39 research outputs found
Characterization of cells transferred to recipient Rag-1−/− mice.
<p>T cells enriched from pooled spleens of CD4−/− mice (Donor CD8<sup>+</sup>) were used for flow cytometric analysis and compared with T cells enriched from spleens of CD8<sup>+</sup> T cell recipient Rag-1−/− mice 48 hours after cell transfer without injury (UI), and 21 days after injury (D21). Representative flow cytometric analysis of CD62L or CD44 gated on CD8b<sup>+</sup> cells from donor mice (A). CD8b<sup>+</sup>CD62L<sup>+</sup> (B) and CD8b<sup>+</sup>CD44<sup>hi</sup> (C) cells T cells were compared between Donors (N = 3), UI recipients (N = 4) and D21 recipients (N = 4). *P<0.05 vs Donors and UI; †P<0.01 vs Donors and UI.</p
T cell activation after arterial injury.
<p>LN = lymph nodes, N≥3; Spl = spleen, N≥5. UI = Uninjured mice; D7 and D21 = 7 days and 21 days after arterial injury, respectively. All values expressed as percent CD4<sup>+</sup> or CD8b<sup>+</sup> gated cells.</p><p>*p<0.05 vs. other time points;</p><p>**p<0.01 vs. other time points;</p>†<p>p<0.01 vs. UI;</p>‡<p>p<0.05 vs. sham.</p
Adoptive T cell transfer.
<p>Representative histogram of CD8<sup>+</sup> T cells homing-into the spleen of a recipient mouse 48 hours after cell transfer, before arterial injury (A). Viable lymphocytes were gated on the FSC/SSC plot. Representative sections of 21-day injured carotid arteries (400× magnification) stained with hematoxylin and eosin from Rag-1−/− mice (B), Rag-1−/− injected with CD8<sup>+</sup> T cells (D), and Rag-1−/− mice injected with CD4<sup>+</sup> T cells (F). Boxed area indicates magnification of the respective cross-sections (C, E, G). Arrows indicate internal elastic lamina. Bar = 50 microns.</p
Neointimal thickening 21 days after arterial injury.
<p>All values are mean ± SD. Rag+CD8 T cells = Rag-1−/− mice injected with CD8<sup>+</sup> T cells from CD4−/− donors; Rag-1+CD4 T cells = Rag-1−/− mice injected with CD4<sup>+</sup> T cells from CD8−/− donors. I/M = intima to media ratio.</p><p>*p<0.05 vs. Rag-1−/−.</p
Lymph node and splenic CD44<sup>+</sup> T cells after arterial injury in WT mice.
<p>Representative scatter plots of lymph node (LN) and spleen cells collected at various times after injury and characterized using CD44 gated on CD4 (A) or CD8b (B). Cells were collected from uninjured (UI) mice, or 7 days (D7) and 21 days (D21) after arterial injury. Sham mice correspond to 7 days after sham surgery. Percentage of cells is indicated on the top right corner of each graph.</p
T cells in the injured arterial wall.
<p>Representative sections of 21-day injured carotid arteries stained for CD4 (A) or CD8b (B) identify their localization (arrows) in the arterial wall. Omission of primary antibody (C) was used as control for staining. N = 4 each; bar = 10 microns.</p
Splenic CD8b<sup>+</sup> T cells after arterial injury in WT mice.
<p>Representative scatter graphs of CD8b-gated CD25<sup>+</sup> (A, top panel) and CD28<sup>+</sup> (A, bottom panel) spleen T cells. Representative histogram of CD28 expression on CD8b-gated cells (B). Geometric mean fluorescence intensity (MFI) of CD28 on CD8b-gated cells (C; N = 3–4 each time point).</p
Effect of MHC-I mAb treatment on CD8<sup>+</sup> T cells of recipient mouse spleens.
<p>Analysis was performed on CD8b gated lymphocytes. All values are mean ± SD. MHC-I mAb (monoclonal antibody) N = 6; IgG isotype N = 5;</p><p>*p<0.05.</p
The cathelicidin protein CRAMP is a potential atherosclerosis self-antigen in ApoE(-/-) mice
<div><p>Auto-immunity is believed to contribute to inflammation in atherosclerosis. The antimicrobial peptide LL-37, a fragment of the cathelicidin protein precursor hCAP18, was previously identified as an autoantigen in psoriasis. Given the reported link between psoriasis and coronary artery disease, the biological relevance of the autoantigen to atherosclerosis was tested in vitro using a truncated (t) form of the mouse homolog of hCAP18, CRAMP, on splenocytes from athero-prone ApoE(-/-) mice. Stimulation with tCRAMP resulted in increased CD8+ T cells with Central Memory and Effector Memory phenotypes in ApoE(-/-) mice, differentially activated by feeding with normal chow or high fat diet. Immunization of ApoE(-/-) with different doses of the shortened peptide (Cramp) resulted in differential outcomes with a lower dose reducing atherosclerosis whereas a higher dose exacerbating the disease with increased neutrophil infiltration of the atherosclerotic plaques. Low dose Cramp immunization also resulted in increased splenic CD8+ T cell degranulation and reduced CD11b+CD11c+ conventional dendritic cells (cDCs), whereas high dose increased CD11b+CD11c+ cDCs. Our results identified CRAMP, the mouse homolog of hCAP-18, as a potential self-antigen involved in the immune response to atherosclerosis in the ApoE(-/-) mouse model.</p></div
Plaque T cells are reactive to tCRAMP simulation.
<p>Aortas from ApoE(-/-) mice at 25 weeks of age were subjected to enzymatic digestion and stimulated for 24 hours with 20μg/ml tCRAMP. Size gate is shown after inclusion of singlets and exclusion of non-viable cells, followed by gating for CD3+ T cells (A). Isotype was used as staining control. Cells were plotted on CD4 vs CD8 and selected for subtype analysis. Results were plotted on bar graphs (B-E). Aortas from 6 mice were pooled and assayed in triplicates. Bars over graph columns indicate statistical significance (P<0.05).</p