Immune checkpoints are predominantly co-expressed by clonally expanded CD4+FoxP3+ intratumoral T-cells in primary human cancers

Delphine Bredel (1) , Edi Tihic (1) , Séverine Mouraud (1) , François-Xavier Danlos (1) , Sandrine Susini (1) , Marine Aglave , Alexia Alfaro , Chifaou Mohamed-Djalim , Mathieu Rouanne (1) , Héloise Halse (1, 2) , Amélie Bigorgne (1, 2) , Lambros Tselikas (1) , Stéphane Dalle (3) , Dana M Hartl (4) , Eric Baudin (5) , Catherine Guettier , Eric Vibert , Olivier Rosmorduc , Caroline Robert (6) , Sophie Ferlicot , Bastien Parier , Laurence Albiges (5) , Olaf Mercier (7) , Vincent Thomas de Montpreville , Benjamin Besse (5) , Caroline Even (5) , Ingrid Breuskin (4) , Marion Classe , Camélia Radulescu , Thierry Lebret , Patricia Pautier , Sébastien Gouy (4) , Jean-Yves Scoazec (8) , Laurence Zitvogel (1) , Aurélien Marabelle (9, 5) , Mélodie Bonvalet (1)

1 ITIC - Immunologie anti-tumorale et immunothérapie des cancers
2 Imagine - U1163 - Imagine - Institut des maladies génétiques (IHU)
3 UNICANCER/CRCL - Centre de Recherche en Cancérologie de Lyon
4 IGR - Institut Gustave Roussy
5 Département de médecine oncologique [Gustave Roussy]
6 PMNCO - Prédicteurs moléculaires et nouvelles cibles en oncologie
7 HPPIT - Hypertension pulmonaire : physiopathologie et innovation thérapeutique
8 Département de biologie et pathologie médicales [Gustave Roussy]
9 DITEP - Département d’Innovation Thérapeutique et essais précoces [Gustave Roussy]

Background In addition to anti-PD(L)1, anti-CTLA-4 and anti-LAG-3, novel immune checkpoint proteins (ICP)-targeted antibodies have recently failed to demonstrate significant efficacy in clinical trials. In these trials, patients were enrolled without screening for drug target expression. Although these novel ICP-targeted antibodies were expected to stimulate anti-tumor CD8 + T-cells, the rationale for their target expression in human tumors relied on pre-clinical IHC stainings and transcriptomic data, which are poorly sensitive and specific techniques for assessing membrane protein expression on immune cell subsets. Our aim was to describe ICP expression on intratumoral T-cells from primary solid tumors to better design upcoming neoadjuvant cancer immunotherapy trials. Methods We prospectively performed multiparameter flow cytometry and single-cell RNA sequencing (scRNA-Seq) paired with TCR sequencing on freshly resected human primary tumors of various histological types to precisely determine ICP expression levels within T-cell subsets. Results Within a given tumor type, we found high inter-individual variability for tumor infiltrating CD45 + cells and for T-cells subsets. The proportions of CD8 + T-cells (~ 40%), CD4 + FoxP3-T-cells (~ 40%) and CD4 + FoxP3 + T-cells (~ 10%) were consistent across patients and indications. Intriguingly, both stimulatory (CD25, CD28, 4-1BB, ICOS, OX40) and inhibitory (PD-1, CTLA-4, PD-L1, CD39 and TIGIT) checkpoint proteins were predominantly co-expressed by intratumoral CD4 + FoxP3 + T-cells. ScRNA-Seq paired with TCR sequencing revealed that T-cells with high clonality and high ICP expressions comprised over 80% of FoxP3 + cells among CD4 + T-cells. Unsupervised clustering of flow cytometry and scRNAseq data identified subsets of CD8 + T-cells and of CD4 + FoxP3-T-cells expressing certain † Delphine Bredel and Edi Tihic contributed equally to this work.

Mots clés: Cancer Immunology Immune checkpoints T-cells Immunotherapy Single-cell RNA-Seq Flow cytometry TCR repertoire; Cancer; Immunology; Immune checkpoints; T-cells; Immunotherapy; Single-cell RNA-Seq; Flow cytometry; TCR repertoire; Cancer Immunology Immune checkpoints T-cells Immunotherapy Single-cell RNA-Seq Flow cytometry TCR repertoire

Domaines: Cancer

Delphine Bredel, Edi Tihic, Séverine Mouraud, François-Xavier Danlos, Sandrine Susini, et al.. Immune checkpoints are predominantly co-expressed by clonally expanded CD4+FoxP3+ intratumoral T-cells in primary human cancers. Journal of experimental & clinical cancer research, 2023, 42, ⟨10.1186/s13046-023-02897-6⟩. ⟨hal-04431918⟩