Исследование гемограммы и субпопуляционного состава лимфоцитов крови у минипигов после кондиционирования облучением, трансплантации аллогенного костного мозга и васкуляризированного композитного аллотрансплантата

Hemogram and blood lymphocyte subpopulations in minipigs after irradiation-based conditioning, allogeneic bone marrow transplantation and vascularized composite allograft

Introduction. Bone marrow transplantation (BMT) is one of the key methods of treating oncological, hematological and immune diseases. The success of BMT largely depends on the restoration of the recipient's immune system, in particular, on the dynamics of lymphocyte subpopulations, which play an important role in the prevention of complications such as graft rejection and graft-versus-host disease (GVHD). Currently, experimental and clinical studies are being conducted on the induction of immunological tolerance in combined allogeneic BMT and transplantation of solid organs or vascularized composite allografts (VCA). The majority of preclinical studies are performed on rodents (mainly on linear mice), the breeding and maintenance of which do not require significant financial costs. However, in terms of proximity to human anatomy and physiology, pigs are a more attractive laboratory object. Aim – to study the mechanisms of immune restoration after irradiation and allogeneic bone marrow transplantation during the induction of immunological tolerance to VСA in MHCmismatched minipigs. Material and methods. The study was conducted on minipigs – hybrids of the Wiesenau and Vietnamese pot–bellied breeds – aged 4–5 months, weighing 10–15 kg. Hemogram and subpopulations of blood lymphocytes were assessed before conditioning, on the day of BMT and VCA (day 0) and on days 2–5, 7–8, 11–14, 21–29, 35–49, 57–124. Conditioning was total body irradiation at a dose of 3 Gy on day-2 and local thymic irradiation at a dose of 7 Gy on day-1 using the GUT-200M gamma-irradiation unit. The leukogram (percentage and absolute amount of young and mature neutrophils, basophils, eosinophils, monocytes and lymphocytes) was assessed by immersion microscopy of smears stained according to Romanowsky-Giemsa, lymphocyte subpopulations – by flow cytometry on a BD FACS Canto-II cytometer (USA). B-lymphocytes (PanB-FITC), T-lymphocytes (CD3-PE-Cy7), T-helpers (CD4-APC), T-killers (CD8-PE) (Invitrogen, USA) were measured. Results. Peripheral blood analysis was performed in 24 minipigs that underwent bone marrow transplantation. Radiation conditioning induced significant leukopenia, with nadir values observed at days 2–14 post-irradiation. While partial recovery occurred by week 3 postBMT, complete leukocyte population restoration was not achieved even after 2 months (p = 0.006). Granulocytic lineages demonstrated the earliest recovery: immature neutrophil counts peaked at weeks 3–4 post–irradiation, basophils reached maximum levels at days 35–49, and eosinophil recovery was observed at 2 months post-conditioning and BMT. Monocyte absolute counts returned to normal values by weeks 3–4 post-BMT. Lymphocyte recovery was markedly delayed – despite their relative predominance over neutrophils in early post-BMT periods, absolute lymphocyte counts remained significantly reduced at 2–4 months – 5.01 (3.66–5.98) • 109/L vs baseline 9.45 (7.07–10.16) • 109/L; p = 0.003). Particularly dangerous was the critical depression of erythroid parameters (erythrocytes, hemoglobin, hematocrit) and thrombocytopenia, necessitating transfusion therapy due to high risks of hemorrhage and hypoxia. Lymphocyte subset analysis revealed profound radiation–induced immunosuppression. Maximum B-cell depletion (a 92-fold reduction) was observed on days 7–14 post-BMT – 0.01 (0–0.01) vs pre–conditioning 0.92 (0.64–1.1) million/mL (p = 0.0003). T-lymphocytes reached their lowest levels on days 2–5 post-BMT (a 32-fold decrease compared to control, p = 0.000003) and again on days 21–29 post-HSCT (a 47-fold decrease, p = 0.0003). The T-helper to T-killer cell ratio changed inversely: the CD4/CD8 index was highest on days 2–5 (T-helper predominance) and lowest on days 11–14 (T-killer predominance). A statistically significant low count of all lymphocyte subsets was observed during the first 1.5 months post-BMT, followed by recovery within 2–4 months. Conclusion. Minipigs after conditioning with sublethal irradiation before allogeneic BMT and VCA showed profound leukopenia, anemia and thrombocytopenia. Leukocyte recovery occurred in stages: granulocytes by 3–4 weeks, lymphocytes only after 2–4 months. The greatest immunosuppression was observed in the first 1.5 months after BMT. The obtained results show the need for careful monitoring of hematopoiesis and the immunity in minipigs after allogeneic BMT, especially in the first 3–4 weeks, when the risk of complications (anemia, bleeding, infections) is highest. © 2025, Geotar Media Publishing Group. All rights reserved.