Journal:

Article Title: T cell-dependent survival of CD20 + and CD20 − plasma cells in human secondary lymphoid tissue

doi: 10.1182/blood-2006-08-043414

Figure Lengend Snippet: Human PCs and T cells persist within tonsil grafts. Immunohistochemistry was used to assess the cellular composition of tonsil grafts recovered at 4 weeks after surgery. (A) Postmortem examination of a xenochimeric mouse at 2 weeks after surgery showing vascularization of tonsil grafts. (B) Staining of tonsil grafts for expression of mouse (m) blood vessels; lower panel shows enlarged view of upper panel; scale bar represents 200 μm. (C) Serial sections of tonsil (before surgery) and tonsil graft (4 weeks after surgery) were stained for expression of human IgD (in red) and human CD38, CD138, CD27, and CD3 (in blue). Scale bar represents 200 μm (left-hand panels) and 60 μm (central and right-hand panels). Staining is representative of 10 tonsils and tonsil grafts.

Article Snippet: Sections were cut, fixed, and embedded as described under “Immunohistochemistry.” The mAbs used were anti–human CD3–Alexa Fluor (AF) 647, CD11c-AF546, CD27-AF647, CD45-AF546, Ki-67-AF546 (BD PharMingen), CD3-AF405, CD138-FITC (Serotec, Raleigh, NC), and anti–mouse CD11b-AF647 (Caltag Laboratories).

Techniques: Immunohistochemistry, Staining, Expressing

Journal:

Article Title: T cell-dependent survival of CD20 + and CD20 − plasma cells in human secondary lymphoid tissue

doi: 10.1182/blood-2006-08-043414

Figure Lengend Snippet: Immunofluorescence analysis of cells within tonsil grafts. Immunofluorescence was used to assess coexpression of multiple markers by the cells within tonsil grafts recovered at 4 weeks after surgery. (A) Three-color staining to detect expression of human CD3 (green), Ki-67 (blue), and mouse (m) CD11b (red). Enlarged view of cells expressing both Ki-67 and CD3 indicated by arrows (far right panel). (B) Three-color staining to detect expression of human CD138 (green), Ki-67 (blue), and human CD27 (red). Enlarged view of cells expressing both Ki-67 and CD138 indicated by arrow (far right panel). (C) Four-color staining (with only 3 of these shown in a given panel) to detect expression of human CD3 (red), human CD138 (green), Ki-67 (blue) in the left 2 panels and human CD138 (green), Ki-67 (red), and mCD11b (blue) in the right 2 panels. Right panel of each pair shows enlarged image. Scale bar represents 50 μm. Staining is representative of 5 tonsil grafts from each of 3 different grafting experiments.

Article Snippet: Sections were cut, fixed, and embedded as described under “Immunohistochemistry.” The mAbs used were anti–human CD3–Alexa Fluor (AF) 647, CD11c-AF546, CD27-AF647, CD45-AF546, Ki-67-AF546 (BD PharMingen), CD3-AF405, CD138-FITC (Serotec, Raleigh, NC), and anti–mouse CD11b-AF647 (Caltag Laboratories).

Techniques: Immunofluorescence, Staining, Expressing

Journal:

Article Title: T cell-dependent survival of CD20 + and CD20 − plasma cells in human secondary lymphoid tissue

doi: 10.1182/blood-2006-08-043414

Figure Lengend Snippet: Treatment with rituximab or alemtuzumab reduced serum immunoglobulin levels. Xenochimeric mice were treated with either rituximab or alemtuzumab and levels of hIg in the circulation of the host mice were assessed by ELISA. (A) Expression of CD20 and CD52 by tonsillar PCs (CD19+CD38bright, CD27bright), GC B cells (CD19+CD38+, CD27+), and naive B cells (‘N', CD19+CD38lo, CD27−). Isotype controls for B cells and for PCs are also shown. (B) Percentage of PCs isolated from blood (BD, n = 8), tonsil (TN, n = 5), LN (n = 5), spleen (SP, n = 6), and BM (n = 4) expressing CD20 or CD138. Error bars represent SEM. (C) Relative fluorescence intensity (MFI-positive cells − MFI-negative cells) of CD20 expression of PCs isolated from blood, tonsil, LN, spleen, and BM. Error bars represent SEM. (D) Frequency of tonsillar cells secreting either IgG, IgA, or IgM, assessed by ELISPOT. Mean of 3 experiments shown, error bars represent SEM. (E) Mean levels of hIgG, hIgM, and hIgA (ng/mL) in groups of untreated (Con), rituximab-treated (Rit), or alemtuzumab-treated (Cam) mice. Error bars represent SEM for treatment groups. (F) Mean levels of hIgG (ng/mL) in groups of untreated mice, xenochimeric mice treated with rituximab or alemtuzumab, and NOD/RAG−/−/perforin−/− mice treated with rituximab or alemtuzumab. Error bars represent SEM for treatment groups. Results are representative of 4 experiments.

Article Snippet: Sections were cut, fixed, and embedded as described under “Immunohistochemistry.” The mAbs used were anti–human CD3–Alexa Fluor (AF) 647, CD11c-AF546, CD27-AF647, CD45-AF546, Ki-67-AF546 (BD PharMingen), CD3-AF405, CD138-FITC (Serotec, Raleigh, NC), and anti–mouse CD11b-AF647 (Caltag Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Isolation, Fluorescence, Enzyme-linked Immunospot

Journal:

Article Title: T cell-dependent survival of CD20 + and CD20 − plasma cells in human secondary lymphoid tissue

doi: 10.1182/blood-2006-08-043414

Figure Lengend Snippet: Depletion of immunoglobulin-secreting PCs from tonsil grafts. Tonsil grafts were recovered form xenochimeric mice at 4 weeks after surgery, and cells were assessed by multiparameter flow cytometry, with live cells sorted for analysis of immunoglobulin secretion by ELISPOT. (A) Expression of human IgD, CD19, CD20, CD27, CD38, CD52 in cells from the tonsil before surgery (tonsil), untreated (control), rituximab-treated (rituximab), and alemtuzumab-treated (Campath) mice. The percentage of positive cells in each quadrant and the percentage of PCs (CD38bright) are shown. Lower panels show expression of human CD19, CD20, CD27, CD52 in PCs. (B) ELISPOT analysis of immunoglobulin secretion showing average spot frequency per 10 000 cells in grafts recovered from control (Con), rituximab-treated (Rit), and alemtuzumab-treated (Cam) mice. Bars represent SEM. (C) The percentage of PCs secreting immunoglobulin was calculated by expressing the number of spots as a percentage of the number of PCs in each well. (D) The mean spot size of cells secreting IgG (□) and IgM (▩) isolated from tonsil before surgery (TN), untreated (Con), rituximab-treated (Rit), or alemtuzumab-treated (Cam) mice was calculated. Bars represent SEM. (E) Sections of tonsil grafts from rituximab-treated mice were stained for expression of human CD138 (green), Ki-67 (blue), CD3 (red). Scale bar represents 50 μm. Results are representative of 4 experiments.

Article Snippet: Sections were cut, fixed, and embedded as described under “Immunohistochemistry.” The mAbs used were anti–human CD3–Alexa Fluor (AF) 647, CD11c-AF546, CD27-AF647, CD45-AF546, Ki-67-AF546 (BD PharMingen), CD3-AF405, CD138-FITC (Serotec, Raleigh, NC), and anti–mouse CD11b-AF647 (Caltag Laboratories).

Techniques: Flow Cytometry, Enzyme-linked Immunospot, Expressing, Isolation, Staining

Journal:

Article Title: T cell-dependent survival of CD20 + and CD20 − plasma cells in human secondary lymphoid tissue

doi: 10.1182/blood-2006-08-043414

Figure Lengend Snippet: T cell-dependent PC survival in tonsil grafts. To assess the role of T cells in PC survival in the grafts, xenochimeric mice were treated with either anti-CD3 (OKT3) or anti-CD154 mAbs. Levels of hIgG and mIgG assessed by ELISA and recovered graft composition assessed by immunofluorescence were analyzed. (A) Mean levels of hIgG (ng/mL) in untreated (Con), OKT3-treated (OKT3), or anti-CD154–treated (CD154) mice. Error bars represent SEM for treatment groups. (B) Mean levels of mIgG (ng/mL) in mice treated with OKT3 or anti-CD154 mAbs. Error bars represent SEM for treatment groups. (C) Tonsil graft sections from control and OKT3-treated and anti-CD154–treated mice stained for expression of human CD138 (green), CD3 (red), and CD45 (blue). Scale bar represents 50 μm. Results are representative of 3 experiments.

Article Snippet: Sections were cut, fixed, and embedded as described under “Immunohistochemistry.” The mAbs used were anti–human CD3–Alexa Fluor (AF) 647, CD11c-AF546, CD27-AF647, CD45-AF546, Ki-67-AF546 (BD PharMingen), CD3-AF405, CD138-FITC (Serotec, Raleigh, NC), and anti–mouse CD11b-AF647 (Caltag Laboratories).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing

Journal: The Journal of Clinical Investigation

Article Title: B cells, antibody-secreting cells, and virus-specific antibodies respond to herpes simplex virus 2 reactivation in skin

doi: 10.1172/JCI142088

Figure Lengend Snippet: (A) B cells: CD20 (red) and CD79b (green) in serial genital skin biopsy samples taken during and after HSV-2 lesion from a single participant (results shown for 1 of 10 patients). No B cells were detected in the control sample. (B) T cells: CD4 (green) and CD8 (red) in serial specimens taken during and after HSV-2 reactivation (result shown for 1 of 8 patients). Scale bars: 100 μm. (C) Densely clustered CD4+CD8+ T cells (left) in this participant were accompanied by CD20+CD79b+ B cells (right); scale bars: 50 μm. (D) CD27 (green) and CD20 (red) in a lesion sample with no evidence of costaining (result shown for 1 of 6 patients); scale bars: 50 μm. (E) In a healing-skin specimen, B cells express CD20 (green) and IgD (red) (result shown for 1 of 5 patients). Arrows indicate sites of coexpression. Inset shows red and green channels separately. Scale bars: 50 μm. (F) IgG-producing cells are loaded with IgG RNA by FISH. IgG mRNA (green) in a healing specimen of HSV-2 reactivation (result shown for 1 of 9 patients). Original magnification, 40× by oil immersion. Scale bar: 25 μm. (G) IgG+ cells by FISH over time, showing distribution within the upper dermis and lymphocyte clusters. In this participant, the control specimen (from the arm) had no IgG+ cells. Dashed line indicates the DEJ. Images obtained at 20×; scale bars: 250 μm. Brightness in G was increased for consistency. wph, weeks after healing.

Article Snippet: To enumerate the relative density of B cells, T cells, ASCs, blood vessels, and/or antibody in tissue biopsy sections, the following primary antibodies were used; rabbit anti–human CD20 (1:100; Abcam ab78237), mouse anti-human CD79b (1:100; Novus Biologicals NBP1-28528), mouse anti-human IgG (1:100; eBioscience 05-4200), sheep anti–human vWF (FITC-conjugated, 1:5000; Abcam ab8822), mouse anti–human CD27 (1:100; BioLegend 356403), mouse anti–human CD20 (1:100; eBioScience 14-0202-82), mouse anti-human IgD (af647-conjugated, 1:50 or 1:100; BioLegend 348227), rabbit anti–HSV-2 (1:1000; Dako B0116), mouse anti-CD4 (1:1000; BioLegend 300502), and mouse anti-CD8 (af647-conjugated, 1:100; BD Pharmingen 557708).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: B cells, antibody-secreting cells, and virus-specific antibodies respond to herpes simplex virus 2 reactivation in skin

doi: 10.1172/JCI142088

Figure Lengend Snippet: (A) ASCs are found infiltrating tissue distant from vascular structures, as well as adjacent to and within small capillaries. IgG-producing cells identified by FISH in a healing biopsy are found independently in tissue (lower left) as well as proximate to small capillaries (lower right) in biopsy specimens taken during healing after HSV-2 reactivation. Faint green signal is due to collagen autofluorescence. Blood vessel endothelial cells identified by vWF IF (green); IgG RNA+ cells, magenta. Scale bars: 200 μm; insets, 25 μm. vWF and IgG colocalization by combination FISH and IF was performed in specimens from 6 participants. While capillaries were identified in all samples, IgG cells were not present within these vascular structures in all samples. (B) IgG-producing cells by FISH (left) clustered in an area of dense T cell infiltration (right). CD4, green; CD8, red. Shown are serial sections of the same specimen. Scale bars: 50 μm. IgG FISH and T cell IF were performed in serial biopsy specimens from 4 participants. (C) Clustered IgG (red) and CD4 (green) by FISH; absence of vWF staining was confirmed. Scale bar: 50 μm. IgG and CD4 FISH was performed in specimens from 8 participants.

Article Snippet: To enumerate the relative density of B cells, T cells, ASCs, blood vessels, and/or antibody in tissue biopsy sections, the following primary antibodies were used; rabbit anti–human CD20 (1:100; Abcam ab78237), mouse anti-human CD79b (1:100; Novus Biologicals NBP1-28528), mouse anti-human IgG (1:100; eBioscience 05-4200), sheep anti–human vWF (FITC-conjugated, 1:5000; Abcam ab8822), mouse anti–human CD27 (1:100; BioLegend 356403), mouse anti–human CD20 (1:100; eBioScience 14-0202-82), mouse anti-human IgD (af647-conjugated, 1:50 or 1:100; BioLegend 348227), rabbit anti–HSV-2 (1:1000; Dako B0116), mouse anti-CD4 (1:1000; BioLegend 300502), and mouse anti-CD8 (af647-conjugated, 1:100; BD Pharmingen 557708).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: B cells, antibody-secreting cells, and virus-specific antibodies respond to herpes simplex virus 2 reactivation in skin

doi: 10.1172/JCI142088

Figure Lengend Snippet: Density of (A) CD20+ cells by IF (n = 11), (B) IgG RNA+ cells by FISH (n = 10), (C) CD4+ T cells by IF (n = 8), and (D) CD8+ T cells by IF (n = 8) in tissue at the biopsy time points (left panels) and by participant over time (right panels). Statistical testing was by Friedman’s test with Dunn’s corrections for multiple comparisons. Each graph on the right presents cell counts from genital-area biopsies over time, as measured from the identified symptomatic lesion. (E) Correlation (r) between B and T cell subsets and between CD20+ and IgG RNA+ cells was calculated by repeated-measure correlation. Participants are labeled by color; results for multiple specimens from genital and control areas from each participant are included in each graph.

Article Snippet: To enumerate the relative density of B cells, T cells, ASCs, blood vessels, and/or antibody in tissue biopsy sections, the following primary antibodies were used; rabbit anti–human CD20 (1:100; Abcam ab78237), mouse anti-human CD79b (1:100; Novus Biologicals NBP1-28528), mouse anti-human IgG (1:100; eBioscience 05-4200), sheep anti–human vWF (FITC-conjugated, 1:5000; Abcam ab8822), mouse anti–human CD27 (1:100; BioLegend 356403), mouse anti–human CD20 (1:100; eBioScience 14-0202-82), mouse anti-human IgD (af647-conjugated, 1:50 or 1:100; BioLegend 348227), rabbit anti–HSV-2 (1:1000; Dako B0116), mouse anti-CD4 (1:1000; BioLegend 300502), and mouse anti-CD8 (af647-conjugated, 1:100; BD Pharmingen 557708).

Techniques: Labeling