Ticks and tick-borne diseases from Mallorca Island, Spain.

Ixodid ticks are obligate blood-feeding arthropods and important vectors of pathogens. In Mallorca, almost no data on the tick fauna are available. Herein, we investigated ticks and tick-borne pathogens in ticks collected from dogs, a cat and humans in Mallorca as result of a citizen science project. A total of 91 ticks were received from German tourists and residents in Mallorca. Ticks were collected from March to October 2023 from dogs, cat and humans, morphologically and genetically identified and tested for pathogens by PCRs. Six tick species could be identified: Ixodes ricinus (n = 2), Ixodes ventalloi (n = 1), Hyalomma lusitanicum (n = 7), Hyalomma marginatum (n = 1), Rhipicephalus sanguineus s.l. (n = 71) and Rhipicephalus pusillus (n = 9). Rhipicephalus sanguineus s.l. adults were collected from dogs and four females from a cat and the 16S rDNA sequences identified it as Rh. sanguineus s.s. Hyalomma lusitanicum was collected from 1 human, 1 dog and 5 specimens were collected from the ground in the community of Santanyi, together with one H. marginatum male. This is the first report of Hyalomma marginatum in Mallorca. Both I. ricinus were collected from humans and I. ventalloi female was collected from a dog. All ticks tested negative for Anaplasma phagocytophilum, Coxiella spp., Francisella spp., and piroplasms. In 32/71 (45%) specimens of Rh. sanguineus s.s., Rickettsia spp. could be detected and in 18/32 (56.2%) sequenced tick DNAs R. massiliae was identified. Ixodes ventalloi female and both I. ricinus tested positive in the screening PCR, but the sequencing for the identification of the Rickettsia sp. failed.

Sensitivity of two SARS-CoV-2 variants with spike protein mutations to neutralising antibodies.

SARS-CoV-2 infections elicit a humoral immune response capable of neutralising the virus. However, multiple variants have emerged with mutations in the spike protein amongst others, the key target of neutralising antibodies. We evaluated the neutralising efficacy of 89 serum samples from patients, infected with SARS-CoV-2 in the beginning of 2020, against two virus variants isolated from acutely infected patients and harbouring spike protein mutations. One isolate was assigned to lineage B.1.351 (MUC-IMB-B.1.351) whilst the other (MUC-484) was isolated from an immunocompromised patient, sharing some but not all mutations with B.1.351 and representing a transitional variant. Both variants showed a significant reduction in neutralisation sensitivity compared to wild-type SARS-CoV-2 with MUC-IMB-B.1.351 being almost completely resistant to neutralisation. The observed reduction in neutralising activity of wild-type-specific antibodies against both variants suggests that individual mutations in the spike protein are sufficient to confer a potent escape from the humoral immune response. In addition, the effect of escape mutations seems to accumulate, so that more heavily mutated variants show a greater loss of sensitivity to neutralisation up to complete insensitivity as observed for MUC-IMB-B.1.351. From a clinical point of view, this might affect the efficacy of (monoclonal) antibody treatment of patients with prolonged infections as well as patients infected with variants other than the donor. At the same, this could also negatively influence the efficacy of current vaccines (as they are based on wild-type spike protein) emphasising the need to thoroughly surveil the emergence and distribution of variants and adapt vaccines and therapeutics accordingly.

A headache with surprising outcome: first case of brucellosis caused by Brucella suis biovar 1 in Germany.

In July 2018, brucellosis was diagnosed in a German patient without a travel history to regions endemic for Brucella. Microbiological analysis, including whole-genome sequencing, revealed Brucella suis biovar 1 as the etiologic agent. Core-genome-based multilocus sequence-typing analysis placed the isolate in close proximity to strains originating from Argentina. Notably, despite a strong IgM response, the patient did not develop Brucella-specific IgG antibodies during infection. Here, we describe the clinical course of infection, the extensive epidemiological investigations, and discuss possible routes of transmission.

CD4(+) FoxP3(+) regulatory T cells suppress fatal T helper 2 cell immunity during pulmonary fungal infection.

The opportunistic fungal pathogen Cryptococcus neoformans causes lung inflammation and fatal meningitis in immunocompromised patients. Regulatory T (Treg) cells play an important role in controlling immunity and homeostasis. However, their functional role during fungal infection is largely unknown. In this study, we investigated the role of Treg cells during experimental murine pulmonary C. neoformans infection. We show that the number of CD4(+) FoxP3(+) Treg cells in the lung increases significantly within the first 4 weeks after intranasal infection of BALB/c wild-type mice. To define the function of Treg cells we used DEREG mice allowing selective depletion of CD4(+) FoxP3(+) Treg cells by application of diphtheria toxin. In Treg cell-depleted mice, stronger pulmonary allergic inflammation with enhanced mucus production and pronounced eosinophilia, increased IgE production, and elevated fungal lung burden were found. This was accompanied by higher frequencies of GATA-3(+) T helper (Th) 2 cells with elevated capacity to produce interleukin (IL)-4, IL-5, and IL-13. In contrast, only a mild increase in the Th1-associated immune response unrelated to the fungal infection was observed. In conclusion, the data demonstrate that during fungal infection pulmonary Treg cells are induced and preferentially suppress Th2 cells thereby mediating enhanced fungal control.

Analysis of asthma patients for cryptococcal seroreactivity in an urban German area.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised patients but is also able to asymptomatically infect immunocompetent individuals. C. neoformans is found ubiquitously especially in urban areas where it is spread by pigeons, and fungal exposure may predispose for asthma development already at an early age, as soon as confronted with pigeon droppings. In the study presented here, we investigated the presence of specific immunoglobulin G (IgG) against C. neoformans in sera from patients suffering from asthma in comparison to a healthy control cohort, accrued from the Leipzig Research Centre for Civilization Diseases (LIFE). For serological analysis we developed a flow cytometry (FACS) based assay specific for an acapsular strain of C. neoformans to comprehensively analyze different cryptococcal serotypes. Compared with the non-asthmatic cohort, asthmatics exhibited, as expected, an elevated level of total serum immunoglobulin E (IgE), whereas the IgG seroreactivity against C. neoformans was not significantly different among the two groups (P = .118). Nevertheless, there was a trend toward increased Cryptococcus-specific IgG antibodies in the serum of asthmatics. Additionally, in male asthmatics an increased IgG-mediated seroreactivity compared to female asthmatics was found. This points to a higher prevalence of subclinical C. neoformans infection in male asthmatics and may support the hypothesis of C. neoformans as a risk factor for the development of asthma in urban areas.

Comparative Analysis of Vaccine-Induced Neutralizing Antibodies against the Alpha, Beta, Delta, and Omicron Variants of SARS-CoV-2.

The SARS-CoV-2 virus has infected more than 660 million people and caused nearly seven million deaths worldwide. During the pandemic, a number of SARS-CoV-2 vaccines were rapidly developed, and several are currently licensed for use in Europe. However, the optimization of vaccination regimens is still ongoing, particularly with regard to booster vaccinations. At the same time, the emergence of new virus variants poses an ongoing challenge to vaccine efficacy. In this study, we focused on a comparative analysis of the neutralization capacity of vaccine-induced antibodies against four different variants of concern (i.e., Alpha, Beta, Delta, and Omicron) after two and three doses of COVID-19 vaccine. We were able to show that both two (prime/boost) and three (prime/boost/boost) vaccinations elicit highly variable levels of neutralizing antibodies. In addition, we did not observe a significant difference in antibody levels after two and three vaccinations. We also observed a significant decrease in the neutralization susceptibility of all but one SARS-CoV-2 variants to vaccine-induced antibodies. In contrast, a SARS-CoV-2 breakthrough infection between the second and third vaccination results in overall higher levels of neutralizing antibodies with a concomitant improved neutralization of all virus variants. Titer levels remained highly variable across the cohort but a common trend was observed. This may be due to the fact that at the time of this study, all licensed vaccines were still based exclusively on wild-type SARS-CoV-2, whereas infections were caused by virus variants. Overall, our data demonstrate the importance of (booster) vaccinations, but at the same time emphasize the need for the continued adaptation of vaccines to induce a protective immune response against virus variants in order to be prepared for future (seasonal) SARS-CoV-2 outbreaks.

Analysis of immune responses in patients with CLL after heterologous COVID-19 vaccination.

Patients with chronic lymphocytic leukemia (CLL) treated with B-cell pathway inhibitors and anti-CD20 antibodies exhibit low humoral response rates following SARS-CoV-2 vaccination. To investigate this observation, a prospective single-institution study was conducted comparing peripheral blood mononuclear cell transcriptional response with antibody and T-cell response rates following heterologous BNT162b2/ChAdOx1 vaccination of 15 patients with CLL/small lymphocytic lymphoma (SLL). Two-dose antibody response rate was 40%, increasing to 53% after booster. Patients on Bruton tyrosine kinase inhibitor (BTKi) and venetoclax ± anti-CD20 antibody within 12 months of vaccination responded inferiorly compared with those under BTKi alone. The 2-dose-T-cell response rate was 80%, which increased to 93% after the booster dose. Key transcriptional findings were that interferon-mediated signaling activation including activation of the JAK-STAT pathway generally occurred within days of vaccination, but was independent from the magnitude of the antibody response. Increasing counts of IGHV genes were associated with B-cell reconstitution and improved humoral response rate in the vaccinated patients. T-cell responses in patients with CLL appeared independent of treatment status, whereas higher humoral response rate was associated with BTKi treatment and B-cell reconstitution. Boosting was particularly effective when intrinsic immune status was improved by CLL treatment. Limitations included studying a relatively small cohort, with different treatments and vaccination schedules.

Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides.

Bioactive peptides are key molecules in health and medicine. Deep learning holds a big promise for the discovery and design of bioactive peptides. Yet, suitable experimental approaches are required to validate candidates in high throughput and at low cost. Here, we established a cell-free protein synthesis (CFPS) pipeline for the rapid and inexpensive production of antimicrobial peptides (AMPs) directly from DNA templates. To validate our platform, we used deep learning to design thousands of AMPs de novo. Using computational methods, we prioritized 500 candidates that we produced and screened with our CFPS pipeline. We identified 30 functional AMPs, which we characterized further through molecular dynamics simulations, antimicrobial activity and toxicity. Notably, six de novo-AMPs feature broad-spectrum activity against multidrug-resistant pathogens and do not develop bacterial resistance. Our work demonstrates the potential of CFPS for high throughput and low-cost production and testing of bioactive peptides within less than 24 h.

Absence of in vitro innate immunomodulation by insect-derived short proline-rich antimicrobial peptides points to direct antibacterial action in vivo.

Some antimicrobial peptides (AMPs) have been described to exert immunomodulatory effects, which may contribute to their in vivo antibacterial activity. Very recently, we could show that novel oncocin and apidaecin derivatives are potently antibacterially active in vivo. Therefore, we studied oncocin and apidaecin derivatives for their effects on murine dendritic cells (DC) and macrophages and compared them with well-known immunomodulatory activities of murine cathelicidin-related antimicrobial peptide (CRAMP). To characterize the immunomodulatory activity of the peptides on key cells of the innate immune system, we stimulated murine DC and macrophages with the oncocin and apidaecin derivatives alone, or in combination with lipopolysaccharide (LPS). We analyzed the secretion of pro-inflammatory cytokines, the expression of surface activation markers, and the chemotactic activity of the AMPs. In contrast to LPS, none of the oncocin and apidaecin derivatives alone has an influence on cytokine or surface marker expression by DC and macrophages. Furthermore, the tested oncocin and apidaecin derivatives do not modulate the immune response after LPS stimulation, whereas CRAMP shows a reduction of the LPS-mediated immune response as expected. All peptides tested are not chemotactic for DC. Together, lack of in vitro immunomodulatory effects by oncocin and apidaecin derivatives on key cells of the innate murine immune system suggests that their potent in vivo antibacterial activity relies on a direct antibacterial effect. This will simplify further pharmaceutical investigation and development of insect peptides as therapeutic compounds against bacterial infections.

Evaluation of Two Rapid Lateral Flow Tests and Two Surrogate ELISAs for the Detection of SARS-CoV-2 Specific Neutralizing Antibodies.

As vaccination against SARS-CoV-2 progresses rapidly around the world, reliable detection of SARS-CoV-2 specific neutralizing antibodies (NAb) has become an indispensable component of serological diagnostics. We evaluated the performance of four commercially available tests, i.e. two lateral flow assays (Coris BioConcept COVID-19 Sero NP/RBD and Concile InfectCheck COVID-19 NAb) and two surrogate ELISA (sELISA) tests (EUROIMMUN SARS-CoV-2 NeutraLISA and AdipoGen SARS-CoV-2 Neutralizing Antibodies Detection Kit) in comparison with an in-house SARS-CoV-2 micro neutralization test as reference. A total of 334 sera were tested, including 30 samples collected prior to the emergence of SARS-CoV-2, 128 sera from convalescent patients as well as 176 sera from partially or fully vaccinated individuals. The overall sensitivity of LFAs differed and was 71.6% for the Coris and 98.4% for the Concile. In contrast, overall sensitivity of the NeutraLISA was 86 and 98% for the AdipoGen. All test showed the highest sensitivity when testing samples from fully vaccinated individuals with both sELISA achieving 100% sensitivity. Overall specificity was 89.3% for the Coris and only 58.3% for the Concile. Similarly, significant differences were observed for both sELISA, with an overall specificity of 82.1% for the NeutraLISA and only 54.8% for the AdipoGen. All tests showed a 100% specificity when testing negative control samples while specificities were lowest when testing samples from only partially vaccinated individuals.

Side-By-Side Evaluation of Three Commercial ELISAs for the Quantification of SARS-CoV-2 IgG Antibodies.

In December 2020, WHO presented the first international standard (WHO IS) for anti-SARS-CoV-2 immunoglobulin. This standard is intended to serve as a reference reagent against which serological tests can be calibrated, thus creating better comparability of results between different tests, laboratories, etc. Here, we have examined three different commercial ELISA kits for the quantification of SARS-CoV-2 IgG antibodies, namely the Anti-SARS-CoV-2 QuantiVac ELISA (IgG) (Euroimmun, Lübeck, Germany), the SERION ELISA agile (Institut Virion Serion, Würzburg, Germany), and the COVID-19 quantitative IgG ELISA (DeMediTec Diagnostics, Kiel, Germany). According to the manufacturers, all are calibrated against the WHO IS and can provide results in either international units (IU) (DeMediTec) or arbitrary antibody units (BAU) per milliliter (Euroimmun, Virion Serion), which are numerically identical, according to the WHO. A total of 50 serum samples from vaccinated individuals were tested side by side and according to the manufacturer's instructions. We compared the test results of all three assays with each other to assess comparability and with a quantitative in-house virus neutralization test (micro-NT). In summary, our data are consistent with other studies published on this topic that tested similar assays from different manufacturers. Overall, the agreement between quantitative ELISAs is variable and cannot be used interchangeably despite calibration against a standard. Therefore, interpretation of results must still be individualized and tailored to each case. More importantly, our results highlight that quantitative ELISAs in their current form cannot replace neutralization tests.

Genetic diversity and spatial distribution of Burkholderia mallei by core genome-based multilocus sequence typing analysis.

Burkholderia mallei is the etiological agent of glanders, a highly contagious and often fatal disease in equids. Due to the high genetic clonality of B. mallei, high-resolution typing assays are necessary to differentiate between individual strains. Here we report on the development and validation of a robust and reproducible core genome-based Multi Locus Sequence Typing Assay (cgMLST) for B. mallei, which is based on 3328 gene targets and enables high-resolution typing at the strain level. The assay was validated using a set of 120 B. mallei genomes from public databases and 23 newly sequenced outbreak strains from in-house strain collections. In this cgMLST analysis, strains from different geographic regions were clearly distinguished by at least 70 allele differences, allowing spatial clustering while closely related and epidemiologically related strains were separated by only zero to three alleles. Neither the different sequencing technologies nor the assembly strategies had an influence on the cgMLST results. The developed cgMLST is highly robust, reproducible and can be used for outbreak investigations, source tracking and molecular characterization of new B. mallei isolates.

Comprehensive analysis of immune responses in CLL patients after heterologous COVID-19 vaccination.

Patients with chronic lymphocytic leukemia (CLL) treated with B-cell pathway inhibitors and anti-CD20 antibodies exhibit low humoral response rate (RR) following SARS-CoV-2 vaccination. To investigate the relationship between the initial transcriptional response to vaccination with ensuing B and T cell immune responses, we performed a comprehensive immune transcriptome analysis flanked by antibody and T cell assays in peripheral blood prospectively collected from 15 CLL/SLL patients vaccinated with heterologous BNT162b2/ChAdOx1 with follow up at a single institution. The two-dose antibody RR was 40% increasing to 53% after booster. Patients on BTKi, venetoclax ± anti-CD20 antibody within 12 months of vaccination responded less well than those under BTKi alone. The two-dose T cell RR was 80% increasing to 93% after booster. Transcriptome studies revealed that seven patients showed interferon-mediated signaling activation within 2 days and one at 7 days after vaccination. Increasing counts of COVID-19 specific IGHV genes correlated with B-cell reconstitution and improved humoral RR. T cell responses in CLL patients appeared after vaccination regardless of treatment status. A higher humoral RR was associated with BTKi treatment and B-cell reconstitution. Boosting was particularly effective when intrinsic immune status was improved by CLL-treatment.

Comparison of two commercial surrogate ELISAs to detect a neutralising antibody response to SARS-CoV-2.

INTRODUCTION: Reliable methods for the detection of SARS-CoV-2 neutralising antibodies (NAbs) are essential for the evaluation of vaccine candidates and for the selection of convalescent plasma donors. Virus neutralisation tests (NTs) are the gold standard for the detection and quantification of NAbs, but they are complex and require BSL3 facilities. In contrast, surrogate enzyme-linked immunosorbent assays (sELISA) offer the possibility of high-throughput testing under standard laboratory safety conditions. In this study, we investigated two commercial sELISA kits (GenScript, AdipoGen) designed for the detection of SARS-CoV-2 NAbs. METHODS: 276 plasma samples were screened using commercial IgG-ELISA and NAbs titres were determined by micro-neutralisation test (micro-NT). In addition, all samples were tested in both sELISA. Sensitivity and specificity for both sELISA were determined in comparison to the micro-NT results. RESULTS: 57 % of the samples were SARS-CoV-2 NAb positive in micro-NT, while 43 % tested negative. Comparison with micro-NT results showed a sensitivity of 98.2 % and a specificity of 69.5 % for the GenScript ELISA. The AdipoGen ELISA had a sensitivity of 83.5 % and a specificity of 97.8 %. False negative results were obtained mainly on samples with low NAbs titres. CONCLUSION: Both sELISA were able to qualitatively detect NAbs in plasma samples. Sensitivity and specificity differed between sELISA with GenScript superior in sensitivity and AdipoGen superior in specificity. Both sELISA were unable to quantify NAbs, thus neither of them can completely replace conventional NTs. However, in a two-step diagnostic algorithm, AdipoGen could potentially replace NT as a subsequent confirmatory test due to its high specificity but only in settings where no exact NAbs quantification is needed.

Emerging SARS-CoV-2 variant B.1.1.7 reduces neutralisation activity of antibodies against wild-type SARS-CoV-2.

Spike-specific antibodies contribute significantly to the neutralising activity against SARS-CoV-2 and are important for the therapeutic effect of convalescent plasma. B.1.1.7 is a recently emerged variant of SARS-CoV-2 that has several mutations in the gene encoding for the spike-protein. To assess the potential effect these mutations could have on the neutralising efficacy of antibodies, we evaluated 96 serum samples from convalescent plasma donors collected before the first occurrence of B.1.1.7 and tested their neutralising effect on wild-type SARS-CoV-2 and B.1.1.7. We found that B.1.1.7 is more resistant to neutralisation by convalescent plasma from patients infected with wild-type SARS-CoV-2 with an overall decrease in neutralising activity of 47.7%. Thus, the neutralising effect of convalescent plasma should be determined against the major circulating virus clades whenever possible to ensure the best possible therapeutic effect.

SARS-CoV-2 Seroprevalence among Health Care Workers-A Voluntary Screening Study in a Regional Medical Center in Southern Germany.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is associated with a potentially severe clinical manifestation, coronavirus disease 2019 (COVID-19), and currently poses a worldwide challenge. Health care workers (HCWs) are at the forefront of any health care system and thus especially at risk for SARS-CoV-2 infection due to their potentially frequent and close contact with patients suffering from COVID-19. Serum samples from 198 HCWs with direct patient contact of a regional medical center and several outpatient facilities were collected during the early phase of the pandemic (April 2020) and tested for SARS-CoV-2-specific antibodies. Commercially available IgA- and IgG-specific ELISAs were used as screening technique, followed by an in-house neutralization assay for confirmation. Neutralizing SARS-CoV-2-specific antibodies were detected in seven of 198 (3.5%) tested HCWs. There was no significant difference in seroprevalence between the regional medical center (3.4%) and the outpatient institution (5%). The overall seroprevalence of neutralizing SARS-CoV-2-specific antibodies in HCWs in both a large regional medical center and a small outpatient institution was low (3.5%) at the beginning of April 2020. The findings may indicate that the timely implemented preventive measures (strict hygiene protocols, personal protective equipment) were effective to protect from transmission of an airborne virus when only limited information on the pathogen was available.

Conventional bone marrow-derived dendritic cells contribute to toll-like receptor-independent production of alpha/beta interferon in response to inactivated parapoxvirus ovis.

Parapoxvirus ovis (PPVO) is a member of the Poxviridae family and belongs to the genus Parapoxvirus. It displays only limited homology with orthopoxviruses and has some molecular features such as an unusual high GC content distinct from orthopoxviruses. Inactivated PPVO (iPPVO) displays strong immunostimulatory capacities mediating antiviral activity in vivo. The role of dendritic cells (DC) and the pattern recognition receptors and signaling requirements responsible for immunostimulation by iPPVO are unknown. We demonstrate here that bone marrow-derived plasmacytoid DC (BM-pDC) and bone marrow-derived conventional DC (BM-cDC) secrete alpha/beta interferon (IFN-alpha/beta) in response to iPPVO. Furthermore, iPPVO induces tumor necrosis factor alpha (TNF-alpha) and interleukin-12/23p40 (IL-12/23p40) release and major histocompatibility complex class II (MHC-II), MHC-I, and CD86 upregulation by bone marrow-derived DC (BMDC). After engulfment, iPPVO is located in endosomal compartments and in the cytosol of BMDC. iPPVO elicits IFN-alpha/beta by Toll-like receptor (TLR)-independent pathways in BM-cDC, since IFN-alpha/beta release does not require myeloid differentiation primary response gene 88 (MyD88) or TIR-domain containing adaptor protein inducing interferon (TRIF). In contrast, iPPVO-induced TNF-alpha release and enhanced expression of MHC-I and CD86 but not of MHC-II by BMDC chiefly requires MyD88 but not TLR2 or TLR4. Induction of IFN-alpha by iPPVO in BM-cDC occurred in the absence of IFN regulatory factor 3 (IRF3) but required the presence of IRF7, whereas iPPVO-triggered IFN-beta production required the presence of either IRF7 or IRF3. These results provide the first evidence that iPPVO mediates its immunostimulatory properties by TLR-independent and TLR-dependent pathways and demonstrate an important role of cDC for IFN-alpha/beta production.

PCR Based Prevalence Study of Francisella Tularensis in Kharkiv, Dnipropetrovsk, and Mykolaiv Oblasts during 2015-2018.

INTRODUCTION: Tularaemia is a zoonotic disease caused by the gram-negative bacterium Francisella tularensis, which is endemic to Ukraine. The aim of this work was to provide screening of different field samples (rodent tails, ticks, pellets, water, and hay) to obtain an actual picture of the tularaemia epizootic situation in the Kharkiv, Dnipropetrovsk, and Mykolaiv oblasts. MATERIAL AND METHODS: Samples were collected using the flag method (for ticks) and break-back traps (for rodents). Also, hay, water and owl pellets were collected for study. The F. tularensis genetic material in samples was detected using a 16S qPCR. RESULTS: It was found that in Kharkiv oblast, 23% of collected samples were positive for F. tularensis, in Dnipropetrovsk oblast 1.9%, and in Mykolaiv oblast 0.4%. CONCLUSION: Among the sample types, 34.7% of ticks, 1.8% of rodents, and 36.4% of pellets were positive for F. tularensis. The most frequent carriers of F. tularensis were the D. reticulatus and I. ricinus ticks (74.2% and 29.3%, respectively, of positive results).

Distinct Features of Canine Non-conventional CD4-CD8α- Double-Negative TCRαß+ vs. TCRγδ+ T Cells.

The role of conventional TCRαß+CD4+ or TCRαß+CD8α+ single-positive (sp) T lymphocytes in adaptive immunity is well-recognized. However, non-conventional T cells expressing TCRαß or TCRγδ but lacking CD4 and CD8α expression [i.e., CD4-CD8α- double-negative (dn) T cells] are thought to play a role at the interface between the innate and adaptive immune system. Dn T cells are frequent in swine, cattle or sheep and predominantly express TCRγδ. In contrast, TCRγδ+ T cells are rare in dogs. In this study, we identified a high proportion of canine dn T cells in the TCRαß+ T cell population of PBMC, lymphatic and non-lymphatic organs. In PBMC, the frequency of this T cell subpopulation made up one third of the frequency of TCRαß+CD4+ sp, and almost half of the frequency of TCRαß+CD8α+ sp T cells (i.e., ~15% of all TCRαß+ T cells). Among TCRαß+CD4-CD8α- dn T cells of PBMC and tissues, FoxP3+ cells were identified indicating regulatory potential of this T cell subset. 80% of peripheral blood FoxP3+TCRαß+CD4-CD8α- dn T cells co-expressed CD25, and, interestingly, also the FoxP3-negative TCRαß+CD4-CD8α- dn T cells comprised ~34% CD25+ cells. Some of the FoxP3-positive TCRαß+CD4-CD8α- dn T cells co-expressed GATA-3 suggesting stable function of regulatory T cells. The frequency of GATA-3 expression by FoxP3-TCRαß+CD4-CD8α- dn T cells was even higher as compared with TCRαß+CD4+ sp T cells (20.6% vs. 11.9%). Albeit lacking FoxP3 and CD25 expression, TCRγδ+CD4-CD8α- dn T cells also expressed substantial proportions of GATA-3. In addition, TCRαß+CD4-CD8α- dn T cells produced IFN-γ and IL-17A upon stimulation. T-bet and granzyme B were only weakly expressed by both dn T cell subsets. In conclusion, this study identifies two dn T cell subsets in the dog: (i) a large (~7.5% in Peyer's patches, ~15% in lung) population of TCRαß+CD4-CD8α- dn T cells with subpopulations thereof showing an activated phenotype, high expression of FoxP3 or GATA-3 as well as production of IFN-γ or IL-17A and (ii) a small TCRγδ+CD4-CD8α- dn T cell subset also expressing GATA-3 without production of IFN-γ or IL-17A. It will be exciting to unravel the function of each subset during immune homeostasis and diseases of dogs.

Canine tissue-associated CD4+CD8α+ double-positive T cells are an activated T cell subpopulation with heterogeneous functional potential.

Canine CD4+CD8α+ double-positive (dp) T cells of peripheral blood are a unique effector memory T cell subpopulation characterized by an increased expression of activation markers in comparison with conventional CD4+ or CD8α+ single-positive (sp) T cells. In this study, we investigated CD4+CD8α+ dp T cells in secondary lymphatic organs (i.e. mesenteric and tracheobronchial lymph nodes, spleen, Peyer's patches) and non-lymphatic tissues (i.e. lung and epithelium of the small intestine) within a homogeneous group of healthy Beagle dogs by multi-color flow cytometry. The aim of this systematic analysis was to identify the tissue-specific localization and characteristics of this distinct T cell subpopulation. Our results revealed a mature extrathymic CD1a-CD4+CD8α+ dp T cell population in all analyzed organs, with highest frequencies within Peyer's patches. Constitutive expression of the activation marker CD25 is a feature of many CD4+CD8α+ dp T cells independent of their localization and points to an effector phenotype. A proportion of lymph node CD4+CD8α+ dp T cells is FoxP3+ indicating regulatory potential. Within the intestinal environment, the cytotoxic marker granzyme B is expressed by CD4+CD8α+ dp intraepithelial lymphocytes. In addition, a fraction of CD4+CD8α+ dp intraepithelial lymphocytes and of mesenteric lymph node CD4+CD8α+ dp T cells is TCRγδ+. However, the main T cell receptor of all tissue-associated CD4+CD8α+ dp T cells could be identified as TCRαß. Interestingly, the majority of the CD4+CD8α+ dp T cell subpopulation expresses the unconventional CD8αα homodimer, in contrast to CD8α+ sp T cells, and CD4+CD8α+ dp thymocytes which are mainly CD8αß+. The presented data provide the basis for a functional analysis of tissue-specific CD4+CD8α+ dp T cells to elucidate their role in health and disease of dogs.