Phenotypic, Transcriptional, and Functional Analysis of Liver Mesenchymal Stromal Cells and Their Immunomodulatory Properties

Abstract

The liver is an immunologically active organ with a tolerogenic microenvironment at a quiescent state. The immunoregulatory properties of the liver appear to be retained after transplantation because liver allografts can reduce alloresponses against other organs that are simultaneously transplanted. Mechanisms of this phenomenon remain unknown. Given the known immunomodulatory properties of mesenchymal stromal cells (MSCs), we hypothesized that liver mesenchymal stromal cells (L‐MSCs) are superior immunomodulators and contribute to liver‐mediated tolerance. L‐MSCs, generated from human liver allograft biopsies, were compared with adipose mesenchymal stromal cells (A‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs). Trilineage differentiation of L‐MSCs was confirmed by immunohistochemistry. Comparative phenotypic analyses were done by flow cytometry and transcriptome analyses by RNA sequencing in unaltered cell cultures. The in vitro functional analyses were performed using alloreactive T cell proliferation assays. The transcriptome analysis showed that the L‐MSCs are different than the A‐MSCs and BM‐MSCs, with significant enrichment of genes and gene sets associated with immunoregulation. Compared with the others, L‐MSCs were found to express higher cell surface levels of several select immunomodulatory molecules. L‐MSCs (versus A‐MSCs/BM‐MSCs) inhibited alloreactive T cell proliferation (22.7% versus 56.4%/58.7%, respectively; P < 0.05) and reduced the frequency of interferon ɤ–producing T cells better than other MSCs (52.8% versus 94.4%/155.4%; P < 0.05). The antiproliferative impact of L‐MSCs was not dependent on cell‐to‐cell contact, could be reversed incompletely by blocking programmed death ligand 1, and required a higher concentration of the competitive inhibitor of indoleamine 2,3‐dioxygenase for complete reversal. In conclusion, L‐MSCs appear to be uniquely well‐equipped immunomodulatory cells, and they are more potent than A‐MSCs and BM‐MSCs in that capacity, which suggests that they may contribute to liver‐induced systemic tolerance.

The Immunological Basis of Liver Allograft Rejection

“…MSCs can reduce the activity of T-cells using different mechanisms, for example by secretion of indoleamine 2,3-dioxygenase (IDO), an enzyme capable of metabolizing the amino acid tryptophan to kynurenine. T-cells rely on this amino acid to become activated, and the depletion of tryptophan induces apoptosis or inhibiting their proliferation and differentiation by cell to cell contact, a process mediated by PD-L1 ( 101 , 102 ).…”

Transplant Tolerance Induction: Insights From the Liver

“…Adult liver-derived MSC are negative for human leukocyte Ag class II (HLA-II) and the co-stimulatory molecules, including CD80 and CD86, which can inhibit the proliferation of T cells activated by mitogen (34). Interestingly, liver graft-derived MSC have greater capacity to suppress allo-reactive T cell proliferation and cytotoxic degranulation than bone marrow-derived MSC (BM-MSC) (35), as well as significantly higher levels of immune-regulatory genes than adipose tissue-derived MSC and BM-MSC, that depend on programmed cell death ligand 1(PDL1) expression (36) for their ability to subvert T cell response.…”

Comparable in vitro Function of Human Liver-Derived and Adipose Tissue-Derived Mesenchymal Stromal Cells: Implications for Cell-Based Therapy

“…The detailed characterization of both the L-MSC and the A-MSC used in the current study was reported previously (Conley et al, 2020;Taner et al, 2020). Both types of MSCs demonstrated plastic-adherent characteristics and spindleshaped morphology when cultured in PL5% MSC media.…”

“…Given the liver's unique tolerogenic microenvironment (Taner et al, 2016(Taner et al, , 2017(Taner et al, , 2018, we had postulated that liver MSCs (L-MSCs) are superior immunomodulators than their counterparts isolated from other tissues. In fact, we demonstrated that L-MSCs inhibited alloreactive T cell proliferation better than MSCs isolated from adipose tissue (A-MSC) and bone marrow (BM-MSC) (Taner et al, 2020). Transcriptome analysis also demonstrated that the L-MSCs have significantly upregulated expression of genes and gene sets associated with immune regulation.…”

“…All the cells used in the current study had been isolated and characterized in our previous studies independently. The six primary L-MSC batches tested herein were generated from deceased donor liver allografts (Taner et al, 2020). Table 1 summarizes the demographics of each group.…”