Enhanced neointima formation following arterial injury in immune deficient Rag-1-/- mice is attenuated by adoptive transfer of CD8 T cells

Abstract

T cells modulate neointima formation after arterial injury but the specific T cell population that is activated in response to arterial injury remains unknown. The objective of the study was to identify the T cell populations that are activated and modulate neointimal thickening after arterial injury in mice. Arterial injury in wild type C57Bl6 mice resulted in T cell activation characterized by increased CD4(+)CD44(hi) and CD8(+)CD44(hi) T cells in the lymph nodes and spleens. Splenic CD8(+)CD25(+) T cells and CD8(+)CD28(+) T cells, but not CD4(+)CD25(+) and CD4(+)CD28(+) T cells, were also significantly increased. Adoptive cell transfer of CD4(+) or CD8(+) T cells from donor CD8-/- or CD4-/- mice, respectively, to immune-deficient Rag-1-/- mice was performed to determine the T cell subtype that inhibits neointima formation after arterial injury. Rag-1-/- mice that received CD8(+) T cells had significantly reduced neointima formation compared with Rag-1-/- mice without cell transfer. CD4(+) T cell transfer did not reduce neointima formation. CD8(+) T cells from CD4-/- mice had cytotoxic activity against syngeneic smooth muscle cells in vitro. The study shows that although both CD8(+) T cells and CD4(+) T cells are activated in response to arterial injury, adoptive cell transfer identifies CD8(+) T cells as the specific and selective cell type involved in inhibiting neointima formation.

Figure 1. Lymph node and splenic CD44⁺ T cells after arterial injury in WT mice.

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.

Figure 2. Splenic CD8b⁺ T cells after arterial injury in WT mice.

Representative scatter graphs of CD8b-gated CD25⁺ (A, top panel) and CD28⁺ (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).

Figure 3. T cells in the injured arterial wall.

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.

Figure 4. Adoptive T cell transfer.

Representative histogram of CD8⁺ 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⁺ T cells (D), and Rag-1−/− mice injected with CD4⁺ T cells (F). Boxed area indicates magnification of the respective cross-sections (C, E, G). Arrows indicate internal elastic lamina. Bar = 50 microns.

Figure 5. CD8b⁺ cells in the injured artery of recipient Rag-1−/− mice.

Detection of CD8b⁺ cells (A; reddish-brown stain) in arteries of recipient Rag-1−/− mice 21 days after injury. Adjacent sections double-stained (B) for CD8b⁺ (orange arrow) and active caspase-3 (dark blue stain, black arrowhead) showed positive cells in close proximity. Omission of primary antibodies was used as control (C). 1000× magnification.

Figure 6. Lytic activity of CD8⁺ T cells from spleens of 21-day injured mice.

Syngeneic aortic SMCs were labeled with CFSE and co-cultured for 4 hours with increasing number of CD8⁺ T cells enriched from CD4−/− mouse spleens. SMC lysis was assessed using 7-AAD⁺ cells gated on CFSE (A) and expressed as % SMC lysis (B) relative to basal lysis (see Methods). N = 3–5 each group. *P<0.05 vs. no T cell; †P<0.01 vs. no T cell and 1∶1.

Figure 7. Characterization of cells transferred to recipient Rag-1−/− mice.

T cells enriched from pooled spleens of CD4−/− mice (Donor CD8⁺) were used for flow cytometric analysis and compared with T cells enriched from spleens of CD8⁺ 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⁺ cells from donor mice (A). CD8b⁺CD62L⁺ (B) and CD8b⁺CD44^hi (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.