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In most of the studies mentioned above, researchers observed that values of IL-1α and IL-1β started to rise at early stages after infection with virulent ASFV isolates, and that serum concentrations often peaked at the end of the observation period [12,13,14,16,17]. The release of these cytokines in vivo should enhance immune surveillance, promoting viral clearance and the development of the acquired immune response . However, steady high serum levels of these proinflammatory cytokines for long periods of time after virus infection were also suggested as indicative of exaggerated, aberrant and failed immune activation with fatal consequences for the outcome of the disease, and with a principal role in the pathogenic mechanisms of ASF lesions [25,26].
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Immunization studies in vivo with attenuated ASFV strains revealed that protection against challenges carried out with virulent isolates was correlated with the development of a cellular immune response led mainly by NK and CD8+ cytotoxic T cells . A hallmark of NK/cytotoxic T cells activation is the production of IFN-γ; thus several research works assessed the production of this antiviral cytokine by using diverse techniques such as ELISA, ELISpot assay or flow cytometry [36,76].
A number of ASFV-specific IFN-γ-producing cells can be monitored using either ELISpot assay or flow cytometry, techniques that can be useful to quantify specific T-cell responses. ELISpot assay to detect ASFV-specific IFN-γ-producing cells has been recently described in detail . This assay is often performed using 96-well plates with peripheral blood mononuclear cells (PBMC) re-stimulated ex vivo with a recall antigen (e.g., virus, peptides) . PBMC from surviving inbred pigs infected with the non-virulent genotype I tissue culture-adapted strain BA71V induced IFN-γ in response to homologous stimulation with ASFV, but not in response to stimulation with heterologous or virulent strains . High numbers of ASFV-specific IFN-γ-producing cells in pigs immunized with the attenuated isolate OURT88/3 and boosted with the virulent isolate OURT88/1 correlated well with protection induced in vivo against homologous and heterologous challenge . Some correlation between virus-specific IFN-γ-producing cells induced after immunization with BeninΔDP148R and protection against the virulent parental Benin97/1 was also observed . In addition, correlation between protection and ASFV-specific IFN-γ-producing cells was recently reported . In detail, the immunization of pigs with the BA71ΔCD2 deletion mutant (developed from the virulent genotype I Badajoz-71) conferred dose-dependent cross-protection against direct-contact challenge with pigs infected with the genotype II virulent isolate Georgia2007/1. Protection was associated with IFNγ-secreting cells, evaluated by ELIspot in PBMC (re-stimulated ex vivo with either BA71ΔCD2 or Georgia2007/1) .
Flow cytometry can be adopted to better characterize ASFV-specific IFN-γ-producing cells. Some studies were only able to speculate on the possible origin of IFN-γ synthesis [54,86,87,88]. In a review, Takamatsu et al.  mentioned some unpublished results in which they observed that IFN-γ+ lymphocytes from ASFV-stimulated immune PBMC displayed mainly the CD4+CD8+ T cell phenotype, of which a third displayed the memory helper T cell (CD4+CD8low) phenotype and the rest, the cytotoxic (CTL) (CD8high) phenotype . So far, few pioneering studies have been carried out. Phenotyping of ASFV-specific IFN-γ-producing cells in pigs after immunization with BeninΔMGF and OURT88/3 have been fully characterized [59,82]. In both studies, the authors observed that the source of IFN-γ in both immunized groups originated from different subsets of CD8+ T cells, mainly cytotoxic (CD8high) T cells, particularly double-positive memory (CD4+CD8+) T cells, suggesting a possible role of these cells in protection. Moreover, they highlighted the importance of other IFN-γ-secreting cells in these mechanisms, such as γδ-T cells or NK cells [59,82]. Goatley et al.  observed how increased numbers of specific IFN-γ+ cells evaluated after the inoculation with the attenuated isolate OURT88/3 in both inbred and outbred pigs did not serve as a protection indicator against challenge with the virulent isolate OURT88/1, and suggested a possible role of antibodies in protection against homologous virulent isolates. However, differences in the cellular responses (mainly of CD8+ T cells) in outbred pigs that survived a second challenge with the genotype II virulent isolate Georgia 2007/1 were observed . In a recent work, both flow cytometry and gene expression analysis were employed to characterize ASFV-specific cytokine-producing cells in domestic pigs immunized with BA71ΔCD2, a candidate vaccine that conferred dose-dependent cross-protection against direct-contact challenge with pigs infected with the genotype II virulent isolate Georgia2007/1. Researchers reported that intracellular cytokine staining of PBMC from BA71ΔCD2-immunized pigs, stimulated ex vivo with BA71ΔCD2, showed elevated percentages of ASFV-specific IFNγ- and TNF-producing CD4+CD8+ T cells, a phenotype characteristic of polyfunctional memory T cells . In the same work, researchers employed bulk and single-cell transcriptomics to characterize immune responses against the deleted mutant BA71ΔCD2. Transcriptomics carried out on PBMC showed that BA71ΔCD2 triggered ASFV-specific activation of Th1 and cytotoxic T cells, concomitant with a rapid IFNγ-dependent triggering of an inflammatory response characterized by TNF-producing macrophages .
CXCL8 is the chemokine whose circulating levels have been most studied during in vivo ASFV infections, although with conflicting findings. On the contrary, there is a paucity of information regarding ASFV modulation of circulating levels of other CXCL chemokines, as reported in Table 4. CXCL8 is broadly known as a potent neutrophil chemoattractant, able to promote the recruitment of neutrophils and other granulocytes to the site of infection . In addition, this chemokine triggers neutrophil degranulation and enhancements in their phagocytic functions [10,92]. CXCL8 is released mainly by macrophages, considered its main source, but also by epithelial cells, endothelial cells and airway smooth muscle cells [10,93].
G-CSF is the major hematopoietic growth factor involved in the control of neutrophil development and supports the maintenance of steady-state neutrophil levels in vivo . In addition, this cytokine enhances several neutrophil effector functions in response to bacterial infection, such as superoxide anion generation and the release of arachidonic acid, as well as the production of leukocyte alkaline phosphatase and myeloperoxidase . In the clinic, G-CSF has been applied in the treatment of various forms of both congenital and acquired neutropenia . 350c69d7ab