In response to infections, complement system can be activated by three different pathways namely classical, alternative, and lectin. Regardless of the pathway involved, activation of C3 is a necessary event in the step-wise progression of the cascade of enzymatic reactions that results in the generation of biologically active molecules, which exert distinct effects during the immune response (8
). In addition to the well studied role in B cell activation, several studies have shown that complement do regulate T cell responses (10
). Although complement activation has been shown to occur in mice infected with LM (26
), the role of complement components in the elicitation of T cell responses is unknown. Here, we show that complement component C3 is essential for optimal activation and expansion of antigen-specific CD8 and CD4 T cells during infection of mice with LM. Further studies to understand the mechanisms demonstrate that reduced T cell responses to LM in C3−/−
mice is not linked to defective maturation of DCs or a deficiency for C5a in vivo. By performing adoptive transfer of wild type CD8 T cells into C3−/−
mice or C3−/−
CD8 T cells into wild type mice, we show that autocrine or paracrine sources of C3 might be sufficient to drive clonal expansion of CD8 T cells in vivo. Finally, we report that C3 augments antigen receptor-triggered proliferation of purified CD8 T cells in vitro, which would suggest that lack of direct effects of C3 on T cells might blunt T cell responses of C3−/− mice to LM infection.
In the present study, complete absence of C3 resulted in a marked reduction in the expansion of antigen-specific CD4 and CD8 T cell responses to LM in mice. Similarly, optimal expansion of virus-specific CD8 T cells during infections with influenza virus and LCMV requires C3 activity (10
). Additionally, T cell-dependent acute rejection of renal grafts is promoted by C3 activity (14
). The mechanism(s) that are involved in promoting T cell expansion by C3 is not completely understood. It has been reported that CD8 T cell responses to influenza virus in mice is significantly reduced by deficiency of either C3 or C5a, which suggested that C3 might promote T cell responses via C5a (12
). In contrast to studies with influenza infection, our studies clearly showed that both CD4 and CD8 T cell responses to LM were normal in the apparent absence of C5aR signaling. These findings indicated that requirement for C5a/C5aR interactions might be dictated by the nature of the infecting organism and the associated pathogenesis. Since C5aR has been found to be expressed locally in tissues such as lung and liver, C5a/C5aR signaling might be more critical for tissue-specific host defense in the peripheral tissues like lung during an influenza virus infection, but not in a systemic LM infection (29
It has been reported that complement might augment vaccine-induced CD8 T cell immunity to leishmaniasis via natural antibodies and IL-4 (13
), and complement activation products iC3b/C3dg can bind to antigen/natural antibody complexes and promote antigen uptake by APCs (54
). Since absence of B cells did not appear to affect CD8 T cell responses to LM in mice, it is unlikely that C3 promotes T cell responses to LM by antibody-dependent mechanisms (56
It is known that innate immune mechanisms, especially those mediated by neutrophiland macrophage-mediated phagocytosis are important in control of LM infection (19
). Complement receptor 3 has been implicated in opsonization, phagocytosis, and killing of C3b–bound LM by listericidal macrophages. Therefore, in C3−/−
mice, lack of C3b–dependent uptake of LM by phagocytes might impede efficient antigen processing and presentation to T cells, resulting in suboptimal expansion of T cells. This hypothesis is supported by reports which show that: 1) efficiency of antigen processing and presentation by professional APCs to T cells is greatly enhanced by tagging antigens to C3 fragments (58
); 2) complement factors can directly interact with T cells and modulate the function of antigen presenting T cells, which are crucial for T cell expansion (10
); 3) C3 deposition on APCs augments T cell proliferation (62
); 4) C3-deficient macrophages and DCs do not effectively stimulate alloreactive T cells in vitro. Our studies clearly show that LM infection-induced maturation of C3-deficient DCs was comparable to +/+ DCs. Notably, the ability of LM-infected DCs from C3−/−
mice to activate naïve CD8 T cells was similar to those of +/+ DCs. Therefore, C3 deficiency does not appear to impair antigen processing and/or presentation of listerial antigens to naïve CD8 T cells at least in vitro
. Similarly, C3-deficient DCs infected with Mycobacterium bovis
strain bacille Calmette-Guérin (BCG) did not exhibit detectable defects in activation of naïve T cells (data not shown). Moreover, the maturation of DCs induced by LM infection appears to be largely normal in C3−/− mice. Importantly, normal activation and expansion of adoptively transferred TCR transgenic CD8 T cells in LM-infected C3−/− mice supports our interpretation that APC-derived C3 may not be required for optimal antigen processing and/or presentation in vivo. It is unknown why DCs require C3 to optimally stimulate alloreactive T cells, but not after LM infection. One possibility is that LM could trigger in infected DCs a broad spectrum of pattern recognition receptors whose downstream effects are redundant with complement-mediated effects. Hence, in LM-infected DCs, C3 function could become redundant and therefore dispensable during T cell activation.
Apart from their ability to enhance antigen presentation by professional APCs, anaphylatoxins C3a and C5a have potent pro-inflammatory and chemotactic effects. Activated T lymphocytes have been shown to express functional receptors for C3a (C3aR) that are known to influence immune cell trafficking in inflammation (50
). Hence, it is possible that deficiency of C3a in C3−/−
mice disrupts proper trafficking of DCs and T cells in vivo during the T cell response to LM. This in turn could adversely influence the recruitment and activation of naïve T cells in the secondary lymphoid organs.
Binding of complement activation products to complement receptors CR1 and CR2 is a strategy by which complement modulates immune responses. CR1/CR2 are predominantly expressed on antigen presenting cells including B cells, and CR1/CR2 signaling in B cells has been shown to regulate activation threshold, antigen uptake, processing and presentation, isotype switching, and generation of memory B cells (4
). In addition to B cells, CR1/CR2 expression on T cells has been reported, and believed to mediate stable binding between the APC and T cells via C3 (62
). Hence, C3 could regulate T cell responses by interacting directly with T cells or indirectly via antigen presenting cells. In other models of infection, C3-promoted CD8 T cell responses are minimally affected by CR1/CR2 deficiency (10
). It remains to be determined whether C3 promotes T cell responses to LM via CR1/CR2.
CD46, membrane-cofactor protein (MCP) is a cell-surface receptor that is expressed on all nucleated cells, and ligands for CD46 include complement products C3b and C4b. The effects on T cell expansion induced by binding of C3b or C4b depends upon the isoform of CD46. Only when both isoforms of CD46 are coexpressed on a T cell, stimulatory effects appear to dominate over the suppressive effects (67
). Therefore, it is possible that in LM-infected mice, C3 deficiency led to abrogation of CD46/C3b interactions, and reduced expansion of antigen-specific CD8 T cells. However, emerging role of CD46-induced regulatory T cells makes it more difficult to separate the effect on T cell activation and regulation (45
To reiterate, reduced expansion of CD8 and CD4 T cells in LM-infected C3−/−
mice could be due to defects in T cells and/or non-T cells. Data presented in this manuscript show that activation and expansion of adoptively transferred wild type TCR transgenic CD8 T cells was largely intact in LM-infected C3−/−
mice, which indicated that C3 deficiency in non-T cells did not significantly affect antigen processing/presentation to wild type CD8 T cells in vivo
. We explored whether impaired T cell responses in C3−/−
) could be a sequel to developmental defects in the peripheral T cell repertoire. However, the peripheral T and B cell compartment, including Foxp3+ve
regulatory T cells and DCs are unaffected by C3 deficiency. Additionally, C3-deficient CD8 T cells do not appear to be intrinsically defective because they exhibit normal activation and expansion upon transfer into wild type mice. These two lines of evidence strongly suggest that lower T cell responses to LM in C3−/− mice is not likely linked to a defective T cell compartment.
How do C3 deficiency blunt T cell responses to LM? First, C3 produced by APCs themselves or other cells could augment their antigen presenting abilities. Second, C3 derived form APCs could act on T cells directly to augment their proliferation. Third, T cell-derived C3 (41
) could activate themselves and/or APCs in an autocrine or paracrine fashion respectively. Our studies indicated that: 1) CD8 T cell activation and expansion can occur when responding T cells can produce C3 but not the APCs; 2) responding CD8 T cells are not dependent upon autocrine C3 for activation and proliferation. It is likely that regardless of the cellular source, autocrine or paracrine C3 will support full expansion of CD8 T cells in vivo. However, it is unclear whether direct effects of C3 on T cells promote T cell responses in vivo. Our in vitro studies showed that C3 might promote the proliferative responses of purified CD8 T cells to TCR stimulation, by a mechanism that is distinct from its effect on professional APCs. This finding suggest that lower CD8 T cell responses to LM in C3−/−
mice might be linked at least in part to the lack of C3-induced effects on antigen-stimulated T cells. Future studies using C3aR-deficient CD8 T cells might be able to resolve this question in vivo.
In summary, in this manuscript we provide strong evidence that C3 plays a critical role in enhancing T cell responses to an intracellular bacterial infection by promoting proliferative expansion of antigen-triggered CD8 T cells. These findings have implications in rational design of effective vaccines and treatment of T cell-dependent autoimmune disorders.