Single‐cell TCR repertoire analysis reveals highly polyclonal composition of human intraepithelial CD8+ αβ T lymphocytes in untreated celiac disease

Celiac disease (CeD) is a chronic inflammatory disease driven by exposure to dietary gluten. The mucosal changes in the small intestine are characterized by infiltration of intraepithelial lymphocytes (IELs) that resolves on a gluten-free diet [1]. In CeD, CD8+ αβ IELs are considered to be central in killing of enterocytes and contribute to tissue destruction [2]. Thus, we wanted to investigate the clonal contribution of these T cells in gut biopsies of CeD affected and non-affected individuals. Early studies in mice showed that the TCRαβ repertoire in the small intestine is oligoclonal [3,4]. Further studies in germfree rats demonstrated that the repertoire is surprisingly broad but transitioned into a more oligoclonal repertoire upon microbial colonization [5]. In humans, it has been reported that the CD8+ αβ repertoire of IELs is oligoclonal in adults but polyclonal during infancy [6–8], suggesting


Letter to the Editor
Single-cell TCR repertoire analysis reveals highly polyclonal composition of human intraepithelial CD8 + αβ T lymphocytes in untreated celiac disease Celiac disease (CeD) is a chronic inflammatory disease driven by exposure to dietary gluten. The mucosal changes in the small intestine are characterized by infiltration of intraepithelial lymphocytes (IELs) that resolves on a gluten-free diet [1]. In CeD, CD8 + αβ IELs are considered to be central in killing of enterocytes and contribute to tissue destruction [2]. Thus, we wanted to investigate the clonal contribution of these T cells in gut biopsies of CeD affected and non-affected individuals. Early studies in mice showed that the TCRαβ repertoire in the small intestine is oligoclonal [3,4]. Further studies in germfree rats demonstrated that the repertoire is surprisingly broad but transitioned into a more oligoclonal repertoire upon microbial colonization [5]. In humans, it has been reported that the CD8 + αβ repertoire of IELs is oligoclonal in adults but polyclonal during infancy [6][7][8] By high-throughput paired single-cell TCRαβ sequencing, we compared the CD8 + αβ IEL repertoire from untreated CeD patients (UCeD, n = 5, 1032 cells), treated CeD patients (TCeD, n = 5, 370 cells) and healthy controls (n = 9, 1499 cells). We found that the IEL repertoire of untreated CeD patients was highly diverse and polyclonal compared to both treated patients and healthy controls. In contrast to previous reports, we found that the IEL infiltration in untreated CeD is characterized by a polyclonal expansion of diverse CD8 + αβ T-cell clonotypes.
Due to the large diversity in TRAV and TRBV gene segments, we next focused on the top 10 expressed TRAV and TRBV genes. The top 10 V genes accounted for approximately 50% of the total V-gene usage in all groups (Fig. 1A). The top 10 TRBV genes used were similar to what have been described for healthy individuals previously [9]. We wanted to look into the TRAV/TRBV gene pairing, as this could differ despite similar gene segment usage. Here we included all available Vgene pairs and found that there were no obvious preferences in V-gene pairing in any group (Fig. 1B). We also compared the relation between expanded clonotypes by dividing each participant group into three; the 10 most expanded clonotypes 1:10, clonotypes 11:100 and 101:1000. Strikingly, the space of the repertoire of which the top 10 clonotypes occupy was found to be markedly lower in the UCeD group compared to the control and the TCeD group ( Fig. 2A). Interestingly, we observed that while the UCeD group had significantly higher Shannon diversity compared with TCeD and control group (p = 0,0079 and p = 0,0016, respectively), the TCeD group was comparable to the controls (Fig. 2B). Furthermore, focusing on each of the top 10 clonotypes for each patient, we observed that the most expanded clonotype (top 1) for both the TCeD patients and controls occupied a large percent of their total TCR repertoires. Moreover, the top 1 clonotypes in all UCeD patients were below 10% of the repertoire in total. The TCeD group resembled the controls with the exception of the top 3 clonotype (Fig. 2C). The sequences of the top 10 clonotypes for all subjects is listed in Table S1.
Overall, these data reveal that the TCR repertoires in untreated CeD are very polyclonal and diverse, but that this increase in diversity is restored to levels comparable with controls when CeD patients are on a GFD. Mainly, we observed that both the TCeD and control subjects had dominating clonotypes that on average accounted for more than 50% of the total repertoires (Fig. 2D). Interestingly, the clonal distribution in the controls, previously reported to be oligoclonal [6,7], varied in a broad range between donors. Importantly, the UCeD group was hallmarked by the contribution of many distinct T-cell clonotypes that occupied a smaller percentage of the total repertoire, and had less dominating expanded clonotypes overall. This feature likely reflects an influx of new CD8 + IEL clonotypes in the active disease state, leading to a polyclonal repertoire that revert to a more oligoclonal repertoire when patients are on a GFD.
In conclusion, our study of single-cell TCRαβ sequencing demonstrates that there is a highly diverse TCR repertoire in CD8 + αβ IELs of untreated CeD without presence of any dominating clonotypes. We also demonstrate that healthy individuals possess more of a mix between oligoclonal and polyclonal IEL repertoires than what has previously been appreciated. This picture is also seen in treated CeD patients. Notwithstanding, the repertoire in CeD patients with active disease is significantly more diverse than what is seen in treated patients and controls. Altogether, these data support the notion that the infiltration of IELs in active CeD is not coming from some selected clones but rather induced by the inflammatory conditions in the small intestine. Ethics declaration: The authors declare no commercial or financial conflict of interests.
Peer review: The peer review history for this article is available at https://pub lons.com/publon/10.1002/eji.202048974

Data availability statement:
The TCR sequencing data supporting this manuscript have been deposited in the European Genome-Phenome Archive (accession number EGAS00001004989).