Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 1. B cell TF staining in mouse spleen and human tonsil. A, Serial sections of immunized mouse spleen are stained for B cell TF and proliferation-associated Ag (MCM7). Low-power (10) images are on top, high power (40) on bottom. oPALS, outer periarteriolar lymphoid sheet; MC, mantle cells; MZ, marginal zone. Note the reciprocal staining pattern of IRF4 vs the other TF in the GC, and the coexpression in the MZ and MC. B, Serial sections of human tonsil are stained for B cell TF and proliferation-associated Ag (MCM7). Low-power (4) images are on top, high power (40) in the middle. Lower panel, The U95 Affymetrix cDNA chip raw values, depicted as bars, indicating cDNA expression for the corresponding transcription factors in memory (Mem), naive, and GC-purified cells. IE, intraepithelial; SE, subepithelial; MC, mantle cell; GC, germinal center. Note the reciprocal staining pattern of IRF4 vs the other TF in the GC, and the coexpression in the MC and IE.

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques: Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 2. Validation of the BCL6 and IRF4 staining. A, A murine wild-type (wt) lymph node (center left) is stained by BCL6 Ab in a GC (strongly) and in the follicles (weakly). The negative control Ab and the BCL6 Ab on a BCL6 knockout (KO) lymph node are negative. A wild-type and knockout mouse spleen are stained by IRF4; the knockout spleen is negative. B, Four mice, each bearing none, one, two BCL6 alleles, and one with one C-terminally truncated allele, are analyzed in the thymus and spleen for BCL6 expression by flow cytometry. The gray histogram is an isotype-matched negative control. Note the increased intensity (rightward shift of the peak fluorescence) of BCL6 staining with increased genetic dosage. The gating for the two B cell subsets are indicated at right. The truncated allele produces increased amounts of protein, because of the lack of autoregulation (Wang et al. (52)). C, Spleen cells from an IRF4 knock- out mouse (KO) and a wild-type littermate (wt) were stained for B220, CD11b, negative control (gray histogram), or IRF4 (black line). Note ab- sence of specific staining in the IRF4 KO mouse.

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques: Biomarker Discovery, Staining, Negative Control, Knock-Out, Expressing, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 3. Coexpression pattern of IRF4 with other B cell TF in human tonsil. A, AID (brown) decorates IRF4-GC cells (✮) and extrafollicular blast with variable amounts of IRF4 (purple). Light zone IRF4 centrocytes are AID. Original magnification, 10; scale bar, 15 m. B, Enlargement of portion of extrafollicular area detailing AID (brown) cells containing variable amounts of IRF4 (purple). The edge of a GC is shown (arrow). Original magni- fication, 40; scale bar, 15 m. C, IRTA1 (brown) lymphocytes express IRF4 (purple) in the mantle/marginal zone (yellow arrows, enlarged in the inset), but not in the intraepithelial location (far right). ✮, GC. Original magnification, 40; scale bar, 15 m. D–G, Low-power (left) and high-power LZ detail (right) of coexpression or IRF4 (green) with PU.1, IRF8, and BCL6 (all red) and of PU.1 (green) and BCL6 (red). Note in D coexpression of IRF4 and PU.1 in mantle zone B cells (✮) but not in centrocytes. where IRF4, BCL6, and IRF8 are mutually exclusive. PU.1 and BCL6 show coexpression in part of the GC cells. Left, Original magnification, 4; scale bar, 100 m. Right, Original magnification, 40; scale bar, 5 m. H, CD30 (blue) interfollicular blasts coexpress IRF4 (green) and Pax5 (red). The boxed area is magnified and the color split on the right. Note two IRF4Pax5CD30 blasts (arrows) at the edge of the GC (✮). Original magnification 40, scale bar 5 m. I, Detail of GC light zone, containing centrocytes variably expressing IRF4 (green) and PRDM1 (red). Two cells also express Pax5 (blue, arrows). One Pax5PRDM1 blast is shown (arrowhead). K, Plasma cells outside the GC coexpress IRF4 (green) and PRDM1 (red) but not Pax5 (blue). Note the heterogeneity in Ag expression. A plasmacytoid cell is PRDM1IRF4. L, Light zone centrocytes are IRF4 (green), PRDM1 (red), and a minority express MUM1 (blue). I, K, and L, magnification, 40; scale bar, 15 m. M, Confocal analysis of IRF4 (green), MUM1 (red), and DAPI (blue) staining in GC light zone, showing two IRF4-only centrocytes (arrows) and, on the right, nonidentical distribution of IRF4 and MUM1 in the nuclei. Original magnification, 40; scale bar, 5 m. N, Light zone centrocytes show relocation of cREL (red, arrows) in IRF4 nuclei (green). Nuclei are stained with DAPI (blue). Original magnification, 40; scale bar, 15 m.

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques: Expressing, Clinical Proteomics, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 5. Coexpression of BCL6, IRF4, and Pax5 in IgD and IgD

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 6. Distribution and coexpression of BCL6 and IRF4 (MUM1 and polyclonal Ab) in DLBCL. A, Bivariate dot plot showing the distri- bution of BCL6 and IRF4 in 40 DLBCL cases, according to the score of positive cells. u, The 10% lower limit for positivity for score (see Mate- rials and Methods). The number of cases in each quadrant is shown in italics. Gray symbols represent cases polysomic for 3q27; asterisks repre- sent cases with 3q27 breaks (BCL6 rearrangement). Other cases are either normal or not tested (n 11). B, The correlation of IRF4 and MUM1 staining in 40 DLBCL cases is shown. u, The 10% lower limit for posi- tivity for score.

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 4. BCL6 expression in human tonsil B cells subsets. Five human B cells subsets, defined by CD38 and IgD staining on CD3-negative, CD20 B cells, are analyzed for BCL6, IRF4, and a negative control (gray histograms). Note expression of both BCL6 and IRF4 in mantle zone B cells (IgDCD38), reduced IRF4 and emergence of a strong BCL6 peak in GC founder cells (IgDCD38), disappearance of IRF4 in GC cells (IgDCD38), expression of IRF4 but minimal BCL6 in memory B cells (IgDCD38) and high IRF4 expression in BCL6- plasmacytoid cells (IgDCD38).

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques: Expressing, Staining, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 8. Comparison of MUM1 and IRF4 polyclonal Ab staining on a sample of low grade non-Hodgkin lymphoma. Selected cases of low grade B cell lymphomas (CLL; MC, mantle cell lymphoma, MZ, marginal zone lymphoma) and tonsil are stained respectively with the MUM1 and the polyclonal IRF4 Ab. Identical fields on serial sections are shown. A low-power field (4) is shown at the sides. The polygon marks the tonsil GC, the star the mantle. Note the more frequent positivity of the polyclonal IRF4 Ab.

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques: Comparison, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance.

doi: 10.4049/jimmunol.177.10.6930

Figure Lengend Snippet: FIGURE 7. Coexpression of BCL6 and IRF4 in DLBCL. Four repre- sentative cases of DLBCL (A–D), double stained for BCL6 and IRF4 (MUM1), are shown, the two stains are split and shown in black and white. Arrowheads indicate nuclei with mutually exclusive staining, arrows co- expression. Note the variety of ratios of the two Ags.

Article Snippet: Therefore, cells were first stained with biotin-conjugated Abs, fixed as above, washed in PBS, fixed in absolute cold methanol for 10 min, Table I. Abs used Ab Clone or Serum Immunogena Reactive onb Species Source Dilution PU.1 (Spi1) sc-352 C-term mouse h, m Rabbit SCBT 1 g/ml PU.1 (Spi1) sc-5949 N-term mouse m Goat SCBT 0.1 g/ml PU.1 (Spi1) G148-74 Full length h, m Mouse BD 1 g/ml IRF4 sc-6059 C-term mouse h, m Goat SCBT 0.2 g/ml IRF4 No. 4964 Asp175 h Rabbit CST 1/100 IRF4 sc-28696 AA 128–267h h, m Rabbit SCBT 1 g/ml IRF4 MUM1 AA 144–451h h Mouse B. Falini 1/50 IRF8 (ICSBP) sc-6058 C-term mouse h, m Goat SCBT 1 g/ml BCL6 PIF6 N-term human h, m Mouse Novocastra 1/100 BCL6 PGB6 276 N-term human h, m Mouse B. Falini 1/10 BCL6 PGB6p 594 N-term human h Mouse B. Falini 1/10 BCL6 sc-858 N-term human h, m Rabbit SCBT 0.1 g BCL6 No. 4242 C-term human h, m Rabbit CST 1/100 Pax-5 24 AA 151–306h h, m Mouse BD 1 g/ml Pax-5 sc-1974 C-term human h, m Goat SCBT 1 g/ml Pax-5 RB-9406 C-term human h, m Rabbit LabVision 1 g/ml Oct-2 sc-233 C-term human h, m Rabbit SCBT 0.2 g/ml CD20 RB-9013 C-term human h Rabbit LabVision 1/200 CD20 L26 Cytoplasm.

Techniques: Staining, Expressing