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Immunity, Volume 31

Supplemental Data

Loss of the LAT Adaptor Converts Antigen-Responsive

T Cells into Pathogenic Effectors

that Function Independently of the T Cell Receptor Michael Mingueneau, Romain Roncagalli, Claude Grégoire, Adrien Kissenpfennig, Arkadiusz Miazek, Cristel Archambaud, Ying Wang, Pierre Perrin, Elodie Bertosio, Amandine Sansoni, Sylvie Richelme, Richard M. Locksley, Enrique Aguado, Marie Malissen, and Bernard Malissen

Supplemental Experimental Procedures

Vector Construction

The Lat targeting vector was assembled using genomic fragments isolated from a 129/Ola

phage library (Aguado et al., 2002; Nunez-Cruz et al., 2003). A 5’ LoxP site was inserted by

PCR during the assembly steps. Briefly, using a 490 bp EcoNI fragment encompassing a

portion of exon 1, exon 2 and exon 3, the 5’ LoxP site was inserted in the first intron of the

Lat gene by two-step PCR amplification using the following oligonucleotides (LAT Ec NIS:

5’ GGC CTG TGG CAG GTG CTC TGC; LAT EcoNIAS: 5’ ACC CTC GAG GAG GGC CAG

AGA C; LATLoxPS: 5’ ATA ACT TCG TAT AAT GTA TGC TAT ACG AAG TTA TGA GCT

CTA CCT GGG CCC TGA TTG A; LATLoxPAS: 5’ ATA ACT TCG TAT AGC ATA CAT TAT

ACG AAG TTA TGG GAC AGT GGA GAG TGC A). The 490 bp EcoNI fragment containing

the 5’ LoxP was then reintroduced into the whole Lat gene. Using ET recombination (Zhang

et al., 2000), a pIRES2-DTREGFP-loxP-Neor-loxP (Kissenpfennig et al., 2005) cassette was

then inserted 114 bp downstream of the Lat stop codon present in exon 12. Briefly, the

chloramphenicol-resistance gene was amplified by PCR from plasmid pACYC184 using a

pair of specific primers (5’ GAT CCC ATT TGA CCT CTG CCT TGC CAC AGC CTG

AGA ATC TTC CCC TAA CTT ATT GTC ACT GTC GAC AAC GAA GCG CTA ACC

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GTT TTT AT and 5’ GAT TCA TTC TCA GGC TTT ATC AGA AGG TAC AGA ATA

TTG GGG ACA CAG ACT GGA CCC CAA GTC GAC AGC GCT GAT GTC CGG CGG

TGC TT). In addition to sequence specific for the chloramphenicol-resistance gene

(underlined), each primer also contained a SalI site (indicated in italics) and a sequence

homologous to the chosen site of integration within the 3' untranslated region of the Lat gene

(specified in bold). Clones containing a correctly inserted chloramphenicol-resistance gene

were selected, the chloramphenicol-resistance gene was excised by SalI digestion, and the

pIRES2-DTREGFP-loxP-Neor-loxP cassette cloned into the SalI sites. The resulting targeting

vector was finally abutted to a HSV Thymidine Kinase (TK) gene.

Isolation of recombinant ES Cell clones

Following electroporation (250V, 500µF) of CK35 129/SV ES cells (Kress et al., 1998) and

selection in G418 and gancyclovir, colonies were first screened for homologous

recombination by PCR utilizing a pair of oligonucleotides corresponding to a sequence

outside of the 5’ homology arm and to the 5’ loxP site. Correctly recombined clones were

then expanded and verified by Southern blot analysis. The 3’ external single-copy probe

corresponding to a 1.4-kb EcoRI-HindIII fragment. When tested on BamHI-digested DNA, it

hybridized either to a 6.6 kb wild-type fragment or to a 7.6 kb recombinant fragment (Figure

S1). Proper recombination at the 5’ end was further verified by Southern blot using a 600 bp

5’ internal BamHI-XbaI probe. Finally, a neomycin probe was used to ensure that adventitious

non-homologous recombination events had not occurred in the selected clones.

Production of mutant mice

Correctly recombined ES cell clones were electroporated with a CRE-EGFP fusion expression

vector (Gagneten et al., 1997) to remove the floxed Cre-NeoR cassette. One day following

transfection, EGFP+ ES cells were sorted by flow cytometry and cloned. Clones were then

screened for the deletion of the floxed NeoR cassette by PCR amplification and Southern blot

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analysis. CRE-mediated deletion of the floxed NeoR cassette can be visualized by the

presence of a 8.6 kb fragment using BamHI-digested DNA hybridized with the 3’ external

single-copy probe (Figure S1). Mutant ES clones were injected into BALB/c blastocysts.

Male chimeras exhibiting over 60% of chimerism, as judged by coat color, were crossed with

C57BL/6 females, and the resulting F1 progeny tested for germline transmission. Tails of F1

progeny were tested by PCR with the following oligonucleotides: BM8589: 5’ GCT CTC

CAT CCA CCT CCA TGG, LATP2: 5’ GAT GAT CAC CCA CAG GTT CAA GAC CAG,

and EGFP: 5’ CAA GTC CGC CAT GCC CGA AGG CTA CG (Figure S1). Although the

DTR sequence present in the IRES2-DTREGFP-loxP cassette was fused in frame to an EGFP

sequence, very low levels of EGFP expression were observed in the resulting Latfl-dtr mice

(data not shown). Thymocytes and T cells from homozygous Latfl-dtr mice expressed

approximately 1.2-fold less LAT molecules than those of wild-type mice (Figure S2) and

showed no impairment in T cell development and peripheral functions (data not shown), a

feature consistent with the normal phenotype of mice that harbor a single functional copy of

the Lat gene and express two-fold less LAT molecules (Nunez-Cruz et al., 2003; Zhang et al.,

1999).

Retroviral vectors

A Cre cDNA was amplified by PCR from the pMC-Cre vector (Gu et al., 1993) and cloned

into the EcoRI-XhoI sites of the pMX-IRES-GFP retroviral vector (Kitamura et al., 2003).

Large-scale production of the resulting pMX-CRE-IRES-GFP vector was performed with the

Endotoxin-free Plasmid Mega kit (Qiagen). PLAT-E cells (Morita et al., 2000) were cultured

in DMEM medium containing 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM

sodium pyruvate, 2 mM glutamine, 50 µM β-mercaptoethanol, 10 µg/ml blasticidin and 1

µg/ml puromycin. PLAT-E cells were transfected with lipofectamin 2000 (Invitrogen)

according to the manufacturer’s instructions. DNA-lipid complexes were applied to PLAT-E

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cells plated one day prior transfection (32 x 106 cells per 175 cm2 flasks) in PLAT-E medium

without antibiotics. After 6 h at 37°C, 18 ml of PLAT-E medium without antibiotics was

added to the flask. Twelve hours later, medium was replaced with complete PLAT-E medium

(30 ml/flask) and cells were incubated at 32°C. After 48 and 72 hours, retroviral supernatants

were collected, centrifuged to eliminate residual cells and sterilized via 0.2 µm filtration.

T cell isolation and activation

CD4+ T cells were isolated from lymph nodes and spleens by magnetic positive selection

(Dynal, Invitrogen). The resulting cell preparations contained ≥95% CD4+ T cells. CD4+ T

cells were cultured in 6-well plate (0.6 x 106 cells/ml) in RPMI-1640 supplemented with 10%

FCS, 1 mM sodium pyruvate, 2 mM glutamine, primocin (Amaxa) and 50 µM β-

mercaptoethanol and stimulated with plate-bound anti-CD3 antibodies (coating solution:

5µg/ml) and soluble anti-CD28 (4µg/ml) antibodies. After 48 h, activated CD4+ T cells were

harvested and infected with CRE-encoding retrovirus. To facilitate Cd28–/– T cell activation,

IL-2 (5-10 U/ml) was added to the culture medium.

Retroviral infection and selection of infected cells

Activated CD4+ T cells (1 x 106 cells) were resuspended in 1.5 ml of supernatant from viral or

mock-infected PLAT-E cells in the presence of IL-2 (5-10 U/ml) and polybrene (8 µg/ml;

Sigma), distributed in 24-well plates (1ml/well) and centrifuged for 2 hours at 520 g in a

centrifuge pre-heated at 32°C and for 2 additional hours in a 32°C incubator. One ml of

medium containing IL-2 was then added to each well and the plates incubated for 12 h at

37°C. CD4+ cells were harvested and “spin-infected” a second time using the same protocol.

The following day, the cells were harvested, distributed in 6-well plates (2 x 106 cells/4 ml) in

the presence of IL-2 (5-10 U/ml) and selected for 48 hours in the presence of DT (240 ng/ml)

(List Biological Laboratories). Prior to adoptive transfer, cells were resuspended in fresh

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medium containing 240 ng/ml DT and 5-10 U/ml IL-2 and incubated for 30min at 37°C to

insure that any residual DTR+ CD4+ T cells will be killed by DT.

Preparation of activated CD4+ T cells deprived of Treg cells. CD4+ cells isolated from

Foxp3EGFP mice (Wang et al., 2008) were activated for 48 hours in the presence of anti-CD3

and anti-CD28 antibodies and further incubated in the presence of IL-2 (5-10U/mL) for 48

hours. EGFP− cells were purified by FACS and mixed with graded numbers of Treg cells

(EGFP+) freshly isolated from Foxp3EGFP mice. Samples were first analyzed by flow

cytometry to verify the frequency of Treg cells and then injected into Cd3eΔ5/Δ5 recipients.

Mice were analyzed 5-weeks after reconstitution.

Vβ repertoire analysis

CD4+ T cells from the specified mice were analyzed by intracellular staining using a mouse

Vβ TCR screening panel (BD Pharmingen).

Antibodies

Fluorescein isothiocyanate-conjugated anti-TCRβ (H57-597), anti-CD5 (53-7-3), anti-CD44

(IM7), anti-CD25 (PC61), anti-CD3ε (145-2C11), anti-CD62L (MEL-14); phycoerythrin-

conjugated anti-CD5 (53-7-3), anti-CD4 (RM4-5), anti-CD3ε (145-2C11), anti-CD95 (Jo2),

anti-IL-2 (IES6-5H4); phycoerythrin-indodicarbocyanine (Cy5)-conjugated anti-CD5 (53-7-

3); peridinin-chlorophyll-protein complex-indodicarbocyanine (Cy5.5)-conjugated anti-CD3ε

(145-2C11), anti-B220 (RA3-6B2); allophycocyanin-conjugated anti-CD3ε (145-2C11), anti-

CD4 (RM4-5), anti-B220 (RA3-6B2), anti-IL-4 (11B11); phycoerythrin-indotricarbocyanine

(Cy7)-conjugated anti-CD4 (RM4-5) ; Pacific Blue-conjugated anti-CD4 (RM4-5) antibodies

were all purchased from BD Biosciences. Phycoerythrin-conjugated anti-CD44 (IM7), anti-

MHC Class II (I-A/I-E) (M5/114.15.2); phycoerythrin-indodicarbocyanine (Cy5.5)-

conjugated anti-TCRβ (H57-597); phycoerythrin-indotricarbocyanine (Cy7)-conjugated anti-

CD44 (IM7) antibodies were all obtained from eBioscience. Biotinylated polyclonal goat anti-

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hHB-EGF (Diphteria Toxin Receptor) antiserum was obtained from R&D Systems. For LAT

intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm and

Perm/Wash buffers (BD Pharmingen), and incubated for 30 min at 4°C in the presence of

supernatant of the B32.8. rat anti-mouse LAT antibody (Ardouin et al., 2005). After three

washes in Perm/Wash buffer, cells were incubated with a polyclonal goat anti-rat Ig

secondary antibody (Multiple adsorption grade; BD Pharmingen) for 30 min at 4°C. Cells

were then washed and labelled with antibodies directed against surface markers.

References

Aguado, E., Richelme, S., Nunez-Cruz, S., Miazek, A., Mura, A.M., Richelme, M., Guo, X.J.,

Sainty, D., He, H.T., Malissen, B., and Malissen, M. (2002). Induction of T helper type 2

immunity by a point mutation in the LAT adaptor. Science 296, 2036-2040.

Ardouin, L., Rolink, A.G., Mura, A.M., Gommeaux, J., Melchers, F., Busslinger, M.,

Malissen, M., and Malissen, B. (2005). Rapid in vivo analysis of mutant forms of the LAT

adaptor using Pax5-Lat double-deficient pro-B cells. Eur J Immunol 35, 977-986.

Gagneten, S., Le, Y., Miller, J., and Sauer, B. (1997). Brief expression of a GFP cre fusion

gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions. Nucleic

Acids Res 25, 3326-3331.

Gu, H., Zou, Y.R., and Rajewsky, K. (1993). Independent control of immunoglobulin switch

recombination at individual switch regions evidenced through Cre-loxP-mediated gene

targeting. Cell 73, 1155-1164.

Kissenpfennig, A., Henri, S., Dubois, B., Laplace-Builhe, C., Perrin, P., Romani, N., Tripp,

C.H., Douillard, P., Leserman, L., Kaiserlian, D., et al. (2005). Dynamics and function of

Langerhans cells in vivo: dermal dendritic cells colonize lymph node areas distinct from

slower migrating Langerhans cells. Immunity 22, 643-654.

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Kitamura, T., Koshino, Y., Shibata, F., Oki, T., Nakajima, H., Nosaka, T., and Kumagai, H.

(2003). Retrovirus-mediated gene transfer and expression cloning: powerful tools in

functional genomics. Exp Hematol 31, 1007-1014.

Kress, C., Vandormael-Pournin, S., Baldacci, P., Cohen-Tannoudji, M., and Babinet, C.

(1998). Nonpermissiveness for mouse embryonic stem (ES) cell derivation circumvented by a

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Morita, S., Kojima, T., and Kitamura, T. (2000). Plat-E: an efficient and stable system for

transient packaging of retroviruses. Gene Ther 7, 1063-1066.

Nunez-Cruz, S., Aguado, E., Richelme, S., Chetaille, B., Mura, A.M., Richelme, M., Pouyet,

L., Jouvin-Marche, E., Xerri, L., Malissen, B., and Malissen, M. (2003). LAT regulates

gammadelta T cell homeostasis and differentiation. Nat Immunol 4, 999-1008.

Wang, Y., Kissenpfennig, A., Mingueneau, M., Richelme, S., Perrin, P., Chevrier, S., Genton,

C., Lucas, B., DiSanto, J.P., Acha-Orbea, H., et al. (2008). Th2 lymphoproliferative disorder

of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive

to the action of Foxp3+ regulatory T cells. J Immunol 180, 1565-1575.

Zhang, W., Sommers, C.L., Burshtyn, D.N., Stebbins, C.C., DeJarnette, J.B., Trible, R.P.,

Grinberg, A., Tsay, H.C., Jacobs, H.M., Kessler, C.M., et al. (1999). Essential role of LAT in

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recombination in Escherichia coli. Nat Biotechnol 18, 1314-1317.

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Figure S1. Generation and identification of Lat fl-dtr mice

(A) Strategy used to produce the Latfl-dtr “knock-in” mice. (1) Partial restriction map of the wild-type Lat locus. Exons are depicted as black filled boxes and untranslated regions as grey filled boxes. Xb: XbaI, B: BamHI, and Bg: BglII. The 3’ single-copy probe used to verify proper homologous recombination events by Southern blot analysis is shown. (2) Targeting vector used for simultaneous introduction of a LoxP site in the first intron of Lat and of an IRES-DTREGFP-loxP-Neor-loxP cassette into the 3’untranslated region of Lat. LoxP sites are shown as filled triangles. TK: thymidine kinase expression cassette, I: IRES, E: EGFP, D: DTR. (3) Structure of the targeted allele following homologous recombination. (4) Structure of the targeted allele following expression of the Cre recombinase in ES clones and excision of the Neor cassette. (B) Southern blot analysis of wild-type (WT) and recombinant ES clones prior to (lane 1) and following (lanes 2 to 4) CRE expression. ES cell DNA was digested by BamHI and hybridized to the 3' single-copy probe shown in (A). (C) Genotyping of tail DNA from wild-type mice and from heterozygous or homozygous mice for the Latfl-dtr allele using the mix of three PCR primers (BM8589, LAT P2 and EGFP) shown as black arrows in (A).

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Figure S2. Latfl-dtr/Y136F and Latfl-dtr/– mice generate peripheral T cells that display a phenotype comparable to wild-type peripheral T cells Splenocytes were isolated from wild-type, Latfl-dtr/Y136F, Latfl-dtr/- and LatY136F/Y136F mice. (A) Expression of CD4 and CD8 on total splenocytes. Numbers in outlined areas indicate percent CD4+ and CD8+ T cells. (B) IL-2 and IL-4 production in single cells. CD4+ splenocytes were cultured for 4 h in the presence of PMA and ionomycin. Numbers in outlined areas indicate percentage of cells. (C) Expression of TCRβ, CD95, CD5, CD44, CD62L and CD25 on CD4+ splenocytes isolated from the specified mice (see key below histograms). Dashed line: isotype control staining. (D) Expression of MHC class II and CD95 molecules on B220+ splenocytes from the specified mice. Dashed line: isotype control staining. (E) CD4+ T cells isolated from Latfl-dtr/Y136F and Latfl-dtr/– mice express a normal Vβ repertoire. Cells were stained with a panel of TCRVβ antibodies and the frequency of positive cells found in two mice of each genotype is shown. Latfl-dtr/Y136F CD4+ T cells activated for 6 days with anti-CD3 and anti-CD28 antibodies are also shown. (F) Flow cytometry of the intracellular expression of LAT in the specified CD4+ T cell populations. Data are representative of two independent experiments.

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Figure S3. Deletion of the Latfl-dtr allele in mature peripheral CD4+ T cells

(A) Experimental protocol used for the preparation of postthymic CD4+ T cells equipped with the sole LATY136F molecules or deprived of wild-type LAT molecules. CD4+ T cells purified from Latfl-

dtr/Y136F or from Latfl-dtr/– mice and activated for 48 hours were exposed to viral or control supernatants and subsequently treated with diphtheria toxin (DT). (B) Expression of diphteria toxin receptor (DTR) and of retroviral-encoded GFP was monitored 4 days after activation and prior to DT treatment in cells exposed to viral (+ Cre) or control (– Cre) supernatants (left panels). After 48 h in the presence (+ DT) or absence (– DT) of DT (day 6 after T cell isolation), surviving CD4+ T cells were gated using SSC and FSC plots and their levels of DTR and GFP were determined (middle and right panels). Numbers in outlined areas indicate percent infected (DTR–EGFP+; red lines) and non-infected cells (DTR+EGFP-; green lines). (C) Cre expression and DT treatment resulted in the complete deletion of the Latfl-dtr allele. Latfl-dtr/Y136F and Latfl-dtr/– CD4+ T cells were subjected to Cre expression and DT selection. DNA prepared from the resulting LatΔ/Y136F and LatΔ/– CD4+ T cells was analyzed using a mix of three PCR primers. Also shown is the expected size for the Cre-unexposed (500 bp) and -exposed (1000 bp) Latfl-dtr allele. In the case of LatY136F allele, the size of the PCR product expected to result from the use of primers 1 and 3 is too long to be detected through the PCR conditions used whereas the Lat– allele gives rise to a 775 bp PCR product. Below, schematic structure of the Latfl-dtr allele prior to or following Cre-mediated recombination. Position of exons (filled boxes), loxP sites (triangles), IRES-hDTR cassette (empty box) and PCR primers (arrows) is indicated. (D) Flow cytometry of the surface expression of hDTR (left panel) and of the intracellular expression of LAT (right panel) in the specified CD4+ T cell populations. Data are representative of 10 (B) and 4 (C-D) independent experiments.

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Figure S4. In vitro activated CD4+ T cells depleted of Treg cells prior to their transfer into Cd3eΔ5/Δ5 host do not give rise to a Th2 lymphoproliferative disorder CD4+ T cells isolated from Foxp3EGFP mice were stimulated for 48 hours with CD3 and CD28 antibodies and then cultured in the presence of IL-2 for 48 hours. At the end of the culture, EGFP− CD4+ T cells were sorted by FACS and transferred into Cd3eΔ5/Δ5 hosts without (0% Treg cell sample) or with graded numbers of Treg cells freshly purified from Foxp3EGFP mice (2%, 10% and 20% Treg cell samples). (A) Frequency of Treg cells (EGFP+) in the various CD4+ T cell samples prior to their injection into Cd3eΔ5/Δ5 hosts. (B) CD4+ T cells found in the spleen of Cd3eΔ5/Δ5 recipients 5-weeks after transfer of the inoculum analyzed in (A) were cultured for 4 hours in the presence of PMA and ionomycin and analyzed for IL-2 and IL-4 production. (C) Quantification of results shown in (B) and presented as % IL-4+ cells among CD4+ T cells. Values obtained for Cd3eΔ5/Δ5 recipients 5 weeks after transfer of Latfl-dtr/Y136F or LatΔ/Y136F CD4+ T cells are also indicated for comparison. (D) Number of CD4+ T cells present in the spleen of Cd3eΔ5/Δ5 recipients 5 weeks after reconstitution with Latfl-

dtr/Y136F, LatΔ/Y136F CD4+ T cells and with the CD4+ T cell samples specified in (A). (E) Expression of TCRβ, CD5 and CD95 on CD4+ splenocytes from wild-type mice (WT), LatY136F/Y136F mice and Cd3eΔ5/Δ5 recipients 5 weeks after reconstitution with the CD4+ T cell samples specified in (A). Data are representative of 2 independent experiments.

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Figure S5. Development of Th2-commited effectors in LatY136F/Y136F 4get newborn mice.

(A) The absolute numbers of CD4+ T cells and percentage of IL-4+ (GFP+) CD4+ T cells present in the spleen of 4get and LatY136F/Y136F 4get mice were studied from 3 to 18 days after birth. Data represent the mean value obtained using 2 to 8 mice per time point. (B) Dot plots show the CD3 versus GFP profile of CD4+ T cells isolated from the spleen and lymph nodes of 15-day-old 4get and LatY136F/Y136F

4get mice. The percentages indicated correspond to IL-4+ (GFP+), CD3– and IL-4– (GFP–), CD3+ CD4+ cells. Data are representative of 2 independent experiments.

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Figure S6. Adoptively transferred LatΔ/– CD4+ T cells convert into pathogenic cells similar to those developing in LatY136F/Y136F mice Analysis of the CD4+ T cells and B220+ B cells found in the spleen of Cd3eΔ5/Δ5 hosts 7- to 8-weeks after reconstitution with Latfl-dtr (control: CTR) or LatΔ/– (floxed:F) CD4+ T cells. (A) Number of total and of CD4+ T cells present in the spleen. (B) Percentage of IL-4-producing CD4+ T cells following a 4-hour stimulation with PMA and ionomycin. (C) Geometric mean of TCR, CD95 and CD5 surface expression on CD4+ T cells. (D) Geometric mean of MHC class II expression (left panel) and frequency of CD95+ cells (right panel) among B220+ splenocytes. (E) Concentrations of IgG1 and IgE antibodies in serum samples. The mean (horizontal bar) and standard deviation are indicated for each panel. Data are representative of 3 independent experiments performed on 9 and 14 recipients reconstituted with Latfl-dtr and LatΔ/– CD4+ T cells, respectively.

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Figure S7. Decreased TCR-induced tyrosine phosphorylation in CD4+ T cells isolated from 6-week-old LatY136F/Y136F mice

CD4+ T cells isolated from 6-week-old wild-type (Lat+/+) and LatY136F/Y136F mice were left untreated (-) or were stimulated (+) with CD3 and CD4 antibodies for 2 min at 37°C or for the specified times. (A) Whole cell lysates were probed with antibody specific for phosphotyrosine (pY) or LAT. (B) TCR-inducible tyrosine phosphorylation of specific proteins was assessed by immunoblotting with the indicated phosphotyrosine-specific antibodies and with antibodies specific for the corresponding proteins. Data are representative of 4 independent experiments. (C) TCR-induced phosphorylation of Akt and of the ERK1 and ERK2 kinases. Cells were left unstimulated or stimulated with anti-CD3 + anti-CD4 for 2, 5, 10 and 30 min, and whole-cell lysates were blotted for phospho-ERK1/2, total levels of ERK1/2, phospho-Akt and total levels of Akt. Note that activation of CD4+ T cells isolated from 6-week-old LatY136F/Y136F mice with anti-CD3 and anti-CD28 was unable to relieve the global decrease in protein phosphorylation observed following activation with anti-CD3 and anti-CD4 (data not shown).

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Figure S8. TCR expressed at the surface of LatΔ/Δ CD4+ T cells are capable of turning on the PI3K lipid kinase

TCR-induced phosphorylation of the Akt kinase and of the glycogen synthase kinase-3β (GSK-3β). LatΔ/Δ and LatΔ/ CD4+ T cells were left unstimulated or stimulated with anti-CD3 and anti-CD4 for 2 min and whole-cell lysates were blotted for phospho-Akt, phospho-GSK-3β and total levels of Akt.