The results of the studies presented here provide strong evidence for a previously undescribed immune suppressive and paradoxical counter-regulatory role for inflammatory monocytes in vaccine immunity. Our studies showed that inflammatory monocytes could be rapidly recruited to vaccine draining LNs in an MCP-1-dependent fashion and once there, monocytes potently suppressed local T cell responses in LNs by sequestering cysteine. These conclusions were based on evidence that 1) immune responses to vaccination were significantly enhanced in CCR2−/− mice, 2) immune responses to vaccination were significantly enhanced when inflammatory monocytes were depleted or their migration blocked, 3) purified vaccine-elicited monocytes directly suppressed T cell responses in vitro, and 4) addition of exogenous cysteine nearly completely reversed the T cell suppressive effects of inflammatory monocytes, both in vitro and in vivo. Therefore, we propose a model wherein inflammation associated with vaccination triggers local production of MCP-1 and induces rapid immune counter-regulatory responses that are mediated by inflammatory monocytes recruited to vaccine draining LNs ().
Proposed model of inflammatory monocyte-mediated counter regulatory immune responses to vaccination and the role of monocyte depletion or blockade in amplifying vaccine immunity.
Inflammatory monocytes are likely to be generated anytime local inflammation develops, resulting in MCP-1 production. Indeed, we saw that depletion of monocytes during vaccination with a variety of adjuvants enhanced vaccine immunity. In fact, it was previously reported that exposure to even very low doses of TLR agonists was sufficient to mobilize inflammatory monocytes from the bone marrow (41
). It is likely therefore that inflammatory monocytes move into circulation shortly after local release of MCP-1 and migrate to sites of inflammation to serve an important homeostatic function. While this response may be helpful during inflammatory disease states, we show that the counter regulatory response elicited by these monocytes can limit immune responses during vaccination. Therefore, transient blockade of monocytes during the vaccination process is an attractive therapeutic target that warrants further study.
Vaccine-elicited inflammatory monocytes appear to use a relatively unusual mechanism to suppress T cell responses. Whereas tumor-generated monocytic myeloid suppressor cells have typically been reported to suppress T cell function by production of arginase or NO, vaccine-elicited inflammatory monocytes suppressed T cell responses by depleting extracellular cysteine, most likely by sequestering its precursor molecule cystine. This mechanism of T cell suppression was reported recently for tumor-elicited myeloid suppressor cells (37
). Since T cells require extracellular cysteine for activation and proliferation, the loss of available cysteine following monocyte sequestration of cystine results in significant suppression of T cell function (see ). This mechanism differs from that reported by Martino et al., which identified NO as the causative component of BCG- elicited, myeloid cell-mediated immunosuppression (9
). Interestingly, Martino and colleagues did not report finding these cells in the draining LN earlier than 3 days after vaccination whereas we identify vaccine elicited monocytes in the node within 24 hours of vaccination suggesting that early responding monocytes that suppress through a cysteine dependent mechanism may actually be a different population than that reported by Martino.
Additionally, our results differ from those of Nakano et al, who concluded that inflammatory monocytes elicited by vaccination actually enhanced immune responses. The immunization models used in our studies and those used by Nakano et al.(10
) were not identical, but we believe the discrepancy can be best explained by the fact that Nakano et al., did not directly evaluate T cell or B cell responses to vaccination, nor did they examine how monocyte blockade or depletion affected those responses. The Nakano publication also noted that since the CCR2 −/− mice lacked a sufficient IFN-γ response then monocytes must be important in augmenting vaccine immunity. However, the known IFN-γ production defect in CCR2−/−
mice would have made it difficult to directly compare IFN-γ production following vaccination of wild type versus CCR2−/−
We believe the monocyte-mediated vaccine counter-regulatory immune responses described here are broadly relevant, since they were found to occur following immunization with a variety of adjuvants and antigens. Recently Friedlender et al., reported that a population of CD11c+ cells were recruited via a CCL2 dependent mechanism following vaccination and augmented cancer immunotherapy (42
). While the vaccine elicited monocytes described in our study did not express CD11c+, it remains possible that they may alter their phenotype over time and therefore may represent be a less differentiated population of cells than those reported by Fridlender et al. In addition, the phenotype of vaccine-elicited inflammatory monocytes may differ significantly in normal animals as in our studies versus their phenotype in animals that already have cancer, as in the Fridlender studies. Finally, the monocyte migration response reported here was most likely TLR independent since it was also observed following immunization with alum adjuvant, though the TLR independence of the effect was not formally examined in our study. Many inflammatory stimuli can induce MCP-1 production and therefore signaling via CCR2 may be the common thread linking vaccine adjuvants and counter-regulatory immune responses mediated by inflammatory monocytes (43
). Our data indicated that MCP-1 was the major chemokine required to mobilize and recruit inflammatory monocytes following vaccination.
The key role played by the MCP-1/CCR2 signaling pathway in regulating vaccine suppression by monocytes may make it possible to use specific chemokine receptor antagonists as a new type of “vaccine adjuvant-adjuvant”. For example, administration of a CCR2 antagonist at the time of vaccination significantly enhanced vaccine humoral and cellular immune responses (see ). Thus, it should be possible to substantially improve vaccine effectiveness using chemokine receptor antagonists already under development for treatment of inflammatory disorders in humans (48
). Finally, vaccine amplification by administration of chemokine receptor antagonists and/or concurrent administration of cysteine may be particularly attractive as strategies to boost vaccine responses in difficult to vaccinate populations such as the very young and the elderly.