Tumor cell surface expression of granulocyte-macrophage colony-stimulating factor elicits antitumor immunity and protects from tumor challenge in the P815 mouse mastocytoma tumor model.

A novel membrane-bound form of GM-CSF (mbGM-CSF) was expressed on the surface of the mouse mastocytoma cell line P815 to target tumor cell-associated Ags to epidermal Langerhans cells. Transfected clones stimulated the proliferation of syngeneic bone marrow cells, indicating that mbGM-CSF is biologically active. We evaluated the in vivo effects of mbGM-CSF by comparing the growth of mbGM-CSF cells (termed 1D6.1E5) to that of wild-type P815 cells in DBA/2 mice. The growth rates of tumors initiated by P815 and 1D6.1E5 were similar until day 12, after which P815 tumors grew to large sizes while 1D6. 1E5 tumors were rejected. In contrast, the growth of both tumors was unimpeded when injected into nude mice, suggesting that a T cell-dependent antitumor response was induced by 1D6.1E5 in normal mice. Lymphocytes from 1D6.1E5-vaccinated mice were able to kill 51Cr-labeled P815 cells in a dose-dependent fashion that was inhibited by anti-CD8 Abs, suggesting that the antitumor response involved CD8+ CTL. We then tested whether vaccination with these cells would elicit a protective antitumor response by injecting mice with either irradiated 1D6.1E5 or P815 cells and challenging them with nonirradiated P815 cells. 1D6.1E5-treated mice grew small tumors that soon disappeared in all animals. In contrast, the majority of animals receiving the irradiated wild-type tumor vaccine grew large tumors, and 50% died. These data demonstrate that mbGM-CSF expressed on the surface of tumor cells is biologically active and elicits protective antitumor immunity.

Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver.

We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-l-lysine and plasmid DNA. We have characterized poly-l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly-l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-l-lysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50-90 nm. In water, poly-l-lysine and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly-l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components.

Persistence of T-cell clones in psoriatic lesions.

BACKGROUND: We previously demonstrated a clonal dominance in the V beta 13.1 messages isolated from the lesional CD8+ T cells of psoriasis vulgaris, which suggested an interaction of V beta 13.1+ CD8+ T cells with skin antigens. OBJECTIVES: To determine whether the clonality observed accurately reflected a clonal population of infiltrating T cells or was skewed by an overabundance of messages from a small number of cells, and to extend our study of V beta gene usage by lesional CD8+ T cells to 9 new patients. DESIGN: Case study. SETTING: Patients were enrolled at the Psoriasis Research Institute in Palo Alto, Calif, and samples were analyzed at The Immune Response Corporation in Carlsbad, Calif. MAIN OUTCOME MEASURES: For the 2 previous patients, skin samples were sorted directly for V beta 13.1+ T cells, for which the T-cell receptors were sequenced. For the 9 new patients, CD8+ T cells were sorted and their T-cell receptor V beta gene usage measured using semiquantitative polymerase chain reaction with V beta-specific primers. RESULTS: The directly sorted V beta 13.1+ T cells exhibited clonal dominance in both patients. The dominant V beta 13.1 clone in each patient was the same as that found in the previous 2 biopsy specimens for which CD8+ T cells were sorted. Additionally, in 8 of the 9 new patients examined, we again found a preferential usage of V beta 3 and/or V beta 13.1 genes by the lesional CD8+ T cells. CONCLUSIONS: The clonality, which was found in the V beta messages of the sorted CD8+ T cells, accurately reflects the dominance of these clones in the infiltrating T cells. Moreover, the persistence in the same patient of the same clone for as long as 15 months and the overrepresentation of V beta 3 and/or V beta 13.1 in lesional CD8+ T cells in the new patients examined support the pathogenic role of T cells bearing these V betas.

T-cell receptor peptides as immunotherapy for autoimmune disease.

The observations in both mouse and rat models of experimental allergic encephalomyelitis (EAE) demonstrating restricted T-cell receptor (TCR) usage among pathogenic T cells has led to the generation of a new class of therapeutic vaccines composed of TCR V region peptides. Whether a similar approach will be of use in the treatment of human autoimmune disorders is still unclear. The experiments performed in our laboratory over the past several years have focused on two aspects of TCR peptide immunoregulation, namely, (1) how to identify the critical T-cell populations involved in the pathology of autoimmune disease, and (2) how to identify biologically relevant TCR peptides--those endogenous TCR peptides presented in association with MHC molecules on the surface of pathogenic T cells that are recognized by immunoregulatory T-cell populations. Results of our recently completed clinical studies regarding TCR V beta expression among CD4+ T cells in the cerebral spinal fluid (CSF) of patients with multiple sclerosis suggests that these cells may be an appropriate T-cell population to be targeted for TCR peptide therapy. In addition, our studies on the immune response to autologous, soluble TCR heterodimers may provide a strategy for the identification of new TCR peptide candidate vaccines.

Insulin-secretory-granule specific T cell clones in human IDDM.

T cell clones reactive to beta-cell antigens prepared from different species were established in order to identify putative pathogenic T cells in human IDDM. We were able to generate T cell clones from patients, but not from controls, reactive specifically to the insulin secretory enriched fraction (ISG) of a rat insulinoma RIN cell line. This finding is suggestive of an in vivo priming by the antigen(s). To examine the relevance of these T cell clones in the pathogenesis of IDDM, we studied their cytokine profile. T cell clones from the newly onset patients had a Th1 cytokine profile, while those from the prediabetic patient were of the Th2 subtype. This segregation suggests that RIN-ISG contains antigen(s) involved in the pathogenesis of this disease, since IDDM is considered a cell-mediated or Th1 disease. Since two of these clones also responded to a hamster insulinoma cell line HIT, at least two antigens in RIN-ISG could be defined by this panel of T cell clones. Examination of CDR3 sequences confirmed the clonality of the dual-reactive T cell clones. The finding of HIT-reactive cells in IDDM patients may be useful in efforts to identify prediabetic patients for immune intervention. Dual reactivity may provide a better prognosis than single reactivity. In contrast to T cell clones reactive to insulinomas, T cell clones reactive to normal human ISG were not found after over 200 clones were screened. In addition, RIN-ISG specific clones did not respond to either normal human or rat ISG, suggesting that IDDM antigens are below detectable levels in normal beta cells.

T cell receptors, immunoregulation, and autoimmunity.

T cell receptor (TCR) peptide vaccines have proven useful in the prevention and treatment of autoimmune disease in animal models. Prospects for developing TCR peptide vaccines for human autoimmune disease are only now being explored. Preliminary indications provide cause for optimism that immunization with TCR peptides eventually will be a viable treatment option for autoimmune pathologies in humans. In the long term, development of this technology may permit reliable manipulation of T cell immunity, leading to treatments for autoimmunity, T lymphoproliferative disorders, and, in the broadest interpretation, any pathogenesis mediated by oligoclonal T cell populations.

Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a disease affecting the synovial membranes of articulating joints that is thought to result from T-cell-mediated autoimmune phenomena. T cells responsible for the pathogenesis of RA are likely present in that fraction of synovial T cells that expresses the interleukin 2 receptor (IL-2R), one marker of T-cell activation. We report herein an analysis of T-cell receptor (TCR) beta-chain gene expression by IL-2R-positive synovial T cells. These T cells were isolated from uncultured synovial tissue specimens by using IL-2R-specific monoclonal antibodies and magnetic beads, and TCR beta-chain transcription was analyzed by PCR-catalyzed amplification using a panel of primers specific for the human TCR beta-chain variable region (V beta). Multiple V beta gene families were found to be transcribed in these patients samples; however, three gene families, V beta 3, V beta 14, and V beta 17, were found in a majority of the five synovial samples analyzed, suggesting that T cells bearing these V beta s had been selectively retained in the synovial microenvironment. In many instances, the V beta 3, V beta 14, or V beta 17 repertoires amplified from an individual patient were dominated by a single rearrangement, indicative of clonal expansion in the synovium and supportive of a role for these T cells in RA. Of note is a high sequence similarity between V beta 3, V beta 14, and V beta 17 polypeptides, particularly in the fourth complementarity-determining region (CDR). Given that binding sites for superantigens have been mapped to the CDR4s of TCR beta chains, the synovial localization of T cells bearing V beta s with significant CDR4 homology indicates that V beta-specific T-cell activation by superantigen may play a role in RA.

Comparative histology of experimental allergic neuritis induced with minimum length neuritogenic peptides by adoptive transfer with sensitized cells or direct sensitization.

Using synthetic peptides representing specific regions of the bovine myelin protein P2, the minimum peptide length requirement for the T-cell epitope necessary for successful production of experimental allergic neuritis (EAN) has been shown to involve residues 61-70 of the P2 protein. In this study, morphometric analysis was used to compare the histologic changes in sciatic nerves of Lewis rats after disease was induced by P2 specific neuritogenic T-cell lines (P(2)3) or, alternatively, by direct inoculation of synthetic peptides representing residues 60-70 or 61-70 of the P2 protein sequence. Inoculation with cell line P(2)3 stimulated with residue 61-70 failed to elicit demyelination in sciatic nerves. However, cells stimulated with residue 60-70 produced inflammation, endoneurial edema, mild demyelination and axonal degeneration within seven days. In contrast, disease induced with either peptide by direct sensitization was more severe. Morphometric analysis revealed that inflammation was most severe in animals sensitized to the decapeptide. In the sciatic nerve, axonal degeneration was proportional in frequency to the extent of inflammation. Inflammation was especially intense in spinal roots with extensive destruction of axons including unmyelinated fibers. Spinal root changes were associated with Wallerian degeneration in the posterior white matter tracts of the spinal cord and were apparently secondary to endoneurial inflammation. Disruption of the blood-nerve-barrier (BNB), evident as physical separation of endothelial cells in association with severe perivascular inflammation, was observed.

P2 specific lymphocyte transformation in Guillain-Barré syndrome and chronic idiopathic demyelinating polyradiculoneuropathy.

Thymidine incorporation proliferation assays to whole bovine P2 protein and its 58-81 and 14-25 synthetic peptides were performed on blood mononuclear cells from ten patients with Guillain-Barré syndrome (GBS), six patients with chronic idiopathic demyelinating polyradiculoneuropathy (CIDP), and age and sex matched normal subjects. The only patients whose cells showed any response were two out of four with very early GBS. One responded to P2 and both synthetic peptides. One responded to P2 but to neither peptide. The results support a role for cell mediated immunity to P2 protein in some patients with Guillain-Barré syndrome.

Vaccination against experimental allergic encephalomyelitis with T cell receptor peptides.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by CD4+ T cells reactive with myelin basic protein (MBP). Rats were rendered resistant to the induction of EAE by vaccination with synthetic peptides corresponding to idiotypic determinants of the beta chain VDJ region and J alpha regions of the T cell receptor (TCR) that are conserved among encephalitogenic T cells. These findings demonstrate the utility of TCR peptide vaccination for modulating the activity of autoreactive T cells and represent a general therapeutic approach for T cell-mediated pathogenesis.

A T cell epitope for experimental allergic neuritis.

A synthetic peptide representing residues 57-81 of the bovine P2 protein produced severe paralytic experimental allergic neuritis (EAN) in Lewis rats. Peptide 57-81 could also be used to stimulate a P2 protein-specific T cell line to transfer paralytic EAN to naive recipient rats. A smaller peptide representing residues 60-81 produced a milder form of clinical disease. Residues 60-81 are the shortest peptide sequence thus far described which can produce clinical EAN. The structural predictions for the sequence represented by these peptides support the contention that T cell antigenic sites tend to be amphipathic alpha-helical structures.

A suppressor T-lymphocyte cell line for autoimmune encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is a model for the in vitro and in vivo study of T-cell activation. It is an autoimmune disease mediated by T lymphocytes of the helper T-cell (Th) subset. After sensitization to guinea-pig myelin basic protein in complete Freund's adjuvant, Lewis rats develop an autoimmune response to central nervous system (CNS) myelin basic protein, manifested clinically as paralysis and histologically by a perivascular mononuclear cell infiltrate of the CNS parenchyma. Suppressor cell regulation of EAE has long been suspected because Lewis rats, which spontaneously recover from active disease, are resistant to reinduction of active EAE, even though effector T-cell lines can be rescued from these recovered rats. Using cyclosporin A, an immunosuppressive agent believed to inhibit Th cell function, suppressor T-cell (Ts) lines have now been generated from recovered Lewis rats. These Ts cells, when admixed with guinea pig myelin basic protein-specific Th cells, will prevent the adoptive transfer of EAE. The Ts cells appear to be CD4+, which explains previous observations that CD8+ lymphocytes are not important in the recovery of EAE in the rat. This is the first direct demonstration of Ts-cell regulation of EAE.

Myelin basic protein gene expression in quaking, jimpy, and myelin synthesis-deficient mice.

Jimpy (jp), myelin synthesis-deficient (jpmsd), and quaking (qk) are mutations which affect myelination to different degrees in the mouse central nervous system (CNS). Total messenger RNA (mRNA) and myelin basic protein (MBP)-specific mRNA from brains of these three mutants have been analyzed by in vitro translation and immunoprecipitation with antibody to MBP. The results indicate that the three mutations do not affect the level of total MBP-specific mRNA in the CNS but do affect the relative proportions of the various MBP-related translation products encoded in vitro. In each case the proportions of 14K and 12K Mr MBP-related translation products are reduced and the proportions of 21.5K, 18.5K, and 17K Mr MBP-related translation products are increased relative to wild type. This effect is most pronounced in jp, less so in jpmsd, and least pronounced in qk animals. The MBP-related polypeptides that accumulate in vivo have also been analyzed in the three mutants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with antibody to MBP. The levels of all the major MBP-related polypeptides that accumulate in vivo are reduced in all three mutations. The reduction is most pronounced in jp, less in jpmsd, and least pronounced in qk animals. These results indicate that the jp, jpmsd, and qk mutations exhibit qualitatively similar phenotypic effects on MBP gene expression but the magnitude of the effect is proportional to the extent of hypomyelination in each mutant.

Structure of bovine P2 basic protein: sequence of a carboxyterminal segment that is a neuritogen in rabbits.

The sequence of the carboxyterminal 18 amino acids released by cyanogen bromide digestion of the bovine P2 protein was determined. It has several interesting structural and immunological properties. It contains the only two half-cystines in the molecule that have the capacity to form an intrachain disulfide bond. Using the rabbit as a test animal, this carboxyterminal peptide was capable of producing experimental allergic neuritis. The sequence of this peptide is Val-Val-Glu-Cys-Lys-Met-Lys-Asp-Val-Val-Cys-Thr-Arg-Ile-Tyr-Glu-Lys-Val.

Bovine peripheral nervous system myelin P2 protein: chemical and immunological characterization of the cyanogen bromide peptides.

Cleavage of bovine P2 protein by cyanogen bromide (CNBr) produced peptide fractions CN1, CN2, and CN3 which were isolated by gel filtration chromatography. CN2 was found to contain two NH2-terminals (lysine and valine) and accounted for both of the cysteine residues of P2. When reduced carboxymethylated P2 (RCM-P2) was digested with CNBr, peptides CN1 and CN3 were obtained as were (1) a peptide with NH2-terminal lysine (Lys) that contained no homoserine and only one cysteine residue and (2) a peptide with NH2-terminal valine (Val) that was co-eluted with CN3. These data and the chemical characterization of all the CNBr peptides obtained from P2 and RCM-P2 suggest that isolated P2 protein has a structure composed of the CNBr peptides in the order CN3-CN1-CN2(Val)-CN2(Lys) with an intrachain disulfide bond between the cysteine residues located in the two constituent peptides of CN2, CN2(Lys) and CN2(Val). To locate the neuritogenic region(s) within the P2 protein structure, CN1, CN2, and CN3 were tested for the ability to induced experimental allergic neuritis (EAN) in Lewis rats. The disease-inducing sites of P2 protein were found only in CN1; neither CN2 nor CN3 produced disease. EAN induced by CN1 was comparable to that induced with P2 protein as determined by disease onset, clinical symptoms, and histologic lesions.

Radioimmunoassay for the P2 protein of bovine peripheral nerve myelin.

The P2 protein of bovine nerve root myelin was radiolabeled with 125I in homogeneous solution by the chloramine-T method and purified by gel filtration on Sephadex G-75 to obtain monomeric 125I-P2. Antigen-antibody complexes were isolated by the silica gel, double antibody, or polyethylene glycol techniques by using rabbit antibody to P2. As little as 1 ng/ml P2 could be detected. The RIA was used to measure the P2 content in nerve tissue and isolated myelin. The presence of P2 in spinal cord as well as in the peripheral nervous system was confirmed. Peptides isolated by CNBr digestion of the P2 protein were tested in the RIA. CN-1, comprising 80 to 90 residues from the interior of the molecule displayed complete immunologic cross-reactivity with intact P2. Neither CN-2, representing 18 amino acids from the COOH terminal, nor CN-3, representing 20 amino acids from the NH2 terminal, showed cross-reactivity. Since the major determinant for experimental allergic neuritis in the rabbit is located in peptide CN-2, our present data suggest that the major neuritogen and the major determinant(s) for humoral antibody response in this species may be at different locations within the P2 molecule.

Allergic encephalomyelitis. Isolation of an encephalitogenic peptide active in the monkey.

A 17-residue peptide (Peptide Y) was isolated from the COOH-terminal end of the basic protein of bovine myelin by peptic digestion. This peptide induced experimental allergic encephalomyelitis in the rhesus monkey. Treatment of Peptide Y with cyanogen bromide released three amino acids from the COOH-terminal end and resulted in a tetradecapeptide (Peptide M) which was also encephalitogenic in the rhesus monkey. The sequence of Peptide M is: Phe-Lys-LEU-Gly-Gly-Arg-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met. Thus a major disease-inducing site active in the rhesus monkey is contained within a 14-residue peptide localized near the COOH-terminal end of the protein. This peptide differs markedly in location and sequence from the 9-residue peptide shown to contain the encephalitogenic determinant for the guinea pig.