Adjuvants are substances added to vaccines to enhance the host’s antigen-specific immune response (36). The commonly used adjuvants (such as aluminum salts) could enhance immune response thought stimulating the NLRP3-inflammasomes and promoting the release of cytokines (such as IL-1β) (37). Other adjuvants (such as MF59 and AS03) exert functions by activating dendritic cells and other antigen-presenting cells (38). Additionally, some adjuvants interact with toll-like receptors (TLRs) or directly target specific immune cell populations. Cytokines, as important immunomodulatory agents, are available to affect any aspect of immune responses in a more or less specific manner (39, 40). As a newly described IFN family, the potential ability of IFN-III as a vaccine adjuvant to affect adaptive immune response has not been fully evaluated until an in vivo study shows that plasmid-encoded IL-28B can increase the virus-specific IgG2a titer in serum. This study reveals the significant potential benefits of IFN-III as an adjuvant in adaptive immune regulation. Moreover, IFN-III has been reported to further enhance splenic CD8⁺ T cell-mediated immunity by reducing regulatory T cell population during DNA vaccination (41). In another study, IL-28B adjuvant can remarkably elevate the antibody levels and neutralization titers of herpes simplex virus 2 in mice by DNA immunization. The IL-28B adjuvant-regulated immune response is significantly more effective than immunization with antigen DNA vaccine alone. Due to the involvement of IL-28B, T helper 1/2 (Th1/Th2) cytokines secreted by activated immune cells are crucial for controlling viral infection and disease progression (42). In addition, IFN-III subtype IFN-λ2 can also influence the production of antibodies against the influenza subunit vaccines. As a result, the influenza virus hemagglutinin (HA)-specific total serum IgG levels and its subclass IgG1 levels are about 15-fold enhanced. Besides, IFN-λ2 vaccination results in a significant increase in IgA levels in the bronchoalveolar lavage (BAL) fluids. Similar phenomenon can also be observed in another influenza vaccine CTA1-3M2e-DD (designated M2e) applied with IFN-λ2 (33). Obviously, IFN-λ2 and IFN-λ3 in mice can potentiate both cellular and humoral immune response in mice, representing promising molecular adjuvants. However, since the potential protection of IFN-III upon virus vaccination exhibits specificity on the mucosal surface, different immune approaches may affect the phenotype of immune responses (16, 22, 33). The characteristics of IFNLR enable IFN-III to serve as a significant front-line defense against viral infections at mucosal sites.

In addition to adjuvant activity, the IFN-III signaling also directly contributes to adaptive immunity against virus infection, which is mediated by direct stimulation of DCs or indirect effects on other immune cells like natural killer (NK) cells, monocytes/macrophages, T cells, and B cells to upregulate cytokines and chemokines (2, 8, 32). IFN-III can indirectly modulate the activation of NK cells and the inflammatory response of monocytes/macrophages, thus exerting auxiliary benefits in the antiviral activity of adaptive immunity against chronic hepatitis C virus (HCV) (43). The common single-nucleotide polymorphisms (SNPs) in the IFN-III signaling are strongly associated with the treatment of chronic HCV infection (31, 32). In the context of yellow fever virus infection, IFN-III deficiency leads to impaired T cell activation and severe perturbation of pro-inflammatory cytokines in mice (44). In response to lymphocytic choriomeningitis virus (LCMV) infection, IFN-III increases CD8⁺ T cell expansion and diminishes weight loss in the chronic pathological stage instead of the acute infective phase, indicating that virus-specific host interactions affect the regulatory mode of IFN-III in the adaptive immune response (45). Further researches investigate the critical role of the IFN-III signaling in combating different subtypes of influenza A virus (IAV) strains (PR8 and X31), which is orchestrated through indirect adaptive immune mechanisms. The adaptive immunomodulation does not rely on the direct attachment of IFN-III to CD8⁺ T cells, but rather on IFN-III triggering DCs, particularly manipulating the migration and function of CD103⁺ DCs, which is essential for optimal antigen uptake and CD8⁺ T cell activation, thus providing permanent IAV protection (46). The activation program of DCs is probably regulated by TSLP, but it has not been confirmed yet.

IFN-III has been demonstrated to possess additional benefits in suppressing tumors in mice by directly reducing the tumorigenicity of tumor cells, inhibiting cell proliferation, and inducing apoptosis (47–49). IFN-III also mediates indirect anti-tumor mechanisms by regulating the tumor microenvironment (50). NK cells and CD8⁺ T cells are recruited into the microenvironment to participate in the anti-tumor mechanism of IFN-III in a murine model of melanoma (51). A murine model of fibrosarcoma further confirms the indirect anti-tumor mechanism of IFN-III. In vivo, antibody depletion of NK cells, neutrophils, and CD8⁺ T cells, but not CD4⁺ T cells, induces IL-28-mediated inhibition of tumor growth. Meanwhile, the inoculation of IL-28-encoded fibrosarcoma cell line in mice advances the production of IFN-γ and evokes the activity of cytotoxic T cells, indicating that the anti-tumor action of IL-28 also partly depends on IFN-γ (52). The IFN-III signaling can target mammary epithelial cells and promote the chemokine CXCL10 production, thereby recruiting CD4⁺ T cells into the tumor microenvironment. Besides, the immune balance between Th1 and Th2 altered by IFN-III may take advantages in the treatment of cancer (53). In addition, IFNLR-deficient (Ifnlr^-/- ) mice are more sensitive to experimental tumor metastasis and carcinogen-induced tumor formation and are also more susceptible to the growth of NK cell-sensitive lymphoma. Administration of IFN-III postpones the fatality in the B16F10 melanoma model mice with adoptive cell transfer and diminishes tumor growth in methylcholanthrene-treated mice (54). Therefore, IFN-III may be utilized as an adjuvant anti-tumor therapy with its ability to modulate tumorigenesis directly and indirectly. Due to the lower expression of IFNLR1 in most cell types, IFN-III offers a more selective targeted anti-tumor ability with fewer side effects compared to IFN-I (55).

Considering that both IFN-III and TSLP are produced by epithelial cells and both play roles in immune response as described above, it is of great significance to investigate the relationship between these two cytokines. Current studies have shown that intranasal inoculation of influenza virus subunit vaccine containing TSLP can effectively reinforce the serum IgG1 levels to a similar extent as IFN-λ2 triggered. However, in Tslpr ^-/- mice, immunization with M2e or HA-based Influsplit Tetra IAV vaccine combined with IFN-III as an adjuvant inhibits the enhancing effect of IgG1, indicating that TSLP can replace the enhancing activity of IFN-III, and TSLP is required for IFN-III-induced immune protection. Simultaneously, the losing function of TSLP in the IAV vaccine in Tslpr-/- mice can’t be compensated even in the presence of IFN-λ2 (33). These results indicate that the positive effects of IFN-III on the production of IgG1 and IgA depend mechanistically on TSLP. Interestingly, thus TSLP-mediated protective antiviral immunity of the IFN-III can similarly be realized through rectal vaccine inoculation. Whereas the stimulating effect of IFN-III as an adjuvant in influenza vaccine is canceled if administered intraperitoneally or subcutaneously, implying that these non-mucosal sites do not express significant IFN-III-responsive cells to produce TSLP-dependent antibodies (35). Taken together, we proved that TSLP was responsible for the IFN-III-related adaptive immune system. The IFN-III/TSLP axis (refer to some immune interactions between TSLP and IFN-III) participated in antiviral adaptive immune regulation at mucosal sites, particularly in the respiratory and gastrointestinal tracts ( Figure 1 ).

Although IFN-III and TSLP are both produced by epithelial cells and exert effects on antiviral and antitumor immune regulation, a direct or well-defined interaction between the two in moderating cancer or hypersensitivity disorder is still lacking. Nevertheless, based on adaptive immunity exerted by the IFN-III/TSLP axis in the context of virus infection, there remains an area of ongoing research.

IFNLR is expressed in human B cells while lacking in mice, which suggests that the IFN-III/TSLP axis may have different immunoregulation functions in mice and humans (14). As mentioned previously, TSLP is a cytokine modulating antibody production towards IgA and IgG via the intranasal route (89). IFN-III vaccination in mice infected with the influenza virus can strongly trigger microfold (M) cells on the upper respiratory tract, which induces TSLP production, CD103⁺ DCs migration, and promotes the production of follicular helper T (Tfh) cells in lymph nodes. Later, TSLP-dependent germinal center (GC) B cells in lymph nodes are activated and thus numerous high-affinity IgA and IgG antibodies are secreted (33, 90). Besides, apart from the critical role of IFN-III in mucosal homeostasis (91), TSLP has also been found highly expressed in antigen-stimulated colonic epithelial cells, which in turn activates Tregs through the mTORC1 pathway to maintain mucosal homeostasis (92). Hence, the IFN-III/TSLP axis may also participate in modulating mucosal homeostasis to some extent ( Figure 2 , left).

In the human species, the IFN-III/TSLP axis seems to show inconsistent roles. A previously in-vitro study has indicated that IFN-III is associated with a decrease in Th2-cytokines, B-cell proliferation, and IgG production in IAV-infected patients (93). By contrast, another study has shown that human naïve B cells interact with IFN-III stimulation and then differentiate into plasmablasts through the mTORC1 pathway (94). In respiratory epithelial cells, human Tslp genes are expressed during respiratory syncytial virus infection (95). TSLP-activated DC cells can induce Tfh cells to differentiate from naïve CD4⁺ T cells into a Th2-skewed environment, mechanistically depending on OX40L, a ligand of OX40 (96). Since previous in vitro studies have demonstrated that human TSLP can modulate GC-B cells’ immune response and B cells from humans directly respond to IFN-III (94, 97), it is currently unknown whether the coordination of the IFN-III/TSLP axis in adaptive antiviral immunity can also be replicated in humans, as observed in mice ( Figure 2 , right).

The reason why IFN-III-stimulated B cells show completely different functions between humans and mice is currently unclear. However, there are indications that the IFNLRI gene in various cell types is correlated with epigenetic changes, including DNA methylation and histone modifications. As has been evidenced previously, the transcriptional regulation of the IFNLR1 gene is dynamic, which may influence IFNLR1 expression and result in multiple functions. Indeed, histone deacetylase inhibitors can cancel the silencing of the IFNLR1 gene and confer IFN-III responsibility in previously inactivated human cell lines. The dynamic expression of IFNLR may explain the conflicting role of IFN-III in adaptive viral protection (98).

Considering that the in-vivo mouse models and in-vitro human studies cannot completely demonstrate the exact operation of IFN-III in the human body, a more relevant model on non-human primates may exhibit closer effects. Interestingly, under IFN-λ3 stimulation, macaques exhibit an adaptive immune response consistent with the immune activity in mice. In rhesus macaques, DNA vaccination encoding IFN-III exerts a positive effect on enhancing the antiviral activity of antigen-specific CD8⁺ T cells. Moreover, IFN-λ3 adjuvant can further induce continuous Th1 cell-biased immune responses (99, 100).

IFN-I can promote adaptive immune response or possess adjuvant activity against virus infection, bacterial burden, and tumor invasion (18, 101–105). The role of IFN-I in autoimmune diseases is bifacial. Though IFN-I is correlated with severe SLE progression, it may also play a positive role in the pathological improvement of RA (106, 107). Here we focus on the adaptive antiviral activity of IFN-I.

IFN-I and IFN-III can commonly be secreted by most nucleated cells, while IFN-III is particularly secreted by epithelial cells of mucosal organs. Although the signaling pathways triggered by these two types of IFNs are similar, their adaptive antiviral immune responses display functionally different (8–12). IFN-I modulates adaptive immunity via direct or indirect mechanism that depends on its impact on B and T cells involved in the adaptive immune response. In indirect mechanism, IFN-I firstly binds to DCs expressing IFNAR (108), thereby stimulating DCs to produce chemokines and cytokines that enhance the survival and proliferation of T cells including CD4⁺ T cells and CD8⁺ T cells, as well as induce the differentiation of T helper cell subsets including Th1 and Tfh cells (109–111). The activated Tfh cells migrate to B cell follicles and participate in GC reactions to generate high-affinity antibodies. Th1 cells on the other way express the master transcription factor T-bet and produce high levels of IFN-γ, which promotes macrophage activation and enhances CD8⁺ T cell responses. However, the effect of IFN-I on CD4⁺ T cell differentiating into Tfh and Th1 may disparate, which depends on the cells sensing these cytokines, the nature of the pathogen, and the stage of the infection (112–115). IFN-I signaling also regulates B cell response indirectly by inducing DCs to secret B cell stimulatory cytokines, thereby promoting immunoglobulin (Ig) isotype switching (116). This pathway acts on the mediation of IFN-I between CD4⁺-differentiated Th1 and Tfh to prosper GC B cell response (117). In the lungs, IFN-I-induced CXCL13 can initiate CXCR5-dependent recruitment of B cells and support ectopic GC formation (118). In direct adaptive immune regulation, IFN-I signaling immediately activates T cell receptor, thus promoting T cell proliferation and differentiation (110). IFNAR-deficient B cells induce an impaired antibody response and subclass switching when treated with IFN-α, indicating a direct effect of IFN-I on B cells (119). Indeed, IFN-I is required for different stages of B cell response, including early B cell activation and GC formation (120). Unlike IFNAR that is extensively expressed on most hematopoietic and non-hematopoietic cells, IFNLR is absent on the surface of mouse B and T cells (30), but specifically activated on the epithelial surface (16). Hence, IFN-I exhibits an adaptive immune response directly by interacting with immune cells, instead of mucosal epithelial cells, which is completely different from IFN-III ( Figure 3 ).

Although IFN-I and IFN-III are engaged in almost the same intracellular signaling pathway, it is of interest that, the functions of IFN-I and IFN-III in their adjuvant activities for influenza vaccines are different. On the one hand, when the influenza vaccine is applied with IFN-αB/D intranasally, both the antigen-specific IgG1 and IgG2c levels in serum and the IgA levels in BAL fluids are elevated. IFN-I can also improve vaccine-induced serum antibodies after intraperitoneal immunization. In contrast, IFN-λ2-enriched influenza vaccine promotes the IgA levels in BAL fluids and IgG1 levels in serum but not IgG2c. Thus IFN-III-induced vaccine immune responses are not observed either through intraperitoneal or subcutaneous routes. On the other hand, while the immune activity of IFN-λ2 plus vaccine is dampened in Tslpr ^-/- mice, antibodies in both serum and BAL fluids remain unaffected in TSLP-deficient mice administrated with IFN-αB/D-involved influenza vaccine (33, 35). In summary, the adjuvant activity of IFN-I is not restricted to mucosal tissue. The vaccine-induced influenza virus resistance mediated by IFN-I does not rely on TSLP immune regulation. On the contrary, TSLP is important for IFN-III-mediated adaptive immune response (34). Hence, IFNs can be selectively used in the application of clinical adjuvants. IFN-III may be more suitable for mucosal immune regulation and epithelial surface protection, whereas IFN-I provides more benefits in systematical immune responses (121). Therefore, the selective use of IFNs contributes to efficient and targeted immunotherapy.