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Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

ABSTRACT: Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model developed using HLA.A2 transgenic mice. Wild-type C57BL/6 mice were sensitized with skin allografts from C57BL/6-Tg (HLA-A2.1)1Enge/J mice and treated with TCZ, BM-MSC, or both TCZ and BM-MSC. We compared HLA.A2-specific IgG levels and subsets of T cells and B cells using flow cytometry among groups. HLA.A2-specific IgG level was decreased in all treated groups in comparison with that in the allo-sensitized control (Allo-CONT) group. Its decrease was the most significant in the TCZ + BM-MSC group. Regarding the B cell subset, combined use of TCZ and BM-MSC increased proportions of pre-pro B cells but decreased proportions of mature B cells in BM (p < 0.05 vs. control). In the spleen, an increase in transitional memory was observed with a significant decrease in marginal, follicular, and long-lived plasma B cells (p < 0.05 vs. control) in the TCZ + BM-MSC group. In T cell subsets, Th2 and Th17 cells were significantly decreased, but Treg cells were significantly increased in the TCZ+BM-MSC group compared to those in the Allo-CONT group in the spleen. Regarding RNA levels, IL-10 and Foxp3 showed increased expression, whereas IL-23 and IFN-γ showed decreased expression in the TCZ + BM-MSC group. In conclusion, combined use of TCZ and BM-MSC can inhibit B cell maturation and up-regulate Treg cells, finally resulting in the reduction of HLA.A2-specific IgG in a highly sensitized mouse model. This study suggests that the combined use of TCZ and BM-MSC can be proposed as a novel strategy in a desensitization protocol for highly sensitized patients.

SUBMITTER: Fang X

PROVIDER: S-EPMC10855827 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

Fang Xianying X Cui Sheng S Lee Hanbi H Min Ji Won JW Lim Sun Woo SW Oh Eun-Jee EJ Yang Chul Woo CW Shin Yoo Jin YJ Chung Byung Ha BH

International journal of molecular sciences 20240123 3

Sensitization to HLA can result in allograft loss for kidney transplantation (KT) patients. Therefore, it is required to develop an appropriate desensitization (DSZ) technique to remove HLA-donor-specific anti-HLA antibody (DSA) before KT. The aim of this research was to investigate whether combined use of the IL-6 receptor-blocking antibody, tocilizumab (TCZ), and bone-marrow-derived mesenchymal stem cells (BM-MSCs) could attenuate humoral immune responses in an allo-sensitized mouse model deve ...[more]

PMID: 38338657

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Project description:BACKGROUND:We investigated in vitro whether HLA highly sensitized patients with end-stage renal disease will be disadvantaged immunologically after a genetically engineered pig kidney transplant. METHODS:Blood was drawn from patients with a calculated panel-reactive antibody (cPRA) 99% to 100% (Gp1, n = 10) or cPRA 0% (Gp2, n = 12), and from healthy volunteers (Gp3, n = 10). Serum IgM and IgG binding was measured (i) to galactose-α1-3 galactose and N-glycolylneuraminic acid glycans by enzyme-linked immunosorbent assay, and (ii) to pig red blood cell, pig aortic endothelial cells, and pig peripheral blood mononuclear cell from α1,3-galactosyltransferase gene-knockout (GTKO)/CD46 and GTKO/CD46/cytidine monophosphate-N-acetylneuraminic acid hydroxylase-knockout (CMAHKO) pigs by flow cytometry. (iii) T-cell and B-cell phenotypes were determined by flow cytometry, and (iv) proliferation of T-cell and B-cell carboxyfluorescein diacetate succinimidyl ester-mixed lymphocyte reaction. RESULTS:(i) By enzyme-linked immunosorbent assay, there was no difference in IgM or IgG binding to galactose-α1-3 galactose or N-glycolylneuraminic acid between Gps1 and 2, but binding was significantly reduced in both groups compared to Gp3. (ii) IgM and IgG binding in Gps1 and 2 was also significantly lower to GTKO/CD46 pig cells than in healthy controls, but there were no differences between the 3 groups in binding to GTKO/CD46/CMAHKO cells. (iii and iv) Gp1 patients had more memory T cells than Gp2, but there was no difference in T or B cell proliferation when stimulated by any pig cells. The proliferative responses in all 3 groups were weakest when stimulated by GTKO/CD46/CMAHKO pig peripheral blood mononuclear cell. CONCLUSIONS:(i) End-stage renal disease was associated with low antipig antibody levels. (ii) Xenoreactivity decreased with increased genetic engineering of pig cells. (iii) High cPRA status had no significant effect on antibody binding or T-cell and B-cell response.

Project description:BackgroundKidney transplantation is the most effective treatment for end-stage kidney disease. Sensitization, the formation of human leukocyte antigen (HLA) antibodies, remains a major barrier to successful kidney transplantation. Despite the implementation of desensitization strategies, many candidates fail to respond. Current progress is hindered by the lack of biomarkers to predict response and to guide therapy. Our objective was to determine whether differences in immune and gene profiles may help identify which candidates will respond to desensitization therapy.Methods and findingsSingle-cell mass cytometry by time-of-flight (CyTOF) phenotyping, gene arrays, and phosphoepitope flow cytometry were performed in a study of 20 highly sensitized kidney transplant candidates undergoing desensitization therapy. Responders to desensitization therapy were defined as 5% or greater decrease in cumulative calculated panel reactive antibody (cPRA) levels, and non-responders had 0% decrease in cPRA. Using a decision tree analysis, we found that a combination of transitional B cell and regulatory T cell (Treg) frequencies at baseline before initiation of desensitization therapy could distinguish responders from non-responders. Using a support vector machine (SVM) and longitudinal data, TRAF3IP3 transcripts and HLA-DR-CD38+CD4+ T cells could also distinguish responders from non-responders. Combining all assays in a multivariate analysis and elastic net regression model with 72 analytes, we identified seven that were highly interrelated and eleven that predicted response to desensitization therapy.ConclusionsMeasuring baseline and longitudinal immune and gene profiles could provide a useful strategy to distinguish responders from non-responders to desensitization therapy. This study presents the integration of novel translational studies including CyTOF immunophenotyping in a multivariate analysis model that has potential applications to predict response to desensitization, select candidates, and personalize medicine to ultimately improve overall outcomes in highly sensitized kidney transplant candidates.

Dataset Information

Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

Publications

Combined Use of Tocilizumab and Mesenchymal Stem Cells Attenuate the Development of an Anti-HLA-A2.1 Antibody in a Highly Sensitized Mouse Model.

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