www.sciencemag.org/cgi/content/full/science.1260668/DC1

Supplementary Materials for

Functional heterogeneity of human memory CD4+ T cell clones primed

by pathogens or vaccines

Simone Becattini, Daniela Latorre,

Federico Mele, Mathilde Foglierini, Corinne De Gregorio,

Antonino Cassotta, Blanca Fernandez, Sander Kelderman, Ton N. Schumacher, Davide Corti,

Antonio Lanzavecchia, Federica Sallusto*

*Corresponding author. E-mail: federica.sallusto@irb.usi.ch

Published 4 December 2014 on Science Express

DOI: 10.1126/science.1260668

This PDF file includes:

Materials and Methods

Figs. S1 to S7

Tables S1 and S2

References and Notes

Other supplementary material for this manuscript includes the following:

Data files (TCR sequences in zipped folder)

2

Materials and Methods

Cells and cell sorting

Blood from healthy donors was obtained from the Swiss Blood Donation Center of

Basel and Lugano and used in compliance with the Federal Office of Public Health

(authorization no. A000197/2 to F.S). Peripheral blood mononuclear cells (PBMCs) were

isolated with Ficoll-Paque Plus (GE Healthcare). Monocytes and total CD4 T cells were

isolated by positive selection using CD14 and CD4 magnetic microbeads, respectively

(Miltenyi Biotech). Memory TH cell subsets were sorted to over 97% purity as follows

and after gating on CD8–CD14

–CD16

–CD19

–CD25

–CD56

–CD45RA

– cells:

CXCR3+CCR4

–CCR6

– cells (defined as TH1), CCR4

+CXCR3

–CCR6

– (defined as TH2),

CCR6+CXCR3

+CCR4

– (defined as TH1*), CCR6

+CCR4

+CXCR3

– (defined as TH17).

Naïve T cells were sorted as CD45RA+CCR7

+CD8

–CD14

–CD16

–CD19

–CD25

–CD56

–

CD45RO–CD95

–. The following fluorochrome-labeled mouse monoclonal antibodies

were used for staining: CD45RA-FITC (ALB11), CD45RO-PE (UCHL1), CD8-PE-Cy5

(B9.11), CD14-PE-Cy5 (RMO52), CD16-PE-Cy5 (3G8), CD19-PE-Cy5 (J3-119), CD25-

PE-Cy5 (B1.49.9), CD56-PE-Cy5 (N901) (from Beckman Coulter), CCR6-PE (11A9),

CCR4-PE-Cy7 (1G1) (from BD Biosciences), CXCR3-AlexaFluor 647 (G025H7),

CCR7-BV421 (G043H7) (from Biolegend), CD95-PerCP-eFluor 710 (DX2) (from

eBioscience). Cells were stained on ice for 15-20 minutes and sorted with FACSAria III

(BD Biosciences). Viable IL-17+ (IFN-

– IL-4

–), IFN-

+ (IL-4

– IL-17

–), and IL-4

+ (IFN-

–

IL-17–) cells were FACS-sorted using the cytokine secretion assay (Miltenyi Biotec).

Briefly, primed T cells were restimulated for 3 hours with PMA and Ionomycin, washed

in cold buffer and incubated with a mix of IFN-γ, IL-4 and IL-17 catch reagents for 5

minutes on ice. Warm medium was added and cells were incubated for 45 minutes at

37°C under slow continuous rotation for cytokine secretion. Cells were then washed and

stained with a cytokine-detection antibody mix containing IFN-γ-FITC, IL-4-PE, IL-17-

APC detection antibodies for 10 minutes on ice.

Ex-vivo T cell stimulation and intracellular staining

T cells were cultured in RPMI 1640 supplemented with 2 mM glutamine, 1% non-

essential amino acids, 1% sodium pyruvate, 1% penicillin/streptomycin (all from Life

Technologies), and 5% human serum (Swiss Red Cross). For some experiments, up to

500 IU/ml IL-2 was added to the medium. Sorted T cells were labeled with

carboxyfluorescein succinimidyl ester (CFSE) or Cell Trace violet (CTV) and cultured at

a ratio of 2:1 with irradiated autologous monocytes pre-pulsed for 3-5h with the antigen

of interest. In the case of C. albicans, cells were stimulated with a mixture of heat-killed

particles and lysate. Proliferating cells were sorted on day 6 (memory) or day 15 (naïve)

based on CFSE or CTV dilution and immediately stained for analysis of surface markers

or stimulated with PMA and ionomycin for 5h in the presence of brefeldin A for the last

2.5 h (all reagents from Sigma-Aldrich). Cells were fixed and permeabilized with

Cytofix/Cytoperm (BD Biosciences), according to the manufacturer’s instructions, and

then stained with the following anti-cytokine antibodies: IL-17A-eFluor660 (64DEC17),

IL-22-PerCP-eFluor710 (22URTI) (eBioscience), IFN- -APC-Cy7 (4S.B3) (Biolegend),

IFN- -FITC (B27), IL-4-PE (MP425D2) (BD Biosciences). Cytokine concentration in the

culture supernatants was assessed using the FlowCytomix assay (eBioscience), according

to manufacturer’s instruction.

3

Microbes and antigens

C. albicans strains SC5314 or ATTC 14053 were used. C. albicans was cultured in

YPD medium for 16 hours at 30°C, extensively washed in PBS and heat-inactivated at

65°C for 30 minutes. Ratio used for stimulation assays was three particles per monocyte.

Lysate was prepared from mixed cultures of conidia and hyphae. Briefly, C. albicans was

cultured in YPD medium for 16 hours at 30°C or in YPD supplemented with 10% human

serum for 16 hours at 37°C. The resulting cultures yielded almost pure populations of

conidia and hyphae, respectively. Cells from the two cultures were extensively washed,

resuspended in PBS with complete protease inhibitor cocktail (Roche), mixed and

sonicated on ice for 30 consecutive cycles (45 seconds on, 90 seconds off, amplitude =

100). The suspension was then centrifuged at max speed for 15 minutes, and supernatant

collected and sterile filtered through 0.22 μm pore membranes. Quantification was

performed using Bradford reagent (Bio-Rad). C. albicans lysate was used at a

concentration of 2.5 μg/ml, M. tuberculosis lysate (strain H37Rv, from Bei Resources)

and Tetanus Toxoid (from Novartis Vaccines, Siena, Italy) were used at 5 μg/ml.

Amplification of TCR genes

Individual T cell clone total cDNA was obtained from 103 – 10

4 cells/reaction.

Reaction was carried out using oligo dT(15) primers (Promega) and Superscript III (Life

Technologies) reverse transcriptase, in a reaction mix containing DTT, NP40, dNTPs,

RNAsin (Promega). Reactions were run with the following conditions: 42°C x 10

minutes, 25°C x 10 minutes, 50°C x 1 hour, 94°C x 5 minutes. T cell receptor (TCR)

sequences from T cells were identified from cDNA (40). DNA libraries were prepared

from the cDNA using the Illumina TruSeq DNA library preparation kit. The resulting

DNA libraries were sequenced on an Illumina MiSeq sequenzer using Paired-end 150bp

chemistry. Sequencing reads in FASTQ files were mapped to the human genome, build

NCBI36/hg18, using BWA and SAMtools (41, 42). PCR duplicates in resulting BAM

files were filtered using Picard (http://picard.sourceforge.net). CDR3 TCR sequences

were identified as previously reported (43). TCRα and β sequences were inferred using

an unpublished python script developed by NKI-AVL. In addition, TCRα and β

sequences were performed at the IRB. Three μl of cDNA were added to a PCR mix (final

volume 25 μl) containing PfuUltra II Fusion HS DNA Polymerase (Agilent Genomics).

Sequences were amplified using one or both the designed TCR Vα or Vβ-specific

forward primer pools (pool fw1, pool fw2) and TAC-rev or TBC-rev reverse primers

pairing to α chain constant region or C1-C2 β chain constant region, respectively. PCR

reactions were performed with the following conditions: 95°C x 1 minute; (95°C x 20

seconds; 50°C x 20 seconds; 72°C x 30 seconds) x 45 cycles; 72°C x 3 minutes.

Sequence amplification was assessed through agarose gel electrophoresis; successfully

amplified fragments were sequenced through Sanger method using TAC-rev or TBC-rev

primer.

RNA extraction and qRT-PCR

Total RNA was extracted using TRIzol reagent (Life Technologies) or E.Z.N.A.

DNA/RNA Isolation Kit (Omega Bio-tek) according to manufacturer’s instructions.

QScript cDNA SuperMix (Quanta Biosciences) was used for cDNA synthesis.

Transcripts were quantified by qRT–PCR on ABI PRISM 7900HT, with predesigned

4

TaqMan Gene Expression Assays: RORC (Hs01076122_m1), TBX21 (Hs00203436_m1),

GATA3 (Hs00231122_m1, all from Life Technologies Applied Biosystems). Reactions

were run on a 7900 Fast Real Time PCR System (Life Technologies Applied

Biosystems). Expression of target genes was normalized to 18S ribosomal RNA or TBP

RNA and expressed as arbitrary units (A.U.).

TCRβ deep sequencing

Antigen-specific T cells were obtained as described above. A minimum number of

106 cells were obtained for all experiments, and each sample was split in two, half of

which was frozen as a backup. In case cells sorted upon stimulation did not reach the

required number, they were expanded for 1 to 5 days in the presence of 50 IU/ml IL-2.

Cells to be analyzed by deep sequencing were centrifuged and washed in PBS, and

genomic DNA was extracted from the pellet using QUIAamp Micro Kit (Qiagen),

according to manufacturer’s instruction. Genomic DNA quantity and purity were

assessed through spectrophotometric analysis. Deep sequencing of TCRβ was performed

by Adaptive Biotechnologies Corp. (Seattle, WA) using the ImmunoSEQ assay

(http://www.immunoseq.com). Briefly, following multiplex PCR reaction designed to

target any TCRβ CDR3 fragments, amplicons were sequenced using the Illumina HiSeq

platform. Raw data consisting of all retrieved sequences of 87 nucleotides or

corresponding amino acidic sequences and containing CDR3 region were exported and

further processed. If not differently stated in the text, the assay was performed at survey

level (detection sensitivity, 1 cell in 40,000); for some samples, a deep level analysis was

used (detection sensitivity, 1 cell in 200,000).

TCRβ sequence analysis

Data sets of TCRβ sequences were analyzed using algorithms written in Java. DNA

sequences containing frame-shift or stop codons were removed prior to analysis. The

experimental noise was determined for each batch of experiments with parallel analysis

of antigen-specific memory T cells (obtained as described above) that were split in two

before DNA extraction and sequencing. For each control, all the sequences (shared and

not shared) were plotted together. The value of the non-shared sequence with the highest

number of reads was set as threshold. Sequences with a value of reads below threshold

level were deleted from the experiment. The percentage of shared clonotypes was

calculated using the Jaccard index [J=(A∩B)/(A∪B)] as number of shared clonotypes

between two subsets divided by the total number of clonotypes present in the same

subsets, and normalized by 100. The percentage of shared reads was calculated as

average of the cumulative frequencies of the shared clonotypes in each of the two

subsets. For instance, if shared clonotypes within the A and B subsets accounted for 30%

and 40% of the total populations, respectively, based on their read count, the size of the

shared population would be calculated as 35% of the A-B compartment. A disparity

index was calculated as previously described (44) as:

Where fi represents the number of reads of the clonotype i divided by the total number of

reads in the sample, and N is the total number of clonotypes present in the same sample.

5

The index ranges from D=0 and D=1, D=0 representing a population composed by

equally expanded clonotypes and D=1 a population where only one clonotype dominates.

Statistical analysis

Statistical analysis was performed with the Prism software (GraphPad). Data in

columns represent means ± SEM values, and significance was assessed by non-

parametric paired Friedman test. Non-parametric Spearman correlation coefficient was

determined for shared sequences.

6

Fig. S1.

Sorting strategy to identify functional memory CD4 T cell subsets. (A and B)

Expression of chemokine receptors identifies four subsets of human memory CD4 T

cells. Cells expressing CD45RA, CD25, CD14, CD19 were excluded from the gate.

Representative dot plot in one donor (A) and percentage of each subset relative to total

memory CD4 T cells in thirteen donors (B). Data are means ± SEM. (C and D) Cytokine

production by the four memory subsets following stimulation with PMA and Ionomycin.

Dot plots are from a representative donor (C). Means ± SEM of four donors (D). (E)

TBX21, GATA3, and RORC mRNA levels in the four memory subsets analyzed

immediately after sorting. Data are the means ± SEM of four donors.

7

Fig. S2

C. albicans–specific clonotype sharing between samples that were split before or

after antigenic stimulation. (A) 1.5x106 TH17 cells were stimulated with C. albicans.

After 6 days, CFSElo

cells were sorted and the sample was divided in two halves that

were separately subjected to genomic DNA extraction and sequencing. Shown are

absolute numbers of reads. The total number of clonotypes in the two samples is

indicated on x and y axis. Values in the lower right corner represent the number of

clonotypes and percentage of reads shared between the two samples. The Spearman

correlation and paired t test value are shown in the upper part. (B and C) Sample split

control experiments were performed for the different donors analyzed and the highest

read value at which a sequence was retrieved in only one of the two split samples was set

as a cutoff. Sequences with read values below the cutoff were removed. Shown in (B) are

the removed sequences (expressed as percentage of total reads) in all analyzed donors (C.

albicans, n = 5; TT n = 4; every dot represents a donor). Shown in (C) is the comparison

between sizes of validated versus removed (below-threshold) sequences, expressed as

average number of reads; every dot represents averaging from one donor. (D) 1.5x106

TH17 cells were sorted and divided in two wells that were stimulated with C. albicans in

the presence of autologous monocytes. TCRβ analysis was performed on day 6 on

genomic DNA from CFSElo

cells that had proliferated independently in the two cultures.

Shown is the number of clonotypes detected in each well, the number of clonotypes and

percent of reads shared between the two wells. Shown is also the Spearman correlation

and paired t test value. Data are representative of three separate experiments performed

with C. albicans and one experiment performed with the four memory subsets (TH1, TH2,

TH1*, and TH17) and TT as antigen.

8

Fig. S3

Clonotypes shared among subsets of C. albicans–specific T cells. Venn diagrams show

for four donors the number of unique and shared clonotypes among C. albicans–specific

T cells isolated from the four memory subsets.

Donor CA-01

5

31 782

24

124

110 171

462

8

17

26

724

6

Th1

Th2 Th1*

Th17

Donor CA-03

4

17 2559

95

433

320 842

1072

2

3

57

1938

10

Th1

Th2 Th1*

Th17

Donor CA-04

11

57 10529

46

160

623 142

702

15

23

43

4050

9

Th1

Th2 Th1*

Th17

Donor CA-05

28

62 22520

224

949

737 1000

1598

11

41

105

18146

23

Th1

Th2 Th1*

Th17

9

Fig. S4

Sorting strategy of CCR6+ memory CD4 T cell subsets. Three subsets of CCR6

+

memory CD4 T cells were sorted according to the expression of CXCR3 and CCR4. (A)

Cytokine production by the indicated memory subsets after sorting from PBMCs and in

vitro stimulation with PMA and Ionomycin for 5 hours. (B) TBX21, GATA3, and RORC

mRNA levels in the indicated memory subsets analyzed immediately after sorting. Data

are the means ± SEM of three donors.

10

Fig. S5

Clonotypic analysis of M. tuberculosis–specific CD4 T cell subsets. (A) CFSE profiles

and percentage of CFSElo

proliferating cells in memory T cell subsets stimulated with M.

tuberculosis in donor MT-01. (B) Cytokine production by CFSElo

cells measured by

intracellular cytokine staining after stimulation for 5 hours with PMA and Ionomycin.

(C) Comparison of clonotype frequency distribution in samples of T cells isolated from

M. tuberculosis–stimulated T cell subsets from donors MT-01 and MT-02. Frequencies

are shown as percentage of total reads. The total number of clonotypes in each sample is

indicated in parenthesis on x axis and y axis. Shown is the number of clonotypes shared

between the two samples; Spearman correlation and paired t test values are shown when

significant. (D) Bar histograms showing for the two donors percentage of clonotypes

(upper panel) and percentage of reads (lower panel) that are shared by the indicated

subsets.

11

Fig. S6

Clonotypes shared among subsets of TT–specific T cells. Venn diagrams show for four

donors the number of unique and shared clonotypes among TT–specific T cells isolated

from the four memory subsets.

5315 5

Donor TT-01

28

106 639

32

293

262 169

161

83

30

77

Th1

Th2 Th1*

Th17

Donor TT-02

24

75 17212

152

688

818 501

536

37

65

28

4587

18

Th1

Th2 Th1*

Th17

5312 4

Donor TT-03

7

26 704

46

166

360 233

280

12

8

48

Th1

Th2 Th1*

Th17

Donor TT-04

11

16 331

77

232

101 350

214

8

11

4

2310

16

Th1

Th2 Th1*

Th17

12

Fig. S7

Sorting of cytokine-secreting cells from cultures of naïve CD4 T cells primed in vitro

by C. albicans. Naïve CD4 T cells were primed in vitro by C. albicans in the presence of

autologous monocytes. On day 15, IL-17+ (IFN-

– IL-4

–), IFN-

+ (IL-17

– IL-4

–), or IL-4

+

(IL-17– IFN-

–) T cells were sorted using the cytokine secretion assay and maintained in

culture with addition of IL-2. Shown is the cytokine profile of cultured cells after 7 days.

IL-17+ (IFN-g–IL-4–) sorted T cells

IL-4+ (IFN-g–IL-17–) sorted T cells

IFN-g+ (IL-17–IL-4–) sorted T cells

15.7 22.3

24.9

36.8 0.8

3.12.2

1.345.9

3.6

0.26.9 55.3 0.7

3.2

51.2 5.3

1.8

13.9 1.6

2.9

11.9 3.2

5454.7

1.72.8

103102 104 1050

103

104

105

0

102

103

104

105

0

103

104

105

0

103

104

105

0

103

104

105

0

103

104

105

0

102

103

104

105

0

102

103

104

105

0

103

104

105

0

103102 104 1050

103102 104 1050

103 104 1050103 104 1050

103 104 1050103 104 1050

103 104 1050103 104 1050

IFN

-g

IL-4

IL-17

IL-4

IFN-gIL-17

IFN

-g

IL-4

IL-17

IL-4

IFN-gIL-17

IFN

-g

IL-4

IL-17

IL-4

IFN-gIL-17

13

Table S1.

T cell counts and number of TCR clonotypes of C. albicans–specific TH cells from five

donors.

14

Table S2.

TCRβ CDR3 sequences of T cell clones primed by C. albicans in vitro and sorted based

on the ability to selectively secrete the indicated cytokine. Data are from one experiment,

representative of three independent experiments performed with different donors.

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