Introduction
We have previously described the conserved butryophilin-like (BTNL)-γδ axis in health, whereby human intestinal Vγ4 intraepithelial lymphocytes (IEL) are regulated by local, epithelial-restricted proteins BTNL 3/81. Mice lacking key Btnls have demonstrable absence of mature Vγ7 IEL, suggesting a T cell selection and maintenance function of these proteins2. Furthermore it has previously been described that the BTNL gd axis is dysregulated in coeliac disease3. However this axis and its potential dysregulation has yet to be elucidated in IBD.
Aims & Methods
We aim to compare the status of BTNL dependent γδ T cells in IBD and in healthy tissue, and to assess the potential impact of the BTNL-γδ IEL axis on IBD incidence and progression.
Biopsies obtained at endoscopy from non-IBD controls and patients with IBD with and without inflammation were analysed by flow cytometry and RNA sequencing. We then undertook a large-scale case control study investigating whether there was an association between IBD and a previously described BTNL variant allele. These studies were performed in a test set and findings were replicated and validated using the UK IBD bioresource.
Results
RNA sequencing of colonic γδ cells demonstrated that a subset expressing the integrin αΕβ7 (CD103) have an intraepithelial transcriptomic programme. Moreover, CD103 expression was reversibly downregulated in gut inflammation. Complete restoration of γδ CD103 expression in IBD patients on treatment (n=7) was associated with significantly lower relapse over 5 years follow up than those who demonstrated an ongoing γδ CD103 dysregulation despite therapy (n=12) (HR 6.54 (1.86 – 22.9) p=0.027). Conventional CD8 T cells also express CD103 which is downregulated by inflammation, but restoration of CD8 CD103 phenotype was not associated with sustained remission (HR 0.72 (0.2 to 2.5) ns).
It was noted that a number of patients and non-IBD controls demonstrated low levels of Vγ4 cells. We investigated whether there may be genetic factors which disrupt communication between Vγ4 IEL and the BTNL3/8 complex. A common 56kb copy number variant (CNV) has previously been described (minor allele frequency 0.310)4, and we showed that this polymorphism is hypomorphic in that it disrupts Vγ4-BTNL3 interactions. By phenotyping our local cohort we found that homozygous carriage of this CNV leads to lower proportions of Vγ4 IEL, and those Vγ4 IEL which remain demonstrated an altered phenotype.
To investigate the effects of this on IBD we performed a large case-control study. While there was no association between this CNV and the development of IBD, there was a significant association between the CNV and the development of penetrating Crohn’s disease (CD) in a test set (n=1817), which was subsequently replicated in an independent cohort from the UK NIHR IBD bioresource (n=4491) (OR 1.38 (1.13-1.68) p=0.0015).
Conclusion
This multi-faceted study demonstrates that γδ IEL are an hitherto undescribed marker of inflammation resolution in IBD and may be a useful biomarker of treatment response and restoration of a functioning epithelium. Furthermore, our genetics data clearly implicate the BTNL-Vγ4 axis in regulating CD severity. Only a few genes are associated with IBD prognosis, and for most, the biological pathway involved remains uncertain. By contrast, our study strongly implicates Vγ4 IEL as regulators of IBD pathobiology, possibly by driving wound healing and tissue repair, as has been implied by murine studies. Practical diagnostic and therapeutic applications of this protective pathway should be further investigated.
References
- Melandri et al Nature Immunology 2018; 19: 1352-1365
- Di Marco Barros et al Cell 2016; 167: 203-218
- Mayassi et al Cell 2019; 176: 967-981
- Aigner et al BMC genetics 2013; 14: 61
Disclosure
R.J.D., I.Z., P.V., N.J.P. and A.C.H. have filed provisional patent applications for work described herein. R.J.D. has received speaking/consulting fees from Janssen, Takeda, Tillotts. All unrelated to this manuscript., C.A.L. declares research support and/or fees for development and delivery of non-promotional education from Janssen, Dr Falk, Takeda, Abbvie, AstraZeneca, Eli Lilly, Orion, Pfizer, Roche, Sanofi Aventis, Ferring, UCB, Biogen and Genentech Inc. All unrelated to this manuscript. P.M.I. declares research support and/or fees for development and delivery of non-promotional education from AbbVie, Arena, Boehringer-Ingelheim, BMS, Celgene, Celltrion, Falk Pharma, Ferring, Genentech, Galapagos, Gilead, Hospira, MSD, Janssen, Lilly, Pfizer, Pharmacosmos, Prometheus, Roche, Sandoz, Samsung Bioepis, Takeda, Tillotts, Topivert, Sapphire Medical, Shire, VH2, Vifor Pharma, Warner Chilcott. A.C.H. is a scientific consultant for Prokarium and eGenesis and receives research funds from Takeda Pharmeceuticals. All other authors report no conflict of interest.
