CD28-B7 Interaction Research Articles

B7-CD28 co-stimulation modulates central tolerance via thymic clonal deletion and Treg generation through distinct mechanisms

The molecular and cellular mechanisms mediating thymic central tolerance and prevention of autoimmunity are not fully understood. Here we show that B7-CD28 co-stimulation and B7 expression by specific antigen-presenting cell (APC) types are required for clonal deletion and for regulatory T (Treg) cell generation from endogenous tissue-restricted antigen (TRA)-specific thymocytes. While B7-CD28 interaction is required for both clonal deletion and Treg induction, these two processes differ in their CD28 signaling requirements and in their dependence on B7-expressing dendritic cells, B cells, and thymic epithelial cells. Meanwhile, defective thymic clonal deletion due to altered B7-CD28 signaling results in the accumulation of mature, peripheral TRA-specific T cells capable of mediating destructive autoimmunity. Our findings thus reveal a function of B7-CD28 co-stimulation in shaping the T cell repertoire and limiting autoimmunity through both thymic clonal deletion and Treg cell generation.

Revisiting the role of B7-CD28 co-stimulation in thymic negative selection: B7-CD28 co-stimulation enforces central tolerance by clonal deletion and Treg generation as independent cellular processes

Abstract Self-Ag-reactive T cells are eliminated from the conventional mature T cell repertoire by clonal deletion and/or fate diversion to the Treg during thymocyte development as a consequence of strong recognition of self-peptide-MHC complex by the TCR, a process termed negative selection. However, the role of the B7-CD28 costimulatory pathway in this process has not been fully elucidated. We analyzed the role of B7-CD28 interaction in thymic negative selection using pMHCII tetramer binding to directly measure the endogenous T cell repertoire specific for distinct self-Ags. Ablation of expression of a self-Ag or of the Aire gene resulted in increase in the number of self-peptide-specific CD4SP thymocytes, consistent with the physiologic process of ongoing negative selection. We found that absence of B7 also resulted in an increase in numbers of conventional CD4SP (non-Treg) thymocytes specific for some but not all self-Ags. Observed increases in conventional CD4SP were far in excess of the concomitant decrease in the number of Treg that occurred in the absence of B7. In addition, the identity of B7-expressing cell populations required for clonal deletion was distinct from the requirements for Treg generation. Thus, the increase of CD4SP cell number specific for self-Ag was largely due to defect in B7 dependent clonal deletion, but not simply conversion of “want-to-be Treg” to conventional CD4SP cells. We propose that 1) thymic clonal deletion occurs through B7-CD28 dependent and independent pathways; 2) B7-CD28 dependent negative selection is mediated by clonal deletion and/or fate diversion to the thymic Treg lineage; and 3) B7-CD28 dependent clonal deletion is a largely independent cellular process from B7-CD28 dependent Treg generation.

Abstract IA08: Mechanisms and signaling pathways involved in regulation of activating and inhibitory T cell responses

Abstract IA08: Mechanisms and signaling pathways involved in regulation of activating and inhibitory T cell responses

Quantification of CD4(+) T Cell Alloreactivity and Its Control by Regulatory T Cells Using Time-Lapse Microscopy and Immune Synapse Detection.

Assays designed to select transplant recipients for immunosuppression withdrawal have met with limited success, perhaps because they measure events downstream of T cell–alloantigen interactions. Using in vitro time‐lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD4+ T cells forming prolonged interactions with donor dendritic cells. By blocking CD4–MHC class II and CD28–B7 interactions, we defined immunologically relevant interactions as those ≥500 s. Using this threshold, T cell–dendritic cell (T‐DC) interactions were examined in rejection, tolerance and T cell control mediated by regulatory T cells. The frequency of T‐DC contacts ≥500 s increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. Regulatory T cells reduced prolonged T‐DC contacts. Importantly, this effect was replicated with human polyclonally expanded naturally occurring regulatory T cells, which we have previously shown can control rejection of human tissues in humanized mouse models. Finally, in a proof‐of‐concept translational context, we were able to visualize differential allogeneic immune synapse formation in polyclonal CD4+ T cells using high‐throughput imaging flow cytometry.

Editorial: Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance.

Editorial: Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance.

Abatacept decreases disease activity in a absence of CD4(+) T cells in a collagen-induced arthritis model.

IntroductionAbatacept is a fusion protein of human cytotoxic T-lymphocyte–associated protein (CTLA)-4 and the Fc portion of human immunoglobulin G1 (IgG1). It is believed to be effective in the treatment of rheumatoid arthritis by inhibiting costimulation of T cells via blocking CD28–B7 interactions as CTLA-4 binds to both B7.1 (CD80) and B7.2 (CD86). However, the interaction of CD28 with B7 molecules is crucial for activation of naive cells, whereas it is unclear whether the action of already activated CD4+ T cells, which are readily present in established disease, also depends on this interaction. The aim of this study was to determine whether the mode of action of abatacept depends solely on its ability to halt T cell activation in established disease.MethodsArthritis was induced in thymectomized male DBA/1 mice by immunisation with bovine collagen type II. The mice were subsequently depleted for CD4+ T cells. Abatacept or control treatment was started when 80 % of the mice showed signs of arthritis. Arthritis severity was monitored by clinical scoring of the paws, and anti-collagen antibody levels over time were determined by enzyme-linked immunosorbent assay.ResultsTreatment with abatacept in the absence of CD4+ T cells resulted in lower disease activity. This was associated with decreasing levels of collagen-specific IgG1 and IgG2a antibodies, whereas the antibody levels in control or CD4+ T cell–depleted mice increased over time.ConclusionsThese results show that abatacept decreased disease activity in the absence of CD4+ T cells, indicating that the mode of action of abatacept in established arthritis does not depend entirely on its effects on CD4+ T cell activation.

0415 : CD28 deletion improves obesity-induced liver steatosis but increases adiposity in mice

Lymphocytes play a critical role in visceral adipose tissue inflammation. The CD28 costimulatory molecule is required for lymphocyte activation and for the development of a functional regulatory T cells (Treg) compartment; however its role during obesity is unknown. During diet-induced obesity, we investigated the effects of selective interference with CD28 signaling using knock-out mice (Cd28KO) and a CTLA4-Ig fusion protein inhibiting CD28-B7 interactions. Cd28 deficiency decreased pathogenic T-cells and Treg content within adipose tissue without changing macrophages number. Cd28KO epididymal but not subcutaneous fat was characterized by enlarged adipocytes, reduced levels of inflammatory cytokines and increased Glut4, adiponectin and lipogenic enzymes mRNA levels. This was associated with reduced inflammation, fat accumulation and enhanced glucose metabolism in liver. Weight gain and fasting glucose tolerance were not affected. CTLA4-Ig injections reduced the number T-cells in epididymal adipose tissue but not inflammatory cytokines levels and failed to improve liver fat accumulation. Deletion of CD28 creates a new pro/anti-inflammatory balance in epididymal adipose tissue and liver and exerts a protective effect against hepatic steatosis.

CD28 deletion improves obesity-induced liver steatosis but increases adiposity in mice.

Lymphocytes have a critical role in visceral adipose tissue (AT) inflammation. The CD28 costimulatory molecule is required for lymphocyte activation and for the development of a functional regulatory T cells (Tregs) compartment; however, its role during obesity is unknown. During diet-induced obesity, we investigated the effects of selective interference with CD28 signaling using knockout mice (Cd28KO) and a CTLA4-Ig fusion protein inhibiting CD28-B7 interactions. Cd28 deficiency decreased pathogenic T cells and Treg content within AT without changing the macrophages number. Cd28KO epididymal but not subcutaneous fat was characterized by enlarged adipocytes, reduced levels of inflammatory cytokines and increased Glut4, adiponectin and lipogenic enzyme mRNA levels. This was associated with reduced inflammation, fat accumulation and enhanced glucose metabolism in liver. Weight gain and fasting glucose tolerance were not affected. CTLA4-Ig injections reduced the number of T cells in epididymal AT (epiAT) but not the inflammatory cytokines levels and failed to improve liver fat accumulation. Deletion of CD28 creates a new pro/anti-inflammatory balance in epiAT and liver and exerts a protective effect against hepatic steatosis.

Unexpected Efficacy of CTLA-4Ig in Controlling Ongoing Allospecific Responses and Treating Acute Rejection.

Once rejection is initiated, halting the immune response becomes much more difficult because of increased frequencies of activated effector and memory alloreactive T and B cells and of plasma cells. Costimulation blockade targeting CD28-B7 and CD40-CD154 interactions is reported to be particularly ineffective at controlling memory T cell and T-dependent B cell responses. Here we report on the unexpected efficacy of CTLA-4Ig in controlling ongoing antibody responses and treating acute rejection. To test the efficacy of CTLA-4Ig at halting ongoing alloimmune response, C57BL/6 mice were sensitized with a subcutaneous injection of BALB/c splenocytes (DST) and treated with biweekly CTLA-4Ig from d0, d7, or d14 post-DST. Without CTLA-4Ig, donor-specific Abs (DSA) were detected on d7 and peaked on d21 post-DST. Delayed treatment with CTLA-4Ig from d7 or d14 post-DST halted the DSA response, and DSA titers tended towards baseline by d42. To test whether delayed CTLA-4Ig prevented development of memory responses, CTLA4-Ig treatment was stopped on d42 and 2 weeks later, the mice were challenged with DST without CTLA4-Ig. Mice receiving d0-42 CTLA-4Ig exhibited no increase in alloantibody production, consistent with tolerance, whereas those receiving d14-42 CTLA-4Ig exhibited recall antibody responses indistinguishable from untreated. Notably, mice receiving d7-42 CTLA-4Ig exhibited a blunted antibody relative to recall as well as to primary response. Finally in a BALB/c into C57BL/6 heart transplantation model, where unmodified rejection occurs on d7-8 post-transplant, biweekly treatment with CTLA4-Ig from 6d post-transplant prevented rejection in 2 of 3 recipients (>42d). A second BALB/c heart transplanted on d21 was also accepted (>21d). These results demonstrate the unexpected efficacy of delayed CTLA4-Ig at halting ongoing alloreactive responses, inhibiting memory formation, facilitating the development of a hyporesponsive state and in treating acute rejection.

Efficacy of CTLA4-Ig in Preventing Acute Rejection of Fully Allogeneic Heart Allografts in Sensitized Recipients.

Belatacept, a high-affinity variant of CTLA4-Ig, is a recently approved immunosuppressive agent for the prevention of acute rejection. It functions as a potent antagonist of the CD28-B7 interactions, thereby inhibiting T cell co-stimulation and activation. Experimental data involving the transient use of CTLA4-Ig to induce allograft tolerance have shaped the current paradigm that CTLA-4Ig is ineffective at inhibiting memory T cell responses, and by extension T-dependent alloantibody responses. This resistance is explained by the acquisition of additional activation-induced costimulatory molecules by memory T cells. We noted that the efficacy of CTLA4-Ig as a continuously administered drug for controlling rejection in sensitized recipients has not been investigated. Here we report on the efficacy of CTLA4-Ig in preventing acute rejection in sensitized C57BL/6 recipients that had received BALB/c spleen cell transfusion 3-6 months previously. We set up four experimental groups. Group 1 (N=5): sensitized mice with HTX and CTLA4-Ig treated (Day -2, Day 0, Day2, then weekly (1 mg/dose) Group 2 (N=3): sensitized mice with HTX and no CTLA4-Ig, Group 3: (N=10) naive mice with HTX and CTLA4-Ig treated, Group 4 (N=10): naive mice with HTX and no CTLA4-Ig. In the untreated Groups 2 & 4, allografts were rejected within 7 days post-transplantation, whereas in Group 3 of naive mice receiving CTLA4-Ig, all the grafts survived long-term (>90 days). In Group 1 of sensitized recipients receiving CTLA4-Ig, the allografts survived for 42, 42, 49, >90, >90 days. The pre-transplant donor-specific antibody (DSA) titers in the recipients that went on to reject their graft was significantly higher compared to those that accepted their grafts. Furthermore, DSA titers increased post-transplantation in the recipients that went on to reject their allografts, whereas DSA titers decreased in those that accepted their grafts. In conclusion, these observations illustrate the efficacy of CTLA-4Ig treatment at controlling rejection in sensitized recipients, and underscore the barrier pre-transplantation DSA poses to long-term graft acceptance. This experimental model also provides a unique opportunity for testing if DSA promotes antibody-mediated rejection or alloreactive memory T cell priming and T cell-mediated rejection in CTLA4-Ig treated recipients, and for identifying therapies that promote long-term graft survival in sensitized recipients.

The Requirement for CD28-B7 Interaction in Sustaining Alloreactive Germinal Center Tfh and B Cell Responses Is Revealed By CTLA-4Ig Treatment.

The Requirement for CD28-B7 Interaction in Sustaining Alloreactive Germinal Center Tfh and B Cell Responses Is Revealed By CTLA-4Ig Treatment.

Foxp3+ regulatory T cells are activated in spite of B7‐CD28 and CD40‐CD40L blockade

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)-cell activity. We previously reported that the blockade of the B7-CD28 and CD40L-CD40 interaction efficiently suppresses allogeneic T-cell activation in vivo. This was characterized by an initial rise in Foxp3(+) cells, followed by depletion of host-reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4(+) cell activation. When CTLA-4Ig and anti-CD40L mAb (MR1) were added to the cultures, T-cell proliferation and IL-2 production were strongly reduced. However, Foxp3(+) cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4(+) cells much more efficiently than did freshly isolated Treg cells. CD4(+) cells activated by allogeneic cells in the presence of MR1 and CTLA-4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4(+) cells when Foxp3(+) Treg cells were removed. We conclude that natural Treg cells are less dependent on B7-CD28 or CD40-CD40L costimulation compared with Foxp3(-) T cells. Reduced costimulation therefore alters the balance between Teff and Treg-cell activation in favor of Treg-cell activity.

CD28 Aptamers as Powerful Immune Response Modulators

CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.

Ex Vivo Enzymatic Treatment of Aged CD4 T Cells Restores Cognate T Cell Helper Function and Enhances Antibody Production in Mice

Previous in vitro studies showed that CD4 T cells from old mice have defects in TCR signaling, immune synapse formation, activation, and proliferation. We reported that removing a specific set of surface glycoproteins by ex vivo treatment with O-sialoglycoprotein endopeptidase (OSGE) can reverse many aspects of the age-related decline in CD4 T cell function. However, the specific mechanism by which this process occurs remains unclear, and it is unknown whether this enzymatic treatment can also restore important aspects of adaptive immunity in vivo. By using an in vivo model of the immune response based on adoptive transfer of CD4 T cells from pigeon cytochrome C-specific transgenic H-2(k/k) TCR-Vα(11)Vβ(3) CD4(+) mice to syngeneic hosts, we demonstrate that aging diminishes CD28 costimulatory signals in CD4 T cells. These age-associated defects include changes in phosphorylation of AKT and expression of glucose transporter type I, inducible T cell costimulatory molecule, and CD40L, suggesting that the lack of CD28 costimulation contributes to age-dependent loss of CD4 function. All of these deficits can be reversed by ex vivo OSGE treatment. Blocking B7-CD28 interactions on T cells prevents OSGE-mediated restoration of T cell function, suggesting that changes in surface glycosylation, including CD28, may be responsible for the age-related costimulation decline. Finally, we show that the age-related decline in CD4 cognate helper function for IgG production and long-term humoral immunity can also be restored by OSGE treatment of CD4 T cells prior to adoptive transfer.

Targeting the Cellular and Molecular Components of CD28 Mediated Survival Signaling in Multiple Myeloma

Abstract 722Very little is known about the cellular or molecular nature of pro-survival interactions between multiple myeloma (MM) and their bone marrow stromal hosts. CD28, the prototypical co-stimulatory molecule on T cells is overexpressed on MM relative to their normal counterparts. Clinical studies have independently shown that expression of CD28 or its ligand CD86 on myeloma cells portends poor survival in patients. We and others have shown that CD28 activation increases MM survival via NFkB accompanied by increased PI3K activity. Previously (ASH2010 and 2011), and since published, we have shown that myeloid dendritic cells (DC) form a vital part of the myeloma microenvironment, and myeloma cells interact with DC via a CD28-CD80/CD86 (B7) interaction. On the myeloma side, this interaction drives a CD28 mediated pro-survival signal, while on the DC side, it drives a B7 mediated induction of growth and immunosuppressive factors that modulate the MM survival niches in the bone marrow. In this work, we delineated key molecules downstream of CD28 that transduce the pro survival signal on the myeloma side as well as explored agents and strategies to target DC that help MM survive in the microenvironment. We also studied the role of DC IL-6 for MM survival in cocultures.We have previously shown that CD28 activation by anti-CD28 mAb involves activation of the multifunctional pro-survival transcription factor NFkB accompanied by increased PI3K activity. Through immunoassays and radioactivity assays, using myeloma cell lines that were transfected with wild type murine CD28 (mCD28 differs only by 3 amino acids from human hCD28) and mutants for PI3K binding site YMNM, we show that the CD28 ligation with anti-mCD28 antibodies induces immediate tyrosine phosphorylation of mCD28 (just as observed by others in T cells), followed by binding of p85, the catalytic subunit of PI3K and activation of VAV1 (Fig 1). Association of p85 and VAV1 activation depended on an intact PI3K binding site on CD28's cytoplasmic tail. Our studies on how this signal is transduced show no involvement of GRB2 or SLP76 more traditionally associated with CD28 signaling. Moreover, silencing SLP76 using siRNA did not affect antibody mediated protection of MM cells against serum starvation. Activation of human CD28 with anti-CD28 mAb also up-regulated the basal tyrosine phosphorylation of human CD28 further just as observed in murine CD28.Through EMSAs, we also show that similar to what we have previously seen with activating antibodies, increase in NFkB activity was observed in myeloma cells when they were cocultured with DCs while being exposed to melphalan, but this spike did not dissipate in the presence of B7 blocking reagents such as CTLA4Ig (Abatacept®) suggesting a low threshold of CD28 activation. Next we tried using agents that have been known to modulate microenvironment such as lenalidomide, a novel drug that is currently used in combination with other chemotherapeutic agents such as dexamethasone and Bortezomib (Velcade®). Survival assays using flow cytometry, show that lenalidomide treated DCs provided significantly lower protection against cell death compared to wild type DC. Lenalidomide DCs also show lower levels of B7 receptors which might be a contributing factor to this phenomenon. Blocking CD28-B7 interaction with CTLA4Ig or CD28(Fab) molecules reduced the pro-survival effect of DC on MM against dexamethasone (Fig 2) melphalan or bortezomib. In order to determine the role of IL-6 in an environment where myeloma cells derive pro-survival signals from their interaction with DC, we used neutralizing antibodies against human IL-6 in cocultures of MM cells with DC. Surprisingly, IL-6 neutralization did not affect the protective effect of DC on MM in cocultures in the presence of chemotherapeutic drugs like melphalan or dexamethasone (Fig 2).All these suggest that targeting DC might be a viable strategy towards augmenting chemotherapy, either by using agents that attenuate DC differentiation or agents that can block the CD28-B7 interaction between DC and MM. Moreover, MM cells do not seem to depend much on IL-6 as a survival factor when they are in contact with DC and its pro-survival effect is more visible when myeloma cells have to survive outside the microenvironment without DC (previous presentation at ASH 2011). [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CD28–B7 Interaction Modulates Short- and Long-Lived Plasma Cell Function

The interaction of CD28, which is constitutively expressed on T cells, with B7.1/B7.2 expressed on APCs is critical for T cell activation. CD28 is also expressed on murine and human plasma cells but its function on these cells remains unclear. There are two types of plasma cells: short-lived ones that appear in the secondary lymphoid tissue shortly after Ag exposure, and long-lived plasma cells that mainly reside in the bone marrow. We demonstrate that CD28-deficient murine short- and long-lived plasma cells produce significantly higher levels of Abs than do their wild-type counterparts. This was owing to both increased frequencies of plasma cells as well as increased Ab production per plasma cell. Plasma cells also express the ligand for CD28, B7.1, and B7.2. Surprisingly, deficiency of B7.1 and B7.2 in B cells also led to higher Ab levels, analogous to Cd28(-/-) plasma cells. Collectively, our results suggest that the CD28-B7 interaction operates as a key modulator of plasma cell function.

A CD28 based bi-directional molecular bridge that support plasma cell survival in the bone marrow. (46.16)

Abstract Dendritic cells (DC) are essential for the long term survival of plasma cells (PC) in the bone marrow and for the maintenance of persistent antibody titers. But the molecular basis for these interactions is not very clear. Both normal and malignant PC (like myeloma) express the T-cell co-stimulatory molecule CD28. Our data show that CD28 expressed on PC play a major role in CD80/CD86 (B7) mediated DC induction of growth factors such as IL-6 and immunosuppressive enzyme indoleamine 2,3 dioxygenase (IDO) as well as in antibody or DC mediated protection of PC in vitro. While IL-6 protected PC in vitro, IDO induced the formation of regulatory T cells in culture. Use of myeloma drug lenalidomide to inhibit DC-B7 expression or anti-CD28(Fab)2 fragments to block CD28-B7 interactions reduced DC ability to protect MM cells or produce IL-6/IDO. Moreover, just like in T-cell models, CD28 activation in malignant PC triggers similar molecular events such as tyrosine phosphorylation and association of p85. We believe that downstream of CD28, while PI3K/Akt governs survival, the Vav1/SLP76 pathway regulate PC proliferation and these form the molecular basis for a bi-directional molecular bridge between the PC and bone marrow microenvironment. Targeting this bridge could form the basis for future therapeutic strategies. Our data suggests a role for CD28-B7 in plasma cell survival by modulating the bone marrow microenvironment.

Restoration of T cell tolerance in primary ITP

Primary immune thrombocytopenia (ITP) has been traditionally thought as an antibody-mediated autoimmune disease involving platelet destruction by macrophages in the reticuloendothelia system. More recently it has become obvious that ITP is a more complex disorder in which T cell mediated immunity plays important roles in platelet destruction. Antiplatelet autoantibody production is under the control of platelet-specific helper T-cells, and loss of tolerance to self antigen by T cells is the critical step of the immune dysregulation in ITP. Dendritic cells (DCs) from ITP patients showed enhanced capacity in stimulating autologous T-cell proliferation in the presence of autologous/allogeneic platelets [1], and ITP patients’ T cells had elevated IL-2 secretion ability compared with controls [2,3], suggesting increased antiplaltelet T-cell reactivity in ITP. The epitopes that recognize platelet glycoprotein (GP) IIIa on T helper (Th) cells has been determined and mapped by several groups [4,5], thus sheding new lights on the “therapeutic vaccination” approach to reinstate tolerance in ITP. Autoreactive T-cell reactivity against platelet antigen in active ITP patients has been observed at polyclonal as well as oligoclonal levels [6,7]. Our group has demonstrated that blocking the B7-CD28 interaction with CTLA4-Ig/CsA could induce platelet GP-specific T-cell anergy, which could exert suppressive effect on GP-reactive T cells via inducing tolerogenic dendritic cells (DCs) [8,9]. It has been well established that apoptotic genes, such as Fas, A20, Bax, Calpastatin, IL2RB, were expressed aberrantly in patients with active ITP [10,11], leading to autoreactive T cells resistant to activation induced cell death (AICD), which could in turn support the expansion of self-reactive T-cell clones. A loss of resistant to AICD might be an important mechanism for the achievement of remission in ITP. Previous studies have revealed that dexamethasone could suppress T-cell proliferation and induce apoptosis of T-cells in ITP [11,12]. In addition, our group has demonstrated that a novel BAFF blocking reagent, BR3-Fc, could restore the apoptosis of both B and T cells [13]. Th polarization in ITP has been attributed to increased Th1 [2,14], and Th17 cells [15] or reduced number or function of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) [16,17]. A parallel body of aberrant cytokine patterns, such as the elevated ratio of interleukin (IL) -18/IL-18 binding protein (BP) [18,19], the increased expression of B cell activating factor (BAFF) has been reported in active ITP patients [20,21]. High-dose dexamthasone (HD-DXM) could not only restore Th1/Th2 [14] or IL-18/IL-18BP balance [19], but also increase the number of Tregs [16], and inhibit the expression of BAFF [12]. Besides HD-DXM, multiple agents, such as rituximab [22], intravenous immunoglobulin (IVIg) [23], romiplostim, eltrombopag [24] as well as indirubin [25], could increase the number or restore the function of Tregs in ITP. Our recently study showed that GP-specific induced Tregs could be successfully generated de novo from nonregulatory CD4+CD25-CD45RA+ cells and could mediate both antigen-specific and linked suppression of proliferating antiplatelet CD4+ Th cells in vitro, and further research revealed that the de novo expanded Tregs mediated their suppressive effects on T cells via actually modulating the T-cell stimulatory capacity of DCs [26], thus providing a clue to the potential of producing antigen-specific Tregs from the patients in vitro for the purpose of antigen-targeted cellular immunotherapy. In conclusion, induction of T-cell tolerance may provide a useful strategy for the management of ITP.

Blockade of NKG2D Synergized With CTLA4-Ig in Promoting Long-Term Graft Survival in Murine Models of Cardiac Transplantation [RETRACTED

Blockade of B7-CD28 interaction with CTLA4-Ig could prolong allograft survival in cardiac transplantation. NKG2D is an activating or coactivating receptor on NK cells, γδ T, and CD8 T cells and played an important role in transplant immunity. C57BL/6 (wild type and γδ or interleukin (IL)-17) mice were transplanted with allogeneic BALB/c hearts and treated with CTLA4-Ig alone or in combination with anti-NKG2D monoclonal antibodies. The survival of grafts was monitored daily by abdominal palpation until the complete cessation of cardiac contractility. We found that wild type recipient treated with anti-NKG2D monoclonal antibodies plus CTLA4-Ig showed significantly prolonged survival cardiac allograft (>90 days, P<0.001). These in vivo results in combined therapy group correlated with low expression of interferon-γ whereas increased expression of IL-4 and alternatively activated macrophage markers. Furthermore, with blockade of NKG2D, the number of IL-17-producing γδ T cells was significantly reduced, which was demonstrated as the main source of IL-17 production. And in our γδ and IL-17 murine cardiac transplantation models, we found that γδ or IL-17 deficiency could significantly prolong cardiac allograft survival. Blockade of NKG2D is effective in synergizing with CTLA4-Ig to promote long-term cardiac allograft survival in mice, and this effect is associated with decreased infiltration of IL-17-producing γδ T cells.

IDO and Regulatory T Cell Support Are Critical for Cytotoxic T Lymphocyte-Associated Ag-4 Ig-Mediated Long-Term Solid Organ Allograft Survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.