Cell surface expression of HLA I molecules as a marker of young platelets.

BACKGROUND: Accurate identification of the proportion of young platelets is important to distinguish peripheral thrombocytopenia from a deficit in platelet production. Young platelets are defined by their higher RNA content and are often assessed as thiazole orange bright (TObright ) by flow cytometry. In clinical practice, their proportion is estimated by automatic blood counter according to their greater RNA content, which identifies a so-called immature platelet fraction (IPF). However, the detected IPFs are not strictly identical to the young TObright platelet population observed by flow cytometry. OBJECTIVES: The aim of this study was to assess the reliability of HLA I/major histocompatibility I (MHC I) cell surface expression as a marker of young platelets. METHODS: The HLA I/MHC I expression was evaluated by flow cytometry after costaining blood with TO and antibodies directed against HLA I/MHC I molecules. RESULTS: We found that platelets with a higher expression of plasma membrane-localized MHC I molecules displayed an increased TO staining and a higher content in ribosomal P-antigen. Transfusion experiments in mice showed that the number of MHC I molecules expressed on the cell surface of young murine platelets decreased during platelet aging, reaching basal levels within 24 h. Finally, we demonstrated that for patients with thrombocytopenias, the identification of young platelets is better assessed by the flow cytometric determination of the level of HLA I expression than by TO staining or the use of hematological blood counter. CONCLUSION: Overall, our results highlight the relevance of MHC I/HLA I expression as a valuable parameter to identify young platelets.

Autologous cellular vaccine overcomes cancer immunoediting in a mouse model of myeloma.

In the Sp6 mouse plasmacytoma model, a whole-cell vaccination with Sp6 cells expressing de novo B7-1 (Sp6/B7) induced anatomically localized and cytotoxic T cell (CTL)-mediated protection against wild-type (WT) Sp6. Both WT Sp6 and Sp6/B7 showed down-regulated expression of MHC H-2 L(d). Increase of H-2 L(d) expression by cDNA transfection (Sp6/B7/L(d)) raised tumour immune protection and shifted most CTL responses towards H-2 L(d)-restricted antigenic epitopes. The tumour-protective responses were not specific for the H-2 L(d)-restricted immunodominant AH1 epitope of the gp70 common mouse tumour antigen, although WT Sp6 and transfectants were able to present it to specific T cells in vitro. Gp70 transcripts, absent in secondary lymphoid organs of naive mice, were detected in immunized mice as well as in splenocytes from naive mice incubated in vitro with supernatants of CTL-lysed Sp6 cell cultures, containing damage-associated molecular patterns (DAMPs). It has been shown that Toll-like receptor triggering induces gp70 expression. Damage-associated molecular patterns are released by CTL-mediated killing of Sp6/B7-Sp6/B7/L(d) cells migrated to draining lymph nodes during immunization and may activate gp70 expression and presentation in most resident antigen-presenting cells. The same could also apply for Mus musculus endogenous ecotropic murine leukaemia virus 1 particles present in Sp6-cytosol, discharged by dying cells and superinfecting antigen-presenting cells. The outcome of such a massive gp70 cross-presentation would probably be tolerogenic for the high-affinity AH1-gp70-specific CTL clones. In this scenario, autologous whole-tumour-cell vaccines rescue tumour-specific immunoprotection by amplification of subdominant tumour antigen responses when those against the immune dominant antigens are lost.

Class II transactivator-induced MHC class II expression in pancreatic cancer cells leads to tumor rejection and a specific antitumor memory response.

OBJECTIVES: The loss of major histocompatibility complex (MHC) classes I and II is a well-known mechanism by which cancer cells are able to escape from immune recognition. In this study, we analyzed the expression of antigen processing and presenting molecules in 2 cell lines derived from mouse models of pancreatic ductal adenocarcinoma (PDA) and the effects of the re-expression of MHC class II on PDA rejection. METHODS: The PDA cell lines were analyzed for the expression of MHC class I, II, and antigen-processing molecules by flow cytometry or polymerase chain reaction. We generated stable PDA-MHC class II transactivator (CIITA) cells and injected them into syngeneic mice. The CD4 and CD8 T-cell role was analyzed in vitro and in vivo. RESULTS: Murine PDA cell lines were negative for MHC and antigen-processing molecules, but their expression was restored by exogenous interferon-γ. CIITA-tumor cells were rejected in 80% to 100% of injected mice, which also developed long-lasting immune memory. In vitro assays and immunohistochemical analyses revealed the recruitment of T effector cells and CD8 T cells into the tumor area. CONCLUSIONS: Overall, these data confirm that immunotherapy is a feasible therapeutic approach to recognize and target an aggressive cancer such as PDA.

The similar expression pattern of MHC class I molecules in human and mouse cerebellar cortex.

The major histocompatibility complex (MHC) class I molecules are considered to be important in the immune system. However, the results reported in the past decade indicate that they also play important roles in the central nervous system. Here we examined the expression of MHC I and ß2-microglobulin (ß2m) in human and mouse cerebellar cortex. The results show that MHC I molecules are expressed both in human and mouse cerebellar cortex during brain development. The expression of H-2K(b)/D(b) is gradually increased with the development of mouse cerebellar cortex, but finally decreased to a very low level. Similarly, the expression of HLA-B/C genes is increased in developing human cerebellar cortex, but decreased after birth. The spatial and temporal expression of ß2m overlaps mostly with that of HLA-B/C molecules, and they are co-expressed in Purkinje cells. Our findings provide a fundamental basis to reveal the functions of neuronal MHC class I molecules in the development of human cerebellum.

The expression pattern of classical MHC class I molecules in the development of mouse central nervous system.

Classical major histocompatibility complex (MHC) class I, first identified in the immune system, is also expressed in the developing and adult central nervous system (CNS). Although the MHC class I molecules have been found to be expressed in the CNS of different species, a necessary step to elucidate the temporal and spatial expression patterns of MHC class I molecules in the brain development has never been taken. Frozen sections were made from the brains of embryonic and postnatal C57BL/6 J mice, and the expression of H-2D(b) mRNA was examined by in situ hybridization. Immunofluorescence was also performed to define the cell types that express H2-D(b) in P15 mice. At E10.5, the earliest stage we examined, H2-D(b) was expressed in neuroepithelium of the brain vesicles. From E12.5 to P0, H2-D(b) expression was mainly located at cerebral cortex, neuroepithelium of the lateral ventricle, neuroepithelium of aquaeductus and developing cerebellum. From P4 to adult, H2-D(b) mRNA was detected at olfactory bulb, hippocampus, cerebellum and some nerve nuclei. The major cell types expressing H-2D(b) in P15 hippocampus, cerebral cortex and olfactory bulb were neuron. H2-K(b) signal paralleled that of H2-D(b) and the expression levels of the two molecules were comparable throughout the brain. The investigation of the expression pattern of H-2D(b) at both embryonic and postnatal stages is important for further understanding the physiological and pathological roles of H2-D(b) in the developing CNS.

Enhanced MHC class I and costimulatory molecules on B16F10 cells by Ganoderma lucidum polysaccharides.

PURPOSE: It is obvious that malignant cells evade from immune system in patients with manifest malignancy. Deficient major histocompatibility complex (MHC) class I and costimulatory molecules on malignant cells partially consist of evasion strategy since antigen bond MHC and costimulatory molecules provide two signals necessary for T cell activation. Therefore, enhancement of MHC-I and costimulatory molecules may favor restraint of the evasion. For this purpose, Ganoderma lucidum Polysaccharides (Gl-PS) was used on B16F10 melanoma cells in this study. METHODS: Immunocytochemistry and flowcytometry were used to determine the H-2K(b) and H-2D(b) (two prominent MHC class I molecules in C57BL mouse) as well as B7-1 and B7-2 (two prominent costimulatory molecules) expression on B16F10 cells after incubation with Gl-PS, while messenger ribonucleic acid (mRNA) of these molecules was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The H-2K(b) and H-2D(b), and B7-1 and B7-2 on B16F10 cells and mRNAs of these molecules were enhanced by Gl-PS, and more efficient antitumor cytotoxicity was induced by the Gl-PS treated cells. CONCLUSIONS: The MHC class I molecules and costimulatory molecules may be enhanced by Gl-PS, and more efficient immune cell mediated cytotoxicity against these B16F10 cells may be induced, which may favor cancer therapy.

Skewing of the NK cell repertoire by MHC class I via quantitatively controlled enrichment and contraction of specific Ly49 subsets.

A major task for the immune system is to secure powerful immune reactions while preserving self-tolerance. This process is particularly challenging for NK cells, for which tolerizing inhibitory receptors for self-MHC class I is both cross-reactive and expressed in an overlapping fashion between NK cells. We show in this study that during an education process, self-MHC class I molecules enrich for potentially useful and contract potentially dangerous NK cell subsets. These processes were quantitatively controlled by the expression level of the educating MHC class I allele, correlated with susceptibility to IL-15 and sensitivity to apoptosis in relevant NK cell subsets, and were linked to their functional education. Controlling the size of NK cell subsets with unique compositions of inhibitory receptors may represent one mechanism by which self-MHC class I molecules generate a population of tolerant NK cells optimally suited for efficient missing self-recognition.

Distinct pathways generate peptides from defective ribosomal products for CD8+ T cell immunosurveillance.

To understand better the endogenous sources of MHC class I peptide ligands, we generated an antigenic reporter protein whose degradation is rapidly and reversibly controlled with Shield-1, a cell-permeant drug. Using this system, we demonstrate that defective ribosomal products (DRiPs) represent a major and highly efficient source of peptides and are completely resistant to our attempts to stabilize the protein. Although peptides also derive from nascent Shield-1-sensitive proteins and "retirees" created by Shield-1 withdrawal, these are much less efficient sources on a molar basis. We use this system to identify two drugs--each known to inhibit polyubiquitin chain disassembly--that selectively inhibit presentation of Shield-1-resistant DRiPs. These findings provide the initial evidence for distinct biochemical pathways for presentation of DRiPs versus retirees and implicate polyubiquitin chain disassembly or the actions of deubiquitylating enzymes as playing an important role in DRiP presentation.

Transplantation tolerance to a single noninherited MHC class I maternal alloantigen studied in a TCR-transgenic mouse model.

The mechanisms underlying tolerance to noninherited maternal Ags (NIMA) are not fully understood. In this study, we designed a double-transgenic model in which all the offspring's CD8(+) T cells corresponded to a single clone recognizing the K(b) MHC class I protein. In contrast, the mother and the father of the offspring differed by the expression of a single Ag, K(b), that served as NIMA. We investigated the influence of NIMA exposure on the offspring thymic T cell selection during ontogeny and on its peripheral T cell response during adulthood. We observed that anti-K(b) thymocytes were exposed to NIMA and became activated during fetal life but were not deleted. Strikingly, adult mice exposed to NIMA accepted permanently K(b+) heart allografts despite the presence of normal levels of anti-K(b) TCR transgenic T cells. Transplant tolerance was associated with a lack of a proinflammatory alloreactive T cell response and an activation/expansion of T cells producing IL-4 and IL-10. In addition, we observed that tolerance to NIMA K(b) was abrogated via depletion of CD4(+) but not CD8(+) T cells and could be transferred to naive nonexposed mice via adoptive transfer of CD4(+)CD25(high) T cell expressing Foxp3 isolated from NIMA mice.

Defective ribosomal products are the major source of antigenic peptides endogenously generated from influenza A virus neuraminidase.

The defective ribosomal product (DRiP) hypothesis of endogenous Ag processing posits that rapidly degraded forms of nascent proteins are a major source of peptide ligands for MHC class I molecules. Although there is broad experimental support for the DRiP hypothesis, careful kinetic analysis of the generation of defined peptide class I complexes has been limited to studies of recombinant vaccinia viruses expressing genes derived from other organisms. In this study, we show that insertion of the SIINFEKL peptide into the stalk of influenza A virus neuraminidase (NA) does not detectably modify NA folding, degradation, transport, or sp. act. when expressed in its natural context of influenza A virus infection. Using the 25-D1.16 mAb specific for K(b)-SIINFEKL to precisely quantitate cell surface complexes by flow cytometry, we demonstrate that SIINFEKL is generated in complete lockstep with initiation and abrogation of NA biosynthesis in both L-K(b) fibroblast cells and DC2.4 dendritic/monocyte cells. SIINFEKL presentation requires active proteasomes and TAP, consistent with its generation from a cytosolic DRiP pool. From the difference in the shutoff kinetics of K(b)-SIINFEKL complex expression following protein synthesis versus proteasome inhibition, we estimate that the t(1/2) of the biosynthetic source of NA peptide is approximately 5 min. These observations extend the relevance of the DRiP hypothesis to viral proteins generated in their natural context.

Cutting edge: antigen presentation to CD8 T cells after influenza A virus infection.

Influenza A virus infection induces massive inflammation and lung damage. Activation of CD8 T cells by dendritic cells (DCs) is necessary to control disease. We undertook studies to track directly Ag presentation to CD8 T cells in vivo through the first 72 h after infection with OVA-expressing influenza A virus. We found that Ag presentation by DCs occurs strictly in the draining lymph nodes and not within the lung itself. Surprisingly, Ag presentation was found to be mediated by a CD11b(+) DC population. Finally, the expression of antigenic complexes on DCs correlated with the location and timing of CD8 T cell activation. These results have implications for approaches to control influenza A virus infection.

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells.

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.

Immunity to growth factor receptor-bound protein 10, a signal transduction molecule, inhibits the growth of breast cancer in mice.

This study describes the application of a unique strategy to identify breast cancer antigens [tumor-associated antigen (TAA)]. In a mouse model, the strategy led to the identification of growth factor receptor-bound protein 10 (Grb10) as a newly identified TAA. Grb10 is a signal transduction molecule associated with multiple transmembrane tyrosine kinase receptors. It was discovered by comparing microarrays of cellular breast cancer vaccines highly enriched for cells that induced breast cancer immunity in tumor-bearing mice with nonenriched vaccines. The vaccines were prepared by transferring a cDNA expression library derived from SB5b cells, a breast cancer cell line C3H/He origin (H-2(k)), into LM mouse fibroblasts (H-2(k)). As the transferred cDNA integrates spontaneously into the genome of the recipient cells, replicates as the cells divide, and is expressed, the vaccine could be prepared from microgram amounts of tumor tissue. Relatively few cells in the transduced cell population, however, incorporated cDNA fragments that included genes specifying TAA. (The vast majority specified normal cellular constituents.) A unique strategy was used, therefore, to enrich the vaccine for immunotherapeutic cells. Twenty genes were overrepresented in the enriched vaccines. One, the gene for Grb10, was approximately 100-fold overrepresented. To determine if Grb10 in the enriched vaccine was partly responsible for its therapeutic benefits, the gene was transferred into the fibroblast cell line, which was then used as a vaccine. Mice with established breast cancer treated solely by immunization with the modified fibroblasts developed robust immunity to the breast cancer cells, which, in some instances, was sufficient to result in tumor rejection.

Repeated BCG treatment of mouse bladder selectively stimulates small GTPases and HLA antigens and inhibits single-spanning uroplakins.

BACKGROUND: Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG) remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following repeated intravesical BCG therapy. METHODS: Mice were transurethrally instilled with BCG or pyrogen-free on days 1, 7, 14, and 21. Seven days after the last instillation, urothelia along with the submucosa was removed and amplified ds-DNA was prepared from control- and BCG-treated bladder mucosa and used to generate suppression subtractive hybridization (SSH). Plasmids from control- and BCG-specific differentially expressed clones and confirmed by Virtual Northern were then purified and the inserts were sequenced and annotated. Finally, chromatin immune precipitation combined with real-time polymerase chain reaction assay (ChIP/Q-PCR) was used to validate SSH-selected transcripts. RESULTS: Repeated intravesical BCG treatment induced an up regulation of genes associated with antigen presentation (B2M, HLA-A, HLA-DQA1, HLA-DQB2, HLA-E, HLA-G, IGHG, and IGH) and representatives of two IFNgamma-induced small GTPase families: the GBPs (GBP1, GBP2, and GBP5) and the p47GTPases (IIGTP1, IIGTP2, and TGTP). Genes expressed in saline-treated bladders but down-regulated by BCG included: the single-spanning uroplakins (UPK3a and UPK2), SPRR2G, GSTM5, and RSP 19. CONCLUSION: Here we introduced a hypothesis-generator approach to determine key genes involved in the urothelium/sumbmucosa responses to BCG therapy. Urinary bladder responds to repeated BCG treatment by up-regulating not only antigen presentation-related genes, but also GBP and p47 small GTPases, both potentially serving to mount a resistance to the replication of the Mycobacterium. It will be of tremendous future interest to determine whether these immune response cascades play a role in the anti-cancer effects exerted by BCG.

Developmental exposure to noninherited maternal antigens induces CD4+ T regulatory cells: relevance to mechanism of heart allograft tolerance.

We hypothesize that developmental exposure to noninherited maternal Ags (NIMA) results in alloantigen-specific natural and adaptive T regulatory (T(R)) cells. We compared offspring exposed to maternal H-2(d) (NIMA(d)) with nonexposed controls. In vitro assays did not reveal any differences in T cell responses pretransplant. Adoptive transfer assays revealed lower lymphoproliferation and greater cell surface TGF-beta expression on CD4(+) T cells of NIMA(d)-exposed vs control splenocytes. NIMA(d)-exposed splenocytes exhibited bystander suppression of tetanus-specific delayed-type hypersensitivity responses, which was reversed with Abs to TGF-beta and IL-10. Allospecific T effector cells were induced in all mice upon i.v. challenge with B6D2F1 splenocytes or a DBA/2 heart transplant, but were controlled in NIMA(d)-exposed mice by T(R) cells to varying degrees. Some (40%) NIMA(d)-exposed mice accepted a DBA/2 allograft while others (60%) rejected in delayed fashion. Rejector and acceptor NIMA(d)-exposed mice had reduced T effector responses and increased Foxp3(+) T(R) cells (CD4(+)CD25(+)Foxp3(+) T(R)) in spleen and lymph nodes compared with controls. The key features distinguishing NIMA(d)-exposed acceptors from all other mice were: 1) higher frequency of IL-10- and TGF-beta-producing cells primarily in the CD4(+)CD25(+) T cell subset within lymph nodes and allografts, 2) a suppressed delayed-type hypersensitivity response to B6D2F1 Ags, and 3) allografts enriched in LAP(+), Foxp3(+), and CD4(+) T cells, with few CD8(+) T cells. We conclude that the beneficial NIMA effect is due to induction of NIMA-specific T(R) cells during ontogeny. Their persistence in the adult, and the ability of the host to mobilize them to the graft, may determine whether NIMA-specific tolerance is achieved.

Xenogeneic beta 2-microglobulin substitution affects functional binding of MHC class I molecules by CD8+ T cells.

NK cells and CD8+ T cells bind MHC-I molecules using distinct topological interactions. Specifically, murine NK inhibitory receptors bind MHC-I molecules at both the MHC-I H chain regions and beta2-microglobulin (beta2m) while TCR engages MHC-I molecules at a region defined solely by the class I H chain and bound peptide. As such, alterations in beta2m are not predicted to influence functional recognition of MHC-I by TCR. We have tested this hypothesis by assessing the capability of xenogeneic beta2m to modify the interaction between TCR and MHC-I. Using a human beta2m-transgenic C57BL/6 mouse model, we show that human beta2m supports formation and expression of H-2K(b) and peptide:H-2K(b) complexes at levels nearly equivalent to those in wild-type mice. Despite this finding, the frequencies of CD8+ single-positive thymocytes in the thymus and mature CD8+ T cells in the periphery were significantly reduced and the TCR Vbeta repertoire of peripheral CD8+ T cells was skewed in the human beta2m-transgenic mice. Furthermore, the ability of mouse beta2m-restricted CTL to functionally recognize human beta2m+ target cells was diminished compared with their ability to recognize mouse beta2m+ target cells. Finally, we provide evidence that this effect is achieved through subtle conformational changes occurring in the distal, peptide-binding region of the MHC-I molecule. Our results indicate that alterations in beta2m influence the ability of TCR to engage MHC-I during normal T cell physiology.

Heterogeneity of effector phenotype for acute phase and memory influenza A virus-specific CTL.

Ag-specific, CD8+ CTLs clear influenza A viruses from the lung via granzyme (Gzm) and perforin-dependent mechanisms. Ex vivo analysis of perforin-Gzm mRNA profiles demonstrated substantial heterogeneity in patterns of effector mRNA transcription of CD8+ D(b)NP(366)- or D(b)PA(224)-specific CTL. The only difference between the two epitope-specific sets was apparent very early after infection with similar molecular profiles seen in peak primary and secondary responses and in long-term memory. Surprisingly, memory T cells also expressed a diverse pattern of effector mRNA profile with an emphasis on GzmB and, surprisingly, GzmK. This analysis thus defines how naive, effector, and memory T cells differ in cytotoxic potential and provides novel insight into the molecular signatures of effector molecules observed at various stages after infection.

NK cells negatively regulate antigen presentation and tumor-specific CTLs in a syngeneic lymphoma model.

NK cells are known to kill tumor cells and produce proinflammatory cytokines that lead to the generation of tumor-specific CTLs. Many studies have used MHC class I-deficient tumor cells and/or adjuvants that induce NK cell responses. In this study, the focus was on less-immunogenic lymphoma cells that express MHC class I as a model to study NK cell responses to tumors that do not directly stimulate NK cell activation. When RMA tumor cells that expressed a truncated version of OVA, or RMA cells alone, were injected into mice that were depleted of NK cells, the mice developed an increased number of tumor-specific CTLs, increased IFN-gamma responses, and a higher amount of Ag presentation in draining LNs compared with mice with intact NK cells. These data suggest that NK cells can inhibit the development of effective adaptive immunity in the absence of signals that trigger NK cell activation.

A hepatitis C virus (HCV) NS3/4A protease-dependent strategy for the identification and purification of HCV-infected cells.

As a tool for the identification and/or purification of hepatitis C virus (HCV)-infected cells, a chimeric form of the Gal4VP16 transcription factor was engineered to be activated only in the presence of the HCV NS3/4A protease and to induce different reporter genes [choramphenical acetyltransferase (CAT), green fluorescent protein (GFP) and the cell-surface marker H-2K(k)] through the (Gal4)(5)-E1b promoter. For this, the NS5A/5B trans-cleavage motif of HCV of genotype 1a was inserted between Gal4VP16 and the N terminus of the endoplasmic reticulum (ER)-resident protein PERK, and it was demonstrated that it could be cleaved specifically by NS3/4A. Accordingly, transient transfection in tetracycline-inducible UHCV-11 cells expressing the HCV polyprotein of genotype 1a revealed the migration of the Gal4VP16 moiety of the chimera from the ER to the nucleus upon HCV expression. Activation of the chimera provoked specific gene induction, as shown by CAT assay, first in UHCV-11 cells and then in Huh-7 cells expressing an HCV replicon of genotype 1b (Huh-7 Rep). In addition, the GFP reporter gene allowed rapid fluorescence monitoring of HCV expression in the Huh-7 Rep cells. Finally, the chimera was introduced into Huh-7.5 cells infected with cell culture-generated HCV JFH1 (genotype 2a), allowing the purification of the HCV-infected cells by immunomagnetic cell sorting using H-2K(k) as gene reporter. In conclusion, the Gal4VP16 chimera activation system can be used for the rapid identification and purification of HCV-infected cells.

Tripeptidyl peptidase II is the major peptidase needed to trim long antigenic precursors, but is not required for most MHC class I antigen presentation.

Recent reports concluded that tripeptidyl peptidase (TPPII) is essential for MHC class I Ag presentation and that the proteasome in vivo mainly releases peptides 16 residues or longer that require processing by TPPII. However, we find that eliminating TPPII from human cells using small interfering RNA did not decrease the overall supply of peptides to MHC class I molecules and reduced only modestly the presentation of SIINFEKL from OVA, while treatment with proteasome inhibitors reduced these processes dramatically. Purified TPPII digests peptides from 6 to 30 residues long at similar rates, but eliminating TPPII in cells reduced the processing of long antigenic precursors (14-17 residues) more than short ones (9-12 residues). Therefore, TPPII appears to be the major peptidase capable of processing proteasome products longer than 14 residues. However, proteasomes in vivo (like purified proteasomes) release relatively few such peptides, and these peptides processed by TPPII require further trimming in the endoplasmic reticulum (ER) by ER aminopeptidase 1 for presentation. Taken together, these observations demonstrate that TPPII plays a specialized role in Ag processing and one that is not essential for the generation of most presented peptides. Moreover, these findings reveal that three sequential proteolytic steps (by proteasomes, TPPII, and then ER aminopepsidase 1) are required for the generation of a subset of epitopes.