Immunization with the apoB-100 peptide p210 have been shown to inhibit the development of atherosclerosis when administered as a vaccine conjugated to cBSA or CTB as well as when administered by subcutaneous slow infusion without carrier or adjuvant [7
]. These observations suggest that it could be possible to develop apoB-100 peptide-based vaccines for prevention of cardiovascular disease. Several lines of evidence support the notion that immunization with p210 inhibits atherosclerosis through activation of Tregs
. Studies based on Treg
depletion or Treg
transfers have established that Tregs
inhibits the development of atherosclerosis [22
]. Moreover, the athero-protective effect of p210 immunization has repeatedly been associated with an activation of Tregs
and removal of Tregs
through CD25-blocking antibodies neutralizes the effect of immunization with p210-cBSA [9
]. However, the mechanisms through which p210 interacts with immune cells have not been previously characterized. The present findings demonstrate that the p210-cBSA conjugate promotes APC-dependent conversion of naïve T effector cells into Tregs
and inhibits the proliferation of pre-activated T effector cells. Exposure of DCs to p210-cBSA also resulted in a down-regulation of MCH class II and the co-stimulatory molecule CD86. The inhibition of naïve T cell proliferation was not dependent on induction of apoptosis and was not explained by a direct effect of p210-cBSA on T effector cells. These findings provide a possible mechanistic explanation to the expansion of Tregs
and suppression of T effector cells observed in previous immunization studies using the p210 antigen and indicates that p210-cBSA functions by inducing a tolerogenic APC phenotype.
are known to be potent suppressors of T effector cells we investigate if the inhibition of T effector cell proliferation by p210-cBSA was mediated by Tregs
. However, we unexpectedly observed that p210-cBSA loaded APCs could suppress the proliferation of naïve T effector cells also in the absence of Tregs
suggesting the involvement of a direct inhibitory effect of APCs. This effect could be due to a reduced release of IL-12 from the APCs. Addition of IL-12 completely restored T effector cell proliferation in cultures with p210-loaded APCs. IL-12 treatment increased the expression of CD86 on MHCII+
cells and this increase could partly be inhibited by p210-cBSA treatment. Since IL-12 is an important polarizing factor for T effector cells, this suggests that p210-cBSA suppresses the proliferation of T effector cells by depletion of an important factor with Th1-polarizing activity. The down-regulation of MHC class II and CD86 that occurred in APCs as a result of the p210-cBSA mediated inhibition of IL-12 expression is also likely to contribute to the reduced activation of T effector cell proliferation. In the present experiment the down-regulation of MHC class II is likely to be of particular importance since activation of T effector cell proliferation in our model was found to be dependent on an interaction with MHC class II. Although the suppression of T effector cells by p210-cBSA was independent of Tregs
in the present study this does not exclude the possibility that Tregs
mediate suppression of T effector cells in both our in vitro assay and in p210-immunized mice. Tregs
are induced to a greater extent by APCs incubated with p210-cBSA compared to un-stimulated cells indicating that the tolerogenic phenotype of APCs that are induced by p210-cBSA have functional properties. Moreover, as addition of IL-12 to the p210-cBSA incubated APCs did not change the frequency of induced Tregs
this indicates that additional mechanisms are also responsible for the changed properties of the APCs. If this also is valid for p210-cBSA in vivo needs to be further clarified. Experiments in which Tregs
depletion has been achieved by treatment with CD25 antibodies have provided evidence for a key role of Tregs
in mediating the athero-protective effect of p210 immunization. These studies suggest that Tregs
generated in response to p210 have the ability to suppress proliferation of T effector cells as well as to inhibit the development of atherosclerosis. However, it remains to be fully clarified if the athero-protective action of these Tregs
involves suppression of plaque antigen-specific T effector cells, a by-stander anti-inflammatory effect through release of IL-10 and TGF-b when encountering the p210 antigen in plaques or a combination of both.
Studies using FITC-labeled p210-cBSA demonstrated that binding and uptake of p210-cBSA primarily occurs in APCs. Inhibition of IL-12 release was identified as the key mediator of the suppressive effects of p210-cBSA. IL-12 is primarily produced by APCs in response to antigen stimulation and plays an important role in activation of Th1 T cells. It consists of two subunits (p40 and p35) and the expression is controlled by transcriptional induction of the p40 subunit by NF-kB. We used splenocytes from MyD88-deficient mice to investigate if p210-cBSA reduced the production of IL-12 through inhibition of TLR-mediated activation of NF-kB but found no evidence for involvement of this pathway. IL-10 is one of the physiologically most important inhibitor of IL-12 production through suppression of c-rel [20
]. However, we observed no increase in IL-10 release in APCs exposed to p210-cBSA and treatment with IL-10 blocking antibodies did not affect the ability of p210-cBSA loaded APCs to inhibit T effector cell proliferation. Collectively, these observations demonstrate that the effect of p210-cBSA on APC function is independent of the NF-kB pathway. The role of the IL-10/c-rel pathway needs to be further characterized, preferably with an antibody blocking the IL-10 receptor. A tolerogenic phenotype of DCs has been shown to be associated with an impaired ability of producing IL-12 [16
]. The tolerogenic DCs may then reduce T effector cell activation directly or induce Treg
formation, both resulting in a reduced proliferation. IL-12 has also directly been implicated in the progression of atherosclerosis and blocking IL-12 production can inhibit atherogenesis in LDL receptor deficient mice [24
]. Inhibition of IL-12 expression in dendritic cells exposed to the p210 antigen could thus play a protective role in atherosclerosis.
An important question is whether the suppressive effect of p210-cBSA loaded APC is antigen-specific in the respect that only the proliferation of p210-specific T effector cell is inhibited. Although pre-activated naive T effector cell started to proliferate when co-cultured with APC in absence of p210 it cannot be excluded that these APCs presented apo B antigens derived from the serum-containing medium. However, p210-cBSA loaded APCs were equally effective in inhibiting the proliferation of T effector cells derived from OTII mice demonstrating that the suppressive effect is not restricted to p210-specific effector cells.
The present observations are of relevance because they provide a better understanding of the mechanisms through which apoB-100 peptide-based vaccines activate immune responses that protect against development of atherosclerosis. The athero-protective potential of immunization with LDL, oxidized LDL, and apoB-100 peptides is well documented from a number of different animal models of atherosclerosis [7
]. Vaccines based on apoB-100 peptides have the best clinical potential because they can be synthesized under conditions that meet regulatory requirements. To make it possible for this type of therapy to enter into clinical testing an improved understanding of the mode-of-action will be required in order to design relevant safety studies, decide on dosing regimens and to monitor the response to treatment.
In summary, results from the present study suggest that p210-cBSA inhibits proliferation of naïve T effector cells trough suppression of IL-12 and a reduced frequency of CD86 on MHCII+
cells and a reduced expression of MHCII+
cells. Moreover, p210-cBSA treated CD11c+
cells have an increased ability to induce Tregs
T cells. This Treg
induction can, however, not be reversed by addition of IL-12. Taken together, these observations suggest that immunization with p210-based vaccines inhibits atherosclerosis by inducing tolerogenic APCs that in turn generate Tregs
that suppress T effector cells and inflammation in atherosclerotic lesions.