Antibody data
- Antibody Data
- Antigen structure
- References [35]
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- Validations
- Flow cytometry [2]
- Other assay [27]
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- Product number
- 56-0038-42 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- CD3 Monoclonal Antibody (UCHT1), Alexa Fluor™ 700, 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.
- Conjugate
- Near infrared dye
- Antibody clone number
- UCHT1
- Concentration
- 5 µL/Test
Submitted references Impact of SARS-CoV-2 variants on the total CD4(+) and CD8(+) T cell reactivity in infected or vaccinated individuals.
Effects of dexmedetomidine on immune response in patients undergoing radical and reconstructive surgery for oral cancer.
A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations.
Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.
High-throughput compound screen reveals mTOR inhibitors as potential therapeutics to reduce (auto)antibody production by human plasma cells.
Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.
Regulatory T cells use arginase 2 to enhance their metabolic fitness in tissues.
CXCR3(+) T Follicular Helper Cells Induced by Co-Administration of RTS,S/AS01B and Viral-Vectored Vaccines Are Associated With Reduced Immunogenicity and Efficacy Against Malaria.
Activation-induced Markers Detect Vaccine-Specific CD4⁺ T Cell Responses Not Measured by Assays Conventionally Used in Clinical Trials.
Safety and Immunogenicity of Malaria Vectored Vaccines Given with Routine Expanded Program on Immunization Vaccines in Gambian Infants and Neonates: A Randomized Controlled Trial.
TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia.
Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities.
HDAC inhibition potentiates immunotherapy in triple negative breast cancer.
Blocking the recruitment of naive CD4(+) T cells reverses immunosuppression in breast cancer.
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.
T-cell activation is an immune correlate of risk in BCG vaccinated infants.
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.
PD-1+Tim-3+ CD8+ T Lymphocytes Display Varied Degrees of Functional Exhaustion in Patients with Regionally Metastatic Differentiated Thyroid Cancer.
Type I IFNs and IL-18 regulate the antiviral response of primary human γδ T cells against dendritic cells infected with Dengue virus.
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.
Transcriptional profile of tuberculosis antigen-specific T cells reveals novel multifunctional features.
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.
Highly heterogeneous, activated, and short-lived regulatory T cells during chronic filarial infection.
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.
Ulcerative colitis impairs the acylethanolamide-based anti-inflammatory system reversal by 5-aminosalicylic acid and glucocorticoids.
CD16+ monocytes control T-cell subset development in immune thrombocytopenia.
Programmed death-1+ T cells and regulatory T cells are enriched in tumor-involved lymph nodes and associated with aggressive features in papillary thyroid cancer.
Epigenetic biomarkers of T-cells in human glioma.
Accumulation of natural killer T cells in progressive nonalcoholic fatty liver disease.
Tarke A, Sidney J, Methot N, Yu ED, Zhang Y, Dan JM, Goodwin B, Rubiro P, Sutherland A, Wang E, Frazier A, Ramirez SI, Rawlings SA, Smith DM, da Silva Antunes R, Peters B, Scheuermann RH, Weiskopf D, Crotty S, Grifoni A, Sette A
Cell reports. Medicine 2021 Jul 20;2(7):100355
Cell reports. Medicine 2021 Jul 20;2(7):100355
Effects of dexmedetomidine on immune response in patients undergoing radical and reconstructive surgery for oral cancer.
Huang L, Qin C, Wang L, Zhang T, Li J
Oncology letters 2021 Feb;21(2):106
Oncology letters 2021 Feb;21(2):106
A Versatile Protocol to Quantify BCR-mediated Phosphorylation in Human and Murine B Cell Subpopulations.
Rip J, Hendriks RW, Corneth OBJ
Bio-protocol 2021 Feb 5;11(3):e3902
Bio-protocol 2021 Feb 5;11(3):e3902
Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.
Tarke A, Sidney J, Kidd CK, Dan JM, Ramirez SI, Yu ED, Mateus J, da Silva Antunes R, Moore E, Rubiro P, Methot N, Phillips E, Mallal S, Frazier A, Rawlings SA, Greenbaum JA, Peters B, Smith DM, Crotty S, Weiskopf D, Grifoni A, Sette A
Cell reports. Medicine 2021 Feb 16;2(2):100204
Cell reports. Medicine 2021 Feb 16;2(2):100204
High-throughput compound screen reveals mTOR inhibitors as potential therapeutics to reduce (auto)antibody production by human plasma cells.
Tuijnenburg P, Aan de Kerk DJ, Jansen MH, Morris B, Lieftink C, Beijersbergen RL, van Leeuwen EMM, Kuijpers TW
European journal of immunology 2020 Jan;50(1):73-85
European journal of immunology 2020 Jan;50(1):73-85
Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.
Tarke A, Sidney J, Kidd CK, Dan JM, Ramirez SI, Yu ED, Mateus J, da Silva Antunes R, Moore E, Rubiro P, Methot N, Phillips E, Mallal S, Frazier A, Rawlings SA, Greenbaum JA, Peters B, Smith DM, Crotty S, Weiskopf D, Grifoni A, Sette A
bioRxiv : the preprint server for biology 2020 Dec 9;
bioRxiv : the preprint server for biology 2020 Dec 9;
Regulatory T cells use arginase 2 to enhance their metabolic fitness in tissues.
Lowe MM, Boothby I, Clancy S, Ahn RS, Liao W, Nguyen DN, Schumann K, Marson A, Mahuron KM, Kingsbury GA, Liu Z, Munoz Sandoval P, Rodriguez RS, Pauli ML, Taravati K, Arron ST, Neuhaus IM, Harris HW, Kim EA, Shin US, Krummel MF, Daud A, Scharschmidt TC, Rosenblum MD
JCI insight 2019 Dec 19;4(24)
JCI insight 2019 Dec 19;4(24)
CXCR3(+) T Follicular Helper Cells Induced by Co-Administration of RTS,S/AS01B and Viral-Vectored Vaccines Are Associated With Reduced Immunogenicity and Efficacy Against Malaria.
Bowyer G, Grobbelaar A, Rampling T, Venkatraman N, Morelle D, Ballou RW, Hill AVS, Ewer KJ
Frontiers in immunology 2018;9:1660
Frontiers in immunology 2018;9:1660
Activation-induced Markers Detect Vaccine-Specific CD4⁺ T Cell Responses Not Measured by Assays Conventionally Used in Clinical Trials.
Bowyer G, Rampling T, Powlson J, Morter R, Wright D, Hill AVS, Ewer KJ
Vaccines 2018 Jul 31;6(3)
Vaccines 2018 Jul 31;6(3)
Safety and Immunogenicity of Malaria Vectored Vaccines Given with Routine Expanded Program on Immunization Vaccines in Gambian Infants and Neonates: A Randomized Controlled Trial.
Mensah VA, Roetynck S, Kanteh EK, Bowyer G, Ndaw A, Oko F, Bliss CM, Jagne YJ, Cortese R, Nicosia A, Roberts R, D'Alessio F, Leroy O, Faye B, Kampmann B, Cisse B, Bojang K, Gerry S, Viebig NK, Lawrie AM, Clarke E, Imoukhuede EB, Ewer KJ, Hill AVS, Afolabi MO
Frontiers in immunology 2017;8:1551
Frontiers in immunology 2017;8:1551
TIGIT expressing CD4+T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia.
Catakovic K, Gassner FJ, Ratswohl C, Zaborsky N, Rebhandl S, Schubert M, Steiner M, Gutjahr JC, Pleyer L, Egle A, Hartmann TN, Greil R, Geisberger R
Oncoimmunology 2017;7(1):e1371399
Oncoimmunology 2017;7(1):e1371399
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
HDAC inhibition potentiates immunotherapy in triple negative breast cancer.
Terranova-Barberio M, Thomas S, Ali N, Pawlowska N, Park J, Krings G, Rosenblum MD, Budillon A, Munster PN
Oncotarget 2017 Dec 26;8(69):114156-114172
Oncotarget 2017 Dec 26;8(69):114156-114172
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
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
T-cell activation is an immune correlate of risk in BCG vaccinated infants.
Fletcher HA, Snowden MA, Landry B, Rida W, Satti I, Harris SA, Matsumiya M, Tanner R, O'Shea MK, Dheenadhayalan V, Bogardus L, Stockdale L, Marsay L, Chomka A, Harrington-Kandt R, Manjaly-Thomas ZR, Naranbhai V, Stylianou E, Darboe F, Penn-Nicholson A, Nemes E, Hatherill M, Hussey G, Mahomed H, Tameris M, McClain JB, Evans TG, Hanekom WA, Scriba TJ, McShane H
Nature communications 2016 Apr 12;7:11290
Nature communications 2016 Apr 12;7:11290
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
PD-1+Tim-3+ CD8+ T Lymphocytes Display Varied Degrees of Functional Exhaustion in Patients with Regionally Metastatic Differentiated Thyroid Cancer.
Severson JJ, Serracino HS, Mateescu V, Raeburn CD, McIntyre RC Jr, Sams SB, Haugen BR, French JD
Cancer immunology research 2015 Jun;3(6):620-30
Cancer immunology research 2015 Jun;3(6):620-30
Type I IFNs and IL-18 regulate the antiviral response of primary human γδ T cells against dendritic cells infected with Dengue virus.
Tsai CY, Liong KH, Gunalan MG, Li N, Lim DS, Fisher DA, MacAry PA, Leo YS, Wong SC, Puan KJ, Wong SB
Journal of immunology (Baltimore, Md. : 1950) 2015 Apr 15;194(8):3890-900
Journal of immunology (Baltimore, Md. : 1950) 2015 Apr 15;194(8):3890-900
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
Transcriptional profile of tuberculosis antigen-specific T cells reveals novel multifunctional features.
Arlehamn CL, Seumois G, Gerasimova A, Huang C, Fu Z, Yue X, Sette A, Vijayanand P, Peters B
Journal of immunology (Baltimore, Md. : 1950) 2014 Sep 15;193(6):2931-40
Journal of immunology (Baltimore, Md. : 1950) 2014 Sep 15;193(6):2931-40
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
Highly heterogeneous, activated, and short-lived regulatory T cells during chronic filarial infection.
Metenou S, Coulibaly YI, Sturdevant D, Dolo H, Diallo AA, Soumaoro L, Coulibaly ME, Kanakabandi K, Porcella SF, Klion AD, Nutman TB
European journal of immunology 2014 Jul;44(7):2036-47
European journal of immunology 2014 Jul;44(7):2036-47
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
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
CD16+ monocytes control T-cell subset development in immune thrombocytopenia.
Zhong H, Bao W, Li X, Miller A, Seery C, Haq N, Bussel J, Yazdanbakhsh K
Blood 2012 Oct 18;120(16):3326-35
Blood 2012 Oct 18;120(16):3326-35
Programmed death-1+ T cells and regulatory T cells are enriched in tumor-involved lymph nodes and associated with aggressive features in papillary thyroid cancer.
French JD, Kotnis GR, Said S, Raeburn CD, McIntyre RC Jr, Klopper JP, Haugen BR
The Journal of clinical endocrinology and metabolism 2012 Jun;97(6):E934-43
The Journal of clinical endocrinology and metabolism 2012 Jun;97(6):E934-43
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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- Staining of normal human peripheral blood cells with Anti-Human CD19 FITC (Product # 11-0199-42) and Mouse IgG1 K Isotype Control Alexa Fluor® 700 (Product # 56-4714-80) (left) or Anti-Human CD3 Alexa Fluor® 700 (right). Cells in the lymphocyte gate were used for analysis.
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- Staining of human peripheral blood mononuclear cells with CD45 Pacific Blue, CD19 FITC and CD3 Alexa Fluor 700. As expected based on known relative expression patterns, CD3 clone UCHT1 stains a subset of lymphocytes (pink), but not monocytes (orange) and granulocytes (blue).
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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
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