Human thymopoiesis produces polyspecific CD8+ α/β T cells responding to multiple viral antigens

  1. Valentin Quiniou
  2. Pierre Barennes
  3. Vanessa Mhanna
  4. Paul Stys
  5. Helene Vantomme
  6. Zhicheng Zhou
  7. Federica Martina
  8. Nicolas Coatnoan
  9. Michele Barbie
  10. Hang-Phuong Pham
  11. Béatrice Clémenceau
  12. Henri Vie
  13. Mikhail Shugay
  14. Adrien Six
  15. Barbara Brandao
  16. Roberto Mallone
  17. Encarnita Mariotti-Ferrandiz
  18. David Klatzmann  Is a corresponding author
  1. Sorbonne Université, INSERM, Immunology‐Immunopathology‐Immunotherapy, France
  2. AP‐HP, Hôpital Pitié‐Salpêtrière, Clinical Investigation Center for Biotherapies (CIC‐BTi) and Immunology‐Inflammation‐Infectiology and Dermatology Department (3iD), France
  3. Université Paris Cité, Institut Cochin, CNRS, INSERM, France
  4. ILTOO pharma, Statistical department, France
  5. CRCINA, INSERM, CNRS, Université d'Angers, Université de Nantes, France
  6. Center of Life Sciences, Skoltech, Russian Federation
  7. Assistance Publique Hôpitaux de Paris, Service de Diabétologie et Immunologie Clinique, Cochin Hospital, France
9 figures and 1 additional file

Figures

Figure 1 with 3 supplements
Thymocyte differentiation produces clustered CDR3s with high generation probability and preferential TRB VJ gene combinations.

(A) Representation of βCDR3aa networks from DPCD3, DPCD3+, and CD8+. Each dot represents a single CDR3. Dots are connected (forming clusters) by edges defined by Levenshtein distance of ≤1 (one AA …

Figure 1—figure supplement 1
Dotplot of thymocytes sorting.

(A) Dotplot of thymocytes sorting. Cells from the different patients were sorted according to their phenotypes: CD3+CD4CD8+ for ThyCD8, CD3+CD4+CD8+ for DPCD3+ and CD3CD4+CD8+ for DPCD3. (B) …

Figure 1—figure supplement 2
Properties of thymocytes’ TCRs.

βCDR3 network during thymopoiesis. Representation of the 18,000 most frequent βCDR3 networks from DPCD3+ and CD8+ thymocytes of eight donors (Pn).

Figure 1—figure supplement 3
Properties of thymocytes’ TCRs.

(A, B) Clustered αCDR3s from DPCD3+ and CD8+ thymocytes. Analyses were performed on the first 18,000 most frequent αCDR3s per sample (n=6 for DPCD3+ and n=10 for CD8+ thymocytes). (A) Percentage of …

Figure 2 with 1 supplement
Thymocyte differentiation produces clustered CDR3s with high publicness.

Mean percentages of public (black) or private (gray) βCDR3s in all, dispersed or clustered nodes (****p<0.0001, Mann–Whitney test, mean ± s.e.m, n=13). (B) Enrichment of public βCDR3s in the CD8+

Figure 2—source data 1

Enrichment of public βCDR3s in CD8+ thymocytes versus DPCD3+.

https://cdn.elifesciences.org/articles/81274/elife-81274-fig2-data1-v1.docx
Figure 2—figure supplement 1
Publicness of thymocytes’ CDR3s.

(A) βCDR3 sharing between individuals. Pie charts represent the sharing between individuals before (DPCD3+) and after thymic selection (CD8+). Colours represent the dispersed (gray) or clustered …

Figure 3 with 3 supplements
Clustered public TCRs are enriched for virus-specific TCRs.

(A) Barplots showing the mean percentages of CDR3s associated with pathogens within DPCD3+ and ThyCD8 cells and for dispersed or clustered CDR3s. (B) Mean generation probability of virus-specific …

Figure 3—source data 1

Enrichment of virus-specific βCDR3s from databases in clustered CD8+ thymocytes.

https://cdn.elifesciences.org/articles/81274/elife-81274-fig3-data1-v1.docx
Figure 3—figure supplement 1
Virus specificities of thymocytes’ CDR3s.

(A) Enrichment of virus-specific βCDR3s in the CD8+ thymocyte repertoire. Representation of the generation probability as a function of βCDR3 connections in individuals (Pn). The contour plots …

Figure 3—figure supplement 2
Sharing of virus specific thymocytes’ CDR3s.

(A) Network of clustered nodes from the first top ranking 18,000 CDR3s. For each individual, we plot CDR3s as viral-associated (red) either if they are in the public database (lines 1 and 2) or if …

Figure 3—figure supplement 3
Binding properties of single-cell TCRs.

(A) Network of tetramer-binding CDR3s from public database (VDJdb) identifying CDR3s with multiple viral specificities. Each dot represents a single CDR3. Dots are connected by an edge defined by a …

Figure 4 with 2 supplements
Identification of polyspecific TCRs.

(A) Analysis of shared T-cell specificities with the GLIPH2 algorithm. 216,000 CDR3β sequences from the CD8 thymocytes and 32,496 sequences from VDJdb that are tetramer specific were analysed to …

Figure 4—source data 1

List of peptides represented on the chord plot from Figure 4C.

https://cdn.elifesciences.org/articles/81274/elife-81274-fig4-data1-v1.docx
Figure 4—figure supplement 1
Binding patterns to A*03 and A*11 dextramers.

(A) Heatmap of the binding score for different dextramers of different TCRs using the same CASSIRSSYEQYF βCDR3, in two donors. The recognition properties are remarkably similar whatever the βCDR3. …

Figure 4—figure supplement 2
Single-cell gene expression analysis.

(A) Six of the most expanded clonotypes were selected from the single-cell data set, of which three were specific to the Influenza_A0201 GILGFVFTL dextramer (A02_GILG9 specific), and the other three …

Figure 5 with 2 supplements
Polyreactivity of polyspecific TCRs.

(A) Schematic representation of the in vitro cross-activation experiment. (B) In vitro activation of polyspecific T cells. Percentage of IFNγ producing emCD8+ cells after activation with …

Figure 5—source data 1

List of the sequences of individual TCRs expressed and analyzed in vitro in Figure 5.

https://cdn.elifesciences.org/articles/81274/elife-81274-fig5-data1-v1.docx
Figure 5—figure supplement 1
Gating strategy for sorting effector memory (emCD8) Dextramer positive cells.

(A, B) CD8 were previously enriched from cytapheresis PBMC. Gating strategy of the sorting of Cytomegalovirus (A) or Epstein-Barr virus (B) dextramère positive effector memory CD8 (emCD8) from CD8 …

Figure 5—figure supplement 2
Polyspecific properties of anti-CMV cytotoxic cells.

(A) PBMC were stimulated in vitro using Peptivator CMV pp65 (that consists mainly of 15-mer peptides with 11-amino acid overlap, covering the complete sequence of the pp65 protein) and cultured for …

Author response image 1
Author response image 2
Author response image 3
multi-GIL are multi-specific clonotypes with the highest specificity being towards the A02-GIL dextramer.

Multi-other refers to multi-specific clonotypes without any differentially high binding to any of the dextramers. Finally, uni-GIL are uni-specific clonotypes that exclusively bind the A02-GIL …

Author response image 4

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