Proliferation Capacity and Cytotoxic Activity Are Mediated by Functionally and Phenotypically Distinct Virus-Specific CD8 T Cells Defined by Interleukin-7Rα (CD127) and Perforin Expression

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127⁺ perforin⁻/CD127⁻ perforin⁺, and CD127⁻/perforin⁻ CD8 T cells, respectively. CD127⁻/perforin⁻ and CD127⁻/perforin⁺ cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin⁺/IL-2⁻ or perforin⁻/IL-2⁺ cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127⁺/IL-2-secreting) and cytotoxic (perforin⁺) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.Cytotoxic CD8 T cells are a fundamental component of the immune response against viral infections and mediate an important role in immunosurveillance (7, 10, 55), and the induction of vigorous CD8 T-cell responses after vaccination is thought to be a key component of protective immunity (37, 41, 49, 50, 58, 60, 69). Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules containing perforin (pore-forming protein) and several granule-associated proteases, including granzymes (Grms) (5, 15, 20, 44). Several studies have recently advanced the characterization of the mechanism of granule-dependent cytotoxic activity and performed a comprehensive investigation of the content of cytotoxic granules in human virus-specific CD8 T cells (2, 19, 29, 44, 53).Heterogeneous profiles of cytotoxic granules have been identified in different virus-specific memory CD8 T cells and associated with distinct differentiation stages of memory CD8 T cells (2, 19, 29, 44). Furthermore, we have observed a hierarchy among the cytotoxic granules in setting the efficiency of cytotoxic activity and demonstrated that perforin (and to a lesser extent GrmB) but not GrmA or GrmK were associated with cytotoxic activity (29). Recently, a novel mechanism of perforin-dependent granule-independent CTL cytotoxicity has also been demonstrated (45).Major advances in the characterization of antigen (Ag)-specific CD4 and CD8 T cells have been made recently and have aimed at identifying functional profiles that may correlate with protective CD8 T-cell responses (1, 3, 4, 12, 13, 24, 28, 36-38, 40, 41, 49, 50, 56-58, 60, 64, 68). In particular, the functional characterization of antigen-specific T cells was mainly performed on the basis of (i) the pattern of cytokines secreted (i.e., gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], or macrophage inflammatory protein 1β [MIP-1β]), (ii) the proliferation capacity, and (iii) the cytotoxic capacity (13, 28, 59). Of note, degranulation activity (i.e., CD107a mobilization following specific stimulation) has been used as a surrogate marker of cytotoxic activity (11, 13).The term “polyfunctional” has been used to define T-cell immune responses that, in addition to typical effector functions such as secretion of IFN-γ, TNF-α, or MIP-1β and cytotoxic activity (measured by the degranulation capacity), comprise distinct T-cell populations able to secrete IL-2 and retain proliferation capacity (13, 28, 49, 50). Some evidence indicates that a hallmark of protective immune responses is the presence of polyfunctional T-cell responses (59). Furthermore, the ability to secrete IL-2 was shown to be linked to proliferation capacity, and both factors have been associated with protective antiviral immunity (13, 28, 49, 50). Although a lack of correlation between degranulation activity and GrmB expression was reported in mice (65), the relationship between degranulation activity and perforin expression has never been comprehensively investigated in mice and in humans.The private α chain of the IL-7 receptor (IL-7Rα, also called CD127) has been suggested to selectively identify CD8 T cells that will become long-lived memory cells (6, 34, 36). Moreover, it was shown in mice (34, 36) and humans (14, 48, 63) that the CD127^high memory-precursor CD8 T cells produced IL-2 in contrast to CD127^low effector CD8 T cells. Of interest, CD127 expression has also been shown to correlate with Ag-specific proliferation capacity in mice (34, 36). A similar correlation was observed in humans, although only for polyclonal stimulations (48). With the exception of studies performed in HIV-1 infection, where an association between CD127 expression and HIV-1 viremia has been shown (21, 22, 42, 48, 54), very limited information is available on the CD127 expression in human virus-specific CD8 T cells other that HIV-1.Although cytotoxic activity and proliferation capacity are key components of the antiviral cellular immune response, the relationship between these functions has been only investigated in nonprogressive HIV-1 infection (46), where these two functions were shown to be related. However, it still remains to be determined whether these functions are mediated by the same or by different T-cell populations.In the present study, we performed a comprehensive characterization of virus-specific CD8 T-cell responses against HIV-1, cytomegalovirus (CMV), Epstein Barr virus (EBV), and influenza virus (Flu) in order to (i) analyze the degree of concordance between degranulation activity and perforin/Grm expression; (ii) identify the relevance of CD127 in identifying virus-specific CD8 T cells endowed with proliferation capacity; (iii) delineate the relationship between proliferation capacity, cytotoxic activity, activation/differentiation stage, and level of exhaustion of CD8 T cells; and (iv) determine the influence of antigen exposure in shaping the functional composition of virus-specific CD8 T cells.Our data indicate that cytotoxic (as defined by perforin expression) and proliferating (as defined by CD127 expression or IL-2 secretion) virus-specific CD8 T cells are contained within distinct CD8 T-cell populations. Furthermore, the proportion of proliferating and cytotoxic T cells within a given virus-specific CD8 T-cell population appears to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferative capacity, differentiation stage, and Ag exposure of memory CD8 T cells.     

Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

Andes Virus Recognition of Human and Syrian Hamster β3 Integrins Is Determined by an L33P Substitution in the PSI Domain

Andes virus (ANDV) causes a fatal hantavirus pulmonary syndrome (HPS) in humans and Syrian hamsters. Human α_vβ₃ integrins are receptors for several pathogenic hantaviruses, and the function of α_vβ₃ integrins on endothelial cells suggests a role for α_vβ₃ in hantavirus directed vascular permeability. We determined here that ANDV infection of human endothelial cells or Syrian hamster-derived BHK-21 cells was selectively inhibited by the high-affinity α_vβ₃ integrin ligand vitronectin and by antibodies to α_vβ₃ integrins. Further, antibodies to the β₃ integrin PSI domain, as well as PSI domain polypeptides derived from human and Syrian hamster β₃ subunits, but not murine or bovine β₃, inhibited ANDV infection of both BHK-21 and human endothelial cells. These findings suggest that ANDV interacts with β₃ subunits through PSI domain residues conserved in both Syrian hamster and human β₃ integrins. Sequencing the Syrian hamster β₃ integrin PSI domain revealed eight differences between Syrian hamster and human β₃ integrins. Analysis of residues within the PSI domains of human, Syrian hamster, murine, and bovine β₃ integrins identified unique proline substitutions at residues 32 and 33 of murine and bovine PSI domains that could determine ANDV recognition. Mutagenizing the human β₃ PSI domain to contain the L33P substitution present in bovine β₃ integrin abolished the ability of the PSI domain to inhibit ANDV infectivity. Conversely, mutagenizing either the bovine PSI domain, P33L, or the murine PSI domain, S32P, to the residue present human β₃ permitted PSI mutants to inhibit ANDV infection. Similarly, CHO cells transfected with the full-length bovine β₃ integrin containing the P33L mutation permitted infection by ANDV. These findings indicate that human and Syrian hamster α_vβ₃ integrins are key receptors for ANDV and that specific residues within the β₃ integrin PSI domain are required for ANDV infection. Since L33P is a naturally occurring human β₃ polymorphism, these findings further suggest the importance of specific β₃ integrin residues in hantavirus infection. These findings rationalize determining the role of β₃ integrins in hantavirus pathogenesis in the Syrian hamster model.Hantaviruses persistently infect specific small mammal hosts and are spread to humans by the inhalation of aerosolized excreted virus (41, 42). Hantaviruses predominantly infect endothelial cells and cause one of two vascular leak-based diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) (41). Hantavirus diseases are characterized by increased vascular permeability and acute thrombocytopenia in the absence of endothelial cell lysis (36, 41, 42, 54). In general, hantaviruses are not spread from person to person; however, the Andes hantavirus (ANDV) is an exception, since there are several reports of person-to-person transmission of ANDV infection (11, 37, 47, 52). ANDV is also unique in its ability to cause an HPS-like disease in Syrian hamsters and serves as the best-characterized hantavirus disease model with a long onset, symptoms, and pathogenesis nearly identical to that of HPS patients (20, 21, 50).Hantavirus infection of the endothelium alters endothelial cell barrier functions through direct and immunological responses (8, 14). Although the means by which hantaviruses cause pulmonary edema or hemorrhagic disease has been widely conjectured, the mechanisms by which hantaviruses elicit pathogenic human responses have yet to be defined. Hantaviruses coat the surface of infected VeroE6 cells days after infection (17), and this further suggests that dynamic hantavirus interactions with immune and endothelial cells are likely to contribute to viral pathogenesis. Hantavirus pathogenesis has been suggested to involve CD8⁺ T cells, tumor necrosis factor alpha or other cytokines, viremia, and the dysregulation of β₃ integrins (7, 8, 13-16, 25-28, 32, 34, 38, 44-46). However, these responses have not been demonstrated to contribute to hantavirus pathogenesis, and in some cases there are conflicting data on their involvement (18, 25-28, 34, 35, 44, 45, 48). Immune complex deposition clearly contributes to HFRS patient disease and renal sequelae (4, 7), but it is unclear what triggers vascular permeability in HPS and HFRS diseases or why hemorrhage occurs in HFRS patients but not in HPS patients (8, 36, 54). Acute thrombocytopenia is common to both diseases, and platelet dysfunction resulting from defective platelet aggregation is reported in HFRS patients (7, 8).Pathogenic hantaviruses have in common their ability to interact with α_IIbβ₃ and α_vβ₃ integrins present on platelets and endothelial cells (13, 16), and β₃ integrins have primary roles in regulating vascular integrity (1, 2, 6, 19, 22, 39, 40). Consistent with the presence of cell surface displayed virus (17), pathogenic hantaviruses uniquely block α_vβ₃ directed endothelial cell migration and enhance endothelial cell permeability for 3 to 5 days postinfection (14, 15). Pathogenic hantaviruses dysregulate β₃ integrin functions by binding domains present at the apex of inactive β₃ integrin conformers (38). α_vβ₃ forms a complex with vascular endothelial cell growth factor receptor 2 (VEGFR2) and normally regulates VEGF-directed endothelial cell permeability (2, 3, 10, 39, 40). However, both β₃ integrin knockouts and hantavirus-infected endothelial cells result in increased VEGF-induced permeability, presumably by disrupting VEGFR2-β₃ integrin complex formation (2, 14, 19, 39, 40). This suggests that at least one means for hantaviruses to increase vascular permeability occurs through interactions with β₃ integrins that are required for normal platelet and endothelial cell functions.α_vβ₃ and α_IIbβ₃ integrins exist in two conformations: an active extended conformation where the ligand binding head domain is present at the apex of the heterodimer and a basal, inactive bent conformation where the globular head of the integrin is folded toward the cell membrane (30, 53, 55). Pathogenic HTN and NY-1 hantaviruses bind to the N-terminal plexin-semaphorin-integrin (PSI) domain of β₃ integrin subunits and are selective for bent, inactive α_vβ₃ integrin conformers (38). Pathogenic hantavirus binding to inactive α_vβ₃ integrins is consistent with the selective inhibitory effect of hantaviruses on α_vβ₃ function and endothelial cell permeability (14, 15, 38). Although the mechanism of hantavirus induced vascular permeability has yet to be defined, there is a clear role for β₃ integrin dysfunction in vascular permeability deficits (5, 6, 22, 29, 39, 40, 51) which make an understanding of hantavirus interactions with β₃ subunits important for both entry and disease processes.The similarity between HPS disease in humans and Syrian hamsters (20, 21) suggests that pathogenic mechanisms of ANDV disease are likely to be coincident. Curiously, other hantaviruses (Sin Nombre virus [SNV] and Hantaan virus [HTNV]) are restricted in Syrian hamsters and fail to cause disease in this animal, even though they are prominent causes of human disease (50). Although the host range restriction for SNV and HTNV in Syrian hamsters has not been defined (33), the pathogenesis of ANDV in Syrian hamsters suggests that both human and Syrian hamster β₃ integrins may similarly be used by ANDV and contribute to pathogenesis.We demonstrate here that ANDV infection of the Syrian hamster BHK-21 cell line and human endothelial cells is dependent on α_vβ₃ and inhibited by α_vβ₃ specific ligands and antibodies. Further, polypeptides expressing the N-terminal 53 residues of human and Syrian hamster β₃ subunits block ANDV infection. This further indicates that ANDV interaction with the N-terminal 53 residues of both human and Syrian hamster β₃ integrins is required for viral entry. We also demonstrate that ANDV recognition of human and Syrian hamster β₃ integrins is determined by proline substitutions at residues 32/33 within the β₃ integrin PSI domain. These results define unique ANDV interactions with human and Syrian hamster β₃ integrins.     

Serine Dephosphorylation of Receptor Protein Tyrosine Phosphatase α in Mitosis Induces Src Binding and Activation

Receptor protein tyrosine phosphatase α (RPTPα) is the mitotic activator of the protein tyrosine kinase Src. RPTPα serine hyperphosphorylation was proposed to mediate mitotic activation of Src. We raised phosphospecific antibodies to the two main serine phosphorylation sites, and we discovered that RPTPα Ser204 was almost completely dephosphorylated in mitotic NIH 3T3 and HeLa cells, whereas Ser180 and Tyr789 phosphorylation were only marginally reduced in mitosis. Concomitantly, Src pTyr527 and pTyr416 were dephosphorylated, resulting in 2.3-fold activation of Src in mitosis. Using inhibitors and knockdown experiments, we demonstrated that dephosphorylation of RPTPα pSer204 in mitosis was mediated by PP2A. Mutation of Ser204 to Ala did not activate RPTPα, and intrinsic catalytic activity of RPTPα was not affected in mitosis. Interestingly, binding of endogenous Src to RPTPα was induced in mitosis. GRB2 binding to RPTPα, which was proposed to compete with Src binding to RPTPα, was only modestly reduced in mitosis, which could not account for enhanced Src binding. Moreover, we demonstrate that Src bound to mutant RPTPα-Y789F, lacking the GRB2 binding site, and mutant Src with an impaired Src homology 2 (SH2) domain bound to RPTPα, illustrating that Src binding to RPTPα is not mediated by a pTyr-SH2 interaction. Mutation of RPTPα Ser204 to Asp, mimicking phosphorylation, reduced coimmunoprecipitation with Src, suggesting that phosphorylation of Ser204 prohibits binding to Src. Based on our results, we propose a new model for mitotic activation of Src in which PP2A-mediated dephosphorylation of RPTPα pSer204 facilitates Src binding, leading to RPTPα-mediated dephosphorylation of Src pTyr527 and pTyr416 and hence modest activation of Src.Protein tyrosine phosphatases (PTPs) are responsible for dephosphorylation of the phosphotyrosyl residues. The human genome contains approximately 100 genes that encode members of the four PTP families, and most of them have mouse orthologues (2, 48). According to their subcellular localization, the classical PTPs, encoded by less than half of the total PTP genes, are divided into two subfamilies: cytoplasmic and receptor protein tyrosine phosphatases (RPTPs). The majority of the RPTPs contain, besides a variable extracellular domain and a transmembrane domain, two highly homologous phosphatase domains (27), with the membrane-proximal domain comprising most of the catalytic activity (33).RPTPα is a typical RPTP with a small, highly glycosylated extracellular domain (13). RPTPα function is regulated by many mechanisms, including proteolysis (18), oxidation (55), dimerization (7, 23, 24, 47, 52), and phosphorylation of serine and tyrosine residues (16, 17, 49). RPTPα is broadly expressed in many cell types, and over the years, RPTPα has been shown to be involved in a number of signaling mechanisms, including neuronal (15) and skeletal muscle (34) cell differentiation, neurite elongation (8, 9, 56), insulin receptor signaling downregulation (3, 28, 30, 31, 35), insulin secretion (25), activation of voltage-gated potassium channel Kv1.2 (51), long-term potentiation in hippocampal neurons (32, 38), matrix-dependent force transduction (53), and cell spreading and migration (21, 45, 57).The majority of the roles played in these cellular processes involve RPTPα''s ability to activate the proto-oncogenes Src and Fyn by dephosphorylating their C-terminal inhibitory phosphotyrosine (5, 15, 39, 45, 61). Normally, this phosphotyrosine (pTyr527 in chicken Src) binds to the Src homology 2 (SH2) domain, keeping the protein in an inactive closed conformation. A displacement mechanism was proposed for RPTPα-mediated Src activation in which pTyr789 of RPTPα is required to bind the SH2 domain of Src before RPTPα dephosphorylates Tyr527 (58). This model is the subject of debate since other studies show that RPTPα lacking Tyr789 is still able to dephosphorylate and activate Src (12, 26, 29, 56). In normal cells, Src reaches its activation peak during mitosis (4, 11, 40, 42), and with the help of overexpressing cells, it was shown that this activation is triggered mainly by RPTPα. The model that emerged is that RPTPα is activated in mitosis due to serine hyperphosphorylation and detaches from the GRB2 scaffolding protein (59, 60) that normally binds most of the pTyr789 of RPTPα via its SH2 domain (14, 17, 46). Two serine phosphorylation sites were mapped in the juxtamembrane domain of RPTPα, Ser180 and Ser204 (49). The kinases that were found responsible for their phosphorylation were protein kinase C delta (PKCdelta) (10) and CaMKIIalpha (9), but there is no clear evidence that these kinases are activated in mitosis. We set out to investigate the role of serine phosphorylation of RPTPα in mitotic activation of Src.We generated phosphospecific antibodies and show that RPTPα pSer204, but not pSer180, is dephosphorylated in mitotic NIH 3T3 and HeLa cells, concomitantly with activation of Src. Selective inhibitors suggested that PP2A was the phosphatase that dephosphorylated pSer204. RNA interference (RNAi)-mediated knockdown of the catalytic subunit of PP2A demonstrated that indeed PP2A was responsible for mitotic dephosphorylation of RPTPα pSer204. It is noteworthy that PP2A is known to be activated in mitosis. Intrinsic PTP activities of RPTPα were similar in unsynchronized and mitotic cells, and mutation of Ser204 did not activate RPTPα in in vitro PTP assays. Yet, Src binding to RPTPα was induced in mitotic NIH 3T3 cells and RPTPα-S204D with a phosphomimicking mutation at Ser204 coimmunoprecipitated less efficiently with Src. Based on our results, we propose a mechanism for mitotic activation of Src that is triggered by dephosphorylation of RPTPα pSer204, resulting in enhanced affinity for Src and subsequent dephosphorylation and activation of Src.     

Gamma Interferon Signaling in Macrophage Lineage Cells Regulates Central Nervous System Inflammation and Chemokine Production

Intracranial (i.c.) infection of mice with lymphocytic choriomeningitis virus (LCMV) results in anorexic weight loss, mediated by T cells and gamma interferon (IFN-γ). Here, we assessed the role of CD4⁺ T cells and IFN-γ on immune cell recruitment and proinflammatory cytokine/chemokine production in the central nervous system (CNS) after i.c. LCMV infection. We found that T-cell-depleted mice had decreased recruitment of hematopoietic cells to the CNS and diminished levels of IFN-γ, CCL2 (MCP-1), CCL3 (MIP-1α), and CCL5 (RANTES) in the cerebrospinal fluid (CSF). Mice deficient in IFN-γ had decreased CSF levels of CCL3, CCL5, and CXCL10 (IP-10), and decreased activation of both resident CNS and infiltrating antigen-presenting cells (APCs). The effects of IFN-γ signaling on macrophage lineage cells was assessed using transgenic mice, called “macrophages insensitive to interferon gamma” (MIIG) mice, that express a dominant-negative IFN-γ receptor under the control of the CD68 promoter. MIIG mice had decreased levels of CCL2, CCL3, CCL5, and CXCL10 compared to controls despite having normal numbers of LCMV-specific CD4⁺ T cells in the CNS. MIIG mice also had decreased recruitment of infiltrating macrophages and decreased activation of both resident CNS and infiltrating APCs. Finally, MIIG mice were significantly protected from LCMV-induced anorexia and weight loss. Thus, these data suggest that CD4⁺ T-cell production of IFN-γ promotes signaling in macrophage lineage cells, which control (i) the production of proinflammatory cytokines and chemokines, (ii) the recruitment of macrophages to the CNS, (iii) the activation of resident CNS and infiltrating APC populations, and (iv) anorexic weight loss.Immune cell recruitment to and infiltration of the central nervous system (CNS) is central to the pathology of a variety of inflammatory neurological diseases, including infectious meningoencephalitis, multiple sclerosis, and cerebral ischemia (59, 60). Chemokines have been shown to be highly upregulated in both human diseases and animal models of neuroinflammation and are thought to be important mediators of immune cell entry into the CNS (59, 60). For example, during experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS), the chemokines CCL2 (monocyte chemoattractant protein 1 [MCP-1α]), CCL3 (macrophage inflammatory protein 1α [MIP-1α]), CCL5 (regulated upon activation, T-cell expressed and secreted [RANTES]), and CXCL10 (gamma interferon [IFN-γ]-inducible protein 10 [IP-10]) are produced by either resident CNS cells or infiltrating cells (27) and serve to amplify the ongoing inflammatory response (25, 28). However, in some EAE studies, neither CCL3 nor CXCL10 were required for disease (72, 73). During CNS viral infection, CXCL10 and CCL5 are highly produced in several models (2, 41, 48, 82). In addition, mice deficient in CCR5, which binds (among others) CCL3 and CCL5, do not display impaired CNS inflammation after certain viral infections (13). Thus, the role of chemokines in CNS inflammation is likely complex and dissimilar between autoimmune and viral infection models.IFN-γ is present in the CNS during autoimmunity and infection (7, 54, 69). Several studies suggest that IFN-γ can be a potent inducer of CNS chemokine expression. Adenoviral expression of IFN-γ in the CNS strongly induced CCL5 and CXCL10 mRNA and protein, and this induction was dependent on the presence of the IFN-γ receptor (50). In EAE and Toxoplasma infection, mice deficient in IFN-γ or the IFN-γ receptor demonstrated reduced expression of several chemokines, including CCL2, CCL3, CCL5, and CXCL10 (26, 69). However, given the near-ubiquitous expression of the IFN-γ receptor (44), the mechanisms by which IFN-γ regulates CNS chemokine production remain to be elucidated.We studied neuroinflammation and immune-mediated disease using a well-studied mouse model of infection with lymphocytic choriomeningitis virus (LCMV). Intracranial (i.c.) injection of mice with LCMV results in seizures and death 6 to 8 days after inoculation. The onset of symptoms is associated with a massive influx of mononuclear cells into the cerebrospinal fluid (CSF), meninges, choroid plexus, and ependymal membranes (6, 8, 18), as well as the presence of proinflammatory cytokines (7, 38). The immune response is critical for disease, since infection of irradiated or T-cell-depleted mice leads to persistent infection with very high levels of virus in multiple tissues without the development of lethal meningitis (18, 34, 64). i.c. LCMV infection of β₂-microglobulin-deficient mice (β₂m^−/− mice) also results in meningitis and production of proinflammatory cytokines and chemokines; however, meningitis occurs with a later onset and lower severity compared to wild-type mice (17, 24, 53, 57). Interestingly, i.c. LCMV infection of these mice also causes severe anorexia and weight loss (33, 38, 46, 52, 57) that is mediated by major histocompatibility complex (MHC) class II-restricted, CD4⁺ T cells (17, 46, 53, 57). Anorexia and weight loss are also observed in wild-type mice, but they succumb to lethal meningitis shortly thereafter (33), making study of this particular aspect of disease difficult. LCMV-induced weight loss, similar to what we have observed in β₂m^−/− mice also occurs in perforin-deficient mice, which possess CD8⁺ T cells (37). Although some reports have observed weight loss after peripheral LCMV infection (11, 45), we note that these studies used high doses of the clone 13 strain of LCMV, in contrast to our studies which have used the Armstrong strain of LCMV and orders of magnitude less virus (33, 38, 46, 52, 57). Although we cannot exclude a contribution of peripheral cells to weight loss in our i.c. Armstrong infection model, we previously showed that this weight loss does not occur with peripheral infection with LCMV Armstrong (33, 38), indicating that interactions between the CNS and the immune system are contribute substantially to disease.During LCMV infection, there is biphasic production of IFN-γ: a small, early peak of IFN-γ (most likely produced by NK or NKT cells), followed by T-cell-mediated production of IFN-γ (23, 75). Further, both CD4⁺ T cells and CD8⁺ T cells produce large amounts of IFN-γ after LCMV infection and T-cell production of IFN-γ is critical for LCMV-induced weight loss (35). Chemokines, especially CXCL10, CCL5, and CCL2, and their receptors, are upregulated in the brain after i.c. LCMV infection (2, 13). Brain chemokine mRNA expression after i.c. LCMV infection is reduced in IFN-γ-deficient mice and relatively absent in athymic mice (2). However, the mechanism(s) by which T cells and IFN-γ mediate the effects on CNS chemokine expression, cellular infiltration into the CNS, and LCMV-induced anorexic weight loss remain unclear.In the present study, we focused on two major questions. The first question concerned the role of IFN-γ on immune cell recruitment to and chemokine/cytokine production within the CNS? We found that macrophages and myeloid dendritic cells (DCs) require IFN-γ for their accumulation within the CNS. Second, since macrophages and myeloid DCs are the predominant cellular infiltrate, we sought to determine whether IFN-γ signaling on these cells was direct with regard to their recruitment and to chemokine/cytokine production. We found that IFN-γ signaling in macrophage lineage cells contributes significantly to their recruitment, to chemokine production in the CNS, and to anorexic weight loss. Together, these data suggest that much of the proinflammatory effects of IFN-γ in the CNS are mediated by the effects of IFN-γ on CD68-bearing cells.     

Potential Application of N-Carbamoyl-β-Alanine Amidohydrolase from Agrobacterium tumefaciens C58 for β-Amino Acid Production

An N-carbamoyl-β-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (βcar_At) has been characterized. βcar_At is most active at 30°C and pH 8.0 with N-carbamoyl-β-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn²⁺, Ni²⁺, and Co²⁺. The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-α-, -β-, -γ-, and -δ-amino acids, with the greatest catalytic efficiency for N-carbamoyl-β-alanine. βcar_At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the β-amino acids taurine and ciliatine, respectively. βcar_At is able to produce monosubstituted β²- and β³-amino acids, showing better catalytic efficiency (k_cat/K_m) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-β-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make βcar_At an outstanding candidate for application in the biotechnology industry.N-Carbamoyl-β-alanine amidohydrolase (NCβAA) (EC 3.5.1.6), also known as β-alanine synthase or β-ureidopropionase, catalyzes the third and final step of reductive pyrimidine degradation. In this reaction, N-carbamoyl-β-alanine or N-carbamoyl-β-aminoisobutyric acid is irreversibly hydrolyzed to CO₂, NH₃, and β-alanine or β-aminoisobutyric acid, respectively (43). Eukaryotic NCβAAs have been purified from several sources (10, 25, 33, 39, 42, 44). Nevertheless, only two prokaryotic NCβAAs, belonging to the Clostridium and Pseudomonas genera (4, 29), have been purified to date, although this activity has been inferred for several microorganisms due to the appearance of the reductive pathway of pyrimidine degradation (38, 45). Pseudomonas NCβAA is also able to hydrolyze l-N-carbamoyl-α-amino acids, and indeed, this activity is widespread in the bacterial kingdom (3, 23, 26, 46).β-Amino acids have unique pharmacological properties, and their utility as building blocks of β-peptides, pharmaceutical compounds, and natural products is of growing interest (14). β-Alanine, a natural β-amino acid, is a precursor of coenzyme A and pantothenic acid in bacteria and fungi (vitamin B₅) (7). β-Alanine is widely distributed in the central nervous systems of vertebrates and is a structural analogue of γ-amino-n-butyric acid and glycine, major inhibitory neurotransmitters, suggesting that it may be involved in synaptic transmissions (20). Another important natural β-amino acid is taurine (2-aminoethanesulfonic acid), which plays an important role in several essential processes, such as membrane stabilization, osmoregulation, glucose metabolism, antioxidation, and development of the central nervous system and the retina (9, 28, 33). 2-Aminoethylphosphonate, the most common naturally occurring phosphonate, also known as ciliatine, is an important precursor used in the biosynthesis of phosphonolipids, phosphonoproteins, and phosphonoglycans (5). β-Homoalanine (β-aminobutyric acid) has been used successfully for the design of nonnatural ligands for therapeutic application against autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, or autoimmune uveitis (30). Substituted β-amino acids can be denominated β², β³, and β^2,3, depending on the position of the side chain(s) (R) on the amino acid skeleton (18). β²-Amino acids are not yet as readily available as their β³-counterparts, as they must be prepared using multistep procedures (17).We decided to characterize NCβAA (β-carbamoylase) from Agrobacterium tumefaciens C58 (βcar_At) after showing that some dihydropyrimidinases belonging to the Arthrobacter and Sinorhizobium genera are able to hydrolyze different 5- or 6-substituted dihydrouracils to the corresponding N-carbamoyl-β-amino acids (18, 22). If βcar_At could decarbamoylate the reaction products of dihydrouracils, different β-amino acids would be obtained enzymatically in the same way that α-amino acids are produced via the hydantoinase process (6, 21). We therefore describe the physical, biochemical, kinetic, and substrate specificity properties of recombinant βcar_At.     

Utilization of Immunoglobulin G Fc Receptors by Human Immunodeficiency Virus Type 1: a Specific Role for Antibodies against the Membrane-Proximal External Region of gp41

Receptors (FcγRs) for the constant region of immunoglobulin G (IgG) are an important link between humoral immunity and cellular immunity. To help define the role of FcγRs in determining the fate of human immunodeficiency virus type 1 (HIV-1) immune complexes, cDNAs for the four major human Fcγ receptors (FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa) were stably expressed by lentiviral transduction in a cell line (TZM-bl) commonly used for standardized assessments of HIV-1 neutralization. Individual cell lines, each expressing a different FcγR, bound human IgG, as evidence that the physical properties of the receptors were preserved. In assays with a HIV-1 multisubtype panel, the neutralizing activities of two monoclonal antibodies (2F5 and 4E10) that target the membrane-proximal external region (MPER) of gp41 were potentiated by FcγRI and, to a lesser extent, by FcγRIIb. Moreover, the neutralizing activity of an HIV-1-positive plasma sample known to contain gp41 MPER-specific antibodies was potentiated by FcγRI. The neutralizing activities of monoclonal antibodies b12 and 2G12 and other HIV-1-positive plasma samples were rarely affected by any of the four FcγRs. Effects with gp41 MPER-specific antibodies were moderately stronger for IgG1 than for IgG3 and were ineffective for Fab. We conclude that FcγRI and FcγRIIb facilitate antibody-mediated neutralization of HIV-1 by a mechanism that is dependent on the Fc region, IgG subclass, and epitope specificity of antibody. The FcγR effects seen here suggests that the MPER of gp41 could have greater value for vaccines than previously recognized.Fc receptors (FcRs) are differentially expressed on a variety of cells of hematopoietic lineage, where they bind the constant region of antibody (Ab) and provide a link between humoral and cellular immunity. Humans possess two classes of FcRs for the constant region of IgG (FcγRs) that, when cross-linked, are distinguished by their ability to either activate or inhibit cell signaling (69, 77, 79). The activating receptors FcγRI (CD64), FcγRIIa (CD32), and FcγRIII (CD16) signal through an immunoreceptor tyrosine-based activation motif (ITAM), whereas FcγRIIb (CD32) contains an inhibitory motif (ITIM) that counters ITAM signals and B-cell receptor signals. It has been suggested that a balance between activating and inhibitory FcγRs coexpressed on the same cells plays an important role in regulating adaptive immunity (23, 68). Moreover, the inhibitory FcγRIIb, being the sole FcγR on B cells, appears to play an important role in regulating self-tolerance (23, 68). The biologic role of FcγRs may be further influenced by differences in their affinity for immunoglobulin G (IgG); thus, FcγRI is a high-affinity receptor that binds monomeric IgG (mIgG) and IgG immune complexes (IC), whereas FcγRIIa, FcγRIIb, and FcγRIIIa are medium- to low-affinity receptors that preferentially bind IgG IC (10, 49, 78). FcγRs also exhibit differences in their relative affinity for the four IgG subclasses (10), which has been suggested to influence the balance between activating and inhibitory FcγRs (67).In addition to their participation in acquired immunity, FcγRs can mediate several innate immune functions, including phagocytosis of opsonized pathogens, Ab-dependent cell cytotoxicity (ADCC), antigen uptake by professional antigen-presenting cells, and the production of inflammatory cytokines and chemokines (26, 35, 41, 48, 69). In some cases, interaction of Ab-coated viruses with FcγRs may be exploited by viruses as a means to facilitate entry into FcγR-expressing cells (2, 33, 47, 84). Several groups have reported FcγR-mediated Ab-dependent enhancement (ADE) of HIV-1 infection in vitro (47, 51, 58, 63, 94, 96), whereas other reports have implicated FcγRs in efficient inhibition of the virus in vitro (19, 21, 29, 44-46, 62, 98) and possibly as having beneficial effects against HIV-1 in vivo (5, 27, 28, 42). These conflicting results are further complicated by the fact that HIV-1-susceptible cells, such as monocytes and macrophages, can coexpress more than one FcγR (66, 77, 79).HIV-1 entry requires sequential interactions between the viral surface glycoprotein, gp120, and its cellular receptor (CD4) and coreceptor (usually CCR5 or CXCR4), followed by membrane fusion that is mediated by the viral transmembrane glycoprotein gp41 (17, 106). Abs neutralize the virus by binding either gp120 or gp41 and blocking entry into cells. Several human monoclonal Abs that neutralize a broad spectrum of HIV-1 variants have attracted considerable interest for vaccine design. Epitopes for these monoclonal Abs include the receptor binding domain of gp120 in the case of b12 (71, 86), a glycan-specific epitope on gp120 in the case of 2G12 (13, 85, 86), and two adjacent epitopes in the membrane-proximal external region (MPER) of g41 in the cases of 2F5 and 4E10 (3, 11, 38, 93). At least three of these monoclonal Abs have been shown to interact with FcRs and to mediate ADCC (42, 43).A highly standardized and validated assay for neutralizing Abs against HIV-1 that quantifies reductions in luciferase (Luc) reporter gene expression after a single round of virus infection in TZM-bl cells has been developed (60, 104). TZM-bl (also called JC53BL-13) is a CXCR4-positive HeLa cell line that was engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly Luc and Escherichia coli β-galactosidase under the control of the HIV-1 Tat-regulated promoter in the long terminal repeat terminal repeat sequence (74, 103). TZM-bl cells are permissive to infection by a wide variety of HIV-1, simian immunodeficiency virus, and human-simian immunodeficiency virus strains, including molecularly cloned Env-pseudotyped viruses. Here we report the creation and characterization of four new TZM-bl cell lines, each expressing one of the major human FcγRs. These new cell lines were used to gain a better understanding of the individual roles that FcγRs play in determining the fate of HIV-1 IC. Two FcγRs that potentiated the neutralizing activity of gp41 MPER-specific Abs were identified.     

Effective Simian Immunodeficiency Virus-Specific CD8+ T Cells Lack an Easily Detectable,Shared Characteristic

The immune correlates of human/simian immunodeficiency virus control remain elusive. While CD8⁺ T lymphocytes likely play a major role in reducing peak viremia and maintaining viral control in the chronic phase, the relative antiviral efficacy of individual virus-specific effector populations is unknown. Conventional assays measure cytokine secretion of virus-specific CD8⁺ T cells after cognate peptide recognition. Cytokine secretion, however, does not always directly translate into antiviral efficacy. Recently developed suppression assays assess the efficiency of virus-specific CD8⁺ T cells to control viral replication, but these assays often use cell lines or clones. We therefore designed a novel virus production assay to test the ability of freshly ex vivo-sorted simian immunodeficiency virus (SIV)-specific CD8⁺ T cells to suppress viral replication from SIVmac239-infected CD4⁺ T cells. Using this assay, we established an antiviral hierarchy when we compared CD8⁺ T cells specific for 12 different epitopes. Antiviral efficacy was unrelated to the disease status of each animal, the protein from which the tested epitopes were derived, or the major histocompatibility complex (MHC) class I restriction of the tested epitopes. Additionally, there was no correlation with the ability to suppress viral replication and epitope avidity, epitope affinity, CD8⁺ T-cell cytokine multifunctionality, the percentage of central and effector memory cell populations, or the expression of PD-1. The ability of virus-specific CD8⁺ T cells to suppress viral replication therefore cannot be determined using conventional assays. Our results suggest that a single definitive correlate of immune control may not exist; rather, a successful CD8⁺ T-cell response may be comprised of several factors.CD8⁺ T cells may play a critical role in blunting peak viremia and controlling human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication. The transient depletion of CD8⁺ cells in SIV-infected macaques results in increased viral replication (26, 31, 51, 70). The emergence of virus-specific CD8⁺ T cells coincides with the reduction of peak viremia (12, 39, 42, 63), and CD8⁺ T-cell pressure selects for escape mutants (6, 9, 13, 28, 29, 38, 60, 61, 85). Furthermore, particular major histocompatibility complex (MHC) class I alleles are overrepresented in SIV- and HIV-infected elite controllers (15, 29, 33, 34, 46, 56, 88).Because it has been difficult to induce broadly neutralizing antibodies (Abs), the AIDS vaccine field is currently focused on developing a vaccine designed to elicit HIV-specific CD8⁺ T cells (8, 52, 53, 82). Investigators have tried to define the immune correlates of HIV control. Neither the magnitude nor the breadth of epitopes recognized by virus-specific CD8⁺ T-cell responses correlates with the control of viral replication (1). The quality of the immune response may, however, contribute to the antiviral efficacy of the effector cells. It has been suggested that the number of cytokines that virus-specific CD8⁺ T cells secrete may correlate with viral control, since HIV-infected nonprogressors appear to maintain CD8⁺ T cells that secrete several cytokines, compared to HIV-infected progressors (11, 27). An increased amount of perforin secretion may also be related to the proliferation of HIV-specific CD8⁺ T cells in HIV-infected nonprogressors (55). While those studies offer insight into the different immune systems of progressors and nonprogressors, they did not address the mechanism of viral control. Previously, we found no association between the ability of SIV-specific CD8⁺ T-cell clones to suppress viral replication in vitro and their ability to secrete gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), or interleukin-2 (IL-2) (18).Evidence suggests that some HIV/SIV proteins may be better vaccine targets than others. CD8⁺ T cells recognize epitopes derived from Gag as early as 2 h postinfection, whereas CD8⁺ T cells specific for epitopes in Env recognize infected cells only at 18 h postinfection (68). Additionally, a previously reported study of HIV-infected individuals showed that an increased breadth of Gag-specific responses was associated with lower viral loads (35, 59, 65, 66). CD8⁺ T-cell responses specific for Env, Rev, Tat, Vif, Vpr, Vpu, and Nef were associated with higher viral loads, with increased breadth of Env in particular being significantly associated with a higher chronic-phase viral set point.None of the many sophisticated methods employed for analyzing the characteristics of HIV- or SIV-specific immune responses clearly demarcate the critical qualities of an effective antiviral response. In an attempt to address these questions, we developed a new assay to measure the antiviral efficacy of individual SIV-specific CD8⁺ T-cell responses sorted directly from fresh peripheral blood mononuclear cells (PBMC). Using MHC class I tetramers specific for the epitope of interest, we sorted freshly isolated virus-specific CD8⁺ T cells and determined their ability to suppress virus production from SIV-infected CD4⁺ T cells. We then looked for a common characteristic of efficacious epitope-specific CD8⁺ T cells using traditional methods.