Journal: Journal of Clinical Investigation

Article Title: CXCL12+ fibroblastic reticular cells in lymph nodes facilitate immune tolerance by regulating T cell–mediated alloimmunity

doi: 10.1172/jci182709

Figure Lengend Snippet: Figure 2. Deletion of LTβR+ signaling on FRCs abrogates transplant tolerance. (A) Comparison of heart allograft survival among WT C57BL/6J, Ccl19CreLtbrfl/fl, and Ccl19CreLtbrfl/fl recipients with FRC injections (n = 7–9 mice/group). Graft survival data were combined from 3 independent experiments. (B) Representative images and comparison of CD3-, CD11b-, and collagen 1–stained heart allografts from WT C57BL/6J and Ccl19CreLtbrfl/fl recipients (n = 4 mice/group). Scale bars: 200 μm. (C) Flow cytometry analysis of DLNs from WT C57BL/6J and Ccl19CreLtbrfl/fl recipients (n = 4 mice/group). (D) Representative images and comparison of Foxp3-stained DLNs (n = 4 mice/group). Scale bars: 200 μm. (E) Flow cytometry analysis of transferred TCR Tg cells in DLNs (n = 4 mice/group). (F) Comparison of heart allograft survival among WT C57BL/6J recipients treated with anti-LTβR agonist mAb alone (20 μg i.v. on day 0), anti-CD40L alone (40 μg i.v. on day 0), and both anti-LTβR agonist mAb and anti-CD40L of BALB/c hearts (n = 6 mice/group). Graft survival data were combined from 2 independent experiments. log-rank test for graft survival. Student’s t test for comparisons between 2 groups. Data are represented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: As the standard protocol in this study, anti-CD40L mAb (MR1, 125 μg i.v. on days 0 and 1, BioXCell, BE0017-1) or CTLA4-Ig (Abatacept, 500 μg i.p. on day 0 and 250 μg i.p. on days 2, 4, 6, 8, Bristol-Myers Squibb) was administered to recipient mice.

Techniques: Comparison, Staining, Flow Cytometry

Journal: Journal of Clinical Investigation

Article Title: CXCL12+ fibroblastic reticular cells in lymph nodes facilitate immune tolerance by regulating T cell–mediated alloimmunity

doi: 10.1172/jci182709

Figure Lengend Snippet: Figure 3. CXCL12hi FRC subsets enriched for genes for T cell migration. (A) Unsupervised clustering of FRC subset clusters visualized with UMAP in LNSCs from naive LNs. (B) Violin plots of Ltbr and Cxcl12 expression among FRC subset clusters in naive LNs. (C) Violin plots of Ltbr and Cxcl12 expres- sion in total FRCs from naive LNs. (D) UMAP of SC populations in naive LNs and DLNs, highlighting Cxcl12 expression on MedRC, TRC, and PRC subsets. (E) Violin plots of Cxcl12 expression in FRC subsets from naive LNs and DLNs. (F) Volcano plot comparing CXCL12hi and CXCL12lo FRCs in mouse LNSCs from heart transplanted recipients with anti-CD40L treatment. (G) Top 20 overrepresented ontology pathways based on DEGs in mouse LNSCs. (H) Volcano plot comparing CXCL12hi and CXCL12lo FRCs in human LNSCs. (I) Top 20 overrepresented ontology pathways in human LNSCs. Common genes shared between mouse and human are highlighted with bold letters surrounded by squares (F and H). Ontology pathways related to chemotaxis and migration are highlighted with bold letters surrounded by squares (G and I). HTx, heart transplantation.

Article Snippet: As the standard protocol in this study, anti-CD40L mAb (MR1, 125 μg i.v. on days 0 and 1, BioXCell, BE0017-1) or CTLA4-Ig (Abatacept, 500 μg i.p. on day 0 and 250 μg i.p. on days 2, 4, 6, 8, Bristol-Myers Squibb) was administered to recipient mice.

Techniques: Migration, Expressing, Chemotaxis Assay, Transplantation Assay

Journal: Journal of Clinical Investigation

Article Title: CXCL12+ fibroblastic reticular cells in lymph nodes facilitate immune tolerance by regulating T cell–mediated alloimmunity

doi: 10.1172/jci182709

Figure Lengend Snippet: Figure 4. Ablation of CXCL12 of FRCs abrogates anti-CD40L mediated transplant tolerance. (A) Flow cytometry analysis of T cell compartments in naive LNs (n = 3 mice/group). (B) Representative intravital images of CMFDA-labeled CD3+ T cells injected into naive LNs. Yellow and white arrows indicate the migration of transferred CMFDA+ CD3+ T cells from inside HEVs into the parenchyma. Representative trajectories of transferred CD3+ T cells (gray, cyan, and magenta lines) and quantitative analysis of cell migration from inside HEVs toward the parenchyma for 20 minutes (n = 3). Scale bars: 100 μm. (C) In vivo T cell migration assay in a skin transplantation model. Representative images and quantitative analysis of CMFDA-labeled CD3+ T cells in DLNs (n = 5). Scale bars: 100 μm. (D) Comparison of heart allograft survival among the recipients under CTLA4 Ig treatment (n = 7 mice/group). Graft survival data were combined from 2 independent experiments. (E) Representative images and comparison of CD3-, CD11c-, and collagen 1–stained heart allografts (n = 5 mice/group). Scale bars: 200 μm. (F) Flow cytometry analysis of DLNs (n = 5 mice/group). log-rank test for graft survival. Student’s t test for comparisons between 2 groups. Data are represented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: As the standard protocol in this study, anti-CD40L mAb (MR1, 125 μg i.v. on days 0 and 1, BioXCell, BE0017-1) or CTLA4-Ig (Abatacept, 500 μg i.p. on day 0 and 250 μg i.p. on days 2, 4, 6, 8, Bristol-Myers Squibb) was administered to recipient mice.

Techniques: Flow Cytometry, Labeling, Injection, Migration, In Vivo, Cell Migration Assay, Transplantation Assay, Comparison, Staining

Journal: bioRxiv

Article Title: Transfusion of allogenic murine HOD red blood cells preferentially induces low-affinity, short-lived IgG antibodies that are germinal center independent

doi: 10.1101/2025.01.16.633377

Figure Lengend Snippet: (A) Experimental approach for CD40L blocking experiments. WT mice were either transfused with HOD RBCs or vaccinated with HEL-OVA/Alum. They received CD40L blocking antibody 4 days after immunization and subsequently on days 7 and 10 pi. Sera were collected 2 weeks after immunization for assessment of IgG levels by limiting dilution ELISA. (B) IgG titers of transfused mice. (C) IgG titers of vaccinated mice. (D) Experimental approach for experiments that utilized BCL6-BKO mice. WT or BCL6-BKO mice were either transfused with HOD RBCs or vaccinated with HEL-OVA/Alum. Sera were collected 2 weeks after immunization for assessment of IgG levels by limiting dilution ELISA. (E) IgG titers of transfused mice. (F) IgG titers of vaccinated mice. Each data point represents on mouse. Bars on scatter plots are median values. Figure shows a representative experiment out of 3. Groups of interest were compared using Mann-Whitney U tests preceded by Kruskal-Wallis tests. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns P>0.5.

Article Snippet: For CD40L blocking experiments, mice were given 250 μg of the anti-mouse CD40L blocking antibody or isotype control via IP injection (BioXCell, BE0017-1 or BE0091) on days 4, 7, 10, and 14 post-immunization.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY