CD4+CD25+ T regulatory cells from FIV+ cats induce a unique anergic profile in CD8+ lymphocyte targets

Abstract Background Using the FIV model, we reported previously that CD4+CD25+ T regulatory (Treg) cells from FIV+ cats are constitutively activated and suppress CD4+CD25- and CD8+ T cell immune responses. In an effort to further explore Treg-mediated suppression, we asked whether Treg cells induce anergy through the alteration of production of cyclins, cyclin-dependent kinases and their inhibitors. Results Lymphocytes were obtained from control or FIV+ cats and sorted by FACS into CD4+CD25+ and CD8+ populations. Following co-culture with CD4+CD25+ cells, CD8+ targets were examined by Western blot for changes in cyclins D3, E and A, retinoblastoma (Rb) protein, as well as the cyclin dependent kinase inhibitor p21cip1. Following co-culture with CD4+CD25+cells, we observed up-regulation of p21cip1 and cyclin E, with down-regulation of cyclin D3, in CD8+ cells from FIV+ cats. As expected, CD8+ targets from control cats were quiescent with little up-regulation of p21cip1 and cyclin E. There was also a lack of Rb phosphorylation in CD8+ targets consistent with late G1 cell cycle arrest. Further, IL-2 mRNA was down regulated in CD8+ cells after co-culture with CD4+CD25+ Treg cells. Following CD4+CD25+ co-culture, CD8+ targets from FIV+ cats also had increased Foxp3 mRNA expression; however, these CD8+Foxp3+ cells did not exhibit suppressor function. Conclusions Collectively, these data suggest that CD4+CD25+ Treg cells from FIV+ cats induce CD8+ anergy by disruption of normal G1 to S cell cycle progression.</p

Modified-live feline calicivirus vaccination elicits cellular immunity against a current feline calicivirus field strain in an experimental geline challenge study

Feline calicivirus (FCV) is a common cat virus associated with oral ulcerations and virulent-systemic disease. Efficacious FCV vaccines protect against severe disease but not against infection. The high genetic diversity of FCV poses a challenge in vaccine design. Protection against FCV has been related to humoral and cellular immunity; the latter has not been studied in detail. This study investigates the cellular and humoral immune response of specified pathogen-free (SPF) cats after modified-live FCV F9 vaccinations and two heterologous FCV challenges by the analysis of lymphocyte subsets, cytokine mRNA transcription levels, interferon (IFN)-γ release assays in peripheral blood mononuclear cells (PBMCs), anti-FCV antibodies, and neutralisation activity. Vaccinated cats developed a Th1 cytokine response after vaccination. Vaccination resulted in antibodies with neutralising activity against the vaccine but not the challenge viruses. Remarkably, IFN-γ-releasing PBMCs were detected in vaccinated cats upon stimulation with the vaccine strain and the first heterologous FCV challenge strain. After the first experimental infection, the mRNA transcription levels of perforin, granzyme B, INF-γ, and antiviral factor MX1 and the number of IFN-γ-releasing PBMCs when stimulated with the first challenge virus were higher in vaccinated cats compared to control cats. The first FCV challenge induced crossneutralising antibodies in all cats against the second challenge virus. Before the second challenge, vaccinated cats had a higher number of IFN-γ-releasing PBMCs when stimulated with the second challenge virus than control cats. After the second FCV challenge, there were less significant differences detected between the groups regarding lymphocyte subsets and cytokine mRNA transcription levels. In conclusion, modified-live FCV vaccination induced cellular but not humoral crossimmunity in SPF cats; innate immune mechanisms, secretory and membranolytic pathways, and IFN-γ-releasing PBMCs seem to be important in the host immune defence against FCV

Precision medicine in cats:novel niemann-pick type C1 diagnosed by whole-genome sequencing

State-of-the-art health care includes genome sequencing of the patient to identify genetic variants that contribute to either the cause of their malady or variants that can be targeted to improve treatment. The goal was to introduce state-of-the-art health care to cats using genomics and a precision medicine approach. To test the feasibility of a precision medicine approach in domestic cats, a single cat that presented to the University of Missouri, Veterinary Health Center with an undiagnosed neurologic disease was whole-genome sequenced. The DNA variants from the cat were compared to the DNA variant database produced by the 99 Lives Cat Genome Sequencing Consortium. Approximately 25× genomic coverage was produced for the cat. A predicted p.H441P missense mutation was identified in NPC1, the gene causing Niemann-Pick type C1 on cat chromosome D3.47456793 caused by an adenine-to-cytosine transversion, c.1322A>C. The cat was homozygous for the variant. The variant was not identified in any other 73 domestic and 9 wild felids in the sequence database or 190 additionally genotyped cats of various breeds. The successful effort suggested precision medicine is feasible for cats and other undiagnosed cats may benefit from a genomic analysis approach. The 99 Lives DNA variant database was sufficient but would benefit from additional cat sequences. Other cats with the mutation may be identified and could be introduced as a new biomedical model for NPC1. A genetic test could eliminate the disease variant from the population

A deletion in GDF7 is associated with a heritable forebrain commissural malformation concurrent with ventriculomegaly and interhemispheric cysts in cats

Publisher Copyright: © 2020 by the authors.An inherited neurologic syndrome in a family of mixed-breed Oriental cats has been characterized as forebrain commissural malformation, concurrent with ventriculomegaly and interhemispheric cysts. However, the genetic basis for this autosomal recessive syndrome in cats is unknown. Forty-three cats were genotyped on the Illumina Infinium Feline 63K iSelect DNA Array and used for analyses. Genome-wide association studies, including a sib-transmission disequilibrium test and a case-control association analysis, and homozygosity mapping, identified a critical region on cat chromosome A3. Short-read whole genome sequencing was completed for a cat trio segregating with the syndrome. A homozygous 7 bp deletion in growth differentiation factor 7 (GDF7) (c.221_227delGCCGCGC [p.Arg74Profs]) was identified in affected cats, by comparison to the 99 Lives Cat variant dataset, validated using Sanger sequencing and genotyped by fragment analyses. This variant was not identified in 192 unaffected cats in the 99 Lives dataset. The variant segregated concordantly in an extended pedigree. In mice, GDF7 mRNA is expressed within the roof plate when commissural axons initiate ventrally-directed growth. This finding emphasized the importance of GDF7 in the neurodevelopmental process in the mammalian brain. A genetic test can be developed for use by cat breeders to eradicate this variant.Peer reviewe

Werewolf, there wolf : Variants in hairless associated with hypotrichia and roaning in the lykoi cat breed

Publisher Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland.A variety of cat breeds have been developed via novelty selection on aesthetic, dermatological traits, such as coat colors and fur types. A recently developed breed, the lykoi (a.k.a. werewolf cat), was bred from cats with a sparse hair coat with roaning, implying full color and all white hairs. The lykoi phenotype is a form of hypotrichia, presenting as a significant reduction in the average numbers of follicles per hair follicle group as compared to domestic shorthair cats, a mild to severe perifollicular to mural lymphocytic infiltration in 77% of observed hair follicle groups, and the follicles are often miniaturized, dilated, and dysplastic. Whole genome sequencing was conducted on a single lykoi cat that was a cross between two independently ascertained lineages. Comparison to the 99 Lives dataset of 194 non‐lykoi cats suggested two variants in the cat homolog for Hairless (HR) (HR lysine demethylase and nuclear receptor corepressor) as candidate causal gene variants. The lykoi cat was a compound heterozygote for two loss of function variants in HR, an exon 3 c.1255_1256dupGT (chrB1:36040783), which should produce a stop codon at amino acid 420 (p.Gln420Serfs*100) and, an exon 18 c.3389insGACA (chrB1:36051555), which should produce a stop codon at amino acid position 1130 (p.Ser1130Argfs*29). Ascertainment of 14 additional cats from founder lineages from Canada, France and different areas of the USA identified four additional loss of function HR variants likely causing the highly similar phenotypic hair coat across the diverse cats. The novel variants in HR for cat hypotrichia can now be established between minor differences in the phenotypic presentations.Peer reviewe

Epigenetic Modulation of CD8+ T Cell Function in Lentivirus Infections: A Review

CD8+ T cells are critical for controlling viremia during human immunodeficiency virus (HIV) infection. These cells produce cytolytic factors and antiviral cytokines that eliminate virally- infected cells. During the chronic phase of HIV infection, CD8+ T cells progressively lose their proliferative capacity and antiviral functions. These dysfunctional cells are unable to clear the productively infected and reactivated cells, representing a roadblock in HIV cure. Therefore, mechanisms to understand CD8+ T cell dysfunction and strategies to boost CD8+ T cell function need to be investigated. Using the feline immunodeficiency virus (FIV) model for lentiviral persistence, we have demonstrated that CD8+ T cells exhibit epigenetic changes such as DNA demethylation during the course of infection as compared to uninfected cats. We have also demonstrated that lentivirus-activated CD4+CD25+ T regulatory cells induce forkhead box P3 (Foxp3) expression in virus-specific CD8+ T cell targets, which binds the interleukin (IL)-2, tumor necrosis factor (TNF)-&alpha;, and interferon (IFN)-&gamma; promoters in these CD8+ T cells. Finally, we have reported that epigenetic modulation reduces Foxp3 binding to these promoter regions. This review compares and contrasts our current understanding of CD8+ T cell epigenetics and mechanisms of lymphocyte suppression during the course of lentiviral infection for two animal models, FIV and simian immunodeficiency virus (SIV)

Antibiotic Therapy Does Not Alter the Humoral Response to Vaccination for Porcine Circovirus 2 in Weaned Pigs

Recent reports suggest that antibiotic therapy may either reduce or enhance the immune response to various porcine vaccines. Based upon these findings, we asked if antibiotic therapy alters immune cell populations, as measured by flow cytometry and/or vaccine-specific humoral immunity, as measured by sample to positive (S/P) antibody ratios. Here, we investigated the immuno-modulatory effects of enrofloxacin, ceftiofur, and tulathromycin on the immune response to a Mycoplasma hyopneumoniae (M. hyopneumoniae) and porcine circovirus type 2 (PCV-2) combination vaccine in weaned pigs. Maternal antibody likely interfered with the induction of immunity to M. hyopneumoniae. Antibiotic administration did not affect immune cell populations, as assessed by flow cytometry and did not affect the induction of humoral immunity to PCV-2

Histone Modulation Blocks Treg-Induced Foxp3 Binding to the IL-2 Promoter of Virus-Specific CD8+ T Cells from Feline Immunodeficiency Virus-Infected Cats

CD8+ T cells are critical for controlling HIV infection. During the chronic phase of lentiviral infection, CD8+ T cells lose their proliferative capacity and exhibit impaired antiviral function. This loss of CD8+ T cell function is due, in part, to CD4+CD25+ T regulatory (Treg) cell-mediated suppression. Our research group has demonstrated that lentivirus-activated CD4+CD25+ Treg cells induce the repressive transcription factor forkhead box P3 (Foxp3) in autologous CD8+ T cells following co-culture. We have recently reported that Treg-induced Foxp3 binds the interleukin-2 (IL-2), interferon-&gamma; (IFN- &gamma;), and tumor necrosis factor-&alpha; (TNF-&alpha;) promoters in virus-specific CD8+ T cells. These data suggest an important role of Foxp3-mediated CD8+ T cell dysfunction in lentiviral infection. To elucidate the mechanism of this suppression, we previously reported that decreased methylation facilitates Foxp3 binding in mitogen-activated CD8+ T cells from feline immunodeficiency virus (FIV)-infected cats. We demonstrated the reduced binding of Foxp3 to the IL-2 promoter by increasing methylation of CD8+ T cells. In the studies presented here, we ask if another form of epigenetic modulation might alleviate Foxp3-mediated suppression in CD8+ T cells. We hypothesized that decreasing histone acetylation in virus-specific CD8+ T cells would decrease Treg-induced Foxp3 binding to the IL-2 promoter. Indeed, using anacardic acid (AA), a known histone acetyl transferase (HAT) inhibitor, we demonstrate a reduction in Foxp3 binding to the IL-2 promoter in virus-specific CD8+ T cells co-cultured with autologous Treg cells. These data identify a novel mechanism of Foxp3-mediated CD8+ T cell dysfunction during lentiviral infection

Micro-RNA 10a Is Increased in Feline T Regulatory Cells and Increases Foxp3 Protein Expression Following In Vitro Transfection

CD4+CD25+Foxp3+ T regulatory (Treg) cells are activated during the course of lentiviral infection and exhibit heightened suppressor function when compared to Treg cells from uninfected controls. Foxp3 is essential to Treg cell function and multiple studies have documented that lentivirus-activated Treg cells exhibit heightened Foxp3 expression when compared to Treg cells from uninfected controls. Our hypothesis was that lentivirus-induced micro-RNAs (miRNAs) contribute to heightened Treg cell suppressor function by stabilizing Foxp3 expression. We demonstrated that CD4+CD25+ T cells from both feline immunodeficiency virus infected (FIV+) cats and uninfected control cats exhibit increased miRNA 10a and 21 levels compared to autologous CD4+CD25− T cells but there was no difference in the levels of these miRNAs when Treg cells from FIV+ cats were compared to Treg cells from uninfected controls. Further, there was no increase in Foxp3 mRNA following transfection of miRNA 10a or 21 into a feline cell line. However, transfection with miRNA 10a resulted in increased Foxp3 protein expression