JP2020092693A - Method for evaluating immunosuppressive action or immune tolerance-inducing action, and immune tolerance inducer - Google Patents
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Abstract
Description
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。 TECHNICAL FIELD The present invention relates to a method for evaluating an immunosuppressive action or an immune tolerance-inducing action using a model mouse of a rejection reaction caused by a minor histocompatibility antigen mismatch, and an immune-tolerance-inducing agent containing an immune cell 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 allograft medicine in which cells or tissues of another person are transplanted to a patient, it is important for the recipient's immune system to overcome the rejection reaction by recognizing the cells or tissues of the donor as a foreign substance, which determines the success or failure of treatment. It is a problem.
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 are to suppress the immune response of the recipient by administering drugs such as cyclosporine and tacrolimus, and to avoid immune type mismatch by autologous transplantation of the patient's own cells or tissues prepared by using cell engineering techniques. Can be mentioned. With the progress of cell transplantation technology using various tissue stem cells or iPS cells, it is expected that the rejection reaction by autologous transplant will be overcome, but in order to rapidly apply cell transplantation medicine to a large number of patients, further tissue or Allogeneic transplantation is still a necessary option for organ transplants, so new means are being sought to suppress rejection.
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 transplantation, the HLA types of the recipient and donor are matched or partially matched to reduce the risk of rejection, but rejection occurs even when the HLA types are exactly matched. Known to get. This rejection is considered to be caused by the mismatch of 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 reaction caused by minor histocompatibility antigen mismatch have become 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, a method of artificially inducing immune tolerance (acquired tolerance) to a non-self antigen has been attracting attention (for example, Patent Documents 1 and 2). It has also been reported that the administration of donor-derived B cells or dendritic cells to a recipient before transplantation improves the tissue colonization rate in allogeneic transplantation (for example, Non-Patent Documents 1 and 2). Induction of immune tolerance is one of the treatments or alleviation of symptoms for allergic diseases and autoimmune diseases, while also suppressing the immune response to non-self antigens or reducing the dose of immunosuppressive agents in allogeneic transplantation. Expected to be connected.
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。 It is an object of the present invention to provide a method for evaluating an immunosuppressive action or an immune tolerance-inducing action on a rejection reaction caused by a 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 show that the recipient mouse shows a different degree of rejection when a recipient mouse having a specific MHC type is transplanted with a tissue derived from a donor mouse partially matching the MHC type, and the foreign cell It was found that a combination of the treatment for facilitating the reception and the administration of the immune cells derived from the donor can induce the immune tolerance of the recipient, and 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) In a recipient mouse whose H-2 antigen type is b/k, the H-2 antigen type is b/b, k/k or b/k and the minor histocompatibility antigen Transplanting cells or tissue from a donor mouse whose type does not match the recipient mouse;
(Ii) subjecting the recipient mouse to a test treatment before, at the same time as, or after the transplantation of the cells or tissue derived from the donor mouse;
(Iii) after the transplantation and the test treatment, a step of evaluating the rejection reaction of the recipient mouse; and (iv) the rejection reaction of the recipient mouse was transplanted with cells or tissue derived from a donor mouse, but the test treatment was performed. When the rejection of the recipient mice treated with the test is weaker than that of the control recipient mice compared with the rejection of the untreated control recipient mice, the test treatment is immunosuppressive or tolerant. A method for evaluating an immunosuppressive action or an immunological tolerance action of a test treatment, which comprises the step of determining that the test treatment has.
(2) (i) a sample containing T cells derived from a recipient mouse in which the H-2 antigen type is b/k, and the H-2 antigen type is b/b, k/k or b/k And a sample containing antigen-presenting cells derived from a donor mouse whose minor histocompatibility antigen type does not match that of the recipient mouse, so that recipient-derived T cells and donor-derived antigen-presenting cells coexist in vitro.
(Ii) subjecting the recipient-derived T cells to a test treatment before, simultaneously with, or after the recipient-derived T cells and the donor-derived antigen-presenting cells coexist;
(Iii) after coexistence with the donor-derived antigen-presenting cells and after the test treatment, the step of evaluating the blastogenic reaction of the recipient-derived T cells; and (iv) the blastogenic reaction of the recipient-derived T cells Compared with the blastogenic response of control recipient-derived T cells that coexisted with antigen-presenting cells but did not carry out the test treatment, the blastogenic response of recipient-derived T cells that underwent test treatment was derived from the control recipient A method for in vitro evaluation of an immunosuppressive effect or an immunotolerance effect of a test treatment, which comprises a step of determining that the test treatment has an immunosuppressive effect or an immunotolerance effect when it is weaker than the T cell blastogenic reaction.
(3) The recipient mouse is a hybrid mouse of 129 strain and C3H strain, and the donor mouse is a C57BL/6 strain, CBA/N strain or a hybrid mouse of C57BL/6 strain and CBA/N strain, (1) Alternatively, 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 C57BL/6 strain or CBA/N strain mouse.
(6) The method according to any one of (2) to (4), wherein the donor mouse is a C57BL/6 strain mouse.
(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 irradiation and T cell ablation treatment on the recipient subject.
(8) The type of major histocompatibility antigen of the donor subject is a perfect match or partial match with that of the recipient subject, and the type of minor histocompatibility antigen of the donor subject is not a perfect match with that of the recipient subject (7 ).
(9) The T cell depleting treatment is administration of one or more anti-T cell antibodies selected from the group consisting of anti-CD4 antibody, anti-CD8 antibody, anti-CD3 antibody, anti-TCR antibody and anti-thymocyte globulin, ( The cell preparation according to 7) or (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 by 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 becomes possible to evaluate an immunosuppressive action or an immune tolerance-inducing action on a rejection reaction caused by a mismatch of minor histocompatibility antigens, and thereby, a treatment capable of suppressing the rejection reaction occurring at the time of allogeneic transplantation. The information can be selected and information that contributes to elucidation of the mechanism can be provided. Further, the cell preparation of the present invention can suppress the rejection reaction of the recipient in the allogeneic transplantation, increase the success rate of transplantation medicine, and reduce the amount of the immunosuppressive agent 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。
Evaluation method of immunosuppressive action or immunological tolerance action (in vivo)
The first aspect of the present invention is
(I) In the recipient mouse in which the H-2 antigen type is b/k, the H-2 antigen type is b/b, k/k or b/k and the minor histocompatibility antigen type is the aforesaid Transplanting cells or tissue from a donor mouse that does not match the recipient mouse;
(Ii) subjecting the recipient mouse to a test treatment before, at the same time as, or after the transplantation of the cells or tissue derived from the donor mouse;
(Iii) after the transplantation and the test treatment, a step of evaluating the rejection reaction of the recipient mouse; and (iv) the rejection reaction of the recipient mouse was transplanted with cells or tissue derived from a donor mouse, but the test treatment was performed. When the rejection of the recipient mice treated with the test is weaker than that of the control recipient mice compared with the rejection of the untreated control recipient mice, the test treatment is immunosuppressive or tolerant. The present invention relates to a method for evaluating an immunosuppressive action or an immunological tolerance action of a test treatment, the method including determining that the test treatment has.
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。 Recipient mice whose H-2 antigen type in this embodiment is b/k are prepared using H-2 antigen type b/b homozygous mice and H-2 antigen type k/k homozygous mice. It is a hybrid mouse that can. H-2 antigen type b/b homozygous mice are 129 strain mice (for example, 129P1/ReJ, 129P3/J, 129P3/JEmsJ, 129P4RrRkJ, 129S1/SvlmJ, 129T2SvEms, 129T2/SvEmsJ, 129X1/SvJ), BXSB/ Mp, C57BL/6, C57BL/10, LP/J, BALN.B and the like can be mentioned. H-2 antigen type k/k homozygous mice are C3H strain mice (for example, C3H/He, C3H/HeN, C3H/Bi, C3HeB/FeJ, etc.), AKR/J, CBA strain mice (for example, 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。 A particularly preferred recipient mouse is the C3H strain mouse, particularly the F1 mouse C3129F1 (H-2 b/k ) having C3H/He as a mother and 129 strain mouse, in particular, 129X1/SvJ as a father.
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 corresponds to a mouse having the same H-2 serotype as the recipient mouse (H-2 b/k ) or at least one allele of the H-2 serotype of the recipient mouse (allele). Allelic mice with alleles (H-2 b/b , H-2 k/k ), in which the types of minor histocompatibility antigens do not completely match those of the recipient mice. Specifically, it is a mouse in which the type of H-2 antigen is b/b, k/k or b/k and the type of minor histocompatibility antigen does not match 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 allogeneic transplantation between MHC-matched donors and recipients, and specifically, on MHC molecules on the cell surface. Among the in-vivo proteins presented in 1., a peptide having an amino acid sequence that differs between a donor and a recipient due to polymorphism and the like, and whose MHC/peptide complex is recognized as non-self by recipient T cells.
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。 As examples of mouse minor histocompatibility antigens, H60 and H4 for H-2K b restriction and H7 for H-2D b restriction are known.
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。 The donor mouse in the present invention, as long as the type of H-2 antigen is b/b, k/k or b/k and the type of mHA does not completely match the recipient mouse, any mouse is selected and used. can do. Donor mice are, for example, mice that are not of the same strain as the recipient parental mouse, H-2 antigen type is b/b, k/k or b/k, examples of which are listed above. .. The donor mouse is preferably a C57BL/6 strain or CBA/N strain mouse.
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 is no particular limitation on the type of cells transplanted into the recipient mouse, various somatic cells derived from the donor mouse, iPS cells produced from the somatic cells and cells differentiated from the iPS cells, tissue stem cells derived from the donor mouse Also, cells derived from the stem cells can be mentioned as an example. In addition, isolation of cells from a donor mouse and induction of differentiation can be performed for each cell by a known method.
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。 The type of tissue transplanted into the recipient mouse is not particularly limited, 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 in which the test treatment is expected to be applied, or a tissue that can be easily handled, 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 is no particular limitation on the specific method for transplantation into the recipient mouse, and it can be carried out by adopting an appropriate method depending on the cells or tissues to be transplanted from the donor mouse. If necessary, the recipient mouse may be subjected to pretreatment such as 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 gives some external stimulus to the mouse, and is typically administration of a substance for which evaluation of immunosuppressive action or immunological tolerance action is desired, or change of external environmental factors. The test treatment may be performed before, at the same time as, or after the transplantation of the cells or tissue derived from the donor mouse. When the test treatment is administration of the test substance, the administration form (liquid, solid, etc.) of the test substance and the administration route (oral intake, intravenous administration, intraperitoneal administration, etc.) are mainly physicochemical properties and organisms of the test substance. It is appropriately selected 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 a donor mouse, both T cell-related rejection and antibody-related rejection can be induced. Therefore, the rejection of recipient mice is specifically determined by observing a phenomenon caused by either T cell-related rejection or antibody-related rejection, specifically, the graft survival rate and engraftment period, It can be evaluated by the degree of infiltration of host lymphocytes into the graft, the measurement of inflammatory markers in the recipient mouse, the measurement of antibody production against the graft, the mixed lymphocyte test, and the like.
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。 This embodiment compares the rejection reaction of the recipient mouse evaluated as described above with the rejection reaction of the control recipient mouse in which cells or tissues derived from the donor mouse are transplanted but the test treatment is not performed, and the test treatment is performed. When the rejection reaction of the recipient mouse that underwent the treatment is weaker than that of the control recipient mouse, the test treatment includes a step of determining that the test treatment has an immunosuppressive effect or an immunotolerance effect.
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 the graft survival time is used as an index of rejection, the change in the graft survival time of each graft of the recipient mouse administered with the test substance and the control recipient not administered with the test substance is observed, and the test substance is It can be determined that the test substance has immunosuppressive ability or immunological tolerance when an extension of the engraftment period is observed upon administration.
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。
Evaluation method of immunosuppressive action or immunological tolerance action (in vitro)
Another aspect of the present invention is (i) a sample containing T cells from a recipient mouse in which the H-2 antigen type is b/k, and the H-2 antigen type is b/b, k/k or b/k minor histocompatibility antigen type is mixed with a sample containing donor mouse-derived antigen-presenting cells that do not match the recipient mouse, in vitro recipient-derived T cells and donor-derived antigen-presenting cells Coexisting with;
(Ii) subjecting the recipient-derived T cells to a test treatment before, simultaneously with, or after the recipient-derived T cells and the donor-derived antigen-presenting cells coexist;
(Iii) after coexistence with the donor-derived antigen-presenting cells and after the test treatment, the step of evaluating the blastogenic reaction of the recipient-derived T cells; and (iv) the blastogenic reaction of the recipient-derived T cells Compared with the blastogenic response of control recipient-derived T cells that coexisted with antigen-presenting cells but did not carry out the test treatment, the blastogenic response of recipient-derived T cells that underwent test treatment was derived from the control recipient It relates to a method for in vitro evaluation of immunosuppressive action or immunotolerance of a test treatment, which comprises a step of determining that the test treatment has an immunosuppressive action or an immunotolerance action when it is weaker than the T cell blastogenic 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 the present embodiment, "type of H-2 antigen is a recipient mouse of b/k" and "type of H-2 antigen is b/b, k/k or b/k is a minor histocompatibility antigen type The “donor mouse which does not match the recipient mouse” is as described in the first embodiment.
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 blastogenic reaction is a reaction that occurs during mixed lymphocyte reaction (MLR; Mixed Lymphocyte Reaction), and mixed lymphocyte culture (MLC; Mixed Lymphocyte Culture) using this is a clinical test for predicting rejection. Known as. This embodiment uses the combination of the recipient mouse and the donor mouse in the mixed lymphocyte culture as the combination of the recipient mouse and the donor mouse of the first embodiment, so that the rejection reaction caused by the minor histocompatibility mismatch (T cell degeneration). It is used as an in vitro evaluation tool reflecting the 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 the present embodiment is a mixed lymph node 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. It can be carried out by performing sphere culture and comparing the resulting blastogenic response of recipient-derived T cells with and without the test treatment. The sample containing the recipient-derived T cells and the sample containing the donor-derived antigen-presenting cells may be T-cells such as CD4 + T cells or CD8 + T cells, and antigen-presenting cells such as dendritic cells, respectively. The lymphocyte fraction may be used as used in mixed lymphocyte culture as a peripheral blood clinical test. The T cells, antigen-presenting cells, or cell groups containing each cell may be prepared according to a method for isolating each cell or a method for preparing each cell group 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 gives some external stimulus to the T cells derived from the recipient, and typically, treatment with a substance for which evaluation of immunosuppressive action or immunological tolerance action is desired or an external environmental factor is changed That is. The test treatment may be performed either before, simultaneously with, or after the recipient-derived T cells and the 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 blastogenic reaction of T cells can be evaluated by labeling the T cells with, for example, the fluorescent dye 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 blastogenic response of recipient-derived T cells evaluated as described above with the blastogenic response of control recipient-derived T cells that coexisted with the donor-derived antigen-presenting cells but did not undergo the test treatment. If the blastogenic response of the recipient-derived T cells that underwent the test treatment was weaker than the blastogenic response of the control recipient-derived T cells, it is determined that the test treatment has an immunosuppressive action or an immunotolerance action. 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。
Cell Preparation for Inducing Immune Tolerance in a Recipient Subject In yet another aspect, the invention provides a cell preparation for inducing immune tolerance in a recipient subject, comprising immune cells from a donor subject, comprising: Provided is a cell preparation for use in combination with irradiation and T cell depletion treatment on an ent subject.
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 an unresponsiveness of the immune system to self-antigens such as self-cells, and is broadly classified into 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 the rejection reaction 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, animals, such as mice, rats, hamsters, rodents including guinea pigs, humans, primates including chimpanzees, pigs, cows, goats, horses, livestock including sheep, dogs, Mammals such as pets including cats are preferable, and humans are particularly preferable. Also, in a preferred embodiment, the types of major histocompatibility antigens of the recipient subject and the donor subject are exact or partial, and the types of minor histocompatibility antigens are not.
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。 Irradiation may be radiation irradiation that is usually performed as one of the pretreatments to be performed on the recipient subject when performing transplantation treatment, such as total body irradiation to the recipient subject or irradiation to the thymus. Is preferred. The irradiation dose can be appropriately adjusted, for example, in the case of whole-body irradiation to humans, which is 1 Gy or more as one irradiation dose, and in the case of irradiation of the thymus, 3 Gy or more, and a dose which does not reach a lethal dose as a guideline. ..
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 depletion treatment may be any treatment that is usually performed as one of the pretreatments performed on the recipient subject when performing transplantation therapy, and includes administration of anti-T cell antibodies and steroids. Examples of anti-T cell antibodies include anti-CD4 antibody, anti-CD8 antibody, anti-CD3 antibody, anti-TCR antibody, and anti-thymocyte globulin (ATG), and one or more of these are administered to a recipient subject in combination. Preferably. In a preferred embodiment, the anti-T cell antibody is a combination of anti-CD4 antibody and anti-CD8 antibody, which are preferably administered intravenously or intraperitoneally to the recipient subject simultaneously or sequentially. The dosage may be an amount sufficient to remove T cells in the recipient'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。 Irradiation and T cell depletion treatment induces immune tolerance after the transplant is performed by the time immediately before the transplant when it is desired to induce the immune tolerance before the transplant is performed, that is, delayed type tolerance (Delayed Tolerance Induction) When induction of is desired, it can be repeated once or multiple times at a desired timing to induce tolerance. The order of irradiation and T cell removal treatment is not particularly limited, but T cell removal treatment is preferably performed prior to irradiation. Hereinafter, 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 it is preferable to administer them to a recipient subject in combination with one or more of them. In a preferred embodiment, the immune cells are antigen-presenting cells such as B cells and dendritic cells, and in a particularly preferred embodiment, the immune cells are B cells and Fms-like tyrosine kinase 3 ligand (Flt3L)-induced dendritic cells. 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 a tissue collected from a donor subject, for example, blood or bone marrow fluid by a method known to those skilled in the art, and if necessary, cell culture is performed to increase the number of cells. Can be used. Alternatively, iPS cells derived from somatic cells derived from a donor subject and differentiated 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 to be administered to the recipient subject should be appropriately adjusted in the range of 1x10 7 to 1x10 9 cells/kg body weight, preferably 1x10 8 to 1x10 9 and more preferably 5x10 8 to 2x10 9 cells/kg body weight. You can The immune cells may be administered to the recipient subject after pre-treatment, and if it is desired to induce immune tolerance prior to transplantation, by induction of immunological tolerance after transplantation, i. When induction of immunological tolerance is desired, it can be administered once or multiple times at a desired timing to induce tolerance. The administration is preferably intravenous administration or intraperitoneal administration.
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 the present aspect can be used in combination with irradiation and T cell depletion treatment on a recipient subject to induce immune tolerance to tissues or cells derived from the donor subject in the recipient subject, whereby the transplantation. Can increase the colonization rate of tissues or cells from a donor subject, and avoid or reduce the dose of cyclosporine and other conventional immune response suppressors to transplant recipient recipients. It will be possible. The cell preparation can be used not only for induction of immunological tolerance before transplantation but also for induction of immunological tolerance after transplantation, that is, delayed type immunological tolerance induction.
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。 Hereinafter, the present invention will be specifically described with reference to examples, but these examples are for helping the understanding of the present invention and do not 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. Generation of minor mismatched skin-transplanted mice
1) Preparation of recipient mouse
F1 mice were prepared from 129X1/SvJ mice (male, purchased from Sankyo Lab Service Co., Ltd.) and C3H/He mice (female, purchased from Sankyo Lab Service Co., Ltd.). The prepared mouse H-2 antigen was used as a peripheral blood flow site using FITC-anti-H-2Kk (36-7-5, Biolegend) and PE-anti-H-2Kb (AF6-88.5, Biolegend). It was confirmed to be H-2 b/k by analysis by means of measurement (FC500, Beckman Coulter). Hereinafter, this F1 mouse is 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 transplant
7 to 10-week-old C3129F1 (male, 6 to 7 mice/group) was used as a recipient mouse, and an 8-mm skin defect portion was prepared on the back of the recipient mouse under anesthesia. Create male BALB/c mice (H-2 d/d ), C57BL/6 mice (H-2 b/b ), CBA/N mice (H-2 k/k ), and skin defects at the same age After removing the auricles from C3129F1 mice of the same individual as the above-mentioned individual under anesthesia, the skin tissue and cartilage tissue were peeled off with a pin, and an 8-mm skin piece was transplanted to the skin defect portion of the recipient mouse. After transplantation, the mice are bred under normal breeding conditions for up to 100 days to 150 days, and the size of the graft is measured over time. The engraftment rate (the number of individuals whose transplants were rejected/the total number of individuals) was calculated (FIG. 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 graft from C3129F1 which is autotransplanted to the recipient mouse, no rejection was observed during the entire breeding period after transplantation, and the graft was completely engrafted at the transplant site. On the other hand, the graft from the BALB/c mouse (H-2 d/d ) whose MHC type was inconsistent with the recipient mouse completely disappeared by 18 days after the transplantation. Grafts from C57BL/6 mice (H-2 b/b ), whose MHC type was partially matched with that of the recipient mouse but whose minor histocompatibility antigens were not matched, also completely disappeared by 18 days after transplantation. Similarly, a graft from a CBA/N mouse (H-2 k/k ) with a minor mismatch remained in the transplant site until day 29 after the transplant.
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 transplants On the 10th day after transplantation, the transplants were excised and collected together with the subcutaneous tissues of the recipient mice, and HE staining was performed. Infiltration of lymphocytes was observed at the transplantation site in mice transplanted with skin pieces from BALB/c mice (H-2 d/d ) and C57BL/6 mice (H-2 b/b ) (Fig. 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 in mice with minor mismatch skin transplantation
1) Tacrolimus In the same manner as in 2) of Example 1, C3129F1 mice were added to BALB/c mice (H-2 d/d ), C57BL/6 mice (H-2 b/b ), or CBA/N mice (H-2). The skin pieces collected from ( k/k ) were transplanted. Every day from the day of transplantation, tacrolimus was intraperitoneally administered to each mouse at 0.5 mg/kg or 2.0 mg/kg for 40 days under normal breeding conditions, and the size of the graft was measured over time. Then, the survival rate of the graft was calculated. Transplanted mice administered with physiological saline instead of tacrolimus were used 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 any of the mice transplanted with any skin piece, there was almost no difference in graft survival rate 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 the complete disappearance of the graft was 8 days for the graft from the BALB/c mouse (H-2 d/d ), and that for the C57BL/6 mouse (H-2 b /b ) grafted for 20 days, each extended. In the grafts from CBA/N mice (H-2 k/k ), no rejection was observed until 40 days after transplantation, and the engraftment rate was 100% (Fig. 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 transplantation was performed on recipient mice in the same manner as in 1) above. Every day from the day of transplantation, 1.0 mg/kg of rapamycin was intraperitoneally administered to each mouse and kept for 100 days under normal breeding conditions, and the graft size was measured over time to determine the graft survival rate. 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）。 By rapamycin administration, the number of days until the graft completely disappeared was extended by 4 days for both the graft from BALB/c mouse (H-2 d/d ) and the graft from C57BL/6 mouse (H-2 b/b ). did. On the other hand, the engraftment rate of grafts from CBA/N mice (H-2 k/k ) decreased to 50% within 30 days after transplantation, but no decrease 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 extracted. The spleen was gently ground with frosted glass to give 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 separated from spleen cells by a separation method using magnetic beads (CD19 MicroBeads, mouse, Miltenyi Biotech) labeled with an anti-mouse CD19 antibody, and designated 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) are sufficient to remove T cells in the
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。 3.0 x 10 7 spleen cells prepared above, 3.0 x 10 7 B cells or the same volume of physiological saline was intravenously administered to the pretreated recipient mouse, and BALB was also administered on the same day. The skin collected from the /c mouse (H-2 d/d ), the C57BL/6 mouse (H-2 b/b ) or the recipient mouse itself was transplanted. The animals were raised under normal breeding conditions for up to 150 days, the size of the graft was measured over time, and the graft survival rate was calculated.
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 was administered, no rejection was observed in the graft from C3129F1 during the entire breeding period after transplantation, and the graft completely adhered to the transplant site. On the other hand, the grafts from BALB/c mice (H-2 d/d ) whose MHC type was inconsistent with the recipient mice showed a prolonged engraftment period by administration of spleen cells. Furthermore, transplants from C57BL/6 mice (H-2 b/b ), whose MHC type partially matches that of the recipient mouse but whose minor histocompatibility antigens do not match, were transplanted by administration of spleen cells or B cells. Completely engrafted on 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。 From Examples 1 and 2, that the minor mismatch skin grafted mice showed a rejection reaction equal to or weaker than that of the MHC type mismatched skin graft mice, reflecting the minor mismatch mismatch rejection reaction in humans, and the minor mismatch skin grafted mice were By using it, it was shown that immunosuppressive effects of immunosuppressive agents such as tacrolimus and rapamycin on the rejection reaction caused by minor inconsistency can be evaluated. Furthermore, it was confirmed that spleen cells and B cells exert a strong immunotolerance in allografts, particularly minor allogeneic allografts, in combination with irradiation and administration of anti-T cell antibody.
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***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 method using anti-mouse CD90.2 antibody-labeled magnetic beads (CD90.2 MicroBeads, mouse, Miltenyi Biotech), and used as T cells. T cells were stained with 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester (CFSE) to observe cell division. Bone marrow cells of BALB/c mouse, C57BL/6 mouse, CBA/N mouse or C3129F1 mouse were cultured for 1 week in the presence of recombinant mouse GM-CSF, and the generated cells were used as dendritic cells. 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 CFSE attenuation in the CD4-positive cells and CD8-positive cells was measured. The cells that were attenuated were regarded as blast cells, and the ratio 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 were little or not proliferated when co-cultured with dendritic cells from C3129F1 or CBA/N mice, whereas those from C57BL/6 mice were derived. dendritic cells CD4 + T cells and CD8 + T cells by co-culture with each of approximately 10%, CD4 + T cells by co-culture with BALB / c mouse-derived dendritic cells about 40%, CD8 + T cells Increased by more than 50% (Fig. 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 the combination of dendritic cells and T cells that causes minor mismatch exhibits T cell blastogenesis that is equal to or weaker than the combination of dendritic cells and T cells that causes MHC type mismatch. It was considered that it can be used for evaluation of minor mismatch rejection reaction in the same manner as the mice with minor mismatch skin graft 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) C57BL/6J (male, donor, H-2 b/b ) aged 7 to 10 weeks were euthanized, and then the humerus, femur and tibia were removed. Both ends of the excised bone were cut, and the inside of the bone was washed with PBS using a syringe to collect bone marrow. The collected bone marrow was filtered using a 70N filter to make 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 to suspend 1.0×10 7 bone marrow cells. After culturing for 7-9 days at 37° C. under 5% CO 2 , floating or weakly adherent cells were collected to prepare FL-DC derived from C57BL/6 mouse.
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) Anti-mouse CD4 antibody (GK1.5) and anti-mouse CD8α antibody (53-6.72) were applied 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) C57BL/6 mouse-derived B cells (3.0×10 7 cells) prepared in 3) of Example 2 against the recipient mouse, and C57BL/6 mouse-derived FL-DC prepared in 1) above. (1.5×10 7 ) or the same volume of physiological saline was administered intravenously. Seven days after the administration, skins collected from BALB/c mice (H-2 d/d ), C57BL/6 mice (H-2 b/b ) or recipient mice themselves (Auto) were transplanted. The size of the graft immediately after transplantation is maintained under normal conditions for up to 100 days (when transferring B cells) or 60 days (when transferring FL-DC) and the size of the graft is measured over time. It was judged to be rejected when the size was 30% or less compared with. The graft survival rate was used as the value (individual graft graft/total number of individuals).
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）。 When any of B cells, FL-DC and physiological saline was administered, no rejection was observed in the transplants of the recipient mice themselves during the entire breeding period after transplantation, and the transplants were completely engrafted at the transplant site. On the other hand, grafts from BALB/c mice (H-2 d/d ) whose MHC type is inconsistent with the recipient mice were treated with B cells, FL-DC and physiological saline at an early stage after transplantation. Was rejected. Grafts from C57BL/6 mice (H-2 b/b ) whose MHC type partially matches the recipient mouse but not the minor histocompatibility antigen type were rejected when saline was administered However, administration of B cells resulted in long-term engraftment at the transplant site for 100 days or longer, and administration of FL-DC at the transplant site for 60 days or longer (FIG. 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, spleen and peripheral blood were collected from the recipient mice, and cell suspensions were prepared according to a conventional method. The cell suspension was subjected to flow cytometric analysis (BD FACSCantoII, Becton Dickinson) to detect donor-derived B cells (CD45.1+) and recipient-derived cells (CD45.2+). Presence of donor-derived cells was confirmed in both 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 into which B cells were transferred in the above 3), a spleen was extracted 50 days after skin transplantation to prepare a cell suspension, which was then CD19 positive using a CD19 cell isolation kit (Miltenyi Biotech). After removing the cells, the cells were stained with CFSE. 2.0 × 10 5 recipient mouse-derived spleen cells and 1.0 × 10 4 BALB/c mouse or C57BL/6 mouse-derived spleen cells irradiated with 30 Gy radiation were co-cultured, and after 6 days, flow cytometry (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 CFSE attenuation in the CD4-positive cells and CD8-positive cells was measured. The cells that were attenuated were regarded as blast cells, and the ratio 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-culture with irradiated spleen cells derived from BALB/c mice, CD4 + T cells derived from recipient mice showed a proliferation rate of about 60%, and CD8 + T cells showed a proliferation rate of about 80%. On the other hand, even when co-cultured with irradiated spleen cells derived from C57BL/6 mice, T cells derived from recipient mice were hardly proliferated, and immunological tolerance was confirmed.
Claims (11)
（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) In the recipient mouse in which the H-2 antigen type is b/k, the H-2 antigen type is b/b, k/k or b/k and the minor histocompatibility antigen type is the aforesaid Transplanting cells or tissue from a donor mouse that does not match the recipient mouse;
(Ii) subjecting the recipient mouse to a test treatment before, at the same time as, or after the transplantation of the cells or tissue derived from the donor mouse;
(Iii) after the transplantation and the test treatment, a step of evaluating the rejection reaction of the recipient mouse; and (iv) the rejection reaction of the recipient mouse was transplanted with cells or tissue derived from a donor mouse, but the test treatment was performed. When the rejection of the recipient mice treated with the test is weaker than that of the control recipient mice compared with the rejection of the untreated control recipient mice, the test treatment is immunosuppressive or tolerant. A method for evaluating an immunosuppressive action or an immunological tolerance action of a test treatment, which comprises the step of determining that the test treatment has.
（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) A sample containing T cells derived from a recipient mouse whose H-2 antigen type is b/k, and a minor tissue whose H-2 antigen type is b/b, k/k or b/k A step of mixing a sample containing antigen-presenting cells derived from a donor mouse whose compatible antigen type 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) subjecting the recipient-derived T cells to a test treatment before, simultaneously with, or after the recipient-derived T cells and the donor-derived antigen-presenting cells coexist;
(Iii) after coexistence with the donor-derived antigen-presenting cells and after the test treatment, the step of evaluating the blastogenic reaction of the recipient-derived T cells; and (iv) the blastogenic reaction of the recipient-derived T cells Compared with the blastogenic response of control recipient-derived T cells that coexisted with antigen-presenting cells but did not carry out the test treatment, the blastogenic response of recipient-derived T cells that underwent test treatment was derived from the control recipient A method for in vitro evaluation of an immunosuppressive effect or an immunotolerance effect of a test treatment, which comprises a step of determining that the test treatment has an immunosuppressive effect or an immunotolerance effect when it is weaker than the T cell blastogenic reaction.
The cell preparation according to any one of claims 7 to 10, wherein the immune cell is a B cell, or a dendritic cell induced by Fms-like tyrosine kinase 3 ligand (Flt3L).
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| WO2023190942A1 (en) * | 2022-03-31 | 2023-10-05 | 学校法人順天堂 | Method for evaluating quality of inducement inhibitory-t-cell formulation |
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