抗原肽/HLA-A2复合物诱导和调节同种反应性T细胞的应答
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摘要
T细胞TCR识别的配体是抗原肽/MHC复合物(pMHC),MHC型别不同的供/受者间进行移植后会发生强烈的排斥反应,其机制主要是同种反应性T细胞(简称同种T细胞)识别同种异体组织抗原(简称同种抗原)并产生应答。关于同种T细胞直接识别的可能模式,以及同种T细胞是否具有pMHC特异性,一直存在争论。本论文拟从单个pMHC(即T细胞识别的抗原表位)角度分析,通过诱导单一pMHC特异性同种T细胞,证实同种T细胞的识别也具有pMHC特异性,并利用同种T细胞识别的特异性探索打破耐受或诱导耐受的方法。论文共分为三部分,其主要内容与结果如下:
一、同种T细胞对抗原的识别具有pMHC特异性
为了研究同种T细胞是否存在对pMHC的特异性,本研究选择Tyr/HLA-A2作为pMHC的代表,利用仅表达HLA-A2、TAP缺陷的T2细胞作为刺激细胞,提呈Tyr/HLA-A2,通过与HLA-A2阴性个体(HLA-A2-ve)的PBLs进行长期混合淋巴细胞培养(LTMLC),诱生Tyr/HLA-A2特异性的同种T细胞,用特异性细胞毒试验和pMHC四聚体染色检测经过LTMLC诱生的同种T细胞。结果显示结合于同种HLA分子上的自身抗原肽与结合于自身HLA分子上的病毒肽一样,可在体外有效的诱导产生特异性的T细胞,显示同种T细胞对抗原的识别亦具有pMHC特异性。
二、pMHC二聚体加载到HLA-A2-ve单核细胞表面诱导单表位特异性的同种T细胞
细胞表面存在种类繁多的pMHC,导致传统实验手段制备的同种T细胞为针对众多pMHC的多克隆T细胞的集合,过继该多克隆T细胞难以避免发生同种T细胞造成的移植物抗宿主反应。本研究利用pMHC二聚体分子中的Fc段与单核细胞的FcR结合,将单一pMHC(Tyr/HLA-A2)加载到HLA-A2-ve个体单核细胞表面,诱导PBLs产生针对该表位的同种T细胞。结果显示短期混合培养体系中,加载Tyr/HLA-A2的HLA-A2-ve单核细胞可刺激自体来源的PBLs明显增殖,并诱导产生针对该表位的同种T细胞,这种经过短期体外扩增的特异性同种反应性T细胞具有更好的细胞活力与功能,有望过继应用打破病理性耐受。
三、可溶性pMHC二聚体特异性抑制同种T细胞应答
在移植免疫研究中,诱导移植物特异性的外周耐受是尚未实现的目标。本研究通过建立HLA-A2-ve PBLs与T2/Tyr混合培养的同种反应体系,研究可溶性抗原肽/HLA-A2二聚体对同种T细胞应答的调节作用。结果显示,Tyr/HLA-A2二聚体特异性抑制HLA-A2-ve PBLs被T2/Tyr刺激后的增殖效应,并特异性抑制同种杀伤性T细胞的功能。Tyr/HLA-A2特异性T细胞频率检测结果提示,Tyr/HLA-A2二聚体可抑制Tyr/HLA-A2特异性同种T细胞的产生或诱导其无能,从而抑制同种反应,为诱导移植受者对移植物特异性免疫耐受提供了新的手段。
本研究的创新点和意义:
1.本研究从单个表位角度证明了长期混合淋巴细胞培养(LTMLC)方法能够诱生pMHC特异性的同种T细胞,同种反应性T细胞与普通抗原反应性T细胞一样具有pMHC特异性。为同种T细胞直接识别机制提供了直接的实验依据,也为后续研究提供了理论基础。
2.本研究利用HLA-A2二聚体,将单一pMHC加载到HLA-A2-ve个体的单核细胞表面,能够有效诱导该个体来源的PBLs产生针对单一pMHC的同种T细胞,为制备单一pMHC特异性的同种T细胞、及其过继治疗提供了可能。
3.本研究从单一pMHC的层次证实可溶性HLA-A2二聚体可有效的抑制同种反应的强度,并且这种抑制作用具有pMHC特异性,为诱导移植受者对移植物特异性免疫耐受提供新的思路。
Alloreactive T cells recognize intact allogeneic MHC molecules expressed on foreign cells and mount vigorous responses as graft rejection in vivo. The role of the bound peptide in alloreactive T cell recognition is controversial, ranging from peptide-independent to peptide-specific. In this study, a single peptide epitope was present to induce alloresponse through mix lymphocyte culture in vitro and prove the pMHC specific character of alloreactive T cells. Meanwhile, novel approaches to induce or inhibit antigen specific allreactive T cells were exploited to aim for tumor tolerance or graft rejection.
1. alloreactive T cell recognize allogeneic peptide/MHC complex (pMHC) in peptide dependent way.
The aim of this part is to find the evidence that there exist pMHC-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the specific target cell (T2/Tyr) and a higher frequency of Tyr/HLA-A2 specific CD8+ T cell was detected by specific tetramer. Our results indicated the LTMLC was able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs.
2. Peptide-specific allogeneic T cell response induced by monocytes-bound HLA-A2 dimer
The allogeneic stimulator cells present a different set of endogenous peptides, leading to potential stimulation of CTLs for the various allogeneic epitopes, which may mount graft-versus-host reaction. In this study, a soluble divalent HLA-A2/IgG molecule (HLA-A2 dimer) was constructed and loaded with a self-protein origin peptide (Tyr368-376) to form Tyr/HLA-A2 dimer, which allowed for exploiting autologous monocytes as single peptide epitope presenting cells to generate Tyr/HLA-A2 specific CTLs from HLA-A2 negative (HLA-A2-ve) donors. The studies described here demonstrate that allogeneic divalent peptide/HLA-A2 presented by autologous monocytes can stimulate and expand peptide-specific allo-restricted CTLs in vitro. It provides a novel approach to enrich peptide-specific donor lymphocytes for the treatment of patients with tumor following HLA-mismatched transplantation.
3. Peptide-dependent inhibition of alloreactive T-cell response by soluble divalent HLA-A2/IgG molecule in vitro
Induction of peripheral tolerance in an Ag-specific manner is currently an unrealized but critical goal of transplant biology. In this study, the soluble divalent HLA-A2/IgG molecule which allowed for specific targeting to Ag-specific alloreactive CTL was used as tolerogen to specific allo-restricted T cells. The Tyr/HLA-A2 dimer suppressed alloreactive T-cell responses by inhibiting its proliferation identified and cytotoxicity against specific target T2/Try in vitro, and it was interesting that the suppression was peptide-specific. The specific Tyr/HLA-A2 tetramer staining results suggested the impaired function of CD8+ T cell was due to deletion of specific alloreactive T cells in 3 subjects. Moreover, the existence of epitope-specific but functional-negative T cells in other two subjects indicated there maight exist other mechanism involved in silencing alloreactive responses by the HLA-A2 dimer. Peptide-loaded dimers offer a novel approach to induce peptide-specific immunosuppression, which may be useful in promoting graft survival.
一、同种T细胞对抗原的识别具有pMHC特异性
为了研究同种T细胞是否存在对pMHC的特异性,本研究选择Tyr/HLA-A2作为pMHC的代表,利用仅表达HLA-A2、TAP缺陷的T2细胞作为刺激细胞,提呈Tyr/HLA-A2,通过与HLA-A2阴性个体(HLA-A2-ve)的PBLs进行长期混合淋巴细胞培养(LTMLC),诱生Tyr/HLA-A2特异性的同种T细胞,用特异性细胞毒试验和pMHC四聚体染色检测经过LTMLC诱生的同种T细胞。结果显示结合于同种HLA分子上的自身抗原肽与结合于自身HLA分子上的病毒肽一样,可在体外有效的诱导产生特异性的T细胞,显示同种T细胞对抗原的识别亦具有pMHC特异性。
二、pMHC二聚体加载到HLA-A2-ve单核细胞表面诱导单表位特异性的同种T细胞
细胞表面存在种类繁多的pMHC,导致传统实验手段制备的同种T细胞为针对众多pMHC的多克隆T细胞的集合,过继该多克隆T细胞难以避免发生同种T细胞造成的移植物抗宿主反应。本研究利用pMHC二聚体分子中的Fc段与单核细胞的FcR结合,将单一pMHC(Tyr/HLA-A2)加载到HLA-A2-ve个体单核细胞表面,诱导PBLs产生针对该表位的同种T细胞。结果显示短期混合培养体系中,加载Tyr/HLA-A2的HLA-A2-ve单核细胞可刺激自体来源的PBLs明显增殖,并诱导产生针对该表位的同种T细胞,这种经过短期体外扩增的特异性同种反应性T细胞具有更好的细胞活力与功能,有望过继应用打破病理性耐受。
三、可溶性pMHC二聚体特异性抑制同种T细胞应答
在移植免疫研究中,诱导移植物特异性的外周耐受是尚未实现的目标。本研究通过建立HLA-A2-ve PBLs与T2/Tyr混合培养的同种反应体系,研究可溶性抗原肽/HLA-A2二聚体对同种T细胞应答的调节作用。结果显示,Tyr/HLA-A2二聚体特异性抑制HLA-A2-ve PBLs被T2/Tyr刺激后的增殖效应,并特异性抑制同种杀伤性T细胞的功能。Tyr/HLA-A2特异性T细胞频率检测结果提示,Tyr/HLA-A2二聚体可抑制Tyr/HLA-A2特异性同种T细胞的产生或诱导其无能,从而抑制同种反应,为诱导移植受者对移植物特异性免疫耐受提供了新的手段。
本研究的创新点和意义:
1.本研究从单个表位角度证明了长期混合淋巴细胞培养(LTMLC)方法能够诱生pMHC特异性的同种T细胞,同种反应性T细胞与普通抗原反应性T细胞一样具有pMHC特异性。为同种T细胞直接识别机制提供了直接的实验依据,也为后续研究提供了理论基础。
2.本研究利用HLA-A2二聚体,将单一pMHC加载到HLA-A2-ve个体的单核细胞表面,能够有效诱导该个体来源的PBLs产生针对单一pMHC的同种T细胞,为制备单一pMHC特异性的同种T细胞、及其过继治疗提供了可能。
3.本研究从单一pMHC的层次证实可溶性HLA-A2二聚体可有效的抑制同种反应的强度,并且这种抑制作用具有pMHC特异性,为诱导移植受者对移植物特异性免疫耐受提供新的思路。
Alloreactive T cells recognize intact allogeneic MHC molecules expressed on foreign cells and mount vigorous responses as graft rejection in vivo. The role of the bound peptide in alloreactive T cell recognition is controversial, ranging from peptide-independent to peptide-specific. In this study, a single peptide epitope was present to induce alloresponse through mix lymphocyte culture in vitro and prove the pMHC specific character of alloreactive T cells. Meanwhile, novel approaches to induce or inhibit antigen specific allreactive T cells were exploited to aim for tumor tolerance or graft rejection.
1. alloreactive T cell recognize allogeneic peptide/MHC complex (pMHC) in peptide dependent way.
The aim of this part is to find the evidence that there exist pMHC-specific CTLs among alloreactive T cells generated with long-term mixed lymphocytes culture (LTMLC). A single pMHC was manipulated by loading the TAP-defective, HLA-A2 expressing T2 cells with a self-peptide (Tyr369-377). The PBLs samples from 4 HLA-A2 negative (HLA-A2-ve) donors were included in this study. The cultural bulk of HLA-A2-ve PBLs with the T2/Tyr showed a more active cytotoxicity against the specific target cell (T2/Tyr) and a higher frequency of Tyr/HLA-A2 specific CD8+ T cell was detected by specific tetramer. Our results indicated the LTMLC was able to expand the viral antigen-specific CTLs as well as allogeneic antigen-specific CTLs.
2. Peptide-specific allogeneic T cell response induced by monocytes-bound HLA-A2 dimer
The allogeneic stimulator cells present a different set of endogenous peptides, leading to potential stimulation of CTLs for the various allogeneic epitopes, which may mount graft-versus-host reaction. In this study, a soluble divalent HLA-A2/IgG molecule (HLA-A2 dimer) was constructed and loaded with a self-protein origin peptide (Tyr368-376) to form Tyr/HLA-A2 dimer, which allowed for exploiting autologous monocytes as single peptide epitope presenting cells to generate Tyr/HLA-A2 specific CTLs from HLA-A2 negative (HLA-A2-ve) donors. The studies described here demonstrate that allogeneic divalent peptide/HLA-A2 presented by autologous monocytes can stimulate and expand peptide-specific allo-restricted CTLs in vitro. It provides a novel approach to enrich peptide-specific donor lymphocytes for the treatment of patients with tumor following HLA-mismatched transplantation.
3. Peptide-dependent inhibition of alloreactive T-cell response by soluble divalent HLA-A2/IgG molecule in vitro
Induction of peripheral tolerance in an Ag-specific manner is currently an unrealized but critical goal of transplant biology. In this study, the soluble divalent HLA-A2/IgG molecule which allowed for specific targeting to Ag-specific alloreactive CTL was used as tolerogen to specific allo-restricted T cells. The Tyr/HLA-A2 dimer suppressed alloreactive T-cell responses by inhibiting its proliferation identified and cytotoxicity against specific target T2/Try in vitro, and it was interesting that the suppression was peptide-specific. The specific Tyr/HLA-A2 tetramer staining results suggested the impaired function of CD8+ T cell was due to deletion of specific alloreactive T cells in 3 subjects. Moreover, the existence of epitope-specific but functional-negative T cells in other two subjects indicated there maight exist other mechanism involved in silencing alloreactive responses by the HLA-A2 dimer. Peptide-loaded dimers offer a novel approach to induce peptide-specific immunosuppression, which may be useful in promoting graft survival.
引文
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[1]Hornick P. Direct and indirect allorecognition. Methods Mol Bio, 2006, 333:145-56
[2]Benichou G, Valujskikh A, heeger P S. Contributions of direct and indirect T cell alloreactivity dudring allograft rejection in mice. J Immunol, 1999,162:352-8.
[3]Whitelegg A, and Barber L D. The structural basis of T-cell allorecognition. Tissue antigens, 2004, 63:101-108.
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[20]Givan AL, Fisher JL, Waugh M, et al. A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens. J. Immunol. Methods, 1999, 230:99-112.
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[23]周晓辉.用Calcein释放实验检测HBsDNA疫苗诱生的特异性CTL活性.中华微生物学和免疫学杂志,2003,23(3):234-247
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[1]Hornick P. Direct and indirect allorecognition. Methods Mol Bio, 2006, 333:145-56
[2]Benichou G, Valujskikh A, heeger P S. Contributions of direct and indirect T cell alloreactivity dudring allograft rejection in mice. J Immunol, 1999,162:352-8.
[3]Whitelegg A, and Barber L D. The structural basis of T-cell allorecognition. Tissue antigens, 2004, 63:101-108.
[4]Suchin E J, Langmuir P B, palmer E, et al. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question. J Immunol, 2001, 166:973-81.
[5]Matzinger P and Bevan M J. Why do so many lymphocytes respond to major histocompatibility antigens? Cell Immunol, 1977, 29:1-5.
[6]Kourilsky P and Claverie J M.MHC-antigen interaction: what does the T cell receptor see.Adv Immunol, 1989,45:107-193.
[7]Wang W, Man S, Gulden P H, et al.Class I-restricted alloreactive cytotoxic T lymphocytes recognize a complex array of specific MHC-associated peptides. J Immunol, 1998,160:1091-1097
[8]Elliott T J and Eisen H N. Cytotoxic T lymphocytes recognize a reconstituted class I histocompatibility antigen (HLA-A2) as an allogeneic target molecule. Proc Natl Acad Sci USA., 1990,346:470-480
[9]Smith P A, Brunmark a, Jackson M R, et al. Peptide-independent recognition by alloreactive cytotoxic T lymphocytes (CTL). J Exp Med, 1997, 185:1023-1033
[10]Jankovic V, Remus K, Maolano A, et al. T cell recognition of an engineered MHC class I molecule: implications for peptide-independent alloreactivity. J Immunol, 2002,169: 1887-1892
[11]Heath W R, Kane K P, Mescher M F, et al. Alloreactive T cells discriminate amoung a diverse set of endogenous peptides. Proc Natl Acad Sci U S A, 1991, 88(12): 5101–5105
[12]Tallquist M D,Yun T J and Pease L R. A single T cell receptor recognizesstructureally distinct MHC/peptide complexes with high specificity. J Exp Med, 1996,184:1017-1026
[13]Alexander-Miller M A, Burke K, Koszinowski U H, et al. Alloreactive cytotoxic T lymphocytes generated in the presence of viral-derived peptides show exquisite peptide and MHC specificity. J Immunol, 1993, 151:1-10
[14]Whitelegg A and Barber L D. the structural basis of T-cell allorecognition.Tissue Antigens, 2004, 63:101-108
[15]Fischer L K and.Wilson DB. Histocompatibility antigen-activated cytotoxic T lymphocytes.Estimates of the absolute frequency of killer cells generated in vitro.J Exp Med, 1977, 145:500-507
[16]McMichael A.J.and C. A. O’Callaghan.A new look at T cells.J. Exp. Med., 1998, 187:1367-1371
[17]van Besouw N M, Zuijderwijk J M,Vaessen L M B, et al.The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation. Clinical & Experimental Immunology, 2005, 141 (3): 534–540.
[18]Jung T, Schauer U, Heusser C, et al.Detection of intracellular cytokines by flow cytometry.J Immunol Methods, 1993,159:197-207
[19]Brosterhus H, Brings S, eyendeckers H L,et al. Enrichment and detection of live antigen-specific CD4 and CD8 T cells based on cytokine secretion. Eur J Immunol, 1999,29:4053.
[20]Givan AL, Fisher JL, Waugh M, et al. A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens. J. Immunol. Methods, 1999, 230:99-112.
[21] Bercovici N, Givan AL, Waugh MG, et al. Multiparameter precursor analysis of T-cell responses to antigen.J Immunol Methods, 2003, 276:5-17
[22]Altman J D, Moss P A, Goulder P J R, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science, 1996, 274(5284):94-96.
[23]周晓辉.用Calcein释放实验检测HBsDNA疫苗诱生的特异性CTL活性.中华微生物学和免疫学杂志,2003,23(3):234-247
[24]Childs R, et al: Regression of metastatic renal-cell carcinoma afternonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Eng J Med, 2000, 343:750.
[25]Gao LQ, et al. Allo-major histocompatibility complex-restricted cytotoxic T lymphocytes engraft in bone marrow transplant recipients without causing graft-versus-host disease. Blood, 1999, 94:2999.
[26]Sadovnikova E and Stauss H J. Peptide-specific cytotoxic T lymphocytes restricted by nonself major histocompatibility complex class I molecules: reagents for tumor immunotherapy. Proc Natl Acad Sci USA, 1996, 93:2198-2203
[27]Gao L, Bellantuono I, Elsasser A, et al. Selective elimination of leukemic CD34(+) progenitor cells by cytotoxic T lymphocytes specific for WT1. Blood, 2000, 95:2198-2203
[28]Sadovnikova E, Jopling L A, Soo K s and Stauss H J. Generation of huamn tumor-reactive cytotoxic T cells against peptides presented by non-self HLA class I molecules. Eur J Immunol., 1998, 28:193-200
[29]Dutoit V, Guillaume P, Romero P, et al. Functional analysis of HLA-A*0201/Melan-A peptide multimer+ CD8+ T cells isolated from an HLA-A*0201-donor: exploring tumor antigen allorestricted recognition. Cancer Immunol, 2002, 2:7
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