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Biomedical subjects

D A Hafler

Publications and source records attributed to D A Hafler.

At least 19 recordsLinked to original sources
PubMed2006

The role of inflammatory bowel disease susceptibility loci in multiple sclerosis and systemic lupus erythematosus.

To date, three loci have been validated to confer susceptibility to inflammatory bowel disease (IBD): the CARD15/NOD2 gene, the discs large homolog 5 gene (DLG5), and the IBD5 locus on 5q31 (IBD5). We have explored the possibility that these loci may also be associated with susceptibility to two other chronic inflammatory diseases, multiple sclerosis (MS) and systemic lupus erythematosus (SLE). As the CARD15 risk alleles had previously been assessed in our collection of 496 MS trios, we focused our efforts on the DLG5 risk allele and the IBD5(risk) haplotype (IBD5(risk)) for MS. While there is no evidence of association within our MS sample with either of these polymorphisms, screening of 1027 subjects with SLE suggests that IBD5(risk) may have a modest contribution to disease risk in the subset of SLE subjects without lupus nephritis. In addition, a pooled analysis of existing published and unpublished data in 1305 cases of SLE genotyped for the CARD15 risk alleles suggests that only the CARD15(908R) IBD risk allele may have a strong effect on risk of SLE. Our data, therefore, suggest that both the CARD15 gene and the IBD5 locus may have a role as general susceptibility loci for certain common, genetically complex inflammatory diseases.

Chromosomes, Human, Pair 5↗
PubMed2003

In vitro evidence that subcutaneous administration of glatiramer acetate induces hyporesponsive T cells in patients with multiple sclerosis.

Glatiramer acetate (GA; Copaxone) is a random sequence polypeptide used in the treatment of relapsing remitting multiple sclerosis (RR MS). We have recently demonstrated that prior to treatment, GA induces proliferation of resting T cells and is not cross-reactive with myelin antigens. Daily GA injections induce a significant loss of this GA responsiveness, which is associated with the induction of highly cross-reactive Th2-type T cells potentially capable of suppressing inflammatory responses. The mechanism of action by which GA induces T cell nonresponsiveness leading to T cell receptor degeneracy in patients with RR MS is unknown. Here, we examined the effects of daily GA administration on the induction of T cell hyporesponsiveness. The frequency of GA-reactive T cells in peripheral blood of seven patients with RR MS was measured by limiting dilution analysis prior to and during 6 months of treatment. In addition, a model in which GA-reactive T cells were stimulated in vitro was developed to better characterize the selection of T cell populations over time. In vivo treatment with GA induced a decrease in GA-reactive T cell frequencies and hyporesponsiveness of CD4(+) T cell reactivity to GA in vitro that was only partially reversed by the addition of IL-2. These data suggest that T cell peripheral tolerance to GA was achieved in vivo during treatment. Thus, our in vitro data suggest that the underlying changes in GA-reactive CD4(+) T cell reactivity could be explained by the induction of T cell anergy and clonal elimination.

Adult↗
PubMed2001

Immunological memory: contribution of memory B cells expressing costimulatory molecules in the resting state.

Traditionally, emphasis has been placed on the roles of Th cells in generating and amplifying both cellular and humoral memory responses. Little is known about the potential contributions of B cell subsets to immunological memory. Resting memory B cells have generally been regarded as poor APC, attributed in part to the relative paucity of costimulatory molecules identified on their surface. We describe a novel subpopulation of human memory B cells that express CD80 in their resting state, are poised to secrete particularly large amounts of class switched Igs, and can efficiently present Ag to and activate T cells. This functionally distinct B cell subset may represent an important mechanism by which quiescent human B cells can initiate and propagate rapid and vigorous immune memory responses. Finally, these studies extend recent observations in the murine system and highlight the phenotypic and functional diversity that exists within the human B cell memory compartment.

Antigen Presentation↗
PubMed2001

In vitro evidence that immunuaffinity-purified MOG contains immunogenic quantities of contaminating mouse IgG; techniques for producing Ig-free MOG.

Myelin oligodendrocyte glycoprotein (MOG) has attracted considerable attention due to its possible role in multiple sclerosis (MS). Previous studies have shown that MOG-reactive T cells are more commonly detected in MS patients than controls. In this report, we studied human MOG-reactive T cell clones generated from healthy individuals as well as MS patients. Our results indicate that immunoaffinity-purified MOG, which was routinely used in prior studies, is contaminated by anti-MOG antibody (mouse IgG), despite the inability to detect IgG by Western blotting. Here, we used continuous SDS-PAGE, which enabled us to isolate highly purified MOG devoid of contaminating mouse IgG.

Animals↗
PubMed2001

CD4+CD25high regulatory cells in human peripheral blood.

Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD(+)CD25(+) T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4(+) population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4(+)CD25(+) regulatory cells isolated in mice. With TCR cross-linking, CD4(+)CD25(high) cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4(+)CD25(-) responder T cells in a contact-dependent manner. The CD4(+)CD25(high) regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RB(low) on murine CD4(+)CD25(+) regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4(+)CD25(high) regulatory cells and CD4(+)CD25(-) responder cells. Whereas higher ratios of CD4(+)CD25(high) T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.

Abatacept↗
PubMed2001

Molecular mimicry in Lyme arthritis demonstrated at the single cell level: LFA-1 alpha L is a partial agonist for outer surface protein A-reactive T cells.

Antibiotic treatment-resistant Lyme arthritis is a chronic inflammatory joint disease that follows infection with Borrelia burgdorferi (BB:). A marked Ab and T cell response to BB: outer surface protein A (OspA) often develops during prolonged episodes of arthritis. Furthermore, cross-reaction between the bacterial OspA and human LFA-1alpha(L) at the T cell level and the inability to detect BB: in the joint implicate an autoimmune mechanism. To analyze the nature of response to OspA and LFA-1alpha(L), we used OspA-specific T cell hybrids from DR4 transgenic mice, as well as cloned human cells specific for OspA(165-184), the immunodominant epitope, from five DRB1*0401(+) patients, using OspA-MHC class II tetramers. Although OspA(165-184) stimulated nearly all OspA-specific human T cell clones tested to proliferate and secrete IFN-gamma and IL-13, LFA-1alpha(L326-345) stimulated approximately 10% of these clones to proliferate and a greater percentage to secrete IL-13. Assays with LFA- or OspA-DR4 monomers revealed that higher concentrations of LFA-DR4 were needed to stimulate dual-reactive T cell hybrids. Our analysis at the clonal level demonstrates that human LFA-1alpha(L326-345) behaves as a partial agonist, perhaps playing a role in perpetuating symptoms of arthritis.

Animals↗
PubMed2001

Uncoupling p70(s6) kinase activation and proliferation: rapamycin-resistant proliferation of human CD8(+) T lymphocytes.

Rapamycin is a fungal macrolide that inhibits the proliferation of T cells. Studies in both animals and humans have found that rapamycin significantly reduces graft rejection. However, though CD8(+) T cells are involved in graft infiltration and rejection, little is known regarding the effects of rapamycin on CD8(+) human T cell responses. In this study, we examined the mechanism of rapamycin-induced inhibition of Ag-driven activation of CD8(+) T cells. Surprisingly, a heterogeneous proliferative response in the presence of rapamycin was observed among different Ag-specific CD8(+) T cell clones; this was also observed in CD8(+) peripheral blood T cells activated with TCR cross-linking ex vivo. Inhibition of T cell proliferation by rapamycin was controlled by both the strength of signal delivered through the Ag receptor as well as the specific costimulatory signals received by the T cell. Rapamycin-resistant proliferation occurred despite inhibition of p70(s6) kinase activity. Moreover, rapamycin-resistant proliferation of the CD8(+) T cell clones was blocked by anti-IL-2 Abs, suggesting that while some of the parallel pathways triggered by IL-2R signaling are sensitive to the effects of rapamycin, others account for the Ag-driven rapamycin resistance. These data provide a new framework for examining the specific mechanism of action of rapamycin in human disease.

Antibodies, Monoclonal↗
PubMed2001

Decreases in interleukin-4 secretion by invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells in peripheral blood of patientswith relapsing-remitting multiple sclerosis.

The cytokine profile of invariant CD4(-)CD8(-)V alpha 24J alpha Q T cells from patients with multiple sclerosis (MS) was compared with that of healthy controls. CD4(-)CD8(-)V alpha 24(+) T cells from the peripheral blood of 12 patients with relapsing-remitting MS (RR-MS), 5 patients with progressive MS (CP-MS), and 9 control individuals were directly sorted into single wells and expanded in vitro for analysis of IL-4 and IFN-gamma secretion; 315 V alpha 24J alpha Q T cell clones were generated and their T cell receptor (TCR) sequenced. T cell functionality was determined by examining cytokine secretion upon TCR cross-linking. RR-MS patients exhibited lower frequencies of IL-4 secreting CD4(-)CD8(-)V alpha 24J alpha Q T cell clones than patients with CP-MS and controls. No differences in IFN-gamma secretion were observed between the groups. An IL-4 positive cytokine profile could be correlated to the cloning efficiency of the V alpha 24J alpha Q T cells. We conclude that alterations in cytokine secretion patterns of CD4(-)CD8(-)V alpha 24J alpha Q T cells may influence the immune system and thus contribute to relapsing-remitting MS.

Adult↗
PubMed2001

The neuroimmunology of multiple sclerosis: possible roles of T and B lymphocytes in immunopathogenesis.

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system white matter. The association of the disease with MHC genes, the inflammatory white matter infiltrates, similarities with animal models, and the observation that MS can be treated with immunomodulatory and immunosuppressive therapies support the hypothesis that autoimmunity plays a major role in the disease pathology. Evidence supports activated CD4+ myelin-reactive T cells as major mediators of the disease. In addition, a renewed interest in the possible contribution of B cells to MS immunopathology has been sparked by nonhuman primate and MS pathological studies. This review focuses on the immunopathology of MS, outlining the hypothetical steps of tolerance breakdown and the molecules that play a role in the migration of autoreactive cells to the CNS. Particular focus is given to autoreactive T cells and cytokines as well as B cells and autoantibodies and their role in CNS pathogenesis in MS.

B-Lymphocytes↗
PubMed2001

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure.

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.

Amino Acid Sequence↗
PubMed2001

Heterophile antibodies indicate progression of autoimmunity in human type 1 diabetes mellitus before clinical onset.

We previously reported serum cytokines in a group of long term non-progressors to Type 1 diabetes; this reactivity detected in ELISA is now identified as heterophile antibody in some sera. Here, we characterize heterophile antibody activity. A 14 kDa-polypeptide from heterophile antibody containing serum bound to an anti-IL-4 column, but IL-4 was not detected by Western blot or by MS/MS sequencing. However, in 2/13 heterophile antibody positive sera, T-cell growth was potentiated and was blocked by an anti-human immunoglobulin. To examine the relationship between low affinity heterophile antibody presence and disease progression, 1100 archived serum samples were analyzed with two pairs of antibodies from 443 diabetes-free first degree relatives of Type 1 diabetes mellitus patients for heterophile antibody; 95 individuals developed diabetes on follow-up. Twenty-two individuals, whose serum was heterophile antibody positive with the second pair of antibodies (but negative with the first pair of antibodies), had a significantly higher incidence of developing diabetes after five years. Thirty-seven individuals with heterophile antibody reactivity with the first pair of antibodies, regardless of reactivity with the second pair of antibodies, had a significantly lower incidence of developing diabetes. While we cannot exclude the presence of genuine cytokine in all sera, these data indicate the presence of distinct groups of heterophile antibodies in patients at high risk to develop diabetes. Thus, anti-Ig heterophilic antibodies with different immunochemical reactivities are linked to the progression of or protection from Type 1 diabetes autoimmunity.

Amino Acid Sequence↗
PubMed2000

Human and murine CD4 T cell reactivity to a complex antigen: recognition of the synthetic random polypeptide glatiramer acetate.

The capacity of glatiramer acetate (GA), a random copolymer of alanine, lysine, glutamic acid, and tyrosine to stimulate primary in vitro human and murine T cell proliferation was examined. PBMCs isolated from healthy humans and relapsing remitting multiple sclerosis patients and spleen cells from inbred strains of mice, expressing different H-2 haplotypes, were used as sources of non-GA-primed lymphocytes. GA functioned as a universal Ag, inducing dose-dependent proliferation of all non-GA-primed human and murine T cell populations tested. Moreover, GA stimulated PBMCs derived ex vivo from human cord blood, strongly suggesting that GA can activate both naive and memory T cells. The human T cell proliferative responses to GA were HLA class II DR-restricted by virtue of the ability of anti-class II Ab to inhibit T cell proliferation, and the demonstration that individual GA specific human T cell clones were HLA class II DR-restricted by either restriction element but not both. Furthermore, GA-reactive T cells secreted Th0 cytokines and expressed a diverse repertoire of TCR. Limiting dilution analysis indicated that the T cell precursor frequency among the healthy human adults tested ranged from 1:5,000 to 1:125,000. Given that all of the T cell populations tested were isolated from non-GA-primed donors, it appears that virtually all humans and murine strains contain significant numbers of T cell populations cross-reactive with GA. These findings may explain the recent clinical finding that daily s.c. administration of GA ameliorates the progression of multiple sclerosis.

Adult↗
PubMed2000

Examination of CD8+ T cell function in humans using MHC class I tetramers: similar cytotoxicity but variable proliferation and cytokine production among different clonal CD8+ T cells specific to a single viral epitope.

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11-19-reactive CD8(+) T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11-19-reactive T cell clones by single cell sorting of HLA-A2/Tax11-19 tetramer binding CD8(+) T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8(+) T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.

Antigens, Viral↗
PubMed2000

Direct enumeration of Borrelia-reactive CD4 T cells ex vivo by using MHC class II tetramers.

We characterized antigen-specific CD4(+) T cells in six patients with treatment-resistant Lyme arthritis, using an HLA-DRB1*0401 major histocompatibility complex (MHC) class II tetramer covalently loaded with OspA(164-175), an immunodominant epitope of Borrelia burgdorferi. Direct analysis of OspA-tetramer binding CD4(+) cells in patients expressing the HLA-DRB1*0401 allele revealed frequencies of between <0.005 and 0.1% in peripheral blood (n = 6), and between <0.005 and 3.1% in synovial fluid (n = 3). OspA-tetramer(+)CD4(+) cells were directly cloned at 1 cell per well and expanded by mitogen and IL-2 on allogeneic feeder cells. As measured by [(3)H]thymidine incorporation, 95% of 168 T cell clones from synovial fluid binding the OspA-tetramer were antigen-reactive. Clones generated from peripheral blood revealed a different pattern of responsiveness when compared with clones generated from synovial fluid, as measured by proliferation, IFN-gamma, and IL-13 secretion. These clones, selected on the basis of their peptide binding, also responded to whole protein, but with a different cytokine profile. Our studies demonstrate that MHC class II tetramers can be used in humans to directly identify, isolate, and characterize antigen-reactive T cells from an inflammatory compartment.

Antigens, Surface↗
PubMed2000

Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes.

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

Diabetes Mellitus, Type 1↗
PubMed2000

A novel population of B7-1+ T cells producing intracellular IL-4 is decreased in patients with multiple sclerosis.

We identified a novel population of human T cells, studied directly ex vivo, that co-express surface B7-1 and intracellular IL-4. These peripheral blood B7-1+/CD4+ T cells expressed cell surface molecules associated with differentiation including CD45RO and MHC class II, yet were CD69(-) and CD25(-). In short-term cultures, T cell receptor (TCR) cross-linking induced further IL-4 production with little IFN-gamma or TNF-alpha. In marked contrast, CD4+ T cells negative for B7-1 expressed intracellular IFN-gamma and high amounts of TNF-alpha but little IL-4 upon TCR cross-linking. The CD4+/B7-1+/IL-4-expressing T cells were of polyclonal origin based on their diverse TCR repertoire. To explore the biological significance of this B7-1+/IL-4+ T cell population and to assess its potential regulatory role in autoimmune disease, we examined whether these T cells isolated ex vivo were altered in subjects with multiple sclerosis (MS). While the frequency of B7-1+ T cells was enhanced in patients with MS as compared to normal subjects, there was a significant diminution of B7-1+/IL-4+ T cells in the patients. The decrease in these IL-4-producing T cells in patients with autoimmune disease is consistent with a possible role as immunoregulatory T cells.

Antigens, CD↗