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

Article Title: CXCL13 Blockade Disrupts B Lymphocyte Organization in Tertiary Lymphoid Structures without Altering B Cell Receptor Bias or Preventing Diabetes in Nonobese Diabetic Mice

doi: 10.4049/jimmunol.0903710

Figure Lengend Snippet: CXCL13 is expressed in inflamed islets of NOD mice. A, Gel electrophoresis showing RT-PCR products from isolated pancreatic islets using cxcl13-specific primers. First three lanes are from inflamed islets of three independent female NOD mice. Last three lanes are from islets of three independent female C57BL/6 mice. Lower lanes are controls using hprt primers from the same islets. B, Real-time PCR showing cxcl13 levels, normalized to hprt, from isolated islets. At least 20 islets were isolated from three mice per group, and pooled for each mouse. Normalized cxcl13 levels are significantly higher in islets from 12-wk-old NOD mice than from any other group. **p < 0.0001, compared with noninfiltrated NOR controls or 3-wk-old NOD mice; p < 0.001 for 6-wk-old NOD mice; p = 0.004 compared with 14-wk-old NOR mice. C, Immunohistochemical staining of frozen NOD pancreatic sections shows CXCL13+ (dark brown) cells among invading lymphocytes in early insulitis. Right panel shows control section processed with secondary reagents, but without primary anti-CXCL13 Ab (original magnification ×20).

Article Snippet: CXCL13 blockade Wild-type or V H 125 transgenic NOD mice were i.p. injected with 100 μg monoclonal, rat-derived, anti-CXCL13 Ab (mAB470, R&D Systems, Minneapolis, MN) in sterile PBS three times weekly, from age 3 wk to the end of each study.

Techniques: Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Control

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

Article Title: CXCL13 Blockade Disrupts B Lymphocyte Organization in Tertiary Lymphoid Structures without Altering B Cell Receptor Bias or Preventing Diabetes in Nonobese Diabetic Mice

doi: 10.4049/jimmunol.0903710

Figure Lengend Snippet: CXCL13 receptor is expressed on B cells in pancreas and spleen. Flow cytometry shows CXCR5 expression on B220+ B cells from pancreas and spleen of the same female NOD mouse. Image is representative of data from six mice.

Article Snippet: CXCL13 blockade Wild-type or V H 125 transgenic NOD mice were i.p. injected with 100 μg monoclonal, rat-derived, anti-CXCL13 Ab (mAB470, R&D Systems, Minneapolis, MN) in sterile PBS three times weekly, from age 3 wk to the end of each study.

Techniques: Flow Cytometry, Expressing

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

Article Title: CXCL13 Blockade Disrupts B Lymphocyte Organization in Tertiary Lymphoid Structures without Altering B Cell Receptor Bias or Preventing Diabetes in Nonobese Diabetic Mice

doi: 10.4049/jimmunol.0903710

Figure Lengend Snippet: CXCL13 blockade does not stop B lymphocyte invasion of pancreatic islets, but scrambles tertiary lymphoid organization. A, Immunofluorescent staining of a typical, untreated, inflamed islet from a 12-wk-old female NOD mouse, with B220+ B cells (pseudocolored pink) surrounding CD3+ T cells (pseudocolored blue) (original magnification ×20). B, Immunofluorescent staining of a typical inflamed islet from a 12-wk-old female NOD mouse treated with CXCL13-blocking Ab from age 3 wk (original magnification ×20). C, Bar chart showing B lymphocytes as proportion of total lymphocytes extracted from pancreata of 12-wk-old female NOD mice treated with anti-CXCL13, isotype control, or not treated, analyzed by flow cytometry. Bar height shows average for three mice per group, with SD as indicated. D, Bar chart showing percent of inflamed islets classified as organized, disorganized, or intermediate from 12-wk-old female NOD mice treated with anti-CXCL13, isotype control Abs, or not treated. p < 0.001 for CXCL13 versus isotype-treated controls; p < 0.001 for CXCL13 versus untreated controls; p = 0.9 for isotype-treated controls versus untreated controls. Independent islets from anti-CXCL13, n = 34; isotype control, n = 27; untreated, n = 18; anti-CXCL13–treated mice, n = 3; isotype control mice, n = 2; untreated controls mice, n = 2.

Article Snippet: CXCL13 blockade Wild-type or V H 125 transgenic NOD mice were i.p. injected with 100 μg monoclonal, rat-derived, anti-CXCL13 Ab (mAB470, R&D Systems, Minneapolis, MN) in sterile PBS three times weekly, from age 3 wk to the end of each study.

Techniques: Staining, Blocking Assay, Control, Flow Cytometry

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

Article Title: CXCL13 Blockade Disrupts B Lymphocyte Organization in Tertiary Lymphoid Structures without Altering B Cell Receptor Bias or Preventing Diabetes in Nonobese Diabetic Mice

doi: 10.4049/jimmunol.0903710

Figure Lengend Snippet: CXCL13 blockade fails to alter selection of the B cell repertoire found in pancreatic islets. A, LC gene families expressed by B cells from pancreata (black bars) and draining PLNs (gray bars) of 12-wk-old prediabetic female VH125/NOD mice treated with anti-CXCL13 from the age of 3 wk. p = 0.002 by Fisher exact test; mice, n = 3; independent clones, n = 53. B, The ratio of predominant B lymphocyte Vκ families found in pancreata, relative to those from PLNs, in untreated VH125/NOD (black bars) and anti-CXCL13–treated VH125/NOD mice (gray bars). p = 0.066; 95% CI 0.77–1.01 by Poisson regression; mice, n = 8; independent clones, n = 104.

Article Snippet: CXCL13 blockade Wild-type or V H 125 transgenic NOD mice were i.p. injected with 100 μg monoclonal, rat-derived, anti-CXCL13 Ab (mAB470, R&D Systems, Minneapolis, MN) in sterile PBS three times weekly, from age 3 wk to the end of each study.

Techniques: Selection, Clone Assay

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

Article Title: CXCL13 Blockade Disrupts B Lymphocyte Organization in Tertiary Lymphoid Structures without Altering B Cell Receptor Bias or Preventing Diabetes in Nonobese Diabetic Mice

doi: 10.4049/jimmunol.0903710

Figure Lengend Snippet: CXCL13 blockade does not significantly decrease CDR mutations found in BCR LCs from pancreata. Gene segments encoding BCR κ LCs cloned from pancreata of VH125/NOD mice were examined for evidence of somatic hypermutation as indicated by mutations in the CDRs. A, A total of 35% of BCR LC gene segments from pancreata of untreated controls have mutations in the CDR-encoding region. B, A total of 34% of BCR LC gene segments from anti-CXCL13–treated mice have mutations in the CDR-encoding regions. Keys show shading corresponding to number of mutations. Untreated, n = 45 clones; anti-CXCL13, n = 29 clones, from at least three mice per group. p = 0.861 by negative binomial regression.

Article Snippet: CXCL13 blockade Wild-type or V H 125 transgenic NOD mice were i.p. injected with 100 μg monoclonal, rat-derived, anti-CXCL13 Ab (mAB470, R&D Systems, Minneapolis, MN) in sterile PBS three times weekly, from age 3 wk to the end of each study.

Techniques: Clone Assay

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

Article Title: CXCL13 Blockade Disrupts B Lymphocyte Organization in Tertiary Lymphoid Structures without Altering B Cell Receptor Bias or Preventing Diabetes in Nonobese Diabetic Mice

doi: 10.4049/jimmunol.0903710

Figure Lengend Snippet: CXCL13 blockade does not protect against development of diabetes. Female VH125/NOD mice were administered monoclonal anti-CXCL13 (◆) or isotype control (■), or were untreated (▲). Blood glucose levels were checked weekly, and mice diagnosed with diabetes at >200 mg/dl. Study was stopped for lack of efficacy when at least 50% of anti-CXCL13–treated mice had become diabetic. Mice, n = 10 per group. Log rank p = 0.94 CXCL13 versus isotype control treated; log rank p = 0.27 anti-CXCL13 versus untreated controls.

Article Snippet: CXCL13 blockade Wild-type or V H 125 transgenic NOD mice were i.p. injected with 100 μg monoclonal, rat-derived, anti-CXCL13 Ab (mAB470, R&D Systems, Minneapolis, MN) in sterile PBS three times weekly, from age 3 wk to the end of each study.

Techniques: Control

Journal: The Journal of Clinical Investigation

Article Title: Bronchus-associated lymphoid tissue–resident Foxp3 + T lymphocytes prevent antibody-mediated lung rejection

doi: 10.1172/JCI122083

Figure Lengend Snippet: (A) Foxp3+ (green), B cells (blue), and CD4+ T cells (red) in BALB/c lungs at least 30 days after transplantation into immunosuppressed B6 Foxp3-IRES GFP recipient (n = 3). Scale bar: 10 μm. CXCR5+ B cells from secondary host (recipient) in BALB/c lungs, initially transplanted into immunosuppressed (B) WT or (C) Foxp3-DTR B6 (CD45.2+) recipient and, at least 30 days later, retransplanted into DT-treated B6 CD45.1+ hosts. Plots are gated on live CD45.2–CD45.1+ cells. (D) CD45.1+CXCR5+ B cells in (circles) control and (inverted triangles) Foxp3+ T cell–depleted lungs 7 days after retransplantation (n = 4 each). CXCL13 (brown) in (E) control and (F) Foxp3+ T cell–depleted grafts 7 days after retransplantation. Scale bars: 100 μm. CD4+ T cells (green) and B cells (blue) in BALB/c lungs, initially transplanted into immunosuppressed B6 Foxp3-DTR recipients and, at least 30 days later, retransplanted into B6 hosts, treated with (G) DT/control-Ig (arrows: CD4+ T–B cell interactions) or (H) DT/anti-CXCL13 (n = 2 each) (red, quantum dots). Scale bars: 20 μm. (I) Contact duration between CD4+ T and B cells, (J) CD4+ T, and (K) B cell mean square displacements and (L) CD4+ T and (M) B cell velocities within retransplanted Foxp3+ T cell–depleted BALB/c lungs with and without CXCL13 inhibition. (N) Gross, (O) histological appearance (H&E), staining for (P) MT (blue), (Q) CCSP (red), and AcT (green) in BALB/c lungs, transplanted into immunosuppressed B6 Foxp3-DTR mice and, at least 30 days later, retransplanted into DT- and anti-CXCL13–treated B6 hosts. Scale bars: 100 μm. (R) Donor-specific IgM titers after retransplantation of BALB/c lungs into DT-treated control (blue) or DT/anti-CXCL13 antibody–treated (red) B6 recipients after initial engraftment into immunosuppressed B6 Foxp3-DTR mice (n = 4 mice per group). Data are expressed as mean ± SEM. Mann-Whitney U test was used to compare the means.

Article Snippet: Primary antibodies used were rabbit monoclonal anti-human Foxp3 (clone SP97, 1:400, Novus), rabbit polyclonal anti-mouse CXCL13 (1:100, Bioss), and rat monoclonal anti-mouse PNAd (clone MECA-79, 1:100, BD Biosciences).

Techniques: Transplantation Assay, Inhibition, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: Peripheral helper-T-cell-derived CXCL13 is a crucial pathogenic factor in idiopathic multicentric Castleman disease

doi: 10.1038/s41467-023-42718-0

Figure Lengend Snippet: a A representative FACS plot of hCD45 + cells in the spleen of iMCD-NOS NSG3 mice. Human CD3 + CD4 + cells were analyzed for PD-1 and CXCR5 expression levels. b The frequencies of hPD-1 high hCXCR5 − cells among CD3 + CD4 + human T cells in NSG mice reconstituted with normal human hematopoiesis via xenotransplantation of CB-derived CD34 + HSPCs (left, black, n = 3) and four independent iMCD-NOS NSG mice (right, red, NSG1: n = 4, NSG2: n = 6, NSG3: n = 6 and NSG5: n = 4). Two-group comparison between the iMCD group and the control group was performed using unpaired two-sided t test ( P = 7.99 × 10 −9 ). Error bar represented as mean ± SD. c Representative FACS plot of hCCR2 expression in hPD-1 high hCXCR5 - Tph cells in the spleen of iMCD-NOS NSG2 mice (P2). d Comparison of the frequencies of CD3 + CD4 + PD-1 high CXCR5 − Tph cells and CD3 + CD4 + PD-1 high CXCR5 + Tfh cells in the reactive LN (C1, C6, C7, C8, and C9: black, n = 5) and iMCD-NOS LN samples (P4, P7, P8, P9, P10, and P11: red, n = 6). Comparison between the two groups was analyzed with two-tailed unpaired t test. (Tph: P = 0.01358 and Tfh: P = 0.6635) Error bar represented as mean ± SE. e Heatmap analysis of human (left) and mouse (right) cytokines and chemokines in the sera of NSG mice reconstituted with normal human hematopoiesis by xenotransplantation of CB-derived CD34 + HSPCs ( n = 3) (left) and LN cells from Patient 2 ( n = 5). f Comparison of the levels of human cytokines and chemokines in the serum of the original patient (P2) and sera of iMCD-NOS NSG2 mice transplanted with LN cells from the same patient. g Representative intracellular staining of CXCL13 expression in CD4 + PD-1 high CXCR5 − Tph cells from the LN of the patient with iMCD-NOS(P1) and PB of patient with iMCD-NOS (P6). ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are provided as a Source Data.

Article Snippet: After permeabilization, the cells were stained with a PE- or Alexa Fluor 647-conjugated anti-human CXCL13 antibody (cat# IC801P, 1:10, R&D Systems and cat#IC8012R, 1:10, R&D Systems, respectively) and for 30 min. After staining, the cells were analyzed using a BD FACS Aria II (BD Biosciences), BD FACS Aria IIIu (BD Biosciences), or Attune NxT Flow Cytometer (Thermo Fisher Scientific).

Techniques: Expressing, Derivative Assay, Comparison, Control, Two Tailed Test, Staining

Journal: Nature Communications

Article Title: Peripheral helper-T-cell-derived CXCL13 is a crucial pathogenic factor in idiopathic multicentric Castleman disease

doi: 10.1038/s41467-023-42718-0

Figure Lengend Snippet: Our xenotransplantation experiments revealed that the T-B interaction is required for the development of iMCD-like systemic inflammation in vivo with the elevation of human gamma-globulin. Aberrantly expanded CD4 + PD-1 high CXCR5 - CCR2 + Tph cells actively secrete CXCL13 and promote the migration of B cells toward inflamed sites. Through interaction with Tph cells, migrated B cells differentiate into plasmablasts and initiate the secretion of human gamma-globulin, cytokines, and chemokines, leading to systemic inflammation. Thus, iMCD-NOS represents an abnormal immunoregulatory disorder in which the aberrant immunological synapse consisting of T-B interactions plays a critical role in disease initiation and exacerbation.

Article Snippet: After permeabilization, the cells were stained with a PE- or Alexa Fluor 647-conjugated anti-human CXCL13 antibody (cat# IC801P, 1:10, R&D Systems and cat#IC8012R, 1:10, R&D Systems, respectively) and for 30 min. After staining, the cells were analyzed using a BD FACS Aria II (BD Biosciences), BD FACS Aria IIIu (BD Biosciences), or Attune NxT Flow Cytometer (Thermo Fisher Scientific).

Techniques: In Vivo, Migration