key: cord-0970238-8wg41v6m
authors: Yánez, Diana C.; Ross, Susan; Crompton, Tessa
title: The IFITM protein family in adaptive immunity
date: 2019-12-22
journal: Immunology
DOI: 10.1111/imm.13163
sha: fb1d3d195ce6f9e6d83a868ef9e9d02c942e976e
doc_id: 970238
cord_uid: 8wg41v6m

Interferon‐inducible transmembrane (IFITM) proteins are a family of small homologous proteins, localized in the plasma and endolysosomal membranes, which confer cellular resistance to many viruses. In addition, several distinct functions have been associated with different IFITM family members, including germ cell specification (IFITM1–IFITM3), osteoblast function and bone mineralization (IFITM5) and immune functions (IFITM1–3, IFITM6). IFITM1–3 are expressed by T cells and recent experiments have shown that the IFITM proteins are directly involved in adaptive immunity and that they regulate CD4(+) T helper cell differentiation in a T‐cell‐intrinsic manner. Here we review the role of the IFITM proteins in T‐cell differentiation and function.

The IFITM family

The family of interferon-inducible transmembrane (Ifitm/ Fragilis) genes encode small homologous proteins localized in the plasma and endolysosomal membranes, which can confer cellular resistance to many viruses in both mice and humans. 1-3 The IFITM family were first discovered as interferon-induced genes in human neuroblastoma cells and their promoters contain one or more interferon-stimulated response elements, making them responsive to type I and type II interferons. [4] [5] [6] [7] However, IFITM expression can be regulated independently of interferon signalling. 7, 8 Ifitm genes are targets of transcriptional repression by Bcl6, and have been shown to be targets of Wnt/b-catenin and Hedgehog (Hh) signal transduction [9] [10] [11] [12] ; and in murine T cells IFITM2 and IFITM3 expression is regulated by T-cell receptor (TCR) signal transduction. 7, 9, 13 In humans, five IFITM genes have been identified, which are located on chromosome 11, whereas in mice there are seven Ifitm genes, six of which are located on chromosome 7, and one on chromosome 16 (illustrated in Fig. 1a) . 6, [14] [15] [16] [17] [18] Homologous IFITM family genes are present in many other species, including marsupials, birds, fish and reptiles, suggesting important conserved roles for IFITM proteins. 19 The topology of the IFITM proteins in the membrane is not certain, several different topologies have been described, which are illustrated in Fig. 1(b) . [20] [21] [22] All IFITM proteins have two transmembrane or intramembrane regions spanning the membrane bi-layer sandwiched between three external regions. The connecting region is highly conserved and is always intracellular, but the N-terminus and C-terminus have been described to be either intracellular or extracellular (Fig. 1c) .

Several distinct functions have been associated with different IFITM family members, including germ cell specification (IFITM1-IFITM3), [14] [15] [16] 23, 24 osteoblast function and bone mineralization (IFITM5), [25] [26] [27] [28] [29] and immune functions (IFITM1-3, IFITM6), 7, 8, 13, [30] [31] [32] [33] [34] [35] [36] [37] [38] in addition to their roles as virus-restriction factors (IFITM1-3, murine IFITM6). The IFITM proteins have also been described to play a role in cell cycle control and apoptosis and their dysregulated expression, over-expression or mutation can be associated with colon cancers and metabolic dysregulation. [39] [40] [41] [42] [43] IFITM10 is highly conserved between species with at least 85% amino acid identity between birds, 3, 20, 46 In vivo studies have confirmed the importance of IFITM proteins in resistance to viruses. In mice, constitutive deletion of the five-gene cluster of Ifitm genes on chromosome 7 (Ifitm1-3, -5 and -6) or of Ifitm3 alone (Ifitm3 À/À ) rendered animals highly sensitive to influenza infection, and in humans genome-wide association studies and sequencing studies have shown that IFITM3 restricts influenza in vivo. 8, 38, [49] [50] [51] [52] [53] [54] [55] Distinct mechanisms have been proposed to explain the ability of different IFITM family members to restrict different classes of viruses, including inhibition of viral entry and also entry-independent effects, such as suppression of viral protein synthesis or viral replication. 3, 21, [44] [45] [46] [47] [48] 56, 57 Differences in the cellular localizations of the IFITM proteins may explain their different activities in the inhibition of entry of diverse viruses at their specific sites of fusion. However, determination of the precise subcellular localization of the different IFITM proteins remains elusive, and their localization within the cell may be dependent on cell type. IFITM1 has been found at the cell surface and also co-localizes with early endolysosomal markers. [30] [31] [32] [33] [58] [59] [60] IFITM2 and IFITM3 also co-localize with endosomal markers, but are found in membranes of different endosomal and lysosomal compartments compared with IFITM1, and co-localize with Rab7, CD63, lysosomal-associated membrane protein 3, 47, 59, 61 (Fig. 1c) .

The way in which IFITM proteins prevent viral entry at different sites is also unclear, and different experimental systems have provided evidence for many mechanisms, such as by changing membrane fluidity or physical properties of cell membranes, or by changing properties of the cytoplasm or lumina. 22, [62] [63] [64] The IFITM proteins in adaptive immunity

Gene and protein expression studies have demonstrated that IFITM1-3 are expressed in murine CD4 + and CD8 + T cells. 7, 11, 13, 37 Expression of Ifitm2 and Ifitm3 are regulated by TCR signalling. 13 In naive CD4 + T cells, RNA sequencing showed that expression of Ifitm3 was rapidly down-regulated during the first 24 hr after activation by anti-CD3/CD28 ligation in T helper type 0 (Th0), Th1 and Th2 culture conditions, whereas Ifitm2 was up-regulated and its expression continued to rise for the first 3 days after activation and levels of Ifitm1 were low and were not changed by TCR signalling. 13 In contrast, expression analysis of IFITM3 protein by Western blotting on naive CD8 + and CD4 + T cells following anti-CD3/CD28 activation demonstrated that IFITM3 protein is up-regulated by day 3 following T-cell activation. 7 This up-regulation was independent of interferon signalling, as naive T cells purified from mice which constitutively lack interferon-c (IFN-c), the IFN-a receptor, or the transcription factors IIrf3 and Irf7, which drive interferon-induced up-regulation of Ifitm3, all showed increased expression of IFITM3 protein 2 days after TCR ligation. 7 The difference in expression patterns of the Ifitm3 gene and the IFITM3 protein on activation of naive CD4 T cells may reflect differences in the strength of the activation signal given in the two experimental systems, or be due to changes in the rate of turnover and ubiquitination of the IFITM3 protein on TCR activation, [65] [66] [67] so that although Ifitm3 gene expression initially decreases, IFITM3 protein levels rise. The cartoon illustrates the proposed models of IFITM protein topology. First model suggests a conserved intracellular loop (CIL) between two transmembrane domains (TM) with extracellular C' and N' terminal domains. Second model shows a CIL between two intramembrane domains (IM) with intracellular C' and N' terminal domains. The third model proposes a CIL between IM and a TM with an intracellular N' and an extracellular C' terminal domain. These three topology models are predominant but alternative models have been proposed for specific IFITM protein topology depending on their function. 1 (c) The cartoon illustrates the cellular localization of IFITM1-3 proteins. IFITM proteins have been shown to span several cellular membranes. IFITM1 is found in different intracellular compartments from IFITM2 and IFITM3 with little overlap. 47,59 IFITM1-3 can all be found on the plasma membrane, but IFITM1 has been shown to be the predominant IFITM associated with the plasma membrane and is also found in early endosomes. 35, 60 IFITM2 and IFITM3 are predominately located intracellularly in late endosomes and lysosomes and co-localize with Rab7, CD63 and lysosomal-associated membrane protein (LAMP1). 21 The illustrations in this figure are cartoons that are not drawn to scale.

Ifitm3 is also regulated by Hh-mediated transcription in murine CD4 + T cells. 11 

The IFITM1-3 genes are expressed in human lymphocytes, and IFITM1 is expressed at their cell surface, where it has been shown to associate with receptor signalling complexes. 12, [31] [32] [33] The IFITM family in T-cell differentiation and function

In mice, IFITM3 protects against influenza, and Ifitm3 À/À mice die when infected with doses of influenza virus that would not be lethal in wild-type (WT) mice. 49, 50 In addition to its protective role in other cell types, such as respiratory epithelial cells and the heart, 49,68 IFITM3 protects cells of the immune system from viral infection, thereby enabling them to mount an effective immune response. 7, 8, 38 During influenza infection, IFITM3 is upregulated in dendritic cells in the lung by type I IFN, allowing them to survive and migrate to the draining lymph node in order to present viral antigens. 38 IFITM3 is then rapidly up-regulated on T cells on their activation in the draining lymph nodes, and high IFITM3 expression is maintained because they migrate to sites of viral infection, providing a survival advantage that enables them to carry out their effector functions. 7, 8 Interestingly, IFITM3 is also constitutively expressed in tissue-resident T cells in lung and airways, and also spleen, skin and brain, suggesting that it promotes their survival at these sites of potential viral infection. 8, 12, 13, 37, 53, 69 Hence, several in vivo and in vitro studies have demonstrated the importance of IFITM3 in immunity to viral infection, by enhancing survival and viral resistance of immune effector populations, but these studies did not demonstrate an additional direct function of the IFITM proteins in the immune function of T cells or dendritic cells.

We investigated the role of IFITM proteins in peripheral CD4 + T-cell function in mice deficient in the five Ifitm genes clustered on chromosome 7. These mice are deficient in Ifitm1-3, -5 and -6 and are referred to as IfitmDel À/À . 17 Whole-genome transcriptome analysis of resting CD4 + T cells from spleens of IfitmDel À/À mice showed that these cells had a Th1-like transcriptional signature compared with their WT counterparts. 13 To investigate if this difference was the result of changes in the immune environment of the cells, or the result of a CD4 + T-cell-intrinsic influence on T helper differentiation, we purified naive (CD62L + CD44 À CD25 À ) CD4 + T cells from IfitmDel À/À and WT littermates and carried out in vitro differentiation experiments in which we activated cells in Th-skewing conditions. These in vitro experiments showed a clear bias in differentiation towards Th1. 13 After 3 days in culture, a greater proportion of the IfitmDel À/À CD4 + T cells cultured in Th0 and Th1 conditions expressed Tbet than WT, whereas in Th2 conditions the proportion of Gata3 + cells was reduced. Expression of the Th1-associated molecules Cxcr3 and CD54 and expression of Tbx21 and Il27ra were also increased in the IfitmDel À/À cells cultured in Th1-skewing conditions compared with their WT counterparts. Bias towards Th1 differentiation was also demonstrated by cytokine production: IfitmDel À/ À cells produced less interleukin-4 (IL-4) and IL-13 than WT when cultured in Th2-skewing conditions, but more IFN-c when cultured in Th1-skewing conditions. Hence, the absence of the IFITM family of proteins led to an overall bias towards Th1 differentiation and a reduction in Th2 differentiation when purified naive CD4 + T cells were activated, suggesting that one or all of the IFITM proteins inhibit Th1 differentiation but promote Th2 differentiation in a T-cell-intrinsic manner 13 (illustrated in Fig. 2a) . As Ifitm2 expression rapidly increased on activation, IFITM2 might be the most likely candidate family member for this function.

In support of this, bias towards Th1 differentiation was not observed when purified naive Ifitm3 À/À CD4 + T cells were activated in Th-skewing conditions in vitro, 13 indicating that IFITM3 was not the sole family member responsible for promotion of Th2 differentiation, although a synergistic or additive effect between the IFITM proteins was not excluded.

The IfitmDel À/À mice also showed reduced Th2 responses and Th2 immunopathology in vivo. 13 On induction of allergic airways disease they had less severe disease and a weaker Th2 response, with lower Il4 expression, cellular infiltration and mucous production in the lung than their WT littermates. In addition to a reduction in eosinophils, myeloid dendritic cells and mast cells, T cells were reduced in the bronchoalveolar lavage and IL-27 secretion was increased but IL-13 production decreased, and the CD4 + population in the mediastinal lymph nodes had a more Th1-like phenotype, with higher cell surface expression of CD27, but lower expression of the Th2-marker T1ST2. Consistent with the in vitro cytokine data, lungs from the allergic airways disease-induced IfitmDel À/À mice had higher expression of Ifng, suggesting that although interferon-inducible, the IFITM family provide negative feedback on IFN-c signalling to dampen Th1 immunity in the lung.

On induction of allergic airways disease in Ifitm3 À/À mice, however, there were no significant changes in eosinophil, mast cell or T-cell infiltration in lung or bronchoalveolar lavage or in T1ST2 expression on T cells compared with WT, although macrophage and neutrophil infiltration was reduced. 13 Therefore, as with the in vitro data, deletion of Ifitm3 alone did not appear to affect the in vivo CD4 + Th2 response in lung; although, additive or synergistic effects between IFITM family members were not excluded.

The role of IFITM proteins in human allergic asthma has to our knowledge not been investigated, but genomewide association studies have linked IFITM2 and/or IFITM3 variants to potentially relevant traits, such as the proportion or count of basophils and eosinophils in blood, and lung function. 70, 71 Given the link between IFITM and Hh signalling and the fact that Hh signalling has also been shown to promote Th2 differentiation and exacerbate allergic asthma, it will be important to investigate the interactions between IFITM proteins and the Hh pathway in allergic asthma. 11, [72] [73] [74] [75] [76] [77] The IFITM proteins are associated with other atopic and inflammatory diseases. In atopic dermatitis patients, IFITM1-3 are up-regulated in lesional skin compared with non-lesional skin from the same individuals, although the functional consequences of their increased expression have not been investigated. 78 Likewise, their expression is up-regulated in inflamed mucosa of ulcerative colitis and Crohn's disease patients, 43, 79 and polymorphisms in IFITM1 and IFITM3 are associated with increased susceptibility to ulcerative colitis. 80, 81 In mice, the IFITM family protect against colitis. 82 IFITM3 deficiency led to exacerbation of chemical-induced colitis, with increased infiltration of macrophages and effector T cells to the colon lamina propria, and biased CD4 + Th differentiation to Th17. 82 That this exacerbated colitis was attributable to cells of the haematopoietic system was confirmed in bone marrow transplantation experiments. Interestingly, IfitmDel À/À mice developed spontaneous chronic colitis, indicating that other IFITM proteins are also protective against colitis, and both Ifitm3 À/À and IfitmDel À/À showed changes in the faecal microbiota. 82

Several studies have shown that IFITM proteins enhance immunity to viral disease by inhibiting viral infection of immune effector cells, thereby enhancing their survival and ability to mount an effective immune response, 7, 8, 12, 38 but the way in which IFITM proteins prevent viral infection is unclear and seems dependent on the cell type and the particular IFITM family member and virus interaction, with experimental evidence supporting many possible mechanisms, including inhibition of viral entry, fusion, transcription and translation. 3 Our recent study highlighted a virus-independent T-cell-intrinsic function for IFITM proteins in Th differentiation 13 and although the mechanism for their T-cell-intrinsic influence on Th differentiation is also unknown and requires investigation, several possibilities arise by extrapolation from the virusrestriction studies and from their cellular localization and expression patterns.

First, it is possible that IFITM proteins influence Th differentiation by influencing the molecular order of membranes and membrane fluidity. 62 In human T cells, high membrane order is associated with Th2 differentiation and IL-4 production, and intermediate membrane order is associated with Th1 cells and IFN-c production. 83 Hence, IFITM deficiency could bias differentiation towards Th1 by reducing membrane order, although how membrane order influences Th differentiation also remains unknown.

Second, it is possible that the IFITM proteins are involved in the regulation of cytokine signalling, and promote IL-4 signal transduction over Th1-cytokine signalling to polarize differentiation. Given their presence in endolysosomal intracellular vesicles, this theory suggests that IFITM proteins are involved in internalization, trafficking or degradation of some cytokine receptors or signalling pathway components, but not others. In support of this, IFN receptor signalling involves internalization by clathrin-dependent endocytosis, 84 so the presence of IFITM2 and IFITM3 in the membrane of late endosomes may modulate IFN signalling.

A third possibility is that the IFITM proteins are involved in enhancing or regulating the transduction of other signalling pathways that regulate Th differentiation. In support of this, they are transcriptional targets of Wnt and Hh signalling, 10, 11 and both these pathways promote Th2 differentiation. 72, 74, 85, 86 Finally, it is possible that the IFITM proteins have unknown direct consequences for Th differentiation through an influence on transcription or translation (as has been described for some viral genes).

Recent studies have identified new roles for the IFITM family in Th differentiation and atopic and inflammatory disease, which are independent of their functions in cellular resistance to viral infection. 13, 36, [80] [81] [82] Mouse studies showed that while IFITM deficiency was protective against induction of Th2 immune pathology and asthma, 13 it exacerbated Th17-driven inflammation in colitis, 82 highlighting the context dependency of their impact on inflammation. Clearly, further studies will be required to investigate the contribution of the different family members to the immune response and inflammation, and the cellular and molecular mechanisms that underlie their functions. It will be important to assess the impact of the IFITM family on the adaptive immune response to infectious disease and cancer.

Functional involvement of interferon-inducible transmembrane proteins in antiviral immunity

IFITM-family proteins: the cell's first line of antiviral defense

Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells

A single DNA response element can confer inducibility by both a-and c-interferons

Molecular analysis of a human interferon-inducible gene family

Rapid interferon independent expression of IFITM3 following T cell activation protects cells from influenza virus infection

Enhanced survival of lung tissueresident memory CD8 + T cells during infection with influenza virus due to selective expression of IFITM3

BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control

Dissecting Wnt/b-catenin signaling during gastrulation using RNA interference in mouse embryos

The transcriptional activator Gli2 modulates T-cell receptor signalling through attenuation of AP-1 and NFjB activity

BCL6 represses antiviral resistance in follicular T helper cells

IFITM proteins drive type 2 T helper cell differentiation and exacerbate allergic airway inflammation

A molecular programme for the specification of germ cell fate in mice

Developmentally regulated expression of mil-1 and mil-2, mouse interferon-induced transmembrane protein like genes, during formation and differentiation of primordial germ cells

The fragilis interferon-inducible gene family of transmembrane proteins is associated with germ cell specification in mice

Normal germ line establishment in mice carrying a deletion of the Ifitm/Fragilis gene family cluster

Evolutionary dynamics of the interferon-induced transmembrane gene family in vertebrates

Evolution of vertebrate interferon inducible transmembrane proteins

Interferon-induced cell membrane proteins, IFITM3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms

S-Palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (IFITM3)-mediated resistance to influenza virus

Interferon-induced transmembrane protein 3 is a type II transmembrane protein

Regulation of expression of mouse interferon-induced transmembrane protein like gene-3, Ifitm3 (mil-1, fragilis), in germ cells

IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion

Bril: a novel bone-specific modulator of mineralization

Characterization of the osteoblast-specific transmembrane protein IFITM5 and analysis of IFITM5-deficient mice

A single recurrent mutation in the 5'-UTR of IFITM5 causes osteogenesis imperfecta type V

A mutation in the 5'-UTR of IFITM5 creates an in-frame start codon and causes autosomal-dominant osteogenesis imperfecta type V with hyperplastic callus

A nonclassical IFITM5 mutation located in the coding region causes severe osteogenesis imperfecta with prenatal onset

TAPA-1, the target of an antiproliferative antibody, is associated on the cell surface with the Leu-13 antigen

Monoclonal antibody to the interferon-inducible protein Leu-13 triggers aggregation and inhibits proliferation of leukemic B cells

Differential modulation of the CD-2 and CD-3 T cell activation pathways by a monoclonal antibody to Leu-13

The CD19/CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules

Expression cloning of an interferon-inducible 17-kDa membrane protein implicated in the control of cell growth

Expression of the mouse fragilis gene products in immune cells and association with receptor signaling complexes

IFITM6 expression is increased in macrophages of tumor-bearing mice

The molecular signature of tissue resident memory CD8 T cells isolated from the brain

Respiratory DC use IFITM3 to avoid direct viral infection and safeguard virus-specific CD8 + T cell priming

Identification of the IFITM family as a new molecular marker in human colorectal tumors

Age-related onset of obesity corresponds with metabolic dysregulation and altered microglia morphology in mice deficient for Ifitm proteins

IFITM1 plays an essential role in the antiproliferative action of interferon-gamma

The human 1-8D gene (IFITM2) is a novel p53 independent pro-apoptotic gene

Interferon-inducible gene family 1-8U expression in colitis-associated colon cancer and severely inflamed mucosa in ulcerative colitis

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus

Identification of five interferon-induced cellular proteins that inhibit West Nile virus and dengue virus infections

Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus

The IFITM proteins inhibit HIV-1 infection

The IFITMs inhibit Zika virus replication

Ifitm3 limits the severity of acute influenza in mice

IFITM3 restricts the morbidity and mortality associated with influenza

Interferon-induced transmembrane protein-3 genetic variant rs12252-C is associated with severe influenza in Chinese individuals

Early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal H7N9 infection

SNPmediated disruption of CTCF binding at the IFITM3 promoter is associated with risk of severe influenza in humans

Evaluation of IFITM3 rs12252 association with severe pediatric influenza infection

IFITM3: how genetics influence influenza infection demographically

ISG56 and IFITM1 proteins inhibit hepatitis C virus replication

IFITM proteins incorporated into HIV-1 virions impair viral fusion and spread

Binding of IFITM1 enhances the inhibiting effect of caveolin-1 on ERK activation

IFITM-2 and IFITM-3 but not IFITM-1 restrict Rift Valley fever virus

A membrane topology model for human interferon inducible transmembrane protein 1

Identification of an endocytic signal essential for the antiviral action of IFITM3

IFITM proteins restrict viral membrane hemifusion

The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication

The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry

Phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (IFITM3) dually regulates its endocytosis and ubiquitination

Regulation of the trafficking and antiviral activity of IFITM3 by post-translational modifications

E3 Ubiquitin ligase NEDD4 promotes influenza virus infection by decreasing levels of the antiviral protein IFITM3

IFITM3 protects the heart during influenza virus infection

T cell memory. Skin-resident memory CD8 + T cells trigger a state of tissue-wide pathogen alert

The allelic landscape of human blood cell trait variation and links to common complex disease

Leveraging polygenic functional enrichment to improve GWAS power

Tissuederived hedgehog proteins modulate Th differentiation and disease

Frontline science: Shh production and Gli signaling is activated in vivo in lung, enhancing the Th2 response during a murine model of allergic asthma

Hedgehog signaling promotes TH2 differentiation in naive human CD4 T cells

Gene expression patterns of Th2 inflammation and intercellular communication in asthmatic airways

Importance of hedgehog interacting protein and other lung function genes in asthma

High expression of Sonic hedgehog in allergic airway epithelia contributes to goblet cell metaplasia

Sonic Hedgehog signaling limits atopic dermatitis via Gli2-driven immune regulation

Genome-wide gene expression differences in Crohn's disease and ulcerative colitis from endoscopic pinch biopsies: insights into distinctive pathogenesis

Identification of the polymorphisms in IFITM1 gene and their association in a Korean population with ulcerative colitis

Identification of the polymorphisms in IFITM3 gene and their association in a Korean population with ulcerative colitis

The anti-inflammatory IFITM genes ameliorate colitis and partially protect from tumorigenesis by changing immunity and microbiota

Primary human CD4 + T cells have diverse levels of membrane lipid order that correlate with their function

Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors

Global regulator SATB1 recruits b-catenin and regulates T H 2 differentiation in Wnt-dependent manner

T cell factor 1 initiates the T helper type 2 fate by inducing the transcription factor GATA-3 and repressing interferon-c

This research was funded by the MRC and GOSHCC.

The authors have no competing interests.