Question

Briefly describe the adaptive immune responses to the African swine fever virus?

Answer 1

ANSWER

African swine fever (ASF) is a devastating viral disease of domestic pigs and wild boar for which there is no vaccine available. The aetiological agent ASF virus (ASFV) has a predilection for cells of the myeloid lineage. Macrophages provide a first line defence against pathogens and are the main target of ASFV, thus several studies analysed their response to infection in terms of cytokine/chemokine expression and modulation of functionality. These studies have typically used macrophages differentiated in vitro from blood or bone marrow progenitors and few studies have focused on responses of polarized macrophages (M1, M2) or functional macrophage subsets isolated from different tissues. ASFV can also infect dendritic cells (DC), but regardless of their central role in the induction of adaptive immune responses, their role in ASFV infection was only partially analysed. Future studies on ASFV-DC interaction are needed, which should take into consideration the heterogeneity within this family, composed of different subsets whose phenotype is also organ specific. Other porcine immune cells such as γδ-T cells, NK cells and fibrocytes, can act as ‘non-conventional’ antigen-presenting cells (APCs). In particular, γδ-T cells from ASFV immune pigs were shown to present viral antigens to T cells, but no studies have further explored the interaction of ASFV with this cell type or other non-conventional APCs.

Macrophages

Macrophages play a central role under steady-state conditions and provide also a first line of defence against pathogens.They clear the interstitial environment from extraneous cellular materials, remove cellular debris generated during tissue remodelling and rapidly clear cells that have undergone apoptosis.Macrophages can also initiate acquired immunity by processing and presenting antigens to naïve T lymphocytes . These cells are equipped with a vast array of sensing molecules, called pathogen-recognition receptor (PRRs), which recognise pathogen-associate molecular patterns (PAMPs) . Macrophages are a heterogeneous population and cells in different anatomical locations show diverse specialization of functions. For example, osteoclasts are specialised in bone remodelling, whereas alveolar macrophages are specialised to clear microorganisms and environmental particles in the lung . Macrophages present remarkable plasticity, changing their phenotype and functionality in response to environmental signals, produced also by antigen-specific immune cells . Macrophage activation results in their polarization into two main different functional subsets: classical activated (M1) and alternative activated (M2) macrophages. M1 polarization can be reached in vitro by exposure of macrophages to IFN-γ and lipopolysaccharide (LPS). M1 macrophages show increased microbicidal and tumoricidal activity compared to un-activated macrophages and their main role is in host defence to intracellular pathogens and in driving Th1 cellular immune responses . Alternative activation of macrophages can instead be achieved in vitro through the stimulation with IL-4 and/or IL-13.

monocytes and macrophages are the main targets of ASFV and are thought to be crucial for viral persistence and dissemination.. The genomes of virulent ASFV isolates encode several non-essential proteins that inhibit apoptosis.

EFFECTS OF ASFV INFECTION POTENTIAL IMPLICATIONS Induction of T cell response Attenuated ASFV: † CCL4, CXCL8, CXCL10, IFN-a, TNF- DODO a, IL12p40, IFN-B, IL-18, IL1-B, IL-1a Mechanisms that limits viral replication and spread MACROPHAGE Dopo Attenuated ASFV: MHCI Induction of NK activation Attenuated and virulent ASFV: CD14/CD16 BODIE Impairment of anti- microbial/viral activity

Dendritic cells

DCs are sentinels of the immune system, which play an important role in the induction of adaptive immune responses against pathogens . DCs are considered professional APCs that detect, uptake and process antigen, and then migrate into secondary lymphoid tissues to present the processed antigens to lymphocytes . They possess the unique capacity to prime naïve T lymphocytes and have a central role in control of T cell activation and regulation. DCs can modulate the type and the homing characteristics of T cell responses and are also involved in B cell responses and NK cell activation . DCs express a variety of specialized PRRs, including Toll like receptors (TLRs), for the recognition of PAMPs . Recognition of PAMPs by TLRs gives rise to ‘danger’ signal essential for DC maturation and activation, which results in a robust cytokine and chemokine response, essential for promoting both innate and adaptive immune responses .

EFFECTS OF ASFV INFECTION POTENTIAL IMPLICATIONS Attenuated ASFV: 1 MHC I (moDC) Induction of NK activation Attenuated and virulent ASFV: 1 CD16 (moDC) BODOC) Impairment of anti- microbial/viral activity DENDRITIC CELL DODOC Attenuated and virulent ASFV: no release of IFN-B, IL-1B, IL-6, IL-8, IL-10, IL-12, IL-18, TNF-a by moDC Covert ASFV replication in moDC Attenuated and virulent ASFV: 1 type I interferon by blood DC PDC are source of type IIFN pathologically high in serum during acute ASF

Other APCs: fibrocytes, γδ-T cells and NK cells

γδ-T cells

T lymphocytes present an antigen receptor (T cell receptor, TcR) consisting of an αβ or γδ heterodimer. As shown in other ungulates, swine possess large numbers of γδ-T cells compared to many other species: in young pigs γδ-T cells represent 50% of the total peripheral blood lymphocyte populations, although their frequency decreases with age . γδ-T cells display characteristics of both T cells and cells of the innate immune system. They are activated not only by TCR agonists, but also through other receptors, such as TLRs and NK receptors (NKp46, NKG2D). A balance between activating and inhibitory signals regulates their response . Activation of this cell type may be direct or mediated by APCs, such as DCs . In pigs these cells can act as cytotoxic cells, lysing virus-infected cells, and can produce several types of cytokines (IFN-γ, TNF-α, and IL-17 A) . Nevertheless, in humans and in pigs it was described that γδ-T cells can also display characteristics of APCs; they can express MHC class II molecules, which have been proposed to be markers of porcine T cell activation . It was observed that porcine γδ-T cells acquired a phenotype of APCs after vaccination against foot-and-mouth disease virus; these cells were able to process and present soluble antigen to CD4+ T cells, likely shaping the outcome of the adaptive immune response . Infection with classical swine fever virus (CSFV) resulted in a partial activation of this cell type, with MHC class II up-regulation, but absence of IFN-γ release or perforin-mediated cytotoxic function .

NK cells

NK cells are cells of the innate immune system. They are able to kill pathogen-infected and malignant cells and are an important source of IFN-γ and TNF-α . Their activation depends on a balance of inhibitory and activating signals received through surface receptors after exposure to diverse ligands exposed on potential target cells . NK activation by most pathogens also occurs through accessory cells, such as DCs . Nevertheless, there are indicators that NK cells display features of APCs. In pigs it was observed that a small proportion of NK cells express MHC class II, which can be up-regulated after in vitro stimulation with cytokines including IL-2, IL-12, IL-18 and IFN-α . In the latter study, it was observed that co-culture of NK cells with pDCs, but not cDCs, resulted in MHC class II upregulation on NK cells, and this was associated with high levels of IFNα released by CSFV-infected pDCs . Increased expression of MHC class II on NK cells after interaction with DCs was also described in mice, where instead NK acquisition of MHC class II was mediated by intercellular membrane transfer (or ‘cross-dressing’) from interacting DCs . A recent study showed that in pigs NK cells can express not only MHC II, but also co-stimulatory molecules CD80/86, especially after in vitro stimulation with IL2/IL-12/Il-18, and these cells were able to induce T-cell proliferation.

Fibrocytes

Fibrocytes are circulating cells of myeloid origin that represent ∼0.5% of peripheral blood leukocytes . They not only play a role in wound healing, rapidly entering sites of injury and synthesising collagen I, collagen III and fibronectin, but also display features of APCs. Human fibrocytes express MHC class II, costimulatory molecules CD80/86 and the adhesion molecules CD11a, CD54, and CD58, and are potent inducers of antigen-specific T cell proliferation . As in humans, porcine fibrocytes can be potent APCs. It was observed that they expressed MHC class I, MHC class II and CD80/86, were able to endocytose and degrade antigens, and efficiently induced cytotoxic T lymphocyte responses using CSFV as a model