PD-1 signaling in HIV and chronic viral infection--potential for therapeutic intervention?

Programmed death-1 (PD-1) is a negative immunoregulatory cell surface receptor molecule whose interaction with its ligands PD-L1 and PD-L2 downmodulates T-cell immune responses. Originally investigated in the context of self-tolerance, PD-1 has more recently been discovered to be upregulated on T cells of HIV-infected individuals. High levels of PD-1 on HIV-infected T cells are correlated with viral load and with a state of cellular anergy, or ' exhaustion' that results in decreased cellular proliferation, cytotoxic function and cytokine secretion. The finding that interruption of PD-1 with its ligand PD-L1 rescues HIV-infected cells from this state of anergy or ' exhaustion' presents the promise for therapeutic intervention. Understanding the molecular signaling pathway(s) of PD-1 may provide opportunities for therapeutic intervention, that may serve as adjunctive therapies to HIV vaccine development. Evidence to date suggests that PD-1 exerts its regulatory effect by interfering with T cell receptor signaling. While certain molecular signals in the PD-1 pathway have been identified, their precise roles and mechanisms of action remain poorly understood. This article reviews what is currently known about PD-1 signaling in human T cells, and more specifically in T cells of individuals chronically infected with certain viruses such as HIV.

A three-item scale for the early prediction of stroke recovery.

BACKGROUND: Accurate assessment of prognosis in the first hours of stroke is desirable for best patient management. We aimed to assess whether the extent of ischaemic brain injury on magnetic reasonance diffusion-weighted imaging (MR DWI) could provide additional prognostic information to clinical factors. METHODS: In a three-phase study we studied 66 patients from a North American teaching hospital who had: MR DWI within 36 h of stroke onset; the National Institutes of Health Stroke Scale (NIHSS) score measured at the time of scanning; and the Barthel Index measured no later than 3 months after stroke. We used logistic regression to derive a predictive model for good recovery. This logistic regression model was applied to an independent series of 63 patients from an Australian teaching hospital, and we then developed a three-item scale for the early prediction of stroke recovery. FINDINGS: Combined measurements of the NIHSS score (p=0.01), time in hours from stroke onset to MR DWI (p=0.02), and the volume of ischaemic brain tissue on MR DWI (p=0.04) gave the best prediction of stroke recovery. The model was externally validated on the Australian sample with 0.77 sensitivity and 0.88 specificity. Three likelihood levels for stroke recovery-low (0-2), medium (3-4), and high (5-7)-were identified on the three-item scale. INTERPRETATION: The combination of clinical and MR DWI factors provided better prediction of stroke recovery than any factor alone, shortly after admission to hospital. This information was incorporated into a three-item scale for clinical use.

Reduced binding and phagocytosis of Pneumocystis carinii by alveolar macrophages from persons infected with HIV-1 correlates with mannose receptor downregulation.

The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIV-infected individuals to bind and phagocytose P. carinii, and to investigate the role of the macrophage mannose receptor in mediating this interaction. Compared with healthy individuals, alveolar macrophage phagocytosis of P. carinii from HIV+ persons was reduced up to 74% (P = 0.02), primarily reflecting a reduction in the number of organisms associated with each macrophage (P = 0.019). Furthermore, macrophages from HIV+ individuals demonstrated up to an 80% (P < 0.05) reduction in mannose receptor surface expression and endocytosis. Mannose receptor affinity was unaltered, and mRNA levels were modestly reduced (P < 0.05). Cells from HIV+ individuals with CD4(+) counts < 200 cells/mm3 (representing individuals at high clinical risk for P. carinii pneumonia) demonstrated the lowest levels of P. carinii phagocytosis and mannose receptor endocytosis. In vitro HIV infection of alveolar macrophages from healthy individuals reduced mannose receptor endocytosis to 53.2% (P < 0.05) and P. carinii binding and phagocytosis to 67.4% (P < 0.05) of control. Our studies suggest that HIV infection may alter innate immunity in the lungs, and that impaired alveolar macrophage mannose receptor-mediated binding and phagocytosis of P. carinii may contribute to the susceptibility of HIV-infected individuals to this opportunistic pulmonary pathogen.

Murine macrophage mannose receptor promoter is regulated by the transcription factors PU.1 and SP1.

The mannose receptor (MR) is a transmembrane protein that functions primarily as a phagocytic receptor for a wide range of microorganisms. Its expression appears to be restricted to tissue macrophages and Langerhans cells. To gain an understanding of the regulation of the gene, we have isolated the 5' flanking sequence of the murine MR gene and have analyzed a 536-bp sequence upstream of the ATG start site for transcriptional activity. This sequence lacks a TATA box but contains an initiator (Inr) consensus element overlapping the single transcriptional start site. Transcription factor binding sites contained within this sequence include PU.1, Sp1, ETS, GATA, and MYB motifs. Serial 100-bp deletions of this promoter fragment fused to a luciferase reporter gene showed various patterns of activity when transfected into different cell types. In myeloid cells, sequence elements upstream of bp -300 appeared to have a silencing effect on promoter activity. Of the four potential PU.1 binding sites contained within the fragment, one site (at -164) bound the PU.1 factor most strongly, whereas the adjacent PU.1 site (at -177 bp) bound PU.1 to a lesser degree. Mutations of these sites decreased transcriptional activity but did not abolish it. However, promoter activity was abrogated when both the -164 bp PU.1 site and the adjacent -177 bp PU.1 site were mutated. In addition, mutation of the Sp1 site also significantly reduced promoter activity. Cotransfection studies in Drosophila Schneider cells indicated that PU.1 and Sp1 may function synergistically in transactivating the murine MR. This study indicates that MR gene expression is regulated in part by the interaction between the ubiquitously expressed factor Sp1 and the lymphoid/myeloid factor PU.1 and provides a basis for studying the regulation of this gene.

p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases.

Cdc25A, a phosphatase essential for G1-S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor beta (TGF-beta). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.

The collectins in innate immunity.

The collectins are proteins with collagen tails and globular lectin domains that appear to play an important role in mammalian first line host defense. Recent insights have clarified the structural basis of ligand recognition, the interactions of collectins with complement cascades, and the association with disease susceptibility.

Rheumatic fever: autoantibodies against a variety of cardiac, nuclear, and streptococcal antigens.

OBJECTIVE: To measure antibody titres to cardiac, nuclear, and streptococcal antigens in different groups of rheumatic fever (n = 60) and control subjects (n = 80) with the aim of identifying cross reactive antigens of potential laboratory diagnostic value. METHODS: Enzyme linked immunosorbent assays (ELISA), immunocytochemical, and electrophoretic techniques were used to measure titres of antibodies to a variety of cardiac, nuclear, and streptococcal antigens in seven groups comprising patients with rheumatic fever and control subjects. RESULTS: Increased concentrations of antibodies to several streptococcal and cardiac antigens, in addition to increased IgA and IgG levels, were noted in sera from patients with acute rheumatic fever and chronic rheumatic heart disease. Autoantibodies to nuclear antigens were evident in three rheumatic fever sera. CONCLUSION: Although we were unable to identify any unique cross reactivity between cardiac and streptococcal antigens, these results demonstrate that there is an exaggerated humoral response to several cardiac, nuclear and streptococcal antigens in patients with rheumatic fever.

Restriction of interferon gamma responsiveness and basal expression of the myeloid human Fc gamma R1b gene is mediated by a functional PU.1 site and a transcription initiator consensus.

The restricted expression of the human Fc gamma R1b gene to myeloid cells is likely to be regulated by a combination of transcription factors that may not be solely expressed in myeloid cells, but act together to restrict the expression of the gene to myeloid cells. Low basal expression of the human Fc gamma R1b gene is specifically upregulated by interferon gamma (IFN-gamma). A 181-bp region of 5' flanking sequence contains several key regulatory motifs that include the extended gamma response region (XGRR) and the PIE region. The XGRR contains the 39-bp gamma response region originally defined in the highly homologous Fc gamma R1a gene. The XGRR is in close proximity to the 21-bp PIE motif that is conserved in the promoters of some other myeloid genes. The PIE motif contains a consensus site for the macrophage and B cell transcription factor, PU.1, and is adjacent to the cluster of transcription start sites. An active transcription initiator, Inr, consensus spans the start sites and appears to direct transcription initiation of this TATA-less gene. In this study, we demonstrate that the PIE region contains a functional PU.1 site that binds a human PU.1-like protein and that associated factors present in myeloid extracts also bind in this PIE region. Mutational analysis reveals an absolute requirement for an intact PU.1 box for both basal and IFN-gamma inducible expression of this gene. In addition, mutations in the Inr greatly reduce basal and inducible transcription. Insertion of a strong TATA box downstream from the Inr or at -30 bp from the transcription start sites restores basal and inducible activity in the presence of a mutated PU.1 site. We also demonstrate that indeed, when the XGRR is positioned in the context of a heterologous TATA containing promoter, it is able to respond equivalently to either IFN-alpha or IFN-gamma. However, IFN-alpha responsiveness does not occur in the context of the physiological Fc gamma R1b TATA-less basal promoter. Our results suggest that a human PU.1-like factor acts as a "bridging factor" between the upstream IFN-gamma enhancer and the Inr dependent preinitiation complex. These findings indicate that the structure of the basal promoter in combination with restricted activators like PU.1 are important in regulating the expression of this gene.

Identification of cardiac autoantigens in human heart cDNA libraries using acute rheumatic fever sera.

Antigenic mimicry or cross-reactivity between Group A streptococcal antigens and cardiac autoantigens may initiate an autoimmune response resulting in cardiovascular damage in acute rheumatic fever. This study describes a molecular biological approach to the identification of such cross-reactive cardiac antigens. Two human heart cDNA libraries were constructed in the expression vector lambda gt11 and screened with patient sera, monoclonal antibodies and rabbit immune sera cross-reactive with streptococcal and cardiac antigens. Using the serum of a patient with a recurrent acute attack of rheumatic fever containing high titres of antibodies cross-reactive with both sets of antigens, we were able to identify three positive clones with insert sizes of 1.0 kb, 1.4 kb and 0.9 kb in these libraries. Acute rheumatic fever (ARF) sera reacted more strongly with these autoantigen clones than did normal sera. Autoantibodies eluted from the purified plaques of all three clones displayed different patterns of cross-reactivity against immunoblots of streptococcal M5, M6, M19 and M24 protein extracts. The cDNA inserts were sequenced and compared with known sequences in the EMBL and Genbank databases. One clone was 98% homologous with human cytokeratin 8 and showed homologies of 40 to 50% with human cardiac heavy chain myosin, tropomyosin and streptococcal M5 protein--all members of the alpha-helical coiled-coil family of proteins. Another clone was completely homologous to the G-protein alpha-subunit of adenyl cyclase, whilst the sequence of the third clone was not found in any of the data banks.

The exon-intron structure and chromosomal localization of the mouse macrophage mannose receptor gene Mrc1: identification of a Ricin-like domain at the N-terminus of the receptor.

The macrophage mannose receptor is a transmembrane protein that is expressed on the surface of mature macrophages. The ectodomain of the receptor contains multiple domains, eight of which belong to the calcium-dependent C-type lectin family. The mannose receptor binds to carbohydrate polymers that have a high content of mannose. This property allows this protein to function as a phagocytic receptor that participates in first-line host defense against invading microorganisms. In this paper we describe the intron-exon structure of the mouse macrophage mannose receptor gene which was found to span at least 70 kilobases. We also report the localization of this gene, termed Mrc1, to mouse Chromosome 2. Like its human counterpart, Mrc1 contains 30 exons and 29 introns. A protein module that resembles a subdomain of the B chain of the plant lectin Ricin has been found within the N-terminal cysteine-rich domain of the mannose receptor.

Definition of interferon gamma-response elements in a novel human Fc gamma receptor gene (Fc gamma RIb) and characterization of the gene structure.

The human Fc gamma RI (CD64) is a high affinity receptor for the Fc portion of immunoglobulin (Ig), and its constitutively low expression on the cell surface of monocyte/macrophage and neutrophils is selectively upregulated by interferon gamma (IFN-gamma) treatment (Perussia, B., E. T. Dayton, R. Lazarus, V. Fanning, and G. Trinchieri. 1983. J. Exp. Med. 158:1092). Three distinct cDNAs have been cloned and code for proteins that predict three extracellular Ig-like domains (Allen, J.M., and B. Seed. 1989. Science [Wash. DC]. 243:378). Several differences in the coding region of these cDNAs suggest that in addition to polymorphic differences a second Fc gamma RI gene could possibly exist. This alternative Fc gamma RI gene (Fc gamma RIb) was defined by the lack of a genomic HindIII restriction site (van der Winkel, J. G. J., L. U. Ernst, C. L. Anderson, and I. M. Chiu. 1991. J. Biol. Chem. 266:13449). We describe the characterization a second gene (Fc gamma RIb) that has a termination codon in the third extracellular domain and therefore predicts a soluble form of a termination codon in the third extracellular domain and therefore predicts a soluble form of the receptor. We also define two distinct IFN-gamma-responsive regions in the 5' flanking sequence of Fc gamma RIb that resemble motifs that have been defined in the class II major histocompatibility complex promoter. The Fc gamma RIb promoter does not possess canonical TATA or CCAAT boxes, but does possess a palindromic motif that closely resembles the initiator sequence identified in the terminal deoxynucleotidyl transferase/human leukocyte IFN/adeno-associated virus type II P5 gene promoters (Smale, S. T., and D. Baltimore. 1989. Cell. 57:103; Seto, E., Y. Shi, and T. Shenk. 1991. Nature [Lond.]. 354:241; Roy, A. L., M. Meisterernst, P. Pognonec, and R. C. Roeder. 1991. Nature [Lond.]. 354:245) virus type II P5 gene promoters raising interesting questions as to its role in the basal and myeloid-specific transcription of this gene.

Human monoclonal antibodies: analysis of two antibodies derived from lymphocytes of a patient with acute rheumatic fever.

Human monoclonal antibodies were produced by fusion of peripheral blood lymphocytes from a patient with acute rheumatic fever, with the HGPRT-non-secreting murine (Balb-c) cell line SP2/0Ag14. Heterohybridomas were selected by screening against rheumatic fever-associated group A streptococci using an ELISA, and against paraffin wax-embedded human heart sections using an immunoperoxidase technique. Two human IgM monoclonal antibodies were selected for further analysis by Western blotting and ELISA. Both antibodies demonstrated multispecificity by immunoblotting and ELISA. One of the monoclonals bound to 48 kD and 83 kD bands common to group A streptococcal and heart antigen preparations. Both human monoclonal antibodies bound to a 43 kD constituent band common to human heart and sarcolemma membrane extract. Inhibition studies performed using a competitive solid phase immunoassay confirmed shared epitopes between group A streptococci and human heart. The significance of these monoclonal antibodies to the pathogenesis of rheumatic fever is uncertain.