Supplementary Figure S1

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1 Supplementry Figure S1 - UTR m - 3HA hgh - 1 Uiquitin *! *! lk distl promoter m K3R/ K121R-3HA UTR hgh founder lines - HA - - founder lines TG- E1 L A2 B1 F9 G6 H4 H6 B C D2 G1 H3 J2 L - 7 IP: lk distl promoter Trnsgeni founder TG expression (ompred to endo. ) Trnsgeni 37 - β-tin d Thymus DP 9 CD4SP Totl expression (TG + endo.) LN CD8SP CD CD HA TG 3 - CD4+ f Trnsgeni CD8+ Control TG ell # e Figure S1. Design, vlidtion nd detetion of trnsgeni onstruts. () Shemti digrms of nd expression onstruts used to generte trnsgeni mie. () COS-7 ells were trnsfeted with either or onstruts. Whole ell lystes were immunopreipitted (IP) with nti- ntiody nd immunolotted with nti-uiquitin. * indite nds orresponding to uiquitinted. Detetion of hemglutinin (HA) nd in whole ell lystes served s protein loding ontrols. Dt re representtive of t lest three independent experiments. () Mthed pirs of independent mouse lines were estlished sed on trnsgene expression levels mesured in CD4+ or CD8+ purified lymph node ells s the rtio of trnsgeni (TG)/endogenous (endo) y Western lotting. Imge orresponds to CD8+ T ells nd the mthed pir of G6 nd D2 nimls. (d) Flow ytometry dot plots of gted thymoytes or lymph node (LN) ells from nd trnsgeni mie stined with nti-cd4+ nd nti-cd8+ ntiodies. Perentge of thymoytes expressing trnsgeni, deteted s HA positive ells, ws mrkedly inresed in mture CD4+ nd CD8+ single-positive (SP) thymoytes ompred with thymoytes undergoing the erlier doule-positive (DP) developmentl stge. The trnsgene ould e deteted in oth CD4+ nd CD8+ peripherl LN ells. (e) nd (f) Detetion y PCR nd FACS, respetively, of endogenous nd trnsgeni.

2 Supplementry Figure S2 Totl Thymoytes CD4SP CD8SP LN Spleen CD CD CD CD CD8 TCRβ CD3ε CD8 Thymus LN CD4 Foxp3 Figure S2. Thymus ellulrity nd development is mintined in mie. () Flow ytometri nlysis of thymoyte differen44on in trnsgene neg4ve ( ), nd trnsgeni mie. Stining of totl thymoytes with n4- CD4 nd n4- CD8 reveled no differenes in the numer (dt not shown) or perentge doule- neg4ve (DN), doule- posi4ve (DP), CD4SP (single- posi4ve) or CD8SP thymoytes. Dot plots orresponding to totl thymoytes stined with n4- CD69 nd n4- TCR reveled tht the numer (dt not shown) nd frequeny of thymoytes entering posi4ve sele4on (CD69 + TCR int ), immture SP ells (CD69 + TCR hi ), nd mture SP ells (CD69 - TCR hi ), were similr in, nd mie. Dot plots of gted CD4SP nd CD8SP thymoytes stined with n4- CD24 nd n4- CD3e showed tht frequenies of mture SP thymoytes (CD24 - CD3e hi ) were similr in nd oth nd trnsgeni nimls. Figures showing frequenies orrespond to represent4ve imges (n 3 nimls per group). () Flow ytometri studies showed similr numer (dt not shown) nd perentge of mture lymphoytes Ter stining lymph node (LN) ells nd splenoytes with n4- CD4 nd n4- CD8. () Flow ytometri studies show similr numer (dt not shown) nd perentge of T regs in thymus nd lymph node (LN) ells stined with n4- CD4 nd n4- Foxp3.

3 Supplementry Figure S3 TG TG- p (light) Trnsgeni 1! Light exposure p (drk) Trnsgeni p AU!!! 2.!! time (min)! PLCγ-1 Trnsgeni Totl p (TG+endo) AU! Drk exposure 1!!! 2.!! time (min)! Figure S3. phosphorylaon in TG-, nd expressing ells. () Western lovng for p (Tyr191), nd PLC- γ1 in whole ell lystes from purified CD4 + lymph node (LN) T ells from TG-, or trnsgeni mie s4multed with 3 μg/ml α- CD3ε + 1 μg/ml α- CD28 n4odies for the indited 4mes. () Qun4t4on of the undnes of p proteins rel4ve to orresponding totl PLC- γ1 protein from the experiment represented in (). AU, ritrry units.

4 Supplementry Figure S4 CD4 + CD ***.8 ell # 2 %perk + ell #.8 %perk + perk perk Figure S4. Lk of uiquianaon enhnes TCR- dependent ERK phosphorylaon in CD4 + T ells. Represent4ve histogrms from the flow ytometri nlysis of phospho- ERK (perk) stining in gted CD4 + or CD8 + lymphoytes tht were either uns4multed (gry, shded) or s4multed with 3 μg/ml α- CD3ε + 1 μg/ml α- CD28 n4odies for 2 minutes. Eh symol orresponds to n individul mouse nd the r denotes the verge + SEM in the grphs.

5 Supplementry Figure S NP-speifi IgG NP-KLH NP-KLH NP-KLH Alum Alum Alum Serum dilution (-log 1 ) NP-speifi IgG NP-KLH NP-KLH NP-KLH Alum Alum Alum Serum dilution (-log 1 ) NP-speifi IgG NP-LPS NP-LPS LNP-LPS PBS PBS PBS Serum dilution (-log 1 ) Figure S. AnAody produaon in, nd expressing mie. () nd () Produ4on of NP- speifi IgG 2 nd IgG 3 n4odies 14 dys Ter T- dependent (NP- KLH) immuniz4on mesured y ELISA (OD t 4nm). Seril dilu4ons: 2-6 = n = 3- mie per group. () NP- speifi n4ody 4ters (IgG 3 ) 2 dys Ter T- independent (NP- LPS in HBBS) immuniz4on. Seril dilu4ons: 2-6 = n = 3- mie per group.

6 Supplementry Figure S6 % survivl Dys post high dose H1N1 infetion CD8 + Foxp3 - CD44 hi Ki67 + ellls (%) Spln Spln MLN **! MLN LP LP 9 dys 9 post T. gondii infetion infetion e CD8 + Foxp3 - CD44 hi IFNγ + ellls (%) EAE Clinil Sore Spln Spln MLN Wt * **! MLN LP LP d f CD8 + Foxp3 - CD44 hi TNFα + ellls (%) EAU Clinil Sore Spln Wt Spln Mln Wt **! Mln 9 dys post T. gondii infetion 9 dys post T. gondii infetion LP Wt LP Dys post immuniztion Figure S6. In vivo models to test enhned funaon of expressing T ells. () Survivl of trnsgene neg4ve ( ), or expressing mie Ter hllenge with mouse- dpted influenz virus. Mortlity is expressed s the perentge of mie tht survived the hllenge. () Prolifer4on () IFNγ produ4on or (d) TNFα produ4on in CD8 + T ells in splenoytes (Spln), mesenteri lymph nodes (MLN) or lmin propri (LP) of mie infeted with 8 ysts of T. gondii. (e) EAE linil sore in indited mie. Dt is represented s men ± SEM. (f) EAU linil sore of histologil se4ons in indited mie 21 dys Ter immuniz4on. Dt is represented s men ± SEM.

7 Supplementry Figure S7 pm TG- Wt effetor Figure S7. Similr suppressive pity of T regs from TG-, nd mie. The suppression pili4es of sorted T reg ells from TG-, nd trnsgeni mie were evluted y o- ulturing effetor ells (,) with the indited numer of sorted CD4 + FoxP3 + ells in the presene of.μg/ml n4- CD3 plus APCs (,). ATer 72 hrs, ultures were pulsed with [ 3 H]thymidine. Dt re the men ± SEM.

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