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In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy for the treatment of congenital stem cell disorders. Despite the purported immaturity of the fetal immune system, the clinical success of this strategy has been limited by poor engraftment of transplanted cells. The fetal host immune system is thought to be the major barrier to achieving successful IUHCTx. Since the fetal immune system is immature, however, we hypothesized that the maternal immune response may instead pose the true barrier to IUHCTx. We have demonstrated that maternal T cells traffic into the fetus after allogeneic in utero transplantation and that these lymphocytes play a critical role in limiting engraftment. Furthermore, we have shown that MHC matching the donor cells to the mother improves engraftment in the unmatched fetus. These results help renew interest in using the fetal environment to treat patients with congenital stem cell disorders.

Purpose of review Regulatory T cells (Treg) are now well established as vital participants in maintaining self-tolerance and preventing autoimmunity. Tregs have already been shown to be effective in preventing graft-versus-host disease in clinical bone marrow transplantation, and numerous animal studies have suggested a therapeutic role for Treg in solid organ transplantation. Recent advances in Treg isolation and expansion have the field poised to perform trials of therapeutic Treg infusion in solid organ transplantation worldwide. An important component of these trials will be the detection of infused cells and the assessment of Treg activity after infusion. Recent findings Several animal studies have demonstrated that infused Treg migrate to transplanted tissue in the early period after transplantation. This finding has important implications for the interpretation of biopsy results in human trials. Recent refinements in Treg identification, quantification, and functional assays will be discussed in the context of immune monitoring. Summary Understanding the migration/localization and persistence of infused Treg into transplanted tissues as well as how they impact the peripheral immune response will be critical to the interpretation of early Treg trials.

Background A recent study by Chen et al described a therapy for diabetes that involved electroporation of primary hepatocytes with human proinsulin cDNA, p3MTChins. Intrahepatic transplantation of treated hepatocytes into streptozotocin (STZ) murine and porcine models led to euglycemia, weight maintenance, and normal insulin production. We tested the repeatability of their basic experiments and transplantation technique and expanded the study to include an autoimmune model. Methods Hepatocytes were isolated from B6 mice, electroporated with p3MTChins, and glucose-challenged or were injected into hepatic or spleen parenchyma of STZ-diabetic B6 and non-obese diabetic mice. Outcomes included survival, serum glucose levels, insulin, and c-peptide release. Untransfected primary hepatocytes and mice transplanted with these cells served as controls. Results p3MTChins-hepatocytes secreted insulin during glucose challenge, but glucose levels did not change with increasing glucose concentrations. Direct hepatic injection led to high mortality rates. Mice that underwent intrasplenic injection survived for >50 days (control = 4 days) and had a mild but stable improvement in hyperglycemia. C-peptide in both mouse models was detectable but eventually declined to baseline in the non-obese diabetic mice. Conclusions Hepatocytes can be transfected with p3MTChins to produce human insulin but may lack the proper glucose-sensing or complex storage and secretion capabilities that allow for a finely tuned dynamic insulin response. Treatment is subtherapeutic, and p3MTChins-hepatocyte function may not endure in an autoimmune model. Without successful preliminary findings, cell therapy involving electroporation of p3MTChins does not appear to be practical as a therapy for diabetes and may not be a strategy to pursue at this time.

Fetal interventions to diagnose and treat congenital anomalies are growing in popularity but often lead to preterm labor. The possible contribution of the maternal adaptive immune system to postsurgical pregnancy complications has not been explored. We recently showed that fetal intervention in mice increases maternal T cell trafficking into the fetus and hypothesized that this process also may lead to increased maternal T cell recognition of the foreign conceptus and subsequent breakdown in maternal-fetal tolerance. In this study, we show that fetal intervention in mice results in accumulation of maternal T cells in the uterus and that these activated cells can produce effector cytokines. In adoptive transfer experiments, maternal T cells specific for a fetal alloantigen proliferate after fetal intervention, escape apoptosis, and become enriched compared with endogenous T cells in the uterus and uterine-draining lymph nodes. Finally, we demonstrate that such activation and accumulation can have a functional consequence: in utero transplantation of hematopoietic cells carrying the fetal alloantigen leads to enhanced demise of semi-allogeneic fetuses within a litter. We further show that maternal T cells are necessary for this phenomenon. These results suggest that fetal intervention enhances maternal T cell recognition of the fetus and that T cell activation may be a culprit in postsurgical pregnancy complications. Our results have clinical implications for understanding and preventing complications associated with fetal surgery such as preterm labor. Copyright © 2014 by The American Association of Immunologists, Inc.

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long-term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor-specific transfusion followed by cyclophosphamide treatment deleted 70-80% donor-reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 10(6) Tregs with direct donor reactivity or 25 × 10(6) polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen-specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8(+) T cells and concomitant increase in endogenous Tregs among graft-infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor-reactive T cells will be an important component of Treg-based therapies in transplantation.

Mechanisms of PDL1-Mediated Regulation of Autoimmune Diabetes Indira Guleria 1✝ , Melanie Gubbels Bupp 2✝ , Shirine Dada 1 , Brian Fife 2 , Qizhi Tang 2 , Mohammed Javeed Ansari 1 , Subbulaxmi Trikudanathan 1, Nidyanandh Vadivel 1 , Paolo Fiorina 1 , Hideo Yagita 3 , Miyuki Azuma 4 , Mark Atkinson 5 , Jeffrey A. Bluestone 2 and Mohamed H. Sayegh 1* Transplantation Research Center, Brigham and Women's Hospital and Children's Hospital Boston, Harvard Medical School UCSF Diabetes Center, University of California, San Francisco, 513 Parnassus Ave. Box 0540 - HSW Room 1114, San Francisco, CA 94143-0540 Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo 113- 8421, Japan Department of Molecular Immunology, Graduate School, Tokyo Medical & Dental University 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, JAPAN University of Florida, Box 100275 JHMHC, 1600 SW Archer Road, Gainesville, FL 32610- ✝ I.G. and M.G.B. contributed equally to this work. Running Title: PDL1 regulates autoimmune diabetes Keywords: NOD, type 1 diabetes, programmed death ligand, negative costimulatory pathways Address correspondence and reprint requests to: Mohamed H. Sayegh, M.D. Transplantation Research Center, 221 Longwood Ave, Boston, MA 02115 Phone: (617) 732-5259; FAX: (617) 732-5254 email: msayegh@rics.bwh.harvard.edu

The large mucin CD43 is actively excluded from T cell/APC interaction sites, concentrating in a membrane domain distal to the site of TCR engagement. The cytoplasmic region of CD43 was necessary and sufficient for this antipodal movement. ERM cytoskeletal adaptor proteins colocalized with CD43 in this domain. An ERM dominant-negative mutant blocked the distal accumulation of CD43 and another known ERM binding protein, Rho-GDI. Inhibition of ERM function decreased the production of IL-2 and IFNgamma, without affecting PKC(theta) focusing or CD69 upregulation. These results indicate that ERM proteins organize a complex distal to the T cell/APC interaction site and provide evidence that full T cell activation may involve removal of inhibitory proteins from the immunological synapse.

Ag-specific immune tolerance in clinical organ transplantation is currently an unrealized but critical goal of transplant biology. The specificity and avidity of multimerized MHC-peptide complexes suggests their potential ability to modulate T cell sensitization and effector functions. In this study, we examined the ability of MHC-peptide dimers to modulate T cell function both in vitro and in vivo. Soluble MHC dimers induced modulation of surface TCR expression and inhibited T cell cytolytic activity at nanomolar concentrations in vitro. Furthermore, engagement of TCR by soluble dimers resulted in phosphorylation of the TCR ζ-chain and recruitment and phosphorylation of ζ-associated protein-70 to the signaling complex, the latter of which increased upon dimer cross-linking. Significantly, Ag-specific inhibition of an alloreactive TCR-transgenic T cell population in vivo resulted in consequent outgrowth of an allogeneic tumor. The prolonged Ag-specific suppression of expansion and/or effector function of cognate T cells in vivo suggests that soluble MHC dimers may be a means of inducing sustained Ag-specific T cell unresponsiveness in vivo.

Anti-CD3 mAbs with low FcR affinity prolong graft survival in the absence of the cytokine-mediated toxicity observed with conventional anti-CD3 treatment. Previous studies have shown that FcR-nonbinding anti-CD3 mAbs suppress immune responses, at least in part, by delivering a partial signal resulting in Th1 unresponsiveness. In this study, the biochemical and functional consequences of FcR-nonbinding anti-CD3 treatment for various activated T cell populations were examined. In contrast to Th1 cells, FcR-nonbinding anti-CD3-treated Th2 cells secreted IL-4 and proliferated. Furthermore, Th2 cells cultured with the mAb were not rendered unresponsive. Mixed “Th0” populations responded to FcR-nonbinding anti-CD3 by producing IL-4, and showed a selective decrease in IL-2 production following preculture with the mAb. The stimulation of IL-4-producing cells did not reflect a more complete TCR signal, since similar defects in ζ, ZAP-70, and MAP kinase phosphorylation were observed in Th1 and Th2 cells. Despite the proximal signaling defects, FcR-nonbinding anti-CD3 induced nuclear translocation of NF-ATc. Thus, Abs that deliver partial TCR signals may promote development of a Th2 phenotype during the course of an immune response via selective effects on different Th subsets.

Dendritic cells initiate immune responses but also influence regulatory T cell activity and homeostasis. Functional outcomes of dendritic cell–T cell interactions depend on the immunological context of their encounter.

Many critical issues remain concerning how best to deploy adoptive regulatory T cell (Treg) immunotherapy to the clinic. These include a determination of their pharmacokinetic characteristics, their optimal dose, their phenotypic stability, and the best therapies with which to pair Tregs. By performing a CFSE-labeled autologous Treg pulse experiment, we determined that the accessible peripheral blood Treg pool in rhesus macaques is quite large (75±11 ×106 Tregs/kg). Pharmacokinetic analysis revealed that Tregs have two phases of elimination: an α phase, with a T1/2 in the peripheral blood of 32.4 +/− 11.3 hours and a β phase with a T1/2 of 120.4 +/− 19.7 hours. In addition to their short initial half-life, Tregs underwent rapid phenotypic shifts after infusion, with significant loss of both CD25 and FoxP3 by Day +6. While tacrolimus stabilized CD25 expression, it did not improve T1/2, nor mitigate the loss of FoxP3. In contrast, rapamycin significantly stabilized both CD25 and FoxP3, and supported an increased half-life, with an α phase of 67.7+/− 6.9 hours and a β phase of 252.1 +/− 54.9 hours. These results suggest that rapamycin may be a necessary addition to Treg immunotherapy, and that tacrolimus may be deleterious to Treg integrity post-transfer.

Regulatory T cells (Tregs) are essential to transplantation tolerance and their therapeutic efficacy is well documented in animal models. Moreover, human Tregs can be identified, isolated, and expanded in short-term ex vivo cultures so that a therapeutic product can be manufactured at relevant doses. Treg therapy is being planned at multiple transplant centers around theworld. In this article,we review topics critical to effective implementation of Treg therapy in transplantation.We will address issues such as Treg dose, antigen specificity, and adjunct therapies required for transplant tolerance induction.We will summarize technical advances in Treg manufacturing and provide guidelines for identity and purity assurance of Treg products. Clinical trial designs and Treg manufacturing plans that incorporate the most up-to-date scientific understanding in Treg biology will be essential for harnessing the tolerogenic potential of Treg therapy in transplantation. © 2013 Cold Spring Harbor Laboratory Press; all rights reserved.

In utero hematopoietic cell transplantation (IUHCTx) is a promising method to induce donor-specific tolerance but the mechanisms of antigen presentation that educate host T cells and the relative importance of deletion vs regulation in this setting are unknown. We studied the roles of direct and indirect antigen presentation (mediated by donor- and host-derived antigen-presenting cells [APCs], respectively) in a mouse model of IUHCTx. We found that IUHCTx leads to precocious maturation of neonatal host dendritic cells (DCs) and that there is early differentiation of donor-derived DCs, even after transplantation of a stem cell source without mature APCs. We next performed allogeneic IUHCTx into donor-specific T-cell receptor transgenic mice and confirmed that both direct and indirect antigen presentation lead to clonal deletion of effector T cells in chimeras. Deletion did not persist when chimerism was lost. Importantly, although the percentage of regulatory T cells (Tregs) after IUHCTx increased, there was no expansion in Treg numbers. In wild-type mice, there was a similar deletion of effector cells without expansion of donor-specific Tregs. Thus, tolerance induction after IUHCTx depends on both direct and indirect antigen presentation and is secondary to thymic deletion, without de novo Treg induction.

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model. © Copyright 2013 The American Society of Transplantation and the American Society of Transplant Surgeons.

Apolipoprotein (apo) E can suppress atherosclerosis by regulating myeloid cell proliferation and monocyte activation. However, little is known about its capacity to regulate adaptive immunity. To explore this question, we studied our recently described HypoE mice deficient in the LDL receptor (Apoeh/hLdlr−/− mice) that spontaneously develop a hyperlipidemia similar to that of Apoe-/-Ldlr-/- mice when fed a chow diet, but accumulate apoE in plasma and display reduced atherosclerosis. Twenty-week old Apoeh/hLdlr-/- mice (n=10) displayed smaller spleen and lymph nodes that contained fewer leukocytes (p

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.

Herpes simplex virus type 1 (HSV-1) corneal infection in immunologically normal mice results in a transient epithelial lesion followed in about 2 weeks by a potentially blinding inflammatory response in the corneal stroma, and a mild blepharitis. Similarly infected T cell-deficient mice do not develop corneal stromal inflammation, but exhibit severe periocular skin disease and succumb to viral encephalitis. The role of certain adhesion molecules in both T cell activation, and in the extravasation of inflammatory cells from the blood into inflammatory sites is now being established. These studies investigated the involvement of the adhesion pair LFA-1/ICAM-1 in the disease that results from HSV-1 corneal infection in mice. Treatment of mice with mAb to LFA-1 beginning 1 day before HSV-1 corneal infection resulted in a delay in the onset of stromal inflammation, but ultimately stromal inflammation developed to a normal extent. This treatment also caused a significant exacerbation of periocular skin disease, but did not render mice susceptible to encephalitis. Treatment with mAb to ICAM-1 beginning 1 day before HSV-1 corneal infection caused an acceleration of both stromal inflammation and periocular skin disease, and rendered mice uniformly susceptible to lethal encephalitis. Treatment with either mAb beginning 6 days after HSV-1 corneal infection did not significantly affect the clinical course of herpetic disease. Our findings suggest that LFA-1 may play a role in the early phase of corneal stromal inflammation following HSV-1 corneal infection. Both LFA-1 and ICAM-1 appear to be important for protection of the skin from HSV-1 infection.(ABSTRACT TRUNCATED AT 250 WORDS)

This study examined the effect of thymoglobulin induction therapy on leukocyte population dynamics in kidney transplant patients. Patients receiving standard immunosuppression were compared with those who received additional thymoglobulin at the time of kidney transplantation. Thymoglobulin induction led to an immediate and significant decrease of all T cells and NK cells, but not B cells or monocytes. CD8(+) T cells recovered to near pretransplant level by 4 weeks post-transplant. CD4(+) T cells remained at less than 30% of pretransplant level for the entire study period of 78 weeks. Both CD4(+) and CD8(+) T cells showed reduced cytokine production after recovery. Deletion of CD4(+) FOXP3(+) HELIOS(+) regulatory T cells (Tregs) was less profound than that of CD4(+) FOXP3(-) cells, thus the relative percentage of Tregs elevated significantly when compared with pretransplant levels in thymoglobulin-treated patients. In contrast, the percentages of Tregs and their expression of FOXP3 in the standard immunosuppression group decreased steadily and by 12 weeks after transplant the average percentage of Tregs was 56% of the pretransplant level. Thus, thymoglobulin-induced deletion of T cells led to significant and long-lasting alterations of the T-cell compartment characterized by a preservation of Tregs and long-lasting reduction in CD4(+) , and potentially pathogenic, T cells.

The presence of maternal cells in offspring may promote tolerance to noninherited maternal antigens (NIMAs). Children with biliary atresia (BA) have increased maternal cells in their livers, which may impact tolerance. We hypothesized that patients with BA would have improved outcomes when receiving a maternal liver. We reviewed all pediatric liver transplants recorded in the SRTR database from 1996 to 2010 and compared BA and non-BA recipients of maternal livers with recipients of paternal livers for the incidences of graft failure and retransplantation. Rejection episodes after parental liver transplantation were examined for patients transplanted at our institution. BA patients receiving a maternal graft had lower rates of graft failure compared to those receiving a paternal graft (3.7% vs. 10.5%, p = 0.02) and, consequently, fewer episodes of retransplantation (2.7% vs. 7.5%, p = 0.04). These differences were not seen among non-BA patients or among BA patients who received female deceased donor grafts. In patients transplanted at our institution, paternal liver transplantation was associated with an increased incidence of refractory rejection compared to maternal liver transplantation only in BA. Our data support the concept that maternal cells in BA recipients promote tolerance to NIMAs and may be important in counseling BA patients who require liver transplantation.

Regulatory T cells (Tregs) are long-lived cells that suppress immune responses in vivo in a dominant and antigen-specific manner. Therefore, therapeutic application of Tregs to control unwanted immune responses is an active area of investigation. Tregs can confer long-term protection against auto-inflammatory diseases in mouse models. They have also been shown to be effective in suppressing alloimmunity in models of graft-versus-host disease and organ transplantation. Building on extensive research in Treg biology and preclinical testing of therapeutic efficacy over the past decade, we are now at the point of evaluating the safety and efficacy of Treg therapy in humans. This review focuses on developing therapy for transplantation using CD4(+)Foxp3(+) Tregs, with an emphasis on the studies that have informed clinical approaches that aim to maximize the benefits while overcoming the challenges and risks of Treg cell therapy.

Lyn kinase deficient mice represent a well established genetic model of autoimmune/autoinflammatory disease that resembles systemic lupus erythematosus. We report that IL-10 plays a crucial immunosuppressive role in this model, modulating the inflammatory component of the disease caused by myeloid and T-cell activation. Double-mutant lyn −/− IL-10 −/− mice manifested severe splenomegaly and lymphadenopathy, dramatically increased proinflammatory cytokine production, and severe tissue inflammation. Single-mutant lyn −/− mice showed expansion of IL-10–producing B cells. Interestingly, WT B cells adoptively transferred into lyn −/− mice showed increased differentiation into IL-10–producing B cells that assumed a similar phenotype to endogenous lyn −/− IL-10–producing B cells, suggesting that the inflammatory environment present in lyn −/− mice induces IL-10–producing B-cell differentiation. B cells, but not T or myeloid cells, were the critical source of IL-10 able to reduce inflammation and autoimmunity in double mutant lyn −/− IL-10 −/− mice. IL-10 secretion by B cells was also crucial to sustain transcription factor Forkhead Box P3 (Foxp3) expression in regulatory T cells during disease development. These data reveal a dominant immunosuppressive function of B-cell–derived IL-10 in the Lyn-deficient model of autoimmunity, extending our current understanding of the role of IL-10 and IL-10–producing B cells in systemic lupus erythematosus.

Background For the clinical applicability of regulatory T cells (Tregs) in transplantation, it is critical to determine if donor antigen specificity is required for their immunosuppressive function. We developed an allospecific CD4 + T cell receptor transgenic (TCR-tg) mouse as a source for large numbers of Tregs with defined allospecificity and tested whether they are more effective than polyclonal Tregs at suppressing allograft rejection. Materials and Methods CD4 + CD25 + CD62L hi T cells were sorted from the spleen and peripheral lymph nodes of wild-type (WT-Tregs) and TCR-tg (Allo-Tregs) mice, and expanded using IL-2 and anti-CD3/anti-CD28 conjugated magnetic beads. Tregs were tested for their ability to suppress the proliferation and cytokine production of alloreactive CD4 + CD25 − T cells in mixed leukocyte assays. Syngeneic WT hosts were adoptively transferred 5 × 10 6 Tregs and transplanted with allogeneic hearts. Results Using anti-CD3/anti-CD28 conjugated beads, Tregs were expanded in vitro 100-fold and maintained their suppressor phenotype and function. Allo-Tregs were 6–8 times more potent on a cell-for-cell basis than WT-Tregs in suppressing allospecific proliferation in vitro . Allo-Tregs were unable to suppress in the absence of allo-antigen. Adoptive transfer of expanded Allo-Tregs into WT recipients prolonged the graft survival in a F1 heart transplant model compared with WT-Treg or no treatment [20.0 ± 4.4 d ( n = 6) versus 10.4 ± 1.2 ( n = 8) and 9.7 ± 1.6 d ( n = 6)]. Conclusions Unlike polyclonal Tregs, allospecific Tregs are able to prolong allograft survival. However, large numbers of Allo-Tregs were unable to induce tolerance, suggesting that Treg therapy in immunocompetent recipients will require conditioning and/or additional immunomodulation for the induction of tolerance.

The remarkable strides made by stem cell biologists and tissue engineers have brought us ever so close to the promised land of having unlimited supplies of cells, tissues, and even organs to cure end-stage organ failure and reverse the course of degenerative diseases. Will our immune system perceive these engineered cells as foreign and target them for destruction as it does for conventional transplants? If so, how do we manage the immune response to prevent rejection, or better yet, to teach the immune system to accept the transplanted stem cells as self? In this ­chapter, we review how the immune system recognizes transplant antigens and analyze current data on immunogenicity of the various types of stem cells. We summarize current strategies for controlling transplant rejection and speculate on future directions in inducing transplant tolerance with the exciting possibilities of using stem cells to reeducate the immune system.

International audience; Regulatory T cells (T reg cells) play a major role in controlling the pathogenic autoimmune process in type 1 diabetes (T1D). Interleukin 2 (IL-2), a cytokine which promotes T reg cell survival and function, may thus have therapeutic efficacy in T1D. We show that 5 d of low-dose IL-2 administration starting at the time of T1D onset can reverse established disease in NOD (nonobese diabetic) mice, with long-lasting effects. Low-dose IL-2 increases the number of T reg cells in the pancreas and induces expression of T reg cell-associated proteins including Foxp3, CD25, CTLA-4, ICOS (inducible T cell costimulator), and GITR (glucocorticoid-induced TNF receptor) in these cells. Treatment also suppresses interferon gamma production by pancreas-infiltrating T cells. Transcriptome analyses show that low-dose IL-2 exerts much greater influence on gene expression of T reg cells than effector T cells (T eff cells), suggesting that nonspecific activation of pathogenic T eff cells is less likely. We provide the first preclinical data showing that low-dose IL-2 can reverse established T1D, suggesting that this treatment merits evaluation in patients with T1D.

Increasing research from preclinical and clinical studies is demonstrating that inflammation with its myriad of heterogeneous mediators and cellular effectors plays a role in the malignant initiation of epithelial cells. Pre-existing inflammation may exist prior to the development of cancer, or on the other hand it may be that oncogenic changes may lead to a certain inflammatory microenvironment that in turn will promote the development of a tumor. Examples of how inflammation may lead to cancer formation include enhanced proliferation of initiated cells, through resistance to apoptosis, induction of genomic instability, alterations in epigenetic events and subsequent inappropriate gene expression, abnormal tumor neovascularization or angiogenesis, and the promotion of metastasis among others.

Programmed death 1 (PD-1) is an inhibitory molecule expressed on activated T cells; however, the biological context in which PD-1 controls T cell tolerance remains unclear. Using two-photon laser-scanning microscopy, we show here that unlike naive or activated islet antigen-specific T cells, tolerized islet antigen-specific T cells moved freely and did not swarm around antigen-bearing dendritic cells (DCs) in pancreatic lymph nodes. Inhibition of T cell antigen receptor (TCR)-driven stop signals depended on continued interactions between PD-1 and its ligand, PD-L1, as antibody blockade of PD-1 or PD-L1 resulted in lower T cell motility, enhanced T cell-DC contacts and caused autoimmune diabetes. Blockade of the immunomodulatory receptor CTLA-4 did not alter T cell motility or abrogate tolerance. Thus, PD-1-PD-L1 interactions maintain peripheral tolerance by mechanisms fundamentally distinct from those of CTLA-4.

The relative contributions of the direct and indirect pathways in alloimmune responses have not been fully elucidated. We report a novel murine TCR transgenic system that can simultaneously track the CD4-direct (CD4-d), CD4-indirect (CD4-i), and CD8-direct (CD8-d) pathways after transplantation. Using this system, we have observed a profoundly greater proliferation of CD4-i T cells relative to CD4-d and CD8-d T cells after transplantation. Furthermore, a much larger proportion of CD4-i T cells attain an effector phenotype. We also analyzed endogenous, wild-type T cells using enzyme-linked immunospot analysis. In naive mice, T cells with indirect reactivity were undetectable, but T cells with direct reactivity were abundant. However, 10 days after skin or heterotopic heart transplantation, CD4-i T cells comprised approximately 10% of the CD4+ response. Consistent with increased priming of the CD4-i pathway, we observed that the CD4-i T cells were further enriched in the effector cells migrating to the allograft and in the memory-like T cells persisting after rejection. Thus, priming of the CD4-i pathway is favored after transplantation, allowing a rare population to rapidly become a major component of the CD4+ T cell response in acute allograft rejection. The generalizability of this observation to other models remains to be determined.

CD4+CD25+ T cells regulate a variety of autoimmune and alloimmune responses including the development of autoimmune diabetes in non-obese diabetic (NOD) mice. We have examined the role of CD28/CTLA4/B7 interactions in the expansion and survival of CD4+CD25+ regulatory T cells (T(reg)) in this setting. CD28/ B7 interactions are essential in the development of T(reg) in the thymus and for their survival in the periphery. The CD28-mediated homeostasis of these cells is independent of Il2, OX40, CD40L, and survival factor Bcl-XI. In addition, analysis of T(reg) from CTLA4-deficient mice suggests that CTLA4 expression is not required for their development or function. However, non-activating anti-CTLA4 antibodies blocked the suppressor activity of regulatory cells in vitro. Thus, clinical application of co-stimulatory blockade using agents such as CTLA4Ig in the treatment of autoimmune disease may result in complicated outcomes.

We appreciate the opportunity to contribute to the interesting discussion initiated by the letter to the editor by Lazarski et al. In their letter, the authors raise the possibility that reduced CD25 expression on T regulatory (Treg) cells is a common feature of inflammation, not a specific autoimmune defect (Lazarski et al., 2008). This hypothesis has critical implications for the basis of CD25 regulation and potential therapeutic use of IL-2 agonists in the clinical setting.

SummaryThe dynamics of CD4+ effector T cells (Teff cells) and CD4+Foxp3+ regulatory T cells (Treg cells) during diabetes progression in nonobese diabetic mice was investigated to determine whether an imbalance of Treg cells and Teff cells contributes to the development of type 1 diabetes. Our results demonstrated a progressive decrease in the Treg cell:Teff cell ratio in inflamed islets but not in pancreatic lymph nodes. Intra-islet Treg cells expressed reduced amounts of CD25 and Bcl-2, suggesting that their decline was due to increased apoptosis. Additionally, administration of low-dose interleukin-2 (IL-2) promoted Treg cell survival and protected mice from developing diabetes. Together, these results suggest intra-islet Treg cell dysfunction secondary to defective IL-2 production is a root cause of the progressive breakdown of self-tolerance and the development of diabetes in nonobese diabetic mice.

The involvement of CD4(+)CD25(+) regulatory T cells (Treg) in general immune homeostasis and protection from autoimmune syndromes is now well established. Similarly, there has been increasing evidence for Treg involvement in allograft rejection and current immunotherapies. However, despite significant advances in understanding the development, function, and therapeutic efficacy of Treg in certain well-defined rodent models, the relevance of Treg to clinical transplantation remains unclear. In this review, we summarize our current understanding of the role of Treg in immunity and organ transplantation in experimental and clinical settings. In addition, we review advances in using Treg as a form of immune therapy. The goal is to highlight the complexities and opportunities in the field and to provide evidence to support the use of antigen-specific Tregs in the context of transplantation to facilitate a robust and selective state of immune tolerance.

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.

This study is aimed at characterizing Treg dynamics at the site of chronic inflammation in the islets of NOD mice. Tregs expressing CD103 were present primarily in the inflamed islets and low in the pancreatic LN and spleen. Both CD103+ and CD103- Tregs from inflamed NOD islets could prevent diabetes when transferred to Treg-deficient CD28KO mice. CD103 was selectively induced and maintained in inflamed islets after transfer. However, fewer CD103+ Tregs were recovered after transfer compared with CD103- Tregs, suggesting that CD103+ Tregs have a survival defect. Indeed, CD103+ Tregs had lower Bcl2 expression despite higher CD25 expression and higher responsiveness to IL-2. CD103+ islet Tregs have reduced TCRb repertoire diversity and higher BrdU incorporation, indicating they clonally expand in the islets. Lastly, in NOD mice expressing a TCR signaling reporter, the Nur77-GFP transgene, CD103+ islet Tregs were found to be uniformly Nur77hi suggesting they were more recently activated by antigens. TCRb sequences used by high frequency clones of CD103+ Tregs could be found in Nur77hiCD103- but not Nur77loCD103- Tregs, consistent with sequential TCR activation and CD103 expression. Together, our results demonstrate that chronically inflamed islets in NOD mice contain functional Tregs, but fail to maintain the highly antigen-specific activated subset. Maintenance of this subset may have important implications for the treatment of chronic inflammatory disease.

The past decade has seen a significant increase in the number of potentially tolerogenic therapies for treatment of new-onset diabetes. However, most treatments are antigen nonspecific, and the mechanism for the maintenance of long-term tolerance remains unclear. In this study, we developed an antigen-specific therapy, insulin-coupled antigen-presenting cells, to treat diabetes in nonobese diabetic mice after disease onset. Using this approach, we demonstrate disease remission, inhibition of pathogenic T cell proliferation, decreased cytokine production, and induction of anergy. Moreover, we show that robust long-term tolerance depends on the programmed death 1 (PD-1)–programmed death ligand (PD-L)1 pathway, not the distinct cytotoxic T lymphocyte–associated antigen 4 pathway. Anti–PD-1 and anti–PD-L1, but not anti–PD-L2, reversed tolerance weeks after tolerogenic therapy by promoting antigen-specific T cell proliferation and inflammatory cytokine production directly in infiltrated tissues. PD-1–PD-L1 blockade did not limit T regulatory cell activity, suggesting direct effects on pathogenic T cells. Finally, we describe a critical role for PD-1–PD-L1 in another powerful immunotherapy model using anti-CD3, suggesting that PD-1–PD-L1 interactions form part of a common pathway to selectively maintain tolerance within the target tissues.

Any scientist opening up an immunology journal today will observe immediately that suppressor T cells, renamed 'regulatory T cells' (Tregs) have become a central concept in the immunology lexicon. Hundreds of Treg publications over the past few years have validated the existence of this unique T cell lineage armed with an ability to regulate autoimmunity. The CD4(+)CD25(+)Foxp3(+) Treg subset develops in the thymus, can be induced in the periphery during the course of normal immune responses and utilizes a T cell repertoire skewed towards autoantigens. Despite these advances, however, there is still controversy over their mechanism of action. This confusion stems from the differences observed in in vitro versus in vivo studies. Recent in vivo analyses support a model in which Tregs directly or indirectly alter the activation and differentiation of pathogenic T cells through an effect on antigen presenting cells.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease.

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.

CD4+CD25+ T cells regulate a variety of autoimmune and alloimmune responses including the development of autoimmune diabetes in non-obese diabetic (NOD) mice. We have examined the role of CD28/CTLA4/B7 interactions in the expansion and survival of CD4+CD25+ regulatory T cells (T(reg)) in this setting. CD28/ B7 interactions are essential in the development of T(reg) in the thymus and for their survival in the periphery. The CD28-mediated homeostasis of these cells is independent of Il2, OX40, CD40L, and survival factor Bcl-XI. In addition, analysis of T(reg) from CTLA4-deficient mice suggests that CTLA4 expression is not required for their development or function. However, non-activating anti-CTLA4 antibodies blocked the suppressor activity of regulatory cells in vitro. Thus, clinical application of co-stimulatory blockade using agents such as CTLA4Ig in the treatment of autoimmune disease may result in complicated outcomes.

The relative importance of the cytokine milieu versus cytolytic T lymphocyte-associated antigen 4 (CTLA-4) and T cell receptor signal strength on T cell differentiation remains unclear. Here we have generated mice deficient for signal transducer and activator of transcription 6 (STAT6) and CTLA-4 to determine the role of CTLA-4 in cytokine-driven T cell differentiation. CTLA-4-deficient T cells bypass the need for STAT6 in the differentiation of T helper type 2 (T(H)2) cells. T(H)2 differentiation of cells deficient for both STAT6 and CTLA-4 is accompanied by induction of GATA-3 and the migration of T(H)2 cells to peripheral tissues. CTLA-4 deficiency also affects the balance of the nuclear factors NFATc1 and NFATc2, and enhances activation of NF-kappaB. These results suggest that CTLA-4 has a critical role in T cell differentiation and that STAT6-dependent T(H)2 lineage commitment and stabilization can be bypassed by increasing the strength of signaling through the T cell receptor.

FcR nonbinding anti-CD3ε mAbs elicit partial TCR signaling that leads to T cell unresponsiveness and tolerance in vivo. In this study, the membrane-proximal events that promote T cell inactivation by FcR nonbinding anti-CD3 mAbs were examined. In the context of FcR nonbinding anti-CD3, TCR complexes did not aggregate and failed to translocate into glycolipid-enriched membrane microdomains. Furthermore, FcR nonbinding anti-CD3 mAbs induced tyrosine phosphorylation of the Fyn substrate Cbl, but not the ZAP-70 substrate linker for activation of T cells. Overexpression of Fyn, but not Lck, restored the mitogenicity of FcR nonbinding anti-CD3 in primary T cells. Taken together, these results suggest that Fyn mediates the partial signaling induced by TCR antagonists.

In order to ensure a standardized and consistent Drug Product freezing and to minimize batch-to-batch differences in cell recovery and viability postthaw, TxCell scientists tested the CoolCell container, a passive freezing device, as an alternative to the classical controlled rate freezer. Results showed that by using a CoolCell freezing device, Ag-Tregs can be successfully cryopreserved and recovered in a standardized way acceptable to the processing and manufacturing of cell therapies. TxCell now intends to use the CoolCell device in its phase IIb clinical study with its lead AgTreg cell product candidate, Ovasave . Use of the CoolCell passive freezing device for cell therapy manufacturing of Ag-Treg represents a new standardized method of cell therapy product cryopreservation. The ability to develop and store functional Treg in a cost-effective and reproducible manner is an important milestone in the ultimate use of these cells in the clinic.

In this report the use of surface-linkage to expand the potential experimental and therapeutic applications of single chain antibody (scFv) constructs is reviewed. A strategy for the generation and functional characterization of surface-linked scFvs that bind selectively to the T-cell proteins CD3epsilon, CD28, and CD152 (CTLA-4) is described in detail. Experimental examples are provided of the use of these constructs to study the positive and negative regulation of T-cell activation and to manipulate the in vivo immunogenicity of tumor cells. In addition, a novel system for Simultaneous T-cell Activation and Retroviral Transduction (START) is described in which retroviral packaging cells are rendered mitogenic for T lymphocytes by combined expression of surface-linked scFvs. Finally, the use of random mutagenesis and yeast surface display to increase the affinity and functional efficacy of scFv constructs is demonstrated.