The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition

The E3/19K protein of human adenovirus type 2 binds to HLA class I antigens and blocks their terminal glycosylation and cell surface expression. The nature of this interaction is non-covalent and involves neither disulfide bridges between the two molecules nor their carbohydrates. The murine H-2 Kd antigen associates with the E3/19K protein in a similar fashion to human HLA antigens whereas the allelic product H-2 Kk does not. Hybrid genes between the Kd and Kk alleles were constructed and their products were expressed in embryonic kidney cells together with the E3/19K protein. This allowed us to identify the alpha 1 and alpha 2 domains as the essential structures of the histocompatibility antigens for binding the viral protein. Interestingly, these domains are also crucial for T cell recognition. The implications for the evolution of adenoviruses and their ability to cause persistent infections are discussed.     

Requirements for the association of adenovirus type 2 E3/19K wild-type and mutant proteins with HLA antigens

The E3/19K protein of human adenovirus type 2 is a resident of the endoplasmic reticulum (ER). Immediately after synthesis, it associates with major histocompatibility complex class I antigens and prevents their intracellular transport and cell surface expression. We have generated several C-terminal deletion mutants of the E3/19K protein that are preterminated at various positions on both sides of the membrane-spanning segment of the protein. One of these mutants is terminated at the luminal side of the membrane (M310), and two are terminated in the hydrophobic segment (M374 and M392), whereas mutant M621 is terminated on the cytoplasmic side of the ER membrane. The M310, M374, and M392 mutants are soluble proteins. They do not associate with HLA antigens in transfected 293 cells, and they are, to some extent, secreted into the medium. The M621 mutant protein is integrated in the ER membrane, associates immediately after its synthesis with HLA antigens, and exits from the ER. By using either an in vitro translation system supplemented with microsomes or overexpression in insect cells, we showed that M374 and E3/19K are able to associate with HLA antigens. These results indicate that the conformation of the luminal part of the E3/19K protein is not grossly altered by the mutations. Rapid transport of the M374 mutant out of the ER and partial degradation of this protein may prevent the interaction with HLA class I antigens in transfected 293 cells.     

Reduced allorecognition of adenovirus-2 infected cells

The early region 3 of adenovirus 2 encodes the membrane glycoprotein E19. This protein specifically binds class I transplantation antigens of a variety of species. Concomitant with this interaction the intracellular transport of newly synthesized class I heavy chains is abrogated. At late stages of the virus infection this leads to a notable decrease in the cell surface expression of class I antigens. We have studied how infection with adenovirus 2 influences target cell recognition by alloreactive cytolytic T lymphocytes. We found that the E19 protein-induced reduction of the HLA-B7 cell surface expression led to a greatly reduced lysis of the infected cells. These findings support our hypothesis that the E19 protein has evolved to facilitate the in vivo replication of the virus by reducing the expression of HLA class I antigens.     

Cell-surface expression and alloantigenic function of a human nonclassical class I molecule (HLA-E) in transgenic mice

We have introduced the gene (E*01033) encoding the heavy chain of the human nonclassical MHC class I Ag, HLA-E, into the mouse genome. Two founder mice carry a 21-kb fragment, the others bear an 8-kb fragment. Each of the founder mice was mated to mice of an already established C57BL/10 transgenic line expressing human beta2-microglobulin (beta2m). Cell surface HLA-E was detected on lymph node cells by flow cytometry only in the presence of endogenous human beta2m. However, HLA-E-reactive mouse CTL (H-2-unrestricted) lysed efficiently the target cells originating from HLA-E transgenic mice without human beta2m, showing that the HLA-E protein can be transported to the cell surface in the absence of human beta2m, presumably by association with murine beta2m. Rejection of skin grafts from HLA-E transgenic mice demonstrates that HLA-E behaves as a transplantation Ag in mice. HLA-E transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal and human classical class I (HLA-B27) transgenic mice. Furthermore, results from split-well analysis indicate that the majority of the primary in vivo-induced CTL recognizes HLA-E as an intact molecule (H-2-unrestricted recognition) and not as an HLA-E-derived peptide presented by a mouse MHC molecule, although a small fraction (ranging from 4 to 21%) of the primary in vivo-induced CTL is able to recognize HLA-E in an H-2-restricted manner. Based on these observations, we conclude that HLA-E exhibits alloantigenic properties that are indistinguishable from classical HLA class I molecules when expressed in transgenic mice.     

The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule K(d)

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.     

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-β and TNF-α production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-α as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-α and IFN-β as well as the dsRNA analog, poly (I:C). Both IFN-β and TNF-α further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.     

Comparison of human memory CD8 T cell responses to adenoviral early and late proteins in peripheral blood and lymphoid tissue

Treatment of invasive adenovirus (Ad) disease in hematopoietic stem cell transplant (SCT) recipients with capsid protein hexon-specific donor T cells is under investigation. We propose that cytotoxic T cells (CTLs) targeted to the late protein hexon may be inefficient in vivo because the early Ad protein E3-19K downregulates HLA class I antigens in infected cells. In this study, CD8+ T cells targeted to highly conserved HLA A2-restricted epitopes from the early regulatory protein DNA polymerase (P-977) and late protein hexon (H-892) were compared in peripheral blood (PB) and tonsils of naturally infected adults. In tonsils, epitope-specific pentamers detected a significantly higher frequency of P-977+CD8+ T cells compared to H-892+CD8+ T cells; this trend was reversed in PB. Tonsil epitope-specific CD8+ T cells expressed IFN-γ and IL-2 but not perforin or TNF-α, whereas PB T cells were positive for IFN-γ, TNF-α, and perforin. Tonsil epitope-specific T cells expressed lymphoid homing marker CCR7 and exhibited lower levels of the activation marker CD25 but higher proliferative potential than PB T cells. Finally, in parallel with the kinetics of mRNA expression, P-977-specific CTLs lysed targets as early as 8 hrs post infection. In contrast, H-892-specific CTLs did not kill unless infected fibroblasts were pretreated with IFN-γ to up regulate HLA class I antigens, and cytotoxicity was delayed until 16-24 hours. These data show that, in contrast to hexon CTLs, central memory type DNA polymerase CTLs dominate the lymphoid compartment and kill fibroblasts earlier after infection without requiring exogenous IFN-γ. Thus, use of CTLs targeted to both early and late Ad proteins may improve the efficacy of immunotherapy for life-threatening Ad disease in SCT recipients.     

A Viral,Transporter Associated with Antigen Processing (TAP)-independent,High Affinity Ligand with Alternative Interactions Endogenously Presented by the Nonclassical Human Leukocyte Antigen E Class I Molecule

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8⁺ lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.     

HLA-E and HLA-G expression on porcine endothelial cells inhibit xenoreactive human NK cells through CD94/NKG2-dependent and -independent pathways

Human NK cells contribute a significant role to host defense as well as xenogeneic cytotoxicity. Previous studies using human 721.221 cell line have shown that peptides derived from the leader sequence of the HLA-G binds and up-regulates the surface expression of HLA-E molecules, which was considered to consequently provide negative signals to human NK cells. However, the direct role of HLA-G in inhibiting human NK cells remains controversial. In this study, we showed that the expression of HLA-G or HLA-E in porcine endothelial cells directly protected sensitive porcine cells from human NK cell-mediated xenogeneic cytotoxicity. Ab blocking assays using F(ab')2 of the HLA class I-specific mAb PA2.6 indicated that the protection was directly mediated by the expression of HLA-G and HLA-E on the porcine cells. The HLA-E-mediated protection was blocked by anti-human CD94 Ab. In addition, the engagement of HLA-E lead to the phosphorylation of the CD94/NKG2 complex and the recruitment of SH2 domain-containing protein phosphatase 1 (SHP-1) to the complex. Therefore, HLA-E protected porcine cells from xenoreactive human NK cells through a CD94/NKG2-dependent pathway. In contrast, HLA-G inhibited human NK cells in the absence of CD94/NKG2 phosphorylation or SHP-1 recruitment, and the inhibition was not blocked by anti-CD94 Ab. Therefore, HLA-G protected porcine cells from human NK cells through a CD94/NKG2-independent pathway. These results demonstrated that both HLA-E and HLA-G could directly inhibit human NK cells in the absence of other endogenous HLA class I molecules. These results also have practical implications in preventing xenograft rejection mediated by human NK cells.     

The Presence of HLA-E-Restricted,CMV-Specific CD8+ T Cells in the Blood of Lung Transplant Recipients Correlates with Chronic Allograft Rejection

The human cytomegalovirus (CMV) immune evasion protein, UL40, shares an identical peptide sequence with that found in the leader sequence of many human leukocyte antigen (HLA)-C alleles and when complexed with HLA-E, can modulate NK cell functions via interactions with the CD94-NKG2 receptors. However the UL40-derived sequence can also be immunogenic, eliciting robust CD8+ T cell responses. In the setting of solid organ transplantation these T cells may not only be involved in antiviral immunity but also can potentially contribute to allograft rejection when the UL40 epitope is also present in allograft-encoded HLA. Here we assessed 15 bilateral lung transplant recipients for the presence of HLA-E-restricted UL40 specific T cells by tetramer staining of peripheral blood mononuclear cells (PBMC). UL40-specific T cells were observed in 7 patients post-transplant however the magnitude of the response varied significantly between patients. Moreover, unlike healthy CMV seropositive individuals, longitudinal analyses revealed that proportions of such T cells fluctuated markedly. Nine patients experienced low-grade acute cellular rejection, of which 6 also demonstrated UL40-specific T cells. Furthermore, the presence of UL40-specific CD8⁺ T cells in the blood was significantly associated with allograft dysfunction, which manifested as Bronchiolitis Obliterans Syndrome (BOS). Therefore, this study suggests that minor histocompatibility antigens presented by HLA-E can represent an additional risk factor following lung transplantation.     

The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor.

We have reported that the E3 14,700-dalton protein (E3 14.7K protein) protects adenovirus-infected mouse C3HA fibroblasts against lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). We have also observed that the E1B 19K protein protects adenovirus-infected human but not mouse cells against TNF lysis (L. R. Gooding, L. Aquino, P. J. Duerksen-Hughes, D. Day, T. M. Horton, S. Yei, and W. S. M. Wold, J. Virol. 65:3083-3094, 1991). We now report that, in the absence of E3 14.7K, the E3 10.4K and E3 14.5K proteins are both required to protect C127 as well as several other mouse cell lines against TNF lysis. The 14.7K protein can also protect these cells from TNF in the absence of the 10.4K and 14.5K proteins. This protection by the 10.4K and 14.5K proteins was not observed in the C3HA cell line. These conclusions are based on 51Cr release assays of cells infected with virus E3 mutants that express the 14.7K protein alone, that express both the 10.4K and 14.5K proteins, and that delete the 14.7K in combination with either the 10.4K or 14.5K protein. The 10.4K protein was efficiently coimmunoprecipitated together with the 14.5K protein by using an antiserum to the 14.5K protein, suggesting that the 10.4K and 14.5K proteins exist as a complex in the infected mouse cells and consistent with the notion that they function in concert. Considering that three sets of proteins (E3 14.7K, E1B 19K, and E3 10.4K/14.5K proteins) exist in adenovirus to prevent TNF cytolysis of different cell types, it would appear that TNF is a major antiadenovirus defense of the host.     

Developmental acquisition of T3-sensitive Na-K-ATPase stimulation by rat alveolar epithelial cells

Late in gestation, the developing air space epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na-K-ATPase acting in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption, and thyroid hormone [triiodothyronine (T3)] stimulates adult alveolar epithelial cell (AEC) Na-K-ATPase. This study explored the developmental regulation of Na-K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) cells. T3 increased Na-K-ATPase activity in primary FDLE cells from gestational day 19 [both primary FDLE cells at embryonic day 19 (E19) and the cell line FD19 derived from FDLE cells at E19]. However, T3 did not increase the Na-K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the cell line FD18 (derived from FDLE cells at E18). Subsequent experiments assessed the T3 signal pathway to define whether it was similar in the late FDLE and adult AEC and to determine the site of the switch in responsiveness to T3. As in adult AEC, in the FD19 cell line, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked the T3-induced increase in Na-K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active mutant of the PI3K catalytic p110 subunit significantly augmented the Na-K-ATPase activity and the cell surface expression of Na-K-ATPase alpha(1) protein. In conclusion, FDLE cells from E17 and E18 lacked T3-sensitive Na-K-ATPase activity but acquired this response at E19. The developmental stimulation of Na-K-ATPase by T3 in rat FDLE cells requires activation of PI3K, and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway.     

Recognition of nonclassical HLA class I antigens by gamma delta T cells during pregnancy

The healthy trophoblast does not express classical HLA-A and HLA-B products; therefore, an MHC-restricted recognition of trophoblast-presented Ags is unlikely. In the decidua and also in peripheral blood of healthy pregnant women, gammadelta T cells significantly increase in number. We investigated the possible role of gammadelta T cells in recognition of trophoblast-presented Ags. PBL and isolated gammadelta T cells from healthy pregnant women as well as from those at risk for premature pregnancy termination were conjugated to choriocarcinoma cells (JAR) transfected with nonclassical HLA Ags (HLA-E, HLA-G). To investigate the involvement of killer-inhibitory/killer-activatory receptors in trophoblast recognition, we tested the effect of CD94 block on cytotoxic activity of Vdelta2(+) enriched gammadelta T cells to HLA-E- and/or HLA-G-transfected targets. Lymphocytes from healthy pregnant women preferentially recognized HLA(-) choriocarcinoma cells, whereas those from pathologically pregnant patients did not discriminate between HLA(+) and HLA(-) cells. Normal pregnancy Vdelta2(+) T cells conjugated at a significantly increased rate to HLA-E transfectants, whereas Vdelta2(+) lymphocytes from pathologically pregnant women did not show a difference between those and HLA(-) cells. Blocking of the CD94 molecule of Vdelta2(+) lymphocytes from healthy pregnant women resulted in an increased cytotoxic activity to HLA-E-transfected target cells. These data indicate that Vdelta2(+) lymphocytes of healthy pregnant women recognize HLA-E on the trophoblast, whereas Vdelta1 cells react with other than HLA Ags. In contrast to Vdelta2(+) lymphocytes from healthy pregnant women, those from women with pathological pregnancies do not recognize HLA-E via their killer-inhibitory receptors and this might account for their high cytotoxic activity.