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opsonin, and also activating complement Bacteria induce macrophages to produce IL-6, which acts on hepatocytes to induce

synthesis of acute-phase proteins

C-reactive protein

Serum amyloid protein serum amyloid protein mannose- binding lectin fibrinogen IL-6 SP-A SP-D liver

Fig. 3.34 The acute-phase response produces molecules that bind pathogens but not host cells. Acute-phase proteins are produced by liver cells in response to cytokines

released by macrophages in the presence of bacteria (top panel). They include serum amyloid protein (SAP) (in mice but not humans), C-reactive protein (CRP), fibrinogen, and mannose-binding lectin (MBL). CRP binds phosphocholine on certain bacterial and fungal surfaces but does not recognize it in the form in which it is found in host cell membranes (middle panel). SAP and CRP are homologous in structure; both are pentraxins, forming five-membered discs, as shown for SAP (lower panel). SAP both acts as an opsonin in its own right and activates the classical complement pathway by binding C1q to augment opsonization. MBL is a member of the collectin family, which also includes the pulmonary surfactant proteins SP-A and SP-D. Like CRP, MBL can act as an opsonin in its own right, as can SP-A and SP-D. Model structure courtesy of J. Emsley.

However, some immune cells seem to be specialized for this task. In Section 3-1 we introduced the plasmacytoid dendritic cell (pDC). Also called interferon- producing cells (IPCs) or natural interferon-producing cells, human plasmacytoid dendritic cells were initially recognized as rare peripheral blood cells that accumulate in peripheral lymphoid tissues during a viral infection and make abundant type I interferons (IFN-α and IFN-β)—up to 1000 times more than other cell types. This abundant production of type I interferon may result from the efficient coupling of viral recognition by TLRs to the pathways of interferon production (see Section 3-7). Plasmacytoid dendritic cells express a subset of TLRs that includes TLR-7 and TLR-9, which are endosomal sensors of viral RNA and of the nonmethylated CpG residues present in the genomes of many DNA viruses (see Fig. 3.11). The requirement for TLR-9 in sensing infections caused by DNA viruses has been demonstrated, for example, by the inability of TLR-9-deficient plasmacytoid dendritic cells to generate type I interferons in response to herpes simplex virus. Plasmacytoid dendritic cells express CXCR3, a receptor for the chemokines CXCL9, CXCL10, and CXCL11, which are produced by T cells. This allows pDCs to migrate from the blood into lymph nodes in which there is an ongoing inflammatory response to a pathogen.

Interferons help defend against viral infection in several ways (Fig. 3.35). IFN-β is particularly important because it induces cells to make IFN-α, thus ampli- fying the interferon response. Interferons act to induce a state of resistance to viral replication in all cells. IFN-α and IFN-β bind to a common cell-surface receptor, known as the interferon-α receptor (IFNAR), which uses the JAK and STAT pathways described in Section 3-16. IFNAR uses the kinases Tyk2 and Jak1 to activate the factors STAT1 and STAT2, which can interact with IRF9 and form a complex called ISGF3 that binds to the promoters of many interferon stimulated genes (ISGs).

One ISG encodes the enzyme oligoadenylate synthetase, which polymerizes ATP into 2ʹ–5ʹ-linked oligomers (whereas nucleotides in nucleic acids are nor- mally linked 3ʹ–5ʹ). These 2ʹ–5ʹ-linked oligomers activate an endoribonuclease that then degrades viral RNA. A second protein induced by IFN-α and IFN-β is a dsRNA-dependent protein kinase called PKR. This serine–threonine kinase phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2α), thus suppressing protein translation and contributing to the inhibition of viral rep- lication. Mx (myxoma resistant) proteins are also induced by type I interfer- ons. Humans and wild mice have two highly similar proteins, Mx1 and Mx2, which are GTPases belonging to the dynamin protein family, but how they interfere with viral replication is not understood. Oddly, most common labo- ratory strains of mice have inactivated both Mx genes, and in these mice, IFN-β cannot act to protect against influenza infection.

In the last few years, several novel ISGs have been identified and linked to antiviral functions. The IFIT (IFN-induced protein with tetratricoid repeats) family contains four human and three mouse proteins that function in restraining the translation of viral RNA into proteins. IFIT1 and IFIT2 can both suppress the translation of normal capped mRNAs by binding to sub units of the eukaryotic initiation factor 3 (eIF3) complex, which prevents eIF3 from interacting with eIF2 to form the 43S pre-initiation complex (Fig. 3.36). This action may be responsible in part for the reduction in cellular proliferation induced by type I interferons. Mice lacking IFIT1 or IFIT2 show increased sus- ceptibility to infection by certain viruses, such as vesicular stomatitis virus. Another function of IFIT1 is to suppress translation of viral RNA that lacks a normal host modification of the 5ʹ cap. Recall that the normal mammalian 5ʹ cap is initiated by linking a 7-methylguanosine nucleotide to the first ribose sugar of the mRNA by a 5ʹ–5ʹ triphosphate bridge, to produce a structure called cap-0. This structure is further modified by cytoplasmic methylation of the 2ʹ hydroxyl groups on the first and second ribose sugars of the RNA. Methylation Immunobiology | chapter 3 | 03_029

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Virus-infected host cells

virus

Activate dendritic cells and macrophages

Activate NK cells to kill virus-infected cells

Induce chemokines to recruit lymphocytes Increase MHC class I expression and antigen

presentation in all cells

Activate STAT1 and STAT2, which combine with IRF9 to form ISGF3

Induce resistance to viral replication in all cells by inducing Mx proteins, 2'–5'-linked adenosine

oligomers, and the kinase PKR Induce expression of IFIT proteins, which

suppress the translation of viral RNA IFN-α, IFN-β

Fig. 3.35 Interferons are antiviral proteins produced by cells in response to viral infection. The interferons IFN-α

and IFN-β have three major functions. First, they induce resistance to viral replication in uninfected cells by activating genes that cause the destruction of mRNA and inhibit the translation of viral proteins and some host proteins. These include the Mx proteins, oligoadenylate synthetase, PKR, and IFIT proteins. Second, they can induce MHC class I expression in most cell types in the body, thus enhancing their resistance to NK cells; they may also induce increased synthesis of MHC class I molecules in cells that are newly infected by virus, thus making them more susceptible to being killed by CD8 cytotoxic T cells (see Chapter 9). Third, they activate NK cells, which then selectively kill virus- infected cells.

Interferon‑γ Receptor Deficiency

of the first ribose sugar produces a structure called cap-1; methylation of the second generates cap-2. IFIT1 has a high affinity for cap-0, but much lower affinity for cap-1 and cap-2. Some viruses, such as Sindbis virus (family Togaviridae), lack 2ʹ-O-methylation, and therefore are restricted by this action of IFIT1. Many viruses, such as West Nile virus and SARS coronavirus, have acquired a 2ʹe-O-methyltransferase (MTase) that produce cap-1 or cap-2 on their viral transcripts. These viruses can thus evade restriction by IFIT1. Members of the interferon-induced transmembrane protein (IFITM) family are expressed at a basal level on many types of tissues but are strongly induced by type I interferons. There are four functional IFITM genes in humans and in mice, and these encode proteins that have two transmembrane domains and are localized to various vesicular compartments of the cell. IFITM proteins act to inhibit, or restrict, viruses at early steps of infection. Although the molecular details are unclear, IFITM1 appears to interfere with the fusion of viral mem- branes with the membrane of the lysosome, which is required for introducing some viral genomes into the cytoplasm. Viruses that must undergo this fusion event in lysosomes, such as the Ebola virus, are restricted by IFITM1. Similarly, IFITM3 interferes with membrane fusion in late endosomes, and so restricts the influenza A virus, which undergoes fusion there. The importance of this mechanism is demonstrated by the increased viral load and higher mortality in mice lacking IFITM3 that are infected with the influenza A virus.

Interferons also stimulate production of the chemokines CXCL9, CXCL10, and CXCL11, which recruit lymphocytes to sites of infection. They also increase the Immunobiology | chapter 3 | 03_105

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C E C E 40S ribosomal subunit methionine tRNA _eIF3 eIF4 eIF2

tRNA, 40S ribosome subunit, eIF2, eIF3, and eIF4 assemble to form a 43S pre-initiation complex

43S pre-initiation complex mRNA 5' cap 40S mRNA IFIT IFIT eIF3

IFIT1 and IFIT2 bind to subunits of eIF3 and prevent formation of 43S

pre-initiation complex

60S ribosomal subunit

Initiation complex forms with 60S ribosome subunit and release of eIF2, 3, and 4

Fig. 3.36 IFIT proteins act as antiviral effector molecules by inhibiting steps in the translation of RNA. Top left panel: formation of a 43S pre-initiation complex is

an early step in the translation of RNA into protein by the 80S ribosome that involves a charged methionine tRNA, the 40S ribosome subunit, and eukaryotic initiation factors (eIFs) eIF4, eIF2, and eIF3. Middle panel: eIFs and a charged methionine tRNA assemble into a 43S pre-initiation complex. Right panel: the pre-initiation complex mRNA recognizes the 5ʹ cap structure and joins with the 60S ribosomal subunit, releasing eIF2, eIF3, and eIF4 and forming a functional 80S ribosome. Lower panel: eIF3 has 13 subunits, a–m. IFIT proteins can inhibit several steps in protein translation. Mouse IFIT1 and IFIT2 interact with eIF3C, and human IFIT1 and IFIT2 interact with eIF3E, preventing formation of the 43S pre-initiation complex. IFITs can also interfere with other steps in translation, and can bind and sequester uncapped viral mRNAs to prevent their translation (not shown). Expression of IFIT proteins is induced in viral infection by signaling downstream of type I interferons.

expression of MHC class I molecules on all types of cells, which facilitates rec- ognition of virally infected cells by cytotoxic T lymphocytes via the display of viral peptides complexed to MHC class I molecules on the infected cell surface (see Fig. 1.30). Through these effects, interferons indirectly help promote the killing of virus-infected cells by CD8 cytotoxic T cells. Another way in which interferons act is to activate populations of innate immune cells, such as NK cells, that can kill virus-infected cells, as described below.

3-23 Several types of innate lymphoid cells provide protection in early infection.

A defining feature of adaptive immunity is the clonal expression of anti- gen receptors, produced by somatic gene rearrangements, that provide the extraordinarily diverse specificities of T and B lymphocytes (see Section 1-11). However, for several decades, immunologists have recognized cells that have lymphoid characteristics but which lack specific antigen receptors. Natural killer (NK) cells have been known the longest, but in the past several years other distinct groups of such cells have been identified. Collectively, these are now called innate lymphoid cells (ILCs) and include NK cells (Fig. 3.37). ILCs develop in the bone marrow from the same common lymphocyte progenitor (CLP) that gives rise to B and T cells. Expression of the transcription factor Id2 (inhibitor of DNA binding 2) in the CLP represses B- and T-cell fates, and is required for the development of all ILCs. ILCs are identified by the absence of T- and B-cell antigen receptor and co-receptor complexes, but they express the receptor for IL-7. They migrate from the bone marrow and populate lymphoid tissues and peripheral organs, notably the dermis, liver, small intestine, and lung.

ILCs function in innate immunity as effector cells that amplify the signals delivered by innate recognition. They are stimulated by cytokines produced by other innate cells, such as macrophages or dendritic cells, that have been acti- vated by innate sensors of microbial infection or cellular damage. Three major subgroups of ILCs are defined, largely on the basis of the types of cytokines that each produces. Group 1 ILCs (ILC1s) generate IFN-γ in response to activation by certain cytokines, in particular IL-12 and IL-18, made by dendritic cells and macrophages, and they function in protection against infection by viruses or intracellular pathogens. NK cells are now considered to be a type of ILC. ILC1s and NK cells are closely related, but have distinct functional properties and differ in the factors required for their development. NK cells are more similar to CD8 T cells in function, while ILC1s resemble more closely the TH1 subset

of CD4 T cells (see Section 3-24). NK cells can be distinguished from recently identified ILC1 cells in several ways. NK cells can be found within tissues, but they also circulate through the blood, while ILC1 cells appear to be largely non-circulating tissue-resident cells. In the mouse, conventional NK cells express the integrin α2 (CD49b), while ILC1 cells, for example in the liver, lack

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Innate lymphoid subgroup

The major categories of innate lymphoid cells (ILCs) and their properties Inducing cytokine Effector molecules produced Function

NK cells IL-12 IFN-γ, perforin, granzyme Immunity against viruses, intracellular pathogens

ILC1 IL-12 IFN-γ Defense against viruses, intracellular pathogens

ILC2 IL-25, IL-33, TSLP IL-5, IL-13 Expulsion of extracellular parasites

ILC3, LTi cells IL-23 IL-22, IL-17 Immunity to extracellular bacteria and fungi

Fig. 3.37 The major categories of innate lymphoid cells (ILCs) and their properties.

CD49b but express the surface protein Ly49a. Both NK and ILC1 cells require the transcription factor Id2 for their development, but NK cells require the cytokine IL-15 and the transcription factors Nfil3 and eomesodermin, while liver ILC1 cells require the cytokine IL-7 and the transcription factor Tbet. ILC2s produce the cytokines IL-4, IL-5, and IL-13, in response to various cytokines, particularly thymic stromal lymphopoietin (TSLP) and IL-33. ILC2 cytokines function in promoting mucosal and barrier immunity and aid in protection against parasites. ILC3s respond to the cytokines IL-1β and IL-23 and produce several cytokines, including IL-17 and IL-22, which increase defenses against extracellular bacteria and fungi. IL-17 functions by stimu- lating the production of chemokines that recruit neutrophils, while IL-22 acts directly on epithelial cells to stimulate the production of antimicrobial pep- tides such as RegIIIγ (see Section 2-4).

The classification of ILC subtypes and the analysis of their development and function is still an active area, and studies to define the relative importance of these cells in immune responses are ongoing. The ILC subgroups identified so far appear to be highly parallel in structure to the subsets of effector CD8 and CD4 T cells that were defined over the last three decades. The transcription factors that control the development of different ILC subsets seem, for now at least, to be the same as those that control the corresponding T-cell subsets. Because of these similarities, we will postpone a detailed description of ILC development until Chapter 9, where we will cover this topic along with the development of T-cell subsets.

3-24 NK cells are activated by type I interferon and macrophage- derived cytokines.

NK cells are larger than T and B cells, have distinctive cytoplasmic granules containing cytotoxic proteins, and are functionally identified by their ability to kill certain tumor cell lines in vitro without the need for specific immunization. NK cells kill cells by releasing their cytotoxic granules, which are similar to those of cytotoxic T cells and have the same effects (discussed in Chapter 9). In brief, the contents of cytotoxic granules, which contain granzymes and the pore-forming protein perforin, are released onto the surface of the target cell, and penetrate the cell membrane and induce programmed cell death. However, unlike T cells, killing by NK cells is triggered by germline-encoded receptors that recognize molecules on the surface of infected or malignantly transformed cells. A second pathway used by NK cells to kill target cells involves the TNF family member known as TRAIL (tumor necrosis factor- related apoptosis-inducing ligand). NK cells express TRAIL on their cell surface. TRAIL interacts with two TNFR superfamily ‘death’ receptors, DR4 and DR5 (encoded by TNFSF10A and B), that are expressed by many types of cells. When NK cells recognize a target cell, TRAIL stimulates DR4 and DR5 to activate the pro-enzyme caspase 8, which leads to apoptosis. In contrast to pyroptosis, induced by caspase 1 following inflammasome activation (see Section 3-9), apoptosis is not associated with production of inflammatory cytokines. We will return to discuss more details of the mechanisms of caspase-induced apoptosis when we discuss killing by cytotoxic T cells in Chapter 9. Finally, NK cells express Fc receptors (see Section 1-20); binding of antibodies to these receptors activates NK cells to release their cytotoxic granules, a process known as antibody-dependent cellular cytotoxicity, or ADCC, to which we will return in Chapter 10.

The ability of NK cells to kill target cells can be enhanced by interferons or cer- tain cytokines. NK cells that can kill sensitive targets can be isolated from unin- fected individuals, but this activity is increased 20- to 100-fold when NK cells are exposed to IFN-α and IFN-β, or to IL-12, a cytokine produced by dendritic cells and macrophages during infection by many types of pathogens. Activated

NK cells serve to contain virus infections while the adaptive immune response is generating antigen-specific cytotoxic T cells and neutralizing antibodies that can clear the infection (Fig. 3.38). A clue to the physiological function of NK cells in humans comes from rare patients deficient in these cells, who are fre- quently susceptible to herpesvirus infection. For example, a selective NK-cell deficiency results from mutations in the human MCM4 (mini chromosome maintenance-deficient 4) protein, which is associated with predisposition to viral infections.

IL-12, acting in synergy with the cytokine IL-18 produced by activated macro- phages, can also stimulate NK cells to secrete large amounts of interferon (IFN)-γ, and this is crucial in controlling some infections before the IFN-γ produced by activated CD8 cytotoxic T cells becomes available. IFN-γ, whose receptor activates only the STAT1 transcription factor, is quite distinct func- tionally from the antiviral type I interferons IFN-α and IFN-β, and is not directly induced by viral infection. The production of IFN-γ by NK cells early in an immune response can directly activate macrophages to enhance their capacity to kill pathogens, augmenting innate immunity, but also influences adaptive immunity through actions on dendritic cells and in regulating the differentiation of CD4 T cells into the pro-inflammatory TH1 subset, which

produces IFN-γ. NK cells also produce TNF-α, granulocyte-macrophage stimulating factor (GM-CSF), and the chemokines CCL3 (MIF 1-α), CCL4, and CCL5 (RANTES), which act to recruit and activate macrophages.

3-25 NK cells express activating and inhibitory receptors to distinguish between healthy and infected cells.

For NK cells to defend against viruses or other pathogens, they should be able to distinguish infected cells from uninfected healthy cells. However, the mechanism used by NK cells is slightly more complicated than pathogen recognition by T or B cells. In general, it is thought that an individual NK cell expresses various combinations of germline-encoded activating receptors and inhibitory receptors. While the exact details are not clear in every case, it is thought that the overall balance of signaling by these receptors determines whether an NK cell engages and kills a target cell. The receptors on an NK cell are tuned to detect changes in expression of various surface proteins on a