Journal: Frontiers in Veterinary Science

Article Title: Cellular and Cytokine Responses in Lymph Node Granulomas of Bacillus Calmette Guérin (BCG)-Vaccinated and Non-vaccinated Cross-Breed Calves Naturally Infected With Mycobacterium bovis

doi: 10.3389/fvets.2021.698800

Figure Lengend Snippet: Immune markers used for immunohistochemistry.

Article Snippet: CD4 , Mouse anti bovine CD4 (monoclonal) , 1/30 , BIO-RAD , NA , Goat vs. mouse (1/200) , RT, 1 h , BIO-RAD , PBS.

Techniques: Immunohistochemistry, Incubation

Journal: Scientific Reports

Article Title: Multiplex gene analysis reveals T-cell and antibody-mediated rejection-specific upregulation of complement in renal transplants

doi: 10.1038/s41598-021-94954-3

Figure Lengend Snippet: Deposition of immune cells in transplant biopsies. Percentage of positive stained area in follow-up biopsies of renal transplants with no rejection or dysfunction (Ctrl, n = 7), delayed graft function (DGF, n = 6), T-cell mediated rejection (TCMR, n = 8) or antibody mediated rejection (ABMR, n = 7) after immunostaining for CD4 ( A ), CD68 ( D ), CD21 ( G ) and ITGB2 ( J ); (Statistical analysis: A , G : ANOVA with Tukey’s; D , J : Kruskal–Wallis with Dunn’s;**p < 0.01; ***p < 0.001; ****p < 0.0001). Examples of biopsies with no rejection or dysfunction (Ctrl) and T-cell mediated rejection (TCMR) after immunostaining for CD4 ( B , C ), CD68 ( E , F ), CD21 ( H , I ) and ITGB2 ( K , L ).

Article Snippet: Following antibodies diluted in 50 mM Tris pH 7.4 were used: a mouse monoclonal antibody against human CD68 (M0876; Dako Deutschland); a mouse monoclonal antibody against human CD21 (M0784; Dako Deutschland); a rabbit monoclonal antibody against human CD4 (Ab133616; Abcam, Cambridge, UK) and a rabbit polyclonal antibody against human integrin beta-2 (ITGB2) (HPA016894; Sigma Aldrich).

Techniques: Staining, Immunostaining

Journal: Cell Reports Medicine

Article Title: T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation

doi: 10.1016/j.xcrm.2022.100559

Figure Lengend Snippet: Analysis of circulating CD4 + and CD8 + T cell subsets from upper extremity recipients over time (A) Skin biopsies and peripheral blood were collected over time (pre-transplantation, 24 h, 1 week, and 3, 6, and 12 months after transplantation) from upper extremity transplant recipients. PBMCs were isolated for posterior flow cytometry analysis. The cartoon was created with BioRender. (B and C) Mean percentages of blood CD4 + (B) and CD8 + (C) naive cells (CCR7 + CD45RA + ), central memory T cells (TCM cells; CCR7 + CD45RA − ), effector memory T cells (TEM cells; CCR7 − CD45RA − ), and effector memory RA cells (TEMRA cells; CCR7 − CD45RA + ) before transplantation and 6 and 12 months after transplantation, represented as pie charts. Data are from all three individuals. (D) Representative contour plots of gating of T helper (Th) 1 (CD4 + CXCR3 + CCR6 − ), Th2 (CD4 + CXCR3 − CCR6 − ), and Th17 (CD4 + CXCR3 − CCR6 + ) cells. (E) Mean percentages of Th1, Th2, and Th17 cells from all three individuals over time. (F–H) Mean percentages of circulating Th17 cells before transplantation and 12 months after transplantation. Statistic by paired t test, ∗p < 0.05. (G and H) Representative contour plots and mean percentages of (G) regulatory T (Treg) cells (CD4 + CD25 + CD127 −/low ) and (H) T follicular helper (Tfh) cells (CD4 + CXCR5 + PD-1 + ) from all three individuals over time. Graphs are displayed as mean ± SD at each time point examined. See also Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 and .

Article Snippet: Sections were incubated with primary antibodies rabbit-anti-human CD4 (Novus Biologicals) at 1:200 and mouse-anti-human CD8 (Abcam) at 1:50 overnight at 4°C.

Techniques: Transplantation Assay, Isolation, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation

doi: 10.1016/j.xcrm.2022.100559

Figure Lengend Snippet: Characterization of CD4 + and CD8 + T cell subsets from allografts and native skin of upper extremity recipients (A–F) Mean percentages per skin area of infiltrating (A) CD4 + TEM cells (CCR7 − CD45RA − ), (B) Th1 cells (CD4 + CXCR3 + CCR6 − ), (C) Th17 cells (CD4 + CXCR3 − CCR6 + ), (D) total CD8 + cells, (E) CD8 + TEM cells, and (F) CD8 + TEMRA cells (CCR7 − CD45RA + ) from the allografts and adjacent native skins. Data are from all three individuals and represented as mean ± SD; statistics by paired t test. (G) Representative H&E staining (left) and immunofluorescence of CD4 + and CD8 + cells (right) from the allograft and adjacent native skin of the same upper extremity transplant recipient; 200× (scale bars, 100 μm). See also Figure S3 .

Article Snippet: Sections were incubated with primary antibodies rabbit-anti-human CD4 (Novus Biologicals) at 1:200 and mouse-anti-human CD8 (Abcam) at 1:50 overnight at 4°C.

Techniques: Staining, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation

doi: 10.1016/j.xcrm.2022.100559

Figure Lengend Snippet: Clinical and histopathological aspects in upper extremity allograft rejection with correlation to peripheral T cell populations (A and B) Clinical photographs of an upper extremity transplant recipient and (B) corresponding H&E graft staining during clinical cellular rejection episodes with graft erythema and edema (grades 2–3) compared with mild rejection on surveillance biopsy (grade 1) without significant erythema or edema. Grade 3 rejection retains lymphocytic vasculopathy (bottom right panel) but also shows epithelial apoptosis associated with lymphoid exocytosis (circled in higher magnification, 400×). Left images, 40×; right images, 400× (scale bars, 50 μm). (C–I) Percentages and (D, F, H, and J) absolute numbers of circulating total CD8 + , CD8 + TEMRA, CD4 + TEMRA, and Treg cells at rejection (grades 2–3, n = 6) and nonrejection (grade 0, n = 6) events. Percentages of (K) IFN-γ + , (L) IL-4 + , (M) IL-17 + CD4 + T cells, and (N) IFN-γ + CD8 + T cells from peripheral blood at rejection and nonrejection time points. (C–N) Data are from all three individuals and represented as mean ± SD; statistics by paired t test. Rejection time points included samples from 1 week to 3 years after transplantation and nonrejection time points from 1 month to 4 years after transplantation.

Article Snippet: Sections were incubated with primary antibodies rabbit-anti-human CD4 (Novus Biologicals) at 1:200 and mouse-anti-human CD8 (Abcam) at 1:50 overnight at 4°C.

Techniques: Staining, Transplantation Assay

Journal: Cell Reports Medicine

Article Title: T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation

doi: 10.1016/j.xcrm.2022.100559

Figure Lengend Snippet: CCL18 attracts human allo-T cells to human skin xenografts (A and B) Discarded human skin was transplanted into NSG recipient mice. Mice were injected weekly with anti-Gr1 to reduce local inflammation and establish an intact vasculature (A). Twenty-eight days after transplantation, recipients were injected with 20 × 10 6 allo-PBMCs. Seven days thereafter, 300 ng of CCL18 or PBS 1× was injected subcutaneously into the skin xenografts for 10 consecutive days. Recipient animals were treated with anti-CCR8 or isotype control daily. Skin xenografts and peripheral blood were analyzed by histology and flow cytometry on day 45 after transplantation. (B–D) Representative macroscopic images of skin xenografts, H&E graft staining, and immunohistochemical staining for human CD31, human CD3, or human CD45 after the CCL18 injections into animals (A) injected (B) or not injected (C) with human PBMCs. Shown are absolute counts of human CD31 + vessels. (E and F) Human CD3 + cells (E) and human CD45 + cells (F) in the skin xenografts, 100×. The number of positive cells was quantified from two representative 400× fields from each transplanted xenograft. Statistics by one-way ANOVA with Tukey’s post-test. (G–J) Representative contour plot and absolute numbers of skin-infiltrating (G) CD4 + and CD8 + cells, (H) skin CD8 + CLA + and CD4 + CLA + cells, (I) skin CD8 + CCR8 + and CD4 + CCR8 + cells, and (J) Th1, Th17, and Th2 in PBS-, CCL18 − , or CCL18 + anti-CCR8-treated groups that received human PBMCs. All data were normalized by square centimeter of tissue. Data represent a pool of two independent experiments (n = 4–6 animals per group) and are represented as mean ± SD. Statistics by one-way ANOVA with Tukey’s post-test. See also Figure S4 .

Article Snippet: Sections were incubated with primary antibodies rabbit-anti-human CD4 (Novus Biologicals) at 1:200 and mouse-anti-human CD8 (Abcam) at 1:50 overnight at 4°C.

Techniques: Injection, Transplantation Assay, Control, Flow Cytometry, Staining, Immunohistochemical staining

Journal: Cell Reports Medicine

Article Title: T cell-attracting CCL18 chemokine is a dominant rejection signal during limb transplantation

doi: 10.1016/j.xcrm.2022.100559

Figure Lengend Snippet:

Article Snippet: Sections were incubated with primary antibodies rabbit-anti-human CD4 (Novus Biologicals) at 1:200 and mouse-anti-human CD8 (Abcam) at 1:50 overnight at 4°C.

Techniques: Purification, Blocking Assay, Plasmid Preparation, Recombinant, Staining, Software