Identification of Stem Cell Transcriptional Programs Normally Expressed in Embryonic and Neural Stem Cells in Alloreactive CD8+ T Cells Mediating Graft-versus-Host.

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Identification of Stem Cell Transcriptional Programs Normally Expressed in Embryonic and Neural Stem Cells in Alloreactive CD8+ T Cells Mediating Graft-versus-Host Disease Koji Kato, Shuaiying Cui, Rork Kuick, Shin Mineishi, Elizabeth Hexner, James L.M. Ferrara, Stephen G. Emerson, Yi Zhang Biology of Blood and Marrow Transplantation Volume 16, Issue 6, Pages 751-771 (June 2010) DOI: 10.1016/j.bbmt.2010.01.012 Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Alloreactive CD8+ TE are highly replicating cells causing GVHD. (A) CFSE-labeled donor CD8+ TN (2 × 106) derived from normal C3H.SW mice were transplanted with B6/SJL TCD BM (0.5 × 106) into lethally irradiated B6/SJL mice. Donor CD8+ T cells were recovered at day 14 after transplantation from the spleen and lymph node of 3 recipients, enumerated, and stained for flow cytometric analysis. Histograms show the cell division based on CFSE dilution and production of IFN-γ by CD8+ TMSC, TCM, and TE. Data are representative of 3 independent experiments. (B) Real-time RT-PCR analysis shows the relative mRNA expression of selected genes in donor day 14 CD8+ TE, TCM and TMSC. Data are representative of 3 independent experiments. (C, D) Day 14 CD8+ TE were relabeled with CFSE and adoptively transferred into lethally irradiated secondary B6/SJL mice. CFSE-labeled C3H.SW CD8+ TN were transferred as controls. Donor T cells were recovered at day 7 after adoptive transfer, enumerated, and analyzed for flow cytometry. Histogram shows the cell division of indicated cells. Nonstimulated CFSE-labeled CD8+ TN were used as nondividing cell control. Contour plots show the expression of Ki67 (C). For BrdU labeling, secondary recipient mice were given drinking water containing BrdU for 3 days prior to the end of the experiments. At day 7, donor cells were recovered, stained with BrdU, and analyzed by flow cytometry. Dot plots show the labeling of BrdU in gated CD8+ TE population (mean ± SD) (D). (E, F) As described in C, the number of donor T cells recovered from these secondary recipients (n = 3 for each group) was calculated (E) and the percentage of apoptotic cell was shown (F). Data are representative of 2 independent experiments. (G) Donor C3H.SW TCD BM (5 × 106) were transplanted alone, or with donor day 14 CD8+ TE (0.5 × 106) and donor CD8+ TN (1.0 × 106) into lethally irradiated secondary B6/SJL mice. Survival of animals was analyzed with the Kaplan-Meier method. ∗P < .05, significant difference. Biology of Blood and Marrow Transplantation 2010 16, 751-771DOI: (10.1016/j.bbmt.2010.01.012) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Proliferation and persistence of alloantigen-specific CD8+ TE depend on the presence of chronic alloantigen. (A) CFSE-labeled donor T cells (CD4+ and CD8+ T cells, 3 × 106 for each) from normal B6/SJL mice (CD45.1) were transplanted, together with B6 TCD BM (CD45.2, 5 × 106), into lethally irradiated (10 Gy) primary BALB/b recipients (CD45.2). Dot plots show percent of host miHA H60-specific CD8+ TE that were recovered at day 12 after transplantation from spleens and livers of primary GVHD BALB/b recipients (n = 4) of B6/SJLTCD BM and B6/SJL T cells. Data are representative of 3 independent experiments. (B, C, D) Donor alloreactive B6/SJL H60+CD8+ TE (5 × 104) and CD4+ T cells (3 × 105) were isolated 12 days after transplantation, respectively, from these primary GVHD BALB/b recipients (CD45.2) of B6/JL T cells, relabeled with CFSE, and adoptively transferred together with B6 TCD BM (5 × 106) into lethally irradiated secondary BALB/b recipients and congenic B6 recipients (CD45.2), respectively. The percentage of donor alloreactive H60+CD8+ TE in the peripheral blood of these secondary recipients at day 14, 28, and 43 after adoptive transfer (B). Dot plots show the fraction of H60+CD8+ TE in the peripheral blood (C). Donor alloreactive H60+CD8+ TE recovered at day 43 from secondary recipients were calculated (D). Data are presented as mean ± SD. ∗P < .01. (E) Histograms show the surface markers of donor H60+CD8+ TE isolated from the secondary allogeneic BALB/b mice (left), and dot plots show the production of IFN-γ and GZMB (right). Data shown in B, C, D, and E are representative of 2 independent experiments. (F) Survival rate of animals was analyzed by the Kaplan-Meier Method. ∗P < .05, significant difference. Biology of Blood and Marrow Transplantation 2010 16, 751-771DOI: (10.1016/j.bbmt.2010.01.012) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Array based mRNA arrays of alloreactive CD8+ T cells. (A) Donor CD44loCD62Lhi CD8+ TN (2 × 106) from C3H.SW mice (CD45.2) were transplanted with B6/SJL TCD BM (0.5 × 106) into lethally irradiated B6/SJL mice (CD45.1). Donor CD8+ T cells were recovered at day 14 after transplantation from the spleens and lymph nodes of these recipients, magnetically purified, and stained with indicated Abs for flow cytometry cell sorting. Affymetrix Mouse Genome 430A 2.0 array was used to broadly compare the transcription profiles of these CD8+ TMSC and TE to that of TN cells. (B) Real-time RT-PCT analysis shows the relative expression of mRNA in each T cell subset. Data are representative of 3 independent preparations of alloreactive T cells. Biology of Blood and Marrow Transplantation 2010 16, 751-771DOI: (10.1016/j.bbmt.2010.01.012) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Acquisition of stem cell transcriptional programs in alloreactive CD8+ T cells. (A) Genes distinctively expressed by CD8+ TMSC and TE relative to TN were analyzed for functional set enrichment analysis using curated gene lists from the Ramalho-Santos's array data. (B) Acquisition of stem cell transcriptional programs in P14 LCMV-gp33-specific CD8+ T cells. Genes differentially expressed by CD8+ TE-KLRG1Hi, CD8+ TE-KLRG1Hint and CD8+ memory T cells relative to TN were analyzed for enrichment for the same stem cell gene lists. Sizes of stem cell gene lists differ slightly from that in Figure 4A because Sarkar's et al. study used Affymetrix Mouse_430_2, which also had slightly different probe-set annotation than our arrays. Biology of Blood and Marrow Transplantation 2010 16, 751-771DOI: (10.1016/j.bbmt.2010.01.012) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Characterization of stem cell genes in alloreactive CD8+ T cells. (A) The relative expression of stem cell genes in alloreactive CD8+ TE and CD8+ TMSC are shown with genes identified by functional set enrichment analysis using curated gene lists from MSigDBv2 (Figure 4). (B) Real-time RT-PCR analysis shows the relative mRNA expression of selected genes in each T cell subset. Data are representative of 3 independent preparations of alloreactive T cells. (C) Western blot analysis shows the expression of EZH2 protein in purified day 14 CD8+ TE, TMSC, and control TN. (D) donor CD8+ TN and day 14 CD8+ T cells were stained with anti-Ezh2 Ab using intracellular staining and analyzed by flow cytometry. Dot plots show the expression of Ezh2 in cell subset of donor T cells. Mean fluorescein intensity shows the amount of testing antigen. Biology of Blood and Marrow Transplantation 2010 16, 751-771DOI: (10.1016/j.bbmt.2010.01.012) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 6 Identification of stem cell gene Ezh2 in alloreactive CD8+ T cells. (A) The relative expression of 23 Ezh2 target or partner genes that were also selected as increased in alloreactive CD8+ TE relative to TN. (B) Western blot shows Ezh2 protein in CD8+ TN expressing with Ezh2-shRNA or Control-shRNA. These Ezh2-shRNA GFP+CD8+ TN were derived from B6 mice reconstituted with HSCs infected by Ezh2-shRNA/GFP-pLVPToff. GFP+CD8+ TN from B6 mice reconstituted with Con-shRNA/GFP-pLVPToff were isolated as controls. (C) Sorted Ezh2-shRNA GFP+CD8+ TN and Control-shRNA GFP+CD8+ TN (1 × 105/well, 96-well plate) were stimulated with anti-CD3Ab and anti-CD28 (2.5 μg/mL for each). Five days later, cells were recovered and analyzed with flow cyotmetry for measuring GFP+CD8+ T cells. (D) Unfractionated Ezh2-shRNA GFP+CD8+ TN were stimulated with BABL/c mouse-derived DCs, or with IL-7 alone. Five days later, cells were recovered and analyzed using flow cytometry for measuring GFP+CD8+ T cells. Fold change of GFP+CD8+ T cells was calculated based on the output number of GFP+CD8+ T cells after culture divided by the input number of GFP+CD8+ T cells before culture. Data shown in B, C, and D are representative of 2 independent experiments. Biology of Blood and Marrow Transplantation 2010 16, 751-771DOI: (10.1016/j.bbmt.2010.01.012) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions