JP7395174B2 - Method for evaluating immunosuppressive effect or immune tolerance inducing effect, and immune tolerance inducing agent - Google Patents
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Description
特許法第３０条第２項適用 第３０条第２項適用、平成３０年２月２３日、第１７回日本再生医療学会総会のプログラム抄録にてウェブサイト上で公表されたApplication of
特許法第３０条第２項適用 第３０条第２項適用、平成３０年３月２１日、第１７回日本再生医療学会総会にて発表されたApplication of
本発明は、マイナー組織適合性抗原不一致により生じる拒絶反応のモデルマウスを利用した、免疫抑制作用又は免疫寛容誘導作用を評価する方法、及び免疫細胞を有効成分とする免疫寛容誘導剤に関する。 The present invention relates to a method for evaluating an immunosuppressive effect or an immune tolerance-inducing effect using a mouse model of rejection caused by minor histocompatibility antigen mismatch, and an immune tolerance-inducing agent containing immune cells as an active ingredient.
患者に他者の細胞又は組織を移植する同種移植医療において、レシピエントの免疫機構がドナー由来の細胞又は組織を異物と認識することによる拒絶反応を克服することは、治療の成否を左右する重要な課題である。 In allogeneic transplantation medicine, in which cells or tissues from another person are transplanted into a patient, overcoming the rejection reaction caused by the recipient's immune system recognizing the donor-derived cells or tissues as foreign is critical to the success or failure of treatment. This is a serious issue.
拒絶反応を克服する主なアプローチとして、シクロスポリン、タクロリムス等の薬剤投与によるレシピエントの免疫応答抑制と、細胞工学的手法を用いて調製した患者自身の細胞又は組織の自家移植による免疫型不適合の回避とを挙げることができる。各種の組織幹細胞又はiPS細胞を利用した細胞移植技術の進展に伴って自家移植による拒絶反応の克服が進むと期待されるが、多数の患者に迅速に細胞移植医療を適用するため、さらに組織又は臓器移植については同種移植は依然として必要な選択肢であるため、拒絶反応を抑制し得る新たな手段が求められている。 The main approaches to overcome rejection include suppressing the recipient's immune response by administering drugs such as cyclosporine and tacrolimus, and avoiding immune type incompatibility by autologous transplantation of the patient's own cells or tissues prepared using cell engineering techniques. can be mentioned. It is expected that the rejection of autologous transplants will be overcome with the advancement of cell transplantation technology using various tissue stem cells or iPS cells. As allogeneic transplantation remains a necessary option for organ transplantation, new means to suppress rejection reactions are needed.
同種移植においては、レシピエントとドナーのHLA型を完全一致又は部分一致させることで拒絶反応の発生リスクを低減させているが、HLA型が完全一致している場合であっても拒絶反応が生じ得ることが知られている。この拒絶反応はマイナー組織適合性抗原の型の不一致により引き起こされるものと考えられているが、マイナー組織適合性抗原の型を全て一致させることは極めて困難である。同種移植医療の展開にあたって、マイナー組織適合性抗原不一致により生じる拒絶反応の解明やその抑制は、重要な課題となっている。 In allogeneic transplants, the risk of rejection is reduced by completely or partially matching the HLA types of the recipient and donor, but even when the HLA types are completely matched, rejection can still occur. known to obtain. This rejection reaction is thought to be caused by a mismatch in the types of minor histocompatibility antigens, but it is extremely difficult to match all the types of minor histocompatibility antigens. In the development of allogeneic transplantation medicine, elucidation and suppression of rejection reactions caused by minor histocompatibility antigen mismatches are important issues.
近年、非自己抗原に対する免疫寛容（獲得寛容）を人工的に誘導する方法が注目されている（例えば特許文献１、２等）。また、ドナー由来のB細胞又は樹状細胞を移植前のレシピエントに投与することで、同種移植における組織定着率が改善されることが報告されている（例えば非特許文献１及び２）。免疫寛容の誘導は、アレルギー性疾患や自己免疫疾患等に対する治療又は症状の緩和策の1つである一方、同種移植においても非自己抗原に対する免疫応答の抑制又は免疫抑制剤の投与量の低減につながり得るものと期待されている。 In recent years, methods of artificially inducing immune tolerance (acquired tolerance) to non-self antigens have attracted attention (eg, Patent Documents 1 and 2). Furthermore, it has been reported that the tissue fixation rate in allogeneic transplantation is improved by administering donor-derived B cells or dendritic cells to the recipient before transplantation (for example, Non-Patent Documents 1 and 2). Induction of immune tolerance is one of the treatments or symptom mitigation measures for allergic diseases and autoimmune diseases, but it is also effective in suppressing immune responses to non-self antigens or reducing the dose of immunosuppressants in allogeneic transplants. It is hoped that they will be able to connect.
本発明は、マイナー組織適合性抗原不一致により生じる拒絶反応に対する免疫抑制作用又は免疫寛容誘導作用を評価する方法、及び免疫寛容を誘導する手段を提供することを目的とするものである。 An object of the present invention is to provide a method for evaluating the immunosuppressive effect or immune tolerance-inducing effect on rejection reactions caused by minor histocompatibility antigen mismatch, and a means for inducing immune tolerance.
本発明者らは、特定のMHC型を有するレシピエントマウスにMHC型が一部一致するドナーマウス由来の組織を移植したときに程度の異なる拒絶反応をレシピエントマウスが示すこと、また外来細胞の受容を容易にするための処置とドナー由来の免疫細胞の投与とを組み合わせることでレシピエントの免疫寛容を誘導することができることを見いだし、以下の発明を完成させた。 The present inventors have demonstrated that when recipient mice with a specific MHC type are transplanted with tissue derived from a donor mouse that partially matches the MHC type, the recipient mice exhibit varying degrees of rejection, and that The inventors have discovered that immune tolerance can be induced in recipients by combining treatment to facilitate acceptance and administration of donor-derived immune cells, and have completed the following invention.
（１）（i）H-2抗原の型がb/kであるレシピエントマウスに、H-2抗原の型がb/b、k/k又はb/kであってマイナー組織適合性抗原の型が前記レシピエントマウスと一致しないドナーマウス由来の細胞又は組織を移植する工程；
（ii）前記ドナーマウス由来の細胞又は組織の移植の前、移植と同時、又は移植の後に、レシピエントマウスに被験処置を行う工程；
（iii）前記移植及び被験処置の後に、レシピエントマウスの拒絶反応を評価する工程；並びに
（iv）前記レシピエントマウスの拒絶反応を、ドナーマウス由来の細胞又は組織を移植したが被験処置を行っていない対照レシピエントマウスの拒絶反応と比較し、被験処置を行ったレシピエントマウスの拒絶反応が対照レシピエントマウスの拒絶反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定する工程
を含む、被験処置の免疫抑制作用又は免疫寛容作用を評価する方法。
（２）（i）H-2抗原の型がb/kであるレシピエントマウス由来のT細胞を含む試料と、H-2抗原の型がb/b、k/k又はb/kであってマイナー組織適合性抗原の型が前記レシピエントマウスと一致しないドナーマウス由来の抗原提示細胞を含む試料とを混合し、レシピエント由来T細胞とドナー由来抗原提示細胞とをインビトロで共存させる工程；
（ii）レシピエント由来T細胞とドナー由来抗原提示細胞とを共存させる前、共存させるのと同時、又は共存させた後に、レシピエント由来T細胞に被験処置を行う工程；
（iii）ドナー由来抗原提示細胞との共存及び被験処置の後に、レシピエント由来T細胞の幼若化反応を評価する工程；並びに
（iv）レシピエント由来T細胞の幼若化反応を、ドナー由来抗原提示細胞と共存させたが被験処置を行っていない対照のレシピエント由来T細胞の幼若化反応と比較し、被験処置を行ったレシピエント由来T細胞の幼若化反応が対照レシピエント由来T細胞の幼若化反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定する工程
を含む、被験処置の免疫抑制作用又は免疫寛容作用をインビトロで評価する方法。
（３）レシピエントマウスが129系統とC3H系統の交雑マウスであり、ドナーマウスがC57BL/6系統、CBA/N系統又はC57BL/6系統とCBA/N系統との交雑マウスである、（１）又は（２）に記載の方法。
（４）レシピエントマウスがC3129F1マウスである、（１）～（３）のいずれか一項に記載の方法。
（５）ドナーマウスがC57BL/6系統又はCBA/N系統のマウスである、（１）、（３）又は（４）のいずれか一項に記載の方法。
（６）ドナーマウスがC57BL/6系統のマウスである、（２）～（４）のいずれか一項に記載の方法。
（７）ドナー対象由来の免疫細胞を含む、レシピエント対象において免疫寛容を誘導するための細胞製剤であって、レシピエント対象に対する放射線照射及びT細胞除去処置と組み合わせて用いるための前記細胞製剤。
（８）ドナー対象の主要組織適合型抗原の型がレシピエント対象のそれと完全一致又は部分一致であり、ドナー対象のマイナー組織適合性抗原の型がレシピエント対象のそれと完全一致ではない、（７）に記載の細胞製剤。
（９）T細胞除去処置が、抗CD4抗体、抗CD8抗体、抗CD3抗体、抗TCR抗体及び抗胸腺細胞グロブリンよりなる群から選択される１又は複数の抗T細胞抗体の投与である、（７）又は（８）に記載の細胞製剤。
（１０）免疫細胞が脾臓細胞、B細胞又は樹状細胞である、（７）～（９）のいずれか一項に記載の細胞製剤。
（１１）免疫細胞がB細胞、又はFms-like tyrosine kinase 3 ligand（Flt3L）で誘導した樹状細胞である、（７）～（１０）のいずれか一項に記載の細胞製剤。
(1) (i) A recipient mouse whose H-2 antigen type is b/k is given a mouse whose H-2 antigen type is b/b, k/k, or b/k and which has a minor histocompatibility antigen. transplanting cells or tissues from a donor mouse whose type does not match the recipient mouse;
(ii) administering a test treatment to the recipient mouse before, simultaneously with, or after transplantation of cells or tissues derived from the donor mouse;
(iii) evaluating the rejection response of the recipient mouse after the transplantation and test treatment; and (iv) evaluating the rejection response of the recipient mouse after transplanting cells or tissue derived from the donor mouse but not receiving the test treatment. The test treatment has an immunosuppressive or tolerogenic effect if the rejection response in the test-treated recipient mice is weaker than the rejection response in the control recipient mice, compared to the rejection response in the control recipient mice without the test treatment. A method for evaluating an immunosuppressive effect or an immunotolerant effect of a test treatment, the method comprising the step of determining that the test treatment has an immunosuppressive effect or an immunotolerant effect.
(2) (i) Samples containing T cells from recipient mice whose H-2 antigen type is b/k and those whose H-2 antigen type is b/b, k/k, or b/k. mixing a sample containing antigen-presenting cells derived from a donor mouse whose type of minor histocompatibility antigen does not match that of the recipient mouse, and allowing the recipient-derived T cells and donor-derived antigen-presenting cells to coexist in vitro;
(ii) performing a test treatment on the recipient-derived T cells before, at the same time as, or after the recipient-derived T cells and donor-derived antigen-presenting cells coexist;
(iii) evaluating the blastogenesis response of recipient-derived T cells after coexistence with donor-derived antigen-presenting cells and the test treatment; and (iv) evaluating the blastogenesis response of recipient-derived T cells Compared to the blastogenesis of T cells derived from a control recipient that coexisted with antigen-presenting cells but not subjected to the test treatment, the blastogenesis of T cells derived from the recipient treated with the test treatment was compared to that of the T cells derived from the control recipient. A method for evaluating the immunosuppressive effect or immunotolerant effect of a test treatment in vitro, comprising the step of determining that the test treatment has an immunosuppressive effect or an immune tolerogenic effect when the response is weaker than the blastogenesis response of T cells.
(3) The recipient mouse is a cross between the 129 strain and the C3H strain, and the donor mouse is a cross between the C57BL/6 strain, the CBA/N strain, or the C57BL/6 strain and the CBA/N strain. (1) Or the method described in (2).
(4) The method according to any one of (1) to (3), wherein the recipient mouse is a C3129F1 mouse.
(5) The method according to any one of (1), (3), or (4), wherein the donor mouse is a mouse of the C57BL/6 strain or CBA/N strain.
(6) The method according to any one of (2) to (4), wherein the donor mouse is a mouse of the C57BL/6 strain.
(7) A cell preparation for inducing immune tolerance in a recipient subject, comprising immune cells derived from a donor subject, said cell preparation for use in combination with radiation irradiation and T cell ablation treatment of the recipient subject.
(8) The donor subject's major histocompatibility antigen type is a perfect or partial match to that of the recipient subject, and the donor subject's minor histocompatibility antigen type is not a perfect match to that of the recipient subject; (7) ).
(9) The T cell depletion treatment is the administration of one or more anti-T cell antibodies selected from the group consisting of anti-CD4 antibodies, anti-CD8 antibodies, anti-CD3 antibodies, anti-TCR antibodies, and anti-thymocyte globulin; 7) or the cell preparation according to (8).
(10) The cell preparation according to any one of (7) to (9), wherein the immune cells are spleen cells, B cells, or dendritic cells.
(11) The cell preparation according to any one of (7) to (10), wherein the immune cells are B cells or dendritic cells induced with Fms-like tyrosine kinase 3 ligand (Flt3L).
本発明によれば、マイナー組織適合性抗原の不一致により引き起こされる拒絶反応に対する免疫抑制作用又は免疫寛容誘導作用の評価が可能になり、これにより同種移植の際に生じる拒絶反応を抑制し得る処置を選択することができ、またその機序解明に資する情報を提供することができる。また、本発明の細胞製剤は、同種移植におけるレシピエントの拒絶反応を抑制することができ、移植医療の成功率を高めることができ、また免疫抑制剤の使用量を低減させることができる。 According to the present invention, it is possible to evaluate the immunosuppressive effect or immune tolerance-inducing effect on rejection reactions caused by mismatching of minor histocompatibility antigens, and thereby to develop treatments that can suppress rejection reactions that occur during allogeneic transplantation. can be selected, and information that contributes to elucidation of the mechanism can be provided. Furthermore, the cell preparation of the present invention can suppress rejection reactions in recipients in allogeneic transplants, increase the success rate of transplant medical treatment, and reduce the amount of immunosuppressants used.
免疫抑制作用又は免疫寛容作用の評価方法（in vivo）
本発明の第１の態様は、
（i）H-2抗原の型がb/kであるレシピエントマウスに、H-2抗原の型がb/b、k/k又はb/kであってマイナー組織適合性抗原の型が前記レシピエントマウスと一致しないドナーマウス由来の細胞又は組織を移植する工程；
（ii）前記ドナーマウス由来の細胞又は組織の移植の前、移植と同時、又は移植の後に、レシピエントマウスに被験処置を行う工程；
（iii）前記移植及び被験処置の後に、レシピエントマウスの拒絶反応を評価する工程；並びに
（iv）前記レシピエントマウスの拒絶反応を、ドナーマウス由来の細胞又は組織を移植したが被験処置を行っていない対照レシピエントマウスの拒絶反応と比較し、被験処置を行ったレシピエントマウスの拒絶反応が対照レシピエントマウスの拒絶反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定する工程
を含む、被験処置の免疫抑制作用又は免疫寛容作用を評価する方法に関する。
Method for evaluating immunosuppressive effect or immune tolerance effect (in vivo)
The first aspect of the present invention is
(i) A recipient mouse whose H-2 antigen type is b/k is given to a recipient mouse whose H-2 antigen type is b/b, k/k, or b/k and whose minor histocompatibility antigen type is transplanting cells or tissue from a donor mouse that does not match the recipient mouse;
(ii) administering a test treatment to the recipient mouse before, simultaneously with, or after transplantation of cells or tissues derived from the donor mouse;
(iii) evaluating the rejection response of the recipient mouse after the transplantation and test treatment; and (iv) evaluating the rejection response of the recipient mouse after transplanting cells or tissue derived from the donor mouse but not receiving the test treatment. The test treatment has an immunosuppressive or tolerogenic effect if the rejection response in the test-treated recipient mice is weaker than the rejection response in the control recipient mice, compared to the rejection response in the control recipient mice without the test treatment. The present invention relates to a method for evaluating the immunosuppressive effect or immunotolerant effect of a test treatment, including the step of determining that the test treatment has an immunosuppressive effect or an immunotolerant effect.
本態様におけるH-2抗原の型がb/kであるレシピエントマウスは、H-2抗原の型がb/bのホモマウスとH-2抗原の型がk/kのホモマウスとを用いて作製することができる、交雑マウスである。H-2抗原の型がb/bのホモマウスは、129系統マウス（例えば129P1/ReJ、129P3/J、129P3/JEmsJ、129P4RrRkJ、129S1/SvlmJ、129T2SvEms、129T2/SvEmsJ、129X1/SvJ）、BXSB/Mp、C57BL/6、C57BL/10、LP/J、BALN.B等を挙げることができる。またH-2抗原の型がk/kのホモマウスは、C3H系統マウス（例えばC3H/He、C3H/HeN、C3H/Bi、C3HeB/FeJ等）、AKR/J、CBA系統マウス（例えばCBA/Ca、CBA/J、CBA/N）、CE/J、HRS/J、MA/MyJ、MRL/Mp、RF/J、ST/bJ、C58/Jを挙げることができる。 In this embodiment, the recipient mouse with the H-2 antigen type b/k is produced using a homozygous mouse with the H-2 antigen type b/b and a homozygous mouse with the H-2 antigen type k/k. It is a crossbred mouse that can be H-2 antigen type b/b homozygous mice include 129 strain mice (e.g. 129P1/ReJ, 129P3/J, 129P3/JEmsJ, 129P4RrRkJ, 129S1/SvlmJ, 129T2SvEms, 129T2/SvEmsJ, 129X1/SvJ), BXSB/ Examples include Mp, C57BL/6, C57BL/10, LP/J, and BALN.B. In addition, homozygous mice with the H-2 antigen type k/k include C3H strain mice (e.g. C3H/He, C3H/HeN, C3H/Bi, C3HeB/FeJ, etc.), AKR/J, CBA strain mice (e.g. CBA/Ca , CBA/J, CBA/N), CE/J, HRS/J, MA/MyJ, MRL/Mp, RF/J, ST/bJ, and C58/J.
特に好ましいレシピエントマウスは、C3H系統マウス、特にC3H/Heを母親とし、129系統マウス、特に129X1/SvJを父親としたF1マウスC3129F1（H-2b/k）である。 Particularly preferred recipient mice are C3H strain mice, especially F1 mice C3129F1 (H-2 b/k ) whose mothers are C3H/He and 129 strain mice, especially 129X1/SvJ fathers.
本態様におけるドナーマウスは、レシピエントマウスと同一のH-2抗原型を有するマウス（H-2b/k）又はレシピエントマウスのH-2抗原型の少なくとも1つのアレル（allele）と一致するアレルを有するアロジェニックマウス（H-2b/b、H-2k/k）であって、いずれもマイナー組織適合性抗原の型がレシピエントマウスと完全一致しないマウスである。具体的には、H-2抗原の型がb/b、k/k又はb/kであって、マイナー組織適合性抗原の型が前記レシピエントマウスと一致しないマウスである。 The donor mouse in this embodiment is a mouse that has the same H-2 serotype as the recipient mouse (H-2 b/k ) or matches at least one allele of the H-2 serotype of the recipient mouse. These are allelic mice (H-2 b/b , H-2 k/k ) that do not completely match the type of minor histocompatibility antigen to the recipient mouse. Specifically, the mouse has an H-2 antigen type of b/b, k/k, or b/k, and a minor histocompatibility antigen type that does not match that of the recipient mouse.
マイナー組織適合性抗原（Minor histocompatibility antigen、mHA）は、MHCが一致するドナー・レシピエント間の同種移植において拒絶反応を惹起する組織適合性抗原であり、具体的には、細胞表面のMHC分子上に提示される生体内タンパク質のうち、ドナーとレシピエント間で多型等により異なるアミノ酸配列をもつペプチドで、そのMHC/ペプチド複合体がレシピエントT細胞に非自己として認識されるものをいう。 Minor histocompatibility antigen (mHA) is a histocompatibility antigen that causes rejection in MHC-matched donor-recipient allografts. A peptide that has an amino acid sequence that differs between the donor and recipient due to polymorphism among the in-vivo proteins presented in the human body, and whose MHC/peptide complex is recognized as non-self by the recipient T cell.
マウスのマイナー組織適合性抗原の例としては、H-2Kb拘束性にH60やH4、H-2Db拘束性にH7等が知られている。 Examples of mouse minor histocompatibility antigens include H60 and H4, which are H-2K b- restricted, and H7, which is H-2D b- restricted.
本発明におけるドナーマウスは、H-2抗原の型がb/b、k/k又はb/kであってmHAの型がレシピエントマウスと完全に一致しないかぎり、任意のマウスを選択して使用することができる。ドナーマウスは、例えば、レシピエントの親マウスと同系統でない、H-2抗原の型がb/b、k/k又はb/kのマウスであり、これらの例は上で挙げたとおりである。ドナーマウスは、好ましくはC57BL/6系統又はCBA/N系統のマウスである。 Any mouse can be selected as the donor mouse in the present invention, as long as the H-2 antigen type is b/b, k/k, or b/k, and the mHA type does not completely match that of the recipient mouse. can do. The donor mouse is, for example, a mouse of the H-2 antigen type b/b, k/k or b/k that is not of the same strain as the recipient parent mouse, examples of which are listed above. . The donor mouse is preferably a mouse of the C57BL/6 strain or CBA/N strain.
レシピエントマウスに移植される細胞の種類には特に制限はなく、ドナーマウス由来の各種体細胞、体細胞から作製されるiPS細胞及び当該iPS細胞から分化誘導された細胞、ドナーマウス由来の組織幹細胞及び当該幹細胞から分化誘導された細胞を例として挙げることができる。また、ドナーマウスからの細胞の分離及び分化誘導等は、細胞毎に公知の方法によって行うことができる。 There are no particular restrictions on the type of cells transplanted into the recipient mouse, including various somatic cells derived from donor mice, iPS cells produced from somatic cells, cells induced to differentiate from the iPS cells, and tissue stem cells derived from donor mice. Examples include cells induced to differentiate from these stem cells. Furthermore, isolation of cells from donor mice, induction of differentiation, etc. can be performed for each cell by known methods.
レシピエントマウスに移植される組織の種類には特に制限はなく、ドナーマウス由来の任意の組織を利用することができる。レシピエントマウスに移植される組織は、例えば、被験処置の適用が予想される移植治療において用いられる組織であってもよく、あるいは取扱の簡便な組織、例えば皮膚片等であってもよい。 There are no particular restrictions on the type of tissue to be transplanted into the recipient mouse, and any tissue derived from the donor mouse can be used. The tissue to be transplanted into the recipient mouse may be, for example, a tissue used in transplantation therapy to which the test treatment is expected to be applied, or a tissue that is easy to handle, such as a piece of skin.
レシピエントマウスへの移植の具体的な方法には特別な制限はなく、ドナーマウスから移植する細胞や組織に応じて適切な方法を採用して行うことができる。また、必要に応じて、移植前にレシピエントマウスに放射線照射等の前処置を行ってもよい。 There are no particular restrictions on the specific method of transplantation into the recipient mouse, and an appropriate method can be adopted depending on the cells and tissues to be transplanted from the donor mouse. Furthermore, if necessary, the recipient mouse may be subjected to pretreatment such as radiation irradiation before transplantation.
被験処置は、マウスに何らかの外部刺激を与える処置であればよく、典型的には、免疫抑制作用又は免疫寛容作用の評価が望まれる物質を投与すること又は外部環境因子を変化させることである。被験処置を行うタイミングは、ドナーマウス由来の細胞又は組織の移植の前、移植と同時、又は移植の後のいずれでもよい。被験処置が被験物質の投与である場合、被験物質の投与形態（液体、固体等）、投与経路（経口摂取、静脈投与、腹腔内投与等）は、主に被験物質の物理化学的性質及び生物学的性質に応じて適宜選択される。 The test treatment may be any treatment that applies some kind of external stimulus to the mouse, typically administering a substance whose immunosuppressive effect or immunotolerant effect is desired to be evaluated, or changing external environmental factors. The test treatment may be performed before, simultaneously with, or after the transplantation of cells or tissues derived from the donor mouse. When the test treatment is the administration of a test substance, the dosage form of the test substance (liquid, solid, etc.) and route of administration (oral ingestion, intravenous administration, intraperitoneal administration, etc.) are mainly determined by the physicochemical properties and biological properties of the test substance. It is selected as appropriate depending on the scientific properties.
ドナーマウス由来の細胞又は組織を移植されたレシピエントマウスにおいては、T細胞関連型拒絶反応、抗体関連型拒絶反応のいずれも惹起され得る。したがって、レシピエントマウスの拒絶反応は、T細胞関連型拒絶反応又は抗体関連型拒絶反応のいずれかにより引き起こされる現象を観察することによって、具体的には移植片の生着率及び生着期間、移植片への宿主リンパ球の浸潤の度合い、レシピエントマウスの炎症マーカーの測定、移植片に対する抗体産生の測定、混合リンパ球試験等によって評価することができる。 In recipient mice transplanted with cells or tissues derived from donor mice, either T cell-related rejection reactions or antibody-related rejection reactions can be induced. Therefore, the rejection reaction in recipient mice can be determined by observing the phenomenon caused by either T cell-related rejection reaction or antibody-related rejection reaction, specifically determining the graft survival rate and survival period. Evaluation can be made by measuring the degree of infiltration of host lymphocytes into the graft, measuring inflammatory markers in recipient mice, measuring antibody production against the graft, mixed lymphocyte test, etc.
本態様は、上記のようにして評価されたレシピエントマウスの拒絶反応を、ドナーマウス由来の細胞又は組織を移植したが被験処置を行っていない対照レシピエントマウスの拒絶反応と比較し、被験処置を行ったレシピエントマウスの拒絶反応が対照レシピエントマウスの拒絶反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定する工程を含む。 In this embodiment, the rejection reaction of the recipient mouse evaluated as described above is compared with the rejection reaction of a control recipient mouse transplanted with cells or tissues derived from the donor mouse but not subjected to the test treatment. The test treatment includes a step of determining that the test treatment has an immunosuppressive effect or an immunotolerant effect when the rejection reaction of the recipient mouse subjected to the treatment is weaker than the rejection reaction of the control recipient mouse.
例えば移植片の生着期間を拒絶反応の指標とする場合、被験物質を投与したレシピエントマウスと被験物質を投与しない対照レシピエントの各移植片の生着期間の変化を観察し、被験物質を投与したときに生着期間の延長が観察されたときに、当該被験物質は免疫抑制能又は免疫寛容能を有すると判定することができる。 For example, when using the survival period of a graft as an indicator of rejection, observe changes in the survival period of each graft in recipient mice administered with the test substance and control recipients not administered the test substance. When an extension of the engraftment period is observed upon administration, the test substance can be determined to have immunosuppressive ability or immunotolerant ability.
免疫抑制作用又は免疫寛容作用の評価方法（in vitro）
本発明の別の態様は、（i）H-2抗原の型がb/kであるレシピエントマウス由来のT細胞を含む試料と、H-2抗原の型がb/b、k/k又はb/kであってマイナー組織適合性抗原の型が前記レシピエントマウスと一致しないドナーマウス由来の抗原提示細胞を含む試料とを混合し、レシピエント由来T細胞とドナー由来抗原提示細胞とをインビトロで共存させる工程；
（ii）レシピエント由来T細胞とドナー由来抗原提示細胞とを共存させる前、共存させるのと同時、又は共存させた後に、レシピエント由来T細胞に被験処置を行う工程；
（iii）ドナー由来抗原提示細胞との共存及び被験処置の後に、レシピエント由来T細胞の幼若化反応を評価する工程；並びに
（iv）レシピエント由来T細胞の幼若化反応を、ドナー由来抗原提示細胞と共存させたが被験処置を行っていない対照のレシピエント由来T細胞の幼若化反応と比較し、被験処置を行ったレシピエント由来T細胞の幼若化反応が対照レシピエント由来T細胞の幼若化反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定する工程
を含む、被験処置の免疫抑制作用又は免疫寛容作用をインビトロで評価する方法に関する。
Method for evaluating immunosuppressive effect or immune tolerance effect (in vitro)
Another aspect of the invention provides (i) a sample comprising T cells from a recipient mouse whose H-2 antigen type is b/k; b/k and a sample containing antigen-presenting cells derived from a donor mouse whose type of minor histocompatibility antigen does not match that of the recipient mouse, and the recipient-derived T cells and donor-derived antigen-presenting cells are in vitro cultured. The process of making them coexist;
(ii) performing a test treatment on the recipient-derived T cells before, at the same time as, or after the recipient-derived T cells and donor-derived antigen-presenting cells coexist;
(iii) evaluating the blastogenesis response of recipient-derived T cells after coexistence with donor-derived antigen presenting cells and the test treatment; and (iv) evaluating the blastogenesis response of recipient-derived T cells after coexistence with donor-derived antigen presenting cells and the test treatment; Compared to the blastogenesis of T cells derived from a control recipient that coexisted with antigen-presenting cells but not subjected to the test treatment, the blastogenesis of T cells derived from the recipient treated with the test treatment was compared to that of the T cells derived from the control recipient. Relates to a method for evaluating in vitro the immunosuppressive or tolerogenic effect of a test treatment, comprising the step of determining that the test treatment has an immunosuppressive or tolerogenic effect if the response is weaker than a T cell blastogenesis response. .
本態様における「H-2抗原の型がb/kであるレシピエントマウス」及び「H-2抗原の型がb/b、k/k又はb/kであってマイナー組織適合性抗原の型が前記レシピエントマウスと一致しないドナーマウス」は、第１の態様において説明したとおりである。 In this embodiment, "a recipient mouse whose H-2 antigen type is b/k" and "a recipient mouse whose H-2 antigen type is b/b, k/k, or b/k and whose minor histocompatibility antigen type is ``donor mouse that does not match the recipient mouse'' is as described in the first aspect.
T細胞の幼若化反応は混合リンパ球反応（MLR；Mixed Lymphocyte Reaction）時に起こる反応であり、これを利用した混合リンパ球培養（MLC；Mixed Lymphocyte Culture）は拒絶反応の予測のための臨床検査として知られている。本態様は、混合リンパ球培養におけるレシピエントとドナーの組み合わせを第一の態様のレシピエントマウスとドナーマウスの組み合わせとすることで、マイナー組織適合性抗原不一致により生じる拒絶反応（T細胞の幼弱化反応）を反映したインビトロでの評価ツールとして利用するものである。 T-cell blastogenesis is a reaction that occurs during mixed lymphocyte reaction (MLR), and mixed lymphocyte culture (MLC), which utilizes this reaction, is a clinical test for predicting rejection. known as. In this embodiment, the combination of recipient and donor in mixed lymphocyte culture is the combination of the recipient mouse and donor mouse of the first embodiment. It is used as an in vitro evaluation tool that reflects the response (reaction).
本態様の方法は、レシピエントをH-2抗原の型がb/kであるマウス、ドナーをH-2抗原の型がb/b、k/k又はb/kであるマウスとした混合リンパ球培養を行い、その結果として起こるレシピエント由来T細胞の幼若化反応を被験処置の有り無しで比較することにより実施することができる。レシピエント由来T細胞を含む試料及びドナー由来抗原提示細胞を含む試料は、それぞれCD4+T細胞又はCD8+T細胞等のT細胞、及び樹状細胞等の抗原提示細胞を含むものであればよく、末梢血臨床検査としての混合リンパ球培養において用いられるようにリンパ球画分を使用してもよい。T細胞、抗原提示細胞又は各細胞を含む細胞群の調製は、当業者において公知の各細胞の単離方法又は各細胞群の調製方法に従って行えばよい。 The method of this embodiment uses a mixed lymphocyte in which the recipient is a mouse whose H-2 antigen type is b/k, and the donor is a mouse whose H-2 antigen type is b/b, k/k, or b/k. This can be carried out by culturing spheres and comparing the resulting blastogenesis of recipient-derived T cells with and without the test treatment. The sample containing recipient-derived T cells and the sample containing donor-derived antigen-presenting cells may each contain T cells such as CD4 + T cells or CD8 + T cells, and antigen-presenting cells such as dendritic cells. , the lymphocyte fraction may be used as used in mixed lymphocyte culture as a peripheral blood clinical test. T cells, antigen-presenting cells, or cell groups containing each cell may be prepared according to cell isolation methods or cell group preparation methods known to those skilled in the art.
被験処置は、レシピエント由来T細胞に何らかの外部刺激を与える処置であればよく、典型的には、免疫抑制作用又は免疫寛容作用の評価が望まれる物質で処理すること又は外部環境因子を変化させることである。被験処置を行うタイミングは、レシピエント由来T細胞とドナー由来抗原提示細胞とを共存させる前、共存させるのと同時、又は共存させた後のいずれでもよい。 The test treatment may be any treatment that provides some external stimulation to recipient-derived T cells, typically treatment with a substance whose immunosuppressive effect or immunotolerant effect is desired to be evaluated, or changes in external environmental factors. That's true. The test treatment may be performed before, at the same time as, or after the recipient-derived T cells and donor-derived antigen-presenting cells coexist.
T細胞の幼若化反応は、当業者において公知の測定方法に従って、例えば蛍光色素CFSEや3Hサイミジン等によりT細胞を標識し、その増殖を観察することによって評価することができる。 The blastogenesis reaction of T cells can be evaluated by labeling T cells with, for example, a fluorescent dye such as CFSE or 3H thymidine, and observing their proliferation according to a measurement method known to those skilled in the art.
上記のようにして評価されたレシピエント由来T細胞の幼若化反応を、ドナー由来抗原提示細胞と共存させたが被験処置を行っていない対照のレシピエント由来T細胞の幼若化反応と比較し、被験処置を行ったレシピエント由来T細胞の幼若化反応が対照レシピエント由来T細胞の幼若化反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定することができる。 Compare the blastogenesis of recipient-derived T cells evaluated as described above with the blastogenesis of control recipient-derived T cells that coexisted with donor-derived antigen-presenting cells but were not subjected to the test treatment. However, if the blastogenesis response of T cells derived from the recipient treated with the test treatment is weaker than the blastogenesis response of T cells derived from the control recipient, the test treatment is determined to have an immunosuppressive effect or an immune tolerogenic effect. can do.
レシピエント対象において免疫寛容を誘導するための細胞製剤
本発明はさらに異なる別の態様として、ドナー対象由来の免疫細胞を含む、レシピエント対象において免疫寛容を誘導するための細胞製剤であって、レシピエント対象に対する放射線照射及びT細胞除去処置と組み合わせて用いるための前記細胞製剤を提供する。
A cell preparation for inducing immune tolerance in a recipient subject In yet another aspect, the present invention provides a cell preparation for inducing immune tolerance in a recipient subject, comprising immune cells derived from a donor subject, the cell preparation comprising immune cells derived from a donor subject. The present invention provides such cell preparations for use in combination with radiation and T cell ablation treatments for human subjects.
免疫寛容は、自己細胞等の自己抗原に対する免疫系の不応答であり、胸腺における中枢性免疫寛容、末梢における末梢性免疫寛容の２つに大別される。本発明において誘導される免疫寛容は、レシピエント対象における拒絶反応を抑制することができるものであれば、上記のいずれであってもよい。 Immune tolerance is the unresponsiveness of the immune system to self-antigens such as self-cells, and is broadly divided into two types: central immune tolerance in the thymus and peripheral immune tolerance in the periphery. The immune tolerance induced in the present invention may be any of the above as long as it can suppress rejection in the recipient subject.
本態様におけるレシピエント対象及びドナー対象は、動物、例えばマウス、ラット、ハムスター、モルモットを含むげっ歯類、ヒト、チンパンジーを含む霊長類、ブタ、ウシ、ヤギ、ウマ、ヒツジを含む家畜、イヌ、ネコを含む愛玩動物といった哺乳動物であり、特に好ましくはヒトである。また、好ましい実施形態において、レシピエント対象及びドナー対象の主要組織適合抗原の型は完全一致又は部分一致であり、マイナー組織適合性抗原の型は完全一致ではない。 Recipient subjects and donor subjects in this embodiment include animals, such as rodents including mice, rats, hamsters, and guinea pigs, humans, primates including chimpanzees, domestic animals including pigs, cows, goats, horses, and sheep, dogs, Mammals such as pets including cats, and humans are particularly preferred. Also, in preferred embodiments, the major histocompatibility antigen types of the recipient subject and the donor subject are an exact match or a partial match, and the minor histocompatibility antigen types are not an exact match.
放射線照射は、移植治療を行う際にレシピエント対象に施される前処置の一つとして通常行われる放射線照射であればよく、レシピエント対象への全身照射（Total Body Irradiation）又は胸腺への照射が好ましい。照射量は、例えばヒトへの全身照射の場合は１回の照射線量として1Gy以上、胸腺への照射の場合は3Gy以上で、かつ致死量に至らない量を目安として、適宜調節することができる。 Radiation irradiation may be radiation irradiation that is normally performed as one of the pretreatments given to the recipient subject during transplantation treatment, and may include total body irradiation of the recipient subject or irradiation to the thymus gland. is preferred. The irradiation dose can be adjusted as appropriate, for example, in the case of whole-body irradiation to humans, it is 1 Gy or more as a single irradiation dose, and in the case of thymus irradiation, it is 3 Gy or more, and the amount does not reach a lethal dose. .
T細胞除去処置は、移植治療を行う際にレシピエント対象に施される前処置の一つとして通常行われる処置であればよく、抗T細胞抗体やステロイドの投与が挙げられる。抗T細胞抗体の例は、抗CD4抗体、抗CD8抗体、抗CD3抗体、抗TCR抗体及び抗胸腺細胞グロブリン（ATG）を包含し、これらの１種又は複数を組み合わせてレシピエント対象に投与することが好ましい。好ましい実施形態において、抗T細胞抗体は、抗CD4抗体及び抗CD8抗体の組み合わせであり、これらを同時に又は連続してレシピエント対象に静脈内投与又は腹腔内投与することが好ましい。投与量は、レシピエント対象の体内においてT細胞を除去するのに十分な量であればよい。 The T cell removal treatment may be any treatment that is normally performed as one of the pretreatments administered to the recipient subject during transplantation treatment, and includes administration of anti-T cell antibodies and steroids. Examples of anti-T cell antibodies include anti-CD4 antibodies, anti-CD8 antibodies, anti-CD3 antibodies, anti-TCR antibodies, and anti-thymocyte globulin (ATG), one or more of which may be administered in combination to a recipient subject. It is preferable. In a preferred embodiment, the anti-T cell antibody is a combination of an anti-CD4 antibody and an anti-CD8 antibody, which are preferably administered to the recipient subject intravenously or intraperitoneally, either simultaneously or sequentially. The dose may be sufficient to eliminate T cells in the recipient subject's body.
放射線照射及びT細胞除去処置は、移植実施前に免疫寛容を誘導することが望まれる場合は移植の直前までに、移植実施後に免疫寛容を誘導すること、すなわち遅延型免疫寛容（Delayed Tolerance Induction）の誘導が望まれる場合は寛容を誘導したい所望のタイミングで、１回又は複数回繰り返して行うことができる。また、放射線照射及びT細胞除去処置の順序に特に制限はないが、T細胞除去処置を放射線照射に先行して行うことが好ましい。以下、放射線照射及びT細胞除去処置を前処置と呼ぶことがある。 If it is desired to induce immune tolerance before transplantation, radiation irradiation and T cell depletion treatment should be carried out immediately before transplantation, or after transplantation (Delayed Tolerance Induction). If induction of tolerance is desired, it can be repeated once or multiple times at the desired timing to induce tolerance. Further, although there is no particular restriction on the order of radiation irradiation and T cell removal treatment, it is preferable to perform T cell removal treatment prior to radiation irradiation. Hereinafter, radiation irradiation and T cell removal treatment may be referred to as pretreatment.
本発明の細胞製剤は、ドナー由来の免疫細胞を有効成分として含む。免疫細胞の例は、脾臓細胞、B細胞及び樹状細胞を包含し、これらの１種又は複数を組み合わせてレシピエント対象に投与することが好ましい。好ましい実施形態において、免疫細胞は、B細胞及び樹状細胞といった抗原提示細胞であり、特に好ましい実施形態において、免疫細胞は、B細胞及びFms-like tyrosine kinase 3 ligand（Flt3L）で誘導した樹状細胞である。 The cell preparation of the present invention contains donor-derived immune cells as an active ingredient. Examples of immune cells include spleen cells, B cells, and dendritic cells, and one or more of these are preferably administered to the recipient subject in combination. In preferred embodiments, the immune cells are antigen-presenting cells, such as B cells and dendritic cells, and in particularly preferred embodiments, the immune cells are B cells and Fms-like tyrosine kinase 3 ligand (Flt3L)-induced dendritic cells. It is a cell.
免疫細胞は、ドナー対象から採取される組織、例えば血液や骨髄液から当業者に知られた方法により調製して使用することができ、必要に応じて細胞培養を行って細胞数を増やしてから使用することができる。また、ドナー対象由来の体細胞からiPS細胞を誘導し、これを免疫細胞に分化誘導したものを使用してもよい。 Immune cells can be prepared and used from tissues collected from a donor subject, such as blood or bone marrow, by methods known to those skilled in the art, and if necessary, after increasing the number of cells by culturing them. can be used. Alternatively, iPS cells derived from somatic cells derived from a donor subject and induced to differentiate into immune cells may be used.
レシピエント対象に投与される免疫細胞の数は、1x107～1x109個／kg体重、好ましくは1x108～1x109、より好ましくは5x108～2x109個／kg体重の範囲で適宜調節することができる。免疫細胞は、前処置の後にレシピエント対象に投与すればよく、移植実施前に免疫寛容を誘導することが望まれる場合は移植の直前までに、移植実施後に免疫寛容を誘導すること、すなわち遅延型免疫寛容の誘導が望まれる場合は寛容を誘導したい所望のタイミングで、１回又は複数回繰り返して投与することができる。投与は、静脈内投与又は腹腔内投与であることが好ましい。 The number of immune cells administered to the recipient subject should be adjusted as appropriate within the range of 1x10 7 to 1x10 9 cells/kg body weight, preferably 1x10 8 to 1x10 9 , more preferably 5x10 8 to 2x10 9 cells/kg body weight. I can do it. Immune cells may be administered to the recipient subject after pretreatment, or immediately before transplantation if it is desired to induce immune tolerance before transplantation; When induction of immune tolerance is desired, the administration can be repeated once or multiple times at the desired timing to induce tolerance. Administration is preferably intravenous or intraperitoneal.
本態様の細胞製剤は、レシピエント対象に対する放射線照射及びT細胞除去処置と組み合わせて用いることで、ドナー対象由来の組織又は細胞に対する免疫寛容をレシピエント対象において誘導することができ、これにより、移植されるドナー対象由来の組織又は細胞の定着率を上昇させることができ、また移植を受けたレシピエント対象へのシクロスポリンその他の従来の免疫応答抑制剤の投与を回避する又は投与量を減らすことが可能となる。細胞製剤は、移植実施前の免疫寛容誘導のみならず、移植実施後の免疫寛容誘導、すなわち遅延型免疫寛容誘導にも用いることができる。 The cell preparation of this embodiment can be used in combination with radiation irradiation and T cell depletion treatment of the recipient subject to induce immune tolerance in the recipient subject to tissues or cells derived from the donor subject, thereby facilitating transplantation. can increase the colonization rate of tissues or cells from donor subjects that have been transplanted, and can avoid or reduce the administration of cyclosporine and other conventional immune response suppressants to transplant recipient subjects. It becomes possible. Cell preparations can be used not only to induce immune tolerance before transplantation, but also to induce immune tolerance after transplantation, that is, to induce delayed immune tolerance.
以下、実施例を示して本発明を具体的に説明するが、これらの実施例は本発明の理解を助けるためのものであって、本発明の技術的範囲を限定するものではない。 EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but these Examples are for helping understanding of the present invention, and are not intended to limit the technical scope of the present invention.
実施例１．マイナー不一致皮膚移植マウスの作製
１）レシピエントマウスの作製
129X1/SvJマウス（雄、三協ラボサービス株式会社より購入）とC3H/Heマウス（雌、三協ラボサービス株式会社より購入）から、F1マウスを作製した。作製されたマウスのH-2抗原を、FITC-anti-H-2Kk（36-7-5、Biolegend）及びPE-anti-H-2Kb（AF6-88.5、Biolegend）を用いた末梢血のフローサイトメトリー（FC500、ベックマンコールター）によって解析し、H-2b/kであることを確認した。以後、このF1マウスをC3129F1と表す。
Example 1. Creation of minor mismatched skin graft mice
1) Preparation of recipient mice
F1 mice were generated from 129X1/SvJ mice (male, purchased from Sankyo Labo Service Co., Ltd.) and C3H/He mice (female, purchased from Sankyo Labo Service Co., Ltd.). The prepared mouse H-2 antigen was transferred to the peripheral blood flow cytoplasm using FITC-anti-H-2Kk (36-7-5, Biolegend) and PE-anti-H-2Kb (AF6-88.5, Biolegend). It was analyzed by metrometry (FC500, Beckman Coulter) and confirmed to be H-2 b/k . Hereinafter, this F1 mouse will be referred to as C3129F1.
２）皮膚片移植
7～10週齢のC3129F1（雄、6～7匹／群）をレシピエントマウスとし、その背部に麻酔下で8 mmの皮膚欠損部を作製した。同週齢の雄のBALB/cマウス（H-2d/d）、C57BL/6マウス（H-2b/b）、CBA/Nマウス（H-2k/k）及び皮膚欠損部を作製した個体と同個体のC3129F1マウスから麻酔下でそれぞれ耳介を摘出したのち、鑷子で皮膚組織と軟骨組織を剥離した後、8 mmの皮膚片をレシピエントマウスの皮膚欠損部に移植した。移植後のマウスを通常の飼育条件下で最大100日間ないし150日間飼育し、移植片のサイズを経時的に測定して移植片サイズが0 mmとなった場合に拒絶と判定し、移植片の生着率（移植片が拒絶された個体数／全個体数）を算出した（図１）。
2) Skin graft
C3129F1 (male, 6 to 7 animals/group) aged 7 to 10 weeks was used as the recipient mouse, and an 8 mm skin defect was created on its back under anesthesia. Male BALB/c mice (H-2 d/d ), C57BL/6 mice (H-2 b/b ), and CBA/N mice (H-2 k/k ) of the same age and skin defects were created. After removing the ear pinna from each C3129F1 mouse from the same individual as the recipient mouse under anesthesia, the skin tissue and cartilage tissue were peeled off with forceps, and an 8 mm piece of skin was transplanted into the skin defect of the recipient mouse. After transplantation, mice are kept under normal breeding conditions for a maximum of 100 to 150 days, and the size of the graft is measured over time. If the graft size becomes 0 mm, rejection is determined. The survival rate (number of individuals whose graft was rejected/total number of individuals) was calculated (Figure 1).
レシピエントマウスに対して自家移植となるC3129F1からの移植片については、移植後の飼育期間全体で拒絶は観察されず、移植部位に完全に生着した。一方、MHC型がレシピエントマウスと不一致であるBALB/cマウス（H-2d/d）からの移植片は、移植後18日目までに完全に消失した。MHC型はレシピエントマウスと部分一致するがマイナー組織適合性抗原の型が一致しないC57BL/6マウス（H-2b/b）からの移植片も移植後18日目までに完全に消失したが、同じくマイナー不一致のCBA/Nマウス（H-2k/k）からの移植片は、移植後29日目まで移植部に残存した。 Regarding the grafts from C3129F1, which were autotransplanted into recipient mice, no rejection was observed during the entire rearing period after transplantation, and the grafts completely engrafted at the transplantation site. On the other hand, grafts from BALB/c mice (H-2 d/d ) whose MHC type was mismatched with the recipient mouse completely disappeared by day 18 after transplantation. Grafts from C57BL/6 mice (H-2 b/b ) whose MHC type partially matches that of the recipient mouse but whose minor histocompatibility antigen types do not match also disappeared completely by day 18 after transplantation. , grafts from CBA/N mice (H-2 k/k ), also with a minor mismatch, remained in the graft site until 29 days posttransplantation.
３）移植片へのリンパ球浸潤の評価
移植後10日目に移植片をレシピエントマウスの皮下組織ごと切除して回収し、HE染色を行った。BALB/cマウス（H-2d/d）及びC57BL/6マウス（H-2b/b）からの皮膚片を移植したマウスにおいて、移植部位でリンパ球の浸潤が観察された（図２）。
3) Evaluation of lymphocyte infiltration into the graft Ten days after transplantation, the graft was excised and collected together with the subcutaneous tissue of the recipient mouse, and HE staining was performed. In mice transplanted with skin pieces from BALB/c mice (H-2 d/d ) and C57BL/6 mice (H-2 b/b ), lymphocyte infiltration was observed at the transplant site (Figure 2). .
実施例２．マイナー不一致皮膚移植マウスを用いた拒絶反応抑制物質の評価
１）タクロリムス
実施例１の２）と同様にしてC3129F1マウスにBALB/cマウス（H-2d/d）、C57BL/6マウス（H-2b/b）又はCBA/Nマウス（H-2k/k）から採取した皮膚片の移植を行った。移植した日から毎日、各マウスに0.5mg/kg又は2.0mg/kgとなるようにタクロリムスを腹腔内投与しながら通常の飼育条件下で40日間飼育し、移植片のサイズを経時的に測定して、移植片の生着率を算出した。タクロリムスに代えて生理食塩水を投与した移植マウスを未処置群とした。
Example 2. Evaluation of anti-rejection substances using mice with minor mismatched skin grafts
1) Tacrolimus In the same manner as in 2) of Example 1, C3129F1 mice were injected with BALB/c mice (H-2 d/d ), C57BL/6 mice (H-2 b/b ), or CBA/N mice (H-2 A skin piece taken from (k/k ) was transplanted. Tacrolimus was intraperitoneally administered to each mouse at 0.5 mg/kg or 2.0 mg/kg every day from the day of transplantation, and the mice were kept under normal breeding conditions for 40 days, and the size of the graft was measured over time. The survival rate of the graft was calculated. Transplanted mice that received physiological saline instead of tacrolimus were designated as an untreated group.
いずれの皮膚片を移植したマウスにおいても、未処置群と0.5mg/kg投与群の間で移植片の生着率は殆ど差異はなかった。一方、タクロリムス2.0mg/kg投与群では、移植片の完全消失までの日数は、BALB/cマウス（H-2d/d）からの移植片で8日、C57BL/6マウス（H-2b/b）からの移植片で20日、それぞれ延長した。また、CBA/Nマウス（H-2k/k）からの移植片では、移植後40日まで拒絶は観察されず、生着率は100％であった（図３） In mice transplanted with any of the skin pieces, there was almost no difference in the survival rate of the grafts between the untreated group and the 0.5 mg/kg administration group. On the other hand, in the tacrolimus 2.0 mg/kg administration group, the number of days until complete graft disappearance was 8 days for grafts from BALB/c mice (H-2 d/d ) and 8 days for grafts from C57BL/6 mice (H-2 b /b ) respectively for 20 days. In addition, for grafts from CBA/N mice (H-2 k/k ), no rejection was observed until 40 days after transplantation, and the engraftment rate was 100% (Figure 3).
２）ラパマイシン
上記１）と同様にしてレシピエントマウスに皮膚移植を行った。移植した日から毎日、各マウスに1.0mg/kgのラパマイシンを腹腔内投与しながら通常の飼育条件下で100日間飼育し、移植片のサイズを経時的に測定して、移植片の生着率を算出した。
2) Rapamycin Skin grafting was performed on recipient mice in the same manner as in 1) above. From the day of transplantation, each mouse was given 1.0 mg/kg of rapamycin intraperitoneally and kept under normal breeding conditions for 100 days, and the size of the graft was measured over time to determine the survival rate of the graft. was calculated.
ラパマイシン投与によって、移植片の完全消失までの日数はBALB/cマウス（H-2d/d）からの移植片、C57BL/6マウス（H-2b/b）からの移植片とも4日延長した。一方、CBA/Nマウス（H-2k/k）からの移植片の生着率は移植後30日以内に50％まで低下するが、その後の低下は認められなかった（図４）。 Rapamycin administration extended the time until complete graft disappearance by 4 days for both BALB/c mice (H-2 d/d ) and C57BL/6 mice (H-2 b/b ). did. On the other hand, the survival rate of grafts from CBA/N mice (H-2 k/k ) decreased to 50% within 30 days after transplantation, but no decrease was observed thereafter (FIG. 4).
３）脾臓細胞又はB細胞
7～10週齢のC57BL/6J（雌）を安楽死させた後、脾臓を摘出した。脾臓をすりガラスを用いて優しくすりつぶし、単一細胞化した。低浸透圧処理により赤血球を破壊し、遠心操作により除去し、残存した細胞を脾臓細胞とした。脾臓細胞から抗マウスCD19抗体で標識した磁気ビーズ（CD19 MicroBeads, mouse、ミルテニーバイオテク）を用いた分離法によりCD19陽性細胞を分離し、B細胞とした。
3) Spleen cells or B cells
After euthanizing 7- to 10-week-old C57BL/6J (female), the spleen was removed. The spleen was gently ground using ground glass to form single cells. Red blood cells were destroyed by low osmotic pressure treatment and removed by centrifugation, and the remaining cells were used as spleen cells. CD19-positive cells were isolated from spleen cells by a separation method using magnetic beads (CD19 MicroBeads, mouse, Miltenyi Biotech) labeled with an anti-mouse CD19 antibody and were used as B cells.
レシピエントマウスであるC3129F1マウスに対し、皮膚移植6日前及び1日前に抗マウスCD4抗体（GK1.5）、抗マウスCD8α抗体（53-6.72）をマウス個体内でT細胞を除去するのに十分な量を腹腔内投与した。更に移植当日に5 Gyの全身放射線照射を実施し、前処置とした。
Anti-mouse CD4 antibody (GK1.5) and anti-mouse CD8α antibody (53-6.72) were administered to
前処置を行ったレシピエントマウスに対して、上で調製した3.0×107個の脾臓細胞、3.0×107個のB細胞又は同容量の生理食塩水を静脈内投与し、さらに同日にBALB/cマウス（H-2d/d）、C57BL/6マウス（H-2b/b）又はレシピエントマウス自身から採取した皮膚の移植を行った。通常の飼育条件下で最大150日間飼育し、移植片のサイズを経時的に測定して、移植片の生着率を算出した。 The pretreated recipient mice were intravenously administered with 3.0 x 10 7 spleen cells, 3.0 x 10 7 B cells, or the same volume of saline prepared above, and on the same day, BALB was administered. Skin grafts were performed from /c mice (H-2 d/d ), C57BL/6 mice (H-2 b/b ), or from the recipient mice themselves. The grafts were kept under normal breeding conditions for up to 150 days, and the size of the grafts was measured over time to calculate the survival rate of the grafts.
脾臓細胞、B細胞及び生理食塩水のいずれを投与した場合も、C3129F1からの移植片は移植後の飼育期間全体で拒絶は観察されず、移植部位に完全に生着した。一方、MHC型がレシピエントマウスと不一致であるBALB/cマウス（H-2d/d）からの移植片は、脾臓細胞の投与により生着期間の延長が認められた。さらに、MHC型はレシピエントマウスと部分一致するがマイナー組織適合性抗原の型が一致しないC57BL/6マウス（H-2b/b）からの移植片は、脾臓細胞又はB細胞の投与によって移植部位に完全に生着した（図５）。 Regardless of whether spleen cells, B cells, or physiological saline were administered, no rejection was observed in the grafts from C3129F1 throughout the post-transplant rearing period, and they completely engrafted at the transplant site. On the other hand, for grafts from BALB/c mice (H-2 d/d ) whose MHC type was mismatched with that of the recipient mouse, the survival period was prolonged by administration of spleen cells. In addition, grafts from C57BL/6 mice (H-2 b/b ) with a partial MHC type but mismatched minor histocompatibility antigen type with the recipient mouse can be transplanted by administration of spleen cells or B cells. The tumor completely engrafted at the site (Fig. 5).
実施例１及び２から、マイナー不一致皮膚移植マウスはMHC型不一致皮膚移植マウスと同等又はそれより弱い拒絶反応を示し、ヒトにおけるマイナー不一致拒絶反応を反映していること、並びにマイナー不一致皮膚移植マウスを用いることで、タクロリムスやラパマイシンといった免疫抑制剤の、マイナー不一致により生じる拒絶反応に対する免疫制御作用を評価することができることが示された。さらに脾臓細胞及びB細胞は、放射線照射及び抗T細胞抗体投与と組み合わせることで、同種移植、特にマイナー不一致の同種移植において強い免疫寛容作用を発揮することが確認された。 Examples 1 and 2 show that mice with minor mismatched skin grafts showed a similar or weaker rejection reaction than mice with MHC type mismatched skin grafts, reflecting minor mismatch rejection in humans, and that mice with minor mismatched skin grafts It has been shown that this method can be used to evaluate the immunoregulatory effect of immunosuppressants such as tacrolimus and rapamycin on rejection reactions caused by minor mismatches. Furthermore, it was confirmed that spleen cells and B cells exert a strong immunotolerogenic effect in allogeneic transplants, especially in minor mismatched allografts, when combined with radiation and anti-T cell antibody administration.
実施例３．混合リンパ球試験
C3129F1マウスの脾臓細胞から抗マウスCD90.2抗体標識磁気ビーズ（CD90.2 MicroBeads, mouse、ミルテニーバイオテク）を用いて磁気ビーズ分離法によりCD90.2陽性細胞を得てT細胞とした。T細胞は細胞分裂を観察するために5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE)で染色した。BALB/cマウス、C57BL/6マウス、CBA/Nマウス又はC3129F1マウスの骨髄細胞を組換えマウスGM-CSF存在下で1週間培養して生成した細胞を樹状細胞とした。2.0×105個のT細胞と1.0×104個の樹状細胞を共培養し、4日後にフローサイトメトリー（BD FACSCantoII、ベクトンディッキンソン）で解析した。共培養した細胞はPE-抗マウスCD4抗体RM4-5（Biolegend）、APC-抗マウスCD8α抗体53-6.72（Biolegend）で染色し、CD4陽性細胞、CD8陽性細胞におけるCFSEの減衰を測定し、CFSEが減衰した細胞を幼若化した細胞とし、その割合を算出した。
Example 3. mixed lymphocyte test
CD90.2-positive cells were obtained from spleen cells of C3129F1 mice by magnetic bead separation using anti-mouse CD90.2 antibody-labeled magnetic beads (CD90.2 MicroBeads, mouse, Miltenyi Biotec) and used as T cells. T cells were stained with 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) to observe cell division. Dendritic cells were generated by culturing bone marrow cells of BALB/c mice, C57BL/6 mice, CBA/N mice, or C3129F1 mice in the presence of recombinant mouse GM-CSF for one week. 2.0×10 5 T cells and 1.0×10 4 dendritic cells were co-cultured and analyzed by flow cytometry (BD FACSCantoII, Becton Dickinson) 4 days later. The co-cultured cells were stained with PE-anti-mouse CD4 antibody RM4-5 (Biolegend) and APC-anti-mouse CD8α antibody 53-6.72 (Biolegend), and the attenuation of CFSE in CD4-positive cells and CD8-positive cells was measured. Cells with attenuated values were considered to be juvenile cells, and the proportion thereof was calculated.
C3129F1マウス由来のCD4+T細胞及びCD8+T細胞は、C3129F1マウス又はCBA/Nマウス由来の樹状細胞と共培養してもほとんど又は全く増殖しなかったのに対し、C57BL/6マウス由来の樹状細胞との共培養によってCD4+T細胞及びCD8+T細胞は各々10%程度、BALB/cマウス由来の樹状細胞との共培養によってCD4+T細胞は40%程度、CD8+T細胞は50%強増加した（図６）。 CD4 + T cells and CD8 + T cells from C3129F1 mice had little or no proliferation when co-cultured with dendritic cells from C3129F1 or CBA/N mice, whereas CD4 + T cells from C3129F1 mice showed little or no proliferation when co-cultured with dendritic cells from C57BL/6 mice. Co-culture with dendritic cells reduces CD4 + T cells and CD8 + T cells to approximately 10% each, and co-culture with BALB/c mouse-derived dendritic cells increases CD4 + T cells to approximately 40% and CD8 + T cells. increased by over 50% (Figure 6).
実施例３から、マイナー不一致となる樹状細胞とT細胞の組み合わせは、MHC型不一致となる樹状細胞とT細胞の組み合わせと同等又はそれより弱いT細胞の幼若化を呈することが示され、実施例１及び２のマイナー不一致皮膚移植マウスと同様にマイナー不一致拒絶反応の評価に利用可能であると考えられた。 Example 3 shows that combinations of dendritic cells and T cells with minor mismatches exhibit the same or weaker T cell juvenileization than combinations of dendritic cells and T cells with MHC type mismatches. , it was thought that it could be used for the evaluation of minor mismatch rejection reaction similarly to the minor mismatch skin grafted mice of Examples 1 and 2.
実施例４．免疫細胞を用いた免疫寛容の誘導
１）7～10週齢のC57BL/6J（雄、ドナー、H-2b/b）を安楽死させた後、上腕骨、大腿骨および脛骨を摘出した。摘出した骨の両端を切断し、シリンジを用いてPBSで骨内部を洗浄して骨髄を回収した。回収した骨髄を70Ｎフィルターを用いてろ過し、単一細胞化した。低浸透圧処理により赤血球を破壊し、遠心操作により除去し、残存した細胞を骨髄細胞とした。10% FBS入りRPMI-1640 10 mlに組換えマウスFlt3L（Biolegend） 2000 ngを加え、骨髄細胞1.0×107を懸濁した。7-9日間、37℃、5% CO2条件下で培養後、浮遊性または弱接着性細胞を回収して、C57BL/6マウス由来のFL-DCを調製した。
Example 4. Induction of immune tolerance using immune cells 1) After euthanizing a 7- to 10-week-old C57BL/6J (male, donor, H-2 b/b ), the humerus, femur, and tibia were removed. Both ends of the extracted bone were cut, and the inside of the bone was washed with PBS using a syringe to collect the bone marrow. The collected bone marrow was filtered using a 70N filter to form single cells. Red blood cells were destroyed by low osmotic pressure treatment and removed by centrifugation, and the remaining cells were used as bone marrow cells. 2000 ng of recombinant mouse Flt3L (Biolegend) was added to 10 ml of RPMI-1640 containing 10% FBS, and 1.0×10 7 bone marrow cells were suspended. After culturing for 7-9 days at 37° C. and 5% CO 2 , floating or weakly adherent cells were collected to prepare FL-DCs derived from C57BL/6 mice.
２）C3129F1マウスに対し、細胞移入6日前及び1日前に抗マウスCD4抗体（GK1.5）、抗マウスCD8α抗体（53-6.72）をマウス個体内でT細胞を除去するのに十分な量を投与した。更に細胞移入当日に3 Gyの全身放射線照射を実施して、前処置したレシピエントマウスを作製した。
2) Apply anti-mouse CD4 antibody (GK1.5) and anti-mouse CD8α antibody (53-6.72) to
３）上記レシピエントマウスに対して、実施例２の３）で調製したC57BL/6マウス由来のB細胞（3.0×107個）、上記１）で調製したC57BL/6マウス由来のFL-DC（1.5×107個）又は同容量の生理食塩水を静脈内投与した。投与から7日後にBALB/cマウス（H-2d/d）、C57BL/6マウス（H-2b/b）又はレシピエントマウス自身（Auto）から採取した皮膚の移植を行った。通常の飼育条件下で最大100日間（B細胞移入の場合）又は最大60日間（FL-DC移入の場合）飼育し、移植片のサイズを経時的に測定して、移植直後の移植片のサイズと比較して30%以下のサイズになった場合に拒絶と判定した。移植片の生着率は（移植片が生着している個体／全個体数）を値として用いた。 3) For the above recipient mouse, B cells (3.0×10 7 cells) derived from C57BL/6 mice prepared in 3) of Example 2, and FL-DCs derived from C57BL/6 mice prepared with 1) above. (1.5×10 7 cells) or the same volume of physiological saline was administered intravenously. Seven days after administration, skin grafts were performed from BALB/c mice (H-2 d/d ), C57BL/6 mice (H-2 b/b ), or the recipient mice themselves (Auto). The size of the graft immediately after transplantation was determined by raising the graft under normal breeding conditions for up to 100 days (for B cell transfer) or for up to 60 days (for FL-DC transfer), and measuring the size of the graft over time. It was judged as rejected if the size was 30% or less compared to the original size. For the graft survival rate, (individuals with graft survival/total number of individuals) was used as a value.
B細胞、FL-DC及び生理食塩水のいずれを投与した場合も、レシピエントマウス自身の移植片は移植後の飼育期間全体で拒絶は観察されず、移植部位に完全に生着した。一方、MHC型がレシピエントマウスと不一致であるBALB/cマウス（H-2d/d）からの移植片はB細胞、FL-DC及び生理食塩水いずれを投与した場合も、移植後早期に拒絶された。MHC型はレシピエントマウスと部分一致するがマイナー組織適合性抗原の型が一致しないC57BL/6マウス（H-2b/b）からの移植片は、生理食塩水を投与した場合では拒絶されたが、B細胞の投与によって移植部位に100日以上、FL-DCの投与によって移植部位に60日以上長期に生着した（図７）。 Regardless of whether B cells, FL-DCs, or physiological saline were administered, the recipient mice's own grafts did not undergo rejection during the entire rearing period after transplantation, and were completely engrafted at the transplant site. On the other hand, grafts from BALB/c mice (H-2 d/d ) whose MHC type is mismatched with that of the recipient mouse will be affected early after transplantation, regardless of whether B cells, FL-DCs, or saline are administered. Rejected. Grafts from C57BL/6 mice (H-2 b/b ) with partial MHC type matching but mismatched minor histocompatibility antigen type with recipient mice were rejected when given saline. However, the cells remained engrafted at the transplant site for over 100 days by administration of B cells, and for over 60 days by administration of FL-DC (Figure 7).
４）上記３）において、C57BL/6マウス由来B細胞投与の1週間後にレシピエントマウスから脾臓及び末梢血を採取し、常法に従って細胞懸濁液を調製した。細胞懸濁液をフローサイトメトリー解析（BD FACSCantoII、ベクトンディッキンソン）に供し、ドナー由来B細胞（CD45.1＋）及びレシピエント由来細胞（CD45.2＋）を検出した。脾臓、末梢血のいずれにおいてもドナー由来細胞の存在が確認された（図８）。 4) In 3) above, one week after the administration of B cells derived from C57BL/6 mice, the spleen and peripheral blood were collected from the recipient mice, and a cell suspension was prepared according to a conventional method. The cell suspension was subjected to flow cytometry analysis (BD FACSCantoII, Becton Dickinson) to detect donor-derived B cells (CD45.1+) and recipient-derived cells (CD45.2+). The presence of donor-derived cells was confirmed in both the spleen and peripheral blood (FIG. 8).
５）上記３）においてB細胞を移入したレシピエントマウスから、皮膚移植後50日目に脾臓を摘出して細胞懸濁液を調製し、CD19 cell isolation kit（ミルテニーバイオテク）を用いてCD19陽性細胞を除去した後、CFSEで染色した。2.0×105個のレシピエントマウス由来脾臓細胞と、30Gy放射線照射を行った1.0×104個のBALB/cマウス又はC57BL/6マウス由来脾臓細胞とを共培養し、6日後にフローサイトメトリー（BD FACSCantoII、ベクトンディッキンソン）で解析した。共培養した細胞はPE-抗マウスCD4抗体RM4-5（Biolegend）、APC-抗マウスCD8α抗体53-6.72（Biolegend）で染色し、CD4陽性細胞、CD8陽性細胞におけるCFSEの減衰を測定し、CFSEが減衰した細胞を幼若化した細胞とし、その割合を算出した。 5) From the recipient mouse to which B cells were transferred in 3) above, the spleen was removed 50 days after skin transplantation, a cell suspension was prepared, and CD19 positive was determined using a CD19 cell isolation kit (Miltenyi Biotec). After removing cells, they were stained with CFSE. 2.0 × 10 5 recipient mouse-derived spleen cells were co-cultured with 1.0 × 10 4 BALB/c or C57BL/6 mouse-derived spleen cells that had been irradiated with 30 Gy, and 6 days later, flow cytometry was performed. (BD FACSCantoII, Becton Dickinson). The co-cultured cells were stained with PE-anti-mouse CD4 antibody RM4-5 (Biolegend) and APC-anti-mouse CD8α antibody 53-6.72 (Biolegend), and the attenuation of CFSE in CD4-positive cells and CD8-positive cells was measured. Cells with attenuated values were considered to be juvenile cells, and the proportion thereof was calculated.
上記の混合リンパ球試験の結果を図９に示す。BALB/cマウス由来の放射線照射脾臓細胞との共培養によってレシピエントマウス由来のCD4+T細胞は60%程度、CD8+T細胞は80%程度の増殖率を示した。一方、C57BL/6マウス由来の放射線照射脾臓細胞と共培養してもレシピエントマウス由来T細胞はほとんど増殖せず、免疫寛容が確認された。
The results of the above mixed lymphocyte test are shown in FIG. By co-culturing with irradiated spleen cells derived from BALB/c mice, CD4 + T cells derived from recipient mice showed a proliferation rate of approximately 60%, and CD8 + T cells exhibited a proliferation rate of approximately 80%. On the other hand, even when co-cultured with irradiated spleen cells derived from C57BL/6 mice, recipient mouse-derived T cells hardly proliferated, confirming immune tolerance.
Claims (6)
（ii）前記ドナーマウス由来の細胞又は組織の移植の前、移植と同時、又は移植の後に、レシピエントマウスに被験処置を行う工程；
（iii）前記移植及び被験処置の後に、レシピエントマウスの拒絶反応を評価する工程；並びに
（iv）前記レシピエントマウスの拒絶反応を、ドナーマウス由来の細胞又は組織を移植したが被験処置を行っていない対照レシピエントマウスの拒絶反応と比較し、被験処置を行ったレシピエントマウスの拒絶反応が対照レシピエントマウスの拒絶反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定する工程
を含む、被験処置の免疫抑制作用又は免疫寛容作用を評価する方法。 (i) A recipient mouse whose H-2 antigen type is b/k is given to a recipient mouse whose H-2 antigen type is b/b, k/k, or b/k and whose minor histocompatibility antigen type is transplanting cells or tissue from a donor mouse that does not match the recipient mouse;
(ii) administering a test treatment to the recipient mouse before, simultaneously with, or after transplantation of cells or tissues derived from the donor mouse;
(iii) evaluating the rejection response of the recipient mouse after the transplantation and test treatment; and (iv) evaluating the rejection response of the recipient mouse after transplanting cells or tissue derived from the donor mouse but not receiving the test treatment. The test treatment has an immunosuppressive or tolerogenic effect if the rejection response in the test-treated recipient mice is weaker than the rejection response in the control recipient mice, compared to the rejection response in the control recipient mice without the test treatment. A method for evaluating an immunosuppressive effect or an immunotolerant effect of a test treatment, the method comprising the step of determining that the test treatment has an immunosuppressive effect or an immunotolerant effect.
（ii）レシピエント由来T細胞とドナー由来抗原提示細胞とを共存させる前、共存させるのと同時、又は共存させた後に、レシピエント由来T細胞に被験処置を行う工程；
（iii）ドナー由来抗原提示細胞との共存及び被験処置の後に、レシピエント由来T細胞の幼若化反応を評価する工程；並びに
（iv）レシピエント由来T細胞の幼若化反応を、ドナー由来抗原提示細胞と共存させたが被験処置を行っていない対照のレシピエント由来T細胞の幼若化反応と比較し、被験処置を行ったレシピエント由来T細胞の幼若化反応が対照レシピエント由来T細胞の幼若化反応よりも弱かった場合に、被験処置は免疫抑制作用又は免疫寛容作用を有すると判定する工程
を含む、被験処置の免疫抑制作用又は免疫寛容作用をインビトロで評価する方法。 (i) Samples containing T cells from recipient mice with H-2 antigen type b/k and minor tissues with H-2 antigen type b/b, k/k or b/k mixing a sample containing antigen-presenting cells derived from a donor mouse whose type of compatible antigen does not match that of the recipient mouse, and allowing recipient-derived T cells and donor-derived antigen-presenting cells to coexist in vitro;
(ii) performing a test treatment on the recipient-derived T cells before, at the same time as, or after the recipient-derived T cells and donor-derived antigen-presenting cells coexist;
(iii) evaluating the blastogenesis response of recipient-derived T cells after coexistence with donor-derived antigen-presenting cells and the test treatment; and (iv) evaluating the blastogenesis response of recipient-derived T cells Compared to the blastogenesis of T cells derived from a control recipient that coexisted with antigen-presenting cells but not subjected to the test treatment, the blastogenesis of T cells derived from the recipient treated with the test treatment was compared to that of the T cells derived from the control recipient. A method for evaluating the immunosuppressive effect or immunotolerant effect of a test treatment in vitro, comprising the step of determining that the test treatment has an immunosuppressive effect or an immune tolerogenic effect when the response is weaker than the blastogenesis response of T cells.
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