Role of the programmed death-1 pathway in regulation of alloimmune responses in vivo

Programmed death-1 (PD-1), an inhibitory receptor up-regulated on activated T cells, has been shown to play a critical immunoregulatory role in peripheral tolerance, but its role in alloimmune responses is poorly understood. Using a novel alloreactive TCR-transgenic model system, we examined the functions of this pathway in the regulation of alloreactive CD4+ T cell responses in vivo. PD-L1, but not PD-1 or PD-L2, blockade accelerated MHC class II-mismatched skin graft (bm12 (I-Abm12) into B6 (I-Ab)) rejection in a similar manner to CTLA-4 blockade. In an adoptive transfer model system using the recently described anti-bm12 (ABM) TCR-transgenic mice directly reactive to I-Abm12, PD-1 and PD-L1 blockade enhanced T cell proliferation early in the immune response. In contrast, at a later time point preceding accelerated allograft rejection, only PD-L1 blockade enhanced T cell proliferation. In addition, PD-L1 blockade enhanced alloreactive Th1 cell differentiation. Apoptosis of alloantigen-specific T cells was inhibited significantly by PD-L1 but not PD-1 blockade, indicating that PD-1 may not be the receptor for the apoptotic effect of the PD-L1-signaling pathway. Interestingly, the effect of PD-L1 blockade was dependent on the presence of CD4+ CD25+ regulatory T cells in vivo. These data demonstrate a critical role for the PD-1 pathway, particularly PD-1/PD-L1 interactions, in the regulation of alloimmune responses in vivo.     

Exacerbation of experimental allergic asthma by augmented Th2 responses in WSX-1-deficient mice

WSX-1 (IL-27R) is a class I cytokine receptor with homology to gp130 and IL-12 receptors and is typically expressed on CD4+ T lymphocytes. Although previous reports have clarified that IL-27/WSX-1 signaling plays critical roles in both Th1 differentiation and attenuation of cell activation and proinflammatory cytokine production during some bacterial or protozoan infections, little is known about the importance of WSX-1 in cytokine-mediated diseases of allergic origin. To this aim, we took advantage of WSX-1-deficient (WSX-1(-/-)) mice and induced experimental asthma, in which Th2 cytokines are central modulators of the pathology. OVA-challenged WSX-1(-/-) mice showed marked enhancement of airway responsiveness with goblet cell hyperplasia, pulmonary eosinophil infiltration, and increased serum IgE levels compared with wild-type mice. Production of Th2 cytokines, which are largely responsible for the pathogenesis of asthma, was augmented in the lung or in the culture supernatants of peribronchial lymph node CD4+ T cells from WSX-1(-/-) mice compared with those from wild-type mice. Surprisingly, IFN-gamma production was also enhanced in WSX-1(-/-) mice, albeit at a low concentration. The cytokine overproduction, thus, seems independent from the Th1-promoting property of WSX-1. These results demonstrated that IL-27/WSX-1 also plays an important role in the down-regulation of airway hyper-reactivity and lung inflammation during the development of allergic asthma through its suppressive effect on cytokine production.     

Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells

Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-kappaB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys(30) and Cys(36) in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-kappaB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.     

Analysis of the role of negative T cell costimulatory pathways in CD4 and CD8 T cell-mediated alloimmune responses in vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.     

NADPH dependent activation of microsomal glutathione transferase 1

Microsomal glutathione transferase 1 (MGST1) can become activated up to 30-fold by several mechanisms in vitro (e.g. covalent modification by reactive electrophiles such as N-ethylmaleimide (NEM)). Activation has also been observed in vivo during oxidative stress. It has been noted that an NADPH generating system (g.s.) can activate MGST1 (up to 2-fold) in microsomal incubations, but the mechanism was unclear. We show here that NADPH g.s treatment impaired N-ethylmaleimide activation, indicating a shared target (identified as cysteine-49 in the latter case). Furthermore, NADPH activation was prevented by sulfhydryl compounds (glutathione and dithiothreitol). A well established candidate for activation would be oxidative stress, however we could exclude that oxidation mediated by cytochrome P450 2E1 (or flavine monooxygenase) was responsible for activation under a defined set of experimental conditions since superoxide or hydrogen peroxide alone did not activate the enzyme (in microsomes prepared by our routine procedure). Actually, the ability of MGST1 to become activated by hydrogen peroxide is critically dependent on the microsome preparation method (which influences hydrogen peroxide decomposition rate as shown here), explaining variable results in the literature. NADPH g.s. dependent activation of MGST1 could instead be explained, at least partly, by a direct effect observed also with purified enzyme (up to 1.4-fold activation). This activation was inhibited by sulfhydryl compounds and thus displays the same characteristics as that of the microsomal system. Whereas NADPH, and also ATP, activated purified MGST1, several nucleotide analogues did not, demonstrating specificity. It is thus an intriguing possibility that MGST1 function could be modulated by ligands (as well as reactive oxygen species) during oxidative stress when sulfhydryls are depleted.     

Activation of protein kinase C epsilon inhibits the two-pore domain K+ channel, TASK-1, inducing repolarization abnormalities in cardiac ventricular myocytes

Activation of the platelet-activating factor (PAF) receptor leads to a decrease in outward current in murine ventricular myocytes by inhibiting the TASK-1 channel. TASK-1 carries a background or "leak" current and is a member of the two-pore domain potassium channel family. Its inhibition is sufficient to delay repolarization, causing prolongation of the action potential duration, and in some cases, early after depolarizations. We set out to determine the cellular mechanisms that control regulation of TASK-1 by PAF. Inhibition of TASK-1 via activation of the PAF receptor is protein kinase C (PKC)-dependent. Using isoform-specific PKC inhibitor or activator peptides in patch clamp experiments, we now demonstrate that activation of PKCepsilon is both necessary and sufficient to regulate murine TASK-1 current in a heterologous expression system and to induce repolarization abnormalities in isolated myocytes. Furthermore, site-directed mutagenesis studies have identified threonine 381, in the C-terminal tail of murine TASK-1, as a critical residue in this regulation.     

Molecular pathogenesis of a novel mutation, G108D, in short-chain acyl-CoA dehydrogenase identified in subjects with short-chain acyl-CoA dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase (SCAD) is a mitochondrial enzyme involved in the β-oxidation of fatty acids. Genetic defect of SCAD was documented to cause clinical symptoms such as progressive psychomotor retardation, muscle hypotonia, and myopathy in early reports. However, clinical significance of SCAD deficiency (SCADD) has been getting ambiguous, for some variants in the ACADS gene, which encodes the SCAD protein, has turned out to be widely prevailed among general populations. Accordingly, the pathophysiology of SCADD has not been clarified thus far. The present report focuses on two suspected cases of SCADD detected through the screening of newborns by tandem mass spectrometry. In both subjects, compound heterozygous mutations in ACADS were detected. The mutated genes were expressed in a transient gene expression system, and the enzymatic activities of the obtained mutant SCAD proteins were measured. The activities of the mutant SCAD proteins were significantly lower than that of the wild-type enzyme, confirming the mechanism underlying the diagnosis of SCADD in both subjects. Moreover, the mutant SCAD proteins gave rise to mitochondrial fragmentation and autophagy, both of which were proportional to the decrease in SCAD activities. The association of autophagy with programed cell death suggests that the mutant SCAD proteins are toxic to mitochondria and to the cells in which they are expressed. The expression of recombinant ACADS-encoded mutant proteins offers a technique to evaluate both the nature of the defective SCAD proteins and their toxicity. Moreover, our results provide insight into possible molecular pathophysiology of SCADD.     

Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2

The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.     

CD4 T-Cell Help Programs a Change in CD8 T-Cell Function Enabling Effective Long-Term Control of Murine Gammaherpesvirus 68: Role of PD-1-PD-L1 Interactions

We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II^−/− (CII^−/−) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII^−/− mice caused a significant reduction in lung viral titers, in contrast to those from control CII^−/− mice. Anti-CD40 treatment also greatly prolonged survival of infected CII^−/− mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII^−/−, CD40^−/−, or CD80/86^−/− mice, compared with that in wild-type or CD28/CTLA4^−/− mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring rodent pathogen which is closely related to Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) (17, 64). Intranasal administration of MHV-68 to mice results in acute productive infection of lung epithelial cells and a latent infection in various cell types, including B lymphocytes, dendritic cells, epithelial cells, and macrophages (18, 19, 52, 53, 61, 65). The virus induces an inflammatory infiltrate in the lungs, lymph node enlargement, splenomegaly, and mononucleosis comprising increased numbers of activated CD8 T cells in the blood (53, 58). It has also been reported to induce lymphoproliferative disease/lymphoma in immunocompromised mice (30, 55, 60). Thus, the pathogenesis resembles that of EBV in humans, although structurally, the virus is more closely related to KSHV.Infectious MHV-68 is cleared from the lungs by a T-cell-dependent mechanism 10 to 15 days after infection (18, 53, 56). In wild-type mice, the lungs remain clear of replicating virus thereafter. Although CD4 T cells are not essential for primary clearance of replicating virus, they are required for effective long-term control (11). Thus, major histocompatibility complex (MHC) class II^−/− mice that lack CD4 T cells or mice rendered CD4 deficient by antibody treatment initially clear infectious virus from the lungs. However, infectious virus reactivates in the lungs 10 to 15 days later and gradually increases in titer (11, 43). The infected CD4-deficient mice eventually die, apparently from long-term lung damage due to continuing lytic viral replication (11). MHC class II^−/− mice do not produce antibody to T-dependent antigens (10). Cytotoxic T-lymphocyte (CTL) epitopes have been identified in open reading frame (ORF) 6 (p56, H-2D^b-restricted), and ORF 61 (p79, H-2K^b-restricted) gene products, which appear to encode early lytic-phase proteins (32, 49). The epitopes are presented during two distinct phases during MHV-68 infection, which changes the pattern of CTL dominance (32, 51). However, there is no significant difference in the numbers of CD8 T cells specific for each epitope in wild-type mice and CD4 T-cell-deficient mice (4, 50). In addition, CTL activity measured in vitro does not differ substantially in the lungs of wild-type mice or CD4 T-cell-deficient mice (4, 11, 50). Furthermore, postexposure vaccination with the p56 epitope failed to prevent viral reactivation in class II^−/− mice, despite dramatically expanding the number of CD8 T cells specific for the peptide (5). In contrast, vaccination of wild-type mice against these epitopes reduced lytic viral titers in the lung dramatically on subsequent challenge with MHV-68. B-cell-deficient mice clear MHV-68 with the kinetics of wild-type mice and do not show viral reactivation in the lungs (13, 61), suggesting that antibody is not essential for control of the virus. Depletion of CD4 T cells during the latent phase of infection in B-cell-deficient mice does not induce viral reactivation, whereas depletion of both CD4 and CD8 T-cell subsets provokes viral reactivation in the lungs (52). Short-term depletion of both CD4 and CD8 T-cell subsets during the latent phase of infection in wild-type mice does not lead to viral reactivation probably due to the presence of neutralizing antibody (11). Taken together, these results suggest that CD4 and CD8 T cells and B cells play overlapping roles in preventing or controlling reactivation of MHV-68 during the latent phase of infection. However, the B-cell- and CD8 T-cell-mediated control mechanisms do not develop in the absence of CD4 T cells.We, and others, have previously shown that the costimulatory molecule CD28 is not required for long-term control of MHV-68 (28, 29). However, interestingly, mice lacking both of the ligands for CD28, CD80 and CD86, show viral reactivation in the lung (21, 35). Our previously published data showed that agonistic antibodies to CD40 could substitute for CD4 T-cell function in the long-term control of MHV-68 (46). CD8 T-cell receptor-positive (TCR+) cells were required for this effect, while antibody production was not restored (45, 46). MHV-68-infected CD40L^−/− mice (7) and CD40^−/− mice (29) also showed viral reactivation in the lungs. However, no change in CD8 CTL activity was detected in in vitro assays following anti-CD40 treatment (46). A key question was whether anti-CD40 treatment (or CD4 T-cell help) caused a direct change in CD8 T-cell function or whether both CD8 T cells and an independent anti-CD40-sensitive step were required for viral control. To address this question, we used adoptive transfer of CD8 T cells from MHV-68-infected wild-type mice, anti-CD40-treated mice, or control MHC class II^−/− mice to MHV-68-infected class II^−/− recipients. We also investigated whether anti-CD40 treatment prolonged survival in addition to reducing lung viral titers. The heterodimeric molecule CD94/NKG2A has been implicated in negatively regulating the CD8 T-cell response to polyomavirus (38) and herpes simplex virus (HSV) (54), while the inhibitory receptor PD-1 (programmed death 1) has been implicated in T-cell exhaustion following infection with several other persistent viruses (2, 15, 20, 22, 26, 36, 39-41, 57, 67). In the present study, we investigated the effect of signaling via various costimulatory molecules on the expression of NKG2A and PD-1 and how these molecules influenced viral control.     

Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis

Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139-151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.