Cryopreservation of mouse thymus depletes thymocytes but supports immune reconstitution on transplantation

Abstract

Cryopreservation of mouse thymus depletes donor thymocytes but preserves thymus function when transplanted after thawing into athymic mice. No differences in immune reconstitution were observed between fresh and frozen/thawed transplants suggesting that donor thymocyte depletion does not affect outcome. Thus, cryopreservation of thymus may improve outcomes in thymus transplant patients.

Keywords: Clinical immunology; Cryopreservation; Thymopoiesis; Thymus transplantation.

Figure 1

Freezing and thawing deplete the thymus of developing T‐cells. One lobe from E17.5 C57BL/6 thymus was cultured in FTOC fresh (NCPL, grey) or after freeze/thaw (CPL, orange), and analyzed by flow cytometry on Day(D)0 (n = 4), D4 (n = 5), and D7 (n = 5). Data representative of three independent experiments. (A) Scatter‐plot: number of cells on D0, D4, and D7. Each shape represents an individual lobe. (B) Flow cytometry (gating in Supporting information Fig. S1A): CD4 versus CD8, D0, D4, and D7. (C) Plots: number of cells in CD4‐CD8−, CD4+CD8+, CD4+CD8−, and CD4‐CD8+ populations on D0, D4, and D7. (D) Bar charts: percentage and number of TEC(CD45‐Epcam+) D0. (E) Bar charts: percentage and number of TEC(CD45‐Epcam+) D7. (F) Bar charts: percentage and number of cTEC(CD45‐Epcam+CD205+CD40+) and mTEC(CD45‐Epcam+CD205−CD40+) D7. Mean ± SEM, Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

Immune reconstitution in blood, spleen, and LN of nude recipients that received fresh or frozen/thawed thymus transplants. C57BL/6 nude mice transplanted subcutaneously with three congenic sex‐matched GFP‐tg E17.5 thymus lobes fresh (NCPL, grey, n = 5) and after freeze/thaw (CPL, orange, n = 5). Data representative of three independent experiments. (A–D) Blood was analyzed by flow cytometry on Week (W) of transplant (W0) and at 2‐week intervals to W12. (A) Plot: percentage GFP+ cells (gating in Supporting information Fig. S2). (B, D) Bar‐charts: percentage NCPLGFP+(green), CPLGFP+(brown), NCPLGFP−(grey), CPLGFP−(orange) cells: CD3+ (B), CD3+CD4+ (C), CD3+CD8+ (D). (E, F) W12 Spleen (E), LN (F). Bar charts: number of CD4 and CD8 T‐cells. Histograms: GFP‐fluorescence. Plots: CD4 and CD8, gated on TCRβ+, in GFP+(upper), and GFP−(lower). Mean±SEM, Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001.