title: [Effect of cryopreservation method on islets specific T cell responses in type 1 diabetic patient].
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.
  
  
  CONCLUSIONS
  More studies are needed to verify a freezing method to bring comparable islets antigen specific T cell responses in T1D patients to fresh PBMCs.
authors: Department of Endocrinology, Second Xiangya Hospital, Changsha, China.
authors: Lin
authors: L
authors: Yang
authors: Chen
authors: C
authors: Chao
authors: Wei
authors: W
authors: Tang
authors: Zhen
authors: Z
authors: Wang
authors: Haibo
authors: H
authors: Yu
authors: Qiuyan
authors: Q
authors: Cui
authors: Zhiguang
authors: Z
authors: Zhou
mesh_terms: D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D015925:Cryopreservation; D003922:Diabetes Mellitus, Type 1 / Q000097:blood* / Q000276:immunology; D005260:Female; D006801:Humans; D007515:Islets of Langerhans / Q000276:immunology*; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008297:Male; D037182:T-Cell Antigen Receptor Specificity / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D055815:Young Adult