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.

Selection of hprt mutant T cells as surrogates for dividing cells reveals a restricted T cell receptor BV repertoire in insulin-dependent diabetes mellitus.

T cells with somatically acquired mutations in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene were isolated from patients with insulin-dependent diabetes mellitus (IDDM) as representatives of populations potentially enriched for in vivo activated T cells. TCRB gene V region usage among mutant isolates from individual IDDM patients, but not from normal controls, showed a pronounced preference for BV14 and, to a lesser extent, BV6. Wild-type (nonmutant) isolates did not show such preferences. Extensive in vivo clonal expansions of the BV14 expressing mutant T cells from IDDM patients were revealed by sequence identity of TCRB chain junctional regions. These data support restricted TCRB gene usage in T cell populations enriched for in vivo activated clones in patients with IDDM.

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.

T cell receptor peptide vaccination in rheumatoid arthritis: a placebo-controlled trial using a combination of Vbeta3, Vbeta14, and Vbeta17 peptides.

OBJECTIVE: Restricted T cell receptor (TCR) gene usage has been demonstrated in animal models of autoimmune disease and has resulted in the successful use of TCR peptide therapy in animal studies. This clinical trial was undertaken to determine the safety and efficacy of a combination of Vbeta3, Vbeta14, and Vbeta17 TCR peptides in Freund's incomplete adjuvant (IFA) in patients with rheumatoid arthritis (RA). METHODS: A double-blind, placebo-controlled, multicenter, phase II clinical trial was undertaken using IR501 therapeutic vaccine, which consists of a combination of 3 peptides derived from TCRs (Vbeta3, Vbeta14, and Vbeta17) in IFA. A total of 99 patients with active RA received either 90 microg (n = 31) or 300 microg (n = 35) of IR501 or IFA alone (n = 33) as a control. The study medication and placebo were administered as a single intramuscular injection (1 ml) at weeks 0, 4, 8, and 20. RESULTS: Treatment with IR501 was safe and well tolerated. None of the patients discontinued the trial because of treatment-related adverse events. Efficacy was measured according to the American College of Rheumatology 20% improvement criteria. Using these criteria, patients in both IR501 dosage groups showed improvement in disease activity. In the most conservative analysis used to evaluate efficacy, an intent-to-treat analysis including all patients who enrolled, the 90-microg dosage group showed a statistically significant improvement compared with control patients at the 20-week time point after the third injection. Trends toward improvement were shown in both the 90-microg and the 300-microg dosage groups at week 24 after the fourth injection. CONCLUSION: IR501 therapeutic vaccine therapy was safe and well tolerated, immunogenic, and demonstrated clinical improvement in RA patients. Additional clinical trials are planned to confirm and extend these observations.

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.

Results of a phase I clinical trial of a T-cell receptor peptide vaccine in patients with multiple sclerosis. I. Analysis of T-cell receptor utilization in CSF cell populations.

To identify a panel of multiple sclerosis patients (MS) for a phase I clinical trial of a T-cell receptor (TCR) peptide vaccine we characterized the T-cell populations present in the cerebrospinal fluids (CSF) of a large group of patients with respect to surface phenotype and state of activation, TCR beta chain utilization, features of the CDR3 junctional region, the extent of clonality and persistence of selected clonotypes over time. These CSF cell populations consist of approximately 60% CD4+ T-cells, half of which bear IL-2 receptors, indicating these activated T-cells may be part of the pathogenic process in MS. When these activated CD4+ T-cells were selectively expanded in IL-2/IL-4 supplemented cultures, an over-representation of several TCRV beta families was noted in 39/47 patients, the most frequent being V beta 6.5, V beta 6.7, V beta 2, V beta 5 and V beta 4. Biased expression of various members of the V beta 6 family was seen in 21 of this group of 39 patients. Clonal analysis of TCR beta 6 CDR3 sequences, revealed two notable features: clonal dominance and clonal persistence. CSF cells from two-thirds of MS patients contained a dominant clone comprising 50% or more of sequences and the same patient-specific clone could be shown to persist for up to 18 months. This clonal prevalence and over representation of V beta 6+TCR raises the possibility that immunization with a V beta 6 peptide vaccine may produce a regulatory immune response leading to a clinical benefit.

Results of a phase I clinical trial of a T-cell receptor vaccine in patients with multiple sclerosis. II. Comparative analysis of TCR utilization in CSF T-cell populations before and after vaccination with a TCRV beta 6 CDR2 peptide.

We report here the results of a phase I trial of a T-cell receptor (TCR) V beta 6 CDR2 region peptide vaccine in 10 patients with multiple sclerosis who showed biased over-representations of V beta 6 mRNA among T-cells in their cerebrospinal fluids (CSF). One group of 5 patients was immunized twice during a four week period with 100 micrograms of the TCRV beta 6 peptide 39-LGQGPEF LTYFQNEAQLEKS-58 emulsified in incomplete Freund's adjuvant (IFA); the second group of 5 MS patients received 300 micrograms of the same peptide in IFA over a similar time period. Patients were monitored for adverse events, immunogenicity of the peptide and changes in their CSF T-cell populations. The results indicate that this peptide was immunogenic (T-cell proliferation assays and recall DTH responses) in some of the patients, although none of the immunized patients produced detectable anti-peptide antibodies. More importantly, we show that the 5 patients treated with higher doses of the vaccine displayed a slight decrease in CSF cellularity, a lack of growth of CSF cells in cytokine supplemented expansion cultures that implies a significant absence of a subset of activated CD4 T-cells and a marked diminution in V beta 6 mRNA levels among T-cells in these cultures. By comparison, in 5 patients receiving the lower dosage of the vaccine, CSF cellularity was the same or slightly increased over pre-vaccination levels, CSF cells from 1 patient failed to grow in expansion cultures and cultured CSF cells from 2 patients underwent a change from an oligoclonal V beta 6 pattern to one that was more polyclonal. These results justify a more through exploration of the use of TCR peptide vaccines as a possible therapeutic treatment for MS.

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.

V beta 17 T cell receptor peptide vaccination in rheumatoid arthritis: results of phase I dose escalation study.

OBJECTIVE: To determine whether modulation of activated T cells occurs in patients with rheumatoid arthritis (RA) after immunization with T cell receptor (TCR) V beta 17 peptides, a phase I trial was initiated to investigate the safety and feasibility of TCR peptide immunization as a therapeutic approach in RA. METHODS: 15 patients with moderate to severe RA were given an intramuscular injection of one of 4 doses (10, 30, 100, and 300 micrograms) of the V beta 17 peptide vaccination, followed by a booster injection of the same dose of vaccine 3 weeks later. Patients were followed for 48 weeks. RESULTS: The product was well tolerated and no serious adverse events attributable to the vaccine were observed. This was an uncontrolled phase I trial, however; decreases in patients joint scores were observed at all followup visits starting at 4 weeks after primary immunization. Activated V beta 17 T cells (IL-2R+) in peripheral blood were decreased (> or = 20%) in 3/5 patients in the 100 micrograms group after initial measurement at Week 2 and 3/4 patients in the 300 micrograms group 3 weeks after immunization. Lymphocyte proliferation in response to the V beta 17 peptide was detected at 6 weeks or later after primary inoculation in 6/15 patients (40%) immunized. CONCLUSION: Further controlled studies are required to assess the biologic and clinical efficacy of this treatment approach.

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 responses to myelin proteins in Guillain-Barré syndrome.

We have investigated the hypothesis that the pathogenesis of Guillain-Barré syndrome (GBS) involves an autoimmune T cell response to P0 and P2 proteins of peripheral nerve myelin. The proliferative responses of blood mononuclear cells (MNC) to myelin proteins and synthetic peptides derived from them were determined in patients with GBS and chronic idiopathic demyelinating polyradiculoneuropathy (CIDP), normal controls (NC) and patients with other neuropathies (ONP). Twelve out of 19 GBS patients responded to P0 or P2, 6 to P0 and its peptides only, 3 to P2 and its peptides only, and 3 to both P0 and P2 antigens. Responses to at least one of the antigens were also found in 6/13 of CIDP patients, but in only 4/17 NC and 2/6 ONP. Immune responses in GBS are heterogeneous. The early T cell responses to P0 protein, described here for the first time, may be important in the pathogenesis of some cases.

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.

Immunoregulation of autoimmune disease by vaccination with T cell receptor peptides.

Restricted TCR gene usage in animal models of autoimmune disease has led to strategies for control of these diseases by targeting the idiotypic determinants within the TCR sequence. Rats can be rendered resistant to EAE by immunization with synthetic peptides representing sequences contained within the V beta, J alpha and VDJ beta regions of the TCR that are conserved among encephalitogenic T cells. We propose that the mechanism of immunoregulation thus produced results from the stimulation of an anticlonotypic response directed at endogenously synthesized TCR peptides presented by Class I MHC on the surface of the autoreactive T cell, and that this mechanism may be part of the natural immunoregulation of T cell responses. The experimental data demonstrate the utility of this therapeutic approach and its potential for treatment of any pathogenic condition mediated by specific, 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.

Response of the axon and barrier endothelium to experimental allergic neuritis induced by autoreactive T cell lines.

Experimental allergic neuritis was induced in Lewis rats by inoculation with autoreactive T cell lines sensitized to residue 57-81 of P2 myelin protein. Control rats received cells derived from immunization to complete Freund's adjuvant alone. Endoneurial fluid pressure (EFP) was measured in both sciatic nerves at 0, 3, 5, 7, 9, and 11 days post-inoculation (PI). The temporal evolution of inflammatory disease was studied by correlating EFP with a morphometric analysis of the nerve microenvironment and with electron microscopic observations. Both edema, as evidenced by increased endoneurial extracellular space, and inflammation paralleled the time course of the EFP increase, reaching peak values at 7 days PI and declining to near-normal values after 11 days. Wallerian degeneration was detectable at 7 days and increased 9 days after inoculation. Axonal damage appeared at the height of the inflammatory process, when edema and increased EFP were maximal. Evidence of demyelination was apparent by 7 days and persisted through 11 days. The onset of edema was associated with changes in venular endothelial cells which tended to lose their normal scaphoid appearance and assumed rhomboid configurations reminiscent of high endothelial venules. At that point, the barrier endothelium was visibly disrupted with the loss of tight junctions and separation of adjacent cells. Specific cell-cell interactions took place between endothelial cells and infiltrating leukocytes as they immigrated into the endoneurial compartment. There was evidence of altered perineurial permeability with fibrin deposition and leukocyte infiltration between the layers of the perineurial sheath.

New minimum length requirement for a T cell epitope for experimental allergic neuritis.

The bovine P2 protein induces experimental allergic neuritis (EAN) in Lewis rats. Using synthetic peptides representing regions of the P2 protein sequence and P2-specific neuritogenic T cell lines, we have demonstrated that the minimum peptide length requirement for the T cell epitope for EAN is residues 61-70 of the P2 protein having the sequence EISFKLGQEF. Adding a threonine at the NH2 terminus of this peptide reproduces residues 60-70 of the P2 protein sequence and significantly enhances the neuritogenic capability of this peptide.