How HLA-DM affects the peptide repertoire bound to HLA-DR molecules

Considerable progress has been made in the field of major histocompatibility complex (MHC) class II-restricted antigen presentation. The analysis of mutant cell lines defective in antigen presentation revealed a central role for the nonclassical MHC class II molecule HLA-DM. Cell biological and biochemical characterization of HLA-DM provided deeper insight into the molecular mechanisms underlying the loading process: HLA-DM accumulates in acidic compartments, where it stabilizes classical class II molecules until a high-stability ligand occupies the class II peptide binding groove. Thus, HLA-DM prevents empty alpha beta dimers from functional inactivation at low endosomal/lysosomal pH in a chaperone-like fashion. In the presence of peptide ligands, HLA-DM acts as a catalyst for peptide loading by releasing CLIP, the residual invariant chain-derived fragment by which the invariant chain is associated with the class II molecules during transport from the endoplasmic reticulum to the loading compartments. Finally, there is accumulating evidence that HLA-DM functions as a peptide editor that removes low-stability ligands, thereby skewing the class II peptide repertoire toward high-stability alpha beta: peptide complexes presentable to T cells.     

Enhanced interaction of HLA-DM with HLA-DR in enlarged vacuoles of hereditary and infectious lysosomal diseases

Following biosynthesis, class II MHC molecules are transported through a lysosome-like compartment, where they acquire antigenic peptides for presentation to T cells at the cell surface. This compartment is characterized by the presence of HLA-DM, which catalyzes the peptide loading process. Here we report that the morphology and function of the class II loading compartment is affected in diseases with a phenotypic change in lysosome morphology. Swollen lysosomes are observed in cells from patients with the hereditary immunodeficiency Chediak-Higashi syndrome and in cells infected with Coxiella burnetii, the rickettsial organism that causes Q fever. In both disease states, we observed that HLA-DR and HLA-DM accumulate in enlarged intracellular compartments, which label with the lysosomal marker LAMP-1. The distribution of class I MHC molecules was not affected, localizing disease effects to the endocytic pathway. Thus, cellular mechanisms controlling lysosome biogenesis also affect formation of the class II loading compartment. Analysis of cell surface class II molecules revealed that their steady-state levels were not reduced on diseased cells. However, in both disease states, enhanced interaction between HLA-DR and HLA-DM was detected. In the Chediak-Higashi syndrome cells, this correlated with more efficient removal of the CLIP peptide. These findings suggest a mechanism for perturbation of Ag presentation by class II molecules and consequent immune deficiencies in both diseases.     

DR/CLIP (class II-associated invariant chain peptides) and DR/peptide complexes colocalize in prelysosomes in human B lymphoblastoid cells

In APCs, MHC class II molecules (MHC class II) bind antigenic peptides after HLA-DM mediated removal of CLIP. To characterize intracellular sites of peptide loading in human B lymphoblastoid cell lines, we conducted immunoelectron microscopy studies with Abs recognizing MHC class II associated with CLIP or bound peptide, respectively, together with Abs to HLA-DM and endocytic markers. The distribution of these molecules indicates that peptide binding occurs in compartments with characteristics of normal late endosomes, and in compartments that show characteristics of late endosomes, but are not detectably accessed by endocytosed BSA-gold. The latter compartments may represent or give rise to recycling vesicles that deliver peptide-loaded class II molecules to the cell surface. In addition, we have compared cells in which HLA-DM and HLA-DR interaction is defective with cells in which this interaction is intact, and find that DM/DR interaction is not required for the proper localization of either molecule to peptide-loading compartments.     

Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80-82 segment of the class II beta chain

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80-82 region of the beta chain were introduced into invariant chain (Ii)-negative fibroblasts with wild-type alpha chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early biosynthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80-82 segment of the MHC class II beta chains.     

The tetraspan protein CD82 is a resident of MHC class II compartments where it associates with HLA-DR, -DM, and -DO molecules

In specialized APCs, MHC class II molecules are synthesized in the endoplasmic reticulum and transported through the Golgi apparatus to organelles of the endocytic pathway collectively called MHC class II compartments (MIICs). There, the class II-associated invariant chain is degraded, and peptides derived from internalized Ag bind to empty class II in a reaction that is facilitated by the class II-like molecule HLA-DM. An mAb raised to highly purified, immunoisolated MIICs from human B lymphoblastoid cells recognized CD82, a member of the tetraspan family of integral membrane proteins. Subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy showed that CD82 is highly enriched in MIICs, particularly in their internal membranes. Coprecipitation analysis showed that CD82 associates in MIICs with class II, DM, and HLA-DO (an inhibitor of peptide loading that binds DM). Similar experiments showed CD63, another tetraspan protein found in MIICs, also associates with these molecules in the compartment and that CD82 and CD63 associate with each other. Preclearing experiments demonstrated that both CD82 and CD63 form complexes with DM-associated class II and DM-associated DO. The ability of CD82 and CD63 to form complexes with class II, DM, and DO in MIICs suggests that the tetraspan proteins may play an important role in the late stages of MHC class II maturation.     

A role for HLA-DO as a co-chaperone of HLA-DM in peptide loading of MHC class II molecules

In B cells, the non-classical human leukocyte antigens HLA-DO (DO) and HLA-DM (DM) are residents of lysosome-like organelles where they form tight complexes. DM catalyzes the removal of invariant chain-derived CLIP peptides from classical major histocompatibility complex (MHC) class II molecules, chaperones them until peptides are available for loading, and functions as a peptide editor. Here we show that DO preferentially promotes loading of MHC class II molecules that are dependent on the chaperone activity of DM, and influences editing in a positive way for some peptides and negatively for others. In acidic compartments, DO is engaged in DR-DM-DO complexes whose physiological relevance is indicated by the observation that at lysosomal pH DM-DO stabilizes empty class II molecules more efficiently than DM alone. Moreover, expression of DO in a melanoma cell line favors loading of high-stability peptides. Thus, DO appears to act as a co-chaperone of DM, thereby controlling the quality of antigenic peptides to be presented on the cell surface.     

Attenuation of HLA-DR expression by mononuclear phagocytes infected with Mycobacterium tuberculosis is related to intracellular sequestration of immature class II heterodimers

MHC class II expression was examined in macrophages infected with Mycobacterium tuberculosis. IFN-gamma increased the surface expression of class II molecules in THP-1 cells and this was markedly reduced in cells infected with M. tuberculosis. Despite this effect, steady state levels of HLA-DRalpha, HLA-DRbeta, and invariant (Ii) chains were equivalent in control and infected cells. Metabolic labeling combined with pulse-chase experiments and biochemical analysis showed that the majority of class II molecules in infected cells became resistant to endoglycosidase H, consistent with normal Golgi processing. However, results of intracellular staining and dual color confocal microscopy revealed a significant defect in transport of newly synthesized class II molecules through the endocytic compartment. Thus, compared with findings in control cells, class II molecules in infected cells colocalized to a minimal extent with a lysosomal-associated membrane protein-1+ endosomal compartment. In addition, in contrast to control cells, class II molecules in infected cells failed to colocalize with endocytosed BSA under conditions where this marker is known to label late endosomes, lysosomes, and the MHC class II compartment. Consistent with defective transport along the endocytic pathway, the maturation of SDS-stable class II alphabeta dimers--dependent upon removal of Ii chain and peptide loading of class II dimers in the MHC class II compartment--was markedly impaired in M. tuberculosis-infected cells. These findings indicate that defective transport and processing of class II molecules through the endosomal/lysosomal system is responsible for diminished cell surface expression of MHC class II molecules in cells infected with M. tuberculosis.     

Exceptional stability of the HLA-DQA1*0102/DQB1*0602 alpha beta protein dimer, the class II MHC molecule associated with protection from insulin-dependent diabetes mellitus

HLA-DQ alleles are closely associated with susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM) but the immunologic mechanisms involved are not understood. Structural studies of the IDDM-susceptible allele, HLA-DQA1*0301/DQB1*0302, have classified it as a relatively unstable dimer, particularly at neutral pH. This is reminiscent of studies in the nonobese diabetic mouse, in which I-A(g7) is relatively unstable, in contrast to other murine I-A alleles, suggesting a correlation between unstable MHC class II molecules and IDDM susceptibility. We have addressed this question by analysis of dimer stability patterns among various HLA-DQ molecules. In EBV-transformed B-lymphoblastoid cell lines and PBL, the protein encoded by the IDDM-protective allele HLA-DQA1*0102/DQB1*0602 was the most SDS stable when compared with other HLA-DQ molecules, including HLA-DQA1*0102/DQB1*0604, a closely related allele that is not associated with protection from IDDM. Expression of six different HLA-DQ allelic proteins and three different HLA-DR allelic proteins in the bare lymphocyte syndrome cell line, BLS-1, revealed that HLA-DQA1*0102/DQB1*0602 is SDS stable even in the absence of HLA-DM, while other HLA class II molecules are not. These results suggest that the molecular property of HLA-DQ measured by resistance to denaturation of the alphabeta dimer in SDS may play a role in IDDM protection.     

A recombinant virus-like particle system derived from parvovirus as an efficient antigen carrier to elicit a polarized Th1 immune response without adjuvant

Hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus (PPV) VP2 capsid protein carrying a CD8+ or CD4+ T cell epitope. Immunization of mice with a single dose of these hybrid pseudo-particles, without adjuvant, induced strong cytotoxic T lymphocyte and T helper (Th) responses against the reporter epitope. The Th response was characterized by a Th1 phenotype. We also analyzed in vitro the uptake mechanism of these parvovirus-like particles and the processing requirements associated with presentation by MHC molecules. Although previously shown to be presented by MHC class I molecules, these particles also enter very efficiently the MHC class II endocytic pathway, and behave as conventional exogenous antigens. Indeed, the processing of chimeric PPV:VLP was performed in endosomal/lysosomal acidic vesicles and the presentation of the foreign epitope carried by these particles was sensitive to brefeldin A and cycloheximide, showing that the foreign peptide was loaded on nascent MHC class II molecules. These results give some indication of how PPV:VLP can be presented by MHC class I and class II molecules, and underscore the wide potency of such VLP system to deliver foreign antigens for vaccine design.     

MHC class II transport from lysosomal compartments to the cell surface is determined by stable peptide binding, but not by the cytosolic domains of the alpha- and beta-chains

Inside APCs, MHC class II molecules associate with antigenic peptides before reaching the cell surface. This association takes place in compartments of the endocytic pathway, more related to endosomes or lysosomes depending on the cell type. Here, we compared MHC class II transport from endosomal vs lysosomal compartments to the plasma membrane. We show that transport of MHC class II molecules to the cell surface does not depend on the cytosolic domains of the alpha- and beta-chains. In contrast, the stability of the alphabeta-peptide complexes determined the efficiency of transport to the cell surface from lysosomal, but not from endosomal, compartments. In murine B lymphoma cells, SDS-unstable and -stable complexes were transported to the cell surface at almost similar rates, whereas after lysosomal relocalization or in a cell line in which MHC class II molecules normally accumulate in lysosomal compartments, stable complexes were preferentially addressed to the cell surface. Our results suggest that when peptide loading occurs in lysosomal compartments, selective retention and lysosomal degradation of unstable dimers result in the expression of highly stable MHC class II-peptide complexes at the APC surface.     

Regulation of MHC class I and II gene transcription: differences and similarities

Major histocompatibility complex (MHC) molecules serve as peptide receptors. These peptides are derived from processed cellular or extra-cellular antigens. The MHC gene complex encodes two major classes of molecules, MHC class I and class II, whose function is to present peptides to CD8+ (cytotoxic) and CD4+ (helper) T cells, respectively. The genes encoding both classes of MHC molecules seem to originate from a common ancestral gene. One of the hallmarks of the MHC is its extensive polymorphism which displays locus and allele-specific characteristics among the various MHC class I and class II genes. Because of its central role in immunosurveillance and in various disease states, the MHC is one of the best studied genetic systems. This review addresses several aspects of MHC class I and class II gene regulation in human and in particular, the contribution to the constitutive and cytokine-induced expression of MHC class I and II genes of MHC class-specific regulatory elements and regulatory elements which apparently are shared by the promoters of MHC class I and class II genes.     

Formulations of sugars with amino acids or mannitol--influence of concentration ratio on the properties of the freeze-concentrate and the lyophilizate

We have identified a novel lysosome-associated membrane glycoprotein localized on chromosome 3q26.3-q27, DC-LAMP, which is homologous to CD68. DC-LAMP mRNA is present only in lymphoid organs and DC. A specific MAb detects the protein exclusively in interdigitating dendritic cells. Expression of DC-LAMP increases progressively during in vitro DC differentiation, but sharply upon activation with LPS, TNFalpha, or CD40L. Confocal microscopy confirmed the lysosomal distribution of the protein. Furthermore, DC-LAMP was found in the MHC class II compartment immediately before the translocation of MHC class II molecules to the cell surface, after which it concentrates into perinuclear lysosomes. This suggests that DC-LAMP might change the lysosome function after the transfer of peptide-MHC class II molecules to the surface of DC.     

Phosphorylation regulates the delivery of MHC class II invariant chain complexes to antigen processing compartments

Transport of newly synthesized MHC class II glycoproteins to endosomal Ag processing compartments is mediated by their association with the invariant chain (Ii). Targeting to these compartments is dependent upon recognition of leucine-based endo. somal/lysosomal targeting motifs in the Ii cytosolic domain. Ii, like many molecules that contain leucine-based endosomal targeting motifs, is phosphorylated in vivo. In this report we demonstrate that the cytosolic domain of the p35 Ii isoform is phosphorylated in class II Ii complexes isolated from human B lymphoblastoid cell lines or freshly obtained PBMC. Mutation of serine residue 6 or 8 prevents phosphorylation of Ii-p35 expressed in HeLa cells. Treatment of B lymphoblastoid cell lines with the serine/threonine kinase inhibitor staurosporine prevented Ii phosphorylation and significantly delayed trafficking of newly synthesized class II Ii complexes to endosomal Ag processing compartments. By contrast, staurosporine had no effect on the rate of transport of class I or class II glycoproteins through the Golgi apparatus and did not inhibit the delivery of the chimeric molecule Tac-DM, to endocytic compartments, suggesting that staurosporine does not nonspecifically inhibit protein transport to the endocytic pathway. These results demonstrate that phosphorylation regulates the efficient targeting of MHC class II Ii complexes to Ag processing compartments and strongly suggest that this effect is mediated by phosphorylation of the MHC class II-associated Ii chain.     

Major histocompatibility complex class II molecules, hemopoiesis and the marrow microenvironment

Currently available data indicate that the earliest identifiable hemopoietic progenitor in normal marrow is CD34+ MHC class II-; subsequent expression of MHC class II antigens is maturation and lineage dependent. Studies on embryonal cells suggest that CD34+DR- cells are actually the common precursors for stromal and hemopoietic elements, with the earliest hemopoietic precursor being CD34+DR+. DQ antigens are apparently not expressed in cells of hemopoietic potential and the expression of DQ appears to be regulated differentially from DR and DP. MHC class II antigens are also expressed on some stromal cells, especially those with endothelial and macrophage features. MHC class II molecules are involved in hemopoietic cell/stroma interaction. The presence of anti-MHC class II monoclonal antibodies (MABs) at early stages of stem cell proliferation/differentiation, at least under conditions of marrow stress, induces signals which may result in final, especially granulocytic, differentiation of later precursors. These may interfere with the survival of those cells which are required for long-term hemopoietic reconstitution. Observations in allogeneic marrow transplant recipients support a role of MHC molecules as expected in allogeneic interactions. Results in autologous models point towards a role of MHC class II molecules other than that of a histocompatibility marker insofar as these molecules or signals transmitted by them appear to be involved in the regulation of hemopoiesis.     

Deficient major histocompatibility complex class II antigen presentation in a subset of Hodgkin's disease tumor cells

Hodgkin's disease is a common malignancy of the lymphoid system. Although the scarce Hodgkin and Reed-Sternberg (HRS) tumor cells in involved tissue synthesize major histocompatibility complex (MHC) class II and costimulatory molecules such as CD40 or CD86, it is unclear whether these tumor cells are operational antigen-presenting cells (APC). We developed an immunofluorescence-based assay to determine the number of MHC class II molecules present on the surface of single living HRS cells. We found that in fresh Hodgkin's disease lymph node biopsies, a subset of HRS cells express a substantial number of surface MHC class II molecules that are occupied by MHC class II-associated invariant chain peptides (CLIP), indicating deficient loading of MHC class II molecules with antigenic peptides. Cultured Hodgkin's disease-derived (HD) cell lines, however, were found to express few MHC class II molecules carrying CLIP peptides on the cell surface and were shown to generate sodium dodecyl sulphate (SDS)-stable MHC class II alphabeta dimers. In addition to showing deficient MHC class II antigen presentation in a subset of HRS cells, our results show that the widely used HD-cell lines are not ideal in vitro models for the disease. The disruption of MHC class II-restricted antigen presentation in HRS cells could represent a key mechanism by which these tumor cells escape immune surveillance.     

Morphology and structure of photosensitive dye J-aggregates adsorbed on AgBr microcrystals grown in gelatin

Our purpose was to determine the expression of the major histocompatibility complex (MHC) class I and class II gene products as well as the costimulatory molecules B7-1 and B7-2 on cervical epithelial cells, and to determine to what extent inflammatory cytokines regulate their expression. Immunohistology and flow cytometry techniques were used to identify and quantify MHC class I and class II molecules, and the costimulatory molecules B7.1 and B7.2, on sections and primary epithelial cell cultures of human endo- and ectocervix. MHC class I but not class II molecules were constitutively expressed on tissue sections and primary epithelial cell cultures derived from endo- and ectocervix. Expression of MHC class I and class II was upregulated in vitro by IFN-gamma in a time and dose dependent fashion. The induction of class II expression was more pronounced on ectocervical cells than on endocervical cells. MHC class I but not class II expression was also enhanced by IFN-alpha as well as TNF-alpha. TNF-alpha and TGF-beta1 inhibited the IFN-gamma induced MHC class II expression. Expression of the costimulatory molecules B7-1 and B7-2 were not detected in tissue sections or on resting or cytokine-treated cervical epithelial cells in vitro. The present results support the concept that endo- and ectocervical epithelial cells, like their counterparts at other mucosal sites. constitutively express MHC class I molecules and can express MHC class II upon cytokine stimulation, indicating that they are capable of presenting antigens to T-cells.     

The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.     

HLA-DM stabilizes empty HLA-DR molecules in a chaperone-like fashion

HLA-DM (DM) is a non-classical major histocompatibility complex (MHC) class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (II)-derived CLIP peptides thereby rendering the peptide binding groove accessible for antigenic peptide loading. However, in situations of peptide scarcity the fate of these DM:DR complexes is not known. We could show that DR molecules incubated at lysosomal pH in the absence of peptide rapidly undergo functional inactivation and aggregation. In the presence of DM, however, empty DR molecules were shown to be stabilised and kept receptive for peptide loading, with the degree of the stabilising effect of DM varying for different DR alleles. In addition, in lysosomal compartments a considerable fraction of DM was found to be stably associated with empty DR alpha beta dimers thereby preserving their functionality. Upon encounter with antigenic peptide the DM-associated DR molecules could be rapidly loaded, whereupon they did no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone that rescues uncharged alpha beta dimers. In view of the suggested shortage of self-peptides in the loading compartment, empty class II molecules that are kept receptive for loading by the chaperone function of DM may enable the antigen processing system to respond promptly to the challenge by newly entering antigens.     

Dual role of dendritic cells in the induction and down-regulation of antigen-specific cutaneous inflammation

We have previously reported that contact sensitivity (CS) to dinitrofluorobenzene (DNFB) in C57BL/6 mice was mediated by MHC class I-restricted CD8+ T cells and down-regulated by MHC class II-restricted CD4+ T cells. In this study, we analyzed the contribution of dendritic cells (DC) in the induction of these two T cell subsets endowed with opposite functions. Hapten-pulsed skin- and bone marrow-derived DC, obtained from either normal C57BL/6 mice or from MHC class II (I+ II-) and MHC class I (I- II+)-deficient mice, were tested for their ability to prime normal mice for CS to dinitrofluorobenzene. Expression of MHC class I molecules by transferred DC was mandatory both for the induction of CS and for the generation of hapten-specific CD8+ T cells in lymphoid organs. I+ II- DC were as potent as I+ II+ DC in priming for CS, demonstrating that activation of effector CD8+ T cells can occur independently of CD4+ T cell help. I- II+ DC could not immunize for CS, although they could sensitize for a delayed-type hypersensitivity reaction to protein Ags. Moreover, I- II+ DC injected simultaneously with cutaneous sensitization down-regulated the inflammatory response, suggesting that hapten presentation by MHC class II molecules could prime regulatory CD4+ T cells. These results indicate that DC can present haptenated peptides by both MHC class I and class II molecules and activate Ag-specific CD8+ effector and CD4+ regulatory T cell subsets, concurrently and independently.     

Monocyte chemoattractant protein 1 is responsible for macrophage recruitment following injury to sciatic nerve

We designed a peptide, H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), that permeabilizes cell membrane at a slightly acidic pH but not at neutral pH. Absorbance, fluorescence, and circular dichroism spectra showed that H5WYG undergoes a dramatic conformational change between pH 7.0 and 6.0 that correlates with the protonation of the histidyl residues. Cell permeabilization studies monitored by flow cytometry on living cells showed that H5WYG permeabilizes the cell membrane with a great efficiency at pH 6.4 but was not active at neutral pH; at pH 6.8, the peptide permeabilized 50% of the cells at 20 degrees C within 10 min. H5WYG increased the expression of genes transferred to cells as glycosylated polylysine-DNA complexes, and the transfection efficiency was not impaired in the presence of serum. Therefore, this peptide containing several histidines that become positively charged when the pH decreased to less than 7.0 is a suitable helper for delivering molecules into the cytosol upon either permeabilization of the plasma membrane induced by lowering the extracellular medium to pH 6.4 or permeabilization of the endosomal membrane induced by acidification of endosomes.