정상 한국인에서 유세포검사법을 이용한 조절 T세포 참고치 설정
Establishing Reference Intervals for Regulatory T-Cell Levels in a Healthy Korean Population Using Flow Cytometry
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초록
배경: 조절 T세포(regulatory T cell)는 인간 면역체계에서 억제 기능을 통해 면역 항상성 및 자가관용을 조절하는 CD4+ T세포 중 하나이다. 과도하게 활성화된 조절 T세포의 기능 이상은 1형 당뇨와 같은 자가면역질환을 유발하기도 하고, 종양질환에서는 종양미세환경 속에서 다양한 면역세포들과 상호작용을 하기도 한다. 최근에는 면역치료제의 표적으로서도 관심을 받고 있지만, 이전 연구들은 특정질환에만 한정되어 있었다. 이에 이번 연구에서는 정상 성인군에서의 참고치를 제안하고자 한다. 방법: 충북대학교병원에서 90명의 건강인 대조군의 말초혈액 검체를 이용하였다. Duraclone IM Treg tube 키트(Becton Dickinson, USA)를 이용해 8-컬러 유세포검사법을 시행하였으며, 각종 형광단클론항체를 사용하여 CLSI EP28-A3c에 따라 Robust 통계 방법을 이용해 참고치를 설정하였다. 결과: 각각의 면역형광항체 조합에 따른 조절 T세포의 참고치는 다음과 같다. CD3+CD4+ T 림프구집단 기준으로 CD3+CD4+CD25+ (1.34–11.42%), CD3+CD4+CD39+ (0.82–9.20%), CD3+CD4+FoxP3+ (0.61–8.77%), CD3+CD4+Helios+ (1.10–14.73%), CD3+CD4+CD25+ FoxP3+ (0.43–3.80%), CD3+CD4+FoxP3+Helios+ (0.37–6.29%)를 나타내었다. 성별과 나이에 따른 유의한 차이는 관찰되지 않았다. 결론: 이번 연구에서는 건강한 한국 성인에서 조절 T세포의 참고치를 최초로 보고하였으며, 다양한 면역형광항체 조합에 따른 결과값에 따라서 자가면역질환 및 암 종양질환에서 유용하게 사용될 것으로 기대된다.
Abstract
Background: Regulatory T (Treg) cells are a small subset of CD4+ T cells that regulate immune homeostasis and self-tolerance. Hyperactivated Treg cells can cause autoimmune diseases such as type 1 diabetes. In the context of cancer, these cells interact with diverse immune cells within the tumor microenvironment. Recently, Treg cells have garnered substantial attention as a potential target for immunotherapy; however, previous studies have been limited to specific disease groups. Therefore, this study aims to propose reference intervals for Treg cells in healthy donors. Methods: Peripheral blood samples were collected from 90 healthy adults at Chungbuk National University Hospital. Eight-color flow cytometry was performed using a DURAClone IM Treg tube kit (Beckman Coulter, USA). The reference intervals for Treg cell detection, using various fluorescent monoclonal antibodies, were established using the Robust statistical method in accordance with the Clinical Laboratory Standards Institute EP28-A3c guidelines. Results: The reference intervals for Treg cells according to the immunofluorescent antibody combination were determined as follows for each subset of CD3+CD4+ T lymphocytes: CD3+CD4+CD25+ (1.34–11.42%), CD3+CD4+CD39+ (0.82–9.20%), CD3+CD4+FoxP3+ (0.61–8.77%), CD3+CD4+Helios+ (1.10–14.73%), CD3+CD4+CD25+FoxP3+ (0.43–3.80%), and CD3+CD4+FoxP3+Helios+ (0.37–6.29%). No significant differences with respect to sex or age were observed. Conclusions: The reference intervals for Treg cells in healthy Korean adults were determined using various immunofluorescent antibody combinations. This approach is useful for detecting abnormal Treg cell expression associated with autoimmune diseases and malignant diseases.
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