Open Access Research article

Elongated TCR alpha chain CDR3 favors an altered CD4 cytokine profile

Catherine Reynolds1, Deborah Chong1, Eleanor Raynsford1, Kathryn Quigley1, Deborah Kelly1, Julia Llewellyn-Hughes2, Daniel Altmann1 and Rosemary Boyton1*

Author Affiliations

1 Lung Immunology Group, Infectious Diseases and Immunity, Department of Medicine, Imperial College, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK

2 Molecular Biology Laboratories, Natural History Museum, Cromwell Road, London SW7 5BD, UK

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BMC Biology 2014, 12:32  doi:10.1186/1741-7007-12-32

Published: 9 May 2014

Additional files

Additional file 1:

Intracellular cytokine staining for antigen specific T cell lines. A representative example of intracellular cytokine staining for antigen specific T cell lines grown in (A) Th1 (n = 6) and (B) Th17 (n = 6) culture media. Cell lines were grown through one re-stimulation in polarizing cell culture medium before intracellular cytokine staining with FITC-conjugated IL-17 and PE-conjugated IFNγ antibodies. Note that IFNγ producing cells readily differentiate within Th17 cultures, notwithstanding clear-cut overall differences in preferential TCR usage (Tables 1 and 3) and binding avidity (Figures 6 and 7).

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Additional file 2:

No bias in the TCR β chain repertoire of naïve TCRVα chain transgenics at baseline or in a primary response in a DLN at Day 10 post-immunization as demonstrated by spectratype analysis. TCR β chain repertoire by spectratype analysis of (A) naïve TCRα transgenic and littermate control splenocytes and (B) primed DLNs at Day 10 post immunization with PLP 56 to 70 in CFA. V region specific primers were used in combination with a FAM labeled constant region primer to amplify TCRβ chain sequences from T cell cDNA templates. Data shown are representative of experiments carried out with 10 TCRα transgenic and 10 littermate controls and three independently performed experiments.

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Additional file 3:

No difference in T-bet transcription between TCRαβ transgenic and littermate control cell lines. TCRαβ transgenic cell lines (black bars) (n = 3) and littermate control lines (white bars) (n = 5) were established from primed DLN cells from mice primed 10 days earlier with PLP56 to 70/CFA and re-stimulated every 10 days through to four cycles in the absence of exogenous polarization. At each re-stimulation the relative expression of T-bet was determined. Error bars indicate SE.

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Additional file 4: Figure S5:

TCRαβ transgenics show strong functional T cell activation and absence of an enhanced apoptotic program. TCRVαβ transgenic (n = 4) and littermate control (n = 5) mice were primed with PLP56 to 70 on Day 0 (footpad, CFA) and Day 28 (flank, IFA). DLN and splenocytes were harvested at Day 10, Day 28 and Day 32. At the Day 32, CD4+ T cells were analyzed for expression of (A) the pro-survival factor Bclxl by real time PCR (Day 32) and (B) CD127 (Day 28), and (C) CD62L (Day 28) by flow cytometry. Statistical significance between groups was determined using an unpaired t test. Error bars indicate SE.

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Additional file 5:

Peptide priming of TCRVα, TCRVαβ transgenics or littermate controls does not result in a systemic cytokine storm or reduced thymocyte numbers. (A) Littermate controls, TCRVα and TCRVαβ transgencis were immunized with 200 μg SEB (striped bars) (littermate controls, n = 5; TCRVα, n = 9; TCRVαβ, n = 9), PBS/CFA (white bars) (littermate controls, n = 4; TCRVα, n = 4; TCRVαβ, n = 4), or 50 μg PLP/CFA (black bars) (littermate controls, n = 4; TCRVα, n = 4; TCRVαβ, n = 4). (B) Serum samples were collected at time points 0, 2, 24 and 72 hours from mice injected with SEB (striped bars), PBS/CFA (white bars) or 50 μg PLP/CFA (black bars) and IFNγ (top row) and TNF-α (middle row) levels measured by ELISA. On Day 7, total thymocyte counts and CD4/CD8 thymocyte ratios were determined (bottom row). CD4 single positive thymocytes were isolated by cell sorting and the CDR3β repertoire of (C) littermate controls and (D) TCRVα transgenic mice immunized with PLP/CFA determined by TCR subcloning and sequencing.

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