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

Article Title: Dual-specificity phosphatase 14 (DUSP14/MKP6) negatively regulates TCR signaling by inhibiting TAB1 activation.

doi: 10.4049/jimmunol.1300989

Figure Lengend Snippet: FIGURE 7. Enhanced in vivo immune responses in DUSP14-deficient (DUSP14-KO) mice. WT and DUSP14-KO mice were immunized with KLH emulsified in alum. Seven days later, enlarged lymph nodes were isolated and restimulated with KLH for 72 h. (A) Cell proliferation was measured using [3H]thymidine incorporation. (B) IL-2, IL-4, and IFN-g levels in supernatants were measured using ELISA. Three mice were an- alyzed for each genotype. Data are mean 6 SEM. (C) WT and DUSP14- KO mice were immunized as before. Sera were collected at 14 d after the primary immunization. NP-specific IgM, IgG1, IgG2a, IgG2b, and IgG3 Abs were determined by ELISA. Results are presented relative to those of normal serum from a WT mouse. Five mice were analyzed for each ge- notype. Data are mean 6 SEM. *p , 0.05, two-tailed t test.

Article Snippet: The titers of anti–NP-specific IgM, IgG1, IgG2a, IgG2b, and IgG3 in the sera were measured by ELISAs (eBioscience) using NP-BSA (Biosearch Technologies) as the coating Ag, followed by incubation with anti-IgM, anti-IgG1, anti-IgG2a, anti-IgG2b, and anti-IgG3 goat anti-mouse Abs (all from Bethyl Laboratories) (19).

Techniques: In Vivo, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 2 | LAG3 þ Treg regulate B-cell functions through Fas. (a–d) Treatment of MRL/lpr mice with adoptive transfer of T-cell subsets. Ten-week-old MRL/lpr mice were injected i.v. with LAG3 þ Treg (n ¼ 9), CD4 þCD25 LAG3 T cells (LAG3 T; n ¼ 9), CD4 þCD25 þ Treg (CD25 þ Treg; n ¼ 9) or CD4 þCD25 CD45RBhigh T cells (naive T; n ¼ 8) from MRL/ þ mice (1 105 cells each). The mice of LAG3 þ Treg x3 group (n ¼ 8) were injected with LAG3 þ Treg (1 105 cells) at 10 weeks of age followed by a twice weekly injection of the same amount of LAG3 þ Treg. The control group received PBS (n ¼ 13). (a) Proteinuria progression. *Po0.05 versus control group (Mann–Whitney U-test). (b) Quantification of serum anti-ds DNA antibodies. *Po0.05 (Bonferroni post-test). (c) Haematoxylin and eosine (H&E) staining (upper panels) and IgG immunofluorescent staining (lower panels) of kidney sections. Scale bars, 50 mm. (d) Glomerular scores. *Po0.05 (Mann–Whitney U-test). (e) LAG3 þ Treg-mediated suppression of in vitro NP-specific antibody responses. B cells and Th cells purified from NP-OVA/alum-pre-immunized B6 mice and OT-II mice, respectively, were incubated with or without LAG3 þ Treg from non-immunized OT-II mice in the presence or absence of anti-FasL blocking antibody, and supernatants were analysed for anti-NP-BSA antibodies using ELISA. See also Supplementary Fig. 1e (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (f) NP-specific antibody responses of Rag1KO mice injected with B6 B cells and OT-II Th cells with or without LAG3 þ Treg from WT, B6/lpr or B6/gld mice, as outlined in Fig. 1e (n ¼ 6 per group). Anti- FasL blocking antibody (200 mg per mouse) was injected i.v. weekly. *Po0.05 (Bonferroni post-test). (g–i) Flow cytometry plots (g) and quantification of splenic CD4 þCD25 CXCR5 þPD-1 þ TFH (h) and B220 þGL-7 þFas þ GCB (i) from the same mice as in f. Statistical significances in h,i were analysed by Bonferroni post-test (*Po0.05). The experiments in e,f were repeated three times. The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Adoptive Transfer Assay, Injection, Control, MANN-WHITNEY, Staining, In Vitro, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 3 | LAG3 þ Treg suppress B-cell activation through TGF-b3. (a) Microarray comparisons of the gene expression profiles between B6 CD25 þ Treg and B6 LAG3 þ Treg. Normalized expression values from B6 CD4 þCD25 CD45RBhigh naive Tcells are depicted according to the colour scale shown. (b) Tgfb3 mRNA expression in sorted T-cell subsets taken from the spleens of B6 mice (n ¼ 3 per group). (c–e) TGF-b1, 2 and 3 protein levels in the culture supernatants of the indicated T-cell subsets from B6 mice determined using ELISA. Cells were seeded at 1 105 cells per well (n ¼ 4 per group). (f) CFSE- labelled B cells were stimulated with or without anti-IgM mAb in the presence or absence of rTGF-b3 (n ¼ 3 per group). (g) Viability of anti-IgM-stimulated B cells in the presence or absence of rTGF-b3 (1 ng ml 1) was assessed by 7-AAD (n ¼ 3 per group). (h) The effects of TGF-b3 on total IgG production in the culture supernatants of anti-CD40/IL-4-stimulated B cells, determined as in Fig. 1d (n ¼ 3 per group). *Po0.05 (unpaired two-tailed Student’s t-test). (i–k) STAT6 (i), Syk (j) and NF-kB p65 (k) phosphorylation in stimulated B cells with or without rTGF-b3, calculated as the ratio of phosphorylated to total protein levels (n ¼ 3 per group). See also Supplementary Fig. 10. *Po0.05 (unpaired two-tailed Student’s t-test). (l) TGF-b3 protein levels in the culture supernatants of freshly isolated B6 LAG3 þ Treg or, naive B6 CD4 þ Tcells cultured under Th0, Th1, Th2 or Th17 conditions determined using ELISA. Cells were seeded at 3 105 cells per well (n ¼ 3 per group). The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Activation Assay, Microarray, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Phospho-proteomics, Isolation, Cell Culture

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 5 | PD-1 expression on B cells is important for the suppressive activity of LAG3 þ Treg. (a) Resistance to TGF-b3-mediated B-cell suppression in PD-1-deficient (PD-1KO) mice. CFSE-labelled B cells from B6, PD-1KO, B6/lpr or B6/gld mice were stimulated in vitro for 72 h with anti-IgM and anti-CD40 in the presence or absence of rTGF-b3 (1 ng ml 1). Histograms are gated on B220 þ B cells. (b) Bcl-xL and Bcl-2a1 mRNA levels in anti-IgM-stimulated B cells from WT or PD-1KO mice in the presence or absence of rTGF-b3 (1 ng ml 1; n ¼ 3 per group). *Po0.05 (unpaired two-tailed Student’s t-test). (c) Blockade of LAG3 þ Treg-mediated suppression of in vitro NP-specific antibody responses by anti-PD-L1 blocking mAb. B cells and Th cells purified from NP-OVA/alum-pre-immunized B6 mice and OT-II mice, respectively, were incubated with or without LAG3 þ Treg from non-immunized OT-II mice in the presence or absence of anti-PD-L1 blocking mAb, and supernatants were analysed for anti-NP-BSA antibodies by ELISA (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (d) NP-specific antibody responses of Rag1KO mice injected with B6 B cells and OT-II Th cells from B6 or PD-1KO mice with or without LAG3 þ Treg from B6 mice. Anti-PD-L1 blocking antibody (200 mg per mouse) was injected i.v. every 3 days. Anti-NP-BSA antibody levels were determined as in Fig. 1e (n ¼ 6 per group). *Po0.05 (Bonferroni post-test). (e) PD-1 expression on B cells. B cells from B6 mice were stimulated in vitro for 72 h with or without anti-IgM and anti-CD40 antibodies in the presence or absence of rTGF-b3 (1 ng ml 1). Histograms are gated on B220 þ B cells. Data are representative of three independent experiments. The means±s.d. are indicated.

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, In Vitro, Two Tailed Test, Blocking Assay, Incubation, Enzyme-linked Immunosorbent Assay, Injection

Journal: Nature communications

Article Title: TGF-β3-expressing CD4+CD25(-)LAG3+ regulatory T cells control humoral immune responses.

doi: 10.1038/ncomms7329

Figure Lengend Snippet: Figure 6 | IL-27 induces TGF-b3-producing Egr2 þ Treg from naive T cells. (a) IL-27-mediated induction of Egr2 and LAG-3 on CD4 þ T cells. Freshly isolated naive CD4 þ T cells were stimulated with anti-CD3/CD28 mAb in the presence or absence of IL-27. Cells were stained for Egr2 and LAG-3 expression on day 5. (b) Quantitative RT–PCR analysis of Tgfb3 mRNA expression in naive WT CD4 þ Tcells activated as in a, assessed on day 3. *Po0.05 (unpaired two-tailed Student’s t-test). (c) ELISA for TGF-b3 in culture supernatants of activated naive WT, Egr2 CKO, STAT1 KO, or Stat3fl/flCD4-Cre þ

Article Snippet: B cells undergoing apoptosis on day 3 and total IgG production in the culture supernatants on day 7 were determined using the Annexin V Apoptosis Detection Kit (BD Pharmingen) and a mouse IgG ELISA Quantitation Set (Bethyl Laboratories), respectively, according to the manufacturer’s protocol.

Techniques: Isolation, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test, Enzyme-linked Immunosorbent Assay

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

Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: Adjuvants impact the durability of protection conferred by eVLP vaccination. (a) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged four (short-term) weeks or twenty-two (long-term) weeks after the vaccine boost. Data in A are pooled from 8 individual studies with 6–10 animals/group. Fisher's exact test: survival in the short-term group was significantly higher than in the long-term group (p < 0.0001). (b) C57BL/6 mice were vaccinated IM two times with 10 μg of eVLP and challenged at the indicated days after the second vaccination. n = 9 or 10/group. Cochran-Armitage test: percentage surviving declined as time to challenge increased (p = 0.0046). There was a significant difference (p = 0.03) between survival on Day 77 and Day 175. (c) Serum samples collected from animals in (B) one week prior to challenge were subjected to an ELISA for the evaluation of anti-GP IgG, IgG1, and IgG2c antibody titers. Red symbols indicate titers of animals that succumbed to challenge while black indicate titers of survivors; red symbols with black outlines indicate that one of these two animals succumbed to challenge, but animal tags were indeterminate after challenge. Median and IQR shown. (d) C57BL/6 mice were vaccinated two times (IM) with VLP, with or without the indicated adjuvants. Animals were challenged twenty-two weeks after the vaccine boost. Data in D are pooled from at least 4 separate studies with a total of at least 35 animals per group. P-values comparing VLP alone to vaccination with VLP and adjuvant are shown, calculated using Fisher's exact tests with stepdown Bonferroni correction. V = VLP, VP = VLP + PolyICLC, VC = VLP + CpG, VM = VLP + MPLA, and VA = VLP + alhydrogel.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Enzyme-linked Immunosorbent Assay, Adjuvant

Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: Adjuvants have variable impact on IgG subclasses and antibody neutralization. (a) Serum was collected 14 days and 147 days after the vaccine boost (days 35 and 168, respectively) and evaluated for anti-GP IgG, IgG1, IgG2c, and IgG3 levels using an ELISA. Data shown are pooled from at least two separate experiments per group. (b) Pairwise comparison using DSCF multiple pairwise comparison was used and p values greater than 0.05 are shown as “ns”. (c) Summary of results shown in A, B and D. (d) Neutralizing antibody titers were evaluated using the PsVNA, with titers giving 80% neutralization shown; median and IQR shown. Samples within each group were selected randomly from three separate studies for evaluation in the assay. Pairwise comparison using post-hoc Tukey's studentized range test procedure was used to evaluate differences between groups at both days 35 and day 168, where “*” indicates 0.01 < p < 0.05, “**” indicates 0.001 < p < 0.01, “***” indicates 0.0001 < p < 0.001, and “****” indicates p < 0.0001.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Comparison

Journal: EBioMedicine

Article Title: Adjuvant-enhanced CD4 T Cell Responses are Critical to Durable Vaccine Immunity

doi: 10.1016/j.ebiom.2015.11.041

Figure Lengend Snippet: CD8 T cell deficiency does not impact short term or long term survival of vaccinated C57BL/6J mice. (a) Mice were treated IM twice with saline, VLP, VLP and polyICLC, or VLP and CpG. Four weeks after the second vaccination, mice were challenged. Closed symbols represent wild type C57BL/6J mice and open symbols indicate CD8-deficient mice. (b) Mice were vaccinated on the same schedule as in A, but challenge occurred 22 weeks after the second vaccination. (c) Two weeks after the second vaccination and 1 week prior to challenge, blood was collected from vaccinated animals and evaluated for anti-GP IgG antibody titers. (D) A subset of vaccinated animals was euthanized 4 days after the vaccine boost to evaluate CD4 + T cell responses. Median response of C57BL/6J mice and CD8-deficient mice is shown. N = 8–10 per treated group for A–C and D presents data pooled from two separate evaluations of 4–8 mice per group each. “*” indicates 0.005 < p < 0.05.

Article Snippet: Secondary antibodies included goat anti-mouse IgG-HRP (Southern Biotech 1030–05), IgG1-HRP (Southern Biotech 1070–05), IgG2c-HRP (Southern Biotech 1079–05), and IgG3-HRP (Southern Biotech 1100–05).

Techniques: Saline