行政院國家科學委員會專題研究計畫 期中進度報告
肌無力病人體內 T 細胞的研究(1/3)
計畫類別： 個別型計畫 計畫編號： NSC91-2320-B-002-189- 執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立臺灣大學生化科學研究所 計畫主持人： 果伽蘭 計畫參與人員： 馬廷維 王穗華 陳威儒 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中 華 民 國 92 年 6 月 2 日
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肌無力病人體內 T 細胞的研究 (1/3)
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中 華 民 國 92 年 5 月 31 日
中文摘要 我們研究肌無力病人內 T 細胞接受器的性質,以幫助了解 T 細胞如何導致此疾病 的發生/延續,並希望由此研究,我們可以為有專一性的免疫治療建立基礎。由鑑定 肌無力病人體內 T 細胞接受器(TCR)的基因序列,我們找到一類在病人間使用頻 率高於正常人很多的 TCR CDR3 基因序列,我們稱此類 TCR CDR3 基因序列為 “MG motifs”,非常令人興奮的是我們居然找到一個 TCR 的蛋白質序列,同時被兩 個 MG 病人所使用,這對變化度極高的 TCR 使用而言,是表示此 TCR 在體內被選 擇使用,尤其我們在文獻找到一有關實驗製造出的肌無力老鼠,它們使用的 TCR 也具有同樣比例的 MG motifs,我們相信此 motifs 與 MG 有關。我們將進一步確定 此 motifs 與 MG 間關係,以幫助了解此與專一性的免疫治療可行度。我們也另外 發現一條 invariant TCR 在病人與非病人間使用有很大差異,此性質在自體免疫機 制的調控所代表意義,值得進一步去了解。 關鍵詞: T 細胞接受器, 肌無力症, 關實驗製造出的肌無力老鼠, Invariant TCR. ABSTRACTTo identify T cell receptor (TCR) features which might provide basis and targets for specific immunotherapy for patients with autoimmune myasthenia gravis (MG) disease, the usage of TCR genes of freshly isolated peripheral blood lymphocytes from several MG patients was determined. Our results indicated that there is an apparent biased usage for certain amino acids in the TCR CDR3 region, we call it the “MG motifs”, among our MG patients when compared with the CDR3 sequences obtained from our non-MG persons. Strikingly one of the motif-containing TCR sequences was found in two MG patients with exactly identical TCR peptide sequence, meaning it was an in vivo selected, functional TCR in the two patients. Importantly, we found by searching through published documents that these motifs are also
apparently present in TCR CDR3 region of in vitro acetylcholine receptor (AChR) epitope-reactive T cell lines and hybridomas made from experimental autoimmune MG (EAMG) mice. We are confident that the biased CDR3 motifs on TCRs are related to this autoimmune disease and further experiments are planned to determine if our findings can form a base for specific immunotherapy on MG patients. Another related finding through this study is that there is an apparent differential usage on an invariant TCR chain gene in MG patients when compared with non-MG persons. The meaning of this differential usage in regulating immune response is worth further pursuing.
Key words: T cell receptor (TCR) gene usage, Myasthenia gravis (MG) disease,
Experimental autoimmune MG (EAMG) mice, Invariant TCR.
Myasthenia gravis is a classical autoantibody-mediated autoimmune disease. The autoantibodies against the neuromuscular junction nicotinic acetylcholine receptor (AChR) destroy AChR on the postsynaptic membrane and cause the muscle weakness seen in patients with MG and murine experimental autoimmune
myasthenia gravis (EAMG) (1, 2). Production of antibodies to AChR is dependent on T cells in human MG and EAMG (3, 4). MG is known to link to HLA/DQ -chain polymorphism (5), and major histocompatibility complex (MHC) class II molecules play a crucial role in the development of EAMG. Either a mutation in MHC class II -chain gene or an MHC class II gene disruption prevents
development of EAMG (6, 7). CD4 T cells play a crucial role in EAMG
pathogenesis because depletion of CD4 cells not only prevented but also treated EAMG (22). Further, CD4 gene knockout (KO) mice are relatively resistant to EAMG development (8).
In human MG or in animals immunized with intact AChR, the anti-AChR antibody response is polyclonal. A small extracellular region within residues 67-76 of the AChR -subunit, the main immunogenic region (MIR), seems to be a major target for anti-AChR antibodies. However, the anti-MIR mAbs are functionally and structurally heterogeneous. Antibodies against other extracellular epitopes on all AChR subunits are present in both experimental and human MG, these include antibodies to the acetylcholine-binding site that against AChR function in various ways and also induce acute experimental MG. Also anti-AChR antibodies
cross-reactive with non-AChR antigens exist, suggesting that MG may result from molecular mimicry (reviewed in 9). Therefore, despite extensive studies, many gaps still remain in the understanding of the antigenic structure of the AChR; especially in relation to human MG.
Among the unsolved problems in myasthenia gravis one is to concerning the origin of the autoimmune disease, as in the other human autoimmune diseases. The thymus has been implicated as a possible site of origin because approximately 75% of patients have thymic abnormalities (10). Of these, 85% have hyperplasia and 15% have thymomas. The myoid cells bearing surface AChR in the thymus gland are probably the source in providing AChR since they were shown particularly vulnerable to immune attack (11). Some alteration of the myoid cells or the lymphocytes, or a breach of immune regulation, may interfere with tolerance and lead to an autoimmune response. The molecular mimicry is regarded also as a potential factor in causing this autoimmune disease because cross-reactivity between bacteria and AChR in Abs isolated from some MG patients and a peptide sequence in herpes simplex virus that is homologous to a sequence of the AChR subunit have been reported (12, 13).
MG is currently treated by nonspecific immunosuppressive drugs with toxic side effects (14-16). An ideal treatment for MG should be directed at elimination of the AChR-specific T and/or B cells that are involved in the pathogenesis. In addition to the identifications of the MHC class II involvement, of MIR and other in vitro T cell epitopes on AChR, and the requirement of CD4+ T cells for MG pathogenesis mentioned above, results obtained from studies focused on the trimolecular
interaction between the MHC-TCR-AChR peptide complex during the development of EAMG showed that more than one TCRV-bearing cell having the affinity for AChR-dominant peptides is involved in pathogenesis (17). Therefore, depletion of a single TCRV with mAb may not be sufficient to completely suppress the response to AChR and development of EAMG/MG. If a similar amino acid sequence in the TCR V(D)J (i.e., CDR3) region among different TCR genes could be identified and proved to be involved in recognizing the AChR epitope(s), then motif-specific mAb reactive to the common motif within the V(D)J region of different TCR would be a more effective method for eliminating the T cells involved in MG development. We have been studying TCR usage in patients with cancers and in MG patients for quite a few years. Nine V genes including the ones showing biased usage in rheumatoid arthritis (RA) patients in the locus (18) were chosen for the studies.
cDNAs were made from freshly isolated peripheral blood lymphocytes from seven MG patients. Sequence-specific primers were used to amplify the TCR messages of interest by PCR using cDNA as templates. PCR products were then cloned,
sequenced and analyzed all done in our lab. Among the MG patients we have been following one patient (called D) for five years and six sets of TCR sequencing data were obtained. Four of the MG patients (MG I, II, III, and IV) were followed two times and samples were taken from the other two patients (called C and Y,
respectively) only once. Patient C is unique in that she had thymectomy for over ten years and been feeling normal for over years when her PBLs were collected.
Interestingly, one TCR sequence persistently appear in patient D in the six sets of data and another sequence with similar CDR3 motif started to appear in the recent two sets of data. We named these CDR3 sequences containing the charged amino acids with similar CDR3 length “MG motifs” and our data analysis showed that the ration of “MG motifs”-containing TCRs in MG patients is significantly higher than that of in non-MG persons (Table 1). We did T cell subset separation; i.e., CD4+, CD8+ or DN, on D’s PBL two times among the six sets of data and the results showed that the reappeared sequences belong to CD8+ and CD4+, respectively. Strikingly, one TCR peptide sequence with MG motif and obtained this week in D’s last set data is identical to one of Y’s peptide sequences obtained and stored in our data base one year ago. There is one nucleotide difference at the CDR3 region, meaning the peptide/TCR was selected in vivo for functionality. No other identical TCR peptide sequence was found to be present at different persons except for the known invariant TCR. Importantly, we found that the MG motif-containing TCRs are present at the similar ratio as our MG patients in the collection of T cells from EAMG mice (Table 1). Thus, we are confident that the presence of MG motifs correlates with the presence of MG. Currently, we are still collecting and analyzing more TCR sequences to determine if there are other CDR3 motifs potentially related to the pathogenesis of autoimmune disease MG. We now plan to generate transgenic TCR mice to determine the direct role of these MG motifs in vivo in the
development of MG. If the in vivo functional role of these motifs can be confirmed, then motif-specific mAb could be used to specifically eliminate the T cells involved in MG development/progression---one of our final goals.
A second unexpected finding during this period of time was that we observed a differential usage on one of the invariant TCRs between MG patients and non-MG persons. Originally this V was added to our TCR usage analysis for testing our “motif” theory whereas this unexpected result was also obtained. Although the function of invariant TCR in immunity in general has been suspected/shown to be
complicated and important, the exact roles and the in vivo antigen(s) are not clear. Now our data indicate that there is a relationship between the usage of this TCR and the presence of the autoimmune disease MG. We will pursue this study to probe the function of this TCR with regard to the immune regulation.
Another intriguing TCR sequence is identified in the PBL of the brother of the
long-time observed MG patient D. This sequence has a unique VJC combination; i.e., V-J-C. This type of combination in TCRs was only identified in mice so far with unknown function (19). With greater than 1000 TCR sequences we have obtained in humans, this type of recombination is only identified in PBL of the MG patient’s brother so far. However so far we do not have time nor persons to pursue the answer to the following questions yet: (1) What subset do the T cells with this type of TCR belong to? (2) What is the function of this subset of T cells? Is it related to the control of MG development in vivo? We would certainly hope that we could have a little more funding support to have some experienced persons to continue this study.
I have been delaying the writing work because my time has been taken by doing the experiments, teaching students about the lab work/how to analyzing the data, solving experimental problems created by students for mostly unknown reasons and the teaching obligation. Essentially, I do hope within next month I could write the first draft about our exciting MG motif to provide the evidence that there exists TCR CDR3 motif in autoimmune disease and to write about our chicken CD8
polymorphic mechanism too. Before ending this report, I do like to take this
opportunity to express our deep appreciation to NSC for supporting our uneasy work and we would also like to ask for more funding, if possible, to support a PhD student and/or a technician to help speed the experiments.
RERERENCES:
1. Drachman, D. B. 1994. Myasthenia gravis. N. Engl. J. Med. 330:1797-1810. 2. Christadoss, P. 1989. Immunogenetics of experimental autoimmune myasthenia
gravis. Crit. Rev. Immunol. 9:247-278.
3. Hohlfeld, R., Toyka, K. V. Heininger, K., Grosse-Wilde, H., and Kalies, I. 1984. Autoimmune human T lymphocytes specific for acetylcholine receptor. Nature 310:244-246.
4. Christadoss, P. and Dauphinee, M. J. 1986. Immunotherapy for myasthenia gravis: A murine model. J. Immunol. 136:2437-2440.
5. Bell, J., Smoot, S., Newly, C., Tokya, K., Rassenti, L., Smith, K., Hohlfeld, R., McDevitt, H. O., and Steinman, L. 1986. HLA-DQ -chain polymorphism linked to myasthenia gravis. Lancet 8489:1058-1060
6. Christadoss, P., Lindstrom J. M., Melvold, R. W., and Talal, N. 1985. Mutation at I-A -chain prevents experimental autoimmune myasthenia gravis.
Immunogenetics 21:33-38.
7. Kaul, R., Shenoy, M., Golusako, E., and Christadoss, P. 1994. Major
histocompatibility complexes class II gene disruption prevents experimental autoimmune myasthenia gravis. J. Immunol. 152:3152-3157.
8. Zhang, G.-X., Xiao, B.-G., Bakhiet, M., van Der Meide, P., Wigzell, H., Link, H., and Olsson, T. 1996. Both CD4+ and CD8+ T cells are essential to induce
experimental autoimmune myasthenia gravis. J. Exp. Med. 184:349-356.
9. Tzartos, S. J., Barkas, T., Cung, M. T., Mamalaki, A., Marraud, M., Orlewski, P., Papanastasiou, D., Sakarellos, C., Sakarellos-Daitsiotis, M., Tsantili, P., and Tsikaris, V. 1998. Anatomy of the antigenic structure of a large membrane autoantigen, the muscle-type nicotinic acetylcholine receptor. Immunol. Rev. 163:89-120.
10. Castleman, B. 1966. The pathology of the thymus gland in myasthenia gravis.
Ann N Y Acad Sci. 135:496-505.
11. Kao, I. and Drachman, D. B. 1977. Thymic muscle cells bear acetylcholine receptors: possible relation to myasthenia gravis. Science 195:74-75.
12. Schwimmbeck, P. L., Dyrberg, T., Drachman, D. B., and Oldstone, M. B. A. 1989. Molecular mimicry and myasthenia gravis: an autoantigenic site of the acetylcholine receptor -subunit that has biologic activity and reacts
immunochemically with herpes simplex virus. J. Clin. Invest. 84:1174-1180. 13. Stefansson, K., Dieperink, M. E., Richman, D. P., Gomez, C. M., and Marton, L.
S. 1985. Sharing of antigenic determinants between the nicotinic acetylcholine receptor and proteins in Escherichia coli, Proteus vulgaris, and Klebsiella pneumoniae: possible role in the pathogenesis of myasthenia gravis. N. Engl. J.
Med. 312:221-225.
14. Drachman, D. B. 1993. Myasthenia gravis. In Current Therapy in Neurologic
Disease. 4th ed. R. T. Johnson and J. W. Griffin, eds. St. Louis: Mosby-year Book, pp, 379-384.
15. Tindall, R., S., Phillips, J. T., Rollins, J. A., Wells, L., and Hall, K. 1993.A clinical therapeutic trial of cyclosporine in myasthenia gravis. Ann. NY Acad.
Sci. 681:539.
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17. Wu, B., Shenoy, M., Goluszko, E., Kaul, R., and Christadoss, P. 1995. TCR gene usage in experimental autoimmune myasthenia gravis pathogenesis: usage of multiple TCRBV genes in the H-2b strains. J. Immunol. 154:3603-3610. 18. Pluschke, G., Ricken, G., Taube, H., Kroniger, S., Melchers, I., Peter, H.,
Eichmann, H., and Krawinkel, K. 1991. Biased T cell receptor V region
repertoire in the synovial fluid of rheumatoid arthritis patients. Eur. J. Immunol. 21:2749-2754.
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Table 1
Ratio of MG motif-containing TCRs among non-MG persons and MG patients. Data shown here were results from TCRs with the specifc V utilized by the repeatly appeared TCR sequences by MG patient D, not from the entire TCR V set.
Non-MG persons T / N1 1/A 2/H 1/L Ratio2 12.5 ( 01 / 08 ) 11.5 ( 03 / 26 ) 20.8 ( 04 / 24 ) Subsum 12.5 11.5 16.7 Total 12.5 11.5 16.7 MG patient “C”3 T / N 1/C Ratio 16.7 ( 06 / 36 ) Subsum 16.7 Total 16.7 MG Patients 4/D 6/D T / N 1/Y 1/D 2/D 3/D DP4 CD45 CD86 DN7 5/D CD4 CD8 DN 2/Ⅲ 2/Ⅳ Ratio 27.8 ( 10 / 36 ) 53.3 ( 08 / 15 ) 25.0 ( 02 / 08 ) 71.4 ( 05 / 07 ) 00.0 ( 00 / 10) 44.4 ( 04 / 09 ) 88.9 ( 08 / 09 ) 100.0 ( 02 / 02 ) 35.7 ( 10 / 28 ) 45.8 ( 11 / 24 ) 48.1 ( 13 / 27 ) 55.6 ( 05 / 09 ) 92.0 ( 22 / 25 ) 31.6 ( 12 / 38 ) Subsum 27.8 53.3 25.0 71.4 46.7 ( 14 / 30 ) 35.7 48.3 ( 29 / 60 ) 88.0 31.6 Total 27.8 45.5 ( 50 / 110 ) 88.0 31.6
EAMG T / N Mouse8 Ratio 50.0 ( 11 / 22 ) Subsum 50.0 Total 50.0 1
T / N：number indicates the order the data were obtained if PBL samples were taken more than once / Name of the person from which PBLs were analyzed
2
No. of sequences containing MG motif in N region of TCR CDR3
Ratio =
---
Total no. of sequences successfully subcloned and sequenced from the time blood sample was analyzed
3
Once a MG patient, by now lives a normal life after thymus had been amputated for years.
4
DP：Double Positive.
5
CD4：T cells with CD4 coreceptor.
6
CD8：T cells with CD8 coreceptor.
7
DN：Double Negative, T cells with no CD4 nor CD8 coreceptor.
8
Data adaped from Ellen Kraig, et al, Restricted T cell receptor repertoire for acetylcholine receptor in murine myasthenia gravis, J. of Neuroimmunology (1996) 71, 87-95.
