Antigen presentation by dendritic cells after immunization with DNA encoding a major histocompatibility complex class II-restricted viral epitope.

Intramuscular and intracutaneous immunization with naked DNA can vaccinate animals to the encoded proteins, but the underlying mechanisms of antigen presentation are unclear. We used DNA that encodes an A/PR/8/34 influenza peptide for CD4 T cells and that elicits protective antiviral immunity. DNA-transfected, cultured muscle cells released the influenza polypeptide, which then could be presented on the major histocompatibility complex class II molecules of dendritic cells. When DNA was injected into muscles or skin, and antigen-presenting cells were isolated from either the draining lymph nodes or the skin, dendritic, but not B, cells presented antigen to T cells and carried plasmid DNA. We suggest that the uptake of DNA and/or the protein expressed by dendritic cells triggers immune responses to DNA vaccines.

Antigen-specific signaling by a soluble, dimeric peptide/major histocompatibility complex class II/Fc chimera leading to T helper cell type 2 differentiation.

Interaction between a T cell receptor (TCR) and various ligands, i.e. , anti-TCR antibodies, superantigens, peptides, or altered peptide ligands in the context of major histocompatibility complex (MHC) molecules can trigger different T helper cell (Th) effector functions. Herein, we studied the T cell response induced by a soluble, dimeric peptide/MHC class II chimera, namely hemagglutinin (HA)110-120/I-E(d)alphabeta/Fcgamma2a (DEF). We have previously demonstrated that the soluble DEF molecule binds stably and specifically to HA110-120-specific TCRs expressed by a T cell hybridoma. Administration of DEF in vivo induced differentiation of resting and activated peptide-specific T cells toward a Th2 response, as indicated by the increase of interleukin (IL)-4, IL-10, and specific immunoglobulin (Ig)G1 antibodies and decrease of IL-2, specific IgG2a antibodies, and cytotoxic T lymphocyte activity. In contrast to HA110-120 peptide presented by the DEF molecule to T cells, the nominal synthetic peptide induced a predominant Th1 response, and the PR8 virus-derived HA110-120 peptides induced a mixed Th1/Th2 response. Independent of antigen processing, soluble DEF was almost 2 logs more potent in stimulating cognate T cells than the nominal peptide. Polarization of cognate T cells toward the Th2 response occurred upon interaction of soluble DEF with TCR and CD4 molecules followed by early activation of p56(lck) and ZAP-70 tyrosine kinases, and negative signaling of the signal transducer and activator of transcription (STAT)4 pathway of Th1 differentiation. DEF-like molecules may provide a new tool to study the mechanisms of signaling toward Th2 differentiation and may also provide a potential immunotherapeutic approach to modulate autoreactive T cells toward protective Th2 immune responses.

Antigen-specific downregulation of T cells by doxorubicin delivered through a recombinant MHC II--peptide chimera.

As the number of drugs with potential therapeutic use for T-cell-mediated diseases increases, there is a need to find methods of delivering such drugs to T cells. The major histocompatibility complex (MHC)--peptide complexes are the only antigen-specific ligands for the T-cell receptor (TCR) expressed on T cells, and they may be an appropriate drug delivery system. We engineered a soluble bivalent MHC class II-peptide chimera on the immunoglobulin scaffold (I-E(d)alpha beta/Fc gamma 2a/HA110-120, DEF) that binds stably and specifically to CD4 T cells recognizing the HA110-120 peptide. Doxorubicin, a powerful antimitogenic anthracycline, was enzymatically assembled on the galactose residues of a DEF chimera. The DEF-gal-Dox construct preserved both the binding capacity to hemagglutinin (HA)-specific T cells, and the drug toxicity. Brief exposure of HA-specific T cells to DEF-gal-Dox construct in vitro was followed by drug internalization in the lysosomes, translocation to the nucleus, and apoptosis. Administration of DEF-gal-Dox to mice expressing the TCR-HA transgene reduced the frequency of TCR-HA T cells in the spleen and thymus by 27% and 42%, and inhibited HA proliferative capacity by 40% and 60%, respectively. It has not been demonstrated previously that pharmacologically active drugs able to modulate T-cell functions can be delivered to T cells in an antigen-specific manner by soluble, bivalent MHC II-peptide chimeras.

Immunopotency of a viral peptide assembled on the carbohydrate moieties of self immunoglobulins.

The T-cell receptor recognizes peptides bound to the major histocompatibility complex antigens. Synthetic peptides corresponding to microbial epitopes can efficiently stimulate the in vitro proliferation of T-cell hybridoma or in vivo primed T cells. However, the in vivo immune responses elicited by synthetic peptides are weak because of their short half-life and poor immunogenicity. We previously showed that a genetically engineered immunoglobulin (Ig-HA), in which the CDR3 region of VH gene was replaced with a viral peptide recognized by CD4+ T cells, was able to deliver this epitope in the correct frame to antigen-processing cells that efficiently presented the peptide to T cells. Recently, we developed an enzymatic method to assemble viral peptides on the sugar moieties of immunoglobulins without alteration of the biological functions of either molecule. The viral peptide carried by these conjugates was twenty times more efficient in activating a T-cell hybridoma than the free peptide as calculated on a molar basis. We show that such conjugates are able to prime in vivo the precursors of peptide-specific T cells and to induce proliferation of naive lymphocytes from transgenic mice expressing a peptide-specific T-cell receptor in both CD4 and CD8 T-cell subsets. Our results suggest that peptides enzymatically linked to the carbohydrate moieties of immunoglobulins, using galactose residues as peptide acceptor, can be used as a safe and efficient delivery system of protective epitopes for the prevention of infectious diseases. The enzymatic engineering of immunoglobulins may also allow the development of immunotherapeutic agents to deliver antagonist peptides to autoreactive T cells or to direct immunomodulatory agents such as interleukins or cytolytic drugs to tumor cells.

Antineoplastic efficacy of doxorubicin enzymatically assembled on galactose residues of a monoclonal antibody specific for the carcinoembryonic antigen.

We have developed a novel procedure to couple enzymatically the antineoplastic agent doxorubicin (Dox) on the galactose residues of a monoclonal antibody specific for the tumor-associated carcinoembryonic antigen. The synthesis of the immunoconjugate consists of covalent attachment of the NH2 terminus of Dox to oxidized galactose residues of desialylated monoclonal antibody, followed by concurrent stabilization of Schiff bases by mild reduction with pyridine borane. The immunoconjugate preserved both antibody specificity and drug cytotoxicity. At equimolar concentrations, the immunoconjugate was 8 times more cytotoxic against two carcinoembryonic antigen-expressing carcinoma cell lines, LoVo and SW-480, than Dox alone. The intracellular drug accumulation was 8-8.5 times higher than that obtained with free Dox, and >50% of the drug delivered by the conjugate was retained for 24 h in the tumor cells. Only 4 days after treatment with a single dose of immunoconjugate carrying 2.5 ng of Dox, LoVo and SW-480 tumor transplants on the chorioallantoic membrane of embryonated hen eggs showed reduced tumor-induced angiogenesis and tumor progression by half, with no detectable damage to surrounding tissues. In contrast, the same amount of free drug induced insignificant changes in tumor progression and tumor-induced angiogenesis. Enzymatically mediated, glycosidic coupling of antineoplastic agents to antibodies specific for tumor-associated antigens may represent a novel platform for the development of more efficient anticancer agents with reduced side effects.

The microsatellite mutator phenotype of colon cancer cells is often recessive.

A new mutator mechanism for tumorigenesis, characterized by somatic genomic instability (SGI) at simple repeated sequences (SRS) or microsatellites, underlies hereditary nonpolyposis colorectal cancer (HNPCC) and some sporadic tumors of the colon and other types. To determine whether the microsatellite mutator phenotype (MMP) is dominant or recessive, we generate somatic cell hybrids between a tumor cell line without SGI at SRS (D98OR) and colon carcinoma cell lines with relative low (HCT-15) and high (LS174-T) SGI at SRS. The normal fidelity of replication of these unstable sequences was observed in each of these cell hybrids. Fusion of HCT-15/DLD-1 low instability cells, with LS174-T, HCT116 and LoVo cell lines, all exhibiting relative high instability, also restored the replication fidelity of SRS in all of the hybrids. Hybrids between the high instability cell lines did not grow possibly because of senescence or apoptosis. These results indicate that, in the cell lines analysed, the characterized mutator phenotype of the mismatch repair system resulting in high SGI at SRS, and the uncharacterized mutator phenotype underlying low SGI at SRS, are both recessive. The results also suggest that different tumor cells of the MMP harbor distinct altered growth-related genes.

Generation of LAK cells in vitro in patients with acute leukemia.

The in vitro stimulation of lymphocytes with interleukin-2 (IL-2) generates lymphokine-activated killer (LAK) cells with tumoricidal potential. In this work we studied the cytolytic capacity of LAK cells in 51 acute leukemia patients in complete remission (CR) after chemotherapy (CT), in 24 acute leukemia patients who had undergone autologous bone marrow transplantation (ABMT), and in a control group of 44 normal donors. In the normal donor control group the effect of non-IL-2-activated peripheral blood mononuclear cells (PBMC) against blast cells was always lower than 10% lysis, which we have taken as a lower limit for positive results. In 95% of post-CT patients, the lytic effect of PBMC was negative. LAK cells produced positive results in 82% of normal donors and in 37.5% of post-CT patients. The effect of PBMC against K562, i.e. natural killer (NK) activity, in post-CT patients as well as in post-ABMT patients was reduced in comparison with the average for normal donors. LAK cells from 25% of post-CT patients had no notable activity against K562 or Raji, nor was there any positive effect against autologous blast cells. In the rest (75%), one-half generated positive activity. We did not observe any correlation between lytic activity in PBMCs or in LAK cells, nor did we observe significant differences between lytic activity in patients with acute lymphoblastic leukemia (ALL) and those with acute myeloblastic leukemia (AML), or between patients who had undergone CT and those receiving ABMTs. These results support the use of IL-2 as a treatment against minimal residual leukemia.

Insights into the pathogenesis of type 1 diabetes: a hint for novel immunospecific therapies.

Type 1 diabetes is an organ-specific autoimmune disease whose incidence is increasing worldwide. At present, there is no effective therapy to prevent or cure this disease. The genetic background (MHC and non-MHC genes) and environmental factors (pathogens, drugs, and diet) are critical for the initiation of the autoimmune response against the pancreatic beta-cells. Recognition of the pancreatic autoantigens by T cells in a predetermined environment of antigen-presenting cells, costimulation, and cytokines is crucial for the selective activation of diabetogenic or protective/regulatory T cells. Once the autoimmune process is triggered, epitope spreading and sustaining the autoimmune responses by continuous antigen stimulation leads to expansion of effector cells, which launch the attack on the beta-cells. Despite of some controversy, most of the studies in humans and animal models suggest that CD4 (Th1) T cells are directly involved in the autoimmune attack by secretion of pro-inflammatory cytokines and recruitment of cytotoxic CD8 T cells. Secretion of anti-inflammatory cytokines by Th2 cells is protective against the disease. Therapy with peptides derived from major target antigens, such as glutamic acid decarboxylase 65 or proinsulin, can prevent the disease in animal models by rising protective Th2 cells. Herein, we review the recent progress in the immunopathogenesis of Type 1 diabetes and insights into the development of new diagnostic tools and antigen-specific immunomodulators, such as MHC-peptide chimeras.

Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK) mice.

Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.

Structure of the mutant fibrillin-1 gene in the tight skin (TSK) mouse.

Mice carrying the tight skin (TSK) mutation harbors a 3.0-kb genomic duplication (exons 17-40) of the fibrillin-1 gene (Fbn-1) located on band F of chromosome 2 as TSK mutation. We cloned and sequenced the mutated Fbn-1 gene, since it is believed to be responsible for TSK syndrome. Sequence analysis showed numerous amino acid differences in the 5' and 3' segments between the TSK mutation and wild-type fbn-1 gene, but any amino acid difference between the TSK mutation and C57BL/6 mice. (TSK and C57B1/6 mice are genetically similar, differing only by TSK mutation.) Four amino acid differences were observed between two copies of TSK's fbn-1 gene encoded by exons 17-40. Our results suggest that the majority of structural differences occurred in the N and C termini segments during strain divergence and only a few after the duplication event.

The temperature dependence of the hydrogen exchange in the SH3 domain of alpha-spectrin.

The amide hydrogen-deuterium exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin has been measured by nuclear magnetic resonance as a function of temperature between 8 and 46 degrees C. The analysis of the temperature dependence of HX from a statistical thermodynamic point of view has allowed us to estimate the enthalpies and entropies of the conformational processes leading to HX. The results indicate that under native conditions the domain undergoes a wide variety of conformational fluctuations, ranging from local motions, mainly located in loops, turns and chain ends and involving only low enthalpy and entropy, to extensive structural disruptions affecting its core and involving enthalpies and entropies that come fairly close to those observed during global unfolding.

pH dependence of the hydrogen exchange in the SH3 domain of alpha-spectrin.

Using nuclear magnetic resonance we have measured the hydrogen exchange (HX) in the Src homology region 3 (SH3) domain of alpha-spectrin as a function of pH*. At very acidic pH* values the exchange of most residues appears to occur via global unfolding, although several residues show abnormally large Gibbs energies of exchange, suggesting the presence of some residual structure in the unfolded state. At higher pH* HX occurs mainly via local or partial unfoldings. We have been able to characterize the coupling between the electrostatic interactions in this domain and the conformational fluctuations occurring under native conditions by analyzing the dependence upon pH* of the Gibbs energy of exchange. The SH3 domain seems to be composed of a central core, which requires large structural disruptions to become exposed to the solvent, surrounded by smaller subdomains, which fluctuate independently.

Structural cooperativity in the SH3 domain studied by site-directed mutagenesis and amide hydrogen exchange.

We have studied the effects produced by site-directed mutagenesis upon energetic and structural cooperativity in the Src homology region 3 domain of alpha-spectrin. The mutation of Asn47 to Gly or Ala in the distal loop brings about significant changes to the global stability of the domain in spite of not affecting its structure to any great extent. The binding affinity for a proline-rich peptide is also largely diminished in both mutant domains. We have compared the apparent Gibbs energies of the amide hydrogen-deuterium exchange (HX) between the wild-type and the Gly47 mutant. The observed changes in the Gibbs energy of HX indicate a remarkable energetic cooperativity in this small domain. Regions of the domain's core have a high cooperativity with the position of the mutation, indicating that their HX occurs mainly in states in which the distal loop is unstructured. More flexible regions, which undergo HX mainly by local motions, show a lower but still considerable cooperativity with the distal loop. We conclude that there is an important correlation between regional stability and cooperativity in this small domain.

A binding event converted into a folding event.

We have designed a chimeric protein by connecting a circular permutant of the alpha-spectrin SH3 domain to the proline-rich decapeptide APSYSPPPPP with a three-residue link. Our aim was to obtain a single-chain protein with a tertiary fold that would mimic the binding between SH3 domains and proline-rich peptides. A comparison of the circular-dichroism and fluorescence spectra of the purified chimera and the SH3 circular permutant showed that the proline-rich sequence occupies the putative SH3 binding site in a similar conformation and with comparable interactions to those found in complexes between SH3 and proline-rich peptides. Differential scanning calorimetry indicated that the interactions in the binding motif interface are highly cooperative with the rest of the structure and thus the protein unfolds in a two-state process. The chimera is more stable than the circular permutant SH3 by 6-8 kJ mol(-1) at 25 degrees C and the difference in their unfolding enthalpy is approximately 32 kJ mol(-1), which coincides with the values found for the binding of proline-rich peptides to SH3 domains. This type of chimeric protein may be useful in designing SH3 peptide ligands with improved affinity and specificity.

Sensitive detection of GM1 lipid rafts and TCR partitioning in the T cell membrane.

The cholesterol-rich lipid rafts on T cell membrane play important role in the formation of T cell receptor (TCR) signalosome upon receptor ligation. Analytical studies on the kinetics of lipid rafts formation and recruitment of protein receptors to lipid rafts are still limited by the use of a large number of cells. Herein, we describe a strategy for detecting fine alterations in the amount and distribution of glycosphingolipid (GM1) lipid rafts, and in the formation of GM1-TCR complexes in detergent-insoluble and -soluble compartments of the T cell membrane from a relative low number of cells. Using this strategy, we found that the GM1 moiety was physically associated with TCR in both detergent-insoluble and -soluble fractions. Shortly after ligation of CD3/TCR complex with a soluble CD3- epsilon mAb, the TCR was found mainly in the detergent-soluble fraction of the T cell membrane.

Protective immunity elicited by vaccination with DNA encoding for a B cell and a T cell epitope of the A/PR/8/34 influenza virus.

Numerous reports have demonstrated that immunization with plasmids bearing influenza virus hemagglutinin (HA) or nucleoprotein (NP) genes elicits humoral and cellular protective responses. Herein we describe the generation of a plasmid (pVH-TB) encoding for a VH region of a self-Ig in which both the major B cell epitope HA150-159 and the immunodominant CD4 T cell epitope HA110-120 of HA of the A/PR/8/34 influenza virus were genetically inserted in the CDR2 and CDR3 loops, respectively. Our results demonstrate unequivocally that i.m. injection of pVH-TB plasmid in BALB/c mice elicited specific cellular and humoral immune responses able to protect against infection with lethal doses of A/PR/8/34 influenza virus.

Induction of antibody response by DNA immunization of newborn baboons against influenza virus.

Previous studies showed that DNA immunization of newborn mice with plasmids expressing influenza virus antigens induced protective immunity. We have now extended the study of neonatal responsiveness to DNA vaccines to nonhuman primates. Baboons immunized as neonates with plasmids expressing type A influenza virus hemagglutinin (HA) and nucleoprotein (NP) in doses ranging from 40 microg to 1 mg per plasmid per dose developed virus-specific humoral responses. The titer and kinetics of appearance of virus-specific IgG antibodies were dose dependent. Specific antibodies were detected by enzyme-linked immunosorbent assay (ELISA) as early as 1 month after birth in baboons immunized with the highest and intermediate doses of vaccine. Virus-neutralizing antibodies were detected in the group of baboons immunized with the highest dose. The specificity of virus-neutralizing antibodies was found to be directed against homologous determinants of HA; however, the IgG antibodies also cross-reacted with HA of a drift variant. Thus, DNA vaccination of newborn baboons with a prototype vaccine against influenza virus resulted in induction of specific humoral immunity.

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras.

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I-, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of alpha-chain in the case of MHC I, or beta-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal 1 to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.

T-cell tolerance and autoimmune diabetes.

Herein we describe the major signaling events that occur in T-cells upon T-cell receptor (TCR) engagement, and the mechanisms responsible for the induction of T-cell anergy that may ultimately lead to the development of immunospecific therapies in T-cell mediated autoimmune diseases. A new type of antigen presenting molecule (dimeric MHC class-II/peptide, DEF) endowed with antigen-specific immunomodulatory effects such as induction of Th2 polarization and T-cell anergy is also described as a potential antidiabetogenic agent. According to our preliminary results, the MHC II/peptide-based approach may provide rational grounds for further development of antigen-specific immunotherapeutic agents such as human-like MHC lI/peptide chimeras endowed with efficient down-regulatory effects in CD4 T-cell-mediated autoimmune diseases such as Type 1 diabetes, multiple sclerosis, primary biliary cirrhosis, and rheumatoid arthritis.

Natural killer cytotoxicity and lymphocyte subpopulations in patients with acute leukemia.

We have studied lymphocyte subpopulations and the NK cytotoxicity of the PBMC from acute leukemia patients in complete remission after chemotherapy or ABMT and from normal donors. We have found a positive linear correlation between the percentage of subpopulations with CD3-CD16+, CD3-CD56+ and CD3-CD8+ phenotypes and the percentage of NK cytotoxicity. The slopes of the regression lines in the two groups of patients were lower than in normal donors, indicating a decreased ability of these cells to operate. The percentages of these subpopulations represent parameters for estimating the percentage of NK cytotoxicity both in normal donors and in patients with acute leukemia.