Recombinant HLA-G as Tolerogenic Immunomodulant in Experimental Small Bowel Transplantation

Abstract

The non-classical MHC I paralogue HLA-G is expressed by cytotrophoblast cells and implicated with fetomaternal tolerance by downregulating the maternal adaptive and innate immune response against the fetus. HLA-G expression correlates with favorable graft outcome in humans and recently promising immunosuppressive effects of therapeutic HLA-G in experimental transplantation (skin allograft acceptance) were shown. Consequently, we examined this novel therapeutic approach in solid organ transplantation. In this study, therapeutic recombinant HLA-G5 was evaluated for the first time in a solid organ model of acute rejection (ACR) after orthotopic intestinal transplantation (ITX). Allogenic ITX was performed in rats (Brown Norway to Lewis) with and without HLA-G treatment. It was found that HLA-G treatment significantly reduced histologically proven ACR at both an early and late postoperative timepoint (POD 4/7), concomitant to a functionally preserved graft contractility at POD 7. Interestingly, graft infiltration by myeloperoxidase+ cells was significantly reduced at POD7 by HLA-G treatment. Moreover, HLA-G treatment showed an effect on the allogenic T-cell immune response as assessed by flow cytometry: The influx of recipient-derived CD8+ T-cells into the graft mesenteric lymphnodes at POD7 was significantly reduced while CD4+ populations were not affected. As a potential mechanism of action, an induction of T-reg populations in the mesenteric lymphnodes was postulated, but flow cytometric analysis of classical CD4+/CD25+/FoxP3+Treg-cells showed no significant alteration by HLA-G treatment. The novel therapeutic approach using recombinant HLA-G5 reported herein demonstrates a significant immunosuppressive effect in this model of allogenic experimental intestinal transplantation. This effect may be mediated via inhibition of recipient-derived CD8+ T-cell populations either directly or by induction of non-classical Treg populations.

Fig 1. Experimental groups and study design.

Fig 2. Morphologic characteristics and histologic grading of ACR using the Wu-score.

(A) Representative H/E stained intestinal specimens with and without HLA-G treatment. Arrows indicate stronger leucocytic infiltration in graft mesentery, sites of crypt epithelial injury, propria fibrosis and increased crypt apoptotic body counts, respectively. (B) Overview of severity of ACR as assessed by Wu-score (one reviewer shown). n = 5 in each group.

Fig 3. MPO⁺ cells in graft muscularis whole-mounts stained by Hanker-Yates reaction.

At seven days after ITX, HLA-G treatment caused a significant reduction in MPO⁺ cell infiltrate in the graft muscularis.

Fig 4. In-vitro graft contractility measured as a dose-response curve under betanechol stimulation.

Dose response of graft contractility to rising bethanechol concentrations, curves fitted to a logistic four-parameter sigmoidal model. Both midpoint (logEC₅₀₎ and slope were significantly ameliorated by HLA-G treatment at seven days after ITX.

Fig 5. Gating strategy and overview of CD4⁺ and CD8⁺ T-cells isolated from gMLN with and without HLA-G treatment after allogenic intestinal transplantation.

A) Representative gating strategy for recipient(LEW)-derived (MHC I/RT1.Ac-negative), CD45⁺ and CD8⁺ and likewise, CD4⁺ T-cells. Percentages of CD4⁺ and CD8⁺ are given as percentages of CD45⁺ parent population, shown is a representative result for CD8⁺ with and without HLA-G treatment at POD 7. (B) Overview of CD4⁺ and CD8⁺ T-cells with and without HLA-G treatment at both timepoints. A significant increase in CD8⁺ T-cells in the CTL groups is shown without treatment, as well as a significant reduction of this CD8⁺ T-cell population at day seven with HLA-G treatment.

Fig 6. Gating strategy and overview of T_reg (CD4⁺/CD25⁺/FoxP3⁺) isolated from gMLN with and without HLA-G treatment after allogenic intestinal transplantation.

(A) Gating strategy and representative result for Treg analysis. (B) Overview of classical Treg (CD4⁺/CD25⁺/FoxP3⁺ Treg), isolated from gMLN. A tendency to higher abundance in HLA-G treated animals, without reaching statistical significance (Mann-Whitney U test p>0.05), was observed.

Fig 7. ACR-related gene expression in graft muscularis.

The first time point at four days after allogenic ITX showed a significant downregulation of both TNFα and IL-10 by HLA-G treatment (Mann-Whitney U test p<0.05). At seven days after allogenic ITX, TNFα expression remained significantly reduced by HLA-G treatment.