To date, Claudia Kemper’s team has taken the lead in conducting most of the research on the complosome and have mostly described it in human T cells, highlighting its connection to inflammasome activation and immunometabolism ( Figure 2A ). As mentioned above, both intracellular C3 and C5 system, together with their interaction with the inflammasome, are indeed required for effective Th1 immunity (3, 5). The complosome is required to induce metabolic reprogramming of immune cells, including increased glycolytic flux and oxidative phosphorylation (OXPHOS), which promote the secretion of the proinflammatory cytokine IFN-γ (25). In resting T cells, CTSL constantly cleaves intracellular C3 into bioactive C3a and C3b, and the intracellular C3a/C3aR engagement maintain T cell survival via low-level mTOR induction (3). Upon toll like receptor activation, the entire intracellular C3 system translocates rapidly to the cell surface, where C3a and C3b signal in an autocrine manner via their receptors, C3aR and CD46, respectively, to induce Th1 immunity and IFN-γ production. Moreover, during T cell activation, CD46-mediated signals permit nutrient influx via expression of the large neutral amino acid transporter 1 (LAT1) and glucose transporter 1 (GLUT1). Autocrine activation of CD46 simultaneously drives induction of late endosomal or lysosomal adaptor and MAPK and mTOR activator 5 (LAMTOR5), which facilitates the assembly of the Amino Acid-sensing Ragulator-Rag-mTORC1 complex and promotes OXPHOS and glycolysis, two processes necessary for IFN-γ production and Th1 lineage commitment (4). This demonstrates a crucial connection between the complement system and immune metabolism guiding human CD4+ T cell effector function. Similarly, upon TCR and CD46 co-stimulation, the intracellular C5a interacts with the mitochondrially expressed C5aR1, resulting in mitochondrial ROS production, intrinsic NLRP3 inflammasome assembly and activation, maintenance of IFN-γ secretion, and subsequent mature IL-1β secretion during T cell migration into inflamed tissues. Additionally, CD4+ T cells express C5aR2 on their surface and intracellularly, and both secreted C5a and the desarginated version of C5a (C5a-desArg) interact with C5aR2 in an autocrine manner. C5aR2 negatively regulates the C5aR1-driven NLRP3 inflammasome activity, which results in a reduction in IFN-γ production, a switch to IL-10 secretion, and a suppression of Th1 responses (5). Moreover, the complosome also participates in the contraction phase of T cell responses. Following effective Th1 induction, CD46-mediated signals interacts with the interleukin (IL)-2 receptor, reduces glycolysis and OXPHOS, promotes IL-10 production in these cells, which induces them to enter a self-regulative contraction phase (8). Moreover, human cytotoxic CD8+ T cells (CTLs) also harbor a complosome system. The TCR and autocrine CD46 costimulation drives IFN-γ production, nutrient influx, and cytotoxic activity in these cells. Interestingly, in CTLs, aside from a strong OXPHOS induction, CD46 is also a potent inducer of fatty acid synthesis. Although CD8+ T cells express NLRP3, the inflammasome is not required for normal IFN-γ secretion or cytotoxicity in CTLs (26, 27). The activated CD8+T cell can also take up C1q, another complement protein, and restrains the response to self-antigens by modulating the mitochondrial metabolism via gC1qR and a yet unknown mechanism (28). Given what we presently know about the complosome in T cells, it is possible that the complosome also plays a role in the development of T cell memory and/or tissue residency, these subjects are being explored currently (29).

This novel pathway of complosome in T cells contributes greatly to disease. For example, Juvenile idiopathic arthritis (JIA) patients’ T cells have increased intracellular C3. In vitro treatment with a cell permeable CTSL inhibitor can normalize the hyper-active intracellular C3 activation and decrease IFN-γ production in patients’ T cells (3). Intracellular C3 system dysregulation contributes to human autoimmune diseases such as scleroderma, systemic lupus erythematosus (SLE), RA, and multiple sclerosis, as pathological intracellular C3 hyperactivation contributes to Th1 hyperactivity in these autoimmune conditions (4, 30). Individuals lacking CD46 expression or C3 secretion by T cells have diminished Th1 immunity (but normal Th2 and TH17 responses) and suffer from recurrent infections (31, 32). Patients with leukocyte adhesion deficiency type 1 (LAD-1) have reduced C3 transcripts and diminished effector activities in immune cells, which have been shown to be rescued proportionally by intracellular C3 provision. It was also shown that C3 transcription is LFA-1 (lymphocyte function-associated antigen 1)-dependent, and perturbations in the LFA-1-C3-axis contribute to primary immunodeficiency (33).

The complosome plays a crucial orchestrating role in cell metabolic processes that regulate T cell effector responses, but the role of the complosome in myeloid immune cells was unknown for several years. There was preliminary evidence showing human monocytes produce IL-1β during infections by relying on intrinsic C3a activity (10). Macrophage populations express C3a receptor (C3aR) intracellularly (34). M2 macrophages highly express intracellular C3/C3b, which modulates the proinflammatory profile during endoplasmic reticulum (ER) stress (35). In general, studies regarding this topic were relatively scarce until recently, when Kemper’s team published a remarkable study that revealed that human monocytes and macrophages continuously synthesized C5 and produced C5a intracellularly via an intrinsic intracellular C5 convertase ( Figure 2B ). C5a/C5aR1 signaling on mitochondrial membranes was shown to alter mitochondrial activity and shift adenosine triphosphate (ATP) production via reverse electron chain flux toward ROS production and aerobic glycolysis, which not only promoted IL-1β gene expression, but also processing of bioactive Il-1β upon damage-associated molecular pattern (DAMP) sensing in monocytes and macrophages (36). These results further elaborated previous findings that C5a augments physiologic inflammasome responses (11, 37) from the perspective of intracellular complement. Unlike the previous conclusions that cell surface anaphylatoxin receptors are indirect regulators of mitochondrial activity (38), evidence has demonstrated that cellular mitochondrial C5aR1 (mtC5aR1) activation can directly reduce mitochondrial ERK1/2 phosphorylation, as well as ATP and cAMP formation and OXPHOS, simultaneously increasing ROS production and glycolysis, thereby establishing a direct link between cellular C5a/C5aR1 signaling and cell metabolic pathways located within the mitochondria. Upon mtC5aR1 blockade, IL-1β suppression was only observed in M2 macrophages, but not M1 macrophages. Conversely, mtC5aR1 inhibition reduced IL-10 production in differentiated M1 macrophages and not M2 macrophages, suggesting that there is still much to learn regarding the interaction between the complosome and macrophages. Furthermore, intracellular factor B was proposed to play a role in this process in macrophages. In diseased states, mice benefited from conditional knockout of C5ar1 in myeloid cells in models of crystal-triggered sterile inflammation (folic acid–induced kidney injury and atherosclerotic cardiovascular disease (ACVD) (36). This research has greatly increased our current knowledge on the complosome in myeloid immune cells and it is of great significance to the follow-up research.

There is growing evidence that the complosome exists and plays a crucial role in other immune cells, even though the majority of research on the topic focuses on T cells and macrophages/monocytes. Human neutrophils, for instance, have intracellular stores of C3, CR1, and FB (2). C3 and FB are released from stores to trigger a local inflammatory process upon neutrophil activation. It is not yet known whether this process involves a traditional cascade reaction or simple proteolytic cleavage to release functional fragments, such as C3a or Ba (24, 39). M-ficolin/Ficolin-1, an activator of the LP pathway, was also found to be localized in neutrophils secretory granules (40).Neutrophil stimulation with C5a resulted in a quick release of the complement proteins FP, C3, and FB, which are critical for the assembly of the AP convertase C3bBb, linking a positive feedback loop between neutrophil and complement activation (41, 42). B cells also harbor a so-called B cell complosome (3), as mentioned above, B cells can transport C3(H2O) from the extracellular milieu into the cell and process it intracellularly via CTSL into C3b and C3a, however the biological implication of this observation remains to be established (20). The endogenous C3 expression is relatively low, and the serum is the main source of intracellular C3 in human B cells. According to research, both serum derived and pure C3 may enter the nucleus of live B cells, and its strong interaction with histone proteins and potential capacity to induce chromatin rearrangement implies that C3 might regulate DNA transcription via chromatin remodeling (43, 44). Natural killer cells (NK cells) express several complement receptors such as C3aR, CR3, CR4, C5aR1 and C5aR2, while C5aR1 and C5aR2 proteins were reported to be only expressed inside NK cells upon permeabilization. The intracellular expression of C5aR1 and C5aR2 was down-regulated in response to Poly (I: C) (a synthetic analogue of double-stranded RNA) or TNF-α (45). A similar pattern of intracellular C5aR1 and C5aR2 expression was also reported in CD3⁺CD56⁺ NKT-like cells (46). However, it is uncertain if this intracellular expression regulates NK cell activity or whether complement components such as C3 or C5 can be generated and secreted by the NK cell.

Extensive studies have highlighted the importance of tumor cell-derived complosome (particularly C3/C3a) in cancer progression (47, 48). Intracellular activation of tumor cell-derived C3 inhibited CD8+ T cell infiltration and function by driving the accumulation and immune-suppressive activity of tumor-associated macrophages (TAMs) in a C3a-C3aR-dependent manner through the C3a-C3aR-PI3Kγ signaling pathway (49). More importantly, deletion of C3 in tumor cells that had high C3 expression enhanced the efficacy of anti–PD-L1 treatment (49). C3 downregulation by siRNA in ovarian cancer cells SKOV3ip1 inhibits tumor cell growth and migration (47). It has been shown that intracellular C3 in human colon carcinoma Caco2 cells can cleave cathepsin L and B and release C3a, just like CTSL can cleave C3 in CD4+ T cells in a non-C3 convertase manner, and C3a secretion can be downregulated by treatment with cathepsin L and B inhibitors in these cells (50). Other tumor cells, such as leukemia cells, express C3a and C5a receptors and respond to C3a and C5a stimulation by phosphorylation of p44/42 MAPK and AKT (51). Additionally, tumor cell derived-FB was found to be upregulated in pancreatic ductal adenocarcinoma (PDAC), promoting cancer cell proliferation by preventing cellular senescence and associated with myeloid-derived suppressor cells (MDSCs), immunosuppressive regulatory T-cells (Tregs), and TAMs in immunological tumor promotion (52). As previously stated, the complosme in CD4+ T cells and monocytes regulates basic metabolic processes and mTOR during T cell activation. Although evidence is lacking, it is very likely that the complosme functions similarly in tumor cells, which rely on aerobic glycolysis for cell survival, proliferation, and metastasis (48).

There is also a growing number of reports about intracellular complement expression from stromal cells. For example, it have been demonstrated to regulate autophagy, energy management, and insulin production by pancreatic β cells (53, 54). Human pancreatic islets highly express intracellular C3, and C3 binds autophagy-related protein 16-1 (ATG16L1), thus regulating autophagy and contributing to β cell survival in human islet inflammation and diabetes (54). The C5b-C9 formation inhibitor protein, CD59, was demonstrated to be localized intracellularly in insulin granules in pancreatic β cells, thereby regulating insulin secretion by exocytosis (55, 56). Adipocytes can also produce complement components such as C3, C1q, properdin, and Components B and D, which have been linked to abnormal adipocyte function, immune cell infiltration, obesity, and insulin resistance (56, 57). Similarly to pancreatic β cells, intracellular C3 also protects human airway epithelial cells from stress-induced cell death (58). Intracellular C3 regulates Paneth cell turnover during repair of intestinal epithelial cells after injury (59). During mesenteric ischemia, C3 activation in a cathepsin-dependent manner in intestinal epithelial cells contribute greatly to intestinal tissue damage (50), confirming the pro-inflammatory role of intracellular C3. During SARS-CoV-2 infection, the complosome also plays an important role in lung epithelial cells by activating intracellular C3a, which can be blocked by a cell-permeable FB inhibitor, confirming the presence of an inducible cell-intrinsic C3 convertase in lung epithelial cells. Mechanically, complement gene transcription in lung epithelial cells was shown to be JAK1/2-STAT1–dependent (60). In retinal pigment epithelial (RPE) cells, intracellular expression of C3, C3a, C3aR, CR3, and FB was reported to be induced by internalized complement factor H-related 3 (FHR-3), in addition to proinflammatory cytokine secretion and inflammasome NLRP3 activation (61).