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Supplementry Figure S1 - UTR m - 3HA - 2-1 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 2.1 2. 3.4 1.1 1.8 2.1 2.7 1.3.1 1.4.6.1 Trnsgeni founder TG expression (ompred to endo. ) Trnsgeni 37 - β-tin 1.8.9 2.3 2.2 3.4 1.3 1.9.9 2.2 2.7 1.7.8 1.9 1.1.1 d Thymus DP 9 CD4SP 8.9 24 Totl expression (TG + endo.) LN CD8SP 73 29 CD4+ 68 12 CD8+ 86 1 84 96.2 1.1 17 8 38.72.23 9 1.4.11 98 1.6 97.4.3.21.18 HA TG 3 - CD4+ f Trnsgeni CD8+ Control TG- 1 1.3 1.7 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.

Supplementry Figure S2 Totl Thymoytes CD4SP CD8SP LN Spleen 13 78 4.7.1 4.7 14 9.7 4 1 4 27 43 16 14 77 1 2 38 4.6 4.8 7.4 48 27 16 CD4 13 77.7 4.9 CD69.6 12 9.1 CD24 1 3 CD4 2 26 47 19 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.

Supplementry Figure S3 TG- 2 2 2 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.

Supplementry Figure S4 CD4 + CD8 + 12 ***.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.

Supplementry Figure S NP-speifi IgG 2.7.6..4.3.2.1. NP-KLH NP-KLH NP-KLH Alum Alum Alum 2 3 4 6 Serum dilution (-log 1 ) NP-speifi IgG 3.2.2.1.1. NP-KLH NP-KLH NP-KLH Alum Alum Alum. 2 3 4 6 Serum dilution (-log 1 ) NP-speifi IgG 3.7.6..4.3.2.1. NP-LPS NP-LPS LNP-LPS PBS PBS PBS 2 3 4 6 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 = 1-2 - 1-6. 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 = 1-2 - 1-6. n = 3- mie per group.

Supplementry Figure S6 % survivl Dys post high dose H1N1 infetion CD8 + Foxp3 - CD44 hi Ki67 + ellls (%) 4 3 2 1 Spln Spln MLN **! MLN LP LP 9 dys 9 post T. gondii infetion infetion e CD8 + Foxp3 - CD44 hi IFNγ + ellls (%) EAE Clinil Sore 4 3 2 1 3 2 1 4 3 2 1 Spln Spln MLN Wt * **! MLN LP LP 1 1 2 2 1 1 2 2 d f CD8 + Foxp3 - CD44 hi TNFα + ellls (%) EAU Clinil Sore 2 1 1 Spln Wt 1. 1... 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.

Supplementry Figure S7 pm 4 4 3 3 2 2 1 1 TG- Wt effetor 2 12 62 312 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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