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.
