4 . 2024

Assessment of hematopoietic chimerism in minipigs during the induction of immune tolerance to vascularized composite allograft

Abstract

Introduction. Quantitative determination of chimerism after allogeneic bone marrow transplantation (aBMT) is one of the main methods for assessing the possibility of engraftment of allografts (including transplantation of vascularized composite tissue complexes – VCA) from the same donor. Complete stable chimerism correlates with long-term engraftment of donor tissues. Dynamic studies of chimerism are necessary to assess the effectiveness of transplantation with further withdrawal of immunosuppressive therapy.

Aim – to study hematopoietic chimerism in minipigs after allogeneic hematopoietic stem cell transplantation (aHSCT) during the induction of immunological tolerance to VCA.

Material and methods. The study was performed on minipig hybrids of the Wisenau and Vietnamese pot-bellied pig. The chimerism was detected using the polymerase chain reaction (PCR) on days «0», «+14», «+21» and «+30» using the COrDIS PIG reagent kit (GORDIZ, Russia). PCR results were studied by capillary electrophoresis using an automatic genetic analyzer (ABI PRISM 3500 Thermo Fisher Scientific, USA). The obtained data were analyzed using GeneMapper software (Thermo Fisher Scientific, USA). Alleles were assessed using 15 microsatellite markers for each minipig blood sample, and the percentage of donor and recipient alleles was calculated.

Results. 9 pairs of experimental animals (Donor–Recipient) were studied. In 5 recipients, on day +30 after aBMT, only their own alleles were determined in the bone marrow aspirate. Mixed donor chimerism was observed in 4 recipients. Determination of the sensitivity of the technique showed a sensitivity of at least 5 % in a model experiment. The determination error depends on the number of loci taken for analysis and does not exceed 10 % when the content of donor alleles is 50 % in the test sample.

Conclusion. Determination of donor chimerism in minipigs after aBMT during the induction of immunological tolerance to VCA demonstrated the dynamics of bone marrow transplant engraftment and the maximum expansion of the recipient’s bone marrow by donor cells up to 80–90 %. The method for determining chimerism using STR loci is highly sensitive and can be used in model experiments with minipigs.

Keywords: allogeneic bone marrow transplantation; hematopoietic chimerism; short tandem repeats; STR; minipigs

For citation: Brilliantova V.V., Kotskiy M.A., Muravskaya M.P., Kozlitina O.V., Tkachuk Yu.V., Sokolova O.N., Bondarenko A.V., Varlachev A.V., Drokov M.U., Mitin A.N., Donetskova A.D. Assessment of hematopoietic chimerism in minipigs during the induction of immune tolerance to vascularized composite allograft. Immunologiya. 2024; 45 (4): 446–55. DOI: https://doi.org/10.33029/1816-2134-2024-45-4-446-455 (in Russian)

Funding. The study was performed within the framework of the Translational Medicine Laboratory of the Advanced Research Foundation (Agreement No. 6/218/2021-2022cpl). Donetskova A.D. was supported by the Strategic Academic Leadership Program from RUDN University named after P. Lumumba, MSHE of Russia.

Conflict of interests. The authors declare no conflict of interests.

Authors’ contribution. Concept and design of the study – Varlachev A.V., Drokov M.Yu., Mitin A.N.; conditioning and maintaining minipigs, collection of material – Kotskiy M.A., Muravskaya M.P., Kozlitina O.V., Tkachuk Yu.V., Sokolova O.N., Bondarenko A.V.; analysis of microsatellite markers – Brilliantova V.V.; writing and editing of the article – Brilliantova V.V., Donetskova A.D.

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