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. Author manuscript; available in PMC: 2016 Sep 1.

Published in final edited form as: Hepatology. 2015 Jul 30;62(3):915–931. doi: 10.1002/hep.27909

(A) Graft hematopoietic NPC promptly became of recipient origin. Liver NPC were isolated at various time points following transplantation of B6 (H-2^b) livers into C3H (H-2^k) recipients (syngeneic transplantation served as control). They were stained for CD45, donor (H-2K^b) and recipient (H-2K^k) MHC class I, and analyzed by flow cytometry. The data was expressed as percentage of H-2K^k+ cells in CD45⁺population. (B) Graft non-hematopoietic NPC remain of graft origin. NPC isolated from long-term surviving liver grafts were analyzed by flow cytometry for CD45, H-2K^b and H-2K^k, and expressed as histograms. Shaded area is isotype control. The number is the percentage of positive cells in the CD45⁺ or CD45⁻ populations, respectively. The data are representative of three separate experiments. (C) Expression of IFN-γR1 on graft non-hematopoietic NPC. CD45⁻ NPC purified from liver grafts (POD 12) were tested for expression of IFN-γR1 in mRNA (q-PCR) (upper panel, n=3) and protein levels (flow cytometry, lower panels). The number is the percentage of IFN-γR1 positive cells in the CD45⁻ cell population. (D) Graft non-hematopoietic cell-mediated T cell inhibition is dependent on intact IFN-γ signaling. CFSE labeled C3H spleen T cells were cultured with B6 DC at a ratio of 20:1 for 3 days. CD45⁻ cells from WT or IFN-γR1^{^−/−} liver allografts (use of DC as regulators for comparison) were added at the beginning into the culture at a regulator: DC ratio of 1:1. A MLR culture without addition of the regulator cells (none) served as control. The proliferation of T cells was determined by CFSE dilution gated on CD4⁺ or CD8⁺ populations (the number is the percentage of dividing cells, upper panels) or by thymidine uptake (n=3, lower panel). (E) Graft CD45⁻ NPC induce T effector cell apoptosis via IFN-γ pathway in vitro. CFSE-labeled H2^b specific CD8⁺ T cells from DES TCR transgenic mice (H2^k) were cultured with B6 DC at a ratio of 20:1 for 3 days. CD45⁻ NPC isolated from WT or IFN-γR1^−/− liver allograft were added at the beginning into the culture at a DC:NPC=1:1. Addition of same number of B6 DC served as control (Cont). The cells were double stained with anti-CD8 and -annexin V mAbs, and analyzed by flow cytometry for the proliferation of specific T cells (CFSE dilution) gated on CD8⁺ population (upper panels). The number is percentage of dividing cells. Expression of annexin V was analyzed on dividing or non-diving CD8⁺ cells. Cell suspensions were stained with TUNEL and examined under a microscope. The number is percentage of annexin V⁺ cells in CD8⁺ cells. The cell suspensions were also stained with TUNEL (red) and examined under microscope, and counted, expressing as mean TUNEL⁺ cell number (in 1000 counted cells) ± SD. The data are representative of two separated experiments.

(A) Graft hematopoietic NPC promptly became of recipient origin. Liver NPC were isolated at various time points following transplantation of B6 (H-2^b) livers into C3H (H-2^k) recipients (syngeneic transplantation served as control). They were stained for CD45, donor (H-2K^b) and recipient (H-2K^k) MHC class I, and analyzed by flow cytometry. The data was expressed as percentage of H-2K^k+ cells in CD45⁺population. (B) Graft non-hematopoietic NPC remain of graft origin. NPC isolated from long-term surviving liver grafts were analyzed by flow cytometry for CD45, H-2K^b and H-2K^k, and expressed as histograms. Shaded area is isotype control. The number is the percentage of positive cells in the CD45⁺ or CD45⁻ populations, respectively. The data are representative of three separate experiments. (C) Expression of IFN-γR1 on graft non-hematopoietic NPC. CD45⁻ NPC purified from liver grafts (POD 12) were tested for expression of IFN-γR1 in mRNA (q-PCR) (upper panel, n=3) and protein levels (flow cytometry, lower panels). The number is the percentage of IFN-γR1 positive cells in the CD45⁻ cell population. (D) Graft non-hematopoietic cell-mediated T cell inhibition is dependent on intact IFN-γ signaling. CFSE labeled C3H spleen T cells were cultured with B6 DC at a ratio of 20:1 for 3 days. CD45⁻ cells from WT or IFN-γR1^{^−/−} liver allografts (use of DC as regulators for comparison) were added at the beginning into the culture at a regulator: DC ratio of 1:1. A MLR culture without addition of the regulator cells (none) served as control. The proliferation of T cells was determined by CFSE dilution gated on CD4⁺ or CD8⁺ populations (the number is the percentage of dividing cells, upper panels) or by thymidine uptake (n=3, lower panel). (E) Graft CD45⁻ NPC induce T effector cell apoptosis via IFN-γ pathway in vitro. CFSE-labeled H2^b specific CD8⁺ T cells from DES TCR transgenic mice (H2^k) were cultured with B6 DC at a ratio of 20:1 for 3 days. CD45⁻ NPC isolated from WT or IFN-γR1^−/− liver allograft were added at the beginning into the culture at a DC:NPC=1:1. Addition of same number of B6 DC served as control (Cont). The cells were double stained with anti-CD8 and -annexin V mAbs, and analyzed by flow cytometry for the proliferation of specific T cells (CFSE dilution) gated on CD8⁺ population (upper panels). The number is percentage of dividing cells. Expression of annexin V was analyzed on dividing or non-diving CD8⁺ cells. Cell suspensions were stained with TUNEL and examined under a microscope. The number is percentage of annexin V⁺ cells in CD8⁺ cells. The cell suspensions were also stained with TUNEL (red) and examined under microscope, and counted, expressing as mean TUNEL⁺ cell number (in 1000 counted cells) ± SD. The data are representative of two separated experiments.