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Both tumour cells and infiltrating T-cells in equine sarcoids express FOXP3 associated with an immune-supressed cytokine microenvironment.

Wilson AD, Hicks C - Vet. Res. (2016)

Bottom Line: Transcription of IL17, which has been shown to have a regulatory function in human papillomavirus-associated tumours, was also elevated in equine sarcoids compared to spleen.In contrast, the levels of mRNA transcripts for effector T cell cytokines IL2, IL4 and interferon-gamma (IFNγ) were not elevated in sarcoids compared to healthy skin or spleen.Similarly neither interferon-alpha (IFNα), interferon-beta (IFNβ) nor IL12 family members were elevated in sarcoids compared to normal skin.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Sciences, University of Bristol, Langford, Bristol, BS40 5DU, UK. doug.wilson@bris.ac.uk.

ABSTRACT
Bovine papillomavirus (BPV) infections of equine species have a central role in the aetiology of equine sarcoids; a common benign skin tumour of horses, zebras and donkeys. Within the lesions, all of the early papillomavirus genes are expressed and promote the excessive replication of fibroblasts which characterise these tumours. Equine sarcoids differ from BPV induced fibro-papillomas of cattle (the natural host of BPV), in that they do not produce high amounts of virus particles, do not usually regress spontaneously and do not sero-convert to BPV; features which suggest that affected horses lack an effective anti-viral immune response to BPV. Equine sarcoids contain large numbers of CD4+ CD8+ dual positive T-cells which uniformly express FOXP3, the key transcription factor of regulatory T-cells, and FOXP3 is also expressed within the BPV infected fibroblasts. Compared to healthy skin, sarcoids showed increased mRNA transcription for FOXP3 and the regulatory cytokine TGFβ. Transcription of IL17, which has been shown to have a regulatory function in human papillomavirus-associated tumours, was also elevated in equine sarcoids compared to spleen. In contrast, the levels of mRNA transcripts for effector T cell cytokines IL2, IL4 and interferon-gamma (IFNγ) were not elevated in sarcoids compared to healthy skin or spleen. Similarly neither interferon-alpha (IFNα), interferon-beta (IFNβ) nor IL12 family members were elevated in sarcoids compared to normal skin. We suggest that the regulatory cytokine micro-environment within sarcoids enables the persistence of the lesions by preventing an effective anti-viral immune response.

No MeSH data available.


Related in: MedlinePlus

FOXP3 expression on sarcoid infiltrating T-cells. A Cryostat sections of sarcoid tissue stained with rabbit anti-FOXP3 (ab 10563) and donkey anti rabbit texas-red with blue DAPI nuclear counterstain. B Same section stained using both mouse monoclonal anti-CD4 and monoclonal anti-CD8α with goat anti mouse FITC conjugate. C Combined image showing CD4 and CD8 stained T-cells and non T-cells all express FOXP3. D Specificity control slide stained with normal rabbit immunoglobulin and isotype matched control mouse monoclonal antibody, DAPI counterstain. E Cryostat sections of sarcoid tissue stained with rat anti-FOXP3 (FJK-16s) and donkey anti rat FITC with blue DAPI nuclear counterstain. F Same section stained using mouse anti equine CD4 with goat anti mouse TRITC conjugate. G Combined image showing both CD4 stained T-cells and non T-cells express FOXP3. H Combined image at higher power with CD4-negative FOXP3 positive cells highlighted (arrow).
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Fig3: FOXP3 expression on sarcoid infiltrating T-cells. A Cryostat sections of sarcoid tissue stained with rabbit anti-FOXP3 (ab 10563) and donkey anti rabbit texas-red with blue DAPI nuclear counterstain. B Same section stained using both mouse monoclonal anti-CD4 and monoclonal anti-CD8α with goat anti mouse FITC conjugate. C Combined image showing CD4 and CD8 stained T-cells and non T-cells all express FOXP3. D Specificity control slide stained with normal rabbit immunoglobulin and isotype matched control mouse monoclonal antibody, DAPI counterstain. E Cryostat sections of sarcoid tissue stained with rat anti-FOXP3 (FJK-16s) and donkey anti rat FITC with blue DAPI nuclear counterstain. F Same section stained using mouse anti equine CD4 with goat anti mouse TRITC conjugate. G Combined image showing both CD4 stained T-cells and non T-cells express FOXP3. H Combined image at higher power with CD4-negative FOXP3 positive cells highlighted (arrow).

Mentions: Formalin fixed tissue samples were stained using rabbit anti-CD3, biotinylated donkey anti-rabbit and avidin biotin peroxidase conjugate as previously described [9] FOXP3 expression was detected using either 1:400 affinity purified polyclonal rabbit anti-FOXP3 (ab10563 abCam, UK) which maps to an epitope at the extreme C-terminal of human and equine FOXP3 or monoclonal rat anti mouse Foxp3 (FJK-16 s eBioscience) which maps to an epitope between residues 75-125 in the region of exon2 of murine Foxp3. For both antibodies heat treatment in Tris–EDTA pH 9.0 was used for antigen recovery. For immunofluorescence the primary antibodies used were 3H5 monoclonal mouse anti-BPV E2 (1 μg/mL) [33], and rabbit anti-CD3 (diluted 1:400; A0452 Dako), 1:400 polyclonal rabbit anti-FOXP3 (ab10563 abCam UK), 1 μg/mL monoclonal mouse anti-equine CD4 (HB61A), CD8 (73/6.9.1), MHC I (H58A) and MHC II H34A) (Kingfisher Biotec). Control sections were incubated with normal rabbit immunoglobulins (diluted 1:400; XO936 Dako), or an isotype matched monoclonal antibody in place of the primary antibody. Secondary antibodies were affinity-purified Fab fragments of donkey FITC-anti-mouse or FITC anti-rat and texas red anti-rabbit immunoglobulins absorbed against multiple species serum proteins; (Jackson Immunoresearch) used at 1:1000 dilution. For dual staining with primary mouse monoclonal antibodies, FITC or TRITC conjugated to goat anti-mouse sub-class specific polyclonal antibodies were used at a dilution of 1:100 (Southern-Biotech). For immunofluorescence staining 6 μm thick frozen sections were cut and mounted onto slides coated with chrome alum gelatine, air dried, then fixed in 100% acetone for 5 min at room temperature. Individually wrapped slides were stored at −70 °C prior to use. Sections were equilibrated to room temperature before unwrapping, then re-hydrated in PBS and non-specific protein binding blocked by incubation in PBS with 10% horse serum. Sections were stained with combinations of rabbit and mouse primary antibodies diluted in PBS with 10% horse serum, washed in PBS, followed by the appropriate florescent conjugate antibodies. Slides were mounted in Vectashield® DAPI aqueous mounting medium (Vector Laboratories). Images were taken using a Leica DC350RX camera and Leica Qfluoro software (Leica microsystems UK Ltd. Miltom Keynes, UK). For each antibody combination, control images, in which the primary antibody is replaced by 1:400 rabbit immunoglobulin or 1 μg/mL isotype matched mouse and rat monoclonal antibodies were taken using the same camera settings and showed no specific staining (see Figure 3D for representative image).


Both tumour cells and infiltrating T-cells in equine sarcoids express FOXP3 associated with an immune-supressed cytokine microenvironment.

Wilson AD, Hicks C - Vet. Res. (2016)

FOXP3 expression on sarcoid infiltrating T-cells. A Cryostat sections of sarcoid tissue stained with rabbit anti-FOXP3 (ab 10563) and donkey anti rabbit texas-red with blue DAPI nuclear counterstain. B Same section stained using both mouse monoclonal anti-CD4 and monoclonal anti-CD8α with goat anti mouse FITC conjugate. C Combined image showing CD4 and CD8 stained T-cells and non T-cells all express FOXP3. D Specificity control slide stained with normal rabbit immunoglobulin and isotype matched control mouse monoclonal antibody, DAPI counterstain. E Cryostat sections of sarcoid tissue stained with rat anti-FOXP3 (FJK-16s) and donkey anti rat FITC with blue DAPI nuclear counterstain. F Same section stained using mouse anti equine CD4 with goat anti mouse TRITC conjugate. G Combined image showing both CD4 stained T-cells and non T-cells express FOXP3. H Combined image at higher power with CD4-negative FOXP3 positive cells highlighted (arrow).
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Related In: Results  -  Collection

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Show All Figures
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Fig3: FOXP3 expression on sarcoid infiltrating T-cells. A Cryostat sections of sarcoid tissue stained with rabbit anti-FOXP3 (ab 10563) and donkey anti rabbit texas-red with blue DAPI nuclear counterstain. B Same section stained using both mouse monoclonal anti-CD4 and monoclonal anti-CD8α with goat anti mouse FITC conjugate. C Combined image showing CD4 and CD8 stained T-cells and non T-cells all express FOXP3. D Specificity control slide stained with normal rabbit immunoglobulin and isotype matched control mouse monoclonal antibody, DAPI counterstain. E Cryostat sections of sarcoid tissue stained with rat anti-FOXP3 (FJK-16s) and donkey anti rat FITC with blue DAPI nuclear counterstain. F Same section stained using mouse anti equine CD4 with goat anti mouse TRITC conjugate. G Combined image showing both CD4 stained T-cells and non T-cells express FOXP3. H Combined image at higher power with CD4-negative FOXP3 positive cells highlighted (arrow).
Mentions: Formalin fixed tissue samples were stained using rabbit anti-CD3, biotinylated donkey anti-rabbit and avidin biotin peroxidase conjugate as previously described [9] FOXP3 expression was detected using either 1:400 affinity purified polyclonal rabbit anti-FOXP3 (ab10563 abCam, UK) which maps to an epitope at the extreme C-terminal of human and equine FOXP3 or monoclonal rat anti mouse Foxp3 (FJK-16 s eBioscience) which maps to an epitope between residues 75-125 in the region of exon2 of murine Foxp3. For both antibodies heat treatment in Tris–EDTA pH 9.0 was used for antigen recovery. For immunofluorescence the primary antibodies used were 3H5 monoclonal mouse anti-BPV E2 (1 μg/mL) [33], and rabbit anti-CD3 (diluted 1:400; A0452 Dako), 1:400 polyclonal rabbit anti-FOXP3 (ab10563 abCam UK), 1 μg/mL monoclonal mouse anti-equine CD4 (HB61A), CD8 (73/6.9.1), MHC I (H58A) and MHC II H34A) (Kingfisher Biotec). Control sections were incubated with normal rabbit immunoglobulins (diluted 1:400; XO936 Dako), or an isotype matched monoclonal antibody in place of the primary antibody. Secondary antibodies were affinity-purified Fab fragments of donkey FITC-anti-mouse or FITC anti-rat and texas red anti-rabbit immunoglobulins absorbed against multiple species serum proteins; (Jackson Immunoresearch) used at 1:1000 dilution. For dual staining with primary mouse monoclonal antibodies, FITC or TRITC conjugated to goat anti-mouse sub-class specific polyclonal antibodies were used at a dilution of 1:100 (Southern-Biotech). For immunofluorescence staining 6 μm thick frozen sections were cut and mounted onto slides coated with chrome alum gelatine, air dried, then fixed in 100% acetone for 5 min at room temperature. Individually wrapped slides were stored at −70 °C prior to use. Sections were equilibrated to room temperature before unwrapping, then re-hydrated in PBS and non-specific protein binding blocked by incubation in PBS with 10% horse serum. Sections were stained with combinations of rabbit and mouse primary antibodies diluted in PBS with 10% horse serum, washed in PBS, followed by the appropriate florescent conjugate antibodies. Slides were mounted in Vectashield® DAPI aqueous mounting medium (Vector Laboratories). Images were taken using a Leica DC350RX camera and Leica Qfluoro software (Leica microsystems UK Ltd. Miltom Keynes, UK). For each antibody combination, control images, in which the primary antibody is replaced by 1:400 rabbit immunoglobulin or 1 μg/mL isotype matched mouse and rat monoclonal antibodies were taken using the same camera settings and showed no specific staining (see Figure 3D for representative image).

Bottom Line: Transcription of IL17, which has been shown to have a regulatory function in human papillomavirus-associated tumours, was also elevated in equine sarcoids compared to spleen.In contrast, the levels of mRNA transcripts for effector T cell cytokines IL2, IL4 and interferon-gamma (IFNγ) were not elevated in sarcoids compared to healthy skin or spleen.Similarly neither interferon-alpha (IFNα), interferon-beta (IFNβ) nor IL12 family members were elevated in sarcoids compared to normal skin.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Sciences, University of Bristol, Langford, Bristol, BS40 5DU, UK. doug.wilson@bris.ac.uk.

ABSTRACT
Bovine papillomavirus (BPV) infections of equine species have a central role in the aetiology of equine sarcoids; a common benign skin tumour of horses, zebras and donkeys. Within the lesions, all of the early papillomavirus genes are expressed and promote the excessive replication of fibroblasts which characterise these tumours. Equine sarcoids differ from BPV induced fibro-papillomas of cattle (the natural host of BPV), in that they do not produce high amounts of virus particles, do not usually regress spontaneously and do not sero-convert to BPV; features which suggest that affected horses lack an effective anti-viral immune response to BPV. Equine sarcoids contain large numbers of CD4+ CD8+ dual positive T-cells which uniformly express FOXP3, the key transcription factor of regulatory T-cells, and FOXP3 is also expressed within the BPV infected fibroblasts. Compared to healthy skin, sarcoids showed increased mRNA transcription for FOXP3 and the regulatory cytokine TGFβ. Transcription of IL17, which has been shown to have a regulatory function in human papillomavirus-associated tumours, was also elevated in equine sarcoids compared to spleen. In contrast, the levels of mRNA transcripts for effector T cell cytokines IL2, IL4 and interferon-gamma (IFNγ) were not elevated in sarcoids compared to healthy skin or spleen. Similarly neither interferon-alpha (IFNα), interferon-beta (IFNβ) nor IL12 family members were elevated in sarcoids compared to normal skin. We suggest that the regulatory cytokine micro-environment within sarcoids enables the persistence of the lesions by preventing an effective anti-viral immune response.

No MeSH data available.


Related in: MedlinePlus