Diversity of T-cell receptor V alpha, V beta, and CDR3 expression by myelin basic protein-specific human T-cell clones.

We sequenced the cDNAs of alpha and beta T-cell receptors (TCRs), including V, J, and CDR3 regions, expressed by 54 myelin basic protein (MBP)-specific T-cell clones generated from the peripheral blood of 15 multiple sclerosis (MS) patients and three normal controls. Thirteen V alpha gene segments, 18 V beta gene segments, 23 CDR3 alpha sequences, and 30 CDR3 beta sequences were represented among these clones. Some CDR3 motifs were common to several clones that shared epitope specificity, while other motifs were common to clones with diverse epitope specificities. The extensive heterogeneity of TCR gene expression in the human immune response to MBP indicates that therapeutic strategies aimed at blocking a limited number of TCRs are unlikely to fully suppress the T-cell response to MBP in vivo.

Evidence for multiple human T cell recognition sites on myelin basic protein.

Myelin basic protein (BP)-specific T cell clones were used to study human T cell recognition sites on the BP molecule. Proliferation assays performed with a panel of xenogeneic BPs of known amino acid sequence and with large peptide fragments of human and guinea pig BPs demonstrated ten different patterns of reactivity. The data provide evidence for at least four different human T cell epitopes within the C-terminal half of the BP molecule, three within the N-terminal half, and three located within the central portion of the molecule. The results indicate that attempts to inhibit anti-BP responses in vivo in an antigen-specific manner will require the suppression of multiple T cell populations.

Measles virus-specific human T cell clones: studies of alloreactivity and antigenic cross-reactivity.

Cross-reactivity between altered self and foreign major histocompatibility complex (MHC) may be of etiologic importance in autoimmune disease. We have studied 29 measles virus-specific cloned and uncloned T cell lines from a patient with multiple sclerosis (MS) and from a normal subject. Two of the T cell clones derived from the normal subject reacted with foreign MHC determinants. No cross-reactivity between measles virus and either myelin basic protein (BP) or galactocerebroside (GC) was detected. T cell clones which are specific for nominal antigen and which also recognize alloantigen were detected with much smaller frequency than that reported in murine systems. Our data do not support a role for alloreactive measles-specific T cells, nor for cross-reactivity between measles virus and either BP or GC, in the pathogenesis of MS.

Measles vaccine failures: lack of sustained measles-specific immunoglobulin G responses in revaccinated adolescents and young adults.

The measles-specific antibody responses of seronegative adolescents and young adults were evaluated after revaccination. Of 1650 previously vaccinated healthy volunteers between the ages of 10 and 30 years, 4.4% were found to be seronegative for measles antibodies and 9.9% had equivocal titers. Seronegative volunteers were revaccinated to measles and followed serially for development of measles-specific IgG. Of 43 subjects followed for at least 1 year, only 58% developed and maintained positive antibody titers; 12% never developed positive titers and 30% initially developed titers that fell below positive levels within 1 year. The peak titers achieved by those subjects who responded transiently were lower than those achieved by subjects who developed sustained responses. Thus even after the recommended two dose schedule of the current measles vaccine, some adolescents and young adults lack protective titers of measles-specific antibody.

A phase I study of FAP (5-Fluorouracil, α-interferon and cisplatin) combined with methotrexate for the treatment of advanced urothelial malignancies.

Combination chemotherapy using 5-fluorouracil and α-interferon is active against advanced urothelial cancers. Its toxcity profile appears favorable such that addition of other active agents (i.e., cisplatin and methotrexate) may improve its therapeutic window. Our goal was to identify the starting dose of FAP (5-fluorouracil-α-interferon-cisplatin) that will permit delivery of methotrexate in patients with advanced chemotherapy-refractory urothelial cancers. This is a phase 1 study in which the dose level of FAP that permitted delivery of two doses of methotrexate in 28 days will be considered the maximum tolerated dose (MTD). This is based on experiences with MVAC chemotherapy in which only half of the patients received two doses of methotrexate in a 28 days cycle. The dose level identified as the MTD is as follows: 5-FU 500 mg/m(2) by intravenous continuous infusion daily for 5 days in weeks 1 and 4; α-interferon 5 MU/m(2) subcutaneously daily for 5 days simultaneously with 5-FU infusion in weeks 1 and 4 and thrice weekly during weeks 2 and 3; and cisplatin 25 mg/m(2) plus methotrexate 30 mg/m(2) intravenously once weekly for 4 weeks. The treatment will be repeated every 6 weeks. Most of the significant toxic effects, including mucositis, neutropenia, and thrombocytopenia, are encountered during weeks 3 and 4. This study showed that the MTD for FAP combined with methotrexate has been determined for phase II studies. Exploration of alterative regimen such as FAP in the treatment of advanced urothelial cancer is warranted. FAP has a unique mechanism of action and acceptable toxicity profile that may allow novel drug combinations and improved therapeutic efficacy.

Effects of calcium ionophore and phorbol ester on class-II-restricted virus-specific human cytotoxic T cell clones.

Calcium ionophore A23187 and phorbol myristate acetate (PMA) were studied for their functional effects on human class-II-restricted measles virus-specific CD4+ cytotoxic T lymphocyte (CTL) clones. A23187 and PMA each dramatically inhibited virus-specific CTL activity. Both PMA and A23187 induced proliferation of resting clones and augmented relatively weak or suboptimal proliferative responses, but inhibited vigorous antigen- or mitogen-induced proliferation. The parallel responses induced by these two agents suggest that they produce their effects via similar mechanisms, most likely through protein kinase C.

Is active skeletal muscle functionally vasoconstricted during dynamic exercise in conscious dogs?

We investigated whether the increase in hindlimb blood flow and vascular conductance in conscious dogs during graded dynamic exercise is functionally restrained by the sympathetic nervous system. Dogs were chronically instrumented to monitor terminal aortic blood flow (TAQ) as an index of hindlimb skeletal muscle blood flow and mean arterial pressure (MAP). The extent of functional sympathetic tone was assessed by measuring the increase in TAQ and terminal aortic vascular conductance (TAC, calculated as TAQ/MAP) in response to intra-arterial infusion of the alpha-adrenergic antagonist prazosin (PZ; 50 micrograms/kg) into the hind-limbs at rest and during steady-state dynamic (treadmill) exercise ranging from mild (3.2 km/h, 0% grade to moderately heavy (8 km/h, 15% grade) workloads. This dose of PZ completely abolished the large hindlimb vasoconstrictor response to phenylephrine (1 microgram/kg ia). At rest, PZ increased TAQ by 0.10 +/- 0.02 l/min and TAC by 1.85 +/- 0.53 ml.min-1.mmHg-1. During exercise, as workload increased and the control levels of TAQ and TAC rose progressively delta TAQ and delta TAC with PZ infusion also increased. At the highest workload, PZ increased TAQ by 0.41 +/- 0.07 l/min and TAC by 4.81 +/- 0.38 ml.min-1.mmHg-1. The increase in TAQ and TAC with PZ were linearly related to the control level of TAQ, indicating that as workload increases progressively greater restraint of muscle vasodilation by the sympathetic nervous system occurs. We conclude that during dynamic exercise in conscious dogs the sympathetic nervous system progressively restrains the normal vasodilation in active skeletal muscle, thereby limiting skeletal muscle perfusion.

Human cytotoxic T-cell recognition of a synthetic peptide of myelin basic protein.

Previous studies with a panel of myelin basic protein (MBP)-specific human T-cell clones suggested a clustering of epitopes in the middle and at the C terminus of the molecule. The current study demonstrates that 19 of 40 clones recognize a synthetic peptide corresponding to residues 152 to 170 of the human MBP molecule and that 9 clones recognize a synthetic peptide corresponding to residues 86 to 105. Myelin basic protein-specific cytotoxic activity was restricted to the clones that recognized peptide 152-170, and this peptide served as a preferential cytotoxic T-cell target when attached to an autologous B-cell line. The specificity of MBP-directed cytotoxic activity appears to be much more restricted than the specificity demonstrated for proliferative activity.

Molecular interactions between transfected human TCR, immunodominant myelin basic protein peptide 152-165, and HLA-DR13.

Chimeric TCR transfectants expressing human extracellular sequences and murine intracellular/transmembrane sequences were generated to analyze the trimolecular interaction between myelin basic protein (MBP) autoantigen, HLA, and a TCR isolated from a patient with multiple sclerosis. Chimeric transfectants responded to TCR activation by CD3- and TCRBV22S1-specific mAbs and by superantigen. Additionally, chimeric transfectants responded to autoantigen-specific activation with MBP 152-165 when presented by DR(alpha,beta1*1301) independent of the CD4 adhesion molecule. Transfectants did not respond to Ag presented by other HLA-DR molecules, including the closely related DR(alpha,beta1*1302). In peptide-binding studies with a panel of serial alanine-substituted MBP peptides, HLA contact residues necessary for anchoring MBP 152-165 to DR(alpha,beta1*1301) were also defined: 154 (F), 159 (R), and 162 (R). The chimeric TCR transfectant's differential response to a similar panel of MBP analogues defined residues that interact with the TCR: 153 (I), 155 (K), 156 (L), 160 (D), and 161 (S). Analysis of molecular interactions, such as those described in this work, may be central to developing new strategies for suppressing Ag-specific responses in human autoimmune disease.

Deficient expression in multiple sclerosis of the inhibitory transcription factor Sp3 in mononuclear blood cells.

To evaluate differential gene expression in multiple sclerosis (MS) patients and control subjects, we used differential display to screen for messenger RNAs that are differentially expressed in peripheral blood mononuclear cells from monozygotic twins who are discordant for MS. We identified a 232-bp complementary DNA fragment, present only in material from the normal twin, that exhibited 100% identity with the inhibitory transcription factor Sp3. Oligonucleotide primers corresponding to Sp3 messenger RNA sequences amplified complementary DNA of appropriate size from 83% of control subjects but from only 21% of MS patients (p < 0.001). These results suggest that Sp3 gene transcription is suppressed in peripheral blood mononuclear cells from most MS patients and that other genes whose expression is normally suppressed by Sp3 in immune cells may consequently be overexpressed.

HLA restriction and TCR usage of T lymphocytes specific for a novel candidate autoantigen, X2 MBP, in multiple sclerosis.

Previous investigations of the major 18.5-kDa isoform of myelin basic protein (MBP) as a target autoantigen in multiple sclerosis (MS) have failed to identify an epitope uniformly recognized with higher frequency in MS patients compared with controls. Because remyelination has been observed in MS plaques, we were prompted to investigate T cells specific for myelin protein isoforms with up-regulated expression during remyelination. We have recently described such T cells that recognize the exon 2-encoded region of MBP (X2 MBP), a sequence included in the 21.5- and 20.2-kDa isoforms of MBP. These cells were shown to be CD4+, HLA class II restricted, and cytolytic. In members of one multiplex MS family, X2 MBP-specific T lymphocytes were as prevalent as T cells specific for immunodominant regions within the major 18.5-kDa isoform of MBP. The present study characterizes X2 MBP-specific T cell responses in additional multiplex MS family members as well as in heterogeneous (non-familial) MS patients and in healthy controls. The frequencies of X2 MBP-specific T cells in each of the affected family members from two of three MS families were significantly increased as compared with both the heterogeneous MS group and the healthy control group. Also, X2 MBP-specific T cell lines from affected family members were primarily restricted to molecules encoded by the DR2/DQw1 allele. Although TCR usage was generally heterogeneous, there was evidence of intraindividual sequence identity. These data suggest that: 1) Myelin proteins with up-regulated expression during the course of disease should be considered as candidate autoantigens in MS. 2) The functional basis for the association of DR2/DQw1 inheritance with MS susceptibility may be related to presentation of autoantigens by this allele. 3) TCR therapy will need to be individually tailored to target the most prevalent autoantigen-specific response.

T cell responses to the paramyxovirus simian virus 5: studies in multiple sclerosis and normal populations.

A recent study suggested that a significant portion of the oligoclonal IgG found in multiple sclerosis (MS) cerebrospinal fluid may be removed by absorption with simian virus 5 (SV5). We have now evaluated the proliferative responses to SV5 generated by peripheral blood mononuclear cells from normal adult subjects and from patients with MS. Positive responses were detected in 16 of 16 subjects in each group. The magnitude of the response was not significantly different in the two groups. There was no correlation between the level of response and the presence or absence of HLA-DR2. No cross-reactivity with SV5 was demonstrated by panels of human T cell clones directed against myelin basic protein or measles virus. More than 60% of normal individuals between 10 and 16 years of age also generated positive T cell proliferative responses to SV5, while fewer than 25% of subjects below the age of 6 generated positive responses. Intermediate percentages of positive responders were detected in subjects 6-9 years of age. We conclude that the adult human population has been widely exposed to this organism and that initial exposure generally occurs in the early to middle school-age years.

Heterogeneity of the T-cell receptor beta gene rearrangements generated in myelin basic protein-specific T-cell clones isolated from a patient with multiple sclerosis.

Seventeen T-cell clones derived from the peripheral blood of a patient with multiple sclerosis and reactive with a synthetic peptide corresponding to residues 152-170 of the human myelin basic protein molecule were previously shown to be cytotoxic for myelin basic protein-coated target cells. Genetic restriction studies have now demonstrated that these clones recognize myelin basic protein in association with human leukocyte antigen DRw13. Studies of the T-cell receptor beta gene rearrangements generated by these clones demonstrated 12 different patterns, as evaluated by Southern blot analysis. Thus, the human T-cell response to myelin basic protein is exceedingly heterogeneous, even among T cells that recognize the same small fragment of the molecule in association with the same class II restriction element.