Supplementary Materials for

Tfr cells lack IL-2Rα but express decoy IL-1R2 and IL-1Ra and

suppress the IL-1–dependent activation of Tfh cells

Paul-Gydeon G. Ritvo, Guillame Churlaud, Valentin Quiniou, Laura Florez,

Faustine Brimaud, Gwladys Fourcade, Encarnita Mariotti-Ferrandiz, David Klatzmann*

*Corresponding author. Email: david.klatzmann@upmc.fr

Published 8 September 2017, Sci. Immunol. 2, eaan0368 (2017)

DOI: 10.1126/sciimmunol.aan0368

The PDF file includes:

Materials and Methods

Fig. S1. Representative flow cytometry gating for Tfol cells.

Fig. S2. IL-2 increases the number of splenic Treg cells.

Fig. S3. Expression of CD25 on Tfr and Tfh cells in three different genetic

backgrounds.

Fig. S4. Tfh and Tfr cells have similar expressions of Bcl6 and CD25.

Fig. S5. Clustering of Treg, Tfr, and Tfh cells based on the expression of the entire

545-gene set from the NanoString mouse immunology panel.

Fig. S6. Cytokines, CD40L, and OX40 expression of Tfr, Treg, and Tfh cells.

Fig. S7. IL-1R2 and OX40 expression of Tfr, Treg, Teff, and Tfh cells.

Fig. S8. IL-1β production in coculture of B and Tfh cells.

Fig. S9. GCB representative gating after INS or OVA immunization.

Table S1. Exact P values of the asterisk symbols shown in figures.

Other Supplementary Material for this manuscript includes the following:

(available at immunology.sciencemag.org/cgi/content/full/2/15/eaan0368/DC1)

Source data (Excel file)

immunology.sciencemag.org/cgi/content/full/2/15/eaan0368/DC1

Supplementary Materials

Materials and Methods

Nanostring

Gene expression analysis based on a NanoString immunology panel

RNA extraction and NanoString procedure: Sorted cells were washed in PBS1X and stored in

RNAquous kit Lysis Buffer (Ambion, Inc/Life Technologies) at -80°C. Total RNA was extracted

according to the manufacturer’s instructions and quality was assessed on a bioanalyzer using the Pico

RNA Reagent Kit (Agilent Technologies). Gene expression was analyzed using the NanoString mouse

immunology panel (NanoString, Seattle, USA). 5 ng of total RNA was pre-amplified (5 cycles of

PCR) using the specific multiprobe approaches following the manufacturer’s instructions. Each

sample was analyzed in a separate multiplexed reaction including in each, eight negative probes and

six concentrations of positive control probes. Negative control analysis was performed to determine

the background for each sample.

Data quality control and normalization: Data were imported into NanoString nSolver analysis

software (version 2.5) for quality control and normalization. A preliminary quality control analysis

was performed on raw data as follows: A first normalization step using the internal positive controls

was applied to correct potential sources of technical variations. To do so, we calculated for each

sample the geometric mean of the positive probe counts. A scaling factor for a sample was the ratio of

the average across all geometric means and the geometric mean of the sample. For each sample, we

multiplied all gene counts by the corresponding scaling factor. Next, for each sample and all genes of

interest, non-specific measurements counted in a water sample (No Template Control) were subtracted

from all measurements for all samples. Finally, to take into account biological variation, 14

housekeeping genes were used to normalize all the 545 genes of interest using the same method as in

the positive control normalization step.

Selection of highly discriminant genes: We further analyzed the genes that better separate the cell

subsets. As 100 transcripts per gene represent the limit for a robust detection of expression, we first

selected genes with such expression in at least one sample. We then selected genes whose mean

expression in one subset was at least twice or half the mean expression in the two other subsets,

leading to a set of 81 genes that were used for Tfr, Tfh and Treg functional profile study.

Fig. S1. Representative flow cytometry gating for Tfol cells.

Flow cytometry contour plots identifying CXCR5hiPD-1hi follicular T cells (Tfol cells) within

CD4+CD19- T cells (middle panel) and CXCR5hiPD-1- status of CD4-CD19+ B cells (right panel).

Fig. S2. IL-2 increases the number of splenic Treg cells.

Fig. S3. Expression of CD25 on Tfr and Tfh cells in three different genetic backgrounds.

Fig. S4. Tfh and Tfr cells have similar expressions of Bcl6 and CD25.

Flow cytometry contour plot identifying CD4+Bcl6+Foxp3+ Tfr (red) and CD4+Bcl6+Foxp3- Tfh (blue)

cells within Tfol cells of A (left) and their expression of Bcl6 (middle panel) and CD25 (right panel).

Fig. S5. Clustering of Treg, Tfr, and Tfh cells based on the expression of the entire 545-gene set

from the NanoString mouse immunology panel.

Heat map comparing the gene expression profiles of Treg, Tfr and Tfh cell subsets from two genetic

backgrounds ten days after immunization. High (red) and low (blue) gene expression.

Fig. S6. Cytokines, CD40L, and OX40 expression of Tfr, Treg, and Tfh cells.

Expression of indicated molecules in Treg (white), Tfh (blue) and Tfr (red) cells.

Fig. S7. IL-1R2 and OX40 expression of Tfr, Treg, Teff, and Tfh cells.

IL-1R2 (left) and OX40 (right) protein expression of Tfr, Treg, Teff and Tfh cells.

Fig. S8. IL-1β production in coculture of B and Tfh cells.

Flow cytometry dot plot showing the IL-1 production of PD-1- B cells and PD-1+ Tfh cells (left) and

of CD4- B cells and CD4+ Tfh cells (right) in co-culture of B and Tfh cells. IL-1 was stained using the

IL-1-APC (eBioscience).

Fig. S9. GCB representative gating after INS or OVA immunization.

Bcl6- GL7- B cells and Bcl6+ GL7+ germinal center B cell (GCB cell) proportions within CD19+ B

cells after INS or OVA immunization

Table S1. Exact P values of the asterisk symbols shown in figures.

Figure Comparison Exact p-value

1 throughout *: p=0.0159, **: p =0.0079

2C **: p = 0.0079

2D *: p=0.0159

2E *: p=0.0256

4C throughout *: p=0.0159, **: p =0.0079

4D throughout ****: p<0,0001

4E *** : p=0.0002 except comparison between Tfh and Treg for OX40 MFI, p=0.0006

5A **: p=0.0022

5B *: p=0.0357

5C *: p=0.0286

5D left *: p=0.0159, **: p =0.0079

5D right *: p=0.0286

5E left *: p=0.0159, **: p =0.0079

5E right *: p=0.0286

6A-D **: p=0.0012

6E **: p=0.0043

7B *: p=0.0238, **:p=0.0079

7D *: p =0.0286