Cross-Talk between TLR4 and FcγReceptorIII (CD16) Pathways

Pathogen-pattern-recognition by Toll-like receptors (TLRs) and pathogen clearance after immune complex formation via engagement with Fc receptors (FcRs) represent central mechanisms that trigger the immune and inflammatory responses. In the present study, a linkage between TLR4 and FcγR was evaluated in vitro and in vivo. Most strikingly, in vitro activation of phagocytes by IgG immune complexes (IgGIC) resulted in an association of TLR4 with FcγRIII (CD16) based on co-immunoprecipitation analyses. Neutrophils and macrophages from TLR4 mutant (mut) mice were unresponsive to either lipopolysaccharide (LPS) or IgGIC in vitro, as determined by cytokine production. This phenomenon was accompanied by the inability to phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) of the FcRγ-subunit. To transfer these findings in vivo, two different models of acute lung injury (ALI) induced by intratracheal administration of either LPS or IgGIC were employed. As expected, LPS-induced ALI was abolished in TLR4 mut and TLR4^−/− mice. Unexpectedly, TLR4 mut and TLR4^−/− mice were also resistant to development of ALI following IgGIC deposition in the lungs. In conclusion, our findings suggest that TLR4 and FcγRIII pathways are structurally and functionally connected at the receptor level and that TLR4 is indispensable for FcγRIII signaling via FcRγ-subunit activation.     

Increased Bone Marrow (BM) Plasma Level of Soluble CD30 and Correlations with BM Plasma Level of Interferon (IFN)-γ, CD4/CD8 T-Cell Ratio and Disease Severity in Aplastic Anemia

Idiopathic aplastic anemia (AA) is an immune-mediated bone marrow failure syndrome. Immune abnormalities such as decreased lymphocyte counts, inverted CD4/CD8 T-cell ratio and increased IFN-γ-producing T cells have been found in AA. CD30, a surface protein belonging to the tumor necrosis factor receptor family and releasing from cell surface as a soluble form (sCD30) after activation, marks a subset of activated T cells secreting IFN-γ when exposed to allogeneic antigens. Our study found elevated BM plasma levels of sCD30 in patients with SAA, which were closely correlated with disease severity, including absolute lymphocyte count (ALC) and absolute netrophil count (ANC). We also noted that sCD30 levels were positively correlated with plasma IFN-γ levels and CD4/CD8 T-cell ratio in patients with SAA. In order to explain these phenomena, we stimulated T cells with alloantigen in vitro and found that CD30⁺ T cells were the major source of IFN-γ, and induced CD30⁺ T cells from patients with SAA produced significantly more IFN-γ than that from healthy individuals. In addition, increased proportion of CD8⁺ T cells in AA showed enhanced allogeneic response by the fact that they expressed more CD30 during allogeneic stimulation. sCD30 levels decreased in patients responded to immunosuppressive therapy. In conclusion, elevated BM plasma levels of sCD30 reflected the enhanced CD30⁺ T cell-mediated immune response in SAA. CD30 as a molecular marker that transiently expresses on IFN-γ-producing T cells, may participate in mediating bone marrow failure in AA, which also can facilitate our understanding of AA pathogenesis to identify new therapeutic targets.     

Genetic polymorphisms of Fas (CD95) and Fas ligand (CD178) influence the rise in CD4+ T cell count after antiretroviral therapy in drug-naïve HIV-positive patients

Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus. To investigate whether the genetic background of the host plays a role in the immunoreconstitution, we studied the influence of different Fas and FasL polymorphisms on CD4+ T lymphocyte count and plasma viral load following initiation of highly active antiretroviral therapy (HAART) in drug-naïve HIV+ patients. We studied 131 individuals, who were compared to 136 healthy donors. Statistical analysis was performed by using X ² test, Fischer's Exact Test, and analysis for repeated measurements. The group of HIV+ patients had an unexpected lower frequency of FasLnt169 polymorphism (delT allele) than healthy controls (p=0.039). We then observed no significant differences in the immune reconstitution, in terms of CD4+ T cell increase, when the influence of single alleles of the gene Fas or FasL was considered. However, the combination of some polymorphisms of Fas or FasL significantly influenced CD4+ T cell production and viral load decrease, showing that these genes can play a role in the immunoreconstitution triggered by antiretroviral therapy.     

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.     

Synthesis and characterization of tris(β-diketonato)technetium(III) and -(IV) complexes

The preparations and properties of tris(dipivaloylmethanato)technetium(III), tris(trifluoroacetylacetonato)technetium(III), and tris(hexafluoroacetonato)technetium(III) are described. The oxidation of the dipivaloyl derivative to tris(dipivaloyl)technetium(IV) hexafluorophosphate was shown to take place readily. Voltammetric studies and magnetic resonance results on the new complexes are reported. The large shifts observed for the complexes seem to be due to a contact interaction.     

Syntheses of (±)-Epoformin (Desoxyepoxydon) and (±)-Epiepoformin (Desoxyepiepoxydon)

Boron is an essential nutrient for plants, but it is toxic in excess. Transgenic rice plants expressing an Arabidopsis thaliana borate efflux transporter gene, AtBOR4, at a low level exhibited increased tolerance to excess boron. Those lines with high levels of expression exhibited reduced growth. These findings suggest a potential of the borate transporter BOR4 for the generation of high-boron tolerant rice.     

Co-localization of CD9 and GPIIb-IIIa (·IIb ·3 integrin) on activated platelet pseudopods and ·-granule membranes

Summary CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of ·-granule membranes. These two antigens also co-localized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from ·-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets. This revised version was published online in November 2006 with corrections to the Cover Date.     

Synthesis of (±)-Homosarkomycin and (±)-Rosaprostol

(±)-Homosarkomycin (2) and (±)-rosaprostol (3) were synthesized from (±)-methyl 2-oxo-bicyclo[3.1.0]hexane-1-carboxylate (1) by using the nucleophilic ring opening reaction on the double-activated cyclopropane ring as the key step.     

Synthesis of (±)-Lamprolobine and (±)-Epilamprolobine

(±)-Lamprolobine, the (+)-enatiomer of which was isolated from the leaves of Lamprolobium fruticosum, and (±)-epilamprolobine were synthesized from δ-valerolactam.     

Cytotoxic functions and susceptibility to apoptosis of human CD56(bright) NK cells differentiated in vitro from CD34? hematopoietic progenitors

Cytotoxic functions and susceptibility to apoptosis are crucial aspects of NK cells suitable to counter cancer after infusion in oncologic patients. To test the feasibility and the usefulness of infusing in vitro generated NK cells, these two features were investigated in NK cells developed in vitro from CD34? hematopoietic progenitors. Purified CD34? cells were cultured for 15-30 days with FLT-3 ligand (FLT3-L) and IL-15 with or without IL-21. To induce terminal differentiation, NK cells were cultured for further 15 days with IL-15, IL-21, or their combination. A CD56(dim) /CD16? NK subset, expressing high level of perforin, granzymes, and LFA-1, appeared early in cultures with FLT3-L, IL-15, and IL-21, but it quickly died, indicating its predisposition to apoptosis. On the contrary, CD56(bright) NK cells generated after 30 days of culture with FLT3-L plus IL-15 did not show a considerable apoptosis, nevertheless only a subset of these cells expressed granzyme-B, perforin, LFA-1, and CD94-CD159a heterodimer, indicating a functional immaturity. Interestingly, further 15 days of culture with IL-21 plus IL-15 did not induce the generation of CD56(dim) cells from the CD56(bright) subset and actually inhibited IL-15-induced maturation/activation of this latter subset. In fact, IL-15 alone upregulated granzyme-B, TRAIL, Fas ligand, CD94-CD159a, LFA-1, CD16, KIRs, and TRAIL-R2 on CD56(bright) NK cells. Our results suggest that during differentiation CD56(bright) NK cells, similarly to mature activated NK cells, become highly cytotoxic and are relatively resistant to apoptosis induced by TNF family members.     

Synthesis and characterization of β-diketiminate germanium(II) and tin(II) bromides

The equimolar reaction of a β-diketiminate lithium salt LLi(OEt₂) [L = HC(CMeNAr)₂; Ar = 2,6-iPr₂C₆H₃] with either GeBr₂ or SnBr₂ in diethyl ether affords the synthetically useful monomeric β-diketiminate-element halides LGeBr (1) and LSnBr (2), respectively. Both are soluble in hydrocarbon solvents, stable in inert atmosphere, and have been characterized by elemental analysis, NMR spectroscopy, and single-crystal X-ray diffraction analysis.     

Inhibition and Activation by CD244 Depends on CD2 and Phospholipase C-��1

Regulation by the NK and T cell surface receptor CD244 in mice and humans depends both on engagement at the cell surface by CD48 and intracellular interactions with SAP and EAT-2. Relevance to human disease by manipulating CD244 in mouse models is complicated by rodent CD2 also binding CD48. We distinguish between contributions of mouse CD244 and CD2 on engagement of CD48 in a mouse T cell hybridoma. CD2 and CD244 both contribute positively to the immune response as mutation of proline-rich motifs or tyrosine motifs in the tails of CD2 and CD244, respectively, result in a decrease in antigen-specific interleukin-2 production. Inhibitory effects of mouse CD244 are accounted for by competition with CD2 at the cell surface for CD48. In humans CD2 and CD244 are engaged separately at the cell surface but biochemical data suggest a potential conserved intracellular link between the two receptors through FYN kinase. We identify a novel signaling mechanism for CD244 through its potential to recruit phospholipase C-γ1 via the conserved phosphorylated tyrosine motif in the tail of the adaptor protein EAT-2, which we show is important for function.The CD2 family of cell surface receptors is differentially expressed on immune cells (1, 2) and is involved in regulating both innate and adaptive immunity (3). These receptors have related extracellular immunoglobulin superfamily domains and interact either homophilically or heterophilically within the CD2 family (1, 2). The CD2 family contains a subgroup of receptors termed the SLAM family that have a conserved tyrosine signaling motif in their cytoplasmic region TXYXX(I/V) referred to as an immunoreceptor tyrosine-based switch motif (ITSM).² The SLAM family of receptors include CD244 (2B4), NTB-A (Ly-108), CD319 (CRACC, CS-1), CD150 (SLAM), CD84, and CD229 (Ly-9). Defects in signaling and aberrant expression of these receptors have been implicated in several immunodeficiency and autoimmune disorders in humans and mice (4–8). Within the SLAM family, CD244 is unusual in that it shares its ligand CD48 with the receptor CD2 in rodents, whereas in humans CD2 has evolved to interact with CD58 (9). The affinity of CD244 for CD48 in rodents is 6–9-fold higher than the still functionally relevant CD2/CD48 interaction (10). CD244 and CD2 have different cytoplasmic regions comprised of tyrosine motifs or proline-rich motifs, respectively.CD244 is predominantly found on NK cells and cytotoxic T cells and primarily characterized as an activating receptor (11–15). CD2 is found on the same cells as CD244 but is also expressed on all T cells, both activated and resting, and has an activating or costimulatory function upon engagement of ligand (9). The tyrosine motifs found in the cytoplasmic tail of CD244 have been shown to bind the SH2 domains of cytoplasmic adaptor proteins SAP and EAT-2 and FYN kinase (16–18) and are important to its function (5, 19–21). In contrast to SH2 interactions of CD244, several SH3 domain-mediated interactions have been reported for the cytoplasmic region of CD2 including CD2AP/CMS, CIN85, FYN, and LCK (22–26).The activating function of CD244 was called into question when a study using cells from a CD244 knock-out mouse showed that CD244 had an inhibitory effect as loss of CD244 resulted in enhanced NK killing of target cells (27). This suggested that previous results in mice where positive effects were seen may have been due to blocking CD244 ligand engagement as opposed to cross-linking with antibodies against CD244 (27). This has led to proposals that there are differences in function between mouse and human CD244 as there is more evidence to suggest that human CD244 is a positive regulator enhancing cytotoxicity and cytokine production (13, 15, 28). However, other more recent studies have shown the mouse CD244/CD48 interaction to be important for cytokine production and effector functions such as cytotoxicity against tumor targets in CD244-deficient mice (29). Long and short forms of CD244 have been cloned from mice with the short form being described as activating and the long form inhibitory (27, 30). Only the long form of CD244 is present in humans and is regarded as activating (14).Positive signaling by CD244 has been attributed to the recruitment of SAP (18), which is a signaling adaptor molecule comprised of a single SH2 domain encoded by the SH2D1A gene and has the ability to recruit the kinase FYN by binding its SH3 domain (31, 32). Loss of the SAP/FYN interaction can lead to X-linked lymphoproliferative disease in humans (17). The molecular basis of in vitro inhibitory effects observed with CD244 in mice on ligation with mAb or ligand remains elusive (33). Protein tyrosine and inositol phosphatases have been reported to associate with CD244 (18, 19, 34) but our studies using surface plasmon resonance found them to be very weak and unlikely to bind competitively compared with the SAP family of adaptors or FYN (16). The SAP-related adaptor EAT-2 has been reported to have an active inhibitory effect that is dependent on tyrosine motifs in the tail of EAT-2 (35) but its mechanism is not understood. The only interaction reported for the tail of EAT-2 is with FYN kinase and studies overexpressing EAT-2 in a T cell hybridoma resulted in increased IL-2 production upon antigen stimulation (16).The conservation between mouse and human CD244 cytoplasmic regions and associated adaptors suggests that both function in a similar way. We have explored the main difference between mouse and human CD244, which is the extracellular interaction through CD48 ligation in the mouse. This has revealed that inhibitory effects of CD244 ligation in mice can be due to competition between CD244 and CD2 for CD48. We have also found that the adaptor protein EAT-2 binds PLCγ1 providing a molecular basis for the important role CD244 plays in regulating cellular cytotoxicity (13, 36). We demonstrate that there is a potentially shared signaling mechanism through the FYN kinase that links CD2 and CD244 intracellularly even though in humans CD2 and CD244 no longer share a cell surface ligand.     

The leucocyte β2 (CD18) integrins: the structure, functional regulation and signalling properties

Leucocytes are highly motile cells. Their ability to migrate into tissues and organs is dependent on cell adhesion molecules. The integrins are a family of heterodimeric transmembrane cell adhesion molecules that are also signalling receptors. They are involved in many biological processes, including the development of metazoans, immunity, haemostasis, wound healing and cell survival, proliferation and differentiation. The leucocyte-restricted β2 integrins comprise four members, namely αLβ2, αMβ2, αXβ2 and αDβ2, which are required for a functional immune system. In this paper, the structure, functional regulation and signalling properties of these integrins are reviewed.     

Praziquantel facilitates IFN-γ-producing CD8+ T cells (Tc1) and IL-17-producing CD8+ T cells (Tc17) responses to DNA vaccination in mice

Background

CD8⁺ cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.

Methodology/Principal Findings

Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8⁺ T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8⁺ T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8⁺ T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.

Conclusions/Significance

Our results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8⁺ T cell-based immunotherapy that breaks tolerance to HBsAg.