Antibody data
- Antibody Data
- Antigen structure
- References [42]
- Comments [0]
- Validations
- Flow cytometry [2]
- Other assay [27]
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- Product number
- 48-0038-42 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- CD3 Monoclonal Antibody (UCHT1), eFluor™ 450, 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 SARS-CoV-2 infection paralyzes cytotoxic and metabolic functions of the immune cells.
Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.
Deficient IL-2 Produced by Activated CD56(+) T Cells Contributes to Impaired NK Cell-Mediated ADCC Function in Chronic HIV-1 Infection.
Two phase kinetics of the inflammatory response from hepatocyte-peripheral blood mononuclear cell interactions.
The Changing Landscape of Naive T Cell Receptor Repertoire With Human Aging.
Establishment of a novel platform cell line for efficient and precise evaluation of T cell receptor functional avidity.
Human cancer evolution in the context of a human immune system in mice.
Human Immunodeficiency Virus Infection Impairs Th1 and Th17 Mycobacterium tuberculosis-Specific T-Cell Responses.
CXCL4 is a novel inducer of human Th17 cells and correlates with IL-17 and IL-22 in psoriatic arthritis.
The NRF2 pathway as potential biomarker for dimethyl fumarate treatment in multiple sclerosis.
Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.
Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities.
Vitamin D deficiency is associated with IL-6 levels and monocyte activation in HIV-infected persons.
Eomesodermin and T-bet mark developmentally distinct human natural killer cells.
Blocking the recruitment of naive CD4(+) T cells reverses immunosuppression in breast cancer.
Failure recovery of circulating NKG2D(+)CD56(dim)NK cells in HBV-associated hepatocellular carcinoma after hepatectomy predicts early recurrence.
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.
Vitamin D deficiency contributes to the reduction and impaired function of naïve CD45RA⁺ regulatory T cell in chronic heart failure.
Follicular regulatory T cells impair follicular T helper cells in HIV and SIV infection.
CD8+ T Cells Can Mediate Short-Term Protection against Heterotypic Dengue Virus Reinfection in Mice.
Antitumor immunity triggered by melphalan is potentiated by melanoma cell surface-associated calreticulin.
A truncation variant of the cation channel P2RX5 is upregulated during T cell activation.
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.
Monocyte-activation phenotypes are associated with biomarkers of inflammation and coagulation in chronic HIV infection.
Evidence for innate immune system activation in HIV type 1-infected elite controllers.
Decreases in colonic and systemic inflammation in chronic HIV infection after IL-7 administration.
Human mesenchymal stromal cells modulate T-cell responses through TNF-α-mediated activation of NF-κB.
Trypan blue exclusion assay by flow cytometry.
Children with asthma by school age display aberrant immune responses to pathogenic airway bacteria as infants.
MHC multimer-guided and cell culture-independent isolation of functional T cell receptors from single cells facilitates TCR identification for immunotherapy.
ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death.
Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells.
Recombinant influenza virus carrying the respiratory syncytial virus (RSV) F85-93 CTL epitope reduces RSV replication in mice.
Memory T cells in latent Mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a CXCR3+CCR6+ Th1 subset.
Ribavirin Does Not Impair the Suppressive Activity of Foxp3(+)CD4(+)CD25(+) Regulatory T Cells.
Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids.
Characterization of Th17 responses to Streptococcus pneumoniae in humans: comparisons between adults and children in a developed and a developing country.
Epigenetic biomarkers of T-cells in human glioma.
Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
Singh Y, Trautwein C, Fendel R, Krickeberg N, Berezhnoy G, Bissinger R, Ossowski S, Salker MS, Casadei N, Riess O, Deutsche COVID-19 OMICS Initiate (DeCOI)
Heliyon 2021 Jun;7(6):e07147
Heliyon 2021 Jun;7(6):e07147
Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.
Zhang L, Li Z, Skrzypczynska KM, Fang Q, Zhang W, O'Brien SA, He Y, Wang L, Zhang Q, Kim A, Gao R, Orf J, Wang T, Sawant D, Kang J, Bhatt D, Lu D, Li CM, Rapaport AS, Perez K, Ye Y, Wang S, Hu X, Ren X, Ouyang W, Shen Z, Egen JG, Zhang Z, Yu X
Cell 2020 Apr 16;181(2):442-459.e29
Cell 2020 Apr 16;181(2):442-459.e29
Deficient IL-2 Produced by Activated CD56(+) T Cells Contributes to Impaired NK Cell-Mediated ADCC Function in Chronic HIV-1 Infection.
Xie Z, Zheng J, Wang Y, Li D, Maermaer T, Li Y, Tu J, Xu Q, Liang H, Cai W, Shen T
Frontiers in immunology 2019;10:1647
Frontiers in immunology 2019;10:1647
Two phase kinetics of the inflammatory response from hepatocyte-peripheral blood mononuclear cell interactions.
Beringer A, Molle J, Bartosch B, Miossec P
Scientific reports 2019 Jun 10;9(1):8378
Scientific reports 2019 Jun 10;9(1):8378
The Changing Landscape of Naive T Cell Receptor Repertoire With Human Aging.
Egorov ES, Kasatskaya SA, Zubov VN, Izraelson M, Nakonechnaya TO, Staroverov DB, Angius A, Cucca F, Mamedov IZ, Rosati E, Franke A, Shugay M, Pogorelyy MV, Chudakov DM, Britanova OV
Frontiers in immunology 2018;9:1618
Frontiers in immunology 2018;9:1618
Establishment of a novel platform cell line for efficient and precise evaluation of T cell receptor functional avidity.
Morimoto S, Fujiki F, Kondo K, Nakajima H, Kobayashi Y, Inatome M, Aoyama N, Nishida Y, Tsuboi A, Oka Y, Nishida S, Nakata J, Hosen N, Oji Y, Sugiyama H
Oncotarget 2018 Sep 25;9(75):34132-34141
Oncotarget 2018 Sep 25;9(75):34132-34141
Human cancer evolution in the context of a human immune system in mice.
Gammelgaard OL, Terp MG, Preiss B, Ditzel HJ
Molecular oncology 2018 Oct;12(10):1797-1810
Molecular oncology 2018 Oct;12(10):1797-1810
Human Immunodeficiency Virus Infection Impairs Th1 and Th17 Mycobacterium tuberculosis-Specific T-Cell Responses.
Murray LW, Satti I, Meyerowitz J, Jones M, Willberg CB, Ussher JE, Goedhals D, Hurst J, Phillips RE, McShane H, Vuuren CV, Frater J
The Journal of infectious diseases 2018 May 5;217(11):1782-1792
The Journal of infectious diseases 2018 May 5;217(11):1782-1792
CXCL4 is a novel inducer of human Th17 cells and correlates with IL-17 and IL-22 in psoriatic arthritis.
Affandi AJ, Silva-Cardoso SC, Garcia S, Leijten EFA, van Kempen TS, Marut W, van Roon JAG, Radstake TRDJ
European journal of immunology 2018 Mar;48(3):522-531
European journal of immunology 2018 Mar;48(3):522-531
The NRF2 pathway as potential biomarker for dimethyl fumarate treatment in multiple sclerosis.
Hammer A, Waschbisch A, Kuhbandner K, Bayas A, Lee DH, Duscha A, Haghikia A, Gold R, Linker RA
Annals of clinical and translational neurology 2018 Jun;5(6):668-676
Annals of clinical and translational neurology 2018 Jun;5(6):668-676
Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.
Roux C, Saviane G, Pini J, Belaïd N, Dhib G, Voha C, Ibáñez L, Boutin A, Mazure NM, Wakkach A, Blin-Wakkach C, Rouleau M
Frontiers in immunology 2017;8:1991
Frontiers in immunology 2017;8:1991
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
Vitamin D deficiency is associated with IL-6 levels and monocyte activation in HIV-infected persons.
Manion M, Hullsiek KH, Wilson EMP, Rhame F, Kojic E, Gibson D, Hammer J, Patel P, Brooks JT, Baker JV, Sereti I, Study to Understand the Natural History of HIV/AIDS in the Era of Effective Antiretroviral Therapy (the ‘SUN Study’) Investigators
PloS one 2017;12(5):e0175517
PloS one 2017;12(5):e0175517
Eomesodermin and T-bet mark developmentally distinct human natural killer cells.
Collins A, Rothman N, Liu K, Reiner SL
JCI insight 2017 Mar 9;2(5):e90063
JCI insight 2017 Mar 9;2(5):e90063
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
Failure recovery of circulating NKG2D(+)CD56(dim)NK cells in HBV-associated hepatocellular carcinoma after hepatectomy predicts early recurrence.
Gao J, Duan Z, Zhang L, Huang X, Long L, Tu J, Liang H, Zhang Y, Shen T, Lu F
Oncoimmunology 2016;5(1):e1048061
Oncoimmunology 2016;5(1):e1048061
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
Vitamin D deficiency contributes to the reduction and impaired function of naïve CD45RA⁺ regulatory T cell in chronic heart failure.
Ma YH, Zhou YL, Yue CY, Zhang GH, Deng L, Xie GH, Xu WP, Shen LS
Journal of immunology research 2015;2015:547697
Journal of immunology research 2015;2015:547697
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
CD8+ T Cells Can Mediate Short-Term Protection against Heterotypic Dengue Virus Reinfection in Mice.
Zellweger RM, Tang WW, Eddy WE, King K, Sanchez MC, Shresta S
Journal of virology 2015 Jun;89(12):6494-505
Journal of virology 2015 Jun;89(12):6494-505
Antitumor immunity triggered by melphalan is potentiated by melanoma cell surface-associated calreticulin.
Dudek-Perić AM, Ferreira GB, Muchowicz A, Wouters J, Prada N, Martin S, Kiviluoto S, Winiarska M, Boon L, Mathieu C, van den Oord J, Stas M, Gougeon ML, Golab J, Garg AD, Agostinis P
Cancer research 2015 Apr 15;75(8):1603-14
Cancer research 2015 Apr 15;75(8):1603-14
A truncation variant of the cation channel P2RX5 is upregulated during T cell activation.
Abramowski P, Ogrodowczyk C, Martin R, Pongs O
PloS one 2014;9(9):e104692
PloS one 2014;9(9):e104692
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
Monocyte-activation phenotypes are associated with biomarkers of inflammation and coagulation in chronic HIV infection.
Wilson EM, Singh A, Hullsiek KH, Gibson D, Henry WK, Lichtenstein K, Önen NF, Kojic E, Patel P, Brooks JT, Sereti I, Baker JV, Study to Understand the Natural History of HIV/AIDS in the Era of Effective Therapy (SUN Study) Investigators
The Journal of infectious diseases 2014 Nov 1;210(9):1396-406
The Journal of infectious diseases 2014 Nov 1;210(9):1396-406
Evidence for innate immune system activation in HIV type 1-infected elite controllers.
Krishnan S, Wilson EM, Sheikh V, Rupert A, Mendoza D, Yang J, Lempicki R, Migueles SA, Sereti I
The Journal of infectious diseases 2014 Mar;209(6):931-9
The Journal of infectious diseases 2014 Mar;209(6):931-9
Decreases in colonic and systemic inflammation in chronic HIV infection after IL-7 administration.
Sereti I, Estes JD, Thompson WL, Morcock DR, Fischl MA, Croughs T, Beq S, Lafaye de Micheaux S, Yao MD, Ober A, Wilson EM, Natarajan V, Imamichi H, Boulassel MR, Lederman MM, Routy JP
PLoS pathogens 2014 Jan;10(1):e1003890
PLoS pathogens 2014 Jan;10(1):e1003890
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
Children with asthma by school age display aberrant immune responses to pathogenic airway bacteria as infants.
Larsen JM, Brix S, Thysen AH, Birch S, Rasmussen MA, Bisgaard H
The Journal of allergy and clinical immunology 2014 Apr;133(4):1008-13
The Journal of allergy and clinical immunology 2014 Apr;133(4):1008-13
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
ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death.
Garg AD, Dudek AM, Ferreira GB, Verfaillie T, Vandenabeele P, Krysko DV, Mathieu C, Agostinis P
Autophagy 2013 Sep;9(9):1292-307
Autophagy 2013 Sep;9(9):1292-307
Comprehensive analysis of dengue virus-specific responses supports an HLA-linked protective role for CD8+ T cells.
Weiskopf D, Angelo MA, de Azeredo EL, Sidney J, Greenbaum JA, Fernando AN, Broadwater A, Kolla RV, De Silva AD, de Silva AM, Mattia KA, Doranz BJ, Grey HM, Shresta S, Peters B, Sette A
Proceedings of the National Academy of Sciences of the United States of America 2013 May 28;110(22):E2046-53
Proceedings of the National Academy of Sciences of the United States of America 2013 May 28;110(22):E2046-53
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
Memory T cells in latent Mycobacterium tuberculosis infection are directed against three antigenic islands and largely contained in a CXCR3+CCR6+ Th1 subset.
Lindestam Arlehamn CS, Gerasimova A, Mele F, Henderson R, Swann J, Greenbaum JA, Kim Y, Sidney J, James EA, Taplitz R, McKinney DM, Kwok WW, Grey H, Sallusto F, Peters B, Sette A
PLoS pathogens 2013 Jan;9(1):e1003130
PLoS pathogens 2013 Jan;9(1):e1003130
Ribavirin Does Not Impair the Suppressive Activity of Foxp3(+)CD4(+)CD25(+) Regulatory T Cells.
Lee J, Choi YS, Shin EC
Immune network 2013 Feb;13(1):25-9
Immune network 2013 Feb;13(1):25-9
Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids.
Suárez J, Romero-Zerbo Y, Márquez L, Rivera P, Iglesias M, Bermúdez-Silva FJ, Andreu M, Rodríguez de Fonseca F
PloS one 2012;7(5):e37729
PloS one 2012;7(5):e37729
Characterization of Th17 responses to Streptococcus pneumoniae in humans: comparisons between adults and children in a developed and a developing country.
Lundgren A, Bhuiyan TR, Novak D, Kaim J, Reske A, Lu YJ, Qadri F, Malley R
Vaccine 2012 Jun 6;30(26):3897-907
Vaccine 2012 Jun 6;30(26):3897-907
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
Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
Syn WK, Oo YH, Pereira TA, Karaca GF, Jung Y, Omenetti A, Witek RP, Choi SS, Guy CD, Fearing CM, Teaberry V, Pereira FE, Adams DH, Diehl AM
Hepatology (Baltimore, Md.) 2010 Jun;51(6):1998-2007
Hepatology (Baltimore, Md.) 2010 Jun;51(6):1998-2007
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Supportive validation
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- Invitrogen Antibodies (provider)
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- Staining of normal human peripheral blood cells with Anti-Human CD19 APC (Product # 17-0199-42) and Anti-Human CD3 eFluor® 450. Cells in the lymphocyte gate were used for analysis.
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- Staining of human peripheral blood mononuclear cells with CD45 PE, CD19 APC and CD3 eFluor 450. As expected based on known relative expression patterns, CD3 clone UCHT1 stains a subset of lymphocytes (pink), but not monocytes (orange) and granulocytes (blue).
Supportive validation
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- Figure 6 Representative high-magnification photomicrographs showing double immunofluorescence for NAAA, CD19, CD3 and CD14 in order to characterize the immune cells in the mucosa infiltrate of UC patients. NAAA immunofluorescence was observed in CD19+ B lymphocytes (A-C), CD3+ T lymphocytes (D-F) and CD14+ macrophages (G-I).
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- Figure 9 Representative high-magnification photomicrographs showing double immunofluorescence for FAAH, NAPE-PLD, CD38 and CD3 in order to characterize the immune cells in the mucosa infiltrate of UC patients. Nearly all FAAH immunofluorescent cells are plasma cell-specific CD38 (A-C). NAPE-PLD immunofluorescence was observed in both CD38+ plasma cells (D-F) and CD3+ T lymphocytes (G-I).
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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