CD8+ T Cells Mediate the Athero-Protective Effect of Immunization with an ApoB-100 Peptide

Immunization of hypercholesterolemic mice with selected apoB-100 peptide antigens reduces atherosclerosis but the precise immune mediators of athero-protection remain unclear. In this study we show that immunization of apoE (-/-) mice with p210, a 20 amino acid apoB-100 related peptide, reduced aortic atherosclerosis compared with PBS or adjuvant/carrier controls. Immunization with p210 activated CD8+ T cells, reduced dendritic cells (DC) at the site of immunization and within the plaque with an associated reduction in plaque macrophage immunoreactivity. Adoptive transfer of CD8+ T cells from p210 immunized mice recapitulated the athero-protective effect of p210 immunization in naïve, non-immunized mice. CD8+ T cells from p210 immunized mice developed a preferentially higher cytolytic response against p210-loaded dendritic cells in vitro. Although p210 immunization profoundly modulated DCs and cellular immune responses, it did not alter the efficacy of subsequent T cell dependent or independent immune response to other irrelevant antigens. Our data define, for the first time, a role for CD8+ T cells in mediating the athero-protective effects of apoB-100 related peptide immunization in apoE (-/-) mice

Immunization with an ApoB-100 Related Peptide Vaccine Attenuates Angiotensin-II Induced Hypertension and Renal Fibrosis in Mice

<div><p>Recent studies suggest the potential involvement of CD8+ T cells in the pathogenesis of murine hypertension. We recently reported that immunization with apoB-100 related peptide, p210, modified CD8+ T cell function in angiotensin II (AngII)-infused apoE (-/-) mice. In this study, we hypothesized that p210 vaccine modulates blood pressure in AngII-infused apoE (-/-) mice. Male apoE (-/-) mice were immunized with p210 vaccine and compared to unimmunized controls. At 10 weeks of age, mice were subcutaneously implanted with an osmotic pump which released AngII for 4 weeks. At 13 weeks of age, p210 immunized mice showed significantly lower blood pressure response to AngII compared to controls. CD8+ T cells from p210 immunized mice displayed a different phenotype compared to CD8+ T cells from unimmunized controls. Serum creatinine and urine albumin to creatinine ratio were significantly decreased in p210 immunized mice suggesting that p210 vaccine had renal protective effect. At euthanasia, inflammatory genes IL-6, TNF-α, and MCP-1 in renal tissue were down-regulated by p210 vaccine. Renal fibrosis and pro-fibrotic gene expression were also significantly reduced in p210 immunized mice. To assess the role of CD8+ T cells in these beneficial effects of p210 vaccine, CD8+ T cells were depleted by CD8 depleting antibody in p210 immunized mice. p210 immunized mice with CD8+ T cell depletion developed higher blood pressure compared to mice receiving isotype control. Depletion of CD8+ T cells also increased renal fibrotic gene expression compared to controls. We conclude that immunization with p210 vaccine attenuated AngII-induced hypertension and renal fibrosis. CD8+ T cells modulated by p210 vaccine could play an important role in the anti-hypertensive, anti-fibrotic and renal-protective effect of p210 vaccine.</p></div

Cholesterol Lowering Modulates T Cell Function <i>In Vivo</i> and <i>In Vitro</i>

<div><p>Background</p><p>The lipid milleu exacerbates the inflammatory response in atherosclerosis but its effect on T cell mediated immune response has not been fully elucidated. We hypothesized that lipid lowering would modulate T cell mediated immune function.</p><p>Methods and Results</p><p>T cells isolated from human PBMC or splenic T cells from apoE-/- mouse had higher proliferative response to T cell receptor (TCR) ligation in medium supplemented with 10% fetal bovine serum (FBS) compared to medium with 10% delipidated FBS. The differences in proliferation were associated with changes in lipid rafts, cellular cholesterol content, IL-10 secretion and subsequent activation of signaling molecule activated by TCR ligation. Immune biomarkers were also assessed in vivo using male apoE-/- mice fed atherogenic diet (AD) starting at 7 weeks of age. At 25 weeks of age, a sub-group was switched to normal diet (ND) whereas the rest remained on AD until euthanasia at 29 weeks of age. Dietary change resulted in a lower circulating level of cholesterol, reduced plaque size and inflammatory phenotype of plaques. These changes were associated with reduced intracellular IL-10 and IL-12 expression in CD4⁺ and CD8⁺ T cells.</p><p>Conclusion</p><p>Our results show that lipid lowering reduces T cell proliferation and function, supporting the notion that lipid lowering modulates T cell function <i>in vivo</i> and <i>in vitro</i>.</p></div

Effects of diet change on apoE-/- mice.

<p>AD = Atherogenic diet; ND = Diet changed from AD to normal diet. All values are mean±SD; n = 10 in each group, t-test for statistic comparison.</p

The effect of p210 vaccine on CD8+ T cell profile and hypertension in AngII infused mice.

<p>(A) CD8+IL-12+ T cells were significantly increased in LNs of p210 immunized mice (N = 8; P<0.05)) compared to PBS (N = 6) and cBSA (N = 8). p210 immunized mice (N = 9) showed significantly lower mean blood pressure (B) and systolic blood pressure (C) compared to controls (PBS N = 11; cBSA N = 10; *P<0.05). Diastolic blood pressure (D) in p210 mice was significantly reduced compared to cBSA (†P<0.05). There were no differences in heart rate (E) among the groups. Expression of inflammatory genes IL-6 (F), MCP-1 (G), and TNF-α (H) in the kidneys analyzed by real-time RT-PCR were significantly reduced in p210 immunized mice. N of mice: (F) = 8 in each group, (G) = 8–9 in each group, (H) = 10 in each group. *P<0.05 vs. PBS and cBSA.</p

Effect of diet change on IL-10 and IL-12 in T cells and their correlation with serum cholesterol.

<p>(A) Flow cytometric analysis of splenocytes revealed that CD4⁺ and CD8⁺ T cells from AD mice express higher level of intracellular IL-10 and IL-12 when compared to those from ND mice. (B) Positive correlation was observed between serum cholesterol and the IL-10 positive or IL-12 positive in CD4⁺ or CD8⁺ T cells. N = 10 in each group. Pearson test was used for correlation.</p

Western blot for pZap70 and IL-10 ELISA.

<p>(A) T cells isolated from naïve apoE-/- mice were cultured in 10% delipidated FBS medium or 10% FBS medium for 2 min with CD3/CD28 activation beads. Western blot analysis of the whole cell lysates revealed a higher expression of pZap-70 from T cells cultured in NM (medium supplemented with 10% FBS) when compared to that from T cells cultured in DM (medium supplemented with 10% delipidated FBS). Upper panel showed a representative Western blot and lower panel is the densitometric analysis from three experiments. N = 3 in each group. (B) IL-10 level was lower in delipidated medium of T cells after activation for 4 days with CD3/CD28 beads when compared to that in normal medium. N = 4.</p

Lipid raft and cholesterol content in T cells cultured in normal or delipidated medium.

<p>T cells isolated from naïve apoE-/- mice were cultured in culture medium supplemented with 10% delipidated FBS or 10% FBS medium for 24 hours with CD3/CD28 activation beads. More unesterified cholesterol contents (A) or lipid rafts (B) were observed in activated CD4⁺ and CD8⁺ T cells cultured in 10% FBS medium when compared to T cells cultured in 10% delipidated FBS medium. Experiments were repeated 4 times using T cells from 3 mice.</p