immunology.sciencemag.org/cgi/content/full/3/25/eaas9103/DC1

Supplementary Materials for

TCR signal strength controls the differentiation of CD4

+ effector and memory T

cells

Jeremy P. Snook, Chulwoo Kim, Matthew A. Williams*

*Corresponding author. Email: matthew.williams@path.utah.edu

Published 20 July 2018, Sci. Immunol. 3, eaas9103 (2018)

DOI: 10.1126/sciimmunol.aas9103

The PDF file includes:

Materials and Methods

Fig. S1. Creation and stimulation of hybridoma T cell lines with NFAT and NFκB reporters.

Fig. S2. Stimulation and characterization of C7 and C26 transgenic CD4+ T cells in vitro.

Fig. S3. Analysis of CD25 expression in an endogenous CD4+ T cell repertoire early after viral

infection.

Fig. S4. Characterization of C7 and C26 transgenic CD4+ T cells via adoptive transfer and

LCMV infection.

Fig. S5. Early expression of CD25 by clone 18 retrogenic CD4+ T cells predicts effector and

memory differentiation.

Fig. S6. SHP-1 KD validation and effect on TCR signaling in vitro.

Fig. S7. Flow cytometry gating strategies.

Other Supplementary Material for this manuscript includes the following:

(available at immunology.sciencemag.org/cgi/content/full/3/23/eaar4135/DC1)

Table S1 (Microsoft Excel format). Primary source data.

Table S2 (Microsoft Excel format). Complete list of genes with significantly changed expression

when comparing CD25lo

to CD25hi

day 5 SMARTA effector cells, as determined by RNA-seq.

Materials and Methods

In vitro stimulations

We subcutaneously injected C57BL/6 mice with 1 x 106 B16 melanoma cells secreting Flt-

3L as previously described (53). 10-14 days later, DCs were isolated from splenocyte single

cell suspensions through passive adherence. Purified DCs were then incubated with 10μM

GP61-80 peptide from LCMV (GLKGPDIYKGVYQFKSVEFD) for 18 hr in cell culture media. DCs

were washed and incubated with hybridoma cell lines at a 1:1 ratio in cell culture media for

up to 24 hours. Alternatively, DCs were incubated with freshly isolated T cells for 3-72

hours at a 1:5 ratio.

Antibodies

The following fluorophore-conjugated antibodies were used for flow cytometry: anti-CD4

(RM4-5), anti-Streptavidin, anti-IL-2 (JES6-5H4), anti-KLRG1 (2F1), anti-Ly6C (HK1.4),

anti-PD-1 (29F.1A12), anti-pZAP70/Syk(pY319/Y352) (n3kobu5), anti-SLAM (TC15-

12FL2.2), anti-T-bet (4B10), anti-TNF (MP6-XT22), anti-V2 (B20.1), and anti-ZAP70

(1E7.2) (Biolegend); anti-Thy1.1 (HIS51), anti-CD44 (IM7), anti-CD62L (MEL-14), and

anti-IFN (XMG1.2) (eBiosciences); anti-TCF-1 (S33-966), anti-Bcl-6 (K112-91), anti-

CD69 (H1.2F3), anti-CXCR5 (2G8), anti-pCD3(pY142) (K25-407.69), anti-V14 (14-2),

anti-V7 (TR310), and anti-CD162 (2PH1) (BD Biosciences); anti-CD25 (PC61.5) (Tonbo

Bioscience); anti-Tim-3 (344823) (R+D Systems); anti-Rat IgG (H+L) (Jackson

Immunoresearch). I-Ab/GP61-80 tetramer conjugated to APC was provided by the NIH

Tetramer Core Facility (Atlanta, GA). Flow cytometry was performed using an LSR Fortessa

(BD Biosciences) and sorted using a FACSAria II (BD Biosciences)(University of Utah Flow

Cytometry Core Facility). Flow cytometry data was analyzed using FlowJo (TreeStar).

Immunoblot Assay

Whole cell extracts were produced by cell lysis in RIPA buffer with protease and

phosphatase inhibitors, as previously described. Protein resolution was achieved through

denaturing SDS-PAGE and subsequently transferred onto a nitrocellulose membrane.

Membranes were probed with anti-GFP (Cell Signaling), anti--tubulin (Cell Signaling),

anti-SHP-1 (Cell Signaling), or anti--actin antibodies in TBS-T + 5% BSA and developed

using HRP conjugated secondary antibodies and SuperSignal West Pico Chemiluminescent

Substrate (Thermo Fisher). Exposures were captured using a Biorad ChemiDoc MP and

Image Lab Software 5.1v (BioRad). Western blot image analysis was performed using

ImageJ 1.48v software.

RT PCR Primers The following primer pairs were used (5’ to 3’): IFNγ: forward-CAAGCGGCTGACTGAACTCA,

reverse-CACTGCAGCTCTGAATGTTTCTTATT; IL-2: forward-

CCTGAGCAGGATGGAGAATTACA, reverse-TCCAGAACATGCCGCAGAG; NFATc1: forward-

CTCGAAAGACAGCACTGGAGCAT, reverse-CGGCTGCCTTCCGTCTCATAG; T-bet: forward-

CCCACAAGCCATTACAGGAT, reverse-CCCTTGTTGTTGGTGAGCTT; GAPDH: forward-

ATTGTCAGCAATGCATCCTG, reverse-ATGGACTGTGGTCATGAGCC; Il2ra: forward-

CGTTGCTTAGGAAACTCCTGGA, reverse-GCTTTCTCGATTTGTCATGGG; Socs2: forward-

GGTTGCCGGAGGAACAGTC, reverse-GAGCCTCTTTTAATTTCTCTTTGGC; Tcf7: forward-

ATCCTTGATGCTGGGATTCTG, reverse-CTTCTCTTGCCTTGGGTTCTG; Pou2af1: forward-

CTGCTTCCACAGTGACAGAGG, reverse-GTCAACACCGAGGAGGGTCC; Egr2: forward-

CTTCAGCCGAAGTGACCACC, reverse-GCTCTTCCGTTCCTTCTGCC. Expression was

normalized to GAPDH and displayed as relative n-fold increase.

Fig. S1. Creation and stimulation of hybridoma T cell lines with NFAT and NFκB

reporters. A) Table of cloned TCRβ genes, amino acid sequences of the CDR3b region,

previously calculated Kd, as determined by tetramer equilibrium binding assays, and

previously calculated off-rate, as determined by tetramer decay assays (Kim et al., 2013).

TCRs were further categorizes as memory “high” (frequency within the GP61-80 population

following LCMV infection of SMα mice at memory time points was the same or higher than

at the peak of the effector response) or memory “low” (frequency within the GP61-80

population following LCMV infection of SMα mice at memory time points was lower than at

the peak of the effector response). Histogram plots indicate the expression of the TCR after

transduction into the 58α-β- TCR hybridoma line. B) Schematic showing the creation of T

cell hybridoma cell lines expressing TCR, and NFAT-GFP reporter and a NFκB-CFP

reporter. C) Western blots showing expression of GFP in whole cell lysates of T cell

hybridomas and DCs after a 24-hour co-incubation. Bar graph shows the ratio GFP in

stimulated versus unstimulated hybridomas, normalized to a-tubulin expression.

Fig. S2. Stimulation and characterization of C7 and C26 transgenic CD4+ T cells in

vitro. A) Naïve TCR transgenic C7 or C26 CD4+ T cells were isolated from the spleen, then

stimulated with pepDCs for the indicated amount of time. Representative flow histograms

indicate C7 (grey) and C26 (black) expression of ZAP-70, phosphorylated ZAP-70 (pZAP-

70) and phosphorylated CD3 zeta chain (pCD3z). B) The bar graph shows induction of

phosphorylated ZAP-70 within C7 and C26 CD4+ T cells over a 6 hour time course. C) The

graph depicts CD25 surface expression measured via flow cytometry over a 24h time

course. D) Gene expression for the indicated transcripts was measured by RT-PCR 24h and

72h after stimulation. The y-axis indicates relative gene expression, normalized to GAPDH.

Fig. S3. Analysis of CD25 expression in an endogenous CD4+ T cell repertoire early

after viral infection. Naïve B6 mice were infected with LCMV. Five days later splenocytes

were harvested and analyzed via flow cytometry. LCMV-specific CD4+ T cells were

identified through tetramer (GP66-77) staining and activation state was assessed via CD44

expression. The representative flow histogram depicts CD25 expression by activated,

LCMV-specific CD4+ T cells.

Fig. S4. Characterization of C7 and C26 transgenic CD4+ T cells via adoptive transfer

and LCMV infection. A) C7 and C26 CD4+ T cells (Thy1.1+) were adoptively transferred (1

x 105) into B6 mice (Thy1.2+) that were subsequently infected (LCMV). CD25 expression by

Thy1.1+ cells in the spleen was measured at days 1, 2 and 3 post-infection. B)

Representative flow plots show the presence of Thy1.1+ transgenic T cells in the spleen,

their surface expression of CD44 and CD62L and cytokine secretion following a 4 hour ex

vivo re-stimulation with 1 uM GP61-80 in the presence of Brefeldin A at day 8 post infection.

Fig. S5. Early expression of CD25 by clone 18 retrogenic CD4+ T cells predicts effector

and memory differentiation. Retrogenic CD4+ T cells were created using the Clone 18

TCR, specific for GP61-80 of LCMV. A) Following adoptive transfer (2 x 105) into B6 mice and

subsequent LCMV infection, Rg+ CD4+ T cells were sorted based on CD25 expression and

transferred into separate infection-matched B6 mice. Representative flow histograms

show the Rg+ CD4+ T cells after CD25 sort but before transfer into infection-matched mice.

B) Peak effector (d8 p.i.) and memory analysis was performed via flow cytometery. Total

Rg+ CD4+ T cells were measured in the spleen at d42 p.i. and normalized to the number of

CD25hi (“High”) and CD25lo (“Low”) Rg+ CD4+ T cells transferred into each mouse at d5. C)

The frequency of Tfh differentiation between CD25hi and CD25lo Rg+ CD4+ T cells at d8 p.i.

was measured by expression of CXCR5 and PD-1 via flow cytometery.

Fig. S6. SHP-1 KD validation and effect on TCR signaling in vitro. A) EL-4 GFP+ cells

were created via transfection with pMigR1-mir30-SHP-1KD-GFP constructs (two different

siRNA sequences, 66 and 68). Western blot shows SHP-1 expression in whole cell lysates

from 3 different EL-4 cell lines. Bar graph shows the relative expression level of SHP-1 in

each cell line compared to the actin control. B) SMARTA SHP-1 KD and WT CD4+ T cells

were co-incubated with pepDCs in a 1:1 ratio for 3 hours. Representative flow cytometry

histograms show the levels of ZAP-70 and CD3𝜁 phosphorylation with SMARTA cells after

co-incubation with DCs (Control) of pepDCs (+gp61-80).

Fig. S7. Flow cytometry gating strategies. Flow cytometry gating for hybridoma cells

post pepDC stimulation (A) and splenocytes from B6 mice that received SMARTA/C7/C26

(B), retrogenic CD4+ T cells (D), and siRNA KD SMARTA cells (E). Gating strategy for the

identification and V subtyping for LMCV-specific CD4+ T cells in SM mice (C).

A.

B.

C.

D.

E.

FSC

FSC

FSC

FSC

FSC

SSC

SSC

SSC

SSC

SSC

SSC

SSC

SSC

SSC

SSC

CD4

CD4

CD4

CD4

mCherry

CD4

CD4

mCherry

Thy1.1

Thy1.1

CD4

Tet

GFP

GFP

GFP

Va2

Vb

Tet

Va2

Live

Live

Live

Live

Live

CD4+

CD4+

CD4+

CD4+

mCherry+

GFP+CD4+

Thy1.1+Va2+

CD4+Tet+

Thy1.1+Va2+

GFP+mCherry+

GFP+Thy1.1+CD4+

Vb+Tet+