Sensitive detection of Foxp3 expression in bovine lymphocytes by flow cytometry

Gerner, Wilhelm; Stadler, Maria; Hammer, Sabine E.; Klein, D.; Saalmüller, Armin

doi:10.1016/j.vetimm.2010.07.009

Cited by 29 publications

(24 citation statements)

References 19 publications

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“…Therefore, we chose the mouse as a model, and compared two clones of anti-mouse Foxp3 antibodies and two Foxp3 staining buffer sets, which were the most frequently used in published studies in various species (Tsang et al, 2006;Izcue et al, 2008;Bolzer et al, 2009;Prochazkova et al, 2009;Gerner et al, 2010;Janikashvili et al, 2011;Lei et al, 2011;Pinheiro et al, 2011;Robbin et al, 2011;Rocchi et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Here, we chose C57BL/6 mice as a model, and compared two kinds of Foxp3 clones: MF23 from BD Pharmingen (San Diego, CA, USA) and FJK-16s. MF23 recognizes an epitope between the 1 and 87 amino acids in the N-terminal domain of the mouse Foxp3 protein, whereas FJK-16s maps the epitope to amino acids 75 and 125 of the mouse, rat, bovine (Gerner et al, 2010), porcine (Bolzer et al, 2009), canine (Pinheiro et al, 2011), ovine (Rocchi et al, 2011, and equine (Robbin et al, 2011) Foxp3 protein. Simultaneously, the Foxp3 buffer sets from the two vendors above were used in our system.…”

Section: Cd4mentioning

confidence: 99%

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Application and effects of mouse Foxp3 antibody and fixation/permeabilization buffer on the detection of CD4+ regulatory T cells in various mammal species

Sun

Lin²,

Zhang³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. CD4+ regulatory T lymphocytes (Treg cells) play a crucial role in maintaining the normal immune homeostasis. Foxp3, as a key marker for Treg cells, is widely used to identify Treg cells, not only in humans but also in other species, like mouse, porcine, ovine, and bovine. To detect reproducible Treg cells is important for evaluating the state of the immune system, and thus, it is necessary to optimize Foxp3 staining. Here, we present a comparative study of MF23 and FJK-16s clones of anti-mouse Foxp3 antibodies, used in combination with two different fixation/permeabilization buffers. For Foxp3 staining, the fixation/permeabilization buffer and Foxp3 antibody FJK-16s clone from eBioscience were better than those from BD Pharmingen, with the best fluorochrome PE. Moreover, when using the best combination, there was a highly significant positive correlation between CD25 + T cells + T cells. Therefore, the CD25 marker can be used as an alternative to the Foxp3 antibody. As FJK-16s is also applicable for detecting bovine, porcine, canine, ovine, and equine Foxp3 antibodies, these results will be helpful not only in quantifying the frequencies of mouse Treg cells, but also in accurately detecting Treg cells of the other species mentioned above by multicolor flow cytometry.

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Section: Discussionmentioning

confidence: 99%

Section: Cd4mentioning

confidence: 99%

Application and effects of mouse Foxp3 antibody and fixation/permeabilization buffer on the detection of CD4+ regulatory T cells in various mammal species

Sun

Lin²,

Zhang³

et al. 2013

Genet. Mol. Res.

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“…Four-color analysis of FoxP3-expressing CD4 T cells was performed by staining cells according to a modification of a previously described protocol (40). Briefly, PBMC or splenocytes were thawed, washed, and incubated with 15 mg/ml MAb CACT138A and CACT108A for 15 min.…”

Section: Expression and Purification Of Recombinant Proteinmentioning

confidence: 99%

Loss of Immunization-Induced Epitope-Specific CD4 T-Cell Response following Anaplasma marginale Infection Requires Presence of the T-Cell Epitope on the Pathogen and Is Not Associated with an Increase in Lymphocytes Expressing Known Regulatory Cell Phenotypes

Brown

Turse

Lawrence

et al. 2015

Clin. Vaccine Immunol.

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We have shown that in cattle previously immunized with outer membrane proteins, infection with Anaplasma marginale induces a functionally exhausted CD4 T-cell response to the A. marginale immunogen. Furthermore, T-cell responses following infection in nonimmunized cattle had a delayed onset and were sporadic and transient during persistent infection. The induction of an exhausted T-cell response following infection presumably facilitates pathogen persistence. In the current study, we hypothesized that the loss of epitope-specific T-cell responses requires the presence of the immunizing epitope on the pathogen, and T-cell dysfunction correlates with the appearance of regulatory T cells. In limited studies in cattle, regulatory T cells have been shown to belong to ␥␦ T-cell subsets rather than be CD4 T cells expressing forkhead box protein P3 (FoxP3). Cattle expressing the DRB3*1101 haplotype were immunized with a truncated A. marginale major surface protein (MSP) 1a that contains a DRB3*1101-restricted CD4 T-cell epitope, F2-5B. Cattle either remained unchallenged or were challenged with A. marginale bacteria that express the epitope or with A. marginale subsp. centrale that do not. Peripheral blood and spleen mononuclear cells were monitored for MSP1a epitope F2-5B-specfic T-cell proliferative responses and were stained for ␥␦ T-cell subsets or CD4 cells before and during infection. As hypothesized, the induction of T-cell exhaustion occurred only following infection with A. marginale, which did not correlate with an increase in either CD4؉ CD25 ؉ FoxP3 ؉ T cells or any ␥␦ T-cell subset examined.A naplasma marginale is a tick-borne intraerythrocytic rickettsial pathogen found in most temperate and tropical regions of the world and causes significant anemia and a mortality rate of up to 30% in naive cattle. Cattle that survive acute disease remain persistently infected for life with cyclic, but microscopically undetectable, levels of bacteremia that do not cause clinical disease (1). Of note, the antigen load in animals during acute and persistent infection is high, reaching 10 9 bacteria per ml of blood during acute infection and 10 7 bacteria per ml of blood in recurrent peaks during persistent infection (2). The mechanisms by which A. marginale is capable of persisting in the immunocompetent host have not been completely elucidated. A. marginale undergoes extensive antigenic variation in immunodominant and abundant major surface protein 2 (MSP2) and MSP3 by gene conversion of whole pseudogenes and segments of pseudogenes into a single expression site (3). Antigenic variation in MSP2, which is rich in T-and B-lymphocyte epitopes, allows the organism to escape specific adaptive immune responses and contributes to persistence (4-7).Our studies have shown that infection of A. marginale in cattle previously immunized with either native MSP2 or recombinant MSP1a resulted in a complete loss of functional CD4 ϩ T-cell responses to the specific immunogen beginning near the peak of acute infection (7,8). The T cells wer...

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“…Whether these cells were bona fide Tregs or activated Tcons, which in some assay systems are capable of mediating non-specific suppression [354,355], remains unclear. A number of subsequent studies presented indirect evidence for populations of CD4 + , CD8 + and γδ T cells with regulatory activity [355][356][357][358][359][360][361], but T cells expressing FOXP3 were first documented in 2009: thus, Seo and colleagues developed novel mAbs against bovine FOXP3, demonstrating that its expression was limited to CD4 + CD25 + T cells [362], while Gerner and colleagues documented cross-reactivity of the antimouse/rat Foxp3 mAb FJK-16s, showing that the majority of FOXP3 + T cells were CD4 + but that minor populations of CD8β + and γδ + FOXP3 + lymphocytes could also be identified [363]. However, the regulatory properties of the CD4 + CD25 high FOXP3 + population were called into question by work performed by Hoek and colleagues, who suggested that these cells were neither anergic nor suppressive; rather, WC1.1 + and WC1.2 + γδ T cells, and CD14 + monocytes, showed regulatory properties in their experiments, supported by the observation that these cells transcribed IL-10 [364].…”

Section: Regulatory T Cells In Other Domestic Animal Species -An Expamentioning

confidence: 99%

All creatures great and small: regulatory T cells in mice, humans, dogs and other domestic animal species

Garden

Pinheiro

Cunningham

2011

International Immunopharmacology

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Sensitive detection of Foxp3 expression in bovine lymphocytes by flow cytometry

Cited by 29 publications

References 19 publications

Application and effects of mouse Foxp3 antibody and fixation/permeabilization buffer on the detection of CD4+ regulatory T cells in various mammal species

Application and effects of mouse Foxp3 antibody and fixation/permeabilization buffer on the detection of CD4+ regulatory T cells in various mammal species

Loss of Immunization-Induced Epitope-Specific CD4 T-Cell Response following Anaplasma marginale Infection Requires Presence of the T-Cell Epitope on the Pathogen and Is Not Associated with an Increase in Lymphocytes Expressing Known Regulatory Cell Phenotypes

All creatures great and small: regulatory T cells in mice, humans, dogs and other domestic animal species

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