Supplemental methods
Immunohistochemistry and quantitative assessment of FoxP3+ and CD4+ cells
Formalin-fixed paraffin-embedded sections from patients diagnosed with gastritis, lowgrade or high grade MALT lymphoma were stained with antibodies directed against
human FoxP3 (clone 236A/E7, Abcam, Cambridge, MA, USA) and CD4 (MT310,
DAKO, Glostrup, Denmark). FoxP3+ and CD4+ positive cells were manually counted in
two to three representative 20x (230 µm x 230 µm) fields of view per sample; a minimum
of 500 cells were counted per specimen. FoxP3-specific real time PCR (LightCycler;
Roche, Basel, CH) was performed with the LightCycler 480 SYBR Green I master kit
(Roche, Basel, CH). GAPDH transcript levels were used for normalization. Human
Foxp3 primers were previously published.1 Primer sequences for murine FoxP3 were:
FoxP3
forward:
5'-GCCCAGACCCCTGTGCT-3'
and
FoxP3
reverse:
5'-
CCGGGAGCACACTGCCC-3'.
Immunomagnetic cell isolation and flow cytometry
The immunomagnetic isolation of B-cells and CD4+CD25+/CD4+CD25- T-cells was
performed using MagCellect mouse B-cell and CD4+CD25+ regulatory T cell isolation
kits (R&D Systems). For FACS staining, the following antibodies were used: anti-CD25
(clone PC61.5, eBioscience, San Diego, CA, USA), anti-CD28 (clone 37.51, Biolegend,
San Diego, CA, USA), anti-CD4 (clone RM4-5, BD Bioscience), anti-CD3 (clone 1452C11, BD Bioscience), anti-CD69 (clone H1.2F3, Santa Cruz Biotech, Santa Cruz, CA,
USA) and anti-CD40L (clone MR1, BD Bioscience). Flow cytometry was performed on
a CyanADP instrument (Dako, Glostrup, Denmark).
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In vitro chemotaxis and T cell suppression assay
The migration of Treg in response to culture supernatants of tumor cell supernatants was
assessed in a 96-well chemotaxis chamber (5µM pore size; ChemoTx System,
NeuroProbe, Gaithersburg, MD, USA). The supernatants from the tumor cultures were
added to the lower chamber of the ChemoTx system alone or in combination with a
neutralizing anti-CCL17 monoclonal antibody and/or anti-CCL22 monoclonal antibody
(2µg/ml, clones 54026 and 57226, R&D Systems). Immunomagnetically isolated
CD4+CD25+ cells were labeled with calcein AM (5µg/ml; BD Bioscience) for 1 hour at
37oC, washed with PBS and seeded into the upper chamber at a density of 1x105 cells.
Cells were allowed to migrate towards the tumor supernatants at 37oC for 2 hours. The
fluorescence of cells that had migrated to the lower chamber was quantified (SpectraMax
M5, Molecular Devices) and absolute cell numbers were calculated based on a 1:2 diluted
standard curve of a known cell number. The suppressive activity of splenic, mesenteric
lymph node-derived and intratumoral Treg was assessed by mixing immunomagnetically
isolated CD4+CD25+ Treg with autologous splenic CD4+CD25- cells at a 1:1 ratio (2.5 x
104 cells each) and stimulating them with anti-CD3/CD28-coated Dynabeads (Invitrogen,
Carlsbad, CA, USA). Proliferation was assessed after 5 days by [3H]-thymidine
incorporation.
Treg conversion assay
For the assessment of Treg conversion from conventional T-cells, splenic CD4+CD25- T
cells were isolated from a mouse strain engineered to express a diphtheria toxin receptoreGFP fusion protein under the control of the foxp3 promoter.2 2x105 CD4+CD25- T cells
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were cultured for 5 days either alone or in the presence of recombinant IL-2 (R&D
Systems), TGF-β1 (Peprotech, London, UK) and anti-CD3 (145-2C11; Bio X Cell) or
supernatants derived from H. felis-stimulated or unstimulated tumor cell or splenocyte
cultures. In all cases, the cultures were examined by FACS for expression of eGFP in the
CD4+ T-cell population and by real time analysis for FoxP3 transcript levels as described
below.
Statistical analysis
Differences between values were determined using the non-paired Student’s t-test. The
difference was considered to be significant if the p-value was < 0.05.
Supplemental references
1. Kelsen J, Agnholt J, Hoffmann HJ, Romer JL, Hvas CL, Dahlerup JF.
FoxP3(+)CD4(+)CD25(+) T cells with regulatory properties can be cultured from colonic
mucosa of patients with Crohn's disease. Clin Exp Immunol 2005; 141: 549-557.
2. Lahl K, Loddenkemper C, Drouin C, Freyer J, Arnason J, Eberl G, et al. Selective
depletion of Foxp3+ regulatory T cells induces a scurfy-like disease. J Exp Med 2007;
204: 57-63.
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