Identification of a specific HLA DR2 Ia molecule as a restriction element for measles virus-specific HLA class II-restricted cytotoxic T cell clones.

By using a panel of HLA-D-defined subtypes of HLA-DR2 HCL with known beta chain structural variabilities, we have demonstrated that HLA-DR2, OKT4+ cytotoxic T lymphocyte (CTL) clones specific for measles virus are apparently restricted to a distinct DR beta chain. The presence of this DR beta 2 molecule correlated precisely with the susceptibility of measles virus-infected HLA-DR2 HCL to lysis by these CTL clones. These studies demonstrate that delineation of HLA-DR2 into various subgroups can have a functional significance that parallels the structural differences within the HLA-D region. These results are discussed in the context of the possible association of HLA class II-restricted, measles virus-specific CTL and multiple sclerosis.

Peptide specificities of myelin basic protein-reactive human T-cell clones.

Forty myelin basic protein (BP)-reactive T-cell clones were isolated from a patient with multiple sclerosis and used to identify human T-cell recognition sites on the BP molecule. At least three sites have been identified: one in the N-terminal half of the molecule (residues 1-97), one in the C-terminal (residues 98-170), and one which spans residues 97-98. The clones exhibited a marked preference for the C-terminal half of the molecule. No cross-reactivity with measles virus was detected. These clones will be useful for both the further delineation of the human T-cell recognition sites on BP and the generation of anticlonotypic monoclonal antibodies.

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.

Cytomegalovirus encephalitis associated with episodic neurologic deficits and OKT-8+ pleocytosis.

After 3 days of symptoms suggesting a viral illness, a 35-year-old man experienced three episodes of aphasia, right-sided sensory symptoms, and bifrontal headache. Each lasted several hours. CSF examination revealed a moderate lymphocytosis consisting of 80% OKT-8+ cells. Serum anti-cytomegalovirus (anti-CMV) antibody titer was elevated at 1:1,024 and subsequently fell to 1:64. Episodic symptoms recurred 5 months later, at which time the anti-CMV titer peaked at 1:8,192. A trial of inhaled oxygen aborted two episodes after several minutes each.

A longitudinal study of T1 hypointense lesions in relapsing MS: MSCRG trial of interferon beta-1a. Multiple Sclerosis Collaborative Research Group.

BACKGROUND: T1 hypointense lesions (T1 black holes) are focal areas of relatively severe CNS tissue damage detected by MRI in patients with MS. OBJECTIVE: To determine the natural history of T1 hypointense lesions in relapsing MS and the utility of T1 hypointense lesions as outcome measures in MS clinical trials. METHODS: MR studies were from the Multiple Sclerosis Collaborative Research Group trial. Longitudinal results are reported in 80 placebo- and 80 interferon beta-1a (IFNbeta-1a)-treated patients with mild to moderate disability relapsing-remitting MS. RESULTS: There was a small but significant correlation between T1 hypointense lesion volume and disability at baseline and on trial (r = 0.22, r = 0.28). In placebo patients there was a 29.2% increase in the mean volume of T1 hypointense lesions (median 124.5 mm3) over 2 years (p < 0.001 for change from baseline), as compared to an 11.8% increase (median 40 mm3) in the IFNbeta-1a-treated patients (change from baseline not significant). These treatment group comparisons did not quite reach significance. The most significant contributor to change in T1 hypointense lesions was the baseline number of enhancing lesions (model r2 = 0.554). Placebo patients with more active disease, defined by enhancing lesions at baseline, were the only group to show a significant increase in T1 hypointense lesion volume from baseline. CONCLUSION: The development of T1 hypointense lesions is strongly influenced by prior inflammatory disease activity, as indicated by enhancing lesions. These results suggest that treatment with once weekly IM IFNbeta-1a (30 mcg) slows the 2-year accumulation of these lesions in the brain.

A longitudinal study of brain atrophy in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG).

OBJECTIVE: To determine if progressive brain atrophy could be detected over 1- and 2-year intervals in relapsing MS, based on annual MR studies from the Multiple Sclerosis Collaborative Research Group (MSCRG) trial of interferon beta-1a (Avonex). METHODS: All subjects had mild to moderate disability, with baseline expanded disability status scores ranging from 1.0 to 3.5, and at least two relapses in the 3 years before study entry. Atrophy measures included third and lateral ventricle width, brain width, and corpus callosum area. RESULTS: Significant increases were detected in third ventricle width at year 2 and lateral ventricle width at 1 and 2 years. Significant decreases in corpus callosum area and brain width were also observed at 1 and 2 years. Multiple regression analyses suggested that the number of gadolinium-enhancing lesions at baseline was the single significant contributor to change in third ventricle width. Atrophy over 1 and 2 years as indicated by enlargement of the third and lateral ventricle and shrinkage of the corpus callosum was greater for patients entering the trial with enhancing lesions. Greater disability increments over 1 and 2 years were associated with more severe third ventricle enlargement. CONCLUSION: In patients with relapsing MS and only mild to moderate disability, significant cerebral atrophy is already developing that can be measured over periods of only 1 to 2 years. The course of cerebral atrophy in MS appears to be influenced by prior inflammatory disease activity as indicated by the presence of enhancing lesions. Brain atrophy measures are important markers of MS disease progression because they likely reflect destructive and irreversible pathologic processes.

Incidence and significance of neutralizing antibodies to interferon beta-1a in multiple sclerosis. Multiple Sclerosis Collaborative Research Group (MSCRG)

BACKGROUND: Interferon beta is an effective treatment for relapsing multiple sclerosis (MS). As with other protein drugs, neutralizing antibodies (NAB) can develop that reduce the effectiveness of treatment. OBJECTIVES: To determine the incidence and biological significance of NAB to interferon beta-la (IFN-beta-1a; Avonex; Biogen, Cambridge, MA) in MS patients. METHODS: A two-step assay for NAB to IFN-beta-1a was developed and used to assay serum samples from participants in the phase III clinical trial of IFN-beta-1a, and from patients in an ongoing open-label study of IFN-beta-1a. The biological significance of NAB to IFN-beta-1a was determined by relating the NAB assay result to in vivo induction of the IFN-inducible molecules neopterin and beta-2 microglobulin, and the clinical significance was determined by comparing clinical and MRI measures of disease activity after 2 years of IFN-beta-1a therapy in patients who were NAB+ and NAB-. The incidence of NAB was compared in MS patients who had used only IFN-beta-1a with the incidence in MS patients who had used only IFN-beta-1b. RESULTS: In patients in the open-label study, development of NAB to IFN-beta-1a resulted in a titer-dependent reduction in neopterin induction after interferon injections. In patients in the phase III study, development of NAB was associated with a reduction in beta-2 microglobulin induction. In the phase III study, a trend toward reduced benefit of IFN-beta-1a on MRI activity in NAB+ versus NAB- patients was observed. The incidence of NAB to IFN-beta-1a in the open-label study was approximately 5% over 24 months of treatment of IFN-beta-1a therapy, but was four- to sixfold higher using the same assay for patients exposed only to IFN-beta-1b for a similar duration. There were no clinical, MRI, or CSF characteristics that were predictive of which patients would develop NAB. CONCLUSIONS: NAB directed against IFN-beta have in vivo biological consequences in patients with MS. The frequency with which MS patients develop NAB against IFN-beta is significantly greater with IFN-beta-1b therapy compared with IFN-beta-1a therapy. Treatment decisions in MS patients treated with IFN-beta should take into account development of NAB.

Impact of interferon beta-1a on neurologic disability in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG).

BACKGROUND AND OBJECTIVE: A phase III double-blind, placebo-controlled clinical trial demonstrated that interferon beta-1a (IFN beta-1a) (Avonex, Biogen) significantly delayed progression of disability in relapsing MS patients. The primary clinical outcome was time from study entry until disability progression, defined as > or = 1.0 point worsening from baseline Kurtzke Expanded Disability Status Scale (EDSS) score persisting for at least two consecutive scheduled visits separated by 6 months. The objective of this study was to examine the magnitude of benefit on EDSS and its clinical significance. METHODS: Post hoc analyses related to disability outcomes using data collected during the double-blind, placebo-controlled phase III clinical trial. RESULTS: (1) Clinical efficacy related to disability did not depend on the definition of disability progression. A significant benefit in favor of IFN beta-1a was observed when > or = 2.0 point worsening from baseline EDSS was required or when worsening was required to persist for > or = 1.0 year. (2) Placebo recipients who reached the primary clinical outcome worsened by a larger amount from baseline EDSS than did IFN beta-1a recipients who reached the primary study outcome. (3) Significantly fewer IFN beta-1a recipients progressed to EDSS milestones of 4.0 (relatively severe impairment) or 6.0 (unilateral assistance needed to walk). (4) Cox proportional hazards models demonstrated that the only baseline characteristic strongly correlated with longer time to disability progression was IFN beta-1a treatment. CONCLUSIONS: The primary clinical outcome for the IFN beta-1a clinical trial underestimated clinical benefits of treatment. Results in this report demonstrate that IFN beta-1a treatment is associated with robust, clinically important beneficial effects on disability progression in relapsing MS patients.

Localization of the human SP3 gene to chromosome 7p14-p15.2. The lack of expression in multiple sclerosis does not reflect abnormal gene organization.

Sp3 belongs to a large family of transcription factors that bind GC/T box elements. We have previously demonstrated the deficient expression of Sp3 in peripheral blood mononuclear cells (PBMC) from most patients with multiple sclerosis (MS). In the current study, the Sp3 gene was assigned to chromosome 7 by using somatic cell hybrid mapping and analysis of a chromosome 7-specific cosmid library. The position of Sp3 was refined to 7p14-p15.2 by fluorescence in situ hybridization (FISH). Southern blot and polymerase chain reaction analysis of genomic DNA failed to demonstrate a detectable difference between MS and control PBMC.

Enhanced transfer of experimental allergic encephalomyelitis with Lewis rat lymph node cells.

Lewis rat lymph node cells (LNC) are greatly enhanced in their ability to transfer experimental allergic encephalomyelitis (EAE) after culture with myelin basic protein (BP). As few as 10(6) LNC transfer disease after culture with antigen. In contrast to spleen cells, enhanced transfer with LNC is not seen after culture with concanavalin A. Neither cell homogenates nor culture supernatants were capable of transferring EAE. Removal of B-cells had no adverse effect on disease transfer. LNC capable of enhanced transfer were found in rats after recovery from, as well as at the height of, clinical disease.

Expansion of antigen-specific T cells from cerebrospinal fluid of patients with multiple sclerosis.

We have expanded cerebrospinal fluid (CSF) T lymphocytes obtained from four patients with multiple sclerosis (MS). Fresh CSF cells were placed into culture with medium, interleukin-2, irradiated autologous peripheral blood mononuclear cells, and either myelin basic protein (BP) or measles virus antigen. Two CSF cell lines demonstrated a mild degree of antigen-specific proliferation to BP, a third reacted with measles virus, and the fourth demonstrated no known antigenic specificity. The percentage of HLA-DR + cells was increased in all four cell lines. The culture procedure has thus selected for a population of activated T cells, some of which demonstrate reactivity with antigens of potential relevance to the pathogenesis of MS.

Myelin basic protein-specific T cell lines and clones derived from SJL/J mice with experimental allergic encephalomyelitis.

Myelin basic protein (BP)-specific T-cell lines and clones have been derived from SJL/J mice which had been sensitized with BP in complete Freund's adjuvant. Cell lines which were initiated and maintained in the presence of BP were specific for this antigen. Cell lines specific for tuberculin-purified protein derivative (PPD) were also established. BP-reactive cell lines maintained for 1 month in culture produced experimental allergic encephalomyelitis (EAE) when transferred to recipient mice. The number of cells required was only slightly less than that necessary for transfer of disease after 3-day culture of sensitized lymph node cells. In contrast, proliferative responses to BP were significantly enhanced after 1 month in culture. Cell lines lost the capacity to transfer EAE after 4 months in culture, but retained a vigorous proliferative response to BP. Similarly, cloned BP-reactive T cells failed to transfer disease, even when recipient mice were treated with IL-2, pertussis vaccine, or low-dose irradiation. Serial FACS analyses demonstrated alterations in cell surface antigen expression, particularly loss of reactivity with anti-Ia antibody, which correlated temporally with loss of ability to transfer disease. Persistence of antigen-induced proliferation by both cloned and uncloned T-cell lines should render these populations suitable for detailed study of the T-cell BP receptor.

Differential recognition of MBP epitopes in BALB/c mice determines the site of inflammatory disease induction.

Although myelin basic protein (MBP)-recognizing T cells are not readily obtained after immunization of BALB/c mice with MBP (reflecting the BALB/c resistance to actively induced experimental autoimmune encephalomyelitis (EAE)), they can be expanded and cloned after several rounds of in vitro culture. The majority of BALB/c-derived clones recognize an epitope defined by MBP peptide 59-76. When transferred to naive BALB/c recipients, these clones cause classical EAE, with characteristic inflammation and demyelination of the central nervous system (CNS). We previously showed that two related clones recognizing a minor epitope, defined by MBP peptide 151-168, cause inflammation and demyelination preferentially of the peripheral nervous system (PNS). Because MBP has alternatively spliced isoforms, residues 151-168 are not present contiguously in all MBP isoforms. In order to determine whether induction of PNS disease is idiosyncratic to these sister clones, or related to their properties of epitope recognition, an independent T-cell line with similar recognition properties was studied. Clone 116F, derived from a BALB/c shiverer mouse, expresses a different T-cell receptor (TCR), with distinct TCR contact residues, but like the previously described T cells, this clone requires residues from both exons 6 and 7 for optimal stimulation. When adoptively transferred to BALB/c recipients, this clone preferentially induces disease of the PNS. A control BALB/c shiverer-derived MBP 59-76-recognizing clone, in contrast, induces CNS disease. These data strongly suggest that the site of disease initiation may correlate with epitope recognition, particularly when alternative isoforms are involved.

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.

Cerebrospinal fluid abnormalities in a phase III trial of Avonex (IFNbeta-1a) for relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group.

BACKGROUND AND OBJECTIVE: This report provides results of CSF analyses done in a subset of relapsing remitting MS patients participating in a placebo-controlled, double-blind, phase III clinical trial of IFNbeta-Studies supported by the National Multiple Sclerosis Society (grants RG2019, RG2827),a (Avonex , Biogen). The clinical trial demonstrated that IFNbeta-1a treatment resulted in significantly reduced disability progression, annual relapse rate, and new brain lesions visualized by cranial magnetic resonance imaging. The objectives of the current study were to determine: (a) whether CSF abnormalities in MS patients correlated with disease or MRI characteristics, and (b) effects of IFNbeta-1a therapy on these CSF abnormalities. METHODS: CSF was analyzed from 262 (87%) of the 301 study subjects at entry into the clinical trial, and a second CSF sample was analyzed from 137 of these 262 subjects after 2 years of therapy. CSF cell counts, oligoclonal bands (OCB), IgG index, and free kappa light chains were measured using standard assays. Baseline CSF results were compared with demographic, disease, and MRI parameters. Differences in on-study relapse rate, gadolinium enhancement, and EDSS change according to baseline CSF status was used to determine the predictive value of CSF for subsequent clinical and MRI disease activity. Change in CSF parameters after 104 weeks were used to determine the effects of treatment. RESULTS: (1) At study baseline, 37% of the subjects had abnormal CSF WBC counts, 61% had abnormal levels of CSF free kappa light chains, 84% had abnormal IgG index values, and 90% were positive for OCB. (2) Baseline IgG index, kappa light chains, and OCB showed weakly positive, statistically significant correlations with Gd-enhanced lesion volume and T2 lesion volume. WBC showed a statistically significant correlation with Gd-enhancing lesion volume but was uncorrelated with T2 lesion volume. (3) There was an associated between baseline CSF WBC counts and on-study clinical and MRI disease activity in placebo recipients. (4) IFNbeta-1a treatment resulted in significantly reduced CSF WBC counts, but there was no treatment-related change in CSF IgG index, kappa light chains, or OCB, which remained relatively stable over time in both patient groups. CONCLUSIONS: The current study documents significant reductions in CSF WBC counts in patients treated with IFNbeta-1a for 104 weeks. This finding is considered relevant to the therapeutic response, since CSF WBC counts were found to be positively correlated with subsequent clinical and MRI disease activity in placebo-treated relapsing MS patients.

Failure of serum from recovered rats to prevent enhanced adoptive transfer of experimental allergic encephalomyelitis.

The rat is unique among species used for research on experimental allergic encephalomyelitis (EAE) because of the spontaneous recovery which occurs routinely after severe, almost fatal disease. The mechanism of recovery has never been adequately explained although it has been suggested that suppressor cells might play a role in this phenomenon. In another immune system (contact sensitivity) anti-idiotypic antibodies obtained during the recovery phase have been shown to have a protective effect. Adoptive transfer of EAE, which can be markedly enhanced by incubation of sensitized cells with antigen in vitro, offers a convenient tool for investigating mechanisms of recovery. With this system, we have attempted to suppress transfer of disease with serum obtained from recovered rats. In spite of various manipulations of the experimental protocol, including the use of serum plus complement before and after incubation of cells with antigen, we have been unable to demonstrate suppression of disease. We and others recently reported that cells from recovered rats are also capable of enhanced transfer. This permitted the use of autologous serum from individual cell donors. Even in this strictly autologous system, however, no inhibitory effect of serum could be detected.

Serum IL-17F does not predict poor response to IM IFNß-1a in relapsing-remitting MS.

OBJECTIVE: There is a significant unmet need for serum biomarkers in relapsing-remitting multiple sclerosis (RRMS) that are predictive of therapeutic response to disease-modifying therapies. Following a recent Stanford study which reported that pretreatment levels of serum interleukin (IL)-17F could predict poor response to interferon-ß (IFNß) therapy, we sought to validate the finding using samples from a large clinical trial. METHODS: The validation cohort included 54 good responders (GR) and 64 poor responders (PR) selected from 762 subjects with RRMS from the IM IFNß-1a dose comparison study (Avonex study C94-805). Subjects were classified as GR and PR based on the number of relapses, Expanded Disability Status Scale score, and new and enlarging T2 lesions on MRI. Serum samples were assayed for IL-17F using a multiplexed Luminex assay and for IL-17F/F using an ELISA. Replicate aliquots from the Stanford study were also assayed to assure reproducibility of methods. RESULTS: Median pretreatment and post-treatment serum IL-17F levels were not statistically significantly different between GR and PR, and serum IL-7/IL-17F ratios were also not predictive of response status. Replicate aliquots from the Stanford study showed good correlation to their original cohort (r = 0.77). CONCLUSIONS: We were unable to validate the finding that serum IL-17F is a predictor of PR in a large independent cohort of subjects with RRMS. Differences in patient populations and methodology might explain the failure to validate the results from the Stanford study.

Short-Term Relapse Quantitation as a Fully Surrogate Endpoint for Long-Term Sustained Progression of Disability in RRMS Patients Treated with Natalizumab.

Time to sustained worsening in the expanded disability status scale as the standard for evaluating the accumulation of disability has been used as a measure of clinical efficacy in many relapsing-remitting multiple sclerosis (RRMS) clinical trials. However, this measurement usually requires a large sample and long-term study to demonstrate the treatment effect. Annualized relapse rate or time to first relapse is also widely used as alternative measurements of clinical efficacy. A formal statistical validation of short-term relapse activity as a surrogate endpoint for long-term sustained progression of disability could potentially permit smaller, shorter, and less expensive clinical trials in RRMS. Four statistical validation/evaluation approaches consistently showed that relapse activity through one year of treatment serves as statistically valid surrogate endpoint for time to sustained progression of disability. The analysis demonstrates that long-term sustained progression of disability can be predicted by short-term relapse measures with 4 consistent validations of statistical approaches, including a formal statistical hypothesis test. This was demonstrated in a large phase III trial of natalizumab and showed that the beneficial clinical effect of natalizumab on sustained progression of disability at 2 years in patients with RRMS can be predicted by the total number of relapses at 1 year.