*Purpose: FOXP3⁺regulatory T cells (Tregs) are not a terminally differentiated population & can, under specific environmental conditions, acquire the phenotype of effector T cells. We have reported that Tregs of liver transplanted (LT) recipients with de novo autoimmune hepatitis (DAIH) display a T_H17-like effector phenotype & demonstrate endoplasmic reticulum (ER) stress with unfolded protein response (UPR) activation. Viruses can induce ER stress; thus given our observation of significantly increased expression of the envelope protein of HERV1_I in Tregs of LT recipients with DAIH, we sought to: (i) investigate for the presence of ENV,HERV1_I in serum & liver of LT recipients with DAIH; & (ii) determine how it induces ER stress.

*Methods: Sera was obtained from: (i) LT patients with: (a) DAIH (n=8), (b) normal graft function (LTC) (n=9), (c) acute rejection (n=2), & (ii) healthy non-transplanted children (HC) (n=10), for measurement of ENV,HERV1_I levels by ELISA. RNA was isolated from archived formalin fixed paraffin embedded (FFPE) liver tissue for determination of ENV,HERV1_I expression by qRT-PCR. FACS isolated Tregs of HC was used for: (i) Measurement of interaction between ATF6α & HERV1 using co-Immunoprecipitation Western Blot; (ii) confirmation of ATF6α binding site on RORC & STAT3 promoter using Chromatin Immunoprecipitation; (iii) transduction with ENV,HERV1_I overexpression particle. Following transduction, protein was isolated for measurement of ENV,HERV1_I, phospho-eIF-2α, CHOP, XBP-1s by WB (n = 10);RNA was harvested for measurement of ENV,HERV1_I, sXBP-1, CHOP expression by qRT-PCR (n = 10).

*Results: Significantly increased ENV,HERV1_I levels observed in sera of LT recipients with DAIH vs. sera of LT recipients with normal graft function (LTC) (p < 0.001). Moreover there is also a trend towards increased ENV,HERV1_I levels in sera of recipients with DAIH vs. LT recipients with acute rejection “$$graphicFigure 1A”. ENV,HERV1_I expression also increased in the liver of LT recipients with DAIH vs. LTC and LT recipients with acute rejection “$$graphicFigure 1B”. This confirms its presence in sera & liver of LT recipients with DAIH. In terms of how it induces ER stress, HERV1 interacts with ATF6α, one of the sentinel proteins known to elicit activation of the UPR “$$graphicFigure 2”; moreover, transduction of Tregs with ENV,HERV1_I overexpression particle results in UPR activation as evidenced by up-regulation of downstream UPR mediators, CHOP, XBP-1s & phospho-eIF-2α (p < 0.001 for CHOP & XBP-1s expression; p < 0.001 for CHOP & XBP-1s levels; p = 0.02 for phospho-eIF-2α level). Lastly, ATF6α binds to promoters in RORC & STAT3 “$$graphicFigure 3”, & drives transcription of IL-17A by Tregs (p < 0.001).

*Conclusions: ENV,HERV1_I reactivation may be the initiating event that drives Treg differentiation to TH17-effector Tregs in LT recipients with DAIH. It does this by inducing UPR activation. The subsequent binding of ATF6α to the promoter of RORC & STAT3 results in transcription of IL-17Afrom Tregs.

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