Name: tailoring in vivo cytotoxicity assays to study immunodominance in tumor-specific cd8+ t cell responses
Uploaded: 2019-05-06T15:00:00
Description: Watch this Scientific Journal Video about Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses at JoVE.com

这项工作得到了加拿大卫生研究所的支持, 向 SMMH 提供了 MOM-130465 和 PJT-156295。JC 得到安大略省培训、学院和大学部伊丽莎白二世科学和技术研究生奖学金的部分支持。CEM 是加拿大自然科学和工程研究理事会 (NSERC) 亚历山大·格雷厄姆·贝尔加拿大研究生奖学金 (博士) 的获得者。

这里描述的实验遵循机构实体批准的动物使用规程, 并遵守既定的国家准则。
1. c57bl6 小鼠 t ag 表达肿瘤细胞的接种
<ol><li>在 Dulbecco 的改性 eagle 培养基 (dmem) 中, 用 4.5 gl d-葡萄糖和 l-谷氨酰胺( 1x), 在 dulbecco 的改性老鹰培养基 (dmem) 中生长 sv40 转化的纤维肉瘤细胞系 c57sv (或类似的 t ag + 粘附细胞系), 并补充 1 mm 的丙酮酸钠和10% 的热失活在含有 10% CO2 的加湿大气中, 37°...

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	<li>Yewdell, J. W., Bennink, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodominance+in+major+histocompatibility+complex+class+I-restricted+T+lymphocyte+responses.">Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses.</a> Annual Review of Immunology. 17, 51-88 (1999).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+in+the+immunodominance+hierarchy+in+secondary+CD8++T+cell+responses+to+influenza+A+virus:+roles+for+cross-presentation+and+lysis-independent+immunodomination.">Reversal in the immunodominance hierarchy in secondary CD8+ T cell responses to influenza A virus: roles for cross-presentation and lysis-independent immunodomination.</a> The Journal of Immunology. 173 (8), 5021-5027 (2004).</li><li>Otahal, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+cross-presentation+limits+the+CD8++T+cell+response+to+a+subdominant+tumor+antigen+epitope.">Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope.</a> The Journal of Immunology. 175 (2), 700-712 (2005).</li><li>Lauron, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-priming+induces+immunodomination+in+the+presence+of+viral+MHC+class+I+inhibition.">Cross-priming induces immunodomination in the presence of viral MHC class I inhibition.</a> PLoS Pathogens. 14 (2), e1006883 (2018).</li><li>Crowe, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+antigen+presentation+regulates+the+changing+patterns+of+CD8++T+cell+immunodominance+in+primary+and+secondary+influenza+virus+infections.">Differential antigen presentation regulates the changing patterns of CD8+ T cell immunodominance in primary and secondary influenza virus infections.</a> The Journal of Experimental Medicine. 198 (3), 399-410 (2003).</li><li>Probst, H. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodominance+of+an+antiviral+cytotoxic+T+cell+response+is+shaped+by+the+kinetics+of+viral+protein+expression.">Immunodominance of an antiviral cytotoxic T cell response is shaped by the kinetics of viral protein expression.</a> The Journal of Immunology. 171 (10), 5415-5422 (2003).</li><li>Gileadi, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+an+immunodominant+CTL+epitope+is+affected+by+proteasome+subunit+composition+and+stability+of+the+antigenic+protein.">Generation of an immunodominant CTL epitope is affected by proteasome subunit composition and stability of the antigenic protein.</a> The Journal of Immunology. 163 (11), 6045-6052 (1999).</li><li>Zanker, D., Waithman, J., Yewdell, J. W., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixed+proteasomes+function+to+increase+viral+peptide+diversity+and+broaden+antiviral+CD8++T+cell+responses.">Mixed proteasomes function to increase viral peptide diversity and broaden antiviral CD8+ T cell responses.</a> The Journal of Immunology. 191 (1), 52-59 (2013).</li><li>Deng, Y., Yewdell, J. W., Eisenlohr, L. C., Bennink, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MHC+affinity,+peptide+liberation,+T+cell+repertoire,+and+immunodominance+all+contribute+to+the+paucity+of+MHC+class+I-restricted+peptides+recognized+by+antiviral+CTL.">MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL.</a> The Journal of Immunology. 158 (4), 1507-1515 (1997).</li><li>Chen, W., Khilko, S., Fecondo, J., Margulies, D. H., McCluskey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinant+selection+of+major+histocompatibility+complex+class+I-restricted+antigenic+peptides+is+explained+by+class+I-peptide+affinity+and+is+strongly+influenced+by+nondominant+anchor+residues.">Determinant selection of major histocompatibility complex class I-restricted antigenic peptides is explained by class I-peptide affinity and is strongly influenced by nondominant anchor residues.</a> The Journal of Experimental Medicine. 180 (4), 1471-1483 (1994).</li><li>Kotturi, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naive+precursor+frequencies+and+MHC+binding+rather+than+the+degree+of+epitope+diversity+shape+CD8++T+cell+immunodominance.">Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance.</a> The Journal of Immunology. 181 (3), 2124-2133 (2008).</li><li>Haeryfar, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminal+deoxynucleotidyl+transferase+establishes+and+broadens+antiviral+CD8++T+cell+immunodominance+hierarchies.">Terminal deoxynucleotidyl transferase establishes and broadens antiviral CD8+ T cell immunodominance hierarchies.</a> The Journal of Immunology. 181 (1), 649-659 (2008).</li><li>Leon-Ponte, M., Kasprzyski, T., Mannik, L. A., Haeryfar, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+immunodominance+hierarchies+of+influenza+A+virus-specific+H-2(b)-restricted+CD8++T+cells+in+the+absence+of+terminal+deoxynucleotidyl+transferase.">Altered immunodominance hierarchies of influenza A virus-specific H-2(b)-restricted CD8+ T cells in the absence of terminal deoxynucleotidyl transferase.</a> Immunological Investigations. 37 (7), 714-725 (2008).</li><li>Kedl, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+compete+for+access+to+antigen-bearing+antigen-presenting+cells.">T cells compete for access to antigen-bearing antigen-presenting cells.</a> The Journal of Experimental Medicine. 192 (8), 1105-1113 (2000).</li><li>Kastenmuller, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-competition+of+CD8++T+cells+shapes+the+immunodominance+hierarchy+during+boost+vaccination.">Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during boost vaccination.</a> The Journal of Experimental Medicine. 204 (9), 2187-2198 (2007).</li><li>Memarnejadian, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-1+Blockade+Promotes+Epitope+Spreading+in+Anticancer+CD8(+)+T+Cell+Responses+by+Preventing+Fratricidal+Death+of+Subdominant+Clones+To+Relieve+Immunodomination.">PD-1 Blockade Promotes Epitope Spreading in Anticancer CD8(+) T Cell Responses by Preventing Fratricidal Death of Subdominant Clones To Relieve Immunodomination.</a> The Journal of Immunology. 199 (9), 3348-3359 (2017).</li><li>Haeryfar, S. M., DiPaolo, R. J., Tscharke, D. C., Bennink, J. R., Yewdell, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+T+cells+suppress+CD8++T+cell+responses+induced+by+direct+priming+and+cross-priming+and+moderate+immunodominance+disparities.">Regulatory T cells suppress CD8+ T cell responses induced by direct priming and cross-priming and moderate immunodominance disparities.</a> The Journal of Immunology. 174 (6), 3344-3351 (2005).</li><li>Rytelewski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+immunodominant+antitumor+and+antiviral+CD8++T+cell+responses+by+indoleamine+2,3-dioxygenase.">Suppression of immunodominant antitumor and antiviral CD8+ T cell responses by indoleamine 2,3-dioxygenase.</a> PLoS One. 9 (2), e90439 (2014).</li><li>Maleki Vareki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+simultaneous+antitumor+and+alloreactive+CD8(+)+T-cell+responses+in+the+same+host+by+rapamycin.">Differential regulation of simultaneous antitumor and alloreactive CD8(+) T-cell responses in the same host by rapamycin.</a> American Journal of Transplantation. 12 (1), 233-239 (2012).</li><li>Irvine, K., Bennink, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+immunodominance+hierarchies+in+TCD8++-mediated+antiviral+responses.">Factors influencing immunodominance hierarchies in TCD8+ -mediated antiviral responses.</a> Expert Review of Clinical Immunology. 2 (1), 135-147 (2006).</li><li>Grossmann, M. E., Davila, T., Celis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+tolerance+against+prostatic+antigens+with+subdominant+peptide+epitopes.">Avoiding tolerance against prostatic antigens with subdominant peptide epitopes.</a> Journal of Immunotherapy. 24 (3), 237-241 (2001).</li><li>Schreiber, H., Wu, T. H., Nachman, J., Kast, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodominance+and+tumor+escape.">Immunodominance and tumor escape.</a> Seminars in Cancer Biology. 12 (1), 25-31 (2002).</li><li>Mylin, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+CD8(+)+T-lymphocyte+responses+to+multiple+epitopes+from+simian+virus+40+(SV40)+large+T+antigen+in+C57BL/6+mice+immunized+with+SV40,+SV40+T-antigen-transformed+cells,+or+vaccinia+virus+recombinants+expressing+full-length+T+antigen+or+epitope+minigenes.">Quantitation of CD8(+) T-lymphocyte responses to multiple epitopes from simian virus 40 (SV40) large T antigen in C57BL/6 mice immunized with SV40, SV40 T-antigen-transformed cells, or vaccinia virus recombinants expressing full-length T antigen or epitope minigenes.</a> Journal of Virology. 74 (15), 6922-6934 (2000).</li><li>Fu, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+endoplasmic+reticulum-targeting+signal+sequence+enhances+the+immunogenicity+of+an+immunorecessive+simian+virus+40+large+T+antigen+cytotoxic+T-lymphocyte+epitope.">An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope.</a> Journal of Virology. 72 (2), 1469-1481 (1998).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-priming+of+CD8++T+cells+by+viral+and+tumor+antigens+is+a+robust+phenomenon.">Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon.</a> European Journal of Immunology. 34 (1), 194-199 (2004).</li><li>Memarnejadian, A., Meilleur, C. E., Mazzuca, D. M., Welch, I. D., Haeryfar, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Alloantibody-Mediated+Cytotoxicity+In+Vivo.">Quantification of Alloantibody-Mediated Cytotoxicity In Vivo.</a> Transplantation. 100 (5), 1041-1051 (2016).</li><li>Aichele, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+antigen+treatment+of+naive+and+virus-immune+mice:+antigen-specific+tolerance+versus+immunopathology.">Peptide antigen treatment of naive and virus-immune mice: antigen-specific tolerance versus immunopathology.</a> Immunity. 6 (5), 519-529 (1997).</li><li>Oehen, S., Brduscha-Riem, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+naive+CTL+to+effector+and+memory+CTL:+correlation+of+effector+function+with+phenotype+and+cell+division.">Differentiation of naive CTL to effector and memory CTL: correlation of effector function with phenotype and cell division.</a> The Journal of Immunology. 161 (10), 5338-5346 (1998).</li><li>Coles, R. M., Mueller, S. N., Heath, W. R., Carbone, F. R., Brooks, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+armed+CTL+from+draining+lymph+node+to+spleen+shortly+after+localized+infection+with+herpes+simplex+virus+1.">Progression of armed CTL from draining lymph node to spleen shortly after localized infection with herpes simplex virus 1.</a> The Journal of Immunology. 168 (2), 834-838 (2002).</li><li>Barber, D. L., Wherry, E. J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+rapid+in+vivo+killing+by+memory+CD8+T+cells.">Cutting edge: rapid in vivo killing by memory CD8 T cells.</a> The Journal of Immunology. 171 (1), 27-31 (2003).</li><li>Meilleur, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+superantigens+expand+and+activate,+rather+than+delete+or+incapacitate,+preexisting+antigen-specific+memory+CD8++T+cells.">Bacterial superantigens expand and activate, rather than delete or incapacitate, preexisting antigen-specific memory CD8+ T cells.</a> The Journal of Infectious Diseases. , (2018).</li><li>Goldszmid, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+charged+with+apoptotic+tumor+cells+induce+long-lived+protective+CD4++and+CD8++T+cell+immunity+against+B16+melanoma.">Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4+ and CD8+ T cell immunity against B16 melanoma.</a> The Journal of Immunology. 171 (11), 5940-5947 (2003).</li><li>Oberg, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+or+mismatch+of+MHC+class+I+is+sufficient+to+trigger+NK+cell-mediated+rejection+of+resting+lymphocytes+in+vivo+-+role+of+KARAP/DAP12-dependent+and+-independent+pathways.">Loss or mismatch of MHC class I is sufficient to trigger NK cell-mediated rejection of resting lymphocytes in vivo - role of KARAP/DAP12-dependent and -independent pathways.</a> European Journal of Immunology. 34 (6), 1646-1653 (2004).</li><li>Wingender, G., Krebs, P., Beutler, B., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-specific+cytotoxicity+by+invariant+NKT+cells+in+vivo+is+CD95/CD178-dependent+and+is+correlated+with+antigenic+potency.">Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency.</a> The Journal of Immunology. 185 (5), 2721-2729 (2010).</li><li>Brinster, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+harboring+SV40+T-antigen+genes+develop+characteristic+brain+tumors.">Transgenic mice harboring SV40 T-antigen genes develop characteristic brain tumors.</a> Cell. 37 (2), 367-379 (1984).</li><li>Tatum, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T+cells+targeting+a+single+immunodominant+epitope+are+sufficient+for+elimination+of+established+SV40+T+antigen-induced+brain+tumors.">CD8+ T cells targeting a single immunodominant epitope are sufficient for elimination of established SV40 T antigen-induced brain tumors.</a> The Journal of Immunology. 181 (6), 4406-4417 (2008).</li><li>Schell, T. D., Tevethia, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+advanced+choroid+plexus+tumors+in+SV40+T+antigen+transgenic+mice+following+priming+of+donor+CD8(+)+T+lymphocytes+by+the+endogenous+tumor+antigen.">Control of advanced choroid plexus tumors in SV40 T antigen transgenic mice following priming of donor CD8(+) T lymphocytes by the endogenous tumor antigen.</a> The Journal of Immunology. 167 (12), 6947-6956 (2001).</li><li>Greenberg, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+cancer+in+a+transgenic+mouse.">Prostate cancer in a transgenic mouse.</a> Proceedings of the National Academy of Sciences of the United States of America. 92 (8), 3439-3443 (1995).</li></ol>

与传统的杀灭检测方法 (如放射性铬 (51cr) 释放和比色乳酸脱氢酶 (ldh) 释放检测) 相比, 基于 cfse 的体内细胞毒性检测具有多种优势。首先, 他们允许监测 CTL 功能在一个建筑完整的二级淋巴器官。
其次, 在体内细胞毒性检测中对靶细胞的特异性杀伤反映了 ag特异性 t Cd8的绝对数量, 这通常是但并不总是存在于脾脏中的 tcd8频率的功能。这与51Cr/ldh...

常规 CD8+ t 细胞 (t cd8) 在抗癌免疫监测中发挥着重要作用。它们主要在细胞分裂 t 淋巴细胞 (Ctl) 的能力, 识别肿瘤特异性或相关的肽抗原 (Ags) 显示在主要组织相容性复合体 (MHC) i 类分子的闭合裂隙中。全副武装的 Ctl 利用其细胞毒性武库来摧毁恶性细胞。抗癌 TCd8 可以检测到在循环中, 甚至在许多癌症患者和肿瘤动物的原发和转移性肿块内。然而, 它们往往是过敏或精疲力竭的, 无法根除癌症。因此, 许多免疫治疗方式旨在增加抗癌 TCd8频率, 并恢复和提高其功能。
肿瘤蛋白含有许多肽, 其中一些可以是免疫原性的, 并具有潜在的免疫保护。然而, 可量化的 TCd8 反应是以不同的大小获得的, 而只是对少数肽的反应。这在 TCd8克隆1之间创建了一个 "免疫调节层次结构"。因此, 免疫球蛋白 (ID)t Cd8 占据显著的等级等级, 这通常是由它们的丰度来判断的。相反, tCd8 细胞的 t 细胞受体 (tcr) 是特定的亚显性 (sd) 表位发生在较低的频率。我们和其他人已经确定了一些因素, 决定或塑造免疫调节在 TCd8反应。除其他外, 这些模式包括对天真的 tcd8的 ag 呈现模式 (即直接呈现、交叉呈现、变装)2、3、4、ag呈现细胞的类型 (apc)参与 tcd8活化5, 蛋白质 ags6,7的丰度和稳定性, 以及蛋白酶体 7,8,与 g 处理 (tap) 相关的转运体对9肽的相对选择性、mhc i 分子9、10的释放肽的亲和力、前体频率和 tcr 多样性。t 细胞池中的同源t cd8 11、12、13、t细胞之间在获得 apc14、15和 tcd8的骨形能力方面的交叉竞争克隆16。此外, TCd8免疫调节是由几种抑制细胞类型介导的免疫调节机制, 如自然发生的调节 t (nTreg) 细胞17, 细胞表面共抑制分子编程死亡-1 (PD-1)16, 以及某些细胞内酶, 如二氯胺酮 2, 3-二氧合酶 (ido ) 18 和雷帕霉素 (mtor)19的哺乳动物靶标。然而, 必须指出, 上述因素并不总是完全解释免疫调节。
除了 TCd8免疫调节的基本生物学外, 对这一有趣现象的检查对癌症免疫学和免疫治疗具有重要意义。首先, ID 状态不一定赋予给定的 TCd8克隆预防肿瘤开始或进展20的能力.ID 和 SDt Cd8 是否以及如何促进抗肿瘤免疫可能取决于恶性肿瘤的类型和程度以及所使用的实验系统。其次, 人们认为, idt cd8克隆可能对免疫系统 "太明显", 因此更容易出现中央和/或外周耐受机制16,21。第三, 异基因肿瘤可能含有肿瘤细胞, 避免被许多 (如果不是大多数的话) Ctl 检测, 只显示狭窄的肽谱: MHC 复合物。在这种情况下, 宽度不足的 t Cd8反应可能会给这些肿瘤细胞提供生存优势, 从而增强其生长优势22。正是出于上述原因, 许多人认为免疫调节是成功的基于 TCd8的疫苗接种和抗癌治疗的障碍。
对 c57bl6 小鼠进行接种, 为 t cd8 免疫调节提供了一个强大的临床前系统, 该小鼠具有 simian 病毒 40 (Sv40) 转化细胞, 表达大肿瘤银 (t ag)。此模型提供了几个好处。首先, 这种临床相关的癌蛋白的肽表位在这个小鼠菌株23中有很好的特征 (表 1)。第二, T Ag 表位, 称为站点 I、II/iii、IV 和 V, 触发 T Cd8 响应, 这些响应一直按以下等级排列: 站点 IV > > 站点i≥ iiieiii > &Gt; 站点 iii. 站点对 t ag 进行最稳健的响应。相反, 站点 i 和 ii/iii 是次优势的, 站点 v特异性 t cd8是最少的, 通常只有在对其他表位 23、24 没有响应的情况下才能检测到。第三, 本文所述协议中使用的 t ag+肿瘤细胞系, 即 c57sv 纤维肉瘤细胞, 以及我们以前的研究中使用的细胞 16、17、18、19 25, 26,用亚基因组 sv40 片段25转化.因此, 它们无法组装和释放可能感染宿主 Apc 的 SV40 病毒。此外, C57SV 细胞缺乏典型的共刺激分子, 如 CD80 (B7-1)、CD86 (B7-1) 和 CD137 配体 (4-1BBL)16。上述属性使这些线成为通过交叉启动检测体内 TCd8激活的理想之选。交叉预防是诱导 TCd8 反应的主要途径, 特别是那些针对非造血源肿瘤细胞启动的非造血源肿瘤细胞, 不能直接提供初级天真的 t 细胞25。
抗肿瘤t Cd8频率和/或功能可以通过 mhc i 四聚体染色、细胞内染色来监测效应细胞因子 (例如, 干扰素 [ifn]-γ) 或裂解分子 (例如, 穿孔)、酶联免疫罐 (elispot) 检测和体内细胞毒性检测。自 20世纪90年代成立以来,基于体内杀灭检测的羧荧光素琥珀酰酯 (cfse) 已经能够评估抗病毒 ctl 29, 30 介导的细胞毒性反应,31、抗肿瘤 ctls16,32, 自然杀伤细胞 (nk) 33, 甘油脂反应不变的自然杀手 t (inkt) 细胞34, 以及预先存在和新的捐献者特有的同种异体抗体26。因此, 它们的应用可能会引起广大读者的兴趣, 包括但不限于在肿瘤免疫学和免疫治疗、抗病原体免疫以及预防和治疗疫苗设计等领域工作的研究人员。
为了评估细胞介导的细胞毒性在典型的情况下, 两个群体的天真的脾细胞, 显示一个不相关的 Ag 或同源 Ag (s) 标记为两种不同剂量的 CFSE, 混合在相同的数量, 并注入天真 (控制) 或杀手细胞窝藏的老鼠。然后用流式细胞仪检查每个目标种群的存在情况。
在我们对抗病毒和抗肿瘤t cd8反应 12、16、17的免疫调节作用研究中, 我们在体内进行了优化和应用。在这里, 我们提供了一个详细的协议, 用于同时评估 ID 和 SDt Cd8对 t ag 表位的响应, 这可以很容易地用于其他实验系统中的类似调查。我们还提供了具有代表性的结果, 表明 nTreg 细胞消耗和 PD-1 封锁可以有选择地增强 ID TCd8-和 Sdt cd8诱导的细胞毒性, 分别。最后, 我们将讨论体内杀灭检测的多重优势以及它们的一些固有局限性。

<table border="0" cellpadding="0" cellspacing="0" style="width:872px;" width="871">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">0.25% Trypsin-EDTA (1X)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:163px;">25200-056</td>
			<td style="width:211px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACK Lysing Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:163px;">A1049201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD25 (clone PC-61.5.3)</td>
			<td>Bio X Cell</td>
			<td>BE0012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse PD-1 (clone RMP1-14)</td>
			<td>Bio X Cell</td>
			<td>BE0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFSE</td>
			<td>Thermo Fisher Scientific</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Wisent Bioproducts</td>
			<td>080-150</td>
			<td>Heat-inactivate prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td>10 mM final concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids Solution (100X)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P0781</td>
			<td>Stock is 100X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG1 (clone KLH/G1-2-2)</td>
			<td>SouthernBiotech</td>
			<td>0116-01</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG1 (clone HRPN)</td>
			<td>Bio X Cell</td>
			<td>BE0088</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG1 (clone TNP6A7)</td>
			<td>Bio X Cell</td>
			<td>BP0290</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat IgG2a (clone 2A3)</td>
			<td>Bio X Cell</td>
			<td>BP0089</td>
			<td>Isotype control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 (1X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Pyruvate (100 mM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>11360-070</td>
			<td>1 mM final concentration</td>
		</tr>
	</tbody>
</table>

tailoring in vivo cytotoxicity assays to study immunodominance in tumor-specific cd8+ t cell responses

羧基荧光素琥珀酰亚胺酯 (CFSE) 在体内细胞毒性检测, 使敏感和准确的定量 Cd8+细胞溶解 t 淋巴细胞 (ctl) 反应获得对肿瘤和病原源肽。与传统的杀虫检测相比, 它们具有几个优势。首先, 他们允许监测 ctl 介导的细胞毒性在建筑学完整的继发性淋巴器官, 通常在脾脏。其次, 它们允许在 CTL 响应的启动、效应和召回阶段进行机械研究。第三, 它们为在真正的体内环境中进行疫苗/药物药效检测提供了有用的平台。在这里, 我们提供了一个优化的方案, 用于检查伴随 CTL 响应的一个以上肽表位的模型肿瘤抗原 (Ag), 即 simian 病毒 40 (SV40) 编码大 T ag (T Ag)。与大多数其他与临床相关的肿瘤蛋白一样, T Ag 含有许多潜在的免疫原肽。然而, 只有四种此类肽可在 c57bl6 小鼠体内诱导可检测到的 CTL 反应。这些响应根据其大小一致地按层次顺序排列, 这构成了在这个强大的系统中 tCd8 "免疫调节" 的基础。因此, T ag 特异性 TCd8反应的大部分集中在单个免疫多瘤表位, 而其他三个表位被识别, 反应很弱。免疫调节损害了抗肿瘤 TCd8反应的广度, 因此被许多人认为是成功接种抗癌疫苗的障碍。因此, 了解决定或塑造Tcd8免疫调节的细胞和分子因素及其机制是非常重要的。我们在这里描述的协议是针对 T Ag 免疫模型中这一现象的研究量身定制的, 但可以很容易地修改和推广到其他肿瘤模型中的类似研究。我们提供了如何使用体内细胞毒性检测来衡量实验性免疫治疗干预措施的影响的例子。

如图 1所示, 实验的目的是确定 nTreg 细胞的存在和功能是否形成或改变 t Ag-t特 cd8的免疫调节层次结构。在 c57bl6 小鼠获得 2 x 10 c57sv 肿瘤细胞 (p. p.) 前四天, 用 PBS 或0.5 毫克抗 cd25 mAb (克隆 pc-61.5.3 [PC61]) 注射了 c57bl6 小鼠.在单独的实验中, 使用了大鼠 IgG1 同型对照代替 PBS。流式细胞仪检测成功 ntreg 细胞消耗的 Pc61。
C57SV ...

Watch this Scientific Journal Video about Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses at JoVE.com

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

活体细胞毒性试验研究肿瘤特异性 CD8+ t 细胞反应中的免疫调节

我们这里描述了一种基于流式细胞仪的体内杀伤检测方法, 该方法能够检查细胞毒性 T 淋巴细胞 (CTL) 对模型肿瘤抗原的反应中的免疫调节。我们提供了如何将这种优雅的检测方法用于机械研究和药物疗效测试的示例。

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

这种测定可用于同时对肿瘤抗原的两种不同肽表位进行定量T细胞培养的细胞毒性。这种测定的体内性质允许在二级淋巴器官的完整结构内测量T细胞的细胞毒性功能。此外，观察到的杀戮是T细胞的绝对数量的准确反映，而不仅仅是它们的频率，这可能具有误导性。此测定能够检查细胞毒性T细胞对免疫主导和亚显性肿瘤抗原的反应。因此，当您设计疫苗时，它提供了有价值的信息，以及癌症的免疫治疗方案。我们的协议是专为研究CD8T细胞反应而定制的。然而，它可以被修改来测量其他杀手细胞类型，如NK细胞和先天类T淋巴细胞引出的细胞毒性。这种测定需要无菌技术、小鼠组织处理、目标细胞准备以及尾静脉注射方面的专业知识。在尝试体内杀戮分析之前，需要获得这种技能。一旦T-抗原阳性粘附肿瘤细胞系完全汇合或稍微过汇，用无菌、温暖的PBS轻轻冲洗单层，并在生物安全柜中用0.25%的胰腺素EDTA分离肿瘤细胞。多次点击培养瓶的两侧以释放剩余的粘附细胞，并在大约五分钟后停止反应，加入5毫升DMEM培养基。在通过70微米孔细胞滤株将细胞悬浮液过滤到50毫升锥形管之前，先将细胞溶液移液几次。通过离心收集细胞，并在相同条件下将颗粒重新在10毫升无菌、冷PBS中重新供应，进行三次洗涤中的第一次。第三次洗涤后，将细胞以每毫升无菌PBS浓度的4倍10重新悬浮到7个细胞，并每6至12周大的C57 Black 6小鼠接受者体内注射500微升T-抗原阳性肿瘤细胞悬浮液。为进行目标飞溅细胞制备，将安乐死供体小鼠放在易发位置，用70%乙醇喷洒动物。使用无菌钳子和剪刀，抬起皮肤，做一个小的腹中线切口。以交叉方式切割皮肤，露出内膜，用钳子以帐篷式的方式拉起内膜，而不抓任何内脏。切开腹膜，露出腹腔，轻轻将脾脏转移到含有5毫升无菌PBS的15毫升Dounce组织研磨机中。使用柱塞施加手动压力，直到血%组织消散成红色均质细胞悬浮液，然后将均质质转移到15毫升管中。通过离心收集悬浮液，将颗粒重新悬浮在四毫升氯化铵碳酸钾解氧水缓冲液中，以消除红细胞。四分钟后，用8毫升全介质停止这个过程，并通过70微米孔细胞滤株将细胞悬浮液过滤到新的50毫升管中。通过离心将白血球从红细胞碎片中分离，并在12毫升的完整介质中重新将颗粒重新暂停。然后将飞毛细胞悬浮液分成三个相等的四毫升等分。对于目标飞溅细胞的肽编码，脉冲一个飞毛细胞等同物与一微摩尔无关肽，一个等同与一个微摩尔的站点一肽，一个等分与一个微摩尔的站点IV肽在37摄氏度和5%二氧化碳。在孵育结束时，通过离心去除多余的肽，每管洗涤12毫升无菌、冷PBS两次，洗涤肽脉冲的飞溅细胞两次。第二次洗涤后，每管将细胞重新用四毫升新鲜、无菌的PBS中重新暂停。将025、0.25和双微摩尔CFSE分别添加到不相关的站点一和站点IV肽脉冲飞溅细胞悬浮液中。将管子在 37 摄氏度下放置 15 分钟，每 5 分钟手动反转一次。在孵化结束时，将三毫升热灭活的胎儿牛血清添加到每个管中，以停止CFSE反应，并将每管的最终体积与PBS一起达到15毫升。然后通过离心收集染料标记的细胞，每次洗涤 12 毫升新鲜、无菌 PBS 中洗两次。要确认目标飞毛细胞的充分CFSE标签，将颗粒重新在PBS的三毫升中，与温和的涡流混合。将每个 CFSE 标记的电池悬浮液中的 10 微升转移到单个五毫升圆形底部聚苯乙烯荧光活性细胞分选或包含 PBS 的 FACS 管中，然后将管加载到装有 488 纳米激光器的流动细胞仪上。在流细胞仪软件中，在获得属于荧光1通道淋巴细胞门内的5，000个事件之前，根据细胞的向前散射和侧散射特性创建淋巴细胞门。在父 CFSE 阳性总体中，绘制额外的直方图门以识别 CFSE 低、CFSE 中间和 CFSE 高亚人口。然后，在三个门内确认其他两个标记单元格管的相等或接近相等的事件编号。对于目标细胞的注射，首先轻轻涡旋源管，然后以相等的比例将三个 CFSE 标记的细胞悬浮液集中到新管中。用无菌PBS对管内装物进行加上，通过离心收集细胞进行计数。然后将体积调整为每接收方每200微升PBS的7个混合靶细胞，将200微升细胞悬浮液注入每个接受者C57 Black 6小鼠的尾静脉。注射后两四个小时，取出并处理每个接受动物的脾脏，并重新将分离的白血球注入每只脾脏三毫升PBS中。将每个加工脾脏的大约 1 倍 10 到 7 个细胞转移到一个干净的 FACS 管中，并立即运行流细胞仪上的细胞，如证明要打开 CFSE 低、中间和高目标细胞群。然后在荧光1通道中获取总共2，000个CFSE-Low事件，并根据文本中详述的公式计算每个共生靶细胞群体的具体解解。在这个具有代表性的实验中，在注射抗CD25单克隆抗体的小鼠中，自然产生的调节T细胞的耗竭成功得到了流细胞学的证实。正如预期的那样，在天真的小鼠中，可检测到与对照细胞和同源靶细胞相对应的接近相等的峰值。相比之下，无论之前使用抗CD25单克隆抗体或PBS治疗，T-抗原-原基因小鼠几乎完全不存在位-IV显示靶细胞。有趣的是，自然发生的T reg细胞耗尽由抗CD25单克隆抗体增强体内细胞毒性T淋巴细胞-细胞细胞-中培养的位点-I脉冲靶细胞。在第二次代表性调查中，编程死亡1封锁增强亚显性CD8阳性T细胞位点1和2斜线-三特异性反应，表明干扰编程死亡1/编程死亡配体1相互作用可能诱发抗癌CD8阳性T细胞反应的表位扩散。在使用 CFSE 标记供体细胞时，确保浓度精确至关重要。否则，这可能会导致直方图峰值重叠，这会使所需的计算和数据解释变得困难（如果不是不可能的话）。优化体内细胞毒性测定非常重要，以量化不同杀手细胞类型在相同宿主中识别肽和非肽抗原所引出的效应器功能。

Research

JoVE Journal

Immunology and Infection

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T细胞受体在小鼠T细胞的逆转录病毒转导

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

科研

免疫与感染

Competitive Homing Assays to Study Gut-tropic T Cell Migration

竞争的原点实验研究肠嗜T细胞迁移

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

酶联免疫吸附法表征小鼠胸腺依赖性和胸腺独立免疫球蛋白同种反应

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination

优化干扰素伽隆弹性体检测在几内亚猪模型接种疫苗后 t 细胞反应

The guinea pig has played a pivotal role as a relevant small animal model in the development of vaccines for infectious diseases such as tuberculosis, ...