CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production

NY-ESO-1 is a tumor-specific shared antigen with distinctive immunogenicity. Both CD8+ T cells and class-switched Ab responses have been detected from patients with cancer. In this study, a CD4+ T cell line was generated from peripheral blood mononuclear cells of a melanoma patient and was shown to recognize NY-ESO-1 peptides presented by HLA-DP4, a dominant MHC class II allele expressed in 43–70% of Caucasians. The ESO p157–170 peptide containing the core region of DP4-restricted T cell epitope was present in a number of tumor cell lines tested and found to be recognized by both CD4+ T cells as well as HLA-A2-restricted CD8+ T cells. Thus, the ESO p157–170 epitope represents a potential candidate for cancer vaccines aimed at generating both CD4+ and CD8+ T cell responses. More importantly, 16 of 17 melanoma patients who developed Ab against NY-ESO-1 were found to be HLA-DP4-positive. CD4+ T cells specific for the NY-ESO-1 epitopes were generated from 5 of 6 melanoma patients with NY-ESO-1 Ab. In contrast, no specific DP4-restricted T cells were generated from two patients without detectable NY-ESO-1 Ab. These results suggested that NY-ESO-1-specific DP4-restricted CD4+ T cells were closely associated with NY-ESO-1 Ab observed in melanoma patients and might play an important role in providing help for activating B cells for NY-ESO-1-specific Ab production.

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing

The spectrum of immunogenic epitopes presented by the H2-IAb MHC class II molecule to CD4+ T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Vα and Vβ chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences (≈20–80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

CD8+ T cells control the TH phenotype of MBP-reactive CD4+ T cells in EAE mice

Trimolecular interactions between the T cell antigen receptor and MHC/peptide complexes, together with costimulatory molecules and cytokines, control the initial activation of naïve T cells and determine whether the helper precursor cell differentiates into either T helper (TH)1 or TH2 effector cells. We now present evidence that regulatory CD8+ T cells provide another level of control of TH phenotype during further evolution of immune responses. These regulatory CD8+ T cells are induced by antigen-triggered CD4+ TH1 cells during T cell vaccination and, in vitro, distinguish mature TH1 from TH2 cells in a T cell antigen receptor Vβ-specific and Qa-1-restricted manner. In vivo, protection from experimental autoimmune encephalomyelitis (EAE) induced by T cell vaccination depends on CD8+ T cells, and myelin basic protein-reactive TH1 Vβ8+ clones, but not TH2 Vβ8+ clones, used as vaccine T cells, protect animals from subsequent induction of EAE. Moreover, in vivo depletion of CD8+ T cells during the first episode of EAE results in skewing of the TH phenotype toward TH1 upon secondary myelin basic protein stimulation. These data provide evidence that CD8+ T cells control autoimmune responses, in part, by regulating the TH phenotype of self-reactive CD4+ T cells.

Stimulation via CD40 can substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus

Reactivation of latent herpesviruses is a particular problem in immunocompromised individuals, such as AIDS patients, who lack effective CD4 T helper cell function. An important question is whether residual immune defenses can be mobilized to combat such opportunistic infections, in the absence of CD4 T cells. In the present study, we used a mouse model of opportunistic infection to determine whether stimulation via CD40 could substitute for CD4 T cell function in preventing reactivation of a latent herpesvirus. Treatment with an agonistic antibody to CD40 was highly effective in preventing reactivation of latent murine gammaherpesvirus (MHV-68) in the lungs of CD4 T cell-deficient mice. CD8+ T cells were essential for this effect, whereas virus-specific serum antibody was undetectable and IFN-γ production was unchanged. This demonstration that immunostimulation via CD40 can replace CD4 T cell help in controlling latent virus in vivo has potential implications for the development of novel therapeutic agents to prevent viral reactivation in immunocompromised patients.

Molecular cloning and sequence analysis of the complestatin biosynthetic gene cluster

Streptomyces lavendulae produces complestatin, a cyclic peptide natural product that antagonizes pharmacologically relevant protein–protein interactions including formation of the C4b,2b complex in the complement cascade and gp120-CD4 binding in the HIV life cycle. Complestatin, a member of the vancomycin group of natural products, consists of an α-ketoacyl hexapeptide backbone modified by oxidative phenolic couplings and halogenations. The entire complestatin biosynthetic and regulatory gene cluster spanning ca. 50 kb was cloned and sequenced. It consisted of 16 ORFs, encoding proteins homologous to nonribosomal peptide synthetases, cytochrome P450-related oxidases, ferredoxins, nonheme halogenases, four enzymes involved in 4-hydroxyphenylglycine (Hpg) biosynthesis, transcriptional regulators, and ABC transporters. The nonribosomal peptide synthetase consisted of a priming module, six extending modules, and a terminal thioesterase; their arrangement and domain content was entirely consistent with functions required for the biosynthesis of a heptapeptide or α-ketoacyl hexapeptide backbone. Two oxidase genes were proposed to be responsible for the construction of the unique aryl-ether-aryl-aryl linkage on the linear heptapeptide intermediate. Hpg, 3,5-dichloro-Hpg, and 3,5-dichloro-hydroxybenzoylformate are unusual building blocks that repesent five of the seven requisite monomers in the complestatin peptide. Heterologous expression and biochemical analysis of 4-hydroxyphenylglycine transaminon confirmed its role as an aminotransferase responsible for formation of all three precursors. The close similarity but functional divergence between complestatin and chloroeremomycin biosynthetic genes also presents a unique opportunity for the construction of hybrid vancomycin-type antibiotics.

Regulatory function of in vivo anergized CD4+ T cells

It has been suggested that anergic T cells may not be only inert cells but may rather play an active role, for example by regulating immune responses. We have previously reported the existence of “anergic” IL-10-producing CD4+ T cells generated in vivo by continuous antigenic stimulation. Using a gene transfer system where the antigen recognized by such T cells is expressed in skeletal muscle by two different DNA viral vectors, we show that these cells not only remain tolerant toward their cognate antigen but also can suppress the immune response of naïve T cells against the immunogenic adenoviral proteins. Furthermore, they can completely inhibit tissue destruction that takes place as a result of an immune response. The system presented here is unique in that the T cells have been anergized in vivo, their antigen specificity and functional status are known, and the amount, form, and timing of antigen expression can be manipulated. This model will therefore permit us to carefully dissect the mechanisms by which these anergic T cells regulate the priming and/or effector function of naïve T cells.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.

Identification of three physically and functionally distinct binding sites for C3b in human complement factor H by deletion mutagenesis.

Human complement factor H controls spontaneous activation of complement in plasma and appears to play a role in distinguishing host cells from activators of the alternative pathway of complement. In both mice and humans, the protein is composed of 20 homologous short consensus repeat (SCR) domains. The size of the protein suggests that portions of the structure outside the known C3b binding site (SCR 1-4) possess a significant biological role. We have expressed the full-length cDNA of factor H in the baculovirus system and have shown the recombinant protein to be fully active. Mutants of this full-length protein have now been prepared, purified, and examined for cofactor activity and binding to C3b and heparin. The results demonstrate (i) that factor H has at least three sites that bind C3b, (ii) that one of these sites is located in SCR domains 1-4, as has been shown by others, (iii) that a second site exists in the domain 6-10 region, (iv) that a third site resides in the SCR 16-20 region, and (v) that two heparin binding sites exist in factor H, one near SCR 13 and another in the SCR 6-10 region. Functional assays demonstrated that only the first C3b site located in SCR 1-4 expresses factor I cofactor activity. Mutant proteins lacking any one of the three C3b binding sites exhibited 6- to 8-fold reductions in affinity for C3b on sheep erythrocytes, indicating that all three sites contribute to the control of complement activation on erythrocytes. The identification of multiple functionally distinct sites on factor H clarifies many of the heretofore unexplainable behaviors of this protein, including the heterogeneous binding of factor H to surface-bound C3b, the effects of trypsin cleavage, and the differential control of complement activation on activators and nonactivators of the alternative pathway of complement.

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was Fas-dependent, as demonstrated by cell survival in the absence of plate-bound anti-Fas antibody, and by the lack of CD4-induced death in cells from Fas-defective lymphoproliferative (lpr) mice. We demonstrate here that CD4 regulates the ability of Fas to induce cell death in Cd4+ T cells.

A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes.

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

Induction of "tissue" transglutaminase in HIV pathogenesis: evidence for high rate of apoptosis of CD4+ T lymphocytes and accessory cells in lymphoid tissues.

Peripheral blood mononuclear cells and lymphoid tissues from HIV-infected individuals display high levels of "tissue" transglutaminase (tTG) with respect to seronegative persons. In asymptomatic individuals, > 80% of the circulating CD4+ T cells synthesize tTG protein and the number of these cells matches the level of apoptosis detected in the peripheral blood mononuclear cells from the same patients. In HIV-infected lymph nodes tTG protein is localized in large number of cells (macrophages, follicular dendritic cells, and endothelial cells), showing distinctive morphological and biochemical features of apoptosis as well as in lymphocytes and syncytia. These findings demonstrate that during the course of HIV infection, high levels of apoptosis also occur in the accessory cells of lymphoid organs. The increased concentration of epsilon(gamma-glutamyl)lysine isodipeptide, the degradation product of tTG cross-linked proteins, observed in the blood of HIV-infected individuals demonstrates that the enzyme accumulated in the dying cells actively cross-links intracellular proteins. The enhanced levels of epsilon(gamma-glutamyl)lysine in the blood parallels the progression of HIV disease, suggesting that the isodipeptide determination might be a useful method to monitor the in vivo rate of apoptosis.

The role of CD4-Lck in T-cell receptor antagonism: evidence for negative signaling.

Small changes in the complex between a peptide and a molecule of the major histocompatibility complex generate ligands able to partially activate (partial agonist) or even inhibit (antagonist) T-cell functions. T-cell receptor engagement of antagonist complex results in a partial zeta chain phosphorylation without activation of the associated ZAP-70 kinase. Herein we show that, despite a strong inhibition of both inositol phospholipid hydrolysis and extracellular increasing antagonist concentrations increased the activity of the CD4-Lck kinase. Addition of anti-CD4 antibody to culture medium prevented inhibitory effects induced by antagonist ligand. We propose that CD4-Lck activation triggered by antagonist complexes may act in a dominant negative mode, thus overriding stimulatory signals coming from agonist ligand. These findings identify a new T-cell signaling profile that may explain the ability of some T-cell receptor variant ligands to inhibit specific biological activities or trigger alternative activation programs.

Multiple forms of complement C3 in trout that differ in binding to complement activators.

In all other species analyzed to date, the functionally active form of complement component C3 exists as the product of a single gene. We have now identified and characterized three functional C3 proteins (C3-1, C3-3, and C3-4) in trout that are the products of at least two distinct C3 genes. All three proteins are composed of an alpha-and a beta-chain and contain a thioester bond in the alpha-chain. However, they differ in their electrophoretic mobility, glycosylation, reactivity with monospecific C3 antibodies, and relative ability to bind to various surfaces (zymosan, Escherichia coli, erythrocytes). A comparison of the partial amino acid sequences of the three proteins showed that the amino acid sequence identity/similarity of C3-3 to C3-4 is 87/91%, while that of C3-3 and C3-4 to C3-1 is 51.5/65.5% and 60/73% respectively. Thus, trout possess multiple forms of functional C3 that represent the products of several distinct genes and differ in their ability to bind covalently to various complement activators.

Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5.

New Zealand black x New Zealand white (NZB/W) F1 mice spontaneously develop an autoimmune syndrome with notable similarities to human systemic lupus erythematosus. Female NZB/WF1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age. Although the development of the immune-complex nephritis is accompanied by abundant local and systemic complement activation, the role of proinflammatory complement components in disease progression has not been established. In this study we have examined the contribution of activated terminal complement proteins to the pathogenesis of the lupus-like autoimmune disease. Female NZB/W F1 mice were treated with a monoclonal antibody (mAb) specific for the C5 component of complement that blocks the cleavage of C5 and thus prevents the generation of the potent proinflammatory factors C5a and C5b-9. Continuous therapy with anti-C5 mAb for 6 months resulted in significant amelioration of the course of glomerulonephritis and in markedly increased survival. These findings demonstrate an important role for the terminal complement cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated C5 inhibition may be a useful approach to the therapy of immune-complex glomerulonephritis in humans.