从外周血人类同种异体抗原特异性T细胞的生成
Summary
This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.
Abstract
The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.
Introduction
T淋巴细胞是适应性免疫系统的关键部件。 T细胞是负责不仅直接通过各种效应机制介导保护性免疫反应的病原体，还积极地维持免疫自身耐受性和指导其他细胞的应答，在免疫系统中。这些功能是通过一些综合信号，包括T细胞受体（TCR）的连接，细胞因子和趋化因子和代谢产物1定向。这些信号的，T细胞受体是特别重要的，因为它提供了特有的特异性，它定义在适应性免疫的T细胞的作用。 TCR的相互作用，在一个高度特异和敏感的态度提出了MHC（人类同源基因HLA）分子（的pMHC复合物），以提供启动T细胞的效应功能的信号线性肽抗原。 TCR互动与配体的pMHC的生化指标不仅提供了特异性对于T细胞的活化，同时也有在随后的T细胞的功能2质的影响。因此，研究T细胞功能往往需要研究克隆T细胞定义的抗原特异性的反应。
人T细胞区室，含有约10 12αβT细胞，包含大约7月10日至八月10日明显αβTCRs3-4。这种多元的曲目为识别肽繁多，从潜在的病原体，将必要的保护性免疫T细胞应答的机会。据估计，T细胞响应一个给定的外源抗原通过自MHC呈现的频率为10 -4的数量级上- 10-7在没有给该抗原5前的免疫应答。幼稚T细胞的剧目是由胸腺选择形状，确保认识自我MHC呈递的抗原肽和极限反应能力ivity对自身抗原肽，最大限度地提高调解的保护性免疫2的潜在效用。然而，违反了此设计的反应性，相对较大的频率，10 -3 – 10 -4，T细胞从免疫幼稚个体对刺激作出反应与同种异体细胞，识别两者的外来MHC分子，以及内源性肽它们呈现6。的同种异基因的pMHC配体的识别是结构上类似于识别通过自MHC呈现外源抗原的; TCR的使得与两个同种异体MHC分子以及所呈现的肽7临界生物化学相互作用。 T细胞以从的pMHC的多样性同种异体刺激的结果的响应的健壮本质络合物本同种异体细胞8的表面上。据估计，每MHC呈现约2×10 4个不同的内源性肽抗原9。这家B响应于同种异体刺激readth是临床相关的病理，如同种异体移植物排斥或移植物抗宿主病（GVHD）的显著方面，从T细胞的同种异体反应性所致。
研究的人T细胞的同种异体反应性的反应时检查下列刺激同种异体细胞幼稚T细胞的多克隆反应传统上依赖。反复刺激具有相同的同种异基因的细胞系结合的有限稀释分析能够产生单克隆T细胞定义的识别同种异体的HLA 10的。然而，这种方法是有问题的用于检查响应个体同种异体的pMHC配位体，如内源性的pMHC的大量不同的曲目络合物存在对于给定的同种异基因的HLA刺激T细胞的广泛的剧目。这种散装人口刺激和有限稀释方法需要大量克隆的筛选以分离的T细胞与所需reactiVITY针对单一的pMHC配体。此外，T细胞响应的个体同种异体的pMHC配体的频率相对低的幼稚T细胞群，其中介绍的障碍有效地产生人T细胞克隆响应于一个给定的抗原之一。
鉴定与多克隆群的抗原特异性T细胞的分离已经通过荧光团标记的pMHC多聚体11上的显影启用。该方法利用装入重组的可溶性生物素化的MHC分子，其通过结合到链霉亲和标记的荧光团标记的特异性肽抗原。的pMHC的多聚化增加了亲和力，补偿TCR的固有的低（微米）可溶性的pMHC配体的亲和力。标记的细胞可被识别和分离通过流式细胞术。但是，这种方法仍然由幼稚T细胞群中的抗原特异性T细胞，这是典型的低频率限制震级小于准确识别和量化大多数流式细胞仪的限制令。为了解决此限制，对的pMHC四聚体标记和随后的磁珠富集为四聚体标记的细胞的方法，已经开发了12。这种方法已经证明可靠检测，计数和低频抗原特异性T细胞的分离。
这个协议描述了一种有效的协议对人T细胞克隆的特异性个体同种异体的pMHC配体响应的产生。该协议适用的pMHC（HLA）的多聚体标记和富集对同种异体抗原-特异性的人T细胞的流式细胞术细胞分选和一个可靠的方法在体外培养的人T细胞的分离，使生产的从单个分选的细胞的T细胞克隆（概述图1）。
Protocol
Representative Results
Discussion
T cell alloreactivity is a long-studied and clinically-relevant phenomenon. The robust proliferative and effector responses of T cells to allogeneic stimulation has enabled extensive analyses of human T cell responses in vitro through relatively straightforward mixed lymphocyte reactions of peripheral blood T cells against inactivated allogeneic cells. However, these primary alloreactive T cell responses are oligoclonal, comprised of a large number of individual T cells responding to specific alloantigens. This …
Disclosures
The authors have nothing to disclose.
Acknowledgements
The author would like to thank the NIH Tetramer Core Facility for tetramer production. The author would also like to thank E.D. O’Connor and K.E. Marquez at the UCSD Human Embryonic Stem Cell Core Facility flow cytometry laboratory for assistance in cell sorting. This work was funded by National Institutes of Health grant K08AI085039 (G.P.M.).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Sodium heparin venous blood collection tube 16 x 100 mm | Becton, Dickenson and Company | 366480 | |
| Lymphoprep | Stemcell Technologies | 7801 | |
| Rosette Sep Human T Cell Enrichment Kit | Stemcell Technologies | 15061 | |
| Dulbecco's PBS, 1x without Ca or Mg | Corning | 21-031-CV | |
| Bovine serum albumin | Sigma-Aldrich | A7906 | |
| EDTA | Sigma-Aldrich | E6635 | |
| Fluorophore-labeled pMHC tetramer | NIH Tetramer Facility | NA | |
| EasySep Biotin Selection Kit | Stemcell Technologies | 18553 | |
| EasySep Selection magnet | Stemcell Technologies | 18000 | |
| TruStain FcX Human Fc blocking solution | Biolegend | 422301 | |
| Anti-CD5 PE-Cy7 (clone UCHT2) | Biolegend | 300621 | |
| Anti-CD14 FITC (clone HCD14) | Biolegend | 325603 | |
| Anti-CD19 FITC (clone HIB19) | Biolegend | 302205 | |
| Iscove's DMEM, without b-ME or L-glutamine | Corning | 15-016-CV | |
| HEPES | Corning | 25-060-CI | |
| b-Mercaptoethanol | Life Technologies | 21985-023 | |
| Glutamax | Life Technologies | 35050061 | |
| Gentamicin sulfate (50 mg/ml) | Omega Scientific | GT-50 | |
| Human AB serum, male donor | Omega Scientific | HS-30 | |
| Recombinant human IL-2 | Peprotech | AF 200-02 | |
| Dynabeads Human T-Activator CD3/CD28 | Life Technologies | 11131D | |
| Media | |||
| Cell sorting buffer | |||
| PBS, pH 7.4 | 1 L | ||
| BSA | 10g | ||
| EDTA (0.5 M) | 2 ml | ||
| Human T Cell Culture Medium | |||
| Iscove's DMEM | 351.6 ml | ||
| Heat-inactivated human AB serum | 40 ml | ||
| HEPES (1 M) | 4 ml | ||
| Glutamax (100 x) | 4 ml | ||
| Gentamicin (50 mg/ml) | 0.4 ml | ||
| b-mercaptoethanol (14.3 M) | 1.4 ml | ||
| Recombinant human IL-2 (1 mg/ml) | 1 ml |
References
- Smith-Garvin, J. E., Koretzky, G. A., Jordan, M. S. T cell activation. Annu. Rev. Immunol. 27 (1), 591-619 (2009).
- Morris, G. P., Allen, P. M. How the TCR balances sensitivity and specificity for the recognition of self and pathogens. Nat. Immunol. 13 (2), 121-128 (2012).
- Arstilla, T. P., et al. A direct estimation of the human αβ T cell receptor diversity. Science. 286 (5441), 958-961 (1999).
- Robbins, H. S., et al. Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells. Blood. 114 (19), 4099-4107 (2009).
- Alanio, C., Lemaitre, F., Law, H. K. W., Hasan, M., Albert, M. L. Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies. Blood. 115 (18), 3718-3725 (2010).
- Suchin, E. J., et al. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question. J. Immunol. 166 (2), 973-981 (2001).
- Felix, N. J., Allen, P. M. Specificity of T-cell alloreactivity. Nat. Rev. Immunol. 7 (12), 942-953 (2007).
- Morris, G. P., Ni, P. P., Allen, P. M. Alloreactivity is limited by the endogenous peptide repertoire. Proc. Natl. Acad. Sci. USA. 108 (9), 3695-3700 (2011).
- Suri, A., et al. In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket. J. Immunol. 168 (3), 1235-1243 (2002).
- Yssl, H., Spits, H. Generation and maintenance of cloned human T cell lines. Curr. Protoc. Immunol. 7, (2002).
- Altman, J. D., et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 274 (5284), 94-96 (1996).
- Moon, J. J., et al. Naive CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity. 27 (2), 203-213 (2007).
- Chicz, R. M., et al. Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles. J. Exp. Med. 178 (1), 27-47 (1993).
- Ni, P. P., Allen, P. M., Morris, G. P. The ability to rearrange dual TCRs enhances positive selection, leading to increased allo- and autoreactive T cell repertoires. J. Immunol. In press, (2014).
- Morris, G. P., Uy, G. L., Donermeyer, D., DiPersio, J. F., Allen, P. M. Dual receptor T cells mediate pathologic alloreactivity in patients with acute graft-versus-host disease. Sci. Transl. Med. 5 (188), (2013).
- Altman, J. D., Reay, P. A., Davis, M. M. Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli. Proc. Natl. Acad. Sci. USA. 90 (21), 10330-10334 (1993).
- Sabatino, J. J., Huang, J., Zhu, C., Evavold, B. D. High prevalence of low affinity peptide-MHC II tetramer-negative effectors during polyclonal CD4+ T cell responses. J. Exp. Med. 208 (1), 81-90 (2011).
