blood mononuclear cells were stained with AAD, annexin, CD3, CD4, CD25, LAP and specific
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1 Supplementary figure legends Figure 1: Selective expression of regulatory markers on CD4 + LAP + T cells. Human peripheral blood mononuclear cells were stained with AAD, annexin, CD3, CD4, CD25, LAP and specific markers including GITR, CTLA-4 and PD-1. Filled blue profiles represent isotype control; empty profiles represent specific staining for different antibodies on selected populations. The percentage positive cells are indicated in each plot. Results are representative of 3 independent experiments. Figure 2: Suppressive activity of non-activated and activated CD4 + LAP + T cells. A, Proliferation of CFSE labeled responder T cells in absence and presence of CD4 + LAP + T cells. B, Sorted CD4 + LAP + T cells were activated with anti-cd3, anti-cd28 and IL-2 and were stained for expression of LAP. Black histograms represent CD4 + LAP - cells, red histograms represent CD4 + LAP + cells on day0 and blue histograms represent expression of LAP on CD4 + LAP + T cells after 48hrs of activation. C, Suppression mediated by activated or non-activated CD4 + LAP + T cells. Results are shown as mean and SD of four independent experiments. D, Effect of CD4 + LAP + T cells on IFN-γ and IL-2 secretion from responder T cells in a co-culture assay. E, Suppression of responder T cell proliferation mediated by CD4 + LAP + T cells in absence and presence of transwell.
2 Supplementary Figure 1
3 Supplementary Figure 2
4 Supplementary Table 1: List of antibodies used to determine signaling pathways in LAP + T cells Target Phospho-Elk-1 (Ser383) Antibody Phospho-p38 MAPK (Thr180/Tyr182) Phospho-p38 MAPK (Thr180/Tyr182) Phospho-p38 MAP Kinase (Thr180/Tyr182) Antibody Phospho-MKK3/MKK6 (Ser189/207) Antibody Phospho-ATF-2 (Thr71) Antibody Phospho-HSP27 (Ser82) Antibody Phospho-MAPKAPK-2 (Thr334) Antibody Phospho-SEK1/MKK4 (Thr261) Antibody Phospho-SAPK/JNK (Thr183/Tyr185) Antibody Phospho-ATF-2 (Thr71) Antibody Phospho-c-Jun (Ser63) II Antibody Phospho-Akt (Ser473) Antibody Phospho-Akt (Thr308) Antibody Akt Antibody Phospho-GSK-3beta (Ser9) Antibody Phospho-Raf (Ser259) Antibody Phospho-PTEN (Ser380) Antibody Phospho-PDK1 (Ser241) Antibody Phospho-Stat1 (Tyr701) Antibody Phospho-Stat2 (Tyr690) Antibody Phospho-Stat3 (Tyr705) Antibody Phospho-Stat3 (Ser727) Antibody Phospho-Stat5 (Tyr694) Antibody Phospho-Stat6 (Tyr641) Antibody Company Millipore
5 Phospho-c-Raf (Ser338) (56A6) Rabbit mab Phospho-Src Family (Tyr416) Antibody Phospho-Pyk2 (Tyr402) Pyk2 Phospho-MEK1/2 (Ser217/221) Antibody Phospho-MEK1/2 (Ser221) Phospho-p44/42 MAPK (Thr202/Tyr204) Phospho-p44/42 MAP Kinase (Thr202/Tyr204) Antibody Phospho-Erk 1/2 (Thr202/Tyr204) Phospho-p90RSK (Ser380) Antibody Phospho-PKC (pan) (BetaII Ser660) Antibody Phospho-PKCalpha/betaII (Thr638/641) Antibody Phospho-PKCdelta (Thr505) Antibody Phospho-PKCdelta (Ser643) Antibody Phospho-PKD/PKCmu (Ser744/748) Antibody Phospho-PKD/PKCmu (Ser916) Antibody PKD/PKCmu Antibody Phospho-PKCtheta (Thr538) Antibody Phospho-PKCzeta/lambda (Thr410/403) Antibody Phospho-Lck (Tyr505) Antibody LAT (phospho Y191) antibody [E205] Phospho-Zap70 (Y315 + Y319) Phospho-ZAP70 (Tyr493) Phospho-CD19 (Tyr513) Antibody Phospho-Syk (Tyr323) Antibody Phospho-Syk (Tyr525/526) Antibody Syk Antibody Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody Phospho-BLNK (Tyr96) Antibody Phospho-PLCgamma2 (Tyr1217) Antibody Phospho-PLCgamma1 (Tyr771) Antibody Phospho-NFKB2 p100 (Ser866/870) Millipore
6 Phospho-NF-kappaB p105 (Ser933) Antibody Phospho-NF-κB p65 (Ser536) Antibody Phospho-IKKalpha/beta (Ser176/180) Antibody II Phospho-IkappaB-alpha (Ser32) Antibody Phospho-Akt Substrate (RXRXXS/T) (110B7) Rabbit mab Phospho-(Ser) PKC Substrate Antibody Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mab Phospho-SHIP1 Phospho-(Ser) CDKs Substrate Antibody Phospho-(Ser/Thr) ATM/ATR Substrate Antibody Phospho-IRAK1 (Ser376) Antibody Phospho-TAK1 (Thr184/187) (90C7) Rabbit mab Phospho-IRF-3 (Ser396) Antibody Phospho-IRAK (Thr209) Phospho-Smad2 (Thr220) Phospho-Smad2 (Ser465/467) Phospho-Smad3 (Ser423/425) Phospho-Smad1/5 (ser463/465) Phospho-Smad2 (Ser465/467) Phospho-mTor (Ser2448) Phospho-mTor (Ser 2481) Phospho-MKK7(Ser271/Thr275) Antibody Phospho-Tpl2 (Ser 400) Phospho-PI3K p85 (Tyr458)/p55 (Tyr199) Phospho-MARCKS (Ser ) Phospho-cFos (Thr232) Tpl2 Antibody PI3K p85 mtor Smad2 Smad5 Phospho-FoxO1 (Ser256) Antibody Prosci Prosci
7 Phospho-FoxO1 (Ser319)/FoxO4 (Ser262) Antibody Phospho-FoxO1 (Thr24)/FoxO3a (Thr32) Antibody Phospho-FoxO3a (Ser318/321) Antibody Phospho-FoxO3a (Ser253) Antibody FoxO1 Antibody FoxO3a Antibody FoxO4 Antibody IRAK4 IRAK-M Antibody IRAK antibody p38 MAP Kinase Antibody PKCα Antibody TAK1 Antibody Phospho-Src (Tyr527) Antibody FOXP3 RORgT Phospho-Stat1 (Ser727) Phospho-Stat5 (Tyr694) Acetyl-Stat3 (Lys685) Antibody Phospho-Stat1 (Ser727) Phospho-Stat5 (Tyr694) Phospho-Stat6 (Tyr641) Stat1 Stat2 Stat3 Stat5 Stat6 Smad3 Smad4 Smad6 Phospho-Smad2 (Ser467) Phospho-Smad3 (Ser423/425) Biolegend
8 Src Phospho-Src (Tyr529) Phospho-Src (Tyr418) Non-phospho-Src (Tyr527) IRF3 IRF4 IRF7 IRF8 Phospho-IRF-3 (Ser396) Aryl hydrocarbon Receptor p44/42 MAPK (Erk1/2) TRAF2 TRAF3 NIK Phospho-IKK alpha/beta (Ser176/180) IKK alpha NFkB2 p100/p52 RelB Raptor Rictor MyD88 Antibody Tollip Antibody TAB1 SOCS1 TRAF6 Phospho-Rac1/cdc42 (Ser71) Antibody Phospho-CREB (Ser133) ATF3 MEF2C MEF2C (phospho S387) MEF2C (phospho T300) Proteintech
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