当前猪到灵长类动物的异种移植中存在的细胞免疫障碍

hyde

% w9 w& R* z) S5 ]8 N; {% L

; n/ \. V( ~: e3 v0 Q原文来自：文献求助 pig-to-primate xenotransplantation5 z  o, V, r+ [; b2 R5 T' [) V3 e
http://www.stemcell8.cn/thread-34426-1-1.html/ _2 g1 v( w' f) v5 C2 Y
由干细胞之家新闻小组成员hyde翻译（转帖请注明）
1 A7 l! f% [2 w4 l. y+ K
) q6 f# Z; {/ z+ m) O当前猪到灵长类动物的异种移植中存在的细胞免疫障碍
& u& l, \6 R, g3 Z2 tCurrent cellular immunological hurdles in pig-to-primate xenotransplantation6 `3 a& U; V$ B! E( B0 G

: K# A, N! \! }% @3 y8 f9 w摘要：" F3 s- y; e# J. W# I2 [8 P
在这篇综述中，我们将总结这几年出版的关于猪到灵长类异种移植模型中存在的细胞免疫障碍的工作成果。这些工作成果涉及先天性细胞免疫反应和获得性细胞免疫反应以及针对它们的措施，这些措施使异种移植物免受排斥反应的损伤。- g- e4 E: F1 L+ A
a b s t r a c t- j  T$ U# v% b$ T
In this review, we summarize the work published over the last few years relative to cellular immunological hurdles encountered specifically in pig-to-primate xenotransplantation models. The works summarized here cover both the innate and adaptative cellular immune response as well as strategies to overcome them and consequently prevent xenograft rejection." J+ Q$ Q# s8 l  D

$ Q3 v' M7 r) L5 e7 B9 U1引言$ J% t6 c* }8 L+ Z' D
现在已经成功地使用了转基因技术敲除猪的半乳糖基转移酶基因获得GalT-KO 猪，在pig-to-primate异种移植过程中能够避免因为异种抗原αGal的存在而引起的体液免疫排斥反应。但是非Gal异种抗原引起的体液免疫排斥反应和细胞性异种排斥反应仍然是异种移植的障碍。建立pig-to-primate异种移植实验模型，可以探索pig-to-human异种移植时存在的各种细胞性机理。6 X( x) j' m. m. R" j$ w; O
1. Introduction6 ?1 Z: G+ P! G& J! `
Now that galactosyltransferase knock-out (GalT-KO) pigs are available,the anti-galactose α1,3-galactose β1,4 GlcNAc-R (αGal) humoral response can be avoided. However, the anti-nonGal humoral as well as cellular xenogeneic responses remain important issues that need solving for xenotransplantation to be successful. The pig-to-primate combination is a suitablemodel to explore the different cellular mechanisms that would be involved in the course of xenotransplantation in humans.1 G8 o" w! [" }( u$ f' e3 `' J( k# r

) c" B& w9 w+ x9 q! _4 t3 |. S2先天性细胞免疫反应6 M1 s6 v" W' z+ r: P
2. The innate cellular immune response+ H( n$ Q9 c2 U

- G) Y# `, D; P' i1 ^2.1NK细胞介导的排斥反应的表征和机制
7 y7 {  O  B3 U, T2.1. Characterization/mechanism of action of natural killer cell-mediated rejection
) W$ x' g/ n5 W/ {/ S  I0 W6 ?- }: e- ~6 P
先天性细胞免疫系统的细胞成分包括：NK细胞、单核/巨噬细胞和树突状细胞。NK细胞上存在着激活和抑制细胞功能的两种受体，这些受体通过不断地与相应配体的结合来维持细胞功能的平衡。抑制性受体识别自身MHC-I后产生的抑制细胞毒作用的信号占优势。如果没有足够的抑制性信号，激活性受体在结合相应配体后会促进NK细胞毒作用。在异种条件下，猪内皮细胞（pEC）在体外对人NK细胞毒作用较敏感，这可能是因为猪白细胞抗原I（SLA-I）不能被人NK细胞上的抑制性受体所识别，即不能产生细胞毒作用的抑制性信号。另一方面，人天然IgG异种抗体（XNA）结合到αGal或其他异种抗原表位后被Nk细胞所识别，NK细胞通过其CD16产生抗体依赖细胞介导的细胞毒作用（ADCC）。除了ADCC，体外实验还发现，抗αGal的IgG能刺激人NK细胞，使其在猪主动脉内皮细胞上粘附和迁移，这是通过CD16的交叉结合来完成的。5 Y; U- G  e$ Y
The cells constituting the innate cellular immune system include natural killer (NK) cells, monocytes/macrophages and dendritic cells. NK function is the result of a continuous equilibrium between inhibitory and activating NK receptors binding their relative ligands. Recognition of self MHC class I molecules by inhibitory NK receptors provides a dominant-negative signal for NK cytotoxicity,whereas in the absence of sufficient inhibitory signals, binding of activating receptors induces NK cytotoxicity. In the xenogeneic context, the in vitro susceptibility of porcine endothelial cells (pEC) to human NK cytotoxicity may be the result of a lack of recognition and inability of swine leukocyte antigen (SLA) class I to signal through human NK inhibitory receptors. On the other hand, the NK recognition of human IgG natural xenoantibodies (XNA) bound to αGal and other epitopes via CD16 leads to antibody dependant cell-mediated cytotoxicity (ADCC) of pEC . In addition to ADCC, anti-αGal IgG antibodies have been shown to stimulate human NK cell adhesion to and migration across porcine aortic endothelial cells (PAEC) via CD16 cross-linking in vitro .
* C$ }7 `. E# A  N' U$ \1 ^
" Y) t+ d4 N- M; \: s/ s此外，使用GalT-KO猪的pEC和PAEC进行实验时，补体依赖细胞毒作用（CDC）和ADCC有所减弱，但是PAEC上持久性存在的人白细胞粘附和NK细胞毒作用说明了αGal不是猪细胞上唯一的靶点。综上所述，NK细胞毒作用、猪内皮细胞上的细胞粘附和迁移与αGal和非αGal异种抗原的识别有关，导致了被免疫排斥的异种移植物中出现NK细胞浸润。7 o+ O  s+ I! q  W' f
Furthermore, studies performed on GalT-KO pEC and on PAEC derived from GalT-KO pigs have shown a reduction in complement dependent cytotoxicity (CDC) and ADCC, but a persistence of human leukocyte adhesion or NK cytotoxicity against PAEC, indicating that αGal is not a unique target on porcine cells. Taken together, these results suggest that the recognition of Gal and non-Gal antigens plays a role in NK cytotoxicity and cell adhesion to and migration across porcine endothelium, leading to NK cell infiltration of the xenograft followed by rejection.. Q6 ]! m/ P0 w
) G- C* S9 y! U: t+ y% a1 W
为了进一步明确αGal抗原在NK细胞介导的异种排斥反应中的作用，Horvath-Arcidiacono等人使用体外实验研究人NK细胞对pEC的识别能力。使用三种pEC，分别是野生型（WT）pEC、EndoGal C处理的野生型（WT）pEC以及GalT-KO猪的pEC。结果显示，EndoGalC-WT组的结果具有多变性，而GalT-KO组被幼稚NK细胞识别发生细胞裂解病变的程度虽然有所减弱但仍能检测到。这种细胞裂解病变取决于NK细胞及其靶点的激活程度，说明了单独清除供体猪上的αGal抗原不足以避免异种NK细胞介导的裂解反应。' R8 a' b& ~1 ~* G. E4 v, W, ^9 W
To further clarify the role of the αGal epitope in NK cell-mediated xenogeneic responses, Horvath-Arcidiacono et al. investigated the in vitro recognition of wild type (WT) pEC, endo-β-galactosidase C (EndoGal C)-treated WT pEC or GalT-KO pEC by human NK cells. The results using EndoGalC were variable, while the use of GalTKO pEC confirmed a reduced but detectable lysis by naive human NK cells. However the lysis was dependent on the activation level of both the NK cells and their target, confirming that elimination of αGal alone from donor pigs would not be sufficient to circumvent xenogeneic NK cell-mediated lysis.
' p- F# u* c6 I% }3 y0 v- E2 Y9 c2 K7 B" s2 x0 F5 B
2.2  针对NK细胞的方案+ a4 v' N1 P$ t6 b
2.2. Natural killer cell strategies
) D# Y* W# A, E0 x1 A9 ^- }( }% {* U2 N4 H3 {( Y5 J
根据这些研究成果，研究者设计出了一些方案用于抑制异种NK细胞介导的裂解反应。这些方案针对NK细胞的激活性和抑制性受体。Forte等人通过阻断激活性受体进行研究，这类受体包括天然细胞毒受体(NCR) NKp30,NKp44, NKp46 和NKG2D。结果显示，只有NKp44和NKG2D参与诱导了NK异种细胞毒作用但不参与ADCC，说明了这一方案可能防止NK细胞排斥反应对异种移植物造成的伤害。9 w1 k' Z" o3 q1 n9 J/ Y( F# w! _
In light of these findings, several strategies have been developed to inhibit xenogeneic NK cell-mediated lysis by acting on NK activating or inhibitory receptors. Forte et al. investigated the blockade of activating NK receptors, including the natural cytotoxic receptors (NCR) NKp30, NKp44, NKp46 andNKG2D.OnlyNKp44 andNKG2Dwere found to be involved in direct NK xenogeneic cytotoxicity, but not in ADCC, indicating that this strategy may prevent direct NK responses against xenografts.2 ?7 S8 U& O- C! @
" y& P* t/ ]7 ^* q
NKG2D受体在NK细胞中被持续表达。现已知它在人体内的配体包括MICA，B（MHC class I chain-related proteins A，B）以及ULBP1-4。pULBP1 和pMIC2是猪体内这些配体的类似物。Lilienfeld等人对pULBP1 和pMIC2进行研究，发现在猪体内pULBP1是人NKG2D受体的主要功能活性配体。他们还进一步发现pULBP1在PAEC表面的表达受许多移植相关刺激因素的调节，人和猪TNF-α的刺激、细胞巨化病毒的感染会减少其表达，人抗猪XNA结合到PAEC会明显增加表达量。因此，pULBP1可能需要被清除，使得猪异种移植物免受人NK细胞毒作用的伤害。) o9 B/ ^" l- g
NKG2D is constitutively expressed by NK cells and its known ligands in humans are MHC class I chain-related proteins A (MICA),B (MICB) and ULBP1-4. Lilienfeld et al. tested the porcine counterparts of these molecules: pULBP1 and pMIC2, and showed that pULBP1 is the predominant functional porcine ligand of human NKG2D . They further showed that pULBP1 expression on the PAEC surface is modulated by various stimuli associated with transplantation, i.e. decreased by human and porcine TNF-α stimulation and cytomegalovirus infection, and strongly increased by the binding of human anti-pig XNA on PAEC . Thus, this molecule may be a target molecule to eliminate, with the aim of protecting porcine xenografts from human NK cytotoxicity.! k; E) P$ O2 Q" z  [
2 @7 F5 j. i9 F  P1 X9 l2 ]
考虑到NK抑制性受体可能不识别SLA-I，Forte等人研究是否能够在猪细胞上稳定表达HLA-E，以此来阻止异种NK细胞介导的细胞毒作用。他们发现，HLA-E转基因的pECs对于人多克隆NK细胞群体介导的细胞裂解具有部分耐受性，对于NKG2Abright NK细胞的杀手作用则具有完全耐受性。另外，对HLA-E进行修饰即单一的氨基酸替代后，其在人和猪细胞表面的表达量更高，对于NK细胞的抑制效果更好。这些修饰措施对于转基因猪的培育具有重要的意义。基于这些发现，Lilienfeld等人分析了猪细胞转基因表达的HLA-E单链三聚体（SCT）对异种NK细胞毒作用的影响。几个连续的序列分别编码了HLA-E binding peptide、HLA-E α chain、human β2microglobulin (β2m)，由它们构成的HLA-E SCT可以减弱多克隆NK细胞介导的细胞毒作用，这与阻断CD94/NKG2A途径的效果明显相反。虽然HLA-E的表达对于人NK细胞粘附到pECs、与猪细胞形成复共轭对配合物没有影响，但结合其他的保护性措施可能有助于临床异种移植的成功。% q4 a# P# Y2 S0 |# a( `
Given that SLA class I may be unable to signal through human NK inhibitory receptors, Forte et al. tested whether stable expression of HLA-E on porcine cells protects against xenogeneic NK-mediated cytotoxicity . They showed that HLA-E-transgenic pECs are partially protected from lysis mediated by human polyclonal NK populations and completely protected from killing by NKG2Abright NK clones. Moreover, modification of the HLA-E molecule by one single amino acid substitution has been shown to result in a higher expression on human and pig cell surface and a greater inhibition of NK cells. Such modifications may have an impact on genetically engineered pigs. Based on these findings, Lilienfeld et al. analyzed the effect of transgenic expression of a HLA-E single chain trimer (SCT) by porcine cells on xenogeneic NK cell cytotoxicity . The HLA-E SCT made up of linked sequences coding for a HLA-E binding peptide, the HLA-E α chain and the human β2microglobulin (β2m) , induced a significant decrease in polyclonal human NK-mediated cytotoxicity that was specifically reversed by blocking CD94/NKG2A . Even though HLA-E expression affected neither the adhesion of human NK cells to pECs nor the formation of heteroconjugates between human NK and porcine cells,this approach may contribute to the success of clinical xenotransplantation if used in combination with other protective strategies.: \+ a1 L' v2 h- b( F( I5 z

% V4 f3 A/ \' m2.3巨噬细胞介导的排斥反应的表征和机制- ^/ |( {9 ?* D
2.3. Characterization/mechanism of action of macrophage-mediated rejection, P! L  c* D! O# f  x6 F
  U9 D5 ]% ^- l2 M* M  S4 K7 b
单核/巨噬细胞被报道在异种移植物有逐渐浸润的现象，同时它们还参与迟发性异种排斥反应。研究发现单核细胞通过公认的C-凝集素（C-type lectin）与异种内皮细胞上的αGal相互作用使其粘附到异种内皮细胞层。这就导致了LFA-1等β2整联蛋白（β2 integrins）快速激活，同时增加了单核细胞对ICAM-1的亲和力。此外Jin等人证实在单核细胞上表达的Galectin-3是C-type lectin受体，能够与PAEC上的αGal结合。综合考虑，这些机制可能解释了单核细胞在异种移植物中大量富集的现象。
3 `7 v, S6 M. h; `  n& v+ cMonocytes/macrophages have also been described to progressively infiltrate xenografts and to participate in delayed xenograft rejection (DXR) . Monocytes have been shown to adhere to the xenogeneic endothelium upon interaction with αGal via a putative Ctype lectin, resulting in a rapid activation of β2 integrins (such as LFA-1) and an increase in their binding affinity to ICAM-1 [19]. Furthermore, Jin et al. confirmed that Galectin-3 expressed on monocytes is the C-type lectin receptor that binds to αGal on PAEC . Taken together, these mechanisms may explain the dramatic accumulation of monocytes in xenografts.: A# l1 Q  J- a$ ~, V) ~
, d  I0 t$ \, o# G' C' C
人巨噬细胞可能也参与了异种排斥反应。Ide等人发现在没有补体和抗体的情况下，单核细胞对缺少αGal的猪红细胞（pRBCs）仍然有吞噬和细胞毒作用，对照的自体以及同种异体的hRBCs则不会出现这种现象。这就说明排斥反应中存在着其他能被识别的异种抗原，也存在着独立于抗体/补体调节系统的调节机制。Rees等人使用枯否氏细胞的一个细胞亚群证实了这一发现。此外，Cantu等人将hCD46转基因猪的肺血管内巨噬细胞(PIMs)耗竭，然后用其肺脏进行异种移植，实验组和对照组的受者动物使用相同的免疫抑制措施进行处理，移植后实验组的存活时间比对照组长5倍，。这说明了供者的巨噬细胞在肺异种移植排斥反应中也起到了重要的作用，它形成的细胞免疫障碍可能也威胁到猪其他器官的异种移植。
0 H; Q/ W2 \* w5 _& g2 eHuman macrophages may also be involved in xenorejection. Ide et al. have shown that monocytes exert phagocytosis and cytotoxicity against porcine red blood cells (pRBCs) lacking the αGal epitope compared to autologous and allogeneic human RBCs, in the absence of Ab and complement, suggesting recognition of other xenoantigens and mechanisms independent of Ab/complement opsonization . Rees et al. confirmed this finding with a subpopulation of Kuppfer cells . Moreover, Cantu et al. showed that pulmonary xenografts from hCD46 transgenic pigs depleted of pulmonary intravascular macrophages (PIMs) survived 5 times longer than control animals under the same immunosuppressive regimen, indicating that donor macrophages also play a critical role in pulmonary xenograft rejection and may constitute a cellular immunological hurdle to the success of other porcine organ xenografts.1 M2 L5 t& n! m7 D9 `& ^6 p) N
2 p2 A1 I, M" x. R1 H
2.4针对巨噬细胞的方案4 s- Q* r; ?' A. j& M% S0 E; W4 b
2.4. Macrophage strategies3 x1 p) r# s! \
巨噬细胞表达的信号调节蛋白SIRPα是CD47的配体。CD47–SIRPα系统参与了许多细胞途径，包括负调节巨噬细胞的吞噬作用。在前期工作的基础上，Ide等人发现猪CD47（pCD47）不能诱导人巨噬细胞样细胞上的SIRPα酪氨酸磷酸化，一种可溶性的hCD47-Fc融合蛋白能够部分但显著地抑制人巨噬细胞吞噬猪细胞。此外，hCD47转染的猪细胞可以避免人巨噬细胞的吞噬。因此，CD47是巨噬细胞介导的异种排斥反应的影响因素，基因工程使猪细胞表达hCD47可能是延长异种移植存活时间的一种有效措施。1 |% g8 E) E3 O8 g6 D! s% `- e  ~
Macrophages express the ligand of CD47: signal regulatory protein (SIRP) α. The CD47–SIRPα system is involved in a variety of cellular process including the negative regulation of macrophage phagocytosis . In a follow-up to their previous report, Ide et al. (see above) demonstrated that pCD47 does not induce SIRPα tyrosine phosphorylation in a human macrophage-like cell line, and that a soluble hCD47-Fc fusionprotein could partially but significantly inhibit the phagocytosis of porcine cells by human macrophages . Furthermore, hCD47- transfected porcine cells are protected from phagocytosis by human macrophages. Thus, CD47 contributes to macrophage-mediated xenograft rejection, and genetic engineering of porcine cells to express hCD47 could represent a novel approach to prolong xenograft survival.
; R9 j- g, ]7 f/ k* O2 r' U. ~
" [$ Y2 \  u2 z  L( Y% |2.5 HMGB1 PAEC 和人T细胞
# h- k/ Q. X# Z, c4 s2.5. HMGB1 PAEC and human T cells0 f2 ~4 F0 o- _+ T
1 Y8 i& v! U7 l+ R# m: U
HMGB1最初被认为是一种核DNA结合蛋白。但是被坏死或激活的细胞释放到细胞外的HMGB1，对细胞内和细胞外有多效性。研究发现移植物中浸润的T细胞能够释放HMGB1。使用抗HMGB1抗体和可溶性的RAGE（receptor for the advanced glycation end-product）进行阻断，能显著延长了心脏移植物在鼠类模型中的存活时间，这说明了HMGB1在移植排斥反应中的作用。在异种移植的情况下，体外研究发现PAEC与T细胞而非B细胞、单核细胞、嗜中性粒细胞的直接相互作用诱导PAEC和T细胞释放HMGB1，这说明HMGB1与移植排斥反应有关联。) j% X! |8 H( V3 V+ q0 c
High mobility group box-1 (HMGB1) was originally identified as a nuclear DNA-binding protein. However, extracellular HMGB1 has pleiotropic effects both inside and outside cells upon its release from necrotic or activated cells. Graft infiltrating T cells have been shown to release HMGB1 and its blockade using anti-HMGB1 Ab and soluble receptor for the advanced glycation end-product (RAGE) significantly prolongs the survival of heart transplants in murine models, suggesting its implication in graft rejection .In the xenogeneic context, results from in vitro studies have shown that under these conditions, direct interactions between PAEC and T cells, but not B cells, monocytes and neutrophils, resulted in HMGB1 release by both PAEC and T cells , suggesting that HMGB1 is associated with graft rejection., G% U8 T& @4 v' ?+ `; W
  D# Y, j5 v  R6 [4 E

) p2 T9 z5 o* g3 F: K6 k8 P3. 获得性细胞免疫反应
6 g/ C* e+ i, V/ `, J& {- r3. The adaptative cellular immune response
, S2 |  a& k5 X  x# r1 g' y9 c0 }+ U- H$ `# K" |8 i4 P5 c
3.1 T细胞介导的反应的表征和机制5 f7 z8 D3 H# K; W8 A+ e. W
3.1. Characterization/mechanism of action of the T cell-mediated response3 X# ~7 o8 P' k5 k! D
5 N4 [! ?/ |# K
Although T cell participation in the elicited B cell Ab response through either anti αGal or non Gal Ab production has been well described, the direct involvement of T cells has not.( x4 A3 {2 g% h& w6 m
T细胞参与诱导B细胞抗体反应产生了针对αGal或非Gal的抗体，但对于T细胞直接参与的反应却知之甚少。
2 M% g* u( R( r* f0 o" H) k, _1 ]3 b9 Y. |9 ?
Davila等人通过体内实验观察灵长类动物T细胞对来自hCD46转染猪的心脏异种移植物做出的激活表现。受者的处理包括：不处理、单独使用TPC（耗竭抗αGal抗体）、联合使用TPC和利妥昔(抗CD20)。T细胞无免疫抑制处理。移植物在移植后5-7天被排斥。免疫组织学分析发现所有移植排斥反应都是由体液和细胞免疫共同参与的，浸润的细胞主要是CD4+ T细胞。在体外分析移植前后受者淋巴细胞对IFNγ激活的供者PAEC的反应性，发现存在对猪异种抗原的敏感性，同时猪特异性CD4+CD28−细胞在外周扩增表明存在着严重的抗PAEC细胞毒作用。这些说明了T细胞直接参与了心脏内皮的损伤过程。
2 x6 U- d, T. aDavila et al. examined the activation of primate T cells in response to hCD46-transfected porcine cardiac xenografts in vivo. Recipients untreated or treated with a αGal polyethylene glycol polymer (TPC, depleting anti-αGal Ab) alone or in combination with rituximab (anti- CD20), in the absence of T-cell immunosuppression, rejected their xenografts between 5 and 7 days post transplant. Immunohistology analyses indicated that all grafts exhibited a mixed humoral and cellular rejection, with infiltrates predominantly made up of CD4+ T cells. In vitro assays of pre- and post-transplant recipient lymphocyte responses to IFNγ-activated donor PAEC indicated a sensitization to pig xenoantigens and an expansion of pig-specific CD4+CD28− cells in the periphery that exhibited a strong anti-PAEC cytotoxicity, suggesting their direct involvement in heart endothelial injury.
% s) @! n$ R% n+ T  Y, s
' ~/ R, _( t4 C( n& p; n# D  N7 W因为胰岛异种移植不涉及到血管化，所以是研究T细胞参与排斥反应的合适模型。+ I' v  u: _, ?; H: r. w4 _  E6 q9 E
Kirchhof等人将成年猪胰岛（API）异种移植到无免疫抑制处理的链脲菌素（STZ）-糖尿病恒河猴上，分析胰岛门静脉的功能和免疫病理。他们发现，API在开始的72小时内发生急性细胞排斥反应，有CD4+和CD8+ T 细胞以及巨噬细胞参与。Lindeborg等人分析了对猪胰岛细胞抗原有活性的T细胞克隆的表型、免疫特异性及其体外功能，结果显示所有的克隆都是αβ CD4+ T细胞，但这些细胞对API的排斥反应在特异性和功能特性方面存在异质性。! O- y  w. E8 A) _* p* B$ n
Because islet xenografts are not vascularized, they are a useful model to observe the T cell involvement in rejection. Kirchhof et al. analyzed the function and immunopathology of intraportal islet xenografts transplanted from adult pigs (API) into streptozotocin (STZ)-diabetic rhesus macaques with no immunosuppressive regimen . They showed that API undergo acute cellular rejection in the first 72 h, involving CD4+ and CD8+ T cells and macrophages.Lindeborg et al. analyzed the phenotype of T cell clones reactive to pig islet cell antigens, their immunological specificity and their functional capacity in vitro, and showed that all clones were αβ CD4+ T cells. However, the clone response against API showed a wide heterogeneity in terms of specificity and functional characteristics .
6 T/ t9 [( ?: E) J$ ?  M' f
. i, r* q9 h- ^2 T/ z/ s根据API异种移植到非免疫抑制灵长类后产生的急性细胞排斥反应的现象，Hering等人将API异种移植到STZ-糖尿病-食蟹猴来评估由抗CD25、抗CD154抗体、他克莫司（tacrolimus）、依维莫司（everolimus）和来氟米特（leflunomide）组成的免疫抑制方案的效果。该方案使有功能API的存在时间由24延长到187天，说明这些措施抑制对供体特异细胞的攻击，抑制抗非Gal猪异种抗原IgG的产生以及随后发生的内移植性炎性细胞因子的表达和单核细胞的浸润。根据这些结果，Cardona等人将新生猪胰岛（NPI）和API移植到胰脏切除的猕猴中，阻断受者共刺激物CD28/CD154进行免疫抑制。异种移植物存活时间延长到140天，期间保持受者正常的血糖水平。该受者不依赖外界供给胰岛素，可以检测到循环水平的猪C肽，移植造成的细胞浸润程度比较小。没有使用该措施的受者在4-5天后就排斥移植物，有大量T细胞、巨噬细胞、和少数B细胞在肝脏浸润。他们还用API移植验证了这些结果，API在处理后的猕猴体内存活达70天，这表明阻断共刺激物对NPI的保护效果比对API好。  According to the description of acute cellular rejection of API transplanted into primates with no immunosuppression , Hering et al. assessed the effect of immunosuppression composed of anti-CD25 and anti-CD154 Ab, tacrolimus, everolimus and leflunomide in the context of API transplanted into streptozotocin-induced diabetic cynomolgus macaques . This regimen led to a prolonged functional API xenograft survival from 24 to up to 187 days, suggesting that this regimen inhibited priming against donor-specific cells, elicited non-Gal pig-specific IgG, and subsequent intragraft expression of proinflammatory cytokines and mononuclear cell infiltration. Based on these results, Cardona et al. showed that neonatal (NPI) and API transplanted into pancreatectomized macaques, under a costimulation (CD28/CD154) blockade-based immunosuppressive regimen, could normalize glycemia with xenograft survival of up to 140 days. The recipients became insulin independent, had detectable circulating levels of porcine C-peptide and their grafts had minimal cellular infiltrates. Non-treated animals on the other hand rejected their grafts after 4 to 5 days,with dense infiltrates of T cells, macrophages and a few B cells in the liver.The same group confirmed these results with API xenografts where survival was up to 70 days in treated macaques , suggesting that this costimulation blockade-based immunosuppression is more efficient at protecting NPI than API xenografts.% P$ `' p. a3 `; f# U) t. h+ ?
$ A) ]7 M2 _7 K
3.2 克服异种器官移植时细胞免疫排斥反应的方案
. n  c' a- e1 Q& q0 H# @3.2. Strategies to overcome the cellular immune response/rejection in xenotransplantation
5 q7 u  m+ q) e& d! c. g" }6 W
  E& k2 ]4 q. [PD-L1（Programmed death ligand 1）属于B7分子家族，在T细胞、B细胞、树突状细胞和巨噬细胞中表达。PD-1是PD-L1的生理受体，在激活后的T细胞中表达。激活PD-1信号途径有利于同种异体移植的存活，当结合共同刺激物的阻断时可以延长同种胰岛移植物的存活时间。为了评估PD-1/PD-L1途径对pig-to-primate异种移植的潜在作用，Jeon等人将克隆的猪PD-L1转染到细胞上，研究其对人CD4 T 细胞增殖的影响。体外功能分析显示，猪PD-L1能够抑制人异种CD4 T细胞的增殖并且诱导T细胞凋亡。PD-1/PDL1途径可能参与调节宿主细胞免疫反应，能够提高异种移植的存活率。5 ]8 j' {8 m8 f3 r  h6 B
Programmed death ligand 1 (PD-L1) is a B7 family molecule expressed by T, B, dendritic cells andmacrophages. Programmed death-1 (PD-1), the physiological receptor of PD-L1, is expressed by activated Tcells.Targeting of PD-1 signaling has been shown to promote allograft survival, and when combined with costimulatory blockade, induces long-term allogeneic islet graft survival. In order to evaluate the possible role of PD-1/ PD-L1 interactions in pig-to-primate xenotransplantation, Jeon et al. cloned porcine PD-L1 and characterized its effect on human CD4 T cell proliferation in response to porcine PD-L1-transfected cells. In vitro functional analyses showed that porcine PD-L1 inhibits human xenogeneic CD4 Tcell proliferation and induces Tcell apoptosis.The PD-1/PDL1 pathway may therefore play a regulatory role in the host cellular immune response and improve graft survival in xenotransplantation.+ T6 y8 h8 P8 g9 c+ P+ V7 @! E: K
3 V1 O( Q9 y$ M
为了确定调节T cells（Treg）在异种反应中是否发挥作用，Porter等人在体外评估人Treg抑制CD4+T细胞介导的抗猪异种移植反应的能力。人Treg能够抑制异种刺激CD4+CD25− T 细胞分泌细胞因子以及CD4+CD25− T细胞产生抗猪细胞的细胞毒性反应。Porter等人已经证实Treg能够促进移植物的存活。他们在体外评估了Treg在狒狒抗猪异种免疫反应中的作用。他们发现新鲜分离和扩增的狒狒Treg能够抑制异种刺激免疫反应，这说明将扩增的Treg选择性地移植到异种移植受者中可能有助于阻止细胞介导的排斥反应以及用于诱导pig-to-baboon异种移植临床前模型的免疫耐受。
- F' {: J$ Q5 m5 W" m, QTo determine whether regulatory T cells (Treg) play a role in the xenogeneic response, Porter et al. assessed the ability of human Treg to suppress CD4+ T cell-mediated anti-porcine xenoresponses in vitro . Human Treg abolished the secretion of cytokines by xenogenicstimulated CD4+CD25− T cells as well as the CD4+CD25− T-cell cytolytic response against porcine cells.Having suggested a role for Treg in promoting xenograft survival, Porter et al. assessed the effects of Treg on baboon anti-porcine xenogeneic immune responses in vitro. They showed that freshly isolated and expanded baboon Treg suppress immune responses to xenogeneic stimulation, suggesting that adoptive transfer of expanded Treg into xenograft recipients may help to prevent cell-mediated rejection and potentially induce tolerance in the pig-tobaboon preclinical model.
7 a6 u% e3 Z, o: S3 o# @4 V0 B. }* v) c) _# E' N
3.3共刺激物和粘附分子的作用特点和机制
6 }: x& Y+ N: E/ n7 J7 a- o' n3.3. Characterization/mechanism of action of costimulation/adhesion molecules* ?5 Q( ?: V5 Z% |& f) b

( G7 ^! P1 s# E. U3 C使跨物种的粘附分子和共刺激物兼容是异种环境下的关键问题。Godwin等人研究了猪ICAM-2 (intercellular adhesion molecule-2)的特点及其与人LFA-1的相互作用，发现ICAM-2在人和猪之间适度保守，单层细胞表达的猪 ICAM-2介导表达人LFA-1细胞的特异性粘附。Rogers等人通过体外功能分析发现猪CD40、CD80和CD86能独立共刺激人CD4 + T细胞，产生的增殖反应规模类似于人同源共刺激的结果。Choi等人在体外研究人T细胞上的 CD40L (hCD40L, hCD154) 和猪内皮CD40 (pCD40)之间的异种反应能否激活pECs。将pECs和人T细胞共培养，结果显示pEC被激活，即趋化因子, SLA-I、II以及pECs上的粘附分子被上调。粘附因子的上调是依靠人T细胞上hCD40L的水平。这个激活反应被证明是由NF-κB介导的。这些结果表明，hCD40L和pCD40之间的相互作用可能通过募集和激活人T细胞来加剧异种移植急性血管性排斥反应。 The compatibility of cross-species adhesion and costimulation molecules is a critical issue in the xenogeneic context. Godwin et al. characterized pig intercellular adhesion molecule-2 (ICAM-2) and its interactions with human LFA-1 and showed that ICAM-2 is moderately conserved between pigs and humans and that pig ICAM-2 expression by a cell monolayer mediates the specific binding of cells expressing human LFA-1 . Using in vitro functional assays, Rogers et al.demonstrated that porcine CD40, CD80 and CD86 are independently capable of costimulating human CD4+ T cells, leading to proliferation responses of a magnitude comparable to that observed upon human homologous costimulation . Choi et al. investigated whether the xenogeneic interaction between human CD40L (hCD40L, hCD154) on T cells and porcine endothelial CD40 (pCD40) can activate pECs in vitro . Co-culture of pECs with human T cells resulted in an up-regulation of chemokines, SLA class I and II, and adhesionmolecules inpECs, whichwas dependent on the level of hCD40L on human T cells, indicating pEC activation. This activation was shown to be mediated by NF-κB. These results suggest that interaction between hCD40L and pCD40 may contribute to acute vascular rejection of xenografts by recruiting and activating human T cells." M% b+ g+ ^, b/ Z2 [  Z  ]7 N# U
9 {+ r4 F/ ?) J+ K- a3 V: Y
3.4阻断共刺激的方案7 K. M7 Q" n% G. H7 x
3.4. Strategies of co-stimulation blockade
& d( _6 e$ {1 P8 o1 u3 E5 k) B" r5 f2 [. q6 O
Kuwaki用可溶性的共轭αGal处理狒狒来耗尽抗乳糖抗体，然后异位移植来自hDAF（human decay-accelerating factor）猪的心脏。狒狒体内的抗CD154抗体结合常规免疫抑制对于维持低抗Gal抗体水平、延缓或阻止急性体液性异种移植排斥反应是有效的。根据这些结果，Wu等人使用相同模型封闭CD40/CD154 和CD28/B7共刺激物途径（有或没有常规免疫抑制剂），发现抗CD154抗体结合手术期ATG（anti-thymocyte globulin）不能完全调节由抗Gal和非Gal引起的排斥反应，然而增加CD28/B7途径的阻断能够防止出现针对这些抗原的致敏反应。& F( K! ?7 |2 ?6 \: Y) @6 Y; O
Kuwaki showed that in baboons that are treated with soluble αGal conjugate to deplete anti-Gal Ab and then receive a heterotopic heart from human decay-accelerating factor (hDAF) donor pigs, anti-CD154 Ab in combination with conventional immunosuppression is effective at maintaining low anti-Gal Ab levels and delaying or preventing acute humoral xenograft rejection . Based on these results, in the same model Wu et al. blocked both the CD40/CD154 and CD28/B7 costimulatory pathways (with or without conventional immunosuppressive agents) and showed that anti-CD154 Ab in combination with perioperative anti-thymocyte globulin (ATG) does not completely modulate either the elicited anti-Gal or non Gal responses,whereas additional blockade of CD28/B7 appeared to prevent sensitization to these antigens.
5 G8 o' H/ c+ m2 p
3 A! K- L$ N; @! {. y% y3.5B细胞介导的反应的表征和机制：产生XNA的B细胞亚群的定义+ d4 z; d, E2 F6 P' J( g
3.5. Characterization/mechanismof action of the B cellmediated response: definition of B-cell subsets producing xenoantibodies
+ z( L+ g0 Y0 o, Z- x2 }# g8 M( \" ]1 k! H, M; K' w
为了确定产生抗Gal产物的细胞，Xu等人分析了非人灵长类（NHP）和人中天然和激活的抗Gal分泌细胞。对人类和狒狒细胞的排序结果显示，在NHP中天然抗-Gal IgM主要由脾脏B细胞产生，而诱导的抗Gal IgM 和IgG主要由B细胞和浆细胞产生。这些发现可能有助于提出新的策略，用于调控异种移植中的异种抗体分泌细胞。: u. p6 c: ^% Y- f2 ]% Y1 s% l' v9 b
Xu et al. characterized anti-Gal-secreting cells in naïve and sensitized NHP and humans to determine which cells contribute to anti-Gal production . Sorting on human and baboon cells showed that natural anti-Gal IgM in primates are produced mainly by splenic B cells whereas elicited anti-Gal IgM and IgG are secreted by both B and plasma cells. These findings may provide new strategies to target xenoreactive Ab-secreting cells in the context of xenotransplantation.
6 `% L* {4 X) ~( u4 r' }# k. _$ V, T
为了能更好地定义NHP和人上的免疫球蛋白反应和免疫球蛋白重链可变区基因（IgVH）的使用，Zahorsky-Reeves等人证实在NHP和人中少数编码针对猪肝细胞的XNA的IgVH基因是高度保守的。他们进一步分析，将hDAF转基因猪心移植给恒河猴，受者使用各种免疫抑制剂以及GAS914。他们发现了一个IgVH3–11基因在免疫抑制的受者猴中编码XNA，使得hDAF猪心脏被排斥。但是IgVH3-11在长期存活动物中的使用率低于5%，这表明IgVH3-11编码的XNA表达缺少可能有助于移植物的长期存活。此外，FischerLougheed等人分析了恒河猴产生XNA的一个B细胞亚型，将产生XNA的外周血B细胞根据结合αGal以及抗个体基因型抗体2G10分类，发现CD11b+CD5−亚型类似于鼠B1b细胞。在异种移植猪恶毒心脏和胰脏之后，这些细胞在NHP中扩增，通过IgVH3-11产生的XNA使得免疫反应更佳剧烈。这些研究表明，类似于小鼠，灵长类的B1b细胞产生XNA。对这一亚群的了解，可能有助于特异性地消耗异种反应B细胞，通过更改免疫反应来更好地控制异种排斥反应。4 `/ @7 M% P& y" c; @( I/ Y: W
In order to better define the Ig response and IgVH gene usage in primates and humans, Zahorsky-Reeves et al. demonstrated that the small number of IgVH genes encoding XNA to porcine hepatocytes in NHP and humans is highly conserved . Thus, they extended their analysis to cynomolgus monkey recipients of pig hearts transgenic for hDAF under a treatment of various immunosuppressive agents together with GAS914, a soluble αGal conjugate that depletes anti-αGal Ab. The authors identified a IgVH3–11 gene encoding XNA in immunosuppressed monkeys that rejected their transplanted hDAF porcine heart whereas the IgVH3–11 usage was less than 5% in long-term surviving animals, suggesting that the lack of expression of XNA encoded by VH3–11 may contribute to long-term graft survival .Furthermore, Fischer- Lougheed et al. characterized a B cell subset producing xenoantibodies (XNA) in rhesus monkeys and showed that XNA producing peripheral blood B cells, sorted by their binding to αGal and to the anti-idiotype Ab 2G10, exhibited a CD11b+ and CD5−.phenotype similar tomouse B1b cells .These cells,which expanded in NHP after transplantation of porcine heart and islet transplantation, increased the active immune response using the IgVH3-11 germline gene to encode XNA. These findings suggest that, like in mice, primate B1b cells produce XNA. The characterization of this subpopulation may allow for specific targeting to deplete these xenoreactive B cells and/or to alter the immune response to better control xenogeneic rejection.
; J% \7 X4 N3 [( g; D( ?# y. ^* M+ H
4结论3 P+ I5 I" b9 K$ \0 `2 f
4. Conclusion
( H" o2 k2 g3 o, V
1 O/ h4 p5 s( v, U; a虽然GalT-KO猪已经成功，但是抗猪免疫反应和抗猪细胞免疫反应仍然是一个很大的挑战。异种移植中使用的免疫抑制措施不仅要控制先天性的细胞免疫，同时还要面对获得新T、B细胞免疫。+ D. V% a2 R3 m& n3 ^6 g$ ~# d
Despite the availability of GalT-KO animals, the anti-pig immune response, and especially the anti-pig cellular response, will be a major challenge. The immunosuppression required in the context of xenotransplantation will have to control not only innate cellular immunity, but also the adaptative B and T cell responses.
4 S' a; C: c7 e0 t2 F2 r
2 I6 W) l! T( ]2 `5 D* W% j7 W, H) D- d  E1 V
个人翻译，理解不准确的语句还望大家积极指出

tpwang

  H" v7 i2 \& F3 \' U3 D

5 W1 Z& E8 _- o4 H% h0 G/ B) B回复 hyde 的帖子# O' h: I$ n2 o( F
- e5 O6 I& \6 C  t) `4 X
这个异种移植系列文章很好，学了很多东西。楼主辛苦。这篇较长较专业，超出本人的能力，仅就个别文字和表达提出些建议（蓝标），供参考。) J! j4 u8 c" k1 H  {

) |4 P2 Y" c  y# `9 U; X当前猪到灵长类动物的异种移植中存在的细胞免疫障碍（猪到灵长类动物异种移植目前所面临的细胞免疫反应问题）
' H) d( N1 n  [, e" |Current cellular immunological hurdles in pig-to-primate xenotransplantation
4 Z: @+ S8 F+ i: [; p3 x5 S! E( w* F" @& s% N2 F3 ]
摘要：
- d! [- O! F6 s9 j9 K* v6 T在这篇综述中，我们将总结这几年出版的关于猪到灵长类异种移植模型中存在的细胞免疫障碍的工作成果。这些工作成果涉及先天性细胞免疫反应和获得性细胞免疫反应以及针对它们的措施，这些措施使异种移植物免受排斥反应的损伤。* W7 g* ~8 D6 U! M, K
（本篇综述总结了近年来所发表的关于猪到灵长类异种移植模型所遇到的细胞免疫反应问题研究，包括先天性及获得性细胞免疫反应，以及克服这些反应从而预防异种移植排斥的相关措施。）7 x1 k; L2 R2 b) O% u
" f0 M" M- t& K* Q0 n
In this review, we summarize the work published over the last few years relative to cellular immunological hurdles encountered specifically in pig-to-primate xenotransplantation models. The works summarized here cover both the innate and adaptative cellular immune response as well as strategies to overcome them and consequently prevent xenograft rejection.
: Y! |- p8 r: [" q
7 n+ f% _$ h! i9 t4结论
3 W% h6 I! j- A( d; ^! Y' l% K4. Conclusion
7 T. d% l; m6 l/ X7 i" R
1 m8 \5 K9 w8 W/ k" ?虽然GalT-KO猪已经成功，但是抗猪免疫反应和抗猪细胞免疫反应仍然是一个很大的挑战。异种移植中使用的免疫抑制措施不仅要控制先天性的细胞免疫，同时还要面对获得新T、B细胞免疫。
8 f1 w. F7 n- S. J  z" A8 R（尽管已有GalT-KO猪，但抗猪免疫反应，特别是抗猪细胞免疫反应仍是一个很大的挑战……）
" I, {0 u& G& b0 A4 d: |3 {! ^" Q" z+ h% ]
Despite the availability of GalT-KO animals, the anti-pig immune response, and especially the anti-pig cellular response, will be a major challenge. The immunosuppression required in the context of xenotransplantation will have to control not only innate cellular immunity, but also the adaptative B and T cell responses.
5 L$ ]# f/ j, ]7 j! z2 w
% {7 d" f9 e# g1 C" |1 B$ a# g（感觉楼主翻译的很努力。本站大部分的网友所翻译的文章或摘要，有一个比较普遍的特点。即技术性细节一般相对比较准确，大概是因为专业熟悉的缘故。而对研究的目的、结论、意义等描述性文字往往把握不是很准，或者翻译的不是很精心。其实，这些"非技术性“内容很重要，关乎研究的价值。例如，研究出发点为何，假设是什么，结果如何解释，对本方向的启示意义如何表达等，很大程度上决定了同一组数据是否能够起到应有的科学交流的作用。从另一个角度来说，如何根据一组数据写出合理的文章，一定要对研究的框架，即出发点，假设，结果解释以及意义阐释等有全面的考虑和精细的表达。所以，读文章和翻译文章的时候一定不要只关注技术细节，忽略对上述内容的把握，其实才是需要大力提升的科学表达能力，中外文都是如此。只有很好地掌握了这些能力，才能从一个技术性实验人员或数据收集人员成为一个具有科学洞察力与表达力的科学工作者。）

shihuijunm

楼主辛苦了 ，感激！！

manybit

感激ing，谢谢

hyde

其实这也算是我的一个作业，希望能借大家的智慧完善之……8 ]1 e& u9 S, f3 s6 W2 J
谢谢各位
* F! G+ G. N. Q) {! x- l" S有些引文中的实验也不是完全明白