A humanized model for multiple sclerosis using HLA-DR2 and a human T-cell receptor.

Multiple sclerosis (MS) is a complex chronic neurologic disease with a suspected autoimmune pathogenesis. Although there is evidence that the development of MS is determined by both environmental influences and genes, these factors are largely undefined, except for major histocompatibility (MHC) genes. Linkage analyses and association studies have shown that susceptibility to MS is associated with genes in the human histocompatibility leukocyte antigens (HLA) class II region, but the contribution of these genes to MS disease development less compared with their contribution to disorders such as insulin-dependent diabetes mellitus. Due to the strong linkage disequilibrium in the MHC class II region, it has not been possible to determine which gene(s) is responsible for the genetic predisposition. In transgenic mice, we have expressed three human components involved in T-cell recognition of an MS-relevant autoantigen presented by the HLA-DR2 molecule: DRA*0101/DRB1*1501 (HLA-DR2), an MHC class II candidate MS susceptibility genes found in individuals of European descent; a T-cell receptor (TCR) from an MS-patient-derived T-cell clone specific for the HLA-DR2 bound immunodominant myelin basic protein (MBP) 4102 peptide; and the human CD4 coreceptor. The amino acid sequence of the MBP 84-102 peptide is the same in both human and mouse MBP. Following administration of the MBP peptide, together with adjuvant and pertussis toxin, transgenic mice developed focal CNS inflammation and demyelination that led to clinical manifestations and disease courses resembling those seen in MS. Spontaneous disease was observed in 4% of mice. When DR2 and TCR double-transgenic mice were backcrossed twice to Rag2 (for recombination-activating gene 2)-deficient mice, the incidence of spontaneous disease increased, demonstrating that T cells specific for the HLA-DR2 bound MBP peptide are sufficient and necessary for development of disease. Our study provides evidence that HLA-DR2 can mediate both induced and spontaneous disease resembling MS by presenting an MBP self-peptide to T cells.

The HLA-DP2 protein binds the immunodominant epitope from myelin basic protein, MBP85-99, with high affinity.

Myelin basic protein (MBP) is a candidate autoantigen in multiple sclerosis (MS). The immunodominant epitope for T-cell responses is assigned to the amino acid sequence MBP84-102, which binds to human leukocyte antigen (HLA)-DR2a (DRB5*0101) and HLA-DR2b (DRB1*1501) of the HLA-DR2 haplotype carrying the strongest genetic association with MS. In contrast with HLA-DR and -DQ molecules, HLA-DP molecules are poorly characterized with respect to the binding of self-peptides. We show here that HLA-DP2 binds MBP85-99 with high affinity, and that the amino acid residues in position MBP91, MBP92 and MBP93 are influencing the binding, as shown by alanine scans. We further used a series of truncated peptides to identify the core of the binding. Moving the frame along the peptide from residues 87-97 to 89-99 progressively decreased the binding affinity for HLA-DP2, while moving further towards the C-terminal completely abrogated the binding of peptides to HLA-DP2. The data suggest that the docking of the MBP85-99 peptide into the HLA-DP2 groove is dependent on MBP88V and MBP89V and may use either of them as primary anchor for the p1 position. HLA-DP2 might thus present the MBP85-99 peptide in the same register as the HLA-DRB1*1501, where the MBP89V is preferred as the p1 anchor. Notably, full-length MBP was able to compete for peptide binding with an affinity similar to that seen for the high-affinity binding peptides, DRα170-83 and IIP53-65. In summary, the HLA-DP2 molecule binds the immunodominant epitope in MS, MBP85-99, possibly in more than one register.

HLA-D restriction of the proliferative response to hapten dinitrophenyl-conjugated cells. I. No indication of cross-reaction between dinitrophenyl-conjugated autologous and nonconjugated allogeneic cells.

Evidence is presented in humans that the dinitrophenylated (DNP) antigen, previously shown to be restricted in cell-mediated cytotoxic assays by the HLA-A and -B antigens, is restricted in the proliferative response by HLA-D antigens. A gene dose effect was observed. Some influence of DNP conjugation on the mixed lymphocyte culture stimulatory capacity is shown, but the restriction phenomenon is obvious with the degree of conjugation chosen. The functional part of HLA-D is more likely to follow DR than D antigens. Unspecific conditioning of alloreactivity in secondary cultures is shown by negative as well as by positive selection procedures to be different from the major part of the DNP-specific clones.

Visualization of myelin basic protein (MBP) T cell epitopes in multiple sclerosis lesions using a monoclonal antibody specific for the human histocompatibility leukocyte antigen (HLA)-DR2-MBP 85-99 complex.

Susceptibility to multiple sclerosis (MS) is associated with the human histocompatibility leukocyte antigen (HLA)-DR2 haplotype, suggesting that major histocompatibility complex class II-restricted presentation of central nervous system-derived antigens is important in the disease process. Antibodies specific for defined HLA-DR2-peptide complexes may therefore be valuable tools for studying antigen presentation in MS. We have used phage display technology to select HLA-DR2-peptide-specific antibodies from HLA-DR2-transgenic mice immunized with HLA-DR2 molecules complexed with an immunodominant myelin basic protein (MBP) peptide (residues 85-99). Detailed characterization of one clone (MK16) demonstrated that both DR2 and the MBP peptide were required for recognition. Furthermore, MK16 labeled intra- and extracellular HLA-DR2-MBP peptide complexes when antigen-presenting cells (APCs) were pulsed with recombinant MBP. In addition, MK16 inhibited interleukin 2 secretion by two transfectants that expressed human MBP-specific T cell receptors. Analysis of the structural requirement for MK16 binding demonstrated that the two major HLA-DR2 anchor residues of MBP 85-99 and the COOH-terminal part of the peptide, in particular residues Val-96, Pro-98, and Arg-99, were important for binding. Based on these results, the antibody was used to determine if the HLA-DR2-MBP peptide complex is presented in MS lesions. The antibody stained APCs in MS lesions, in particular microglia/macrophages but also in some cases hypertrophic astrocytes. Staining of APCs was only observed in MS cases with the HLA-DR2 haplotype but not in cases that carried other haplotypes. These results demonstrate that HLA-DR2 molecules in MS lesions present a myelin-derived self-peptide and suggest that microglia/macrophages rather than astrocytes are the predominant APCs in these lesions.

Identification of new sensitive biomarkers for the in vivo response to interferon-beta treatment in multiple sclerosis using DNA-array evaluation.

OBJECTIVE: Neutralizing antibodies (NAbs) occur in a proportion of multiple sclerosis (MS) patients treated with interferon (IFN)-beta. NAbs impair the effect of treatment. The biological effect of IFN-beta can be measured as the induction of the myxovirus resistance protein A (MxA) molecule. However, other markers could be more sensitive for evaluating the response to IFN-beta. We used DNA array analysis to identify genes that are strongly induced in blood cells by IFN-beta, and measured their expression in MS patients with different NAb levels. METHODS: Gene expression was studied on DNA arrays in untreated patients, in NAb negative patients, and in MS patients with varying NAb levels 9-12 h and 36-48 h after IFN-beta administration. The expression of selected genes was measured by real-time PCR. NAb levels were assessed by a cytopathic effect assay. RESULTS: Several hundred genes were induced 9-12 h after an injection of IFN-beta. The molecules CXCL10, CCL2 and IFI27 were among the most strongly induced. Gene induction was generally much less pronounced after 36-48 h, but IFI27 remained strongly induced. The strong induction of these molecules and MxA was confirmed by real-time PCR. Induction of MxA, CCL2, CXCL10 and IFI27 was reduced in patients with low NAb levels and lost in patients with intermediate/high NAb levels. CONCLUSION: We identify IFI27, CCL2 and CXCL10 as sensitive biomarkers for the response to IFN-beta. The expression of these markers adequately reflects bioactivity of IFN-ss as documented by the decreased induction in low NAb-positive patients and the lost induction in patients with moderate/high NAb levels.

Haematopoietic cell transplantation with non-myeloablative conditioning in Denmark: disease-specific outcome, complications and hospitalization requirements of the first 100 transplants.

We analysed the outcome and hospitalization requirements of the first 100 patients (Hodgkin's disease (HD), N=13; multiple myeloma (MM), N=14; CLL, N=12; non-Hodgkin's lymphoma (NHL), N=17; myelodysplastic syndrome (MDS), N=18; AML, N=24 and CML, N=2) treated in Denmark with haematopoietic cell transplantation after non-myeloablative conditioning with TBI 2 Gy+/-fludarabine. The cumulative incidence of acute GVHD grade II-IV and extensive chronic GVHD was 67 and 49%. After a median follow-up of 534 days, the overall survival, PFS, relapse-related mortality and treatment-related mortality were 59, 50, 25 and 17%, respectively. Patients with CLL, NHL, AML and MDS with <5% blasts at any time had a favourable outcome with a PFS of 61-71%. Patients with MM, HD and MDS and a history of > or =5% blasts had a less favourable outcome with a PFS of 19-38% (P=0.001). The cumulative incidence of discontinuation of immunosuppression was 37%. During the first and second year post transplant, patients experienced a mean of 41 and 13 outpatient clinic visits, and 53 and 16 days of hospitalization. Sixteen patients were admitted to the intensive care unit, of whom eight are still alive. In conclusion, transplantation outcomes were encouraging, but complications requiring admission and outpatient clinic visits occur frequently post transplant.

Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis.

Mannose-binding lectin (MBL) is a key factor in innate immunity, and lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Accordingly, we investigated whether MBL variant alleles, which are associated with recurrent infections, might be risk factors for CF patients. In 149 CF patients, different MBL genotypes were compared with respect to lung function, microbiology, and survival to end-stage CF (death or lung transplantation). The lung function was significantly reduced in carriers of MBL variant alleles when compared with normal homozygotes. The negative impact of variant alleles on lung function was especially confined to patients with chronic Pseudomonas aeruginosa infection. Burkholderia cepacia infection was significantly more frequent in carriers of variant alleles than in homozygotes. The risk of end-stage CF among carriers of variant alleles increased 3-fold, and the survival time decreased over a 10-year follow-up period. Moreover, by using a modified life table analysis, we estimated that the predicted age of survival was reduced by 8 years in variant allele carriers when compared with normal homozygotes. Presence of MBL variant alleles is therefore associated with poor prognosis and early death in patients with CF.

Genetic polymorphisms in the genes encoding human interleukin-7 receptor-alpha: prognostic significance in allogeneic stem cell transplantation.

Interleukin-7 (IL-7) is essential for T-cell development in the thymus and for the maintenance of peripheral T cells. IL-7 signals through IL-7R, that consists of the gammac-chain and an alpha-chain. Sequencing of IL-7Ralpha has revealed the existence of four single nucleotide polymorphisms (SNPs) (+510C/T, +1237 A/G, 2087T/C and +3110A/G), which all give rise to amino-acid substitutions. The aim of the present investigation was to evaluate the significance of IL-7Ralpha SNPs for the outcome in allogeneic stem cell transplantation (SCT). IL-7Ralpha polymorphisms were determined in 195 recipient and donor pairs from either matched sibling donors or matched unrelated donors (MUD). Genotyping of 173 normal controls was performed in parallel. In MUD transplants, the +1237 genotype of the donor was associated with survival after SCT, the mortality being highest and intermediate for the GG and AG genotypes, respectively (P = 0.023). This pattern was more pronounced with respect to treatment-related mortality (P = 0.003), while IL-7Ralpha genotypes were unrelated to the risk of relapse of leukaemia. The IL-7Ralpha +1237 genotype of the recipient and the genotypes of the other three polymorphisms, were not significantly associated with the outcome of SCT. These findings suggest that the IL-7Ralpha polymorphisms may be of importance for treatment-related mortality after SCT.

HLA, islet cell antibodies, and types of diabetes mellitus.

Insulin-dependent diabetes mellitus, in contrast to non-insulin-dependent diabetes mellitus, is associated with HLA factors B8, BW15, and B18. Recent studies have shown the association to be even stronger with HLA, DW3, and DW4 and have produced evidence for the existence of two "diabetogenic" genes predisposing to insulin-dependent diabetes in different ways. Evidence to suggest the existence of a gene--associated with DW2--that protects against the disease is accumulating. Islet cell antibodies are a feature of insulin-dependent diabetes mellitus and can be seen, in most cases, at the time of diagnosis.

Histocompatibility antigens and manic-depressive disorders.

Forty-seven unrelated Danish patients considered to be manic-depressive, according to strict diagnostic, symptomatic, and course criteria, were typed for histocompatibility (HLA) antigens. Significantly more manic-depressive patients than controls were found to have HLA-A3, HLA-B7, and HLA-Bw16, while significantly fewer manic depressives than controls had HLA-B8. All eight of the patients with HLA-Bw16 were bipolar patients, and none were unipolar depressive patients. We emphasize the need to consider the results with caution in view of the large number of antigens considered and the relatively small number of patients involved. When statistical corrections are made for the large number of antigens investigated, only the difference between bipolar patients and controls remains significant. The best way to determine if our findings are really significant is to attempt to confirm them in other series of patients. The importance of utilizing strict symptomatic and course criteria for the selection and polarization of proband is stressed.

Post-induction residual leukemia in childhood acute lymphoblastic leukemia quantified by PCR correlates with in vitro prednisolone resistance.

Most prognostic factors in childhood acute lymphoblastic leukemia (ALL) are informative for groups of patients, whereas new approaches are needed to predict the efficacy of chemotherapy for the individual patient. The residual leukemia following 4 weeks of induction therapy with prednisolone, vincristine, doxorubicin and i.t. methotrexate and the in vitro resistance to prednisolone, vincristine, and doxorubicin were measured in 30 boys and 12 girls with B (n = 34) or T lineage (n = 8) ALL. The residual leukemia was quantified after 2 (MRD-D15, n = 29) and 4 weeks (MRD-PI, n = 42) of induction therapy with a precise and reproducible clone-specific PCR technique. The median MRD-D15 and MRD-PI were 0.50% (75% range 0.0088.1%) and 0.014% (75% range 0.001-2.0%), respectively, and these levels correlated significantly (n = 29, rs = 0.75, P < 0.001). Both the MRD-D15 and the MRD-PI were related to the age of the patient (MRD-D15: rs= 0.48, P= 0.009; MRD-PI: rs = 0.45, P = 0.003). Patients with T lineage ALL had higher MRD-PI than those with B lineage ALL (median MRD-PI: 0.5% vs 0.01%, P = 0.05). The median LC50 (concentration lethal to 50% of cells) for prednisolone was 2.3 microg/ml (75% range 0.05-668). Both MRD-D15 and MRD-PI correlated significantly with the in vitro resistance to prednisolone (MRD-D15: rs = 0.41, P = 0.03; MRD-PI: rs = 0.39, P = 0.01); but not to in vitro vincristine or doxorubicin resistance. The correlations between MRD and in vitro prednisolone resistance were even more pronounced when B cell precursor and T cell leukemia were analyzed separately (B cell precursor ALL: MRD-PI vs prednisolone LC50: n = 33, rs = 0.47, P = 0.006; T cell ALL: MRD-PI vs prednisolone resistance: n = 8, rs = 0.84, P = 0.009). After a median follow-up of 5.0 years (75% range 3.2-6.9) eight patients have relapsed. All of the 21 patients with a MRD-PI < or =0.5% and a prednisolone LC50 < or =10 microg/ml have remained in remission whereas the 7 year event-free survival for the remaining 20 patients was 0.45 +/- 0.16 (P= 0.002) Prospective studies in childhood ALL are needed to clarify whether combined monitoring of in vitro drug resistance and residual leukemia early during chemotherapy could offer new ways to classify patients and stratify the intensity of therapy.

Studies of the HLA system and insulin-dependent diabetes mellitus.

The relationship between the HLA system and insulin-dependent diabetes mellitus (IDDM) is reviewed. Data compiled by the HLA and Disease Registry reveal that HLA-B8 and/or Dw3 are associated with IDDM in all populations studied so far, but further population studies in non-Caucasian populations should be performed. In Caucasians, HLA-Dw2 renders protection against IDDM while HLA-Dw3 and Dw4 are associated with susceptibility to IDDM. The exact mode of inheritance of susceptibility to IDDM remains to be established. Involvement of at least two genes is likely. Heterogeneity of IDDM is highly possible and should be a matter of major interest in diabetes research.

Heterozygosity for a deletion in the CKR-5 gene leads to prolonged AIDS-free survival and slower CD4 T-cell decline in a cohort of HIV-seropositive individuals.

OBJECTIVE: Recently, it has been shown that a homozygous 32 base-pair deletion in the gene encoding CKR-5, a major coreceptor for HIV-1, leads to resistance to infection with HIV-1. We have investigated whether HIV-seropositive individuals who were heterozygous for the CKR-5 deletion had a different course of the disease. DESIGN: Thirty-five high-risk HIV-seronegative and 99 HIV-seropositive Danish homosexual men followed form 1985 to 1996 and 37 blood donors were analysed for their CKR-5 genotype by polymerase chain reaction. RESULTS: Two (6%) of the 35 HIV-seropositive subjects at high-risk of infection were homozygous and seven (20%) were heterozygous for the CKR-5 deletion. This was not significantly different from the distribution in normal donors. Twenty-two (22%) of the 99 HIV-seropositive subjects were heterozygous and none was homozygous. Two subgroups of patients who had an opposite course of the HIV disease were identified. Of nine long-term non-progressors, six (66%) were heterozygous for the deletion. This frequency is significantly higher than in nine rapid progressors of whom non was heterozygous. The frequency of heterozygotes in long-term non-progressors was also significantly higher than in the cohort as a whole. A Kaplan-Meier plot of the HIV-seropositive subjects, of whom 57 developed AIDS, showed a significantly better prognosis within the first 7 years of follow-up for those who were heterozygous for the deletion. Heterozygous individuals also had a significantly slower decrease in CD4 T-cell count per year. CONCLUSION: Individuals who are heterozygous for the 32-base-pair deletion in the CKR-5 gene have a slower decrease in their CD4 T-cell count and a longer AIDS-free survival than individuals with the wild-type gene for up to 11 years of follow-up.

ABO blood groups in unipolar and bipolar manic-depressive patients.

The authors determined ABO blood groupings for 66 manic-depressive patients diagnosed and divided into bipolar and unipolar groups according to strict symptomatic and course criteria. A significantly higher percentage of bipolar patients than unipolar patients had blood group O, while a significantly higher percentage of unipolar patients than bipolar patients had blood group A. THese findings provide support for the validity of the unipolar-bipolar distinction and are consistent with the concept that vulnerability to manic-depressive disorders may be related to membrane disturbances.

HLA typing in acute optic neuritis. Relation to multiple sclerosis and magnetic resonance imaging findings.

OBJECTIVE: To study the association of brain magnetic resonance imaging (MRI) findings and HLA findings to clarify the relationship between monosymptomatic optic neuritis (ON) and ON as part of clinically definite multiple sclerosis (CDMS). DESIGN: Population-based cohort of patients with ON referred prospectively during 6 years by neurologists and ophthalmologists within 4 weeks of onset of ON. SETTING: Referral center in the general community of greater Copenhagen (Denmark) (population, 1.5 million). PATIENTS: A consecutive sample of 199 patients aged 12 to 59 years with ON (133 with idiopathic ON, 66 with ON + CDMS), ethnically matched with 192 healthy volunteers. MAIN OUTCOME MEASURES: Relation between the HLA-DR15, -DR17, -DQA-1B, and -DQB-1B polymorphisms as defined by restriction fragment length polymorphism analysis, and presence of plaques on T2-weighted brain MRI. RESULTS: The frequency of HLA-DR15 was significantly increased in patients with ON + CDMS (52%) and ON (47%) compared with control subjects (31%). The frequency of HLA-DR17 was almost equal in the ON + CDMS (18%), ON (23%), and control (23%) groups. The frequencies of HLA-DQA-1B (55% in ON + CDMS, 58% in ON) and HLA-DQB-1B (49% in ON + CDMS, 59% in ON) were significantly increased compared with control subjects (41%, HLA-DQA-1B; 37%, HLA-DQB-1B). Brain MRI was abnormal in 48 of 56 examined patients with ON + CDMS and in 64 of 120 examined patients with ON (P < .001). In contrast, the frequencies of HLA alleles did not differ between patients with and without demyelinating lesions. However, patients with ON and normal MRI findings did not show association with HLA-DR15. CONCLUSIONS: The frequencies of alleles were similar in patients with ON and ON + CDMS, confirming that they are not 2 immunogenetically distinct disease entities. The heterogeneity within the group of patients with ON suggests that the HLA-DR15 molecule is involved in susceptibility to initial demyelinating lesion formation.

Technical aspects of typing for HLA-DP alleles using allele-specific DNA in vitro amplification and sequence-specific oligonucleotide probes. Detection of single base mismatches.

The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells was extracted with either a simple salting out method or phenol/chloroform. Both DNAs could be readily used for PCR. The MgC2 concentration of the PCR buffer and the annealing temperature of the thermal cycle of the PCR were the two most important variables. The MgCl2 concentration and the temperature must be carefully titrated for each primer pair in the PCR. The influence of mismatches between the primer and the DNA template were studied and we found that, by using primers differing only from each other at the 3' end, cross-amplification of closely homologous alleles could be avoided. Thus, single base mismatches may be detected in the PCR and typing for HLA-DP gene variants, which differ for only one base, may be performed.

A standard microcytotoxicity technique for quantitative analysis of lymphocyte subsets. A comparison with indirect immunofluorescence, evaluated by microscopy or flow cytometry.

A standard complement-dependent microcytotoxicity (CDC) technique was used for quantitative analysis of T-lymphocyte subsets in human peripheral blood and the results compared to those obtained by indirect immunofluorescence microscopy and flow cytometry. The monoclonal antibodies OKT3, OKT4 and OKT8 were used in the CDC method for detection of total-T cells, T-helper and T-suppressor cells respectively. The CDC technique provided reproducible results (CV, 3-7%) correlating well with both immunofluorescence techniques. This observation was valid both for healthy persons (n = 21) and for patients (n = 10) with immunological disorders. The correct antibody dilution, correction for background and the use of eosin staining are considered critical for the usefulness of this technique. The method has several advantages: it is widely used for histocompatibility testing, only simple equipment is necessary, and the amount of monoclonal antibody required per test is small.

Competitive PCR for quantification of minimal residual disease in acute lymphoblastic leukaemia.

A very precise and reproducible polymerase chain reaction (PCR) method was developed in order to quantify minimal residual disease (MRD) in children with acute lymphoblastic leukaemia (ALL). A clone-specific competitor was constructed by introducing a restriction site in a PCR product identical to parts of the highly specific rearranged T-cell receptor delta (TCR-delta), T-cell receptor gamma (TCR-gamma), or immunoglobulin heavy chain (IgH) genes of the malignant clone. Using primers located externally to the restriction site the competitor and the DNA from the malignant clone will be amplified under identical conditions. After restriction enzyme cleavage, the PCR products originating from the competitor and the malignant clone can be distinguished by size in a gel electrophoresis step and the amount of residual disease can be determined. The method is very sensitive with a detection limit of at least one malignant cell in 10(5) normal cells. This method may be used for treatment stratification based on the early response to antileukaemic therapy.

Tumor necrosis factor alpha gene polymorphism in multiple sclerosis and optic neuritis.

The NcoI tumor necrosis factor (TNF alpha) polymorphism was studied in relapsing/remitting multiple sclerosis and monosymptomatic optic neuritis. The frequency of the NcoI marker phenotypes did not differ between healthy controls and the two disease groups. No extra or missing DNA fragments were observed in the disease groups when compared with controls.