The original hypothesis that neutrophils can acquire an antigen-presenting function is based on the observations that MHC-II and costimulatory molecules (e.g., CD80 or CD86) can be induced on their cell surface by exposure to specific cytokines such as IFN-γ or GM-CSF (13). Furthermore, it was demonstrated that cytokine-exposed neutrophils gained the ability to stimulate T cells in an MHC-II-restricted manner (14). Human and murine bone marrow neutrophils exposed to GM-CSF can differentiate into neutrophil–DC hybrids, exhibiting a DC-like phenotype and antigen-presenting function, while still maintaining several neutrophil features (15). Both immature and mature neutrophils in mice can acquire DC-like properties, which is in line with both human mature neutrophils and immature neutrophil precursors acquiring DC-like characteristics after exposure to cytokines (GM-CSF, IFN-γ, IL-4, and TNF) (16, 17). Therefore, the plasticity of neutrophils to become APCs may not be restricted to a particular stage of differentiation. In addition to the findings that neutrophils can differentiate to APCs in cytokines-conditioned cultures, neutrophils isolated from patients receiving GM-CSF or IFN-γ treatment have shown well-detectable MHC-II expression (18–20). Induction of MHC-II on neutrophils was also observed in patients with chronic inflammatory diseases associated with high levels of cytokines; for example, in patients with rheumatoid arthritis or Wegener’s granulomatosis (11, 21, 22).

Recent studies demonstrated that T cells can deliver signals to neutrophils to differentiate into APCs (23). Fresh human neutrophils cocultured with T cells were shown to acquire surface expression of CD80 and CD86 (14). Neutrophils without any stimulation can induce proliferation of antigen-specific T cells when cultured together with T cells and antigens (14). Murine neutrophils purified from peritoneal exudate cells were shown to express MHC-II and costimulatory molecules after coculture with T cells. Neutrophils isolated from this site could also present OVA peptide antigens to OVA-specific T cells in vitro without exogenous cytokines (24). This raises the question of whether the induction of an APC function in neutrophils is in fact initiated by interaction with activated T cells. The very first trigger of neutrophil differentiation may be dependent on bystander activation from adjacent APCs, that present antigens and result in stimulation of antigen-specific T cells, that in turn produce cytokines which subsequently differentiate neutrophils. Upregulation of MHC-II and costimulatory receptors at sufficient levels to present antigen may therefore be induced on neutrophils only if they are present in a milieu where activated T cells secrete cytokines as a consequence of responding to antigen presentation by neighboring APCs (Figure 1). This is supported by that neutrophils exposed to supernatants from cytokine-producing T cells upregulate MHC-II and costimulatory receptors (6). It is well known that toll-like receptor (TLR) activation of DCs promotes their antigen-presenting ability (25). Neutrophils express the majority of TLRs and can be activated via TLR ligation (26). However, we recently showed that TLR activation of neutrophils was not sufficient to induce HLA-DR despite upregulation of other markers such as CD11b and CD83 (6).

Although the classical receptors involved in antigen presentation are absent on the surface of resting neutrophils, studies have showed that normal human neutrophils contain cytoplasmic CD80 and CD86 stored in secretory vesicles or granules (11). Cytoplasmic MHC-II in neutrophils has also been detected, although only in about 10% of healthy donors (27). MHC-II, CD80, and CD86 can rapidly translocate to the cell surface upon stimulation of neutrophils (11, 28). In addition to intracellular storage, de novo synthesis of MHC-II can also be induced by cytokine exposure, i.e., IFN-γ as described earlier. This IFN-γ-induced MHC-II expression is mediated by the induction of the independent promoter IV, which regulates expression of the class II transactivator, a master regulator controlling MHC-II transcription, as recently reviewed in Ref. (29).

As discussed earlier, neutrophils have been shown to act as APCs after coculture with antigens and memory T cells. Blocking MHC-II on neutrophils was shown to abolish their antigen-presenting function (6, 14). However, as discussed earlier, it is not entirely clear how memory T cells activate neutrophils to express MHC-II. We recently showed that fresh human neutrophils can present cognate protein antigens (cytomegalovirus pp65 or influenza hemagglutinin) to autologous antigen-specific CD4⁺ T cells (6). Acquisition of MHC-II and costimulatory molecules on neutrophils in this system required both the presence of the antigen-specific memory T cells and the specific antigens. While it is plausible that antigens alone could induce neutrophil activation to gain APC function, this appears to be less likely since neutrophils cultured with antigens or even strong stimuli such as TLR agonists do not show significant upregulation of MHC-II (6). The presence of T cells may therefore be required to deliver signals to neutrophils, either by cell-to-cell contact or secretion of mediators, to induce proper cell differentiation to become APCs. Among the T cell-derived cytokines, IFN-γ, in particular, has been shown to be potent at inducing neutrophil differentiation as mentioned earlier (2, 23). Importantly, the antigen-presenting function of neutrophils appears to be provided exclusively by antigen-specific memory T cells and not by naïve T cells. Memory T cells produce IFN-γ more rapidly and in larger quantities. They also have less stringent requirements for activation than naïve T cells such as needing lower levels of costimulatory signals (30). Beyond this, memory T cells may not be fully quiescent cells, especially in situations with inflammation or persistent stimuli present in the microenvironment. In addition, detectable mRNA levels of IFN-γ have been reported in human resting neutrophils (27). A constitutive storage of IFN-γ proteins has also been observed in resting neutrophils, which is spontaneously released during culture or upon stimulation (31). Neutrophils may therefore activate themselves by IFN-γ produced in an autocrine manner.

In addition to secreted mediators from memory T cells, a direct cell-to-cell contact between T cells and neutrophils may be sufficient to induce the antigen-presenting function of neutrophils. In a recent study, T cell-induced expression of MHC-II on neutrophils was abolished when neutrophils and T cells were separated by a transwell system (24). The receptors that are critical in the T cell–neutrophil interaction remain as yet unknown. However, the classical costimulatory receptor–ligand interactions that facilitate the formation of the immunological synapse are potential candidates. As discussed with IFN-γ production, memory T cells express comparatively higher levels of several receptors than naïve T cells, which may again explain the inability of the naïve T cells to induce phenotypic differentiation of neutrophils. For example, memory T cells express higher levels of intercellular adhesion molecule I (ICAM-1) than naïve T cells (32). ICAM-1 predominantly binds to the integrin molecule Mac-1 (CD11b/CD18), which is constitutively expressed by neutrophils. CD66b, a molecule expressed exclusively on neutrophils can function as a receptor for galectin-3, which is constitutively expressed by human CD4⁺ memory T cells but only at a low level on naïve T cells (33–35). The aforementioned receptor–ligand interactions between memory T cells and neutrophils may provide the requisite signals to initiate neutrophils to express MHC-II on their surface. With expression of MHC-II on neutrophils, there will be a further amplification of the MHC–TCR ligation that would activate more T cells to secrete cytokines, and augment MHC-II expression on neutrophils. This positive feedback loop may be central for induction and maintenance of antigen presentation by neutrophils. This potential mechanism may also apply to other granulocyte subsets (Figure 2).

Details on the pathways involved in antigen processing in neutrophils remains largely unknown. MHC-II associated invariant chain (Ii), one of the critical components binding to nascent MHC-II molecules to regulate the loading of MHC-II with peptides during antigen processing, has not been detected in neutrophils (36). However, cathepsin S, the lysosomal enzyme that degrades Ii to facilitate MHC-II-mediated antigen presentation, was shown to be expressed in neutrophils (37). In addition, the formation and maturation of phagosomes in neutrophils differ from classical APCs (i.e., macrophages) in many respects (38). Therefore, whether the phagocytic and endocytic pathways that are involved in MHC-II-mediated antigen presentation by classical APCs are fully operational in neutrophils remains to be investigated. Aside from the molecular mechanisms, the types of antigens that neutrophils are able to process and present also need further clarification. As described earlier, neutrophils can present soluble protein or peptide antigens to CD4⁺ T cells. Limited evidence is available regarding presentation of particulate antigens or immune complexes such as whole microorganisms, virosomes, or antigens formulated in nanoparticles via MHC-II, although this seems quite likely to occur. It has been shown that neutrophils can cross-present particulate bead-conjugated OVA antigens as well as heat-killed bacteria to induce CD8⁺ T cell responses (39, 40).

Although the antigen-presenting function of neutrophils has been well documented in experimental settings, it is not clear to what extent their relative contribution is compared to other classical APCs. Consequently, there is still much left to understand regarding the physiological or pathological significance. It is clear that there are conditions and treatments associated with inflammation and high levels of cytokines that result in neutrophils expressing an APC feature; but whether this also holds true under normal circumstances needs more investigation. Clinical findings showed an upregulation of MHC-II on neutrophils in patients with active Wegener’s granulomatosis or rheumatoid arthritis (21, 22). Since aberrant expression of MHC-II is associated with development of autoimmune diseases (41), a possibility is that neutrophils play a role in the pathogenesis of the disease by presenting autoantigens.

Infiltration of neutrophils to inflammatory sites and their rapid migration to secondary lymphoid organs [i.e., draining lymph nodes (dLNs)] have been reported in different contexts (42). In BCG-immunized mice, neutrophils were the predominant cells to capture BCG at injection sites and transport the vaccine to T cell areas in dLNs (43). Migration of antigen-bearing neutrophils from peripheral sites to dLNs likely contributes to T cell stimulation. We recently evaluated this after vaccination in non-human primates and found that neutrophils represented the largest population of cells internalizing vaccine antigens at both the vaccine injection site and in the vaccine dLNs (6, 44). Vaccine⁺ neutrophils in the dLNs could present the vaccine antigen to memory CD4⁺ T cells. Although DCs and monocytes demonstrated a superior antigen-presenting capacity, the overall impact of neutrophil-derived responses may be substantial since the neutrophils vastly outnumber the conventional APCs. An indication of neutrophils being able to differentiate into APCs in a transient inflammatory state is that neutrophils isolated from vaccine-draining dLNs showed higher MHC-II expression than neutrophils isolated from contralateral control dLNs from the same animal (6, 44).

In addition to conventional antigen presentation via MHC-II, there is also evidence of the ability of cross-presentation by neutrophils (40). Studies have demonstrated that the machinery and pathways associated with antigen cross-presentation are operative in neutrophils (45–47). Human circulating neutrophils isolated from patients with acute sepsis could cross-present proteins to CD8⁺ T cells (48). In addition, tumor-infiltrating neutrophils isolated from patients with lung cancer could cross-present tumor antigens to promote anti-tumor T cell responses (49). Based on these data, a cross-presenting function in neutrophils may also be induced under special circumstances and elevate the importance of these cells in maintaining robust T cell responses.