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1 第 6 回認定医集中教育セミナー抗 HLA 抗体のモニタリング広島大学大学院医歯薬保健学研究院応用生命科学部門消化器移植外科学大段秀樹 Anti-HLA Abs 非自己 HLA 抗原の移入により産生輸血妊娠移植歴など抗体関連型拒絶反応のハイリスク群グラフト廃絶の主要な原因 1

2 Methods for HLA-specific Ab screening and donor cross-matching testing Cell-Based Assays Complement-dependent cytotoxicity (CDC) =Lymphocyte cytotoxicity test (LCT) Anti-human immunoblobulin-lct (AHG-LCT) Flow cytometry crossmatch (FCXM) Solid-Phase Immunoassays Enzyme-linked immunosorbent assay (ELISA) Bead-based array assays (Flow cytometry or Luminex) Complement-dependent cytotoxicity (CDC) =Lymphocyte cytotoxicity test (LCT) 2

3 Complement-dependent cytotoxicity (CDC) =Lymphocyte cytotoxicity test (LCT) Kidneys transplanted across a positive crossmatch failed immediately. Higher risk: multiparous females patients reciving secondary transplants Immediate failures occurred among transplants from unrelated than among those from related donors. Complement-dependent cytotoxicity (CDC) =Lymphocyte cytotoxicity test (LCT) The presence of preformed cytotoxic antibodies against the donor appears to be a strong contraindication for transplantation 3

4 Complement-dependent cytotoxicity (CDC) =Lymphocyte cytotoxicity test (LCT) ント血 1 L ナーリン 1 LL 反応体反応ン定 Complement-dependent cytotoxicity (CDC) =Lymphocyte cytotoxicity test (LCT) 4

5 Complement-dependent cytotoxicity (CDC) =Lymphocyte cytotoxicity test (LCT) 血 dithiothreitol DTT IgM 抗体の Anti human immunoglobulin AHG L-chain Type L-chain Type より Interpretation of crossmatch result (Mulley WR, Kanellis J. Nephrology ) 5

6 Points at LCT-XM, AHG-LCT-XM 定の主に抗体ななリンのViabilityによ大非抗体によな血中の抗体の体のに IgGのisotypeによ体なに Flow cytometry crossmatch (FCXM) ント血 100 L ナーリン 100 L 200, 反応 Goat F(ab )2 anti-human IgG-FITC, CD19- PE, CD3-PerCP 4 30 反応フーイトトリー定 6

7 Flow cytometry crossmatch (FCXM) B-FCXM に B のBCR 抗体 cross reaction pronase 反応 pronase BCR 外の抗原ににりなフーイトーター要 Points at FCXM HLA 抗体の歴のな定要定によなントーナー血 AB 型のー血定 MFI 絶ントー 10 のフト血の MFI ントーの MFI のナー血 FCXM 用の B 7

Luminex LABScreen Mixed LABScreen PRA LABScreen Single Antigen 抗体の %PRA %PRA

8 Solid-Phase Immunoassays Enzyme-linked immunosorbent assay (ELISA) Bead-based array assays Flow cytometry Flow PRA Screening Test Flow PRA Specific Flow PRA Single Antigen Luminex LABScreen Mixed LABScreen PRA LABScreen Single Antigen 抗体の %PRA %PRA Differences between Flow PRA & LABScreen Flow PRA LABScreen 体な器要要定自自要要器要薬 non-hla 抗体な 8

9 Flow PRA Screening Test Class I Beads Class I PRA = 99% Class II Beads Class II PRA = 66% Flow PRA Specific Specificity: B57, 58 PRA: 9.4% Flow PRA Specific Flow PRA Single Antigen 9

10 Flow PRA Single Antigen LABScreen Mixed HLA 抗体 MICA 抗体のスクリーニング Luminex ー 1 り 3 5 の (HLA 抗原 ) の HLA 抗原 MICA 抗原ーング Class l: 12beads (3 panels/bead) Class ll: 5beads (5panels/bead) MICA-Ag: 2beads (5panels/bead) Class I: Class II: 10

11 LABScreen PRA HLA 抗体の PRA 定 Luminex ー 1 り 1 のの HLA 抗原ーング Class I 55 のー Class II 35 のーに HLA 抗原ーング LABScreen Single Antigen HLA 抗体の定ー 1 り 1 の HLA 抗原ーング 11

12 ICFA (Immunocomplex Capture Fluorescence Analysis ) HLA HLA Ab non-hla Ab + Donor Lymphocyte Patient serum Ag Ab reaction Solubilization Anti-HLA class I or II Ab Immune Complex Wash Capture Second Ab reaction PE labeled anti-human IgG Ab HLA class I or II specific beads Analysis by Luminex Determination by index (Positive >2.0) Variation in Results by Choice of Anti-HLA Antibody Detection Technique Method Positive Negative CDC AHG-CDC 116 (+13%) 148 ELISA 127 (+10%) 137 Flow PRA 139 (+10%) 125 (Gebel, HM & Bray, RA. Transplantation. 2000) 12

13 Preformed DSA Preformed DSA class I/II に関廃絶の因 Lefaucheur C, et al. J Am Soc Nephrol 2010; 21:

14 ハイタイター preformed 抗 HLA-DSA 移植廃絶のリスク Lefaucheur C, et al. J Am Soc Nephrol 2010; 21: 1398 IgG クラスの Complement fixing subclass C1 complex IgG3 >> IgG1 > IgG2 > IgG4 Complement tivati Fc-receptor binding properties IgG3 > IgG1 >> IgG2 > IgG4 Cell mediated cytotoxicity 14

(University Hospital Basel, Switzerlan) Transplantation 2011;

15 Pretransplant IgG subclasses of donor-specific HLA Abs and development of AMR Hönger G, et al. (University Hospital Basel, Switzerlan) Transplantation 2011; 92(1):41 ニン IgG2/IgG4 subclass DSA のント AMR グラフト生 Hönger G, et altransplantation 2011; 92(1):41 15

16 Preformed DSA に関ンセンス Preformed DSA class I/II に関廃絶の因 Cross Reactive Epitope Group (CREG) Abs の AMR の (Nainani N, et al. Transpl Immunol 2009; 20:113) ハイタイター preformed DSA 廃絶のリスク IgG2/IgG4 subclass DSA のグラフト生 De novo DSA 16

Transplantation 2003; 75(9): 1601. 2. Zhu L, et al. Clin Transpl 2008: 171. 3. Kobayashi T, et al. Hum Immunol 2011; 72(1): 11. 4. Wiebe C, et al.

17 DSA 生 -- グラフトスのハイリスク De novo DSA AMR の因 Non-DSA 因な De novo DSA の因に Wiebe C, et al. Am J Transplant 2012;12(5):1157 Significant association between chronic AMR and DR-DSA in renal transplantation 1. Iniotaki-Theodoraki AG, et al. Transplantation 2003; 75(9): Zhu L, et al. Clin Transpl 2008: Kobayashi T, et al. Hum Immunol 2011; 72(1): Wiebe C, et al. Am J Transplant 2012;12(5):1157. De novo DQ-DSA are associated with a significant risk of AMR and transplant glomerulopathy 1. Worthington JE, et al. Transplantation 2003; 75(7): Willicombe M, et al. Transplantation. 2012;94(2): Devos JM, et al. Kidney Int 2012;82(5):

18 De novo DSA に関ンセンス De novo DSA AMR/ グラフトスの因 Non-DSA HLA 抗体因な HLA class II >> class I DR? DQ? DP? De novo DSA の生に? De novo DSA Antibody-mediated rejection an ounce of prevention is worth a pound of cure oz の lb のに Bradley JA, Baldwin WM, Bingaman A, Ellenrieder C, Gebel HM, Glotz D, Kirk AD. Am J Transplant 2011;11(6):

19 Functional properties of IgG isotypes Complement fixing subclass C1 complex IgG3 >> IgG1 > IgG2 > IgG4 Complement tivati Fc-receptor binding properties IgG3 > IgG1 >> IgG2 > IgG4 Cell mediated cytotoxicity IgG3 subclass に 19

20 T H によ IgG クラススイのターン T H 1-biased isotype (IFN-γ, TGF-β) IgG2, IgG3 T H 2-biased isotype (IL-4) IgG1, IgG4 MMF/mTORi STAT1 のリン化 STAT5 のリン化 T H 応応な IgG3 subclass の CNI の minimization に IgG3-DSA の MMF/mTORi 20

21 Algorithmic approach to sensitized patients (Marfo K, et al. Clin J Am Soc Nephrol. 2011) Basic Concepts in Desensitization Removal of existing antibodies Inhibition of residual antibody and Complement system cascade t ( V (C5 ) Depletion of antibody producing cells y B Rituxim ( ti-cd20) - B ) Suppression of the T cell response T wi MMF t 21

8 2 生 placebo 群 75%, IVIG 群 80% な移植 20 IVIG (2g/kg 2 2 dose)

22 移植 48 1 回 2g/BWのグリン 4 placebo 群に PRA に移植 placebo 群 17% IVIG 群 35% placebo 群 10.3 IVIG 群生 placebo 群 75%, IVIG 群 80% な移植 20 IVIG (2g/kg 2 2 dose) + Rituximab (1g 2 dose) PRA 77±19% 44±30% 移植の144±89 5±6 移植 80% IVIGの 35% 1 生 100%, 1 生 94% 22

23 Outcomes in Kidney Recipients Receiving Desensitization Treatment 広島大学院にな FCXM (-) HLA(-) り FCXM (-) HLA (+) り FCXM (-~+) CDC-T (-~+) り FCXM (++) CDC-T (++) DSA (-) DSA (-) DSA (+) DSA (+) な DSA(+): MFI>1000 Rituximab & DFPP Rituximab & Bortezomib & DFPP Rituximab リンよ Bortezomib イの用に学の認 23

24 B リの化 Germinal Center Follicular B cell 抗 HLA 抗体産生に関主な Plasma blast cell Long-lived Plasma cell Memory B cell Naïve B cell Plasma blast cell Short-lived Plasma cell ミ Germinal Center Follicular B cell 抗 HLA 抗体産生に関主な Plasma blast cell Long-lived Plasma cell Memory B cell Naïve B cell Plasma blast cell Short-lived Plasma cell 24

42 血型輸血により HLA typing: Full mismatch CDC T-warm B-warm B-cold T-cell FCXM LABScreen Single Antigen Class I 抗体 : HLA-B*1501 (MFI: 12966) 34 Class II 抗体 : 3 CFSE-MLR: の抗

25 42 血型輸血により HLA typing: Full mismatch CDC T-warm B-warm B-cold T-cell FCXM LABScreen Single Antigen Class I 抗体 : HLA-B*1501 (MFI: 12966) 34 Class II 抗体 : 3 CFSE-MLR: の抗ナー応血型移植にクス化 Control Patient Cell number 移植 5 0 AMR( ), ( ) S-Cr 1.20 mg/dl DSA( ), NDSA(+) MFI 3.43 MFI 2.84 MFI 2.93 MFI MFI 9.54 MFI 4.15 Anti human IgG-FITC 25

26 58 血型移植 HLA typing: Full mismatch CDC T-warm B-warm B-cold T-cell FCXM LABScreen Single Antigen Class I 抗体 : HLA-A2 (MFI: 14958) 11 Class II 抗体 : 6 CFSE-MLR: の抗ナー応血型移植にクス化な Control Patient Anti human IgG-FITC 26

27 血中に B Memory B な認ミミ 1 イク 1.3mg/m 2 体 2 回 2 1, 4, 8, 11 タン用 2 に DFPP ミ CDC / FCXM 化移植 3 0 AMR( ), ( ) S-Cr 1.27 mg/dl DSA( ), NDSA( ) 27

61 血型妊娠によ HLA typing: Full mismatch A A B B DRB1 DRB1 Recipient i 0201 0201 3901 1510 0401 0801 Donor 2402 2402 0702 5201 0101 1501 CDC T-warm B-warm B-cold T-cell FCXM

28 61 血型妊娠によ HLA typing: Full mismatch A A B B DRB1 DRB1 Recipient i Donor CDC T-warm B-warm B-cold T-cell FCXM LABScreen Single Antigen Class I 抗体 : HLA-B7 (MFI: 5317) 16 Class II 抗体 : な CFSE-MLR: の抗ナー応血型にクス化な移植 1 2 AMR( ), ( ) S-Cr 0.88 mg/dl DSA( ), NDSA( ) ミクス DSA 消 28

ミ DSA な移植移植 1 移植 6 移植 1 スイス IgG 2.0 87.9 80.2 120 81.8 78.2 イス IgG 2.0 9.2 67.5 23.3 26.

29 ミ DSA な移植移植 1 移植 6 移植 1 スイス IgG イス IgG イス IgG イス IgG ンスイス IgG イトイス IgG EB イス抗 VCA IgG 中な認な Possible cause of differential depression of antibody DSA vs. protective immunity Everly MJ, et al. Transplantation 2010; 90:

30 Technical Group (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch. (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group (a)risk categories should be established based on the antibody and the XM results obtained. (b)dsa detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c)a renal transplantation can be performed in the absence of a prospective XM if singleantigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d)the presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. 30

31 Posttransplantation Group (a)high-risk patients (i.e., desensitized or DSA positive/xm negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b)intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c)low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results. DSA に short-lived plasma cell long-lived lived plasma cell のの要 Rituximab Bortezomib 段にによりな認なに 31

日本組織適合性学会誌第20巻2号

日本組織適合性学会誌第20巻2号 Major Histocompatibility Complex 2013; 20 (2): 121 129 平成 25 年度認定 HLA 検査技術者講習会テキスト HLA 1) 1) HLA HLA HLA PRA Panel reactive antibody DSA Donor specific alloantibody class subclass non-hla HLA キーワード :Human