Liver homing of clinical grade Tregs after therapeutic infusion in patients with autoimmune hepatitis

Abstract

Autoimmune hepatitis (AIH) is an immune-mediated disease with no curative treatment. Regulatory T cell (Treg) therapy is potentially curative in AIH given the critical role of Tregs in preventing autoimmunity. To work effectively, adoptively transferred Tregs must migrate to and survive within the inflamed liver. We conducted a proof-of-concept study aiming to assess the safety and liver-homing properties of good manufacturing practice (GMP)-grade autologous Tregs in patients with AIH.

Methods: Autologous polyclonal GMP-grade Tregs were isolated using leukapheresis and CliniMACS, labelled with indium tropolonate and re-infused intravenously to 4 patients with AIH. GMP-Treg homing to the liver was investigated with longitudinal gamma camera and SPECT-CT scanning. GMP-Treg immunophenotype, function and immunometabolic state were assessed during the study.

Results: We observed that the isolated Tregs were suppressive and expressed CXCR3, a chemokine receptor involved in recruitment into the inflamed liver, as well as Treg functional markers CD39, CTLA-4 and the transcription factor Foxp3. Serial gamma camera and SPECT-CT imaging demonstrated that 22-44% of infused Tregs homed to and were retained in the livers of patients with autoimmune hepatitis for up to 72 h. The infused cells did not localise to any off-target organs other than the spleen and bone marrow. GMP-Tregs were metabolically competent and there were no infusion reactions or high-grade adverse effects after Treg infusion.

Conclusion: Our novel findings suggest that the liver is a good target organ for Treg cellular therapy, supporting the development of clinical trials to test efficacy in autoimmune hepatitis and other autoimmune liver diseases.

Lay summary: Autoimmune liver diseases occur when the body's immune cells target their own liver cells. Regulatory T cells (Tregs) prevent autoimmunity, thus they are a potential therapy for autoimmune liver diseases. In patients with autoimmune hepatitis, Treg infusion is safe, with nearly a quarter of infused Tregs homing to the liver and suppressing tissue-damaging effector T cells. Thus, Tregs are a potentially curative immune cell therapy for early autoimmune liver diseases.

Keywords: Autoimmune hepatitis; cell therapy; homing; human liver; regulatory T cells.

Graphical abstract

Fig. 1

Graphical representation of AUTUMN Study. (A) Schedule of activities during intervention week between days 1 and 5. (B) Study profile indicating the number of patients to complete each stage of the study from screening, enrollment and leukapheresis to GMP-Treg infusion and 16 weeks follow-up. GMP, Good Manufacturing Practice; Treg, regulatory T cell.

Fig. 2

Immunophenotype and viability of the autologous GMP-Tregs. (A) CXCR3 expression. (B) Viability post indium labelling and immediately before infusion. (C) Purity (CD3⁺CD4⁺CD25⁺CD127^neg Tregs expressed as percentage of total). (D) Memory/naïve phenotype showing the proportions of CD45RA⁺CCR7⁺ (naïve), CD45RA^negCCR7⁺ (central memory), CD45RA^negCCR7^neg (effector memory) and CD45RA⁺CCR7^neg (tissue-resident effector memory RA-positive, TEMRA). (E) Expressions of Treg functional surface proteins. (F) Suppressive potential. GMP, Good Manufacturing Practice; Treg, regulatory T cell.

Fig. 3

Immunometabolic profile of leukapharesis Tregs. (A) OXPHOS profiles; (B) respiratory parameters including: basal respiration, maximal respiration, ATP-linked respiration and spare respiratory capacity; (C) mitochondrial content (MitoTracker) and membrane potential (TMRE) of CD4 non-Treg, Treg and CD8 T cells are shown. (D) Long chain fatty acid levels in normal donor (n = 4) and autoimmune diseased (n = 7) liver tissue supernatants. Statistical significance was tested by Kruskal-Wallis test with Dunn’s multiple comparisons post-hoc test (A-B); 2-way ANOVA with Tukeys multiple comparisons post hoc test (C) and Mann-Whitney U test with Benjamin-Hochberg correction for multiple testing. Data expressed as mean *p ≤0.05. Treg, regulatory T cell.

Fig. 4

Serial Gamma camera scanning of indium labelled GMP-Treg after infusion. Serial Gamma camera imaging performed at 4 h (Ai), 24 h (Aii) and 72 h (Aiii) post GMP-Treg infusion demonstrates the presence of indium tropolonate labelled GMP-Tregs in the liver, spleen and bone marrow. GMP-Tregs were present and remained in the liver for up to 72 h (Aiii). (Anterior = Gamma camera imaging scan from the anterior view; posterior = Gamma camera imaging scan from posterior view). GMP, Good Manufacturing Practice; Treg, regulatory T cell.

Fig. 5

SPECT-CT axial, coronal and sagittal imaging of GMP-Tregs at 24 h after infusion and cell distribution. (A) Fused images showing CT (black and white) with functional colours overlay of SPEC-CT data. Top-bottom axial, coronal and sagittal. Moderate to intense uptake of indium labelled GMP-Tregs is shown in the liver and the spleen. (B) Anatomical delineation of brain, heart, lungs, liver, gut and spleen on multi-planar CT. (C) Relative uptake of indium labelled GMP-Tregs in different organs expressed as the percentage of injected activity. GMP, Good Manufacturing Practice; Treg, regulatory T cell.

Fig. 6

Longitudinal changes in circulating immune cell subsets before and after leukapheresis and Treg infusion. Central memory, effector memory and tissue-resident effector memory RA-positive (TEMRA) subsets of CD8 T cells, CD4 non-Treg and Treg. (B) Granzyme B and perforin-expressing CD4 non-Treg and CD8 T cells. Each patient’s profile between day 1 (D1) and week 16 (W16) is illustrated by a different line: Black: Patient 2; red: Patient 3; blue: Patient 4; green: Patient 5. Significance was assessed using Friedman’s test with Dunn’s Multiple Comparisons post hoc analysis comparing to baseline (D1) (indicated by the box), *p ≤0.05. Treg, regulatory T cell.