Inflammatory chemokine transport and presentation in HEV: a remote control mechanism for monocyte recruitment to lymph nodes in inflamed tissues.

Interstitial fluid is constantly drained into lymph nodes (LNs) via afferent lymph vessels. This conduit enables monocyte-derived macrophages and dendritic cells to access LNs from peripheral tissues. We show that during inflammation in the skin, a second recruitment pathway is evoked that recruits large numbers of blood-borne monocytes to LNs via high endothelial venules (HEVs). Inhibition of monocyte chemoattractant protein (MCP)-1 blocked this inflammation-induced monocyte homing to LNs. MCP-1 mRNA in inflamed skin was over 100-fold upregulated and paralleled MCP-1 protein levels, whereas in draining LNs MCP-1 mRNA induction was much weaker and occurred only after a pronounced rise in MCP-1 protein. Thus, MCP-1 in draining LNs was primarily derived from inflamed skin. In MCP-1(-/-) mice, intracutaneously injected MCP-1 accumulated rapidly in the draining LNs where it enhanced monocyte recruitment. Intravital microscopy showed that skin-derived MCP-1 was transported via the lymph to the luminal surface of HEVs where it triggered integrin-dependent arrest of rolling monocytes. These findings demonstrate that inflamed peripheral tissues project their local chemokine profile to HEVs in draining LNs and thereby exert "remote control" over the composition of leukocyte populations that home to these organs from the blood.

CCR5, CXCR4, and CD4 are clustered and closely apposed on microvilli of human macrophages and T cells.

The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences.

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.

Functional significance of varied quantitative and qualitative expression of HLA-A2.1 antigens in determining the susceptibility of cells to cytotoxic T lymphocytes.

To determine whether varied quantitative HLA expression affects the susceptibility of target cells to CTLs, a panel of 15 EBV-transformed lymphoblastoid cell lines expressing a fivefold difference of surface HLA-A2.1 antigens were employed. The susceptibility of these cell lines to HLA-A2.1-restricted and influenza virus matrix peptide-specific CTLs was correlated with the amounts of HLA-A2.1 antigens expressed on their surface. The results show a linear correlation between both parameters using exogenous viral peptide. The same linear correlation was observed when target cells infected with influenza virus were studied. These findings support the hypothesis that the amount of HLA antigens expressed on the cell surface is functionally significant in determining the susceptibility of target cells to CTLs. During our study, we also found that two HLA-A-2.1-positive cell lines were unresponsive to the CTL. Further investigation of the amino acid sequences of these cell lines reveals that their HLA-A2.1 antigens belong to the HLA-A0207 subtype which is different from HLA-A0201(A2.1) by one nucleotide. This difference results in an amino acid substitution from tyrosine to cysteine at position 99 of HLA-A2.1 heavy chains. Using a peptide-induced reconstitution assay, it was shown that failure of the peptide binding is responsible for the absence of cytotoxicity. This finding supports the hypothesis that amino acid 99 plays an important role in determining the peptide-binding specificity of HLA-A2 molecules.

Mutation of the alpha 2 domain disulfide bridge of the class I molecule HLA-A*0201. Effect on maturation and peptide presentation.

A combination of saturation and site-directed mutagenesis was utilized to disrupt the alpha 2 domain disulfide bridge of HLA-A*0201. Mutation of cysteine 101 to a serine (C101S) or of cysteine 164 to alanine (C164A) decreased the rate of maturation of the heavy chain, the total amount of mature heavy chain within the cell, and the level of surface expression. Cells expressing these genes and loaded with a synthetic peptide derived from the influenza A matrix protein (58-66) were recognized poorly by HLA-A*0201-restricted, peptide-specific CTLs. Cells expressing mutant HLA-A*0201 loaded with a synthetic peptide derived from the HIV-1 pol protein (476-484) were not recognized by pol IV-9-specific CTLs. Mutant C164A cells infected with influenza virus were partially recognized by influenza matrix peptide-specific CTLs, while C101S cells were not lysed. Surprisingly, endogenous peptide loading of cells expressing mutant HLA-A*0201 using a minigene coding for either the influenza A matrix peptide 58-66, or HIV-1 pol peptide 476-484, resulted in efficient CTL recognition. This suggests different structural constraints for peptide binding in the endoplasmic reticulum during biosynthesis and for binding to exported molecules on the cells surface.

Presentation of endogenous peptides to MHC class I-restricted cytotoxic T lymphocytes in transport deletion mutant T2 cells.

The ability of minigene-encoded viral peptide epitopes to be presented by class I molecules in the absence of MHC-encoded transporters has been evaluated in mutant T2 cells. These cells have a large deletion in the class II MHC region that includes the known transporter protein for antigenic peptides and proteasome genes and they are defective in presenting viral epitopes to CTL. T2 cells that express minigenes encoding the influenza virus matrix peptide 58-66 (GILGFVFTL) and two HTLV 1 Tax peptides 11-19 (LLFGYPVYV) and 12-19 were lysed by HLA-A2-restricted peptide-specific CTL. Minigene expression of a HLA-A2-restricted HIV reverse transcriptase peptide 476-484 (ILKEPVHGV) with three charged residues sensitized T2 cells poorly for lysis by HIV-specific CTL unless the peptide was preceded by an endoplasmic reticulum translocation signal sequence. Expression of an influenza virus nucleoprotein peptide 383-391 (SRYWAIRTR) with three charged arginine residues did sensitize HLA-B27+ T2 cells for lysis by peptide-specific CTL. These and other results with endogenously expressed peptide analogs in which hydrophobic and charged amino acids were interchanged demonstrate that antigenic peptides can be translocated from the cytoplasm into the class I Ag presentation pathway independent of MHC-encoded transporters; and that peptide hydrophobicity appears not to be a major determinant in selecting peptides for this alternate pathway.

Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2.

Previous studies have indicated that most HLA-A2-binding peptides are 9 amino acid (aa) residues long, with a Leu at position 2 (P2), and a Val or Leu at P9. We compared the binding properties of different peptides by measuring the rate of dissociation of beta 2-microglobulin from peptide-specific HLA-A2 complexes. The simplest peptide that we identified that could form HLA-A2 complexes had the sequence (in single letter aa code) GLFGGGGGV, indicating that three nonglycine aa are sufficient for binding to HLA-A2. To determine whether most nonapeptides that contained Leu at P2 and Val or Leu at P9 could bind to HLA-A2, we tested the binding of nonapeptides selected from published HIV and melanoma protein sequences, and found that six of seven tested formed stable HLA-A2 complexes. We identified an optimal antigenic undecapeptide from the cytomegalovirus gB protein that could form stable HLA-A2 complexes that contained apparent anchor residues at P2 and P11 (sequence FIAGN-SAYEYV), indicating that the spacing between anchor residues can be somewhat variable. Finally, we tested the importance of every aa in the influenza A matrix peptide 58-66 (sequence GILGFVFTL) for binding to HLA-A2, by using Ala-substituted and Lys-substituted peptides. We found that multiple positions were important for stable binding, including P2, P3, P5-P7, and P9. We conclude that the P2 and P9 anchor residues are of prime importance for peptide binding to HLA-A2. However, other peptide side chains (especially at P3) contribute to the stability of the interaction. In certain cases, the optimal length for peptide binding can be longer than 9 residues.

Endogenous loading of HLA-A2 molecules with an analog of the influenza virus matrix protein-derived peptide and its inhibition by an exogenous peptide antagonist.

Episomal plasmids (p8901) with minigenes coding for the influenza virus matrix peptide amino acids 57-68 (KGILGFVFTLTV; referred to as M57-68) or coding for a modified peptide were introduced into HLA-A2-positive target cells. The association of these peptides, synthesized in the cytoplasm, with HLA-A2 and the expression of this complex at the cell surface was evaluated with HLA-A2-restricted CTL specific for the influenza virus matrix peptide M57-68. Cells expressing M57-68 were lysed effectively, as were cells expressing a peptide that retained residues 60-64 with seven flanking alanine residues (AAALGFVFAAAA). An exogenously added synthetic analog of peptide M57-68 that inhibited sensitization of targets with synthetic peptide M57-68 also inhibited lysis of cells expressing the minigene coding for the peptide with seven alanine substitutions. These results demonstrate the utility of minigene DNA constructs in creating experimental systems to develop agents to diminish the severity of CTL-mediated tissue damage in autoimmune diseases and graft rejection.

The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2.

Influenza virus matrix protein-derived peptides were synthesized based on the amino acid motifs for HLA-A2 bound self peptides. Among these peptides a nonamer (amino acids 58 through 66: G I L G F V F T L) was found to be 100 to 1000 times more effective than the commonly used peptide 57-68 (K G I L G F V F T L T V) in sensitizing HLA-A2+ target cells to lysis by influenza virus specific cytotoxic T lymphocytes. The sensitizing activity of the 12-mer 57-68 was not due to contamination with shorter and more active peptides. Intracellular expression of peptide 58-66 (mediated by a stable expression plasmid with DNA coding for this peptide) also sensitized HLA-A2+ cells to lysis. Peptide 58-66 stimulated human PBMC to generate CTL that recognized peptides 58-66 and 57-68 in association with HLA-A2.

Endogenously synthesized peptide with an endoplasmic reticulum signal sequence sensitizes antigen processing mutant cells to class I-restricted cell-mediated lysis.

The HLA-A2-positive human mutant cell line T2 is not lysed by influenza virus-specific HLA-A2-restricted cytotoxic lymphocytes after virus infection. However, lysis does occur when cells are incubated with the antigenic influenza matrix protein-derived peptide M57-68. To examine the nature of this defect, T2 cells were transfected with two different plasmids. One plasmid encoded the peptide M57-68, and the other encoded the same peptide preceded by an endoplasmic reticulum translocation signal sequence. Mutant T2 cells expressing the M57-68 peptide without the signal sequence were not susceptible to lysis by M57-68-specific HLA-A2-restricted cytotoxic T lymphocytes, whereas T2 cells expressing the M57-68 peptide plus signal sequence were lysed effectively. Lysis of parental T1 cells with either plasmid was equally effective. These results suggest that the T2 mutant cells are defective in the transport of antigenic peptides from the cytosol into the secretory pathway.

Soluble HLA-A2.1 restricted peptides that are recognized by influenza virus specific cytotoxic T lymphocytes.

The influenza A virus matrix protein derived peptide with amino acids 57-68 (Lys-Gly-Ileu-Leu-Gly-Phe-Val-Phe-Thr-Leu-Thr-Val) is recognized by influenza virus HLA-A2 restricted CTL. Because of the large number of hydrophobic residues this peptide is very insoluble. Substitution with a number of polar amino acids resulted in a soluble peptide (Lys-Lys-Ala-Leu-Gly-Phe-Val-Phe-Thr-Leu-Asp-Lys) that was very effective in sensitizing HLA-A2 positive target cells. Further substitution of threonine in position 65 with lysine resulted in a soluble antagonist peptide that inhibited sensitization. Both agonist and antagonist peptides retained 20% of their biological activity when tyrosine was added at the N terminus. Soluble radio-iodinated peptides can now be prepared that will be useful reagents to study the interaction of peptides and class I molecules.

Human cytotoxic lymphocyte granzyme B. Its purification from granules and the characterization of substrate and inhibitor specificity.

Granzyme B has been purified to homogeneity from the granules of a human cytolytic lymphocyte line, Q31, in an enzymatically active form by a three-step procedure. Q31 granzyme B hydrolyzed Na-t-butyloxycarbonyl-L-alanyl-L-alanyl-L-aspartyl (Boc-Ala-Ala-Asp) thiobenzyl ester with a kcat of 11 +/- 5 mol/s/mol enzyme and catalytic efficiency kcat/Km of 76,000 +/- 44,000 M-1 s-1. The hydrolysis of Boc-Ala-Ala-Asp thiobenzyl ester by crude Q31 Percoll fractions paralleled the tryptase activity for granule-containing fractions, which showed that granzyme B was associated with granules. When chromatographed on Sephacryl S-300, Q31 granzyme B eluted in two broad bands corresponding to dimer and monomer, both of which electrophoresed at 35 kDa in reducing NaDodSo4 polyacrylamide, and both of which showed a lag phase in assays. The lag phase in assays could be extended with 0.03 mM pepstatin. Upon elution from ion-exchange chromatography Q31 granzyme B electrophoresed at 32 kDa in reducing NaDodSO4 polyacrylamide and did not have a lag phase in assays. The amino-terminal sequence of the 32-kDa Q31 granzyme B was identical to four other human cytotoxic T-lymphocyte granzymes B in 18 of 18 positions sequenced. Purified Q31 granzyme B had a preference for substrates with Glu or Asp as the residue amino-terminal to the scissile bond; little or no activity was noted with oligopeptide substrates for trypsin-like, chymotrypsin-like, and elastase-like proteases. Human plasma alpha 1-protease inhibitor, human plasma alpha 2-protease macroglobulin, soybean and lima-bean trypsin inhibitors, bovine aprotinin, phosphoramidon, and chymostatin inhibited Q31 granzyme B. The inhibition by alpha 1-protease inhibitor was rapid enough to be of physiological significance.

The enzymatic activity of human cytotoxic T-lymphocyte granzyme A and cytolysis mediated by cytotoxic T-lymphocytes are potently inhibited by a synthetic antiprotease, FUT-175.

The synthetic antiprotease, FUT-175 (6-amidino-2-naphthyl-4-guanidinobenzoate), was found to be an extraordinarily potent and rapid inhibitor of human Q31 cytotoxic T-lymphocyte granzyme A. The granzyme A was inhibited in a time-dependent manner with kobs/i = 430,000 +/- 80,000 M-1 s-1. Four other FUT-175 analogs were also found to be potent, rapid Q31 granzyme A inhibitors. All five compounds inhibited Q31 cytotoxic T-lymphocyte-mediated cytolysis of human JY lymphoma cells, but at concentrations far in excess of those needed for granzyme A inhibition. The data presented suggest that postmarketing surveillance of FUT-175 should include a review of possible immunosuppressive side-effects, such as increased susceptibility to viral infections and to neoplastic transformations.

Evidence of widespread binding of HLA class I molecules to peptides.

We have tested the binding of HLA class I proteins to peptides using a solid-phase binding assay. We tested 102 peptides, mostly derived from the HIV gag and HIV pol sequences. Most peptides did not bind to any class I protein tested. The pattern of binding among the three class I proteins tested, HLA-A2, -B27, and -B8, was approximately 85% concordant. Further, all five of the known HIV-1 gag T cell epitopes detected by human CTL bound at least one class I protein. Binding of class I to the peptides could be detected either by directly iodinated class I proteins, or indirectly using monoclonal antibodies specific for class I. The binding to the plates could be blocked with MA2.1, which binds in the alpha 1 region of A2, but not by W6/32, which binds elsewhere. The data presented here show that binding of class I to peptides is specific, but that many peptides bind to more than a single class I protein.

A human monoclonal antibody that protects mice against Pseudomonas-induced pneumonia.

X-linked immunodeficient (xid) (CBA/N female x DBA/2 male) F1 male mice, when treated with cyclophosphamide, were much more susceptible to challenge with aerosolized Pseudomonas aeruginosa serotype 11 than were control F1 female littermates. Mortality of F1 males was decreased significantly after intravenous administration of human P. aeruginosa serotype 11 O-specific monoclonal antibody. Antibody treatment reduced bacterial titers in the lungs as well as the severity of Pseudomonas-induced lung histopathology.

Glycolipids as host resistance stimulators.

6-(5-Cholesten-3 beta-yloxy)hexyl 1-thio-beta-D-mannopyranoside (L-644,257) enhances natural host resistance in cyclophosphamide-treated mice against Pseudomonas aeruginosa in a dose-dependent manner. It is active sc, im, and ip but not orally. L-644,257 is substantially more protective against P. aeruginosa than its alpha anomer. The beta-L-fucose glycolipid is more effective when given im and ip than sc. The lactose and beta-D-glucose glycolipids were only marginally effective to nonprotective. The 17 beta-steroidal side chain of L-644,257 can be modified without substantial loss of protective activity.

Steroidal glycolipid, L-644,257, is a potent enhancer of nonspecific host resistance.

A steroidal glycolipid that enhances the nonspecific cellular response to opportunistic infection in an immunocompromised host has been discovered. A dose dependent response with 6-(5-cholesten-3 beta-yloxy)hexyl 1-thio-beta-D-mannopyranoside, L-644,257, was observed against several infective agents including bacterial, fungal, and viral pathogens in cyclophosphamide-treated mice. A mechanism for this protective action is proposed.

Heterohybridomas that secrete high levels of pseudomonas-specific therapeutic human monoclonal antibodies: their generation and large scale growth in an automated hollow fiber cell culture system.

Fusion of lymphoblastoid cell lines that produce human monoclonal antibodies against Pseudomonas aeruginosa with the human/mouse heteromyeloma SHM-D33 generated heterohybrids that were stable and secreted antibody in the range of 20 to 300 micrograms/ml. One of the hybridoma cell lines ws adapted to serum-free medium and maintained for 60 days in an automated hollow fiber system. During that time, 3 g of antibody was produced. Such yields make it possible to evaluate these monoclonals for their therapeutic potential in patients at risk for Pseudomonas infections.

In vitro and in vivo activity of polyclonal and monoclonal human immunoglobulins G, M, and A against Pseudomonas aeruginosa lipopolysaccharide.

We evaluated the in vitro opsonophagocytic killing activity of monoclonal human immunoglobulin G (IgG), IgM, and IgA specific for Pseudomonas aeruginosa lipopolysaccharide and the in vivo protective capacity in neutropenic mice of both monoclonal and purified polyclonal IgG, IgM, and IgA. Monoclonal IgM was efficacious in mediating opsonophagocytic killing only in conjunction with complement, whereas monoclonal IgG opsonic killing was potentiated by complement, and monoclonal IgA opsonic killing was independent of complement. These findings are similar to those previously reported for purified polyclonal IgM, IgG, and IgA. The monoclonal and polyclonal immunoglobulins had comparable 50% protective doses in neutropenic mice (range, 0.28 to 0.46 microgram per mouse). The protective activity of IgM in neutropenic mice was abolished by cobra venom factor treatment, whereas IgG and IgA maintained efficacy in cobra venom factor-treated mice. These data indicate that all three major human serum immunoglobulin isotypes have opsonophagocytic and protective activities against P. aeruginosa, with a critical role for complement in the function of IgM.

Suppression of in vitro granulocytopoiesis by captopril and penicillamine.

The mechanisms underlying drug-induced neutropenia are poorly characterized. We have examined the mechanism of suppression of granulocytopoiesis by captopril and penicillamine using human and canine bone marrow cells in an in vitro culture system. Addition of captopril caused no significant change in granulocyte-macrophage colony formation at concentrations up to 30 micrograms/ml. In the presence of CuSO4 (1-3 micrograms/ml), however, captopril caused significant inhibition of colony growth (p less than 0.05). Penicillamine, another agent associated with neutropenia and, like captopril, having a reactive thiol group, also inhibited colony formation in the presence of copper. Chemical congeners of captopril lacking a reactive thiol group and enalaprilic acid, an alternative angiotensin-converting enzyme (ACE) inhibitor, failed to show inhibition, suggesting that the thiol group and not ACE inhibition was responsible. Analysis of day-7 colonies (98% neutrophilic) and day-21 colonies (37% neutrophilic, 30% macrophagic, 27% eosinophilic, and 6% mixed) showed that neutrophil-containing colonies, but not nonneutrophilic colonies were inhibited by the addition of captopril plus copper. Catalase totally reversed the inhibition of colony formation caused by these agents. Direct measurement of oxygen consumption in the presence of captopril showed marked enhancement with the addition of CuSO4 and a 48% reduction in the presence of added catalase. These data indicate that drugs with a reactive thiol group can interact with copper to generate H2O2, which can be toxic to neutrophilic progenitor cells. We postulate that this may be an important mechanism for drug-associated neutropenia and a general mechanism for drug-induced marrow cell injury.