Journal: The Journal of Experimental Medicine

Article Title: Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

doi: 10.1084/jem.20081633

Figure Lengend Snippet: Characterization of resident dermal APC. (A) Strategy used in the characterization of resident CD45 + HLA-DR + dermal cells showing successive gating on DAPI-live cells, CD45 + cells, and HLA-DR + cells. Progressive refinement of SSC lo AF lo and SSC hi AF hi fractions is depicted at each step by the corresponding panel below. CD45 + cells include macrophages, DC, mast cells, and lymphocytes. Mast cells (high SSC) are HLA-DR − , as are the majority of lymphocytes (low SSC), and are easily excluded by the final HLA-DR gate. AF was recorded in the FL1 channel (488-nm laser and 530/30 band-pass filter). Similar results were found in >50 independent preparations of skin. (B) Further analysis of SSC lo AF lo and SSC hi AF hi fractions of CD45 + HLA-DR + cells by expression of CD14 + and CD1a + . Blue indicates the position of isotype controls and red the expression of CD14/CD1a by each fraction. Three principal populations were sorted and show distinct morphological appearances by Giemsa staining (right). Bar, 20 µm. A representative example of three experiments is shown. (C) Surface phenotype of resident CD45 + HLA-DR + dermal cells. Gates were placed as described in A and B using CD45, HLA-DR, SSC, AF CD1a, and CD14 to define macrophages (mac), CD14 + DC, and CD1a + DC. CD14 expression on DC cells was analyzed by gating on CD1a + and CD1a − cells. All other markers for CD1a + DC and CD14 + DC were determined by gating on CD14 − and CD14 + populations, respectively. Staining was performed at least six times on different skin preparations with similar results. (D) Identification of large melanosome-laden macrophages by CD45, HLA-DR, and FXIIIa. Freshly isolated APC prepared on cytospin were simultaneously stained with antibodies to CD45 (green), HLA-DR (red), and FXIIIa (blue). After immunofluoresence imaging the coverslip was removed and the slide stained with Giemsa. FXIIIa staining is restricted to macrophages that are a subset of the HLA-DR+ APC in the preparation (red). A number of small agranular HLA-DR+ DC with reniform nuclei are FXIIIa negative. All HLA-DR + cells and a number of small lymphocytes in the field are highlighted by CD45 staining. Bar, 20 µm. This finding was reproduced three times.

Article Snippet: The following antibodies were supplied by BD unless stated otherwise and are denoted as antigen fluorochrome (clone): CD1a FITC (NA1/34; Dako); CD1a APC (HI 149); CD1c APC (AD5-8E7; Miltenyi Biotec); CD3 PE (SK7); CD11b PE (ICRF 44); CD11c APC (B-ly6); CD14 PE and PECy7 (M5E2); CD45 APC Cy7 (2D1); CD80 PE (L307.4); CD83 APC (HB15); CD86 APC (2331:FUN-1); CD163 APC (215927; R&D Systems); HLA-DR FITC, APC, and PerCP Cy5.5 (L243); CCR7 APC (150503; R&D Systems); IL-17A Alexa Fluor 647 (eBio64DEC17; eBioscience); IFN-γ FITC (25723.11); FXIIIa (sheep polyclonal; Enzyme Research Laboratories) with APC-conjugated donkey anti–sheep (Invitrogen); CD52 PE (YTH34.5; AbD Serotec).

Techniques: Expressing, Staining, Isolation, Imaging

Journal: The Journal of Experimental Medicine

Article Title: Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

doi: 10.1084/jem.20081633

Figure Lengend Snippet: Functional properties of dermal myeloid cells. (A) Differential migration of dermal myeloid cells. 1-cm square dermal sheets were freshly digested with collagenase or incubated in RPMI 10% FCS for 72 h in low adhesive culture plates, after which migrant cells were collected and the remnant dermis was digested to release nonmigrating cells. CD45 + HLA-DR + gated cells were analyzed by SSC and AF and further separated into CD14 +/− fractions as described in . Populations a–e were sorted by flow cytometry for morphological analysis as shown. CCR7 expression was determined on freshly isolated populations. The experiment was repeated 12 times with similar findings. Bar, 20 µm. (B) Phagocytosis of FITC dextran by dermal myeloid cells. 5 × 10 5 collagenase-digested dermal cells were incubated with 1 mg/ml of FITC-coated dextran particles at 37 or 4°C for 1 h in RPMI containing 10% FCS. Cells were harvested and washed before immunostaining. Fluorescence histograms show a representative example. For the bar charts, all three are independent experiments. ΔMFI, change in geometric mean fluorescence compared with no dextran control. (C) Adherence of dermal myeloid cells. 5 × 10 5 collagenase-digested cell suspensions were cultured overnight in RPMI containing 10% FCS. Nonadherent cells and washes were collected and pooled. Adherent cells were removed by trypsinization. Flow cytometric analysis was performed using SSC, AF, CD14, and CD1a according to . A representative example is shown with the cumulative results of five independent experiments. Bars represent the means.

Article Snippet: The following antibodies were supplied by BD unless stated otherwise and are denoted as antigen fluorochrome (clone): CD1a FITC (NA1/34; Dako); CD1a APC (HI 149); CD1c APC (AD5-8E7; Miltenyi Biotec); CD3 PE (SK7); CD11b PE (ICRF 44); CD11c APC (B-ly6); CD14 PE and PECy7 (M5E2); CD45 APC Cy7 (2D1); CD80 PE (L307.4); CD83 APC (HB15); CD86 APC (2331:FUN-1); CD163 APC (215927; R&D Systems); HLA-DR FITC, APC, and PerCP Cy5.5 (L243); CCR7 APC (150503; R&D Systems); IL-17A Alexa Fluor 647 (eBio64DEC17; eBioscience); IFN-γ FITC (25723.11); FXIIIa (sheep polyclonal; Enzyme Research Laboratories) with APC-conjugated donkey anti–sheep (Invitrogen); CD52 PE (YTH34.5; AbD Serotec).

Techniques: Functional Assay, Migration, Incubation, Adhesive, Flow Cytometry, Expressing, Isolation, Immunostaining, Fluorescence, Control, Cell Culture

Journal: The Journal of Experimental Medicine

Article Title: Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

doi: 10.1084/jem.20081633

Figure Lengend Snippet: Effect of conditioning therapy on recipient dermal DC and macrophages. (A) Representative flow cytometry analysis of dermal cell suspensions derived from clinical biopsies by collagenase digestion before and after conditioning. Selective depletion of SSC lo AF lo CD14 − CD1a + DC is evident. (B) Paired analysis of dermal cells obtained before and after conditioning from a total of 27 patients showing a decline in CD1a + DC but preservation of CD14 + DC and macrophages. Enumeration of cells per unit area was achieved using a standard 2-mm punch of shaved dermis freshly digested into single cell suspension with collagenase. The total number of cells present in each fraction was quantified with the aid of Trucount fluorescent beads. (C) Subgroup analysis of 17 patients treated with reduced intensity and 10 patients with full intensity conditioning showing similar depletion of dermal DC in both groups. P <0.001 for before and after counts (Wilcoxon rank sum test). There is also a slight but nonsignificant increase in CD14 + DC with full intensity conditioning. Error bars indicate SD. (D) Immunofluorescence images of skin before and after conditioning showing maintenance of CD163 + perivascular macrophages. AF in the green channel can be seen in some cells (arrows). Bar, 50 µm. Two representative examples of six patients are shown. (E) Expression of CD52 antigen by dermal APC (red) compared with isotype controls (blue). Numbers indicate median fluorescence intensity ratios. Dermal T cells in the same preparation are shown for comparision. Normal skin was prepared stained and gated as described in .

Article Snippet: The following antibodies were supplied by BD unless stated otherwise and are denoted as antigen fluorochrome (clone): CD1a FITC (NA1/34; Dako); CD1a APC (HI 149); CD1c APC (AD5-8E7; Miltenyi Biotec); CD3 PE (SK7); CD11b PE (ICRF 44); CD11c APC (B-ly6); CD14 PE and PECy7 (M5E2); CD45 APC Cy7 (2D1); CD80 PE (L307.4); CD83 APC (HB15); CD86 APC (2331:FUN-1); CD163 APC (215927; R&D Systems); HLA-DR FITC, APC, and PerCP Cy5.5 (L243); CCR7 APC (150503; R&D Systems); IL-17A Alexa Fluor 647 (eBio64DEC17; eBioscience); IFN-γ FITC (25723.11); FXIIIa (sheep polyclonal; Enzyme Research Laboratories) with APC-conjugated donkey anti–sheep (Invitrogen); CD52 PE (YTH34.5; AbD Serotec).

Techniques: Flow Cytometry, Derivative Assay, Preserving, Suspension, Immunofluorescence, Expressing, Fluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

doi: 10.1084/jem.20081633

Figure Lengend Snippet: Allostimulatory properties of dermal DC and macrophages. (A) [ 3 H]thymidine incorporation of allogeneic CD4 + T cells stimulated by CD1a + DC or macrophages. The left and right show representative examples of six experiments in which APC were titrated against naive and memory T cells as indicated. Stimulation with anti-CD3/28 beads + naive CD4 produced 442 × 10 3 cpm and with anti-CD3/28 beads + memory CD4 produced 430 × 10 3 cpm. Results are the mean ± SEM of triplicate wells. The middle shows the cumulative results and mean of at least seven independent experiments at an APC/T cell ratio of 1:10 compared with medium alone controls (T). The stimulation index (SI) was normalized to the proliferation of naive T cells with DC. (B) Intracellular IL-17 and IFN-γ production by memory CD4 + T cells in response to DC, macrophages, anti-CD3/28 beads, or medium alone. Representative examples of four independent experiments are shown on the left, with gating for IL-17–expressing Th17 cells and IFN-γ–expressing Th1 cells. The mean and cumulative results of four independent experiments are shown on the right, expressed as the percentage of Th17, Th17/1, or Th1 cells in each. (C) [ 3 H]thymidine incorporation of allogeneic CD8 + T cells stimulated by dermal DC or macrophages or medium alone (T). The left shows a representative example of the data at an APC/T cell ratio of 1:10. Results are the mean ± SEM of triplicate wells. The right shows the cumulative results and mean of six independent experiments. (D) IFN-γ secretion into the medium of CD8 + T cells stimulated with DC, macrophages, medium alone (T), or anti-CD3/CD28 beads (beads). Results show the mean ± SEM of six independent experiments. (E) Activation antigen expression by CD8 + T cells stimulated by DC, macrophages, anti-CD3/28 beads, or medium alone. Representative examples of the data with gating for positive cells are shown on the left. The mean and cumulative results of four independent experiments are shown on the right, expressed as the percentage of positive cells in each.

Article Snippet: The following antibodies were supplied by BD unless stated otherwise and are denoted as antigen fluorochrome (clone): CD1a FITC (NA1/34; Dako); CD1a APC (HI 149); CD1c APC (AD5-8E7; Miltenyi Biotec); CD3 PE (SK7); CD11b PE (ICRF 44); CD11c APC (B-ly6); CD14 PE and PECy7 (M5E2); CD45 APC Cy7 (2D1); CD80 PE (L307.4); CD83 APC (HB15); CD86 APC (2331:FUN-1); CD163 APC (215927; R&D Systems); HLA-DR FITC, APC, and PerCP Cy5.5 (L243); CCR7 APC (150503; R&D Systems); IL-17A Alexa Fluor 647 (eBio64DEC17; eBioscience); IFN-γ FITC (25723.11); FXIIIa (sheep polyclonal; Enzyme Research Laboratories) with APC-conjugated donkey anti–sheep (Invitrogen); CD52 PE (YTH34.5; AbD Serotec).

Techniques: Produced, Expressing, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Degradation of Transcription Factor Rfx5 during the Inhibition of Both Constitutive and Interferon γ–Inducible Major Histocompatibility Complex Class I Expression in Chlamydia-Infected Cells

doi:

Figure Lengend Snippet: Chlamydia inhibits both constitutive and IFN-γ–inducible MHC class I expression in infected cells. HeLa cells with or without chlamydial infection were stimulated with IFN-γ or unstimulated and collected for flow cytometry (A), Western blot (B and C), and RT-PCR (E) analysis. Chlamydia prevents both constitutive and IFN-γ–inducible HLA-A, -B, and -C heavy chain and β2M surface expression (A). Chlamydia suppresses the total cellular protein level of both constitutive and IFN-γ–inducible β2M (B) and HLA-A and -B heavy chains (C) in various human cell lines. Chlamydia inhibits the mRNA expression of both MHC class I heavy chains and β2M (E). For pulse–chase labeling experiment (D), HL cells with or without chlamydial infection were metabolically labeled with S 35 –methionine/cysteine for 30 min, and the pulsed cell samples were aliquoted and chased for various times as indicated. Mature or immature bands correspond to proteins resistant or sensitive to EndoH digestion as determined in a separate experiment (data not shown).

Article Snippet: Mouse antibodies were used to detect HLA-A and -B (HB296; ATCC), β2M (HB149; ATCC), β1 integrin (provided by Dr. J. Wilkins, University of Manitoba, Manitoba, Canada), 20S proteasome α subunit HC2 (MCP20; AFFINITI), 20S proteasome α subunit HC3 (MCP21; AFFINITI), 20S proteasome subunits α1, 2, 3, 5, 6, and 7 (MCP231; AFFINITI), 20S proteasome subunit β7 (MCP205; AFFINITI), and a chlamydial major outer membrane protein (MOMP; clone MC22; our unpublished data).

Techniques: Expressing, Infection, Flow Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Pulse Chase, Labeling, Metabolic Labelling

Journal: The Journal of Experimental Medicine

Article Title: Degradation of Transcription Factor Rfx5 during the Inhibition of Both Constitutive and Interferon γ–Inducible Major Histocompatibility Complex Class I Expression in Chlamydia-Infected Cells

doi:

Figure Lengend Snippet: Inhibition of MHC class I expression correlates with RFX5 degradation. (A) HeLa cells with or without chlamydial infection and with or without IFN-γ stimulation were analyzed on Western blot for the levels of RFX5, HLA-A and -B heavy chains, β2M, β1 integrin (as a control membrane protein), and a chlamydial MOMP. Chlamydia-infected cell samples showed reduced levels of HLA heavy chain and β2M and complete loss of RFX5. (B) Cell-free assay analysis of HLA-A and -B heavy chain, β2M, and RFX5 degradation. Cytosolic S100 fractions from either normal HeLa (HeLa S100) or chlamydia-infected HeLa cells (chlamydia S100) were incubated with the corresponding target proteins (either a membrane protein extract as the source of HLA-A and -B heavy chains and β2M or a nuclear protein extract as the source of RFX5). The incubated mixtures were analyzed on Western blot for the levels of the residual target proteins with the corresponding antibodies. Chlamydia S100 selectively degrades RFX5 but not HLA-A and -B heavy chains and β2M.

Article Snippet: Mouse antibodies were used to detect HLA-A and -B (HB296; ATCC), β2M (HB149; ATCC), β1 integrin (provided by Dr. J. Wilkins, University of Manitoba, Manitoba, Canada), 20S proteasome α subunit HC2 (MCP20; AFFINITI), 20S proteasome α subunit HC3 (MCP21; AFFINITI), 20S proteasome subunits α1, 2, 3, 5, 6, and 7 (MCP231; AFFINITI), 20S proteasome subunit β7 (MCP205; AFFINITI), and a chlamydial major outer membrane protein (MOMP; clone MC22; our unpublished data).

Techniques: Inhibition, Expressing, Infection, Western Blot, Control, Membrane, Cell-Free Assay, Incubation

Journal: The Journal of Experimental Medicine

Article Title: Degradation of Transcription Factor Rfx5 during the Inhibition of Both Constitutive and Interferon γ–Inducible Major Histocompatibility Complex Class I Expression in Chlamydia-Infected Cells

doi:

Figure Lengend Snippet: Chlamydial but not host protein synthesis is required for both the degradation of RFX5 and suppression of HLA-A and -B heavy chains and β2M. (A) Correlation between infection dose and RFX5 degradation. 30 h after chlamydial infection at various MOI, HeLa cells were analyzed for the levels of chlamydial MOMP and host RFX5, USF-1, and USF-2 proteins on Western blot. The anti–USF-2 antibody used in this experiment can bind to both USF-1 and -2. These two USF isoforms can be separated on blots if gels are run far enough. Since we have previously shown that USF-1 is degraded in chlamydia-infected cells , USF-1 is used as a positive control. Since USF-2 protein level is usually not altered by chlamydial infection, it serves as a negative control. ns denotes nonspecific binding. (B) Time course relationship between chlamydial growth and RFX5 degradation. At various time points after infection, HeLa cell samples were analyzed on Western blot as described in A. (C) Inhibition of chlamydial but not host protein synthesis prevents RFX5 degradation and restores HLA-A and -B heavy chain and β2M expression. Rifampin (RF; final concentration = 0.1 μg/ml), chloramphenicol (CH; 60 μg/ml) and penicillin (PG; 100 μg/ml) were added at the beginning of chlamydial infection and maintained throughout the culture. Cycloheximide (CY; 10 μg/ml) was added to the culture 10 h before IFN-γ treatment and maintained during the IFN-γ stimulation. The treated HeLa cells were analyzed for protein levels of HLA-A and -B heavy chains, β2M, RFX5, USF-1, and chlamydial MOMP on Western blot. ns denotes nonspecific binding.

Article Snippet: Mouse antibodies were used to detect HLA-A and -B (HB296; ATCC), β2M (HB149; ATCC), β1 integrin (provided by Dr. J. Wilkins, University of Manitoba, Manitoba, Canada), 20S proteasome α subunit HC2 (MCP20; AFFINITI), 20S proteasome α subunit HC3 (MCP21; AFFINITI), 20S proteasome subunits α1, 2, 3, 5, 6, and 7 (MCP231; AFFINITI), 20S proteasome subunit β7 (MCP205; AFFINITI), and a chlamydial major outer membrane protein (MOMP; clone MC22; our unpublished data).

Techniques: Infection, Western Blot, Positive Control, Negative Control, Binding Assay, Inhibition, Expressing, Concentration Assay