Antibody data
- Antibody Data
- Antigen structure
- References [40]
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- Validations
- Flow cytometry [1]
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- Product number
- 47-0038-41 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- CD3 Monoclonal Antibody (UCHT1), APC-eFluor™ 780, eBioscience™
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- Description: The UCHT1 monoclonal antibody reacts with human CD3e, a 20 kDa subunit of the TCR complex. Along with the other CD3 subunits gamma and delta, the epsilon chain is required for proper assembly, trafficking and surface expression of the TCR complex. CD3 is expressed by thymocytes in a developmentally regulated manner and by all mature T cells. Crosslinking of TCR via immobilized UCHT1 initiates an intracellular biochemical pathway resulting in cellular activation and proliferation.
- Antibody clone number
- UCHT1
- Concentration
- 5 µL/Test
Submitted references A controlled human Schistosoma mansoni infection model to advance novel drugs, vaccines and diagnostics.
Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection.
Toll like receptor 7 expressed by malignant cells promotes tumor progression and metastasis through the recruitment of myeloid derived suppressor cells.
Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting.
A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers.
T cell phenotypes in women with Chlamydia trachomatis infection and influence of treatment on phenotype distributions.
A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism.
Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70.
Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells.
Biallelic mutations in DNA ligase 1 underlie a spectrum of immune deficiencies.
Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities.
Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells.
Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.
DNA Vaccination by Electroporation Amplifies Broadly Cross-Restricted Public TCR Clonotypes Shared with HIV Controllers.
CD151 Expression Is Associated with a Hyperproliferative T Cell Phenotype.
Staphylococcus aureus Downregulates IP-10 Production and Prevents Th1 Cell Recruitment.
Inflammatory Ly6C(high) Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation.
Distinct peripheral vs mucosal T-cell phenotypes in chlamydia-infected women.
Blocking the recruitment of naive CD4(+) T cells reverses immunosuppression in breast cancer.
Evidence for Resident Memory T Cells in Rasmussen Encephalitis.
CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment.
MAIT cells are activated during human viral infections.
Interaction among activated lymphocytes and mesenchymal cells through podoplanin is critical for a high IL-17 secretion.
B cells from African American lupus patients exhibit an activated phenotype.
Expansion of Inefficient HIV-Specific CD8 T Cells during Acute Infection.
Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.
Follicular regulatory T cells impair follicular T helper cells in HIV and SIV infection.
Induction of type I and type III interferons by Borrelia burgdorferi correlates with pathogenesis and requires linear plasmid 36.
Novel lentiviral vectors with mutated reverse transcriptase for mRNA delivery of TALE nucleases.
A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.
A polysaccharide virulence factor of a human fungal pathogen induces neutrophil apoptosis via NK cells.
CX₃CR1⁺ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22.
Human mesenchymal stromal cells modulate T-cell responses through TNF-α-mediated activation of NF-κB.
Trypan blue exclusion assay by flow cytometry.
MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.
Recombinant influenza virus carrying the respiratory syncytial virus (RSV) F85-93 CTL epitope reduces RSV replication in mice.
Mass spectrometry reveals changes in MHC I antigen presentation after lentivector expression of a gene regulation system.
Brefeldin A, but not monensin, enables flow cytometric detection of interleukin-4 within peripheral T cells responding to ex vivo stimulation with Chlamydia trachomatis.
Changes in antigen-specific T-cell number and function during oral desensitization in cow's milk allergy enabled with omalizumab.
Epigenetic biomarkers of T-cells in human glioma.
Langenberg MCC, Hoogerwerf MA, Koopman JPR, Janse JJ, Kos-van Oosterhoud J, Feijt C, Jochems SP, de Dood CJ, van Schuijlenburg R, Ozir-Fazalalikhan A, Manurung MD, Sartono E, van der Beek MT, Winkel BMF, Verbeek-Menken PH, Stam KA, van Leeuwen FWB, Meij P, van Diepen A, van Lieshout L, van Dam GJ, Corstjens PLAM, Hokke CH, Yazdanbakhsh M, Visser LG, Roestenberg M
Nature medicine 2020 Mar;26(3):326-332
Nature medicine 2020 Mar;26(3):326-332
Immunotherapeutic Blockade of CD47 Inhibitory Signaling Enhances Innate and Adaptive Immune Responses to Viral Infection.
Cham LB, Torrez Dulgeroff LB, Tal MC, Adomati T, Li F, Bhat H, Huang A, Lang PA, Moreno ME, Rivera JM, Galkina SA, Kosikova G, Stoddart CA, McCune JM, Myers LM, Weissman IL, Lang KS, Hasenkrug KJ
Cell reports 2020 Apr 14;31(2):107494
Cell reports 2020 Apr 14;31(2):107494
Toll like receptor 7 expressed by malignant cells promotes tumor progression and metastasis through the recruitment of myeloid derived suppressor cells.
Dajon M, Iribarren K, Petitprez F, Marmier S, Lupo A, Gillard M, Ouakrim H, Victor N, Vincenzo DB, Joubert PE, Kepp O, Kroemer G, Alifano M, Damotte D, Cremer I
Oncoimmunology 2019;8(1):e1505174
Oncoimmunology 2019;8(1):e1505174
Enhanced Anti-lymphoma Activity of CAR19-iNKT Cells Underpinned by Dual CD19 and CD1d Targeting.
Rotolo A, Caputo VS, Holubova M, Baxan N, Dubois O, Chaudhry MS, Xiao X, Goudevenou K, Pitcher DS, Petevi K, Kachramanoglou C, Iles S, Naresh K, Maher J, Karadimitris A
Cancer cell 2018 Oct 8;34(4):596-610.e11
Cancer cell 2018 Oct 8;34(4):596-610.e11
A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers.
Claireaux M, Galperin M, Benati D, Nouël A, Mukhopadhyay M, Klingler J, de Truchis P, Zucman D, Hendou S, Boufassa F, Moog C, Lambotte O, Chakrabarti LA
mBio 2018 May 8;9(3)
mBio 2018 May 8;9(3)
T cell phenotypes in women with Chlamydia trachomatis infection and influence of treatment on phenotype distributions.
Ogendi BMO, Bakshi RK, Gupta K, Kapil R, Brown LT, Jordan SJ, Sabbaj S, Press CG, Lee JY, Geisler WM
Microbes and infection 2018 Mar;20(3):176-184
Microbes and infection 2018 Mar;20(3):176-184
A human anti-IL-2 antibody that potentiates regulatory T cells by a structure-based mechanism.
Trotta E, Bessette PH, Silveria SL, Ely LK, Jude KM, Le DT, Holst CR, Coyle A, Potempa M, Lanier LL, Garcia KC, Crellin NK, Rondon IJ, Bluestone JA
Nature medicine 2018 Jul;24(7):1005-1014
Nature medicine 2018 Jul;24(7):1005-1014
Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70.
Zhao M, Svensson MND, Venken K, Chawla A, Liang S, Engel I, Mydel P, Day J, Elewaut D, Bottini N, Kronenberg M
Nature communications 2018 Jul 6;9(1):2627
Nature communications 2018 Jul 6;9(1):2627
Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells.
Li Z, Zeppa JJ, Hancock MA, McCormick JK, Doherty TM, Hendy GN, Madrenas J
Journal of immunology (Baltimore, Md. : 1950) 2018 Feb 15;200(4):1471-1479
Journal of immunology (Baltimore, Md. : 1950) 2018 Feb 15;200(4):1471-1479
Biallelic mutations in DNA ligase 1 underlie a spectrum of immune deficiencies.
Maffucci P, Chavez J, Jurkiw TJ, O'Brien PJ, Abbott JK, Reynolds PR, Worth A, Notarangelo LD, Felgentreff K, Cortes P, Boisson B, Radigan L, Cobat A, Dinakar C, Ehlayel M, Ben-Omran T, Gelfand EW, Casanova JL, Cunningham-Rundles C
The Journal of clinical investigation 2018 Dec 3;128(12):5489-5504
The Journal of clinical investigation 2018 Dec 3;128(12):5489-5504
Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities.
Van Caeneghem Y, De Munter S, Tieppo P, Goetgeluk G, Weening K, Verstichel G, Bonte S, Taghon T, Leclercq G, Kerre T, Debets R, Vermijlen D, Abken H, Vandekerckhove B
Oncoimmunology 2017;6(3):e1283460
Oncoimmunology 2017;6(3):e1283460
Creation of an immunodeficient HLA-transgenic mouse (HUMAMICE) and functional validation of human immunity after transfer of HLA-matched human cells.
Zeng Y, Liu B, Rubio MT, Wang X, Ojcius DM, Tang R, Durrbach A, Ru Z, Zhou Y, Lone YC
PloS one 2017;12(4):e0173754
PloS one 2017;12(4):e0173754
Expression Profiles of Ligands for Activating Natural Killer Cell Receptors on HIV Infected and Uninfected CD4⁺ T Cells.
Tremblay-McLean A, Bruneau J, Lebouché B, Lisovsky I, Song R, Bernard NF
Viruses 2017 Oct 12;9(10)
Viruses 2017 Oct 12;9(10)
DNA Vaccination by Electroporation Amplifies Broadly Cross-Restricted Public TCR Clonotypes Shared with HIV Controllers.
Mukhopadhyay M, Galperin M, Patgaonkar M, Vasan S, Ho DD, Nouël A, Claireaux M, Benati D, Lambotte O, Huang Y, Chakrabarti LA
Journal of immunology (Baltimore, Md. : 1950) 2017 Nov 15;199(10):3437-3452
Journal of immunology (Baltimore, Md. : 1950) 2017 Nov 15;199(10):3437-3452
CD151 Expression Is Associated with a Hyperproliferative T Cell Phenotype.
Seu L, Tidwell C, Timares L, Duverger A, Wagner FH, Goepfert PA, Westfall AO, Sabbaj S, Kutsch O
Journal of immunology (Baltimore, Md. : 1950) 2017 Nov 1;199(9):3336-3347
Journal of immunology (Baltimore, Md. : 1950) 2017 Nov 1;199(9):3336-3347
Staphylococcus aureus Downregulates IP-10 Production and Prevents Th1 Cell Recruitment.
Li Z, Levast B, Madrenas J
Journal of immunology (Baltimore, Md. : 1950) 2017 Mar 1;198(5):1865-1874
Journal of immunology (Baltimore, Md. : 1950) 2017 Mar 1;198(5):1865-1874
Inflammatory Ly6C(high) Monocytes Protect against Candidiasis through IL-15-Driven NK Cell/Neutrophil Activation.
Domínguez-Andrés J, Feo-Lucas L, Minguito de la Escalera M, González L, López-Bravo M, Ardavín C
Immunity 2017 Jun 20;46(6):1059-1072.e4
Immunity 2017 Jun 20;46(6):1059-1072.e4
Distinct peripheral vs mucosal T-cell phenotypes in chlamydia-infected women.
Ogendi BMO, Bakshi RK, Sabbaj S, Brown L, Lee JY, Kapil R, Geisler WM
American journal of reproductive immunology (New York, N.Y. : 1989) 2017 Dec;78(6)
American journal of reproductive immunology (New York, N.Y. : 1989) 2017 Dec;78(6)
Blocking the recruitment of naive CD4(+) T cells reverses immunosuppression in breast cancer.
Su S, Liao J, Liu J, Huang D, He C, Chen F, Yang L, Wu W, Chen J, Lin L, Zeng Y, Ouyang N, Cui X, Yao H, Su F, Huang JD, Lieberman J, Liu Q, Song E
Cell research 2017 Apr;27(4):461-482
Cell research 2017 Apr;27(4):461-482
Evidence for Resident Memory T Cells in Rasmussen Encephalitis.
Owens GC, Chang JW, Huynh MN, Chirwa T, Vinters HV, Mathern GW
Frontiers in immunology 2016;7:64
Frontiers in immunology 2016;7:64
CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment.
Hotta R, Ohira M, Matsuura T, Muraoka I, Tryphonopoulos P, Fan J, Tekin A, Selvaggi G, Levi D, Ruiz P, Ricordi C, Vianna R, Ohdan H, Waldmann H, Tzakis AG, Nishida S
PloS one 2016;11(8):e0161618
PloS one 2016;11(8):e0161618
MAIT cells are activated during human viral infections.
van Wilgenburg B, Scherwitzl I, Hutchinson EC, Leng T, Kurioka A, Kulicke C, de Lara C, Cole S, Vasanawathana S, Limpitikul W, Malasit P, Young D, Denney L, STOP-HCV consortium, Moore MD, Fabris P, Giordani MT, Oo YH, Laidlaw SM, Dustin LB, Ho LP, Thompson FM, Ramamurthy N, Mongkolsapaya J, Willberg CB, Screaton GR, Klenerman P
Nature communications 2016 Jun 23;7:11653
Nature communications 2016 Jun 23;7:11653
Interaction among activated lymphocytes and mesenchymal cells through podoplanin is critical for a high IL-17 secretion.
Noack M, Ndongo-Thiam N, Miossec P
Arthritis research & therapy 2016 Jun 23;18:148
Arthritis research & therapy 2016 Jun 23;18:148
B cells from African American lupus patients exhibit an activated phenotype.
Menard LC, Habte S, Gonsiorek W, Lee D, Banas D, Holloway DA, Manjarrez-Orduno N, Cunningham M, Stetsko D, Casano F, Kansal S, Davis PM, Carman J, Zhang CK, Abidi F, Furie R, Nadler SG, Suchard SJ
JCI insight 2016 Jun 16;1(9):e87310
JCI insight 2016 Jun 16;1(9):e87310
Expansion of Inefficient HIV-Specific CD8 T Cells during Acute Infection.
Eller MA, Goonetilleke N, Tassaneetrithep B, Eller LA, Costanzo MC, Johnson S, Betts MR, Krebs SJ, Slike BM, Nitayaphan S, Rono K, Tovanabutra S, Maganga L, Kibuuka H, Jagodzinski L, Peel S, Rolland M, Marovich MA, Kim JH, Michael NL, Robb ML, Streeck H
Journal of virology 2016 Apr;90(8):4005-4016
Journal of virology 2016 Apr;90(8):4005-4016
Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets.
Wendt ER, Ferry H, Greaves DR, Keshav S
PloS one 2015;10(3):e0119532
PloS one 2015;10(3):e0119532
Follicular regulatory T cells impair follicular T helper cells in HIV and SIV infection.
Miles B, Miller SM, Folkvord JM, Kimball A, Chamanian M, Meditz AL, Arends T, McCarter MD, Levy DN, Rakasz EG, Skinner PJ, Connick E
Nature communications 2015 Oct 20;6:8608
Nature communications 2015 Oct 20;6:8608
Induction of type I and type III interferons by Borrelia burgdorferi correlates with pathogenesis and requires linear plasmid 36.
Krupna-Gaylord MA, Liveris D, Love AC, Wormser GP, Schwartz I, Petzke MM
PloS one 2014;9(6):e100174
PloS one 2014;9(6):e100174
Novel lentiviral vectors with mutated reverse transcriptase for mRNA delivery of TALE nucleases.
Mock U, Riecken K, Berdien B, Qasim W, Chan E, Cathomen T, Fehse B
Scientific reports 2014 Sep 18;4:6409
Scientific reports 2014 Sep 18;4:6409
A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.
Wiencke JK, Bracci PM, Hsuang G, Zheng S, Hansen H, Wrensch MR, Rice T, Eliot M, Kelsey KT
Epigenetics 2014 Oct;9(10):1360-5
Epigenetics 2014 Oct;9(10):1360-5
A polysaccharide virulence factor of a human fungal pathogen induces neutrophil apoptosis via NK cells.
Robinet P, Baychelier F, Fontaine T, Picard C, Debré P, Vieillard V, Latgé JP, Elbim C
Journal of immunology (Baltimore, Md. : 1950) 2014 Jun 1;192(11):5332-42
Journal of immunology (Baltimore, Md. : 1950) 2014 Jun 1;192(11):5332-42
CX₃CR1⁺ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22.
Longman RS, Diehl GE, Victorio DA, Huh JR, Galan C, Miraldi ER, Swaminath A, Bonneau R, Scherl EJ, Littman DR
The Journal of experimental medicine 2014 Jul 28;211(8):1571-83
The Journal of experimental medicine 2014 Jul 28;211(8):1571-83
Human mesenchymal stromal cells modulate T-cell responses through TNF-α-mediated activation of NF-κB.
Dorronsoro A, Ferrin I, Salcedo JM, Jakobsson E, Fernández-Rueda J, Lang V, Sepulveda P, Fechter K, Pennington D, Trigueros C
European journal of immunology 2014 Feb;44(2):480-8
European journal of immunology 2014 Feb;44(2):480-8
Trypan blue exclusion assay by flow cytometry.
Avelar-Freitas BA, Almeida VG, Pinto MC, Mourão FA, Massensini AR, Martins-Filho OA, Rocha-Vieira E, Brito-Melo GE
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas 2014 Apr;47(4):307-15
Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas 2014 Apr;47(4):307-15
MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.
Dössinger G, Bunse M, Bet J, Albrecht J, Paszkiewicz PJ, Weißbrich B, Schiedewitz I, Henkel L, Schiemann M, Neuenhahn M, Uckert W, Busch DH
PloS one 2013;8(4):e61384
PloS one 2013;8(4):e61384
Recombinant influenza virus carrying the respiratory syncytial virus (RSV) F85-93 CTL epitope reduces RSV replication in mice.
De Baets S, Schepens B, Sedeyn K, Schotsaert M, Roose K, Bogaert P, Fiers W, Saelens X
Journal of virology 2013 Mar;87(6):3314-23
Journal of virology 2013 Mar;87(6):3314-23
Mass spectrometry reveals changes in MHC I antigen presentation after lentivector expression of a gene regulation system.
Vogel R, Al-Daccak R, Drews O, Alonzeau J, Mester G, Charron D, Stevanovic S, Mallet J
Molecular therapy. Nucleic acids 2013 Feb 12;2(2):e75
Molecular therapy. Nucleic acids 2013 Feb 12;2(2):e75
Brefeldin A, but not monensin, enables flow cytometric detection of interleukin-4 within peripheral T cells responding to ex vivo stimulation with Chlamydia trachomatis.
Vicetti Miguel RD, Maryak SA, Cherpes TL
Journal of immunological methods 2012 Oct 31;384(1-2):191-5
Journal of immunological methods 2012 Oct 31;384(1-2):191-5
Changes in antigen-specific T-cell number and function during oral desensitization in cow's milk allergy enabled with omalizumab.
Bedoret D, Singh AK, Shaw V, Hoyte EG, Hamilton R, DeKruyff RH, Schneider LC, Nadeau KC, Umetsu DT
Mucosal immunology 2012 May;5(3):267-76
Mucosal immunology 2012 May;5(3):267-76
Epigenetic biomarkers of T-cells in human glioma.
Wiencke JK, Accomando WP, Zheng S, Patoka J, Dou X, Phillips JJ, Hsuang G, Christensen BC, Houseman EA, Koestler DC, Bracci P, Wiemels JL, Wrensch M, Nelson HH, Kelsey KT
Epigenetics 2012 Dec 1;7(12):1391-402
Epigenetics 2012 Dec 1;7(12):1391-402
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Supportive validation
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- Staining of normal human peripheral blood cells with Anti-Human CD19 APC (Product # 17-0199-42) and Mouse IgG1 K Isotype Control APC-eFluor® 780 (Product # 47-4714-82) (left) or Anti-Human CD3 APC-eFluor® 780 (right). Cells in the lymphocyte gate were used for analysis.
Supportive validation
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- Figure 6 Profile of T-lymphocytes stained with monoclonal antibody anti-CD3-FITCfollowed by treatment with propidium iodide (PI) and trypan blue (TB) at 0.002and 0.4% (w/v) or PBS (untreated control).
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- Figure 7 A , Percentage of dead human T-lymphocytes(CD3-FITC + cells) submitted to cell culture at temperatures (T)of 37degC (physiologic temperature) or 50degC (high-stress temperature) followed bystaining with trypan blue (TB) or propidium iodide (PI). B ,Dot-plot graph profile between human lymphocytes submitted to pretreatment withhigh-stress temperature (50degC) followed by staining with TB and PI andmonoclonal antibody anti-CD3-FITC + . C , Pearson'scorrelation test between dead CD3 + lymphocytes using PI and TB flowcytometry assays.
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- Figure 3 Functional analysis of NRTLV-delivered TALEN constructs. (a) Schematic vector design of I: LeGO-iTALEN-iG2 (wPRE), II: LeGO-iTALEN-iG2-wPRE-BGH-p(A) and III: LeGO-iTALEN-iG2-wPRE-SV40-p(A) 3 rd generation LVV derived from LeGO-system 11 . CMV = CMV-ie promoter; Delta (DeltaU3), R, U5 = elements of SIN-LTR, self-inactivating long terminal repeat; Psi = Psi , packaging signal; RRE = Rev response element; SFFV = promoter of spleen focus-forming virus; wPRE = Woodchuck hepatitis virus posttranscriptional regulatory element; IRES = internal ribosome entry site; eGFP = enhanced green fluorescent protein; p(A) = polyadenylation signal; BGH = bovine growth hormone; SV40 = simian virus 40. (b) Knockout of CCR5 in reporter cell line CCR5+/293T-cell clones were co-transduced with non-concentrated NRTLVs delivering different iTALEN-constructs with either no internal polyadenylation (p(A)) signal (iTALEN-wPRE), or internal BGH-p(A) (iTALEN-wPRE-BGH-p(A)) or SV40-p(A) (iTALEN-wPRE-SV40-p(A)) signals downstream of the wPRE-element, respectively. Mock-transduction and transduction of left or right TALEN-arms, only, served as negative controls (homodimers only). Measured for 3 independently produced vector preparations, each time in duplicates, *p
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- Figure 2 Single-cell PCR delivers sequences of functional CMV-specific TCRs. (A) PBMCs from Donor 1 were recovered and stained with HLA-B7/pp65 417-426 multimers. Dot plot shows the further analyzed CD8 and MHC multimer double-positive cell population. Cells were pre-gated on live lymphocytes (propidium iodide-negative, and CD3-positive). (B) TCR SCAN as described in Figure 1 and agarose gel electrophoresis of the resulting PCR products was performed. The photography shows the agarose gel. Upper row shows alpha-chain products matched with the respective beta-chains in the lower row. White boxes indicate alpha- and matched beta-chain-products derived from identical single cell samples. (C) The table summarizes the V- D- J- segment type and amino acid sequences of TCRs identified from CMV-multimer positive T cells in Figure 2A . TCR1A was identified 11 times and TCR 1B was detected once. (D) MHC multimer-positive T cells from the same donor were in vitro expanded and six T cell clones were successfully maintained. All clones contained TCR1A as confirmed by PCR and sequencing. The left FACS plot shows HLA-B7/pp65 417-426 staining and the right FACS plot shows staining with an irrelevant MHC multimer. (E) PBMCs from donor 2 were recovered and stained with HLA-B8/IE-1 199-207 multimers. The dot plot shows the further analyzed CD8 and MHC multimer double-positive cell population. Cells were pre-gated on living lymphocytes (propidium iodide negative and CD3 positive). (F) TCR SCAN a
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- Figure 3 Characterization of a small diverse T cell repertoire and transgenic expression of detected TCRs. (A) PBMCs from donor 3 were stained with HLA-B8/IE-1 88-96 multimers. Dot plot shows the further analyzed CD8 and MHC multimer double-positive cell population. Cells were pre-gated on living lymphocytes (propidium iodide-negative and CD3-positive). (B) A PCR slide with single antigen-specific T cells from Figure 3A were FACS-isolated. TCR SCAN as described in Figure 1 and agarose gel electrophoresis of the resulting PCR products was performed. Upper row shows alpha-chain products matched with the respective beta-chains in the lower row. White boxes indicate samples alpha- and matched beta-chain-products derived from identical single cell samples. (C) The table summarizes the V- D- J- segment type and amino acid sequences of TCRs identified from CMV-multimer positive T cells in Figure 3A . In three independent experiments we identified nine different TCRs (TCR 3A-I) (D) Pie chart indicates the prevalence of identified TCRs from donor 3. Percentages represent incidence of respective TCR divided by total number of positive samples. (E) Sequences from TCR 3D, 3E and 3G were expressed in Jurkat76 T cells by retroviral gene transfer. Non-transduced (left FACS plot) and transduced Jurkat76 T cells were analyzed for expression of CD3 and MHC multimer binding.
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- Figure 5 lp36 contributes to the association of B. burgdorferi with specific populations of dendritic cells. Human PBMCs (4x10 6 ) were co-incubated with 4x10 7 GFP-tagged B31 (black bars), A3-M9 lp36- (white bars) or A3-M9 lp36-/lp36+ (cross-hatched bars) B. burgdorferi for 6 hours at 4degC or 37degC. The percentages of GFP + mDC1s (CD19 - CD3 - BDCA2 - BDCA1 + ) ( A ), pDCs (CD19 - CD3 - BDCA2 + BDCA1 - ) or ( B ) mDC2s (CD19 - CD3 - BDCA3 + BDCA2 - ) ( C ) were determined by multiparameter flow cytometry. Dot plots representing 500,000 collected events are provided to illustrate gating strategies (left). Column graphs represent the mean and standard deviation of three biological replicates (right). Statistical analysis was performed using a one-way ANOVA with a Tukey's post-test for multiple comparisons.
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- Figure 2 T FR expansion in lymphoid tissues during chronic SIV infection. ( a ) Disaggregated lymph node and spleen cells from SIV uninfected ( n =9) or chronically SIV-infected rhesus macaques ( n =11) were analysed by flow cytometry. Representative examples of flow cytometry gating are shown. Of viable CD3 + CD8 - cells, follicular subsets were defined as CXCR5 + cells (F) and germinal centre subsets were defined as CXCR5 hi PD-1 hi cells (GC). Of these subsets, regulatory cells were defined as CD25 hi CD127 - . T FR (CXCR5 + CD25 hi CD127 - ) were Foxp3 + , whereas T FH (CXCR5 + CD25 lo/- ) were Foxp3 - . ( b ) The percentages of each rhesus macaque regulatory subset, as analysed in a are shown. ( c ) The ratios of each regulatory cell population to its non-regulatory cell counterpart are shown. ( d ) The percentage of total CTLA-4 expression is shown in SIV-uninfected ( n =9) and chronically SIV-infected ( n =8) rhesus macaques. The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed by Mann-Whitney (Wilcoxon) tests to compare unpaired, nonparametric values and significance is denoted by asterisks where * P
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- Figure 3 HIV entry and replication promote T FR expansion. Disaggregated tonsil cells were spinoculated with X4 or R5 HIV and T FR populations were analysed by flow cytometry ( n =15). ( a ) A representative example of tonsil cell flow gating. From viable CD3 + CD8 - cells, T FR are defined as CXCR5 + and CD25 hi CD127 - . T FR cells contain Foxp3 + cells, whereas remaining T FH (CXCR5 + CD25 lo/- ) cells are Foxp3 - . ( b ) Percentages of T FR determined by gating strategies in a are shown. Experimental conditions include mock-spinoculated cells cultured with PMA (50 ng ml -1 ) and ionomycin (1 mug ml -1 ) or exogenous TGF-beta (100 ng ml -1 ) for 24 h and cells pretreated to block CXCR4 (AMD, 200 muM) and CCR5 (MVC, 2 muM). ( c ) Using flow cytometry counting beads, the number of cells per mul were determined for total (CD3 + CD8 - ), T FH (CXCR5 + CD25 lo/- ) and T FR (CXCR5 + CD25 hi CD127 - ) subsets in mock- and X4-spinoculated samples ( n =3). ( d ) Bcl-6 expression is shown in CXCR5- (grey), T FH (blue) and T FR (red) populations after mock-, X4- or R5-spinoculation ( n =5). ( e ) Blimp-1 expression was also determined as in d . The horizontal bars of each graph indicate the median value and are listed where appropriate for clarity. Statistical analyses were performed by Friedman nonparametric tests ( b , d , e ) and significance is denoted by asterisks where * P
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- Figure 3. Phenotype and endogenous TCR expression of CD34 + HPC-derived transgenic AR + T cells. Flow cytometric analysis of the AR-transgenic T cells. (A) CAR-transgenic GFP + cells of cultures transduced to express either the CAR:zeta or the CAR:28zeta were analyzed on day 26 of OP9-DL1 culture for CD3 and TCRalphabeta expression. As a control, GFP - cells are shown from the OP9-DL1 culture transduced to express the CAR:zeta ( N = 5). (B) Dot plots show CD3 expression of cells from the OP9-DL1 cultures transgenic for the wtTCR, TCR:zeta and TCR:28zeta. Vbeta14 staining is used to mark transgene expression, as no GFP is expressed by the transgenic cells ( N = 5). (C) Surface and cytoplasmic staining for CD3 of in vitro generated mature T cells that were expanded for one cycle on feeder cells in the presence of cytokines. (D) Expression of various membrane markers by the CD27 + CD1a - mature T cells at the end of OP9-DL1 culture (46 d) ( N = 2). (E) Day 0: fresh cord blood after MACS CD34 enrichment sorted using the sorting window shown. Day 13: cord blood cells cultured on OP9-DL1 were sorted for CD5 CD7 double positive cells, using the indicated sorting window. The cells were then transduced to express CAR:28zeta and further differentiated on OP9-DL1 feeder layer. Day 21: analysis of the transgenic GFP + cultured cells for DP cells and CD27 + CD1a - mature cells. (F) Flow cytometric analysis of GFP + CAR:28zeta-transgenic cultures, gated on GFP + CD27 + CD1a - mature AR + c
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- Figure 3 Naive CD4 + T cells are converted to functional Tregs by tumor-infiltrating DCs and tumor conditioned medium (CM). (A-C) Naive CD4 + T cells from peripheral blood of patients with invasive breast carcinoma were co-cultured with or without autologous pDCs isolated from tumor (TI) or peripheral blood (PB) for 9 days in the presence or absence of 30% CM from autologous tumor slices or adjacent normal tissue slices. (A , B) Non-adherent cells from co-cultures were stained for CD3, CD4, CD25 and intracellular Foxp3, and analyzed by flow cytometry. Representative plots of gated CD3 + CD4 + cells (A) and quantification of percentage of Foxp3 + CD25 + cells among CD3 + CD4 + cells (B) are shown (mean +- SEM, n = 19; * P < 0.05, ** P < 0.01, *** P < 0.001 by Student's t -test). (C) Expression of Treg-associated genes, assessed by qRT-PCR normalized to GAPDH , in sorted CD4 + T cells, relative to expression in cultures without DCs or CM (mean +- SEM, n = 19; * P < 0.05, ** P < 0.01, *** P < 0.001 compared with naive CD4 + T cells cultured alone by Student's t -test). (D-G) Effect of naive CD4 + T cell-derived Tregs, obtained by co-culture with TI pDCs and tumor CM as above, on function of autologous tumor-specific CD8 + T cells. Tumor-specific CD8 + T cells were generated for each subject by stimulating autologous PB CD8 + T cells with autologous tumor lysate-pulsed autologous DCs. Tregs were recovered from co-cultures by magnetic sorting. (D) CFSE-labeled CD8 + T ce
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- Figure 6 In vivo knockdown of PITPNM3 in CD4 + T cells reverses immunosuppression and inhibits tumor progression in humanized mice. (A) Humanized mice bearing palpable MDA-MB-231 orthotopic xenografts were intraperitoneally injected daily for 14 days with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 (sequence in A , AsiC-PI) to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Experimental schematic is provided in Supplementary information, Figure S9A . (B) Representative immunoblots showing selective knockdown of PITPNM3 protein in PB CD4 + T cells, but not tumor xenografts ( n = 3). (C) PITPNM3 knockdown did not affect the distribution of human CD45 + hematopoietic cells, CD4 + and CD8 + T cells, and CD14 + monocytes in the peripheral blood of humanized mice. Representative flow plots are shown ( n = 3). (D , E) Effect of PITPNM3 knockdown on TI naive CD4 + , Tregs and CD8 + T cell numbers, and apoptosis by TUNEL assay in xenografts. D shows representative immunofluorescence microscopy images. Top row indicates CD4 + naive T cells by arrows; the second row indicates CD4 + CD45RO + Foxp3 - CD4 + memory T cells (yellow arrows) and Foxp3 + Tregs (white arrows). Scale bar, 50 mum. E shows number of cells of each subtype/high power field in eight mice ( ** P < 0.01, *** P < 0.001 compared to PBS group by Student's t -test). (F) Flow cytometry analysis of gated human CD3 + CD4 + cells isolated from xenogra
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- Figure 7 CD4-aptamer-siRNA targeting PITPNM3 reduces TI Tregs and inhibits tumor progression in humanized mice with circulating human Tregs. Humanized mice, implanted with MDA-MB-231 tumors and concurrently injected intravenously with autologous Tregs, were intraperitoneally injected daily for 14 days after tumors became palpable with PBS, 1 nmol CD4-aptamer-control siRNA (AsiC-con) or CD4-aptamer-siRNA targeting PITPNM3 to assess the role of PITPNM3 in TI Tregs, and other T cells and tumor control. Tregs were administered every 10 days after the initial injection and mice were sacrificed 30 days after tumor cell inoculation. (A) Experimental schematic. (B , C) Peripheral blood cells of humanized mice were stained for human CD3, CD4 and Foxp3, and analyzed by flow cytometry. A representative flow plot (B) and the percentage (mean +- SEM) of PB CD4 + cells that are CFSE + Tregs in six mice per group (C) are shown. (D , E) Isolated cells from xenografts were stained for human CD3, CD4 and Foxp3. The percentage (mean +- SEM) of six mice per group (D) and representative flow plot (E) of FoxP3 + Tregs are shown. Most Tregs were CFSE - (i.e., did not come from infused Tregs) and the number of TI Tregs was reduced by knocking down PITPNM3 in CD4 + T cells ( *** P < 0.001 compared to the PBS group by Student's t -test). (F) Tumor size (mean +- SEM, n = 6 per group; *** P < 0.001 by two-way ANOVA with Bonferroni multiple comparison tests). (G) Lung metastases assessed by qRT-PCR