Single Cell approaches for the study of oncogenic processes during coeliac disease.

Project Information

SingCelCD

Grant agreement ID: 843042

DOI

10.3030/843042

Project closed

EC signature date 5 April 2019

Start date 1 May 2019

End date 30 April 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 196 707,84

EU contribution

€ 196 707,84

Coordinated by

IMAGINE INSTITUT DES MALADIES GENETIQUES NECKER ENFANTS MALADES FONDATION
France

Periodic Reporting for period 1 - SingCelCD (Single Cell approaches for the study of oncogenic processes during coeliac disease.)

Reporting period: 2019-05-01 to 2021-04-30

Summary of the context and overall objectives of the project

This project is part of the long-term effort of the team to elucidate how chronic autoimmune-like intestinal inflammation driven by dietary gluten in coeliac disease (CD), can ultimately result into the development of enteropathy-associated T lymphoma (EATL), a rare but severe malignancy. Our past work has demonstrated the origin of EATL from gut intraepithelial lymphocytes (IEL) and shown that, in approximately 70% of CD patients, invasive EATL is preceded by a clonal low-grade intraepithelial lymphoproliferation, usually called type II refractory CD (RCDII).
In the present complementary project, we intended to: 1- to search for an anti-tumor T cell response in RCDII; and 2- to analyze the functional intra-clonal heterogeneity of RCDII IEL and the peri-tumor microenvironment (figure 1).
The results should provide further insight into the mechanisms, which control disease progression and will help us to assess therapeutic strategies. They will notably help to assess if the JAK1/STAT3 pathway is a pertinent therapeutic target.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project has progressed despite some delay due to the Covid epidemics which has considerably restricted digestive endoscopy. Despite this, we have initiated the project by performing single cell transcriptomic approaches on 8 RCDII, and as controls 4 type I refractory CD (RCDI) and 1 healthy donor. RCDI, here used as controls for RCDII, is defined as CD refractory to diet but without evidence of the clonal population of lymphoma cells observed in RCDII. Its pathogenesis is not yet well understood and may be due to a switch from gluten-driven immunity to bona fide autoimmunity. We established a robust pipeline to acquire viable cells from cryopreserved duodenal biopsies with intact transcriptomes and cell surface phenotypes. This protocol yielded immune and stromal cell lineages with preserved surface markers, minimized RNA-seq transcriptomes variability and a high frequency of viable cells compare to fresh cells.
Single-cell RNAseq data allowed us to identify an amplified tumor clone in the biopsy sharing characteristics of malignant RCDII IELs. Interestingly in 3 out of 6 RCDII donors, single cell V(D)J revealed a unique clonotype shared by malignant RCDII IELs expressing an α chain and no in-frame re-arrangement of the β chain. In parallel we used a graph-based clustering algorithm to define tumor heterogeneity. This method allowed us to pinpoint heterogeneity in the transcriptional profile of cells belonging to the cluster of malignant RCDII IELs. The next step will be to define functional signatures of key signaling pathways expressed in individual cells and thus to anticipate their possible contribution to disease progression and resistance to treatment by JAK inhibitors. Finally, to get better insight into the natural history of the malignant transformation induced by CD-associated chronic autoimmune-like activation, we analyzed samples from RCDI donors. Interestingly, in one patient, we found a strongly amplified clone of CD8+ T cells. Further investigations are needed to understand if these cells represent an amplified autoreactive clonotype or may be malignant T cells.
Unfortunately the Covid epidemics has considerably restricted digestive endoscopy causing major delays in the completion of the project. Collection and processing of additional healthy donor biopsies will be necessary to confirm our results and consider their publication.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The present project allowed us to identify amplified tumor clone in RCDII biopsies expressing abnormal V(D)J rearrangements. Furthermore, single cell approches revealed functional intra-clonal heterogeneity of RCDII IELs transcriptomic profils. These results raise the question of the presence of subclones, which may be selected during tumour progression and or treatment. Indeed, studies in many cancers have provided compelling evidence that intra-tumour heterogeneity is a key factor contributing to the lethal outcome of cancer, therapeutic failure, drug resistance and relapsed disease.

In parallel, we identified a putative anti-tumoral T cell responses in RCDII. Interestingly, in the 8 patients so far studied, analysis of paired α and β V(D)J sequences at single-cell level allowed us to pinpoint oligoclonal expansion of CD8+ TCRab+ T cells find both in the biopsy and in the blood, with a transcriptomic profile of exhausted CD8+ T cells that is compatible with tumor infiltrating T cells. Altogether our data suggest that amplified T cells clones might be engaged in anti-tumor response.

The results should provide further insight into the mechanisms, which control disease progression and will help to assess therapeutic strategies. It will notably help to assess if the JAK1/STAT3 pathway is a pertinent therapeutic target and the possible interest of using check-point inhibitors.

Figure 1