the third round of selection with vTreg53. (D) Positional frequency matrix based on the deep sequencing of the fourth round of the peptide-MHC yeast selection. (E) A single peptide-MHC yeast clone, displaying the top enriched peptide, LMFKGPHAVQAVG (Fat 7; *right panel; left panel* shows staining for Eα control peptide), shows a positive Fat-TCR tetramer staining (500 nM final tetramer concentration). Data shown are representative of at least two independent experiments.

Figure 2—figure supplement 1

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Evolution of peptide-Ab yeast display.

(A) Size exclusion chromatography of the vTreg53 TCR following purification by Ni-NTA column and overnight biotinylation using BirA enzyme. (B) Reducing and non-reducing SDS-PAGE of the vTreg53 TCR fractions collected in (A).

Figure 3 with 1 supplement

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Identification of robust vTreg53 TCR agonists.

Single-point mutants based on the Fat7 (A) or Fat15 (B) peptides were used to stimulate Fat-TCR transduced Jurkat T cells. Data shown are mean fluorescence intensity for CD69 up-regulation following overnight stimulation with the indicated peptides at 100 µM. (C) Potent SPs based on Fat15. Single-residue mutations from the Fat15 are shown in red. Anchor positions are shown in grey and potential TCR-facing positions are shown in green. A peptide titration of single- and double-point mutants from the Fat15 SP show up to ~4- (D; CD69) or ~8 fold (E; CD25) increase in E_max. (F) Surface plasmon resonance for Fat7, Fat15, Fat1562 and Fat2564 was used to determine the pMHC/Fat TCR affinity. All biotinylated pMHC were purified by SEC and immobilized in a streptavidin-coated SPR sensor chip from 200 up to 600 RU. Data are mean ± SD from n = 2 (**A, D, E**) or 3 (B) biological replicates from 1 representative of 3 independent experiments. Data shown in (F) is representative of 2 independent experiments.

Figure 3—figure supplement 1

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In vitro peptide stimulation of the vTreg53 TCR cells.

(A) Expression of wild-type Ab MHC in stably transduced K562 cells. (B) Expression of vTreg53 TCR in stably transduced Jurkat T cells (TCRαβ-/-). (C) Fraction of CD25+/CD69+ cells following overnight stimulation with Fat mimotopes at the indicated concentrations. (D) Peptide-A^b - streptavidin PE (pAb -SAPE) titration of vTreg53 transduced Jurkat T cells. Cells were stained with 250 nM of pA^b-SAPE tetramer for 1 hr. (**C, D**) Data are mean ± SD from n = 2 biological replicates from 1 representative of 3 independent experiments.

Figure 4 with 1 supplement

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SPs induce proliferation and activation of vTreg53 Treg cells in vitro and in vivo.

(**A–D**) Proliferation and activation of vTreg53 Treg cells by different concentrations of SPs for 3 days in vitro (n = 3). (A) Representative flow cytometric plot of cell division. (B) Summary of cell proliferation. (C) Mean flourescence intensity (MFI) of CD44 staining in clonotype⁺ Treg cells. (D) MFI of CD62L staining in clonotype⁺ Treg cells. (E) Proliferation of transferred CD45.2⁺ vTreg53 Treg cells in the spleen of CD45.1⁺ B6 recipient mice immunized *s.c.* with CFA or CFA/Fat1562 at day 3 (n = 3). (F) Expansion of transferred CD45.2⁺ vTreg53 Treg cells in the Spl/LNs or VAT of CD45.1⁺ B6 recipient mice that were primed with CFA/fat1562 *s.c.* and boosted with IFA/Fat1562 *i.p*. (n ≥ 3). Data are mean ± SD. Data shown are representative of at least two independent experiments.

Figure 4—figure supplement 1

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Single cell TCR sequencing (scTCR-seq) analyses of fat1562-reactive endogenous Treg cells following immunization.

(A) Scheme of the experiment. (B) Flow cytometric analysis of fat1562/A^b -PE tetramer+ Treg cells in Spl/LNs (enriched by anti-PE beads) or VAT (not enriched) of mice immunized with adjuvant alone or adjuvant/fat1562. Plot is a representative of mice that did show detectable expansion of tetramer+ Tregs. (C) Numbers of fat1562/A^b -PE tetramer+ Treg cells in Spl/LNs or VAT of mice immunized with adjuvant alone or adjuvant/fat1562 (D) Summary of the frequency and CDR3 sequences of expanded fat1562/A^b -PE tetramer+ Treg clones.

Figure 5

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Transcriptional analyses of vTreg53 Treg cells stimulated by Fat1562 in vivo.

(**A–C**) CD45.2⁺ vTreg53 Treg cells were transferred *i.v.* into CD45.1⁺ B6 mice. 6 weeks later, the recipient mice were immunized with CFA alone or CFA/Fat1562 *s.c.*, and one week later, boosted with IFA alone or IFA/Fat1562 *i.p*. CD45.2⁺ clonotype⁺ Treg cells were sorted from Spl/LNs or VAT of the recipient mice one week later for RNA-Seq. (A) Volcano plot comparing gene expression of transferred clonotype⁺ Treg cells in the VAT and Spl/LNs of recipient mice immunized with adjuvant alone (left) or with adjuvant/Fat1562 (right). VAT-Treg signature genes are highlighted in red (induced) or blue (repressed). The number of genes from each signature preferentially expressed by one or the other population are shown at the top. (B) GSEA analysis of top KEGG pathways (p<0.05) that are enriched in cells activated by adjuvant/Fat1562 compared with adjuvant alone in VAT (left) or Spl/LN (right). NES, normalized enrichment score. FDR, false discovery rate. (C) Normalized reads of *Pparg* transcript in CD45.2⁺ clonotype⁺ Treg cells sorted from mice immunized with adjuvant alone or adjuvant/Fat1562. Data are mean ± SD. (D) Volcano plot comparing gene expression of transferred clonotype⁺ Treg cells in mice immunized with adjuvant/Fat1562 and adjuvant alone in the VAT (left) and spleen (right). Representative transcripts are highlighted.

Figure 6 with 1 supplement

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Immunization with Fat1562 expands transferred vTreg53 Treg cells and suppresses HFD-induced VAT inflammation.

(A) Scheme of the transfer and immunization protocol. (B) Frequencies of CD45.2⁺ cells in Treg cells (n ≥ 7). (C) Frequencies of clonotype⁺ cells in transferred CD45.2⁺ Treg cells (n ≥ 7). (D) Number of clonotype⁺ CD45.2⁺ Treg cells in the spleen (n ≥ 7). (E) Number of clonotype⁺ CD45.2⁺ Treg cells in the VAT (n ≥ 7). (F) Number of total Treg cells in the spleen (n ≥ 7). (G) Number of total Treg cells in VAT (n ≥ 7). (H) Numbers of eosinophils and CD11c^hi inflammatory macrophages in VAT (n ≥ 7). Data are mean ± SD. Data shown are representative of at least two independent experiments.

Figure 6—figure supplement 1

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Gating strategy.

Gating strategy used for detection of VAT Treg cells (top) and, eosinophils or macrophage cells (bottom) by flow cytometry.

Figure 7 with 1 supplement

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Immunization with Fat1562 following vTreg53 Treg transfer ameliorates HFD-induced insulin resistance.

(**A–F**) Metabolic indices of mice treated as in Figure 6A (n ≥ 7). (A) Body weight. (B) Insulin tolerance test (ITT). (C) Area over curve for ITT analysis. (D) Glucose tolerance test (GTT). (E) Area under curve for GTT analysis. (F) Plasma insulin levels after 6 hr of fasting. Data shown are representative of at least two independent experiments. (G) Scheme of the anti-TNFα experiment. (H) Number of total Treg cells in VAT (n ≥ 5). (I) Number of CD11c^hi inflammatory macrophages in VAT (n ≥ 5). (J) ITT (n ≥ 5). (K) Area over curve for ITT analysis (n ≥ 5). Data are mean ± SD.

Figure 7—figure supplement 1

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Absolute blood glucose levels.

ITT analysis of mice transferred with vTreg53 TCR-tg Tregs and immunized with adjuvant/Fat1562 or adjuvant alone. Data shown are representative of at least two independent experiments (n ≥ 5).

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (Mus. musculus)	C57BL/6(J)	Jackson lab	Stock No: 000664
Genetic reagent (Mus. musculus)	CD45.1. C57BL/6	Jackson lab	Stock No: 002014
Genetic reagent (Mus. musculus)	vTreg53 TCR-tg	Li et al., 2018
Genetic reagent (Mus. musculus)	Foxp3-GFP	Gift of Vijay. Kuchroo, (Brigham and Women’s Hospital)
Cell line (Homo sapiens)	Jurkat E6.1	ATCC	Cat# TIB-152 RRID:CVCL_0367
Cell line (Homo sapiens)	Lenti-X 293T	Takara Bio	Cat# 632180
Cell line (Homo sapiens)	K-562	ATCC	Cat# CCL-243, RRID:CVCL_0004
Antibody	anti-CD45.1 (clone A20)	Biolegend	Cat# 110724 RRID:AB_493733	FACS (1:100)
Antibody	anti-CD45.2 (clone 104)	Biolegend	Cat# 109824 RRID:AB_830789	FACS (1:100)
Antibody	anti-CD3 (clone 17A2)	Biolegend	Cat#: 100214 RRID:AB_493645	FACS (1:100)
Antibody	anti-CD4 (clone GK1.5)	Biolegend	Cat#: 100422 RRID:AB_312707	FACS (1:100)
Antibody	anti-TCR Vα2 (clone B20.1)	Biolegend	Cat#: 127806 RRID:AB_1134188	FACS (1:100)
Antibody	anti-CD44 (clone IM7)	Biolegend	Cat#: 103032 AB_2076204	FACS (1:100)
Antibody	anti-CD62L (clone MEL-14)	Biolegend	Cat#: 104418 RRID:AB_313103	FACS (1:100)
Antibody	anti-CD45 (clone 30-F11)	Biolegend	Cat#: 103126 RRID:AB_493535	FACS (1:100)
Antibody	anti-CD11b (clone M1/70)	Biolegend	Cat#: 101228 RRID:AB_893232	FACS (1:100)
Antibody	anti-CD11c (clone N418)	Biolegend	Cat#: 117318 RRID:AB_493568	FACS (1:100)
Antibody	anti-F4/80 (clone BM8)	Biolegend	Cat#: 123116 RRID:AB_893481	FACS (1:100)
Antibody	anti-c-Myc Alexa Fluor 488	Cell Signaling Technology	Cat# 9402 RRID:AB_2151827	FACS (1:100)
Antibody	anti-CD69 (clone FN40)	Biolegend	Cat# 104508, RRID:AB_313111	FACS (1:100)
Antibody	anti-CD25 (clone M-A25)	Biolegend	Cat# 356110, RRID:AB_2561977	FACS (1:100)
Recombinant protein	Streptavidin APC	Biolegend	Cat#: 405243	FACS (40–400 nM)
Antibody	anti-TCR Vβ4 (KT4)	BD Biosciences	Cat#: 553366 RRID:AB_394812	FACS (1:100)
Antibody	anti-Siglec-F (E50-2440)	BD Biosciences	Cat#: 552126 RRID:AB_394341	FACS (1:100)
Antibody	anti-Foxp3 (clone FJK-16s)	ThermoFisher	Cat#: 17-5773-82 RRID:AB_469457	FACS (1:100)
Antibody	anti-TNFα (clone XT3.11)	BioXCell	Cat#: BE0058 RRID:AB_1107764	In vivo 10 μg/g
antibody	isotype control IgG (clone HRPN)	BioXCell	Cat#: BE0088 RRID:AB_1107775	In vivo 10 μg/g
Commercial assay or kit	Streptavidin microbeads	Miltenyi	Cat#: 130-048-101	50 μl/sample
Commercial assay or kit	Foxp3/Transcription Factor Staining Buffer Set	ThermoFisher	Cat#: 00-5523-00
Commercial assay or kit	CellTrace Violet dye	ThermoFisher	Cat#: C34557
Commercial assay or kit	Ultra Sensitive Mouse Insulin ELISA Kit	Crystal Chem	Cat#: 90080
Chemical compound, drug	D-(+)-Glucose	Sigma Aldrich	Cat#: G8270-1KG
Chemical compound, drug	Humulin R	Lilly	Cat#: U-100
Chemical compound, drug	Freund’s Adjuvant, Complete	Sigma	Cat#: F5881
Chemical compound, drug	Freund’s Adjuvant, Incomplete	Sigma	Cat#: F5506
Software, algorithm	Multiplot Studio	Genepattern	https://www.genepattern.org/modules/docs/Multiplot/2
Software, algorithm	GSEA	Broad Institute	https://www.gsea-msigdb.org/gsea/index.jsp
Software, algorithm	V-QUEST	IMGT	http://www.imgt.org/IMGT_vquest/user_guide