Journal: Journal of Cellular and Molecular Medicine

Article Title: Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells

doi: 10.1111/j.1582-4934.2009.00923.x

Figure Lengend Snippet: Haematopoietic reconstitution potential of spleen HSC for dendritic cells. Chimeras were prepared by adoptive transfer of marker-distinct neonatal spleen (SPL) cells along with host-type bone marrow (BM) cells into lethally irradiated mice. Whole splenocytes (1 × 10 5 ) from 8-day-old B6.SJL mice (CD45.1 + ) were co-injected into lethally irradiated (9.5Gy) C57BL/6J mice (CD45.2 + ) along with 1 × 10 5 host-type (CD45.2 + ) adult BM cells. Control BM chimeras were prepared by injection of only 1 × 10 5 dissociated CD45.1 + (B6.SJL) adult BM cells. For ‘short-term’ reconstitution, spleens were collected at 8 weeks, enriched for DC by depletion of T and B cells, and stained with antibodies specific for CD45.1, CD11c, CD11b, CD8 and MHC-II. Prior to flow cytometry, cells were incubated with propidium iodide (PI; 1 mg/ml) for gating of live (PI − ) cells. (A) Spleen APC subsets were gated as follows: L-DC (CD11c lo CD11b hi MHC-II − FSC hi ), myeloid cells (CD11c − CD11b hi MHC-II − FSC lo ), CD8a − cDC (CD11c hi CD11b + CD8 − MHC-II + ) and CD8a + cDC (CD11c hi CD11b − CD8 + MHC-II + ). (B) Spleen APC subsets were assessed for donor (CD45.1 + ) cell composition. Numbers in gates indicate per cent positive cells. C57BL/6J and B6.SJL splenocytes were included as negative and positive controls, respectively, in order to set gates to define CD45.1 − and CD45.1 + cell populations. Labelled chimeras (*) indicate mice showing only partial donor cell reconstitution.

Article Snippet: Antibodies specific for CD8 (53–6.7; PE) and MHC-II (25–9-17; biotin) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Adoptive Transfer Assay, Marker, Irradiation, Injection, Staining, Flow Cytometry, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells

doi: 10.1111/j.1582-4934.2009.00923.x

Figure Lengend Snippet: Dendritic cell reconstitution by neonatal spleen HSC. Spleen (SPL) and bone marrow (BM) chimeras were prepared as described in . For analysis of long-term (.25 week) reconstitution, spleens were collected, enriched for DC by depletion of T and B cells, and stained with antibodies specific for CD45.1, CD11c, CD11b, CD8 and MHC-II. Prior to flow cytometry, cells were incubated with propidium iodide (PI; 1 mg/ml) for gating of live (PI − ) cells. C57BL/6J splenocytes were stained as a CD45.1 − control. (A) Individual spleen APC subsets (L-DC, myeloid cells, CD8 − cDC and CD8 + cDC) were gated as shown in and assessed for donor-cell (CD45.1 + ) composition. Numbers in gates indicate per cent positive cells. Labelled chimeras (*) indicate mice showing only partial donor reconstitution with some evidence of host-type cell development. (B) The relative prevalence of donor- to host-type cells was calculated for each spleen APC subset in animals showing partial donor reconstitution as indicated in and (*). The fold-increase of donor- to host-type cells was calculated relative to CD8 − cDC assigned a fold-increase of 1.0. Data represent mean 6 SE ( n = 4). Cell subsets having significantly higher representation of donor- over host-type cells, compared with CD8 − cDC, are indicated (*) ( P ≤ 0.02; Wilcoxon Rank Sum Test).

Article Snippet: Antibodies specific for CD8 (53–6.7; PE) and MHC-II (25–9-17; biotin) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Staining, Flow Cytometry, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Haematopoietic stem cells in spleen have distinct differentiative potential for antigen presenting cells

doi: 10.1111/j.1582-4934.2009.00923.x

Figure Lengend Snippet: Neonatal spleen HSC give multilineage long-term reconstitution. Mouse chimeras described in and were analysed for donor-derived (CD45.1 + ) subsets of L-DC, myeloid cells, CD8 − cDC, CD8 + cDC, CD8 + T cells, B cells, and ckit + haematopoietic progenitors in spleen, as well as CD8 + T cells and B cells in mesenteric lymph nodes (MLN). APC subsets were identified by antibody staining and flow cytometry as described in and . Lymphoid subsets in spleen were identified amongst the enriched subset discarded after T/B cell depletion to enrich for APC subsets. Cells were then stained for CD45.1, CD19 and CD8. Analysis of cells in MLN involved direct staining with antibodies specific for CD45.1, CD19 and CD8. For detection of haematopoietic progenitors, T/B cell depleted splenocytes were stained with antibodies specific for CD45.1, CD11c, CD11b, ckit and MHC-II. In all analyses, propidium iodide (PI; 1 mg/ml) staining was performed for gating of live (PI − ) cells. (A) The prevalence of donor-derived spleen L-DC, myeloid cells, CD8 − cDC and CD8 + cDC subsets in SPL and BM chimeras was calculated as a percentage of total (DC plus myeloid cells) in spleen. Chimeras were compared with control C57BL/6J mice for distribution of spleen APC subsets. (B) The presence of lymphoid cell subsets was analysed in spleen and MLN. For spleen, total [CD8 + T cells and CD19 + CD8 − B cells] are shown as per cent amongst T/B cell enriched spleen cells. For MLN, total [CD8 + T cells and CD19 + CD8 − B cells] are represented as per cent amongst total MLN. (C) Spleens collected from long-term SPL chimeras were analysed for the presence of Lin − ckit + progenitors. T/B cell depleted splenocytes (CD19 − Thy1.2 − ) were gated as CD11c − CD11b − MHC-II − (Lin − ) cells, prior to analysis of donor (CD45.1 + ) ckit expression. Numbers in gates indicate per cent positive cells.

Article Snippet: Antibodies specific for CD8 (53–6.7; PE) and MHC-II (25–9-17; biotin) were purchased from BD Biosciences (San Jose, CA, USA).

Techniques: Derivative Assay, Staining, Flow Cytometry, Expressing

Journal: AIDS (London, England)

Article Title: Activation of the signal transducer and activator of transcription 1 signaling pathway in thymocytes from HIV-1-infected human thymus

doi: 10.1097/01.aids.0000060415.18106.1a

Figure Lengend Snippet: (a) Quantitative measurement of differential gene expression. RNA from six different yeast-specific genes was mixed with the HIV or mock-infected RNA samples in the microarray assay. RNA from each yeast gene was added at a known ratio (3, 10 or 25) in the Cy3 (HIV) or Cy5 (mock) sample (Cy3/Cy5 added). The six yeast genes were arrayed on the slides in quadruplets. Positive values indicate threefold, 10-fold or 25-fold more RNA were used in the Cy3 (HIV) sample. Negative values (−3, −10 or −25) indicate that threefold, 10-fold or 25-fold more RNA were included in the Cy5 (mock) sample. An equal amount of RNA in Cy3 and Cy5 samples is indicated as 1 (+1 or −1). The X axis shows the added ratios and the Y axis indicates Cy3/Cy5 readout data from the assay. Error bars indicate standard deviations. (b) RNA sample and reaction control. RNA samples from HIVJD (Cy3 labeled) or mock (Cy5 labeled) infected severe combined immunodeficient (SCID)-hu Thy/Liv mice were analysed using the microarray assay. Relative expression levels of 23 000 Mr highly basic protein (HBP), ribosomal subunit S9 (S9 rRNA) and α-tubulin were quantified in quadruplets. Three sets of human genes known to express in multiple tissues (complex target 1–3) were also included as controls. Positive values (Cy3 signal/Cy5 signal) indicate more RNA in the Cy3 (HIV) sample, thus induction of expression by HIV. Negative values (Cy5 signal/Cy3 signal) indicate more RNA in the Cy5 (mock) sample, thus suppressed expression by HIV. Error bars indicate standard deviations from the quadruplet arrays. The relative expression of three T cell markers (TCRβ, CD8 and Thy-1) from the microarray assay was also shown. Comparison of HIV (JD or NL4-3) and mock infected samples from different donor tissues showed similar results. (c) Alteration of gene expression in SCID-hu Thy/Liv mice after HIV-1 infection. A small number (< 0.2%) of genes were induced (> threefold) in HIV-1-infected Thy/Liv organs over mock infected samples. The majority of genes were in the +2.5 to −2.5 range (99.5%). Error bars indicate standard deviations from three experiments (two with JD and one with NL4-3) with different donor tissues.

Article Snippet: Monoclonal antibodies reactive with human CD4 and CD8 cells were purchased from Becton Dickinson (San Jose, CA, USA).

Techniques: Gene Expression, Infection, Microarray, Control, Labeling, Expressing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Mutated nucleophosmin 1 as immunotherapy target in acute myeloid leukemia

doi: 10.1172/JCI97482

Figure Lengend Snippet: CD8+ T cells specific for ΔNPM1 were single cell isolated from PBMCs from HLA-A*02:01–positive healthy individuals using a mix of ΔNPM1-CLA and ΔNPM1-C*LA pHLA tetramers. (A) T cell clones were stained with pHLA tetramers. T cell clones 1A2 (left) and 4A8 (right) were both positive for the ΔNPM1-CLA tetramer (red), whereas only clone 1A2 stained with ΔNPM1-C*LA (orange). (B) T cell clones 1A2 (upper panel) and 4A8 (lower panel) were tested for reactivity against HLA-A*02:01–positive T2 cells exogenously loaded with titrated concentrations of ΔNPM1 peptide CLAVEEVSL (red circles), the cysteinylated variant C*LAVEEVSL (orange squares), or an irrelevant HLA-A*02:01–binding CMV peptide NLVPMVATV (blue triangles) by IFN-γ ELISA. Both T cell clones showed recognition of CLAVEEVSL, and clone 1A2 also showed recognition of C*LAVEEVSL. Release of IFN-γ (ng/ml) in duplicate wells is shown. (C) Clones 1A2 (upper panel) and 4A8 (middle panel) were tested for reactivity against 5 HLA-A*02:01–positive primary AMLs by IFN-γ ELISA. The panel included 3 AMLs with ΔNPM1 and 2 AMLs with WTNPM1. T cell clone 1A2 reacted against all 3 AMLs with ΔNPM1, whereas clone 4A8 recognized 2 AMLs. An HLA-A*02:01–specific alloreactive T cell clone (Allo-A2 clone) was included as a positive control. Release of IFN-γ (ng/ml) in duplicate wells is shown; bars represent mean IFN-γ release.

Article Snippet: T cells were stained with FITC-conjugated antibodies against CD3 (catalog 555339), CD4 (catalog 555346), CD8 (catalog 555366) (BD Biosciences), and TCR-Vβ5.1 (catalog IM1552, Beckman Coulter); APC-conjugated antibodies against CD3 (catalog 555342), CD4 (catalog 555349), CD8 (catalog 555369), and mouse TCR-Cβ (catalog 553174); and PE-conjugated pHLA tetramers and antibodies against CD3 (catalog 555340), CD4 (catalog 555347), and CD8 (catalog 555367) (BD Biosciences).

Techniques: Isolation, Clone Assay, Staining, Variant Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control

Journal: The Journal of Clinical Investigation

Article Title: Mutated nucleophosmin 1 as immunotherapy target in acute myeloid leukemia

doi: 10.1172/JCI97482

Figure Lengend Snippet: CD8+ and CD4+ T cells isolated from HLA-A*02:01–positive healthy individuals were transduced with the TCR for ΔNPM1 from clone 1A2 and a TCR for HLA-A*02:01–binding CMV peptide NLVPMVATV. TCR-transduced T cells were purified by MACS. (A) TCR-transduced T cells were analyzed by flow cytometry using ΔNPM1-CLA (red) and CMV-NLV (blue) pHLA tetramers. CD8+ (CD8ØNPM1) and CD4+ (CD4ØNPM1) T cells transduced with the TCR for ΔNPM1 stained with the ΔNPM1-CLA tetramer, whereas CD8+ (CD8ØCMV) and CD4+ (CD4ØCMV) T cells transduced with the CMV-specific TCR showed binding to the CMV-NLV tetramer. Results are shown for donor 1. (B) TCR-transduced T cells were analyzed for reactivity against T2 cells exogenously loaded with titrated concentrations of CLAVEEVSL (red circles) or NLVPMVATV (blue squares) by IFN-γ ELISA. CD8ØNPM1 (upper left panel) and CD4ØNPM1 (lower left panel) showed half-maximum recognition of CLAVEEVSL at concentrations of 30–100 nM (dotted lines), but no recognition of NLVPMVATV. Conversely, CD8ØCMV (upper right panel) and CD4ØCMV (lower right panel) reacted against NLVPMVATV, but not CLAVEEVSL. Release of IFN-γ (ng/ml) in duplicate wells is shown for donor 1. (C) TCR-transduced T cells were tested for recognition of HLA-A*02:01–positive AML cell lines with ΔNPM1 (OCI-AML3) or WTNPM1 (OCI-AML2) in the absence or presence of blocking antibodies against HLA class I (αHLA-I) or HLA class II (αHLA-II) by IFN-γ ELISA. Recognition of OCI-AML3 by CD8+ and CD4+ T cells transduced with the TCR for ΔNPM1 is mediated by HLA class I. Release of IFN-γ (ng/ml) in duplicate wells is shown for donor 2. Bars represent mean IFN-γ release.

Article Snippet: T cells were stained with FITC-conjugated antibodies against CD3 (catalog 555339), CD4 (catalog 555346), CD8 (catalog 555366) (BD Biosciences), and TCR-Vβ5.1 (catalog IM1552, Beckman Coulter); APC-conjugated antibodies against CD3 (catalog 555342), CD4 (catalog 555349), CD8 (catalog 555369), and mouse TCR-Cβ (catalog 553174); and PE-conjugated pHLA tetramers and antibodies against CD3 (catalog 555340), CD4 (catalog 555347), and CD8 (catalog 555367) (BD Biosciences).

Techniques: Isolation, Transduction, Binding Assay, Purification, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Blocking Assay