协同刺激分子CD80和CD86在实验性变态反应性神经炎发病中的作用
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摘要
目的 探讨协同刺激分子在实验性变态反应性神经炎发病中的作用。
方法 以18只2月龄的、体重1000克左右的雄性新西兰大白兔为研究对象,随机分为3组:FKG组背部多点皮下注射完全弗氏佐剂(FCA)、血蓝蛋白(KLH)和神经节苷脂(GM-1)混合制成的抗原乳剂;FK组注射完全弗氏佐剂和血蓝蛋白;FG组注射完全弗氏佐剂和神经节苷脂。各组动物均为6只,共注射3次。分别于注射前及第一次注射后每隔10天抽血提取淋巴细胞和血浆一次。采用逆转录多聚酶链反应技术(Reverse Transcription Polymerase Chain Reaction,RT-PCR)来半定量分析外周血淋巴细胞CD80和CD86 mRNA的表达水平;通过酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)方法检测血浆抗GM1-IgG抗体滴度。
结果 FKG组、FK组CD80 mRNA的表达在免疫后10天开始增加,免疫后40天达高峰;免疫后各个时间点上FKG组、FK组CD80 mRNA的表达与FG组比较,差异有显著性(P<0.01),但FKG组与FK组比较差异无显著性(P>0.05)。FKG组、FK组CD86 mRNA的表达在免疫后10天开始增加,免疫后30天达高峰;免疫后各个时间点上FKG组、FK组CD86mRNA的表达与FG组比较,差异有显著性(P<0.01),但FKG组与FK组比较只有在免疫后40天差异有显著性(P<0.05),在其它时间点比较差异无显著性(P>0.05)。FKG组抗GM1-IgG抗体滴度在免疫后10天开始增加,免疫后40天达高峰;免疫后各个时间点上FKG组与FK组、FG组比较,差异有显著性(P<0.01),但FK组与FG组比较差异无显著性(P>0.05)。
结论 协同刺激分子CD80和CD86的表达通过载体蛋白活化T细胞,进而导致抗GM1-IgG抗体产生,在EAN的发病中起重要作用。
Objective To investigate the roles of costimulatory molecules in experimental allergic neuritis(EAN).Methods EAN modles were established on 18 male New Zealand white rabbits. They were randomly divided into 3 groups. Subcutaneous injection took place on the rabbits of FKG with antigen emulsion that was mixed by FCA, KLH and GM1. FCA and KLH in FK group, FCA and GM1 in FG . There were 6 rabbits in each group and 3 injections on each rabbit. Lymphocyte and blood serum were collected before the first injection and every 10 days after the first injection. We analyse the expression of CD80 and CD86 mRNA in peripheral blood lymphocyte by Reverse Transcription Polymerase Chain Reaction(RT-PCR) and detect anti-GM1-IgG antibody titre in peripheral blood serum by Enzyme-linked immunosorbent assay (ELISA).Results The expression of CD80 mRNA in FKG and FK began to rise on the 10th day after immunity .It reached the peak on the 40th day after the injection.In FKG and FK it had a significant difference at each time point comparied with FG (P<0. 01),but there was no difference between FKG and FK(P>0.05). The expression of CD86 mRNA in FKG and FK began to rise on the 10th day after immunity .It reached the peak on the 30th day after the injection. In FKG and FK it had a significant difference at each time point comparied with FG (P<0. 01), between FKG and FK there was different only on the 40th day(P<0. 05),on the other time point there was no difference (P>0.05). Anti-GM1-IgG antibody titre in FKG began to rise on the 10th day after immunity. It reached the peak on the 40th day after the injection. In FKG it had a significant difference at each time point comparied with FK and FG (P<0. 01), but between FK and FG there was no difference (P>0.05).Conclusion Costimulatory molecules CD80 and CD86 play an important role in the activation of T cell mediated by protein carrier, then induced the production of anti-GM1 antibody, and then induced EAN..
方法 以18只2月龄的、体重1000克左右的雄性新西兰大白兔为研究对象,随机分为3组:FKG组背部多点皮下注射完全弗氏佐剂(FCA)、血蓝蛋白(KLH)和神经节苷脂(GM-1)混合制成的抗原乳剂;FK组注射完全弗氏佐剂和血蓝蛋白;FG组注射完全弗氏佐剂和神经节苷脂。各组动物均为6只,共注射3次。分别于注射前及第一次注射后每隔10天抽血提取淋巴细胞和血浆一次。采用逆转录多聚酶链反应技术(Reverse Transcription Polymerase Chain Reaction,RT-PCR)来半定量分析外周血淋巴细胞CD80和CD86 mRNA的表达水平;通过酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)方法检测血浆抗GM1-IgG抗体滴度。
结果 FKG组、FK组CD80 mRNA的表达在免疫后10天开始增加,免疫后40天达高峰;免疫后各个时间点上FKG组、FK组CD80 mRNA的表达与FG组比较,差异有显著性(P<0.01),但FKG组与FK组比较差异无显著性(P>0.05)。FKG组、FK组CD86 mRNA的表达在免疫后10天开始增加,免疫后30天达高峰;免疫后各个时间点上FKG组、FK组CD86mRNA的表达与FG组比较,差异有显著性(P<0.01),但FKG组与FK组比较只有在免疫后40天差异有显著性(P<0.05),在其它时间点比较差异无显著性(P>0.05)。FKG组抗GM1-IgG抗体滴度在免疫后10天开始增加,免疫后40天达高峰;免疫后各个时间点上FKG组与FK组、FG组比较,差异有显著性(P<0.01),但FK组与FG组比较差异无显著性(P>0.05)。
结论 协同刺激分子CD80和CD86的表达通过载体蛋白活化T细胞,进而导致抗GM1-IgG抗体产生,在EAN的发病中起重要作用。
Objective To investigate the roles of costimulatory molecules in experimental allergic neuritis(EAN).Methods EAN modles were established on 18 male New Zealand white rabbits. They were randomly divided into 3 groups. Subcutaneous injection took place on the rabbits of FKG with antigen emulsion that was mixed by FCA, KLH and GM1. FCA and KLH in FK group, FCA and GM1 in FG . There were 6 rabbits in each group and 3 injections on each rabbit. Lymphocyte and blood serum were collected before the first injection and every 10 days after the first injection. We analyse the expression of CD80 and CD86 mRNA in peripheral blood lymphocyte by Reverse Transcription Polymerase Chain Reaction(RT-PCR) and detect anti-GM1-IgG antibody titre in peripheral blood serum by Enzyme-linked immunosorbent assay (ELISA).Results The expression of CD80 mRNA in FKG and FK began to rise on the 10th day after immunity .It reached the peak on the 40th day after the injection.In FKG and FK it had a significant difference at each time point comparied with FG (P<0. 01),but there was no difference between FKG and FK(P>0.05). The expression of CD86 mRNA in FKG and FK began to rise on the 10th day after immunity .It reached the peak on the 30th day after the injection. In FKG and FK it had a significant difference at each time point comparied with FG (P<0. 01), between FKG and FK there was different only on the 40th day(P<0. 05),on the other time point there was no difference (P>0.05). Anti-GM1-IgG antibody titre in FKG began to rise on the 10th day after immunity. It reached the peak on the 40th day after the injection. In FKG it had a significant difference at each time point comparied with FK and FG (P<0. 01), but between FK and FG there was no difference (P>0.05).Conclusion Costimulatory molecules CD80 and CD86 play an important role in the activation of T cell mediated by protein carrier, then induced the production of anti-GM1 antibody, and then induced EAN..
引文
1. Peach RJJ, Baiorath J, Naemura G, et al. Both extracellular immunoglobulin like domains of CD80 contain residues critical for binding T cell surface receptors CTLA-4 and CD28. J Bid Chem, 1995,270:21181-21187.
2. Taylor WA, Brostoff SW, Hughs RA. p2 specific lymphocyte transformation in Guillain-Barre syndrome and chronic idiopathic demyelinating polyradiculoneuropathy. J Neuro Sci,1991,104:52.
3.王强,王虹蛟,等.实验性轴索型格林—巴利结合征动物模型的建立.中华医学杂志,2000,80(12):947-949。
4.王强,孟威宏,等.急性运动性轴索型GBS的免疫发病机制研究进展.神经疾病与精神卫生杂志,2001,1(3):53-54。
5. Yuki N, Taki T, et al. Penner's serotype 4 of Campylobacterjejuni has a lipopoly saccharide that bears a GM_1 ganglioside epitope as well as on that bear a GD1a epitope. Infect Immun, 1994,62:2101.
6. Yuki N, Taki T, et al. A bacterium lipopolysaccharide that elicits Guillain-Barre syndrome has a GM_1 ganglioside-like structure. J Exp Med, 1993,178:1771.
7. Fujimoto S, Amako K. Guillain-Barre syndrome and campylobacter jejuni infection. Lancet, 1990,335; 1350.
8. Speed BR, Kaldor T, et al. Campylobacter jejuni/Campylobacter coli associated Guillain-Barre syndrome. Med J Aust, 1987,147:13-6
9.赵晓燕,薛平,等.运动轴索型格林-巴利结合征患者血清对神经轴索损害作用的研究.脑与神经疾病杂志,1998,6(4):200-203
10.赵晓燕,薛平,等.空肠弯曲菌抗体对兔周围神经致病作用的研究.中风与神经疾病杂志,1999,16(6):325-327
11.张杰敏,蔡方成.不同血清型空肠弯曲菌免疫大鼠的血清及单个核细胞转移诱发周围神经病变的比较研究.中华儿科杂志,2002,1,40(1):32-36.
12. Yuki N, Yoshino H, et al, Acute axonal polyneuropathy associated wih ani-GM_1 antibodies following Campylobacter jejuni enteritis. Neurology, 1990,40:1900-1902.
13. Santro M, Thomas FP, et al. IgM deposits at nodes of Ranvier in a patient with amyotrophic lateral sclerosis, anti-GM_1 antibodies and multiforcal motor conduction block. Ann Neurol, 1990,28:373.
14. Thomas FP, Trojaborg W, et al. Experimental autoimmune neuropathy with anti-GM_1 antibodies and immunoglobulin deposits at nodes of Ranvier. Acta Neuropathol, 1991,82:378.
15. Santoro M, Uncini A, et al. Experimentul conduction block induced by serum from a patients with anti- GM_1 antibodies. Ann Nourol, 1992,31:385.
16. Willison HJ, O' Hanlon GM, et al. Mechanism of action of anti-GM_1 and anti-GQ_(1b) ganglioside antibodies in Guillain-Barre syndrome. J Infections Disease,1997, 176(Supple 2):S144.
17. Kusunoki S, Mashiko H, et al. Binding of antibodies against GM_1 and GD_(1b) in human peripheral nerve. Muscle Nerve, 1997,20:840.
18. Ledeen RW, Cannella MS, et al. Neurobiology of ganghosides. Clin Neuropharmacol, 1986, 9(Supple4):331-3.
19. Ouarler RH, Ilyas AA, et al. Antibodies to glycolipids in demyelinating disease of the human peripheral nervous systems. Chem Phys lipids, 1986,42:235.
20. Ilyas AA, Wilson HJ, et al. Serum antibodies to gangliosides in Guillain-Barre syndrome. Ann Neurol, 1988,23:440.
21. Sadiq SA, Thomas FP, et al. The spectrum of neurologic disease associated with anti-GM_1 antibodies. Neurology, 1990,40:1067-72.
22. Walsh FS, Cronin M, et al. Association between glycoconjugate antibodies and Campylobacter infection in patients with Guillain-Barre syndrome. J Neuroimmunol, 1991,34:43-51.
23. Ender U, karch H, et al. The spetrum of immune responses to Campylobacter jejuni and glycoconjugates in Guillain- Barre syndrome and in other neuroimmunological disorders. Ann Neurol, 1993,34:136-44.
24. Jacobs BC, Meulstee J, et al. Electrodiagnostic finding related to anti-GM_1 and anti-GQ_(1b) antibodies in Guillain-Barre syndrome. Muscle Nerve.1997.20(4): 446-52.
25.闫晓波,王维治.格林—巴利综合征患者血清及脑脊液中GM_1抗体及IgG指数检测。中国神经免疫学和神经病学杂志,1997,12,4(4):230-3.
26.忻琪珺,程源深.格林—巴利综合征临床、C-jejuni和GM_1抗体的相关性.中国临床神经科学,1999,7(1):4-9.
27.忻琪珺,程源深.血清Cj抗体、GM_1抗体和格林巴利综合征的相关性研究.中国神经免疫学和神经病学杂志,1999,6(1):27-31.
28.萧兴军,陈琳,等.格林—巴利综合征患者血清和脑脊疫中GM_1抗体的检测及意义.哈尔滨医科大学学报,1996,6,33(3):170-2.
29.李春岩,龚燕平,等.格林—巴利综合征患者血清中抗神经节昔脂抗体的检测.脑与神经疾病杂志,2001.9(5):274-6.
30. Thomas FP, Trojaborg W, et al. Experimental autoimmune neuropathy with-GM_1 antibodies and immunoglobulin deposits at node of Ranvier. Acta Neuropathol, 1991,82:378-383.
31. Illa I, Ortiz N, et al, Acute axonal Guillain-Barre syndrome nith IgG antibodies against motor axons following parenteral gangliosides.Ann Neurol,1995,38,218-24.
32. Yuki N, Yamada M, et al. Animal model of Guillain-Barre syndrome induced by sensitization with GM, ganglioside. Ann Neurol ,2001, 49(6):712-20.
33. Hathcock KSG,Laszlo HB,Dickler J,et al.Identification of an alternative CTLA-4 ligand costimulatory for T cell activation.Science, 1993,262:905.
34. Freeman GJ,Gribben JG,Boussiotis VA,et al. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science. 1993 ,262 :909-l 1.
35. Thompson CB, Distinct roles for the costimulatory ligands B7-1 and B7-2 in T helper cell differentiation?Cell. 1995 Jun 30;81(7):979-82.
36. Cross AH,Girard TJ,,Giacoletto KS,et al. Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. J Clin Invest. 1995 Jun;95(6):2783-9.
37. Djukanovic R.The role of co-stimulation in airway inflammation.Clin Exp Allergy. 2000 Jun;30 Suppl 1:46-50.
38. Hernandez HJ,Sharpe AH,Stadecker MJ,et al. Experimental murine schistosomiasis in the absence of B7 costimulatory molecules: reversal of elicited T cell cytokine profile and partial inhibition of egg granuloma formation. J Immunol. 1999 Mar l;162(5):2884-9.
39. Linsley RS,Clark EA.Ledbetter JA.T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-l.Proc Natl Acad Sci USA. 1990 Jul;87(13):5031-5.
40. Lazetic S,Leong SR,Chang JC,et al.Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells.J Biol Chem. 2002 Oct ll;277(41):38660-8.
41. Jeannin P,Magistrelli G,Aubry JP,et al. Soluble CD86 is a costimulatory molecule for human T lymphocytes.lmmunity. 2000 Sep;13(3):303-12.
42. Blazevic V.Trubey CM,Shearer GM. Analysis of the costimulatory requirements for generating human virus-specific in vitro T helper and effector responses.J Clin Immunol. 2001 Jul;21(4):293-302.
43. Lanier LL,O' Fallon S,Somoza C,et al.CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL. J Immunol. 1995 Jan l;154(1):97-105.
44. Azuma M,Yssel H,Phillips JH,et al. Functional expression of B7/BB1 on activated T lymphocytes.J Exp Med. 1993 Mar l;177(3):845-50.
45. Ramarathinam L,Castle M,Wu Y,et al. T cell costimulation by B7/BB1 induces CD8 T cell-dependent tumor rejection: an important role of B7/BB1 in the induction, recruitment, and effector function of antitumor T cells.J Exp Med. 1994 Apr l;179(4):1205-14.
2. Taylor WA, Brostoff SW, Hughs RA. p2 specific lymphocyte transformation in Guillain-Barre syndrome and chronic idiopathic demyelinating polyradiculoneuropathy. J Neuro Sci,1991,104:52.
3.王强,王虹蛟,等.实验性轴索型格林—巴利结合征动物模型的建立.中华医学杂志,2000,80(12):947-949。
4.王强,孟威宏,等.急性运动性轴索型GBS的免疫发病机制研究进展.神经疾病与精神卫生杂志,2001,1(3):53-54。
5. Yuki N, Taki T, et al. Penner's serotype 4 of Campylobacterjejuni has a lipopoly saccharide that bears a GM_1 ganglioside epitope as well as on that bear a GD1a epitope. Infect Immun, 1994,62:2101.
6. Yuki N, Taki T, et al. A bacterium lipopolysaccharide that elicits Guillain-Barre syndrome has a GM_1 ganglioside-like structure. J Exp Med, 1993,178:1771.
7. Fujimoto S, Amako K. Guillain-Barre syndrome and campylobacter jejuni infection. Lancet, 1990,335; 1350.
8. Speed BR, Kaldor T, et al. Campylobacter jejuni/Campylobacter coli associated Guillain-Barre syndrome. Med J Aust, 1987,147:13-6
9.赵晓燕,薛平,等.运动轴索型格林-巴利结合征患者血清对神经轴索损害作用的研究.脑与神经疾病杂志,1998,6(4):200-203
10.赵晓燕,薛平,等.空肠弯曲菌抗体对兔周围神经致病作用的研究.中风与神经疾病杂志,1999,16(6):325-327
11.张杰敏,蔡方成.不同血清型空肠弯曲菌免疫大鼠的血清及单个核细胞转移诱发周围神经病变的比较研究.中华儿科杂志,2002,1,40(1):32-36.
12. Yuki N, Yoshino H, et al, Acute axonal polyneuropathy associated wih ani-GM_1 antibodies following Campylobacter jejuni enteritis. Neurology, 1990,40:1900-1902.
13. Santro M, Thomas FP, et al. IgM deposits at nodes of Ranvier in a patient with amyotrophic lateral sclerosis, anti-GM_1 antibodies and multiforcal motor conduction block. Ann Neurol, 1990,28:373.
14. Thomas FP, Trojaborg W, et al. Experimental autoimmune neuropathy with anti-GM_1 antibodies and immunoglobulin deposits at nodes of Ranvier. Acta Neuropathol, 1991,82:378.
15. Santoro M, Uncini A, et al. Experimentul conduction block induced by serum from a patients with anti- GM_1 antibodies. Ann Nourol, 1992,31:385.
16. Willison HJ, O' Hanlon GM, et al. Mechanism of action of anti-GM_1 and anti-GQ_(1b) ganglioside antibodies in Guillain-Barre syndrome. J Infections Disease,1997, 176(Supple 2):S144.
17. Kusunoki S, Mashiko H, et al. Binding of antibodies against GM_1 and GD_(1b) in human peripheral nerve. Muscle Nerve, 1997,20:840.
18. Ledeen RW, Cannella MS, et al. Neurobiology of ganghosides. Clin Neuropharmacol, 1986, 9(Supple4):331-3.
19. Ouarler RH, Ilyas AA, et al. Antibodies to glycolipids in demyelinating disease of the human peripheral nervous systems. Chem Phys lipids, 1986,42:235.
20. Ilyas AA, Wilson HJ, et al. Serum antibodies to gangliosides in Guillain-Barre syndrome. Ann Neurol, 1988,23:440.
21. Sadiq SA, Thomas FP, et al. The spectrum of neurologic disease associated with anti-GM_1 antibodies. Neurology, 1990,40:1067-72.
22. Walsh FS, Cronin M, et al. Association between glycoconjugate antibodies and Campylobacter infection in patients with Guillain-Barre syndrome. J Neuroimmunol, 1991,34:43-51.
23. Ender U, karch H, et al. The spetrum of immune responses to Campylobacter jejuni and glycoconjugates in Guillain- Barre syndrome and in other neuroimmunological disorders. Ann Neurol, 1993,34:136-44.
24. Jacobs BC, Meulstee J, et al. Electrodiagnostic finding related to anti-GM_1 and anti-GQ_(1b) antibodies in Guillain-Barre syndrome. Muscle Nerve.1997.20(4): 446-52.
25.闫晓波,王维治.格林—巴利综合征患者血清及脑脊液中GM_1抗体及IgG指数检测。中国神经免疫学和神经病学杂志,1997,12,4(4):230-3.
26.忻琪珺,程源深.格林—巴利综合征临床、C-jejuni和GM_1抗体的相关性.中国临床神经科学,1999,7(1):4-9.
27.忻琪珺,程源深.血清Cj抗体、GM_1抗体和格林巴利综合征的相关性研究.中国神经免疫学和神经病学杂志,1999,6(1):27-31.
28.萧兴军,陈琳,等.格林—巴利综合征患者血清和脑脊疫中GM_1抗体的检测及意义.哈尔滨医科大学学报,1996,6,33(3):170-2.
29.李春岩,龚燕平,等.格林—巴利综合征患者血清中抗神经节昔脂抗体的检测.脑与神经疾病杂志,2001.9(5):274-6.
30. Thomas FP, Trojaborg W, et al. Experimental autoimmune neuropathy with-GM_1 antibodies and immunoglobulin deposits at node of Ranvier. Acta Neuropathol, 1991,82:378-383.
31. Illa I, Ortiz N, et al, Acute axonal Guillain-Barre syndrome nith IgG antibodies against motor axons following parenteral gangliosides.Ann Neurol,1995,38,218-24.
32. Yuki N, Yamada M, et al. Animal model of Guillain-Barre syndrome induced by sensitization with GM, ganglioside. Ann Neurol ,2001, 49(6):712-20.
33. Hathcock KSG,Laszlo HB,Dickler J,et al.Identification of an alternative CTLA-4 ligand costimulatory for T cell activation.Science, 1993,262:905.
34. Freeman GJ,Gribben JG,Boussiotis VA,et al. Cloning of B7-2: a CTLA-4 counter-receptor that costimulates human T cell proliferation. Science. 1993 ,262 :909-l 1.
35. Thompson CB, Distinct roles for the costimulatory ligands B7-1 and B7-2 in T helper cell differentiation?Cell. 1995 Jun 30;81(7):979-82.
36. Cross AH,Girard TJ,,Giacoletto KS,et al. Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation. J Clin Invest. 1995 Jun;95(6):2783-9.
37. Djukanovic R.The role of co-stimulation in airway inflammation.Clin Exp Allergy. 2000 Jun;30 Suppl 1:46-50.
38. Hernandez HJ,Sharpe AH,Stadecker MJ,et al. Experimental murine schistosomiasis in the absence of B7 costimulatory molecules: reversal of elicited T cell cytokine profile and partial inhibition of egg granuloma formation. J Immunol. 1999 Mar l;162(5):2884-9.
39. Linsley RS,Clark EA.Ledbetter JA.T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-l.Proc Natl Acad Sci USA. 1990 Jul;87(13):5031-5.
40. Lazetic S,Leong SR,Chang JC,et al.Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells.J Biol Chem. 2002 Oct ll;277(41):38660-8.
41. Jeannin P,Magistrelli G,Aubry JP,et al. Soluble CD86 is a costimulatory molecule for human T lymphocytes.lmmunity. 2000 Sep;13(3):303-12.
42. Blazevic V.Trubey CM,Shearer GM. Analysis of the costimulatory requirements for generating human virus-specific in vitro T helper and effector responses.J Clin Immunol. 2001 Jul;21(4):293-302.
43. Lanier LL,O' Fallon S,Somoza C,et al.CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production, and generation of CTL. J Immunol. 1995 Jan l;154(1):97-105.
44. Azuma M,Yssel H,Phillips JH,et al. Functional expression of B7/BB1 on activated T lymphocytes.J Exp Med. 1993 Mar l;177(3):845-50.
45. Ramarathinam L,Castle M,Wu Y,et al. T cell costimulation by B7/BB1 induces CD8 T cell-dependent tumor rejection: an important role of B7/BB1 in the induction, recruitment, and effector function of antitumor T cells.J Exp Med. 1994 Apr l;179(4):1205-14.