Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.

Phenotypic effects of mutations observed in the neuraminidase of human origin H5N1 influenza A viruses.

Global spread and regional endemicity of H5Nx Goose/Guangdong avian influenza viruses (AIV) pose a continuous threat for poultry production and zoonotic, potentially pre-pandemic, transmission to humans. Little is known about the role of mutations in the viral neuraminidase (NA) that accompanied bird-to-human transmission to support AIV infection of mammals. Here, after detailed analysis of the NA sequence of human H5N1 viruses, we studied the role of A46D, L204M, S319F and S430G mutations in virus fitness in vitro and in vivo. Although H5N1 AIV carrying avian- or human-like NAs had similar replication efficiency in avian cells, human-like NA enhanced virus replication in human airway epithelia. The L204M substitution consistently reduced NA activity of H5N1 and nine other influenza viruses carrying NA of groups 1 and 2, indicating a universal effect. Compared to the avian ancestor, human-like H5N1 virus has less NA incorporated in the virion, reduced levels of viral NA RNA replication and NA expression. We also demonstrate increased accumulation of NA at the plasma membrane, reduced virus release and enhanced cell-to-cell spread. Furthermore, NA mutations increased virus binding to human-type receptors. While not affecting high virulence of H5N1 in chickens, the studied NA mutations modulated virulence and replication of H5N1 AIV in mice and to a lesser extent in ferrets. Together, mutations in the NA of human H5N1 viruses play different roles in infection of mammals without affecting virulence or transmission in chickens. These results are important to understand the genetic determinants for replication of AIV in mammals and should assist in the prediction of AIV with zoonotic potential.

The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens.

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2's effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.

HPLC-MS/MS-based quantification of human monoclonal antibodies targeting SARS-CoV-2 in the presence of endogenous SARS-CoV-2 antibodies in human serum.

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC-MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC-MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC-MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC-MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC-MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals.

Pyrroloquinoline Quinone Aza-Crown Ether Complexes as Biomimetics for Lanthanide and Calcium Dependent Alcohol Dehydrogenases*.

Understanding the role of metal ions in biology can lead to the development of new catalysts for several industrially important transformations. Lanthanides are the most recent group of metal ions that have been shown to be important in biology, that is, in quinone-dependent methanol dehydrogenases (MDH). Here we evaluate a literature-known pyrroloquinoline quinone (PQQ) and 1-aza-15-crown-5 based ligand platform as scaffold for Ca2+ , Ba2+ , La3+ and Lu3+ biomimetics of MDH and we evaluate the importance of ligand design, charge, size, counterions and base for the alcohol oxidation reaction using NMR spectroscopy. In addition, we report a new straightforward synthetic route (3 steps instead of 11 and 33 % instead of 0.6 % yield) for biomimetic ligands based on PQQ. We show that when studying biomimetics for MDH, larger metal ions and those with lower charge in this case promote the dehydrogenation reaction more effectively and that this is likely an effect of the ligand design which must be considered when studying biomimetics. To gain more information on the structures and impact of counterions of the complexes, we performed collision induced dissociation (CID) experiments and observe that the nitrates are more tightly bound than the triflates. To resolve the structure of the complexes in the gas phase we combined DFT-calculations and ion mobility measurements (IMS). Furthermore, we characterized the obtained complexes and reaction mixtures using Electron Paramagnetic Resonance (EPR) spectroscopy and show the presence of a small amount of quinone-based radical.

Elucidating essential kinases of endothelin signalling by logic modelling of phosphoproteomics data.

Endothelins (EDN) are peptide hormones that activate a GPCR signalling system and contribute to several diseases, including hypertension and cancer. Current knowledge about EDN signalling is fragmentary, and no systems level understanding is available. We investigated phosphoproteomic changes caused by endothelin B receptor (ENDRB) activation in the melanoma cell lines UACC257 and A2058 and built an integrated model of EDNRB signalling from the phosphoproteomics data. More than 5,000 unique phosphopeptides were quantified. EDN induced quantitative changes in more than 800 phosphopeptides, which were all strictly dependent on EDNRB. Activated kinases were identified based on high confidence EDN target sites and validated by Western blot. The data were combined with prior knowledge to construct the first comprehensive logic model of EDN signalling. Among the kinases predicted by the signalling model, AKT, JNK, PKC and AMP could be functionally linked to EDN-induced cell migration. The model contributes to the system-level understanding of the mechanisms underlying the pleiotropic effects of EDN signalling and supports the rational selection of kinase inhibitors for combination treatments with EDN receptor antagonists.

The Epoxyeicosatrienoic Acid Pathway Enhances Hepatic Insulin Signaling and is Repressed in Insulin-Resistant Mouse Liver.

Although it is widely accepted that ectopic lipid accumulation in the liver is associated with hepatic insulin resistance, the underlying molecular mechanisms have not been well characterized.Here we employed time resolved quantitative proteomic profiling of mice fed a high fat diet to determine which pathways were affected during the transition of the liver to an insulin-resistant state. We identified several metabolic pathways underlying altered protein expression. In order to test the functional impact of a critical subset of these alterations, we focused on the epoxyeicosatrienoic acid (EET) eicosanoid pathway, whose deregulation coincided with the onset of hepatic insulin resistance. These results suggested that EETs may be positive modulators of hepatic insulin signaling. Analyzing EET activity in primary hepatocytes, we found that EETs enhance insulin signaling on the level of Akt. In contrast, EETs did not influence insulin receptor or insulin receptor substrate-1 phosphorylation. This effect was mediated through the eicosanoids, as overexpression of the deregulated enzymes in absence of arachidonic acid had no impact on insulin signaling. The stimulation of insulin signaling by EETs and depression of the pathway in insulin resistant liver suggest a likely role in hepatic insulin resistance. Our findings support therapeutic potential for inhibiting EET degradation.

Acriflavine Is More than It Seems: Resolving Optical Properties of Multiple Isomeric Constituents at 5 K.

Ion mobility spectrometry at room temperature was combined with vibrationally resolved electronic spectroscopy of mass-selected ions at 5 K to study the well-known cationic fluorophore acriflavine. One- and two-color photodepletion action spectra recorded in gas-phase (by helium tagging) as well as dispersed fluorescence spectra obtained in neon matrix (after soft-landing deposition) indicate that the primary cation mass electrosprayed from solution comprises two isomers with different optical properties. Theory at the TD-DFT level allowed full spectral assignment. The results have implications for the preparation of novel thin film photonic materials by low-energy ion beam deposition.

Apoe, Mbl2, and Psp plasma protein levels correlate with diabetic phenotype in NZO mice--an optimized rapid workflow for SRM-based quantification.

Male New Zealand Obese (NZO) mice progress through pathophysiological stages similar to humans developing obesity-associated type 2 diabetes (T2D). The current challenge is to establish quantitative proteomics from small plasma sample amounts. We established an analytical workflow that facilitates a reproducible depletion of high-abundance proteins, has high throughput applicability, and allows absolute quantification of proteins from mouse plasma samples by LC-SRM-MS. The ProteoMiner equalizing technology was adjusted to the small sample amount, and reproducibility of the identifications was monitored by spike proteins. Based on the label-free relative quantification of proteins in depleted plasma of a test set of NZO mice, assays for potential candidates were designed for the setup of a targeted selected reaction monitoring (SRM) approach and absolute quantification. We could demonstrate that apolipoprotein E (Apoe), mannose-binding lectin 2 (Mbl2), and parotid secretory protein (Psp) are present at significantly different quantities in depleted plasma of diabetic NZO mice compared to non-diabetic controls using AQUA peptides. Quantification was validated for Mbl2 using the ELISA technology on non-depleted plasma. We conclude that the depletion technique is applicable to restricted sample amounts and suitable for the identification of T2D signatures in plasma.

Tissue-based proteomics reveals FXYD3, S100A11 and GSTM3 as novel markers for regional lymph node metastasis in colon cancer.

Regional lymph node metastasis negatively affects prognosis in colon cancer patients. The molecular processes leading to regional lymph node metastasis are only partially understood and proteomic markers for metastasis are still scarce. Therefore, a tissue-based proteomic approach was undertaken for identifying proteins associated with regional lymph node metastasis. Two complementary tissue-based proteomic methods have been employed. MALDI imaging was used for identifying small proteins (≤25 kDa) in situ and label-free quantitative proteomics was used for identifying larger proteins. A tissue cohort comprising primary colon tumours without metastasis (UICC II, pN0, n = 21) and with lymph node metastasis (UICC III, pN2, n = 33) was analysed. Subsequent validation of identified proteins was done by immunohistochemical staining on an independent tissue cohort consisting of primary colon tumour specimens (n = 168). MALDI imaging yielded ten discriminating m/z species, and label-free quantitative proteomics 28 proteins. Two MALDI imaging-derived candidate proteins (FXYD3 and S100A11) and one from the label-free quantitative proteomics (GSTM3) were validated on the independent tissue cohort. All three markers correlated significantly with regional lymph node metastasis: FXYD3 (p = 0.0110), S100A11 (p = 0.0071), and GSTM3 (p = 0.0173). FXYD3 and S100A11 were more highly expressed in UICC II patient tumour tissues. GSTM3 was more highly expressed in UICC III patient tumour tissues. By our tissue-based proteomic approach, we could identify a large panel of proteins which are associated with regional lymph node metastasis and which have not been described so far. Here we show that novel markers for regional lymph metastasis can be identified by MALDI imaging or label-free quantitative proteomics and subsequently validated on an independent tissue cohort.

Vibrationally Resolved Absorption, Fluorescence, and Preresonance Raman Spectroscopy of Isolated Pyronin Y Cation at 5 K.

Exploring how charge-changing affects the photoluminescence of small organic dyes presents challenges. Here, helium tagging photodissociation (PD) action spectroscopy in the gas phase and dispersed laser-induced fluorescence (DF) spectroscopy in the solid Ne matrix are used to compare the intrinsic photophysical properties of pyronin Y cation [PY]+ and its one electron-reduced neutral radical [PY]• at 5 K. Whereas the cation shows efficient visible photoluminescence, no emission from the neutral, in line with theoretical predictions, was detected. B3LYP/aug-cc-pVDZ calculations based on the TD-DFT/FCHT method allow for unambiguous assignment of recorded vibrationally resolved absorption and emission spectra. Surprisingly, our experimental sensitivity was high enough to also observe electronic preresonance Raman (ePR-Raman) spectra of [PY]+, with a significant efficiency factor (EF). These characteristics of the [PY]•/[PY]+ pair suggest that appropriately functionalized derivatives may open new perspectives in the area of in vivo bioimagining microscopy and find applications in various sophisticated stimulated-Raman spectroscopies.

Functional and spatial proteomics profiling reveals intra- and intercellular signaling crosstalk in colorectal cancer.

Precision oncology approaches for patients with colorectal cancer (CRC) continue to lag behind other solid cancers. Functional precision oncology-a strategy that is based on perturbing primary tumor cells from cancer patients-could provide a road forward to personalize treatment. We extend this paradigm to measuring proteome activity landscapes by acquiring quantitative phosphoproteomic data from patient-derived organoids (PDOs). We show that kinase inhibitors induce inhibitor- and patient-specific off-target effects and pathway crosstalk. Reconstruction of the kinase networks revealed that the signaling rewiring is modestly affected by mutations. We show non-genetic heterogeneity of the PDOs and upregulation of stemness and differentiation genes by kinase inhibitors. Using imaging mass-cytometry-based profiling of the primary tumors, we characterize the tumor microenvironment (TME) and determine spatial heterocellular crosstalk and tumor-immune cell interactions. Collectively, we provide a framework for inferring tumor cell intrinsic signaling and external signaling from the TME to inform precision (immuno-) oncology in CRC.

Dynamic modulation of intrinsic functional connectivity by transcranial direct current stimulation.

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique capable of modulating cortical excitability and thereby influencing behavior and learning. Recent evidence suggests that bilateral tDCS over both primary sensorimotor cortices (SM1) yields more prominent effects on motor performance in both healthy subjects and chronic stroke patients than unilateral tDCS over SM1. To better characterize the underlying neural mechanisms of this effect, we aimed to explore changes in resting-state functional connectivity during both stimulation types. In a randomized single-blind crossover design, 12 healthy subjects underwent functional magnetic resonance imaging at rest before, during, and after 20 min of unilateral, bilateral, and sham tDCS stimulation over SM1. Eigenvector centrality mapping (ECM) was used to investigate tDCS-induced changes in functional connectivity patterns across the whole brain. Uni- and bilateral tDCS over SM1 resulted in functional connectivity changes in widespread brain areas compared with sham stimulation both during and after stimulation. Whereas bilateral tDCS predominantly modulated changes in primary and secondary motor as well as prefrontal regions, unilateral tDCS affected prefrontal, parietal, and cerebellar areas. No direct effect was seen under the stimulating electrode in the unilateral condition. The time course of changes in functional connectivity in the respective brain areas was nonlinear and temporally dispersed. These findings provide evidence toward a network-based understanding regarding the underpinnings of specific tDCS interventions.

Two-dimensional peptide separation improving sensitivity of selected reaction monitoring-based quantitative proteomics in mouse liver tissue: comparing off-gel electrophoresis and strong cation exchange chromatography.

Protein expression analysis is one of the most powerful tools to further the understanding of biological systems. Progress in the field of mass spectrometry has shifted focus from gel-based approaches to the upcoming LC-selected reaction monitoring (SRM) technique which combines high technical accuracy with absolute quantification of proteins and the capability for high-throughput analyses. Due to these properties, LC-SRM has the potential to become the foundation for biomarker analysis, targeted hypothesis driven proteomic studies and contribute to the field of systems biology. While the performance of LC-SRM applied to samples from various bodily fluids, particularly plasma, and microorganisms has been extensively investigated, there is only little experience with its application to animal tissue samples. Here, we show that a conventional one-dimensional LC-SRM workflow applied to mouse liver tissue suffers from a shortcoming in terms of sensitivity for lower abundance proteins. This problem could be solved through the extension of the standard workflow by an additional dimension of separation at the peptide level prior to online LC-SRM. For this purpose, we used off-gel electrophoresis (OGE) which is also shown to outperform strong cation exchange (SCX) in terms of resolution, gain of signal intensity, and predictability of separation. The extension of the SRM workflow by a high resolving peptide separation technique is an ideal combination as it allows the addition of stable isotope standards directly after trytic digestion and will increase the dynamic range of protein abundances amenable by SRM in animal tissue.

Adaptive Cellular Immunity against African Swine Fever Virus Infections.

African swine fever virus (ASFV) remains a threat to global pig populations. Infections with ASFV lead to a hemorrhagic disease with up to 100% lethality in Eurasian domestic and wild pigs. Although myeloid cells are the main target cells for ASFV, T cell responses are impacted by the infection as well. The complex responses remain not well understood, and, consequently, there is no commercially available vaccine. Here, we review the current knowledge about the induction of antiviral T cell responses by cells of the myeloid lineage, as well as T cell responses in infected animals, recent efforts in vaccine research, and T cell epitopes present in ASFV.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ T cells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ T cells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

Ion Mobility Studies of Pyrroloquinoline Quinone Aza-Crown Ether-Lanthanide Complexes.

Lanthanide-dependent enzymes and their biomimetic complexes have arisen as an interesting target of research in the past decade. These enzymes, specifically, pyrroloquinoline quinone (PQQ)-bearing methanol dehydrogenases, efficiently convert alcohols to the respective aldehydes. To rationally design bioinspired alcohol dehydrogenation catalysts, it is imperative to understand the species involved in catalysis. However, given the extremely flexible coordination sphere of lanthanides, it is often difficult to assess the number and nature of the active species. Here, we show how such questions can be addressed by using a combination of ion mobility spectrometry, mass spectrometry, and quantum-chemical calculations to study the test systems PQQ and lanthanide-PQQ-crown ether ligand complexes. Specifically, we determine the gas-phase structures of [PQQH2]-, [PQQH2+H2O]-, [PQQH2+MeOH]-, [PQQ-15c5+H]+, and [PQQ-15c5+Ln+NO3]2+ (Ln = La to Lu, except Pm). In the latter case, a trend to smaller collision cross sections across the lanthanide series is clearly observable, in line with the well-known lanthanide contraction. We hope that in the future such investigations will help to guide the design and understanding of lanthanide-based biomimetic complexes optimized for catalytic function.

Clinical, neuropathological, and immunological short- and long-term feature of a mouse model mimicking human herpes virus encephalitis.

Herpes simplex encephalitis (HSE) is one of the most serious diseases of the nervous system in humans. However, its pathogenesis is still only poorly understood. Although several mouse models of predominantly herpes simplex virus 1 (HSV-1) infections mimic different crucial aspects of HSE, central questions remain unanswered. They comprise the specific temporofrontal tropism, viral spread within the central nervous system (CNS), as well as potential molecular and immunological barriers that drive virus into latency while only rarely resulting in severe HSE. We have recently proposed an alternative mouse model by using a pseudorabies virus (PrV) mutant that more faithfully represents the striking features of human HSE: temporofrontal meningoencephalitis with few severely, but generally only moderately to subclinically affected mice as well as characteristic behavioral abnormalities. Here, we characterized this animal model using 6- to 8-week-old female CD-1 mice in more detail. Long-term investigation over 6 months consistently revealed a biphasic course of infection accompanied by recurring clinical signs including behavioral alterations and mainly mild meningoencephalitis restricted to the temporal and frontal lobes. By histopathological and immunological analyses, we followed the kinetics and spatial distribution of inflammatory lesions as well as the underlying cytokine expression in the CNS over 21 days within the acute phase of infection. Affecting the temporal lobes, the inflammatory infiltrate was composed of lymphocytes and macrophages showing a predominantly lymphocytic shift 15 days after infection. A strong increase was observed in cytokines CXCL10, CCL2, CCL5, and CXCL1 recruiting inflammatory cells to the CNS. Unlike the majority of infected mice, strongly affected animals demonstrated extensive temporal lobe edema, which is typically present in severe human HSE cases. In summary, these results support the validity of our animal model for in-depth investigation of HSE pathogenesis.

A semisynthetic glycoconjugate provides expanded cross-serotype protection against Streptococcus pneumoniae.

Streptococcus pneumoniae (S. pneumoniae)infections are the leading cause of child mortality globally. Currentvaccines fail to induceaprotective immune response towards a conserved part of the pathogen,resulting in newserotypescausing disease. Therefore, new vaccinestrategies are urgently needed.Described is atwo-pronged approach combiningS. pneumoniaeproteins, pneumolysin (Ply) and pneumococcal surface protein A (PspA),with aprecisely defined synthetic oligosaccharide,wherebythe carrier protein actsas a serotype-independent antigen to provideadditional protection. Proof of concept in mice and swine modelsrevealed thatthe conjugatesinhibited colonization of the nasopharynx, decreased the bacterial load and reduced disease severity in the bacteria challenge model. Immunization of piglets provided the first evidence for the immunogenicity and protective potential of synthetic glycoconjugate vaccine in a large animal model.Acombination of synthetic oligosaccharides with proteins from the target pathogen opens the path to create broadly cross-protective ("universal") pneumococcal vaccines.

T-cell responses in domestic pigs and wild boar upon infection with the moderately virulent African swine fever virus strain 'Estonia2014'.

Infection with African swine fever virus (ASFV) causes a highly lethal haemorrhagic disease in domestic and Eurasian wild pigs. Thus, it is a major threat to pig populations worldwide and a cause of substantial economic losses. Recently, less virulent ASFV strains emerged naturally, which showed higher experimental virulence in wild boar than in domestic pigs. The reason for this difference in disease progression and outcome is unclear but likely involves different immunological responses. Unfortunately, besides the importance of CD8α+ lymphocytes, little is known about the immune responses against ASFV in suids. Against this background, we used a multicolour flow cytometry platform to investigate the T-cell responses in wild boar and domestic pigs after infection with the moderately virulent ASFV strain 'Estonia2014' in two independent trials. CD4- /CD8α+ and CD4+ /CD8α+ αß T-cell frequencies increased in both subspecies in various tissues, but CD8α+ γδ T cells differentiated and responded in wild boar only. Proliferation in CD8α+ T cells was found 10 days post infectionem only. Frequencies of T-bet+ T cells increased in wild boar but not in domestic pigs. Of note, we found a considerable loss of perforin expression in cytotoxic T cells, 5 and 7 dpi. Both subspecies established a regulatory T-cell response 10 dpi. In domestic pigs, we show increasing levels of ICOS+ and CD8α+ invariant Natural Killer T cells. These disparities in T-cell responses might explain some of the differences in disease progression in wild boar and domestic pigs and should pave the way for future studies.