Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment.

The major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response is important in the clearance of viral infections in humans. After influenza A infection, a peptide from the matrix protein, M58-66, is presented in the context of the MHC allele HLA-A0201 and the resulting CTL response is detectable in most HLA-A0201 subjects. An initial study suggested that M58-66-specific CTL clones show conserved T cell receptor (TCR) alpha and beta gene segments. We have addressed the significance of this observation by determining the expression of V beta 17 during the development of M58-66-specific CTL lines in 21 unrelated HLA-A0201 subjects, and analyzing TCR usage by M58-66-specific CTL clones. TCR V beta 17 was the dominant V beta segment used and CD8 V beta 17 expansion correlated with M58-66-specific lysis. Limiting dilution analysis from five subjects showed the M58-66 CTL precursor frequency to vary between 1/54,000 and less than 1/250,000, and that up to 85% of the matrix peptide (M58-66)-specific CTL used the V beta 17 gene segment. The M58-66 specific CTL response was dependent on previous viral exposure and specific V beta 17 expansion, as it was not found in cord blood, despite a readily expandable V beta 17+ CD8+ T cell subpopulation. Sequence analysis of 38 M58-66-specific V beta 17 transcripts from 13 subjects revealed extensive conservation in the CDR3 region including conservation of an arginine-serine motif. To test the dependence of this CTL response on the V beta 17 gene segment, peripheral blood lymphocytes were depleted of CD8+ TCR V beta 17+ cells, before the generation of M58-66-specific CTL. In most cases such depletion blocked or severely reduced the generation of the M58-66-specific response, and under limiting dilution conditions could abolish M58-66-specific CTL precursors. These studies reveal the dependence of this natural human immune response on a particular TCR gene segment.

A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes.

Newly assembled major histocompatibility complex (MHC) class I molecules, together with the endoplasmic reticulum chaperone calreticulin, interact with the transporter associated with antigen processing (TAP) through a molecule called tapasin. The molecular cloning of tapasin revealed it to be a transmembrane glycoprotein encoded by an MHC-linked gene. It is a member of the immunoglobulin superfamily with a probable cytoplasmic endoplasmic reticulum retention signal. Up to four MHC class I-tapasin complexes were found to bind to each TAP molecule. Expression of tapasin in a negative mutant human cell line (220) restored class I-TAP association and normal class I cell surface expression. Tapasin expression also corrected the defective recognition of virus-infected 220 cells by class I-restricted cytotoxic T cells, establishing a critical functional role for tapasin in MHC class I-restricted antigen processing.

Orthotopic liver transplantation for subacute hepatic failure following partial treatment of isoniazid-resistant tuberculosis.

The management of patients with pre-existing tuberculosis (TB) undergoing liver transplantation is challenging. Cautious immunosuppression is required to prevent reactivation of disease, and second-line anti-tuberculous treatment may be necessary to prevent graft hepatotoxicity. Furthermore, liver transplantation in the context of isoniazid-resistant TB has seldom been reported. We report on a 44-year-old man with recent isoniazid-resistant extra-pulmonary TB who developed subacute hepatic failure requiring emergency liver transplantation and treatment with second-line anti-tuberculous therapy. We demonstrate that patients who have pre-existing TB can be successfully treated with alternative anti-tuberculous medication while under immunosuppression post transplantation. Pre-existing TB, including resistant strains, should not be an absolute contraindication to liver transplantation.

Antigen presentation: coming out gracefully.

Efficient assembly of antigen-presenting class I MHC molecules requires the formation of a complex between the class I molecule and the TAP peptide transporter. The complex has been found to contain an additional four proteins, which help to ensure optimal peptide loading onto the class I molecules.

Antigen presentation: peptides and proteins scramble for the exit.

The fate of peptides that fail to bind to major histocompatibility complex class I molecules in the endoplasmic reticulum (ER)has remained unclear. A recent study has revealed that these peptides exit the ER via the Sec61 channel and compete for this pathway with misfolded proteins.

Antigen presentation: TAP dances with ATP.

Assembly of antigen-presenting complexes between class I MHC molecules and peptide requires formation of a complex between the 'ABC' peptide transporter, TAP, and newly synthesized class I molecules. Recent studies have provided new insights into the role of ATP in peptide binding, transport and release.

Processing and delivery of peptides presented by MHC class I molecules.

Effective MHC class I peptide loading requires the proteolytic degradation of cytosolic proteins and the TAP-mediated translocation of peptides across the membrane of the endoplasmic reticulum. The proteasome is emerging as the main cytosolic protease generating class I binding peptides. The recent elucidation of the proteasome crystal structure, together with the use of functional inhibitors, has enhanced our understanding of proteasome function. Genetic analysis of a novel mutant cell line emphasizes the importance of the TAP-class I interaction in the assembly of mature class I heterotrimers, and suggests that additional MHC-encoded components are required.

Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries.

UNLABELLED: Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. IMPORTANCE: Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.

Identifying the ERAD ubiquitin E3 ligases for viral and cellular targeting of MHC class I.

The human cytomegalovirus (HCMV) US2 and US11 gene products hijack mammalian ER-associated degradation (ERAD) to induce rapid degradation of major histocompatibility class I (MHC-I) molecules. The rate-limiting step in this pathway is thought to be the polyubiquitination of MHC-I by distinct host ERAD E3 ubiquitin ligases. TRC8 was identified as the ligase responsible for US2-mediated MHC-I degradation and shown to be required for the cleavage-dependent degradation of some tail-anchored proteins. In addition to MHC-I, plasma membrane profiling identified further immune receptors, which are also substrates for the US2/TRC8 complex. These include at least six α integrins, the coagulation factor thrombomodulin and the NK cell ligand CD112. US2's use of specific HCMV-encoded adaptors makes it an adaptable viral degradation hub. US11-mediated degradation is MHC-I-specific and genetic screens have identified TMEM129, an uncharacterised RING-C2 E3 ligase, as responsible for US11-mediated degradation. In a unique auto-regulatory loop, US11 readily responds to changes in cellular expression of MHC-I. Free US11 either rebinds more MHC-I or is itself degraded by the HRD1/SEL1L E3 ligase complex. While virally encoded US2 and US11 appropriate mammalian ERAD, the MHC-I complex also undergoes stringent cellular quality control and misfolded MHC-I is degraded by the HRD1/SEL1L complex. We discuss the identification and central role of E3 ubiquitin ligases in ER quality control and viral degradation of the MHC-I chain.

Tartrate-resistant acid phosphatase (Acp 5): identification in diverse human tissues and dendritic cells.

Histochemical demonstration of tartrate-resistant acid phosphatase (TRAP) is used for the specific identification of osteoclasts. The enzyme, which we have shown to be critical for normal bone development in mice, is also characteristic of monohistiocytes, including alveolar macrophages, and is associated with diverse pathological conditions such as Gaucher's disease and hairy cell leukemia. TRAP activity is enhanced in serum when bone resorption is increased, and the activity is used routinely to monitor treatment responses in Gaucher's disease. We have lately shown widespread expression of the enzyme in murine tissues with particular reference to the skin, thymus, gut epithelia, and isolated dendritic cells, suggesting a possible role in immunity. To further clarify the significance of TRAP in human physiology, we have examined its distribution in non-skeletal human tissues and in CD34+ -derived human dendritic cells. TRAP mRNA determined by Northern blotting analysis was expressed abundantly in spleen, liver, colon, lung, small intestine, kidney, stomach, testis, placenta, lymph node, thymus, peripheral blood leukocyte, bone marrow, and fetal liver. Expression of TRAP protein was investigated by immunohistochemistry, with which the enzyme was identified in multiple tissues. Histochemical staining detected enzymatically active protein in spleen, lung, skin, colon, stomach, and ileum. Active TRAP was identified in CD34+ -derived immature dendritic cells and co-localized to intracellular CD63 positive organelles. When these cells were matured by induction with LPS, the TRAP activity increased fivefold and remained within the cell during the phase associated with CD63 surface expression. Our findings demonstrate widespread expression of TRAP in human tissues. Its abundant expression in epithelia and dendritic cells suggests a potential role in antigen processing and in immune responses.

Dual infection with Pneumocystis carinii and respiratory viruses complicating bone marrow transplantation.

Pneumonia due to dual infection with Pneumocystis carinii and respiratory viruses is a rare but formidable complication of bone marrow transplantation. We report here two cases of viral infections complicating P. carinii pneumonia in bone marrow transplant recipients who, at the time of infection, were not taking P. carinii prophylaxis. Both patients died following the pneumonia. Potential factors contributing to the dual infection included graft-versus-host disease, high-dose steroids and cyclosporin A. P. carinii prophylaxis should be continued for 12 months, or longer in bone marrow transplant recipients requiring prolonged immunosuppressive therapy. As specific antiviral therapy becomes available for some respiratory viral infections, performing regular viral surveillance cultures and responding with active early treatment may help improve the outcome in these immunocompromised patients.

Congenital malaria in Papua New Guinea.

The incidence of malarial infection in pregnant women at delivery, their corresponding infants and umbilical cords and a control group of non-pregnant women were investigated in the Madang region of Papua New Guinea. Anti-malarial antibody titres were measured in maternal and paired cord sera. Parasitaemia occurred in 18/73 (24.7%) of non-pregnant females compared with 15/51 (29.4%) of pregnant females. Malarial parasites were found in 7/48 (14.6%) cord blood samples and in 4/52 (7.7%) samples of the infant's peripheral blood, indicating transplacental transmission. Infection with Plasmodium falciparum was commoner in pregnant than non-pregnant females, and accounted for all the cord and infant infections. A significant correlation was found between anti-malarial IgG antibodies in paired maternal and cord bloods. There was an association between umbilical cord infection and low levels of cord antibody. Clinical malaria developed in at least one out of the 7 cases in which placental transfer of parasites was known to have occurred. This study suggests that transfer of parasites across the placenta is a common event in Papua New Guinea. Further consideration should be given to treatment with anti-malarial drugs of infants with cord or peripheral blood parasitaemia or, indeed, of all infants of mothers with parasitaemia.

An in vitro model for the regulation of human cytomegalovirus latency and reactivation in dendritic cells by chromatin remodelling.

Human cytomegalovirus (HCMV) is a frequent cause of major disease following primary infection or reactivation from latency in immunocompromised patients. Infection of non-permissive mononuclear cells is used for analyses of HCMV latency in vitro. Using this approach, it is shown here that repression of lytic gene expression following experimental infection of CD34+ cells, a site of HCMV latency in vivo, correlates with recruitment of repressive chromatin around the major immediate-early promoter (MIEP). Furthermore, long-term culture of CD34+ cells results in carriage of viral genomes in which the MIEP remains associated with transcriptionally repressive chromatin. Finally, specific differentiation of long-term cultures of infected CD34+ cells to mature dendritic cells results in acetylation of histones bound to the MIEP, concomitant loss of heterochromatin protein 1 and the reactivation of HCMV. These data are consistent with ex vivo analyses of latency and may provide a model for further analyses of the mechanisms involved during latency and reactivation.

Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers.

Human cytomegalovirus (HCMV) persists as a subclinical, lifelong infection in the normal human host, but reactivation from latency in immunocompromised subjects results in serious disease. Latency and reactivation are defining characteristics of the herpesviruses and are key to understanding their biology; however, the precise cellular sites in which HCMV is carried and the mechanisms regulating its latency and reactivation during natural infection remain poorly understood. Here we present evidence, based entirely on direct analysis of material isolated from healthy virus carriers, to show that myeloid dendritic cell (DC) progenitors are sites of HCMV latency and that their ex vivo differentiation to a mature DC phenotype is linked with reactivation of infectious virus resulting from differentiation-dependent chromatin remodeling of the viral major immediate-early promoter. Thus, myeloid DC progenitors are a site of HCMV latency during natural persistence, and there is a critical linkage between their differentiation to DC and transcriptional reactivation of latent virus, which is likely to play an important role in the pathogenesis of HCMV infection.

The human cytomegalovirus gene product US6 inhibits ATP binding by TAP.

Human cytomegalovirus (HCMV) encodes several genes that disrupt the major histocompatibility complex (MHC) class I antigen presentation pathway. We recently described the HCMV-encoded US6 gene product, a 23 kDa endoplasmic reticulum (ER)-resident type I integral membrane protein that binds to the transporter associated with antigen processing (TAP), inhibits peptide translocation and prevents MHC class I assembly. The functional consequence of this inhibition is to prevent the cell surface expression of class I bound viral peptides and their recognition by HCMV-specific cytotoxic T cells. Here we describe a novel mechanism of action for US6. We demonstrate that US6 inhibits the binding of ATP by TAP1. This is a conformational effect, as the ER lumenal domain of US6 is sufficient to inhibit ATP binding by the cytosolic nucleotide binding domain of TAP1. US6 also stabilizes TAP at 37 degrees C and prevents conformational rearrangements induced by peptide binding. Our findings suggest that the association of US6 with TAP stabilizes a conformation in TAP1 that prevents ATP binding and subsequent peptide translocation.

Soluble tapasin restores MHC class I expression and function in the tapasin-negative cell line .220.

Tapasin forms a bridge between TAP (transporters associated with antigen processing) and MHC class I molecules and plays a critical role in class I assembly. In its absence, TAP and class I do not associate, and class I cell surface expression is reduced. We now identify two independent functions for tapasin. Tapasin increases TAP levels and allows more peptide to be translocated to the endoplasmic reticulum. Furthermore, when expressed in the tapasin-negative .220 cell line, recombinant soluble tapasin retains its association with class I and restores class I cell surface expression and function, even though it no longer binds TAP or increases TAP levels. This finding suggests that the association of tapasin with class I is sufficient to facilitate loading and assembly of class I molecules.

Inhibition of MHC class I-restricted antigen presentation by gamma 2-herpesviruses.

The gamma-herpesviruses, in contrast to the alpha- and beta-herpesviruses, are not known to inhibit antigen presentation to CD8(+) cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine gamma-herpesvirus 68 causes a chronic lytic infection in CD4(+) T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, gamma-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two gamma-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Roles for calreticulin and a novel glycoprotein, tapasin, in the interaction of MHC class I molecules with TAP.

Assembly of MHC class I-beta 2 microglobulin (beta 2m) dimers in the endoplasmic reticulum involves two chaperones. Calnexin has previously been shown to interact with free class I heavy chains. Here, we show that the related chaperone, calreticulin, binds human class I-beta 2m dimers prior to peptide loading. Calreticulin remains associated with at least a subset of class I molecules when they, in turn, bind to TAP. Further evidence suggests that the interaction of class I-beta 2m dimers with TAP occurs via a novel uncharacterized 48 kDa glycoprotein, tapasin, which can bind independently to TAP and class I-beta 2m-calreticulin complexes. Tapasin is absent from the mutant cell line .220, in which class I-TAP association and peptide loading is defective.

Interferon-gamma rapidly increases peptide transporter (TAP) subunit expression and peptide transport capacity in endothelial cells.

Human cytotoxic T lymphocytes (CTL) recognize specific complexes of HLA class I molecules and peptides, which assemble when nascent class I molecules bind peptides transported from the cytoplasm into the endoplasmic reticulum by the heterodimeric transporter associated with antigen processing (TAP). Increased class I molecule expression on the cell surface increases the efficiency of CTL lysis. The kinetics of interferon (IFN)-gamma induction of TAP, peptide transport capacity, and HLA class I molecule expression was determined in endothelial cells, which are targets of CTL following transplantation or viral infection. TAP mRNAs are induced rapidly, increasing 20-fold (TAP1) or 10-fold (TAP2) by 12 h, whereas HLA class I mRNA is induced more slowly, increasing 10-fold in 24 h. TAP1 and TAP2 proteins are also induced rapidly, increasing 10-fold in 24 h, whereas HLA class I heavy chain proteins and surface expression increase more slowly. Peptide transport capacity in endothelial and HeLa cells increases within 6 h of IFN-gamma treatment, suggesting that the IFN-gamma-induced TAP heterodimers are functional. Therefore, the IFN-gamma-induced increase in TAP proteins is accompanied by an increased peptide transport capacity, which may be important in supporting the subsequent rise in HLA class I protein expression.