Lessons on Conditional Gene Targeting in Mouse Adipose Tissue

Conditional gene targeting has been extensively used for in vivo analysis of gene function in adipocyte cell biology but often with debate over the tissue specificity and the efficacy of inactivation. To directly compare the specificity and efficacy of different Cre lines in mediating adipocyte specific recombination, transgenic Cre lines driven by the adipocyte protein 2 (aP2) and adiponectin (Adipoq) gene promoters, as well as a tamoxifen-inducible Cre driven by the aP2 gene promoter (iaP2), were bred to the Rosa26R (R26R) reporter. All three Cre lines demonstrated recombination in the brown and white fat pads. Using different floxed loci, the individual Cre lines displayed a range of efficacy to Cre-mediated recombination that ranged from no observable recombination to complete recombination within the fat. The Adipoq-Cre exhibited no observable recombination in any other tissues examined, whereas both aP2-Cre lines resulted in recombination in endothelial cells of the heart and nonendothelial, nonmyocyte cells in the skeletal muscle. In addition, the aP2-Cre line can lead to germline recombination of floxed alleles in ∼2% of spermatozoa. Thus, different “adipocyte-specific” Cre lines display different degrees of efficiency and specificity, illustrating important differences that must be taken into account in their use for studying adipose biology.Adipose tissue plays an important role in metabolism through its storage and release of triglycerides, peptide hormones (adipokines) and other proteins, and in the case of brown fat, for its role in thermogenesis (1). Excess adipose tissue (i.e., obesity) is a risk factor for numerous comorbidities, including type 2 diabetes, coronary heart disease, hypertension, hepatosteatosis, and even cancer (2).Analysis of adipocyte function in vivo has benefited from the development of mouse lines that use the Cre/LoxP site-specific recombination system to inactivate specific genes in fat (3). The use of such targeting systems has allowed researchers to clarify the relative contribution of the adipose tissue in many metabolic phenotypes and circumvent lethality that might be associated with inactivation of genes at the whole-body level. Several different Cre transgenes have been used for this purpose. The most common use the promoter of the mouse adipocyte protein-2 (aP2) gene, which encodes fatty acid-binding protein-4 (Fabp4). A 5.4-kb piece of the aP2 promoter/enhancer has been shown to be sufficient to direct expression in adipocytes (4,5). At least three independent laboratories have developed aP2-Cre transgenic mice. The first aP2-Cre line was created by Kleanthis Xanthopoulos (6); subsequently, the aP2-Cre^BI line was created by Barbara Kahn (Beth Israel, Boston, MA) (7), and the aP2-Cre^SI was created by Ronald Evans (Salk Institute, San Diego, CA) (8). In addition, the aP2 promoter has been used by the Chambon laboratory (Institut de Génétique et Biologie Moléculaire et Cellulaire, Paris, France) to drive the expression of a tamoxifen-inducible Cre transgene (aP2-CreERT2), which is only able to recombine floxed alleles in the presence of 4-hydroxytamoxifen (4-OHT) (9,10).Although aP2/Fabp4 was originally identified as an adipocyte-specific protein, recent studies have shown that Fabp4 is also expressed in other cell types (11), including macrophages (12–14), the lymphatic system (15), and during embryogenesis (16). To circumvent the possible side effects of gene deletion of the aP2-Cre in tissues other than adipocytes, two laboratories have developed adiponectin-Cre transgenic mice (Adipoq-Cre), with expression of a Cre recombinase driven by the promoter/regulatory regions of the mouse adiponectin locus using a bacterial artificial chromosome (BAC) transgene (17) or by a 5.4-kB promoter fragment (18).In the current study, we have directly compared the specificity and efficacy of three mouse transgenic Cre lines—the aP2-Cre^BI, aP2-CreERT2, and Adipoq-Cre BAC transgenic mouse lines—in mediating adipocyte-specific recombination using a number of different floxed alleles as well as by breeding these mice to the LacZ-Gt(ROSA)26Sor^tm1Sor (termed R26R-lacZ) reporter mouse, in which Cre-mediated recombination irreversibly activates a lacZ reporter gene (19). We find that all of the Cre lines induce recombination in the adipose tissue. In addition, the aP2-Cre^BI and aP2-CreERT2 lines both induce recombination in the capillary endothelium in the heart and in intermyofibrillar cells in the skeletal muscle, but not in macrophages in adipose tissue. Interestingly, we find that different floxed gene loci display differential sensitivity to Cre-mediated recombination and that different adipose depots recombine to different extents. The aP2-Cre^BI can also lead to germline recombination of floxed alleles. These results illustrate the differences between “adipose-specific” Cre lines and caveats in their use that are critical for interpretation of research using these models.     

Modification and Repression of Genes Expressed in the Mammary Gland Using Gene Targeting and Other Technologies

Transgenic experiments using oocytemicro-injection methodology are often performed in orderto target expression of a foreign gene in a specifictissue or, to a lesser extent, to study the regulationof gene expression. However, the isolation ofembryonic stem cells in mice and the development ofantisense and ribozyme technologies have allowed moresubtle alterations of endogenous gene expression to be achieved. The mammary gland is one of the feworgans able to undergo several cycles of development,differentiation and apoptosis through complexmultihormonal regulation during adult life. It is thusan attractive model to assess the in vivo functionof some genes potentially involved in these mechanisms,either by silencing them or by partially repressingtheir expression. Furthermore, such alterations of gene expression have also been performed formore applied objectives such as the modification of milkcomposition for nutritional and technological purposes.This review will describe the experimental procedures used toward these aims and theresults already obtained in this field. Some potentialnew targets will be suggested.     

Preventing Renal Ischemia–Reperfusion Injury Using Small Interfering RNA by Targeting Complement 3 Gene

The complement system is one of the important mediators of renal ischemia-reperfusion injury (IRI). We hypothesized that efficient silencing of C3, which is the central component on which all complement activation pathways converge, could be achieved using small interfering RNA (siRNA), and that this would result in overall inhibition of complement activation, thereby preventing IRI in kidneys. A series of experiments was conducted, using a mouse model of IRI and vector-delivered C3-specific siRNA. We demonstrated the following: (1) renal expression of C3 increases as a result of IRI; (2) by incorporation into a pRNAT U6.1 vector, siRNA can be delivered to renal cells in vivo; (3) systemically delivered siRNA is effective in reducing the expression of C3 in an experimentally induced mouse kidney model of IRI; (4) similarly, siRNA reduces complement-mediated IRI-related effects, both in terms of renal injury (as evidenced by renal function and histopathology examination) and mouse mortality and (5) silencing the production of C3 diminishes in vivo production of TNF-alpha. This study implies that siRNA represents a novel approach to preventing IRI in kidneys and might be used in a variety of clinical settings, including transplantation and acute tubular necrosis.     

Targeting Cells Causing Split Tolerance Allows Fully Allogeneic Islet Survival With Minimal Conditioning in NOD Mixed Chimeras

Donor‐specific tolerance induced by mixed chimerism is one approach that may eliminate the need for long‐term immunosuppressive therapy, while preventing chronic rejection of an islet transplant. However, even in the presence of chimerism it is possible for certain donor tissues or cells to be rejected whereas others from the same donor are accepted (split tolerance). We previously developed a nonmyeloablative protocol that generated mixed chimerism across full major histocompatability complex plus minor mismatches in NOD (nonobese diabetic) mice, however, these chimeras demonstrated split tolerance. In this study, we used radiation chimeras and found that the radiosensitive component of NOD has a greater role in the split tolerance NOD mice develop. We then show that split tolerance is mediated primarily by preexisting NOD lymphocytes and have identified T cells, but not NK cells or B cells, as cells that both resist chimerism induction and mediate split tolerance. Finally, after recognizing the barrier that preexisting T cells impose on the generation of fully tolerant chimeras, the chimerism induction protocol was refined to include nonmyeloablative recipient NOD T cell depletion which generated long‐term mixed chimerism across fully allogeneic barriers. Furthermore, these chimeric NOD mice are immunocompetent, diabetes free and accept donor islet allografts.     

丝状真菌基因敲除技术研究进展

丝状真菌在自然界分布广泛，与人类的生产、生活密切相关。近年来，对于其基因功能的研究取得了较大的进展，一系列转化和基因操作技术已在不同的丝状真菌中得到运用。许多对于工业、农业和医药卫生具有重要意义的丝状真菌已经完成或正在进行全基因组序列测定。作者简述了丝状真菌基因敲除的历史，重点介绍了近年来基因敲除技术在丝状真菌研究中的进展，并对冀在丝状真菌基因功能研究、工业菌株改良等方面进行了展望。     

Report from the 1st International NOD Mouse T-Cell Workshop and the follow-up mini-workshop.

A workshop on autoreactive T-cell responses in NOD mice was held to optimize autoreactive T-cell detection methodologies. Using different proliferation assay protocols, 1 of the 11 participating laboratories detected spontaneous T-cell responses to GAD(524-543) and insulin(9-23) in their NOD mice. Two other laboratories were able to detect autoreactive responses when using enzyme-linked immunospot assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA) analysis of cytokines in culture supernatants, suggesting that these assays provided greater sensitivity. To address the divergent findings, a follow-up mini-workshop tested NOD mice from four different colonies side-by-side for T-cell proliferative responses to an expanded panel of autoantigens, using the protocol that had enabled detection of responses in the 1st International NOD Mouse T-Cell Workshop. Under these assay conditions, 16 of 16 NOD mice displayed proliferative responses to whole GAD65, 13 of 16 to GAD(524-543), 9 of 16 to GAD(217-236), 7 of 16 to insulin(9-23), and 5 of 16 to HSP277. Thus, spontaneous proliferative T-cell responses can be consistently detected to some beta-cell autoantigens and peptides thereof. Overall, the results suggest that more sensitive assays (e.g., ELISPOT, ELISA analysis of cytokines in supernatants, or tetramer staining) may be preferred for the detection of autoreactive T-cells.     

Gene expression profiles define a key checkpoint for type 1 diabetes in NOD mice

cDNA microarrays with >11,000 cDNA clones from an NOD spleen cDNA library were used to identify temporal gene expression changes in NOD mice (1-10 weeks), which spontaneously develop type 1 diabetes, and changes between NOD and NOD congenic mice (NOD.Idd3/Idd10 and NOD.B10Sn-H2(b)), which have near zero incidence of insulitis and diabetes. The expression profiles identified two distinct groups of mice corresponding to an immature (1-4 weeks) and mature (6-10 weeks) state. The rapid switch of gene expression occurring around 5 weeks of age defines a key immunological checkpoint. Sixty-two known genes are upregulated, and 18 are downregulated at this checkpoint in the NOD. The expression profiles are consistent with increased antibody production, antigen presentation, and cell proliferation associated with an active autoimmune response. Seven of these genes map to confirmed diabetes susceptibility regions. Of these seven, three are excellent candidate genes not previously implicated in type 1 diabetes. Ten genes are differentially expressed between the NOD and congenic NOD at the immature stage (Hspa8, Hif1a, and several involved in cellular functions), while the other 70 genes exhibit expression differences during the mature (6-10 week) stage, suggesting that the expression differences of a small number of genes before onset of insulitis determine the disease progression.     

Targeting the LRP5 Pathway Improves Bone Properties in a Mouse Model of Osteogenesis Imperfecta

The cell surface receptor low‐density lipoprotein receptor‐related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5^p.A214V) that is orthologous to a human HBM‐causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5^+/p.A214V mice to Col1a2^+/p.G610C mice, which model human type IV OI. We found that Col1a2^+/p.G610C;Lrp5^+/p.A214V offspring had significantly increased bone mass and strength compared to Col1a2^+/p.G610C;Lrp5^+/+ littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2^+/p.G610C mice with a monoclonal antibody that inhibits sclerostin activity (Scl‐Ab). We found that antibody‐treated mice had significantly increased bone mass and strength compared to vehicle‐treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. © 2014 American Society for Bone and Mineral Research.     

Unexpected Acceleration of Type 1 Diabetes by Transgenic Expression of B7-H1 in NOD Mouse Peri-Islet Glia

OBJECTIVE

Autoimmune target tissues in type 1 diabetes include pancreatic β-cells and peri-islet Schwann cells (pSC)—the latter active participants or passive bystanders in pre-diabetic autoimmune progression. To distinguish between these alternatives, we sought to suppress pSC autoimmunity by transgenic expression of the negative costimulatory molecule B7-H1 in NOD pSC.

RESEARCH DESIGN AND METHODS

A B7-H1 transgene was placed under control of the glial fibrillary acidic protein (GFAP) promoter. Transgenic and wild-type NOD mice were compared for transgene PD-1 affinities, diabetes development, insulitis, and pSC survival. Mechanistic studies included adoptive type 1 diabetes transfer, B7-H1 blockade, and T-cell autoreactivity and sublineage distribution.

RESULTS

Transgenic and endogenous B7-H1 bound PD-1 with equal affinities. Unexpectedly, the transgene generated islet-selective CD8⁺ bias with accelerated rather than suppressed diabetes progression. T-cells of diabetic transgenics transferred type 1 diabetes faster. There were no earlier pSC losses due to conceivable transgene toxicity, but transgenic pSC loss was enhanced by 8 weeks, preceded by elevated GFAP autoreactivity, with high-affinity T-cells targeting the major NOD K^d-GFAP epitope, p253–261. FoxP3⁺ regulatory T- and CD11c⁺ dendritic cell pools were unaffected.

CONCLUSIONS

In contrast with transgenic B7-H1 in NOD mouse β-cells, transgenic B7-H1 in pSC promotes rather than protects from type 1 diabetes. Here, ectopic B7-H1 enhanced the pathogenicity of effector T-cells, demonstrating that pSC can actively impact diabetes progression—likely through modification of intraislet T-cell selection. Although pSC cells emerge as a new candidate for therapeutic targets, caution is warranted with regard to the B7-H1–PD1 axis, where B7-H1 overexpression can lead to accelerated autoimmune disease.The NOD mouse is a spontaneous model of type 1 diabetes, with genetic and pathophysiological roots comparable with the human disease (1). Pancreatic islets of Langerhans are tightly enveloped by peri-islet Schwann cells (pSC) that express glial fibrillary acidic protein (GFAP), a marker of Schwann cells and astrocytes (2). During pre-diabetes progression, T-cell infiltrates accumulate at the endocrine/exocrine border, constituted by the pSC mantle, where lengthy “peri”-insulitis lasts for weeks to months in NOD mice and likely for years in humans with islet autoimmunity. Eventual breakdown of the pSC mantle initiates pathogenic islet invasion, progressive β-cell loss, insulin deficiency, and overt diabetes development. In NOD mice, CD8⁺ T-cells predominate islet attack until late in this process (3).Islet T-cell infiltrations are heterogeneous in their target autoantigen specificities for not only β-cell–selective autoantigens (e.g., insulin) but also autoantigens shared by β-cells and nervous system tissue, islet-associated autoantigens shared by pSC and β-cells (e.g., S100β) or those that are pSC specific (e.g., GFAP) (4). pSC functions and their importance in type 1 diabetes development have yet to be fully characterized. In NOD mice, pSC-specific T-cell autoreactivities are present by 5 weeks of age. GFAP target epitopes were recently mapped to residues 79–87 and 253–261 for K^d and 96–110, 116–130, and 216–230 for NOD-IA^g7, and fresh ex vivo CD8⁺ cells mediate direct lysis of primary pSC cultures from diabetic NOD mice (5).pSC cells likely have physiological functions similar to conventional Schwann cells of the peripheral nervous system, providing neurotrophic support for islet-innervating neurons as well as the neural crest-derived β-cell (2). For example, nerve growth factor, glial cell—derived neurotrophic factor, and insulin-like growth factor-1 promote β-cell survival and probably regeneration (6–8). Loss of these factors with pSC destruction may amplify β-cell stress, enhancing β-cell susceptibility to inflammatory insults (7). Anatomically, pSC provide a physical barrier to infiltrating T-cells, accumulating at the endo-exocrine islet border and impeding direct β- and T-cell contact.B7-H1, a ligand for programmed death (PD)-1, is expressed by CD4⁺ and CD8⁺ T-cells, B-cells, dendritic cells (DCs), macrophages, mast cells, and nonhemopoietic tissues (9). In nonlymphoid tissue, DC-B7-H1 supports peripheral tolerance, limiting randomly arising autoaggressive lymphocytes and their inflammatory tissue damage (10,11). In tumors, expression of B7-H1 contributes to immune evasion, inducing anergy or apoptosis of tumor-specific T-cells (12–14). Consistently with an inhibitory role, treatment of NOD mice with blocking antibodies to either PD-1 or B7-H1 accelerates diabetes (15), with analogous scenarios in autoimmune (16) and other (12,17,18) models. These systemic manipulations of the PD-1/B7-H1 axis generated the consensus view that B7-H1 ligation keeps potentially damaging autoimmune T-cells in check and serves to downregulate lymphoid effector functions (19).However, conflicting data exist. The B7-H1 pathway can promote T-cell activation and autoimmunity in certain experimental settings, including transgenic expression of B7-H1 in β-cells of C57Bl/six mice (20–22). For these exceptions, an alternative receptor for B7-H1 has been proposed but not identified to date (23,24). We nevertheless felt that the weight of evidence, specifically in NOD mice, suggested that B7-H1 might serve as a tool to selectively suppress NOD pSC autoimmunity, allowing us to learn whether and how pSC cells impact on the β-cell autoimmune progression program: transgenic expression of B7-H1 in NOD β-cells protects from type 1 diabetes (19). We here describe the effects of a pSC B7-H1 transgene. Our finding of type 1 diabetes acceleration emphasizes the complexity of this costimulatory pathway, while the selective, intraislet CD8⁺ bias of high-affinity T-cells demonstrates that pSC cells do impact the β-cell destruction program, culminating in type 1 diabetes.     

Gene delivery using human cord blood-derived CD34+cells into inflamed glomeruli in NOD/SCID mice

BACKGROUND: Bone marrow reconstitution using genetically-modified hematopoietic stem cells has been reported to confer resistance to inflammation and prevent renal injury in glomerulonephritis. Although this strategy has potentials for clinical use, taking hematopoietic stem cells from bone marrow is highly stressful for patients. In this regard, umbilical cord blood may be a useful alternative and, therefore, we focused on their suitability as a source of hematopoietic stem cells for transplantation-based therapy for glomerulonephritis. METHODS: CD34+ cells were obtained from human umbilical cord blood, retrovirally transduced with human beta-glucuronidase (HBG) gene, and transplanted into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After confirming the successful chimerism, these mice were treated with lipopolysaccharide (LPS), and local HBG expression in glomeruli was examined using immunohistochemical analysis, HBG bioassay, and Western blot analysis. RESULTS: Clonogenic assay showed that 88.4 +/- 5.9% burst-forming unit-erythroid (BFU-E), 79.7 +/- 11.4% in colony-forming unit-macrophage (CFU-M), and 81.1 +/- 14.1% in colony-forming unit-granulocyte (CFU-G), respectively, possessed the transgene after transfection, suggesting that precommited cells were susceptible to retroviral infection. Flow cytometric analysis revealed that 24.1 +/- 14.5% of bone marrow cells in these chimera mice expressed human lymphocyte antigen (HLA) 8 weeks after transplantation. Also, clonogenic assay showed that a sustained engraftment of human hematopoietic cells expressed HBG. CD14-positive cells were recruited into the glomeruli upon LPS treatment and they secreted bioactive HBG, suggesting that cord blood-derived CD34+cells may differentiate into monocyte lineage while maintaining the expression of the transgene. CONCLUSION: These data indicate that umbilical cord blood cells can be utilized as a source of hematopoietic stem cells for the transplantation-based therapy of glomerulonephritis.     

Interrogating Mouse Mammary Cancer Models: Insights from Gene Expression Profiling

Numerous mouse models for mammary cancer have been developed and characterized based upon their biological, molecular, and histopathological features. In an effort to dissect the molecular anatomy of such models and compare their gene expression profiles to those of human breast cancer, six models representing various oncogenic pathways have been investigated using cDNA microarray technology. Results of these analyses are presented and discussed in the context of technological challenges presented by analyzing data on such a large scale. Further expression profiling coupled with emerging proteomic technologies will more completely define and distinguish mouse models of mammary cancer from each other and provide a comprehensive basis for comparing such models with the human disease they are intended to represent.     

稳定整合靶向EGFL7基因的shRNA载体的胶质瘤细胞系的建立

目的构建针对EGFL7基因的特异性RNA干扰载体,建立稳定转染该干扰载体的人胶质瘤细胞系。方法根据EGFL7基因的编码序列设计并合成针对EGFL7基因的特异性shRNA,将其克隆入pSilencer3.1-H1neo载体中,重组载体经脂质体LipofectamineTM2000介导转染胶质瘤细胞株U251;转染细胞经G418筛选,采用Westernblot方法在蛋白水平检测筛选出的G418抗性的克隆细胞。结果酶切鉴定和测序证实,成功构建了靶向EGFL7基因的shRNA真核表达载体pSilencer-shEGFL7,并获得了稳定表达靶向EGFL7基因的shRNA的胶质瘤细胞株。结论 EGFL7基因特异性RNA干扰载体能够显著抑制EGFL7基因在U251细胞中的表达,这为进一步研究EGFL7在胶质瘤细胞系U251中的生物学功能和作用机制奠定了基础。     

小鼠睾丸新基因TSEG-2的克隆及表达分析

目的:利用生物信息学手段克隆小鼠睾丸特异性基因TSEG-2。方法:从二级表达序列标签(EST)数据库ZooDDD中获得小鼠正常睾丸表达的EST,通过dbEST数据库检索出与其高度同源的EST序列,构建EST叠加群,Biolign软件拼接,GeneScan软件预测contigs对应的基因组序列中的外显子、内含子;针对开放阅读框设计引物序列,采用RT-PCR从小鼠睾丸组织中克隆新基因的cDNA,分析该基因在小鼠睾丸不同发育阶段、隐睾中及各脏器中的表达,并对测序结果进行生物信息学分析。结果:成功克隆了小鼠睾丸新基因TSEG-2,全长451bp,开放阅读框为267bp,编码88个氨基酸。RT-PCR证实该基因开放阅读框正确,TSEG-2在小鼠睾丸组织中高表达,在4、9、14、18、21、38日龄和2、6月龄小鼠睾丸组织中呈现规律性表达,在隐睾组织中表达减弱,且与小鼠其他cDNA无同源性,获得GenBank登录号EU079025。功能区分析发现,小鼠TSEG-2cDNA序列定位于染色体15qE3,为可溶性非分泌型蛋白,有2个可能的蛋白激酶C(PKC)磷酸化位点,1个酪蛋白激酶Ⅱ(CK2)磷酸化位点,并可能是一种核蛋白。结论:获得小鼠睾丸特异性表达基因TSEG-2,诱导小鼠隐睾发生的17-β雌二醇可引发基因TSEG-2的下调,为进一步研究该基因的生物学功能和表达调控奠定了基础。     

Regulatory T Cells Are Critical to Tolerance Induction in Presensitized Mouse Transplant Recipients Through Targeting Memory T Cells

Memory T cells are a significant barrier to induction of transplant tolerance. However, reliable means to target alloreactive memory T cells have remained elusive. In this study, presensitization of BALB/c mice with C57BL/6 skin grafts generated a large number of OX40⁺CD44^hieffector/memory T cells and resulted in rapid rejection of donor heart allografts. Recognizing that anti‐OX40L monoclonal antibody (mAb) (α‐OX40L) monotherapy prolonged graft survival through inhibition and apoptosis of memory T cells in presensitized recipients, α‐OX40L was added to the combined treatment protocol of LF15–0195 (LF) and anti‐CD45RB (α‐CD45RB) mAb—a protocol that induced heart allograft tolerance in non‐presensitized recipients but failed to induce tolerance in presensitized recipients. Interestingly, this triple therapy restored donor‐specific heart allograft tolerance in our presensitized model that was associated with induction of tolerogenic dendritic cells and CD4⁺CD25⁺Foxp3⁺ T regulatory cells (Tregs). Of note, CD25⁺ T cell depletion in triple therapy recipients prevented establishment of allograft tolerance. In addition, adoptive transfer of donor‐primed effector/memory T cells into tolerant recipients markedly reduced levels of Tregs and broke tolerance. Our findings indicated that targeting memory T cells, by blocking OX40 costimulation in presensitized recipients was very important to expansion of Tregs, which proved critical to development of tolerance.