What is an interferon?

Interferon (IFN) proteins are a family of cytokines secreted by host cells to modulate the immune response. As the first class of cytokines discovered, they were named “interferon” due to the protein’s ability to interfere with viral replication. These signaling proteins are typically released by the host cell in the presence of a pathogen and function to eradicate pathogens by alerting neighboring uninfected cells to activate proper cell defense mechanisms. IFNs are classified into three types (Type I, Type II, and Type III), according to the different receptors to which they bind (Table 1); each IFN type induces a specific immune response. Additionally, IFN-mediated signaling promotes upregulation of major histocompatibility class I and II molecules (MHC I, MHC II) and activates many downstream signaling cascades, resulting in anti-viral defense machineries. IFNs have since been used as therapeutic treatment for viral infections such as hepatitis C and B virus; however, several viruses have evolved to withstand IFN activity [1].

Interferon types

Type I, II, III

Type I IFNs bind to a specific cell surface receptor known as IFN-α/β (IFNAR1, IFNAR2) and function as a warning system for uninfected cells. In humans, Type I IFNs are the largest IFN family and include IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω; they are produced by many cell types, including plasmacytoid dendritic cells and fibroblasts. One major function of Type I IFN is the inactivation of eukaryotic translation initiation factor 2a (eIF-2a), thereby inhibiting the synthesis of viral protein (Figure 1). Additionally, Type I IFN activates RNase L, which cleaves any ssRNA within the cytoplasm, further inhibiting viral replication [2,3]. IFN-α has been used to treat hairy cell leukemia, while IFN-β has been used as treatment to slow the progression of multiple sclerosis.

Type II IFNs (IFN-γ in humans) bind to the IFN-γ receptor complex (IFNGR1, IFNGR2) and are involved in immune and inflammatory responses; they are produced by activated T cells and natural killer (NK) cells. When Type II IFNs are released by T helper cells, type 1 (Th1 cells), leukocytes are recruited to the sites of infection, leading to increased inflammation. Due to their role in the immune response, unregulated Type II IFNs can lead to autoimmune diseases.

Type III IFNs include IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4 and are implicated in the inhibition of viral infections similar to Type I IFNs. IFN-λ1, IFN-λ2, and IFN-λ3 were originally named IL-29, IL-28a, and IL-28b, respectively; IFN-λ4 is the newest Type III IFN to be discovered [4]. Type III IFNs bind to the receptors IFRL1 and IL-10R2, which are distinct from Type I receptors. Although not as well understood as Type I and II, Type III IFNs have been associated with the JAK-STAT pathway and are synthesized when the host detects pathogen-associated molecular patterns (PAMPs), similar to Type I IFNs [4,5].

Table 1. Interferon types and their receptors.

Type Subtype Receptors
Type I IFN-α IFNAR1 and IFNAR2
Type III IFN-λ IFNLR1 and IL-10R2
Abbreviations: IFN, interferon; IFNAR1, interferon alpha receptor 1; IFNAR2, interferon alpha receptor 2; IFNGR1, interferon gamma receptor 1; IFNGR2, interferon gamma receptor 2; IFNLR1, interferon lambda receptor 1; IL-10R2, interleukin-10 receptor 2.
Artistic illustration of type 1 interferons binding to receptor and inducing transcription of PKR and OAS proteins

Figure 1. Intracellular pathways induced by Type 1 IFNs. Type I IFNs bind to their respective receptors (Table 1) and induce transcription of PKR and OAS proteins. Once outside of the nucleus, PKR and OAS proteins contribute to an anti-viral state within the cell. Upon activation, PKR inactivates eIF-2α, therefore inhibiting viral protein translation. OAS presence in the cell contributes to activation of RNase L, which functions to cleave any viral ssRNA found in the cytosol.

Interferon induction

Given the anti-viral functions of IFNs, much focus was directed at viral RNA being the likely inducer of IFN expression. However, the discovery of the Toll-Dorsal pathway in Drosophila lead to the search for a Toll-like receptor (TLR) homolog in humans. TLRs are a class of pattern-recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) [6]. These membrane-spanning receptors are found on the cell surface, as well as within endosomes. Once bound to a PAMP, the TLR initiates a signaling cascade resulting in IFN gene activation, as well as subsequent protein secretion from the cell. RIG-I–like receptors (RLRs) are another class of PRRs that function as cytosolic sensors for viral RNA detection [6]. Activation of RLRs leads to upregulation of IFN regulatory factor-3 (IRF-3), IRF-7, and NF-κB, which are transcription factors that result in the induction of Type I IFN and inflammatory cytokines [7,8].

Artistic illustration of interferon induction by Toll-like receptor (TLR)/RIG-1 like receptor (RLR) mediated signaling

Figure 2. Interferon (IFN) induction by Toll-like receptor (TLR)/RIG-1–like receptor (RLR)–mediated signaling. TLR2 and TLR4 are located on the plasma membrane of cells, whereas TLR3, TLR 7/8, and TLR9 are located intracellularly within endosomes. RLRs circulate freely in the cytosol. Once bound, each receptor type leads to eventual upregulation of IFN and inflammatory genes.

Interferon antagonism

Viruses have developed many strategies to avoid or circumvent detection. Some RNA viruses such as influenza simply replicate in the nucleus to avoid cytoplasmic DNA/RNA sensors. Other viruses use techniques such as preventing viral products from binding cellular sensors or inhibiting the IFN response downstream. For example, the ORF52 protein on Kaposi’s sarcoma-associated herpesvirus directly inhibits cGAS enzymatic activity, therefore inhibiting the STING complex, a known promoter of IFNβ production [9]. RNA viruses have incorporated viral proteins that bind to and inhibit RLRs, thus limiting cytosolic detection, and many viruses have also evolved strategies to interfere with host transcription and translational effects induced by IFNs. West Nile virus has been shown to redistribute cholesterol to the viral replication membranes, resulting in inhibition of JAK-STAT activation by IFN [10]. These are just a few examples of how viruses have evolved to survive within host cells despite the many mechanisms and pathways meant to terminate or limit their proliferation.

Tools to study interferons

Staining for IFN-γ expression

Identification of IFN-γ is a key marker for inflammation and the engagement of the immune system during infection. One of the most common methods for detecting IFN-γ is flow cytometry. In vitro stimulation of splenocytes or PBMCs in presence of brefeldin A protein transport inhibitor of IFN-gamma production is required to find detectable levels of protein. Cells should be first stained for extracellular markers and then fixed with the IC Fixation and Permeabilization Buffer Set. Thereafter, the cells are ready to be stained for intracellular markers including IFN-γ. To help easily detect IFN-γ, we recommend using brighter fluorophores including PE. OMIPs and flow cytometry methods papers can help in panel design [11, 12, 13].

Two panel graph depicting intracellular staining of stimulated human peripheral blood cells for interferon gamma versus isotype control

Figure 3. Intracellular staining of stimulated human peripheral blood cells for IFN-γ. HPBCs were stimulated in both figures and stained for PerCP-eFluor 710 (46-0087-42) with either Mouse IgG1 K Isotype Control PE (12-4714-81) (left) or IFN gamma PE (412-7319-4).


  1. Pestka S, Krause CD, Walter MR (2004) Interferons, interferon-like cytokines, and their receptors. Immunol Rev 202:8–32.
  2. Louten J,van Rooijen N, Biron CA (2006) Type 1 IFN deficiency in the absence of normal splenic architecture during lymphocytic choriomeningitis virus infection. J Immun 177:3266–3272.
  3. Gil MP, Salomon R, Louten J et al. (2006) Modulation of STAT1 protein levels: A mechanism shaping CD8 T-cell responses in vivo. Blood 107:987–993.
  4. Zhou JH, Wang YN, Chang QY et al. (2018) Type III interferons in viral infection and antiviral immunity. Cell Physiol Biochem 51: 173–185.
  5. Fish EN, Platanias LC (2014) Interferon receptor signaling in malignancy: A network of cellular pathways defining biological outcomes. Mol Cancer Res 12:1691–1703.
  6. Baccala R, Gonzalez‐Quintial R, Lawson BR et al. (2009) Sensors of the innate immune system: Their mode of action. Nat Rev Rheumatol 5:448–456.
  7. Libermann TA, Baltimore D (1990) Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol 10:2327–2334.
  8. Fan X, Jin T (2019) Structures of RIG-I-like receptors and insights into viral RNA sensing. Adv Exp Med Biol 1172:157–188.
  9. Ma Z, Jacobs SR, West JA et al. (2015) Modulation of the cGAS-STING DNA sensing pathway by gammaherpesviruses. Proc Natl Acad Sci U S A 112:E4306–E4315.
  10. Mackenzie JM, Khromykh AA, Parton RG (2007) Cholesterol manipulation by West Nile virus perturbs the cellular immune response. Cell Host Microbe 2:229–239.
  11. Mendes R, Bromelow KV, Westby M, et al. (2000) Flow cytometric visualization of cytokine production by CD3-CD56+ NK cells and CD3+CD56+ NK-T cells in whole blood. Cytometry. 39(1):72-78.
  12. Vitale M, Caruso A, Licenziati S, et al. (2000) Differential production of IFN-gamma, analyzed at the single-cell level, by specific subsets of human NK and T cells from healthy and HIV(+) subjects. Cytometry. 39(3):189-194.
  13. Caraher EM, Parenteau M, Gruber H, Scott FW. (2000) Flow cytometric analysis of intracellular IFN-gamma, IL-4 and IL-10 in CD3(+)4(+) T-cells from rat spleen. J Immunol Methods. 244(1-2):29-40.

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