Effect of cryopreservation method on islets specific T cell responses in type 1 diabetic patient.
10.3969/j.issn.1672-7347.2013.02.010
- Author:
Lin YANG
1
;
Chen CHAO
;
Wei TANG
;
Zhen WANG
;
Haibo YU
;
Qiuyan CUI
;
Zhiguang ZHOU
Author Information
1. Department of Endocrinology, Second Xiangya Hospital, Changsha, China.
- Publication Type:Journal Article
- MeSH:
Adult;
CD4-Positive T-Lymphocytes;
immunology;
CD8-Positive T-Lymphocytes;
immunology;
Cryopreservation;
Diabetes Mellitus, Type 1;
blood;
immunology;
Female;
Humans;
Islets of Langerhans;
immunology;
Leukocytes, Mononuclear;
cytology;
Male;
T-Cell Antigen Receptor Specificity;
immunology;
T-Lymphocytes;
cytology;
immunology;
Young Adult
- From:
Journal of Central South University(Medical Sciences)
2013;38(2):169-175
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To explore the better freezing protocol to preserve peripheral blood monuclear cells (PBMCs), islets antigen-specific T cells responses compared with freshly isolated samples in type 1 diabetic (T1D) patients.
METHODS:The T cell Workshop Committee of the Immunology of Diabetes Society (IDS-TCW) organized the Freezing Study I and we were one of the 9 centers in the world to participate in the study. According to the two standardized T cell freezing protocols (warm and cold) to freshly isolated PBMCs in terms of recovery, viability, cell subset composition (FACS) and performance in Enzyme-linked immunospot (ELISPOT) assays, we chose 5 newly onset T1D patients and 5 age and sex matched healthy controls. Besides the protocols, all the freezing reagents and antigens were also centralized. The antigens used in ELISPOT were labeled blindedly.
RESULTS:1) Although warm frozen-thawed (W) samples had a slightly higher recovery rate (61.2% vs 60.1%, P>0.05) and viability (77.5% vs 74.9%, P>0.05) as compared with the cold frozen ones (C), the difference was not significant. 2) Both protocols led to a relative loss in monocytes as compared with the fresh samples (F) [3.2±1.1% (C) and 3.0±0.9% (W) vs 7.0±1.1% (F), both P<0.05], while other subsets including CD4+T, CD8+T, B cells, NK cells and NKT cells didn't. 3) Freezing and fresh samples showed similar IFN-γ secretion responses to polystimuli in ELISPOT. Irrespective of the freezing protocol, recall antigen Pediacel and islet antigen-reactive responses were both lower in the frozen cells compared with fresh PBMCs. The stimuli index (SI) of GADspecific T cell response in the fresh samples from T1D patients was 5.1, higher than that of frozen samples with either cold protocol (1.3) or warm one (1.4) (both P<0.05). Only fresh samples from T1D showed significantly higher GAD-specific T cell responses than the healthy controls no matter in SI (5.1 vs 0.9, P<0.05) or spot forming cells (8.1 vs 0.1, P<0.05), whereas the frozen samples did not show such difference.
CONCLUSION:More studies are needed to verify a freezing method to bring comparable islets antigen specific T cell responses in T1D patients to fresh PBMCs.
