1. Immunology and Inflammation

CD8+ T cell self-tolerance permits responsiveness but limits tissue damage

  1. Emily N Truckenbrod
  2. Kristina S Burrack
  3. Todd P Knutson
  4. Henrique Borges da Silva
  5. Katharine E Block
  6. Stephen D O'Flanagan
  7. Katie R Stagliano
  8. Arthur A Hurwitz
  9. Ross B Fulton
  10. Kristin R Renkema  Is a corresponding author
  11. Stephen C Jameson  Is a corresponding author
  1. Center for Immunology, University of Minnesota, United States
  2. Minnesota Supercomputing Institute, University of Minnesota, United States
https://doi.org/10.7554/eLife.65615
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Cite this article
  1. Emily N Truckenbrod
  2. Kristina S Burrack
  3. Todd P Knutson
  4. Henrique Borges da Silva
  5. Katharine E Block
  6. Stephen D O'Flanagan
  7. Katie R Stagliano
  8. Arthur A Hurwitz
  9. Ross B Fulton
  10. Kristin R Renkema
  11. Stephen C Jameson
(2021)
CD8+ T cell self-tolerance permits responsiveness but limits tissue damage
eLife 10:e65615.
https://doi.org/10.7554/eLife.65615
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6 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
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Trp2/Kb-specific CD8+ T cells in pre-immune wild-type (WT) and Dct-/- mice share a naïve phenotype while showing modest differences in frequency and tetramer staining.

(A) Tetramer enrichment was performed to enumerate Trp2/Kb-specific CD8+ T cells per mouse. Median tetramer fluorescence intensity (MFI) was used to estimate the avidity of enriched Trp2/Kb-specific …

Figure 1—source data 1

Data file related to Figure 1.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig1-data1-v2.pzfx
Figure 1—source data 2

Primers used to screen prospective clones for homologous recombination of the Dct knockout construct.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig1-data2-v2.docx
Figure 1—source data 3

Primers used to screen mice to confirm deletion of the Dct gene.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig1-data3-v2.docx
Figure 1—figure supplement 1
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Generation of Dct-/- mouse model by deleting exons 2 through 6.

(A) The intact Dct gene is shown schematically with eight exons (E1–E8) in green and large intronic regions shown in yellow. The Hurwitz lab designed a new knockout model, which involved deleting …

Figure 1—figure supplement 2
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Additional analysis of the Trp2/Kb-specific population and cells specific for other melanocyte epitopes in pre-immune mice.

(A) Tetramer staining of pre-immune lymphocytes (enriched fraction, gated on live, dump-negative CD8+ T cells). Dual tetramer staining was used to facilitate more accurate gating on antigen-specific …

Figure 2 with 3 supplements
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Differences in the magnitude of the response to Trp2 immunization in wild-type (WT) and Dct-/- mice.

Mice were primed with TriVax (50 μg each of Trp2 and B8R peptides; A–E). The number (A) or percent (B) of splenic Trp2/Kb or B8R/Kb-specific cells was assessed at day 7. (C, D) The tetramer …

Figure 2—source data 1

Data file related to Figure 2.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig2-data1-v2.pzfx
Figure 2—figure supplement 1
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Additional phenotyping and functional analysis of Trp2/Kb-specific cells at day 7 after TriVax.

The median fluorescence intensity of (A, B, D) or frequency of cells expressing (C, D) the indicated marker are shown for Trp2/Kb tetramer-positive cells from the spleens of mice treated with TriVax …

Figure 2—figure supplement 2
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Analysis of Trp2/Kb-specific cells in the skin of wild-type (WT) and Dct-/- mice at day 7 after TriVax.

Mice were treated with TriVax containing Trp2 and B8R peptides (100 μg each) and sacrificed at day 7. An ~2 cm2 piece of skin from the flank of each mouse was shaved, collected, and processed to a …

Figure 2—figure supplement 3
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Response to infection with Listeria monocytogenes strain expressing Trp2 (LmTrp2).

Mice were infected with a recombinant LmTrp2. The percent (A) or number (B, C) of splenic Trp2/Kb-specific cells was assessed at the indicated day. (D) Day 7 splenocytes were stimulated for 4–6 hr …

Figure 3 with 1 supplement
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Wild-type (WT) Trp2/Kb-specific cells exhibit cell-intrinsic tolerance.

(A) We performed negative enrichment for CD8+ T cells from WT or Dct-/- donors and transferred bulk CD8+ T cells into congenically distinct WT or Dct-/- recipients. One day later, mice were …

Figure 3—source data 1

Data file related to Figure 3.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig3-data1-v2.pzfx
Figure 3—figure supplement 1
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Response to infection with Listeria monocytogenes strain expressing Trp2 (LmTrp2) after adoptive transfer.

(A,B) CD8+ T cells from pre-immune wild-type (WT) (A) or Dct-/- (B) donors were negatively enriched and bulk CD8+ T cells were transferred into congenically distinct WT or Dct-/- recipients. One day …

Figure 4 with 1 supplement
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Wild-type (WT) Trp2/Kb-specific cells are capable of an initial response to Trp2 similar to that of Dct-/- cells.

WT and Dct-/- mice received intravenous injections of TriVax with 200 μg Trp2 peptide. Tetramer enrichment was used to enumerate Trp2/Kb-specific cells and assess their phenotype at the indicated …

Figure 4—source data 1

Data file related to Figure 4.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig4-data1-v2.pzfx
Figure 4—figure supplement 1
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Tetramer staining kinetics and response to peptide immunization.

WT and Dct-/- mice received intravenous injections of 200 μg Trp2 peptide as part of TriVax (A) or alone (C–E). (A) Histograms of tetramer staining for Trp2/Kb-binding cells at the indicated day …

Figure 5 with 2 supplements
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Wild-type (WT) Trp2/Kb-specific cells show proliferative defects in the early effector phase.

Trp2/Kb-specific CD8+ T cells were isolated from WT and Dct-/- mice on day 3 after TriVax and submitted for scRNA-seq. After initial processing, the WT and Dct-/- datasets were merged and further …

Figure 5—source data 1

Data file related to Figure 5.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig5-data1-v2.pzfx
Figure 5—figure supplement 1
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scRNAseq analysis of Trp2/Kb-specific cells at day 7 after TriVax.

Wild-type (WT) and Dct-/- mice were treated with TriVax (50 μg Trp2), and Trp2/Kb-specific cells were isolated at day 7 and submitted for scRNA-seq. After initial processing, the WT and Dct-/- …

Figure 5—figure supplement 2
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Gene set enrichment analysis (GSEA) of day 3 single-cell data and response of wild-type (WT) and Dct-/- Trp2/Kb-specific cells to IL-2C.

(A) GSEA plots show enrichment of the following gene sets in Group A: Myc targets, MTORC1 signaling, G2M checkpoint, E2F targets. (B) Enrichment between clusters and a dataset of genes involved in …

Figure 6 with 2 supplements
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Wild-type (WT) Trp2/Kb-specific cells are unable to mediate efficient anti-melanocyte activity.

(A) WT mice were monitored for vitiligo after receiving 50,000 Trp2/Kb-specific cells from WT or Dct-/- donors primed with TriVax 7 days prior; recipient mice received TriVax (100 μg Trp2) on the …

Figure 6—source data 1

Data file related to Figure 6.

https://cdn.elifesciences.org/articles/65615/elife-65615-fig6-data1-v2.pzfx
Figure 6—figure supplement 1
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Vitiligo scoring metric and correlation between the average vitiligo score and the number of transferred Trp2/Kb-specific cells.

(A) Vitiligo scoring metric used to quantify the degree of vitiligo. (B, C) Examples of vitiligo. (D) Average vitiligo score per mouse (days 0–93) relative to the number of transferred Trp2/Kb …

Figure 6—figure supplement 2
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Vitiligo development and skin infiltration in P14 mice receiving cell transfers from wild-type (WT) or Dct-/- donors.

Rag-positive P14 T cell receptor (TCR) transgenic mice were monitored for vitiligo after receiving 50,000 Trp2/Kb-specific cells from WT or Dct-/- donors primed with TriVax 7 days prior; recipient …

Tables

Key resources table
Reagent type
(species) or
resource		DesignationSource or
reference		IdentifiersAdditional
information
Strain, strain background (Mus musculus)Dct-/- (Exon 2–6-/-)Dr. A. Andy Hurwitz, NCI
Strain, strain background (Mus musculus)C57BL/6NCrl (C57BL/6)NCI Charles RiverStrain code: 556
Strain, strain background (Mus musculus)
B6.SJL-PtprcaPepcb/BoyCrCrl (CD45.1)NCI Charles RiverStrain code: 564
Strain, strain background (Mus musculus)P14Dr. R. Ahmed, Emory University
Chemical compound, drugPoly(I:C)InvivogenCat. #: vac-pic 
AntibodyInVivoMAb anti-mouse CD40BioXCellCat. #: BE0016-2;
RRID:AB_1107647		Clone FGK4.5
Peptide, recombinant proteinTrp2 peptideNew England PeptideTrp2180-188 H2N-SVYDFFVWL-OH
Chemical compound, drug1-Fluoro-2,4-dinitrobenzene (DNFB)Sigma AldrichCat. #: D-1529
AntibodyAnti-mouse CD8a FITCTonbo BiosciencesCat. #: 35-0081;
RRID:AB_2621671		Clone 53-6.7
AntibodyAnti-mouse CD8a vf450Tonbo BiosciencesCat. #: 75-0081; RRID:AB_2621931Clone 53-6.7
AntibodyAnti-mouse CD8a PerCP-Cy5.5Tonbo BiosciencesCat. #: 65-0081; RRID:AB_2621882Clone 53-6.7
AntibodyAnti-mouse CD8a APCef780eBioscienceCat. #: 47-0081-80; RRID:AB_1272221Clone 53-6.7
AntibodyAnti-mouse CD4 BV605BioLegendCat. #: 100548; RRID:AB_2563054Clone RM4-5
AntibodyAnti-mouse CD4 PE-Cy7Tonbo BiosciencesCat. #: 60-0042; RRID:AB_2621829Clone RM4-5
AntibodyAnti-mouse CD44 BV786BD BiosciencesCat. #: 563736; RRID:AB_2738395Clone IM7
AntibodyAnti-mouse CD44 FITCeBioscienceCat. #: 11-0441-85; RRID:AB_465046Clone IM7
AntibodyAnti-mouse CD44 rf710Tonbo BiosciencesCat. #:80-0441; RRID:AB_2621985Clone IM7
AntibodyAnti-mouse CD45.2 FITCTonbo BiosciencesCat. #: 35-0454; RRID:AB_2621692Clone 104
AntibodyAnti-mouse CD45.1 PE-Cy7Tonbo BiosciencesCat. #: 60-0453; RRID:AB_2621850Clone A20
AntibodyAnti-mouse CD90.1 ef450eBioscienceCat. #: 48-0900-82; RRID:AB_1272254Clone HIS51
AntibodyAnti-mouse CD90.2 PE-Cy7Tonbo BiosciencesCat. #: 60-0903; RRID:AB_2621857Clone 30-H12
AntibodyAnti-mouse MHC Class II (I-A/I-E) APC ef780Thermo Fisher ScientificCat. #: 47-5321-82; RRID:AB_1548783Clones M5/114.15.2
AntibodyAnti-mouse MHC Class II (I-A/I-E) BV510BioLegendCat. #: 107635; RRID:AB_2561397Clones M5/114.15.2
AntibodyAnti-mouse F4/80 BV510BioLegendCat. #: 123135; RRID:AB_2562622Clone BM8
AntibodyAnti-mouse F4/80 APC ef780Thermo Fisher ScientificCat. #: 47-4801-82; RRID:AB_2735036Clone BM8
AntibodyAnti-mouse CD122 BV421BD BiosciencesCat. #: 562960; RRID:AB_2737918Clone TM-β1
AntibodyAnti-human Granzyme B PEInvitrogen/ThermoCat. #: GRB04; RRID:AB_2536538Clone GB11
AntibodyAnti-mouse IFN-g PE-Cy7Tonbo BiosciencesCat. #: 60-7311-U100; RRID:AB_2621871Clone XMG1.2
Antibody
Anti-mouse TNFa APCeBioscienceCat. #: 17-7321-81; RRID:AB_469507Clone MP6-XT22
AntibodyAnti-mouse CD107a APCBioLegendCat. #: 121614; RRID:AB_2234505Clone ID4B
AntibodyAnti-mouse IL-2 APCBioLegendCat. #: 503810; RRID:AB_315304Clone JES6-5H4
AntibodyAnti-mouse CD62L BV510BD BiosciencesCat. #: 563117; RRID:AB_2738013Clone MEL-14
AntibodyAnti-mouse CD62L PerCP-Cy5.5Tonbo BiosciencesCat. #: 65-0621Clone MEL-14
AntibodyAnti-mouse KLRG1 PE-Cy7eBioscience/ThermoCat. #: 25-5893-82; RRID:AB_1518768Clone 2F1
AntibodyAnti-mouse CD127 BV786BD BiosciencesCat. #: 563748; RRID:AB_2738403Clone SB/199
AntibodyAnti-mouse CD69 FITCTonbo BiosciencesCat. #: 35-0691; RRID:AB_2621698Clone H1.2F3
AntibodyAnti-mouse CD25 PE-Cy7Thermo FisherCat. #: 25-0251-82; RRID:AB_469608Clone PC61.5
AntibodyAnti-mouse CD103 BV510BD BiosciencesCat. #: 563087; RRID:AB_2721775Clone M290
AntibodyAnti-mouse PD-1 PerCP-Cy5.5BioLegendCat. #: 135208; RRID:AB_2159184Clone 29F.1A12
AntibodyAnti-mouse PD-1 FITCBioLegendCat. #: 135214; RRID:AB_10680238Clone 29F.1A12
AntibodyAnti-mouse Tim-3 BV421BioLegendCat. #: 119723; RRID:AB_2616908Clone RMT3-23
AntibodyAnti-mouse CTLA-4 PE-Cy7BioLegendCat. #: 106314; RRID:AB_2564238Clone UC10-4B9
AntibodyAnti-mouse CD5 APCef780Thermo Fisher scientificCat. #: 47-0051-82; RRID:AB_2573940Clone 53-7.3
AntibodyAnti-mouse TCRβ APCef780Thermo Fisher ScientificCat. #: 47-5961-82; RRID:AB_1272173Clone H57-597
AntibodyAnti-mouse LAG3 PE-Cy7BioLegendCat. #: 125226; RRID:AB_2715764Clone C9B7W
AntibodyAnti-mouse CD49a BUV395BD BiosciencesCat. #: 740262; RRID:AB_2740005Clone Ha31/8
AntibodyAnti-mouse CXCR3 PerCP-Cy5.5BioLegendCat. #: 126514; RRID:AB_1186015Clone CXCR3-173
AntibodyInVivoMab anti-mouse IL-2 antibodyBioXCellCat. #: BE0043-1; RRID:AB_1107705Clone S4B6
Chemical compound, drugRecombinant Mouse IL-2 Protein, CFR&D SystemsCat. #: 402-ML-500/CF
Chemical compound, drugStreptavidin, R-Phycoerythrin Conjugate, premium gradeInvivogenCat. #: S21388
Chemical compound, drugStreptavidin, Allophycocyanin Conjugate, premium gradeInvivogenCat. #: S32362
Chemical compound, drugH-2K(b) SVYDFFVWL MonomerNIH Tetramer CoreBiotinylated Monomer
Chemical compound, drugGhost Dye Red 780 Viability DyeTonbo BiosciencesCat. #: 13-0865-T100
Chemical compound, drugLIVE/DEAD Fixable Aqua Dead Cell Stain KitThermo FisherCat. #: L34966
Chemical compound, drugAnnexin V FITC Apoptosis Detection KitBD BiosciencesCat no. 556570; RRID:AB_2869085

Additional files

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