The peptide-binding motif of HLA-DR8 shares important structural features with other type 1 diabetes-associated alleles.

The objective of this study was to characterize the peptide-binding motif of the major histocompatibility complex (MHC) class II HLA-DR8 molecule included in the type 1 diabetes-associated haplotype DRB1(*)0801-DQA1(*)0401/DQB1(*)0402 (DR8-DQ4), and compare it with that of other diabetes-associated MHC class II alleles; DR8-bound peptides were eluted from an HLA-DR homozygous lymphoblastoid cell line. The repertoire was characterized by peptide sequencing using a LTQ ion trap mass spectrometer coupled to a multidimensional liquid chromatography system. After validation of the spectra identification, the definition of the HLA-DR8 peptide-binding motif was achieved from the analysis of 486 natural ligands, based on serial alignments of all possible HLA-DR-binding cores. The DR8 motif showed a strong similarity with the peptide-binding motifs of other MHC class II diabetes-associated alleles, HLA-DQ8 and H-2 I-A(g7). Similar to HLA-DQ8 and H-2 I-A(g7), HLA-DR8 preferentially binds peptides with an acidic residue at position P9 of the binding core, indicating that DR8 is the susceptibility component of the DR8-DQ4 haplotype. Indeed, some DR8 peptides were identical to peptides previously identified as DQ8- or I-A(g7) ligands, and several diabetes-specific peptides associated with DQ8 or I-A(g7) could theoretically bind to HLA-DR8. These data further strengthen the association of HLA-DR8 with type I diabetes.

An endogenous processing pathway in vaccinia virus-infected cells for presentation of cytoplasmic antigens to class II-restricted T cells.

The recognition of virus-infected cells by class I MHC-restricted cytotoxic T cells requires endogenous processing of antigen for presentation. It is still unclear whether endogenous processing of antigen can be utilized by class II MHC molecules for presentation. To test this possibility, a human B cell line expressing HLA-A2 and HLA-DR1 was infected with a recombinant vaccinia virus expressing the Influenza A virus M1 matrix protein (VAC-M1) and was assayed for lysis by different M1-specific cytolytic T cell lines, restricted by either HLA-A2 or by HLA-DR1. Class II-restricted lysis of VAC-M1-infected cells did occur. This lysis required de novo M1 synthesis and was not due to exogenous antigen. Several properties of the endogenous processing pathway for class II-restricted presentation were different from those of the pathway utilized by class I molecules. First, class II-mediated recognition of VAC-M1 infected cells was less efficient, requiring higher doses of virus and longer infection times, than the class I-mediated recognition. Second, chloroquine completely blocked presentation of endogenous M1 to class II-restricted T cells but had no effect on the class I-restricted presentation. Third, the class II-restricted presentation of M1 was only mildly affected by Brefeldin A, a drug that prevents transport from the endoplasmic reticulum to the Golgi, whereas the class I-restricted presentation of M1 was completely abrogated by this drug. These data demonstrate the existence of an endogenous processing pathway for the presentation of cytosolic antigen by class II molecules and show that this pathway is distinct from the one used for presentation by class I molecules.

Alloreactive cytolytic T cell clones with dual recognition of HLA-B27 and HLA-DR2 antigens. Selective involvement of CD8 in their class I--directed cytotoxicity.

HLA-B27- responder cells were stimulated in vitro with HLA-B27.1+ lymphoblastoid cell lines, and alloreactive CTL clones were obtained by limiting dilution. Three of these clones specifically lysed B27.1+ targets. In addition, they also lysed homozygous DR2 targets with various degrees of efficiency, depending on the Dw specificity of the target cell. All three clones possessed a homogeneous CD3+,CD8+,CD4- phenotype and were also homogeneous upon subcloning. Cold-target inhibition analyses showed mutual inhibition of B27.1 target lysis by DR2 targets and vice versa. Lysis of B27.1 targets was selectively inhibited by anti-class I mAbs. In contrast, lysis of DR2 targets was inhibited only by anti-class II and anti-DR monomorphic antibodies, but not by anti-class I, anti-DQw1, or anti-DP antibodies. The results indicate that these clones display dual recognition for HLA-B27.1 and for HLA-DR2 and suggest that HLA-B27.1 may share at least one epitope that is closely related to some stimulatory Dw determinants present on the HLA-DR2 antigens. Lysis of both B27+ and DR+ targets was inhibited by an anti-CD3 mAb. In contrast, an anti-CD8 antibody selectively inhibited the B27- but not the DR2-directed killing by these clones. The data support a stabilizing role of CD8 through its binding to the same class I (but not class II) molecule on the target cell bound by the T cell antigen receptor.

Structural requirements for pairing of alpha and beta chains in HLA-DR and HLA-DP molecules.

To test for the assembly of human MHC class II molecules having an alpha chain from one isotype (HLA-DR, -DQ, or -DP) and the beta chain of another (mixed isotypic pairs), murine fibroblasts were transfected with expressible cDNAs encoding the different class II alpha and beta chains. A rapid and efficient transient transfection system was developed using a polyoma virus-based vector. Typically, 30-50% of cells transfected using this system expressed high levels of class II molecules on their surface, but only with matched isotypic pairs. Biochemical analysis of cells transfected with matched or mixed isotypic pairs of the DR and DP molecules revealed that only matched chains could pair efficiently inside the cell. Thus, the lack of expression of the two mixed isotypic pairs is due to inefficient primary assembly of the class II molecule and not to a processing or transport defect. To define what region of the beta chains controlled their assembly with alpha chains, a series of chimeric cDNA molecules containing both DR and DP beta chain sequences were constructed. Expression of these chimeric beta chains with DR and DP alpha chains was determined by cytofluorimetry and biochemical analysis. Both alpha chains paired with beta chains in which only the beta 1 domain was isotypically matched. In contrast, the pattern of expression of chimeras made at other points within the beta 1 domain was different for DR and DP. These data show that different areas of primary sequence are important for the assembly of different human class II isotypes, and suggest that HLA-DR and -DP molecules have different secondary or tertiary structures in their NH2-terminal domains.

A myelin basic protein peptide is recognized by cytotoxic T cells in the context of four HLA-DR types associated with multiple sclerosis.

We have examined previously the peptide specificity of the T cell response to myelin basic protein (MBP) in patients with multiple sclerosis (MS) and healthy controls, and demonstrated that an epitope spanning amino acids 87-106 was frequently recognized. Because this region is encephalitogenic in some experimental animals, it has been postulated that the response to the epitope may have relevance to MS. In this study, the fine specificity of this response is studied using four well-characterized, monospecific T cell lines from three MS patients and an identical twin of a patient. Each of the lines recognized a peptide with the same core sequence, amino acids 89-99, although the responses were affected to various degrees by truncations at the COOH- or NH2 terminal ends of the 87-106 epitope. Importantly, the epitope was recognized in conjunction with four different HLA-DR molecules. Also, the T cell receptor beta chain usage was heterogeneous, and each line expressed a different VDJ sequence. The four HLA-DR molecules restricting the response to this epitope have been shown to be overrepresented in MS populations in various geographic areas, suggesting that the response to this region of the MBP molecule may be relevant to the pathogenesis of MS. These findings may have important implications in designing therapeutic strategies for the disease.

Influx of recent thymic emigrants into autoimmune thyroid disease glands in humans.

Autoimmune thyroid diseases (AITD) are considered as prototypic organ-specific autoimmune diseases, yet their underlying aetiology remains poorly understood. Among the various pathophysiological mechanisms considered, a failure of central tolerance has received little attention. Here we present evidence in favour of dysregulated thymic function playing a role in AITD. Flow-cytometric analyses conducted in peripheral blood lymphocytes from 58 AITD patients and 48 age- and-sex-matched controls showed that AITD patients have significantly higher blood levels of CD4(+)CD45RA(+), CD4(+)CD31(+) and CD4/CD8 double-positive T lymphocytes, all markers of recent thymic emigrants (RTE). In addition, the alpha-signal joint T cell receptor excision circles (TRECs) content (a molecular marker of RTEs) was higher in the group of AITD patients older than 35 years than in age-matched controls. This was independent from peripheral T cell expansion as assessed by relative telomere length. Comparisons of TREC levels in peripheral blood lymphocytes and intrathyroidal lymphocytes in paired samples showed higher levels within the thyroid during the initial 30 months of the disease, indicating an influx of RTE into the thyroid during the initial stages of AITD. Additionally, a lack of correlation between TREC levels and forkhead box P3 expression suggests that the intrathyroidal RTE are not natural regulatory T cells. These results uncover a hitherto unknown correlation between altered thymic T cell export, the composition of intrathyroidal T cells and autoimmune pathology.

Effect of glatiramer acetate (Copaxone) on the immunophenotypic and cytokine profile and BDNF production in multiple sclerosis: a longitudinal study.

We assessed the effect of glatiramer acetate (GA) on the immunophenotypic and cytokine profile and the BDNF production by peripheral blood mononuclear cells, and their association with the clinical response in 19 naïve-treated MS patients prospectively followed-up after GA therapy. Two patients withdrew the therapy. After a median follow-up of 21 months, twelve were considered responders and five as non-responders. Non-responder patients had significant longer disease duration and a higher EDSS score at baseline. In the responder group, a significant decrease in the percentage of INF-gamma producing total lymphocytes, CD4+ and CD8+ T cells, and reduced percentage of IL-2 producing CD4+ and CD8+ T cells were observed at 12, 18 and 24 months. These changes were associated with a significant increase in the percentage of CD3+, CD4+ and CD4(+) CD45RA(+) T cells, and BDNF production from month 6 that remained significant throughout the study. We did not observe significant changes in the nonresponder group for any of the parameters studied. Our data suggest that GA treatment induces a downmodulation of proinflammatory cytokines associated with the regulation of the peripheral T cell compartment and with increased production of BDNF that might be related to the clinical response.

Tissue-specific self-peptides bound by major histocompatibility complex class I molecules of a human pancreatic beta-cell line.

The process of beta-cell destruction in IDDM is mediated, in part, by CD8+ T-cells. Structural characterization of HLA-I-bound self-peptides presented by the human beta-cell line HP-62 was performed to identify possible tissue-specific autoantigens in the context of CD8+ T-cell/HLA-I interactions. The sequences of the beta-cell line HLA-I-bound peptides were compared with sequence databases. Six of the obtained sequences showed homology to known precursor proteins, three of which--GLUT2 receptor, phosphatidylinositol-glycan-specific phospholipase D, and 5-hydroxytryptamine-1F receptor--have a limited, tissue-specific expression. These HLA-bound self-peptides may be part of a pool of autoantigens recognized by beta-cell reactive cytotoxic T-cells.

Expression of transporter associated with antigen processing-1 in the endocrine cells of human pancreatic islets: effect of cytokines and evidence of hyperexpression in IDDM.

A possible role of transporter associated with antigen processing (TAP)-1 in the pathogenesis of IDDM has been investigated by examining the level of TAP-1 expression in the islets of IDDM pancreas and by studying in vitro the effect of interferon (IFN)-gamma, IFN-alpha, and tumor necrosis factor-alpha in TAP-1 expression by cultured islet cells. A remarkable hyperexpression of TAP-1 has been found in the endocrine cells (beta and non-beta) of IDDM islets, which constitutes first evidence of hyperexpression of this molecule in the target organ of an autoimmune disease. TAP-1 hyperexpression correlated clearly with HLA class I hyperexpression but only very partially with HLA class II ectopic expression. IFN-gamma and IFN-alpha, both cytokines putatively implicated in IDDM pathogenesis, were capable of inducing TAP-1 protein (as assessed by immunofluorescence flow cytometry) and message (by Northern blot analysis and reverse transcription polymerase chain reaction). These findings suggest that under the influence of cytokines (most probably IFN-alpha) beta-cells may express in their surface a high density of HLA class I-peptide complexes that may facilitate their recognition and lysis by low-affinity CD8+ T-cells.

Evolution of brain-derived neurotrophic factor levels after autologous hematopietic stem cell transplantation in multiple sclerosis.

A neuroprotective role of inflammation has been suggested based on that immune cells are the main source of brain-derived neurotrophic factor (BDNF). We investigated the 3-year evolution of BDNF levels in serum, CSF and culture supernatant of peripheral blood mononuclear cells (PBMC), unstimulated and stimulated with anti-CD3 and soluble anti-CD28 antibodies, in 14 multiple sclerosis patients who underwent an autologous hematopoietic stem cell transplantation (AHSCT). BDNF levels were correlated with previously reported MRI measures that showed a reduction of T2 lesion load and increased brain atrophy, mainly at first year post-transplant. A significant decrease of serum BDNF levels was seen at 12 months post-transplant. BDNF values were found significantly lower in stimulated but not in unstimulated PBMC supernatants during the follow-up, supporting that AHSCT may induce a down-regulation of BDNF production. The only significant correlation was found between CSF BDNF levels and T2 lesion load before and 1 year after AHSCT, suggesting that BDNF reflects the past and ongoing inflammatory activity and demyelination of these highly active patients. Our study suggests that AHSCT can reduce BDNF levels to values associated with lower activity. This decrease does not seem to correlate with the brain atrophy measures observed in the MRI.

On the relative immunogenicity of DR alloantigens: T cell recognition of HLA-DR2a and HLA-DR2b.

The HLA-DR2 haplotype encodes two highly polymorphic DR molecules, DR2a and DR2b. Because little is known regarding the relative immunogenicity of different HLA-DR molecules, we have studied the T-cell recognition of DR2a and DR2b molecules from the DRw15, Dw2 haplotype. A series of DR2-specific alloreactive T-cell clones were analyzed with murine L-cell transfectants expressing either the DR2a or the DR2b molecules as stimulator cells in proliferation assays. Somewhat surprisingly, both DR2a and DR2b were capable of stimulating DR2-specific T-cell clones with equal magnitude and similar frequency. In addition, DR2a and DR2b are functionally distinct, that is, no clone was identified which was stimulated by both DR2a and DR2b molecules.

HLA-Dw specificity assignments are independent of HLA-DQ, HLA-DR, and other class II specificities and define a biologically important segregant series which strongly activates a functionally distinct T cell subset.

Several lines of evidence indicate that HLA-Dw, as defined by HTC typing, is not the result of the combined stimulatory effect of HLA-DR and DQ. Therefore, responder cells do not have to share HLA-DQ antigens with the stimulator HTCs to give a typing response. The common HLA-DR-DQ associations observed in HTCs correspond to different patterns of linkage disequilibrium in different populations. HLA-DQ and HLA-Dw are functionally heterogeneous. Although HLA-DQ molecules may play a role in primary stimulation, this role is distinct from that of Dw determinants which have strong lymphocyte activating properties. The role of the HLA-DQ determinants on the other hand, is one of modulating the total T cell response by controlling the proliferation of suppressor and cytotoxic cells. The primary MLC response is the result of the proliferative effect of HLA-Dw, DR, DP, and other associated determinants, in conjunction with a modulatory effect of DQ molecules. However, HLA-Dw (as detected by HTC typing) are DR associated determinants which are immunodominant in primary MLR. The genes of the HLA-DR subregion have been named DR by the WHO nomenclature committee. This subregion encodes the HLA-DR specificities and the DRw52 and DRw53 determinants. Unfortunately this nomenclature does not take into account the need to define the genetic basis of the HLA-Dw determinants--whether they are encoded by separate genes within the HLA-DR subregion or whether they are encoded by as yet unspecified genes in the HLA class II region in linkage disequilibrium with HLA-DR DRw52/53. There are at least three and possibly four beta chain genes in the HLA-DR subregion, all in strong linkage disequilibrium with each other. Some of these are expressed in most haplotypes while others are not; some behave as pseudogenes in some haplotypes and in others, all the genes are expressed. All the genes of the class II region have not been fully characterized. HLA-Dw determinants may be specified by one or more of these genes. When more information becomes available, the genetic and molecular basis of the HLA-Dw series as well as the functional heterogeneity and antigenic strength of the various class II determinants will be better understood.

Efficient cDNA expression vectors for stable and transient expression of HLA-DR in transfected fibroblast and lymphoid cells.

cDNA expression vectors with several useful features were constructed. First, the long terminal repeat of Rous sarcoma virus was used as a promoter to obtain high levels of expression in various cells of human and mouse origin. Second, cis-linked expression units that confer resistance either to mycophenolic acid or the neomycin analog G418 were inserted to facitate the isolation of transfected cells expressing the cDNA of interest. Third, by replicating in simian COS cells, these vectors can be used for efficient transient expression. cDNA fragments encoding the DR alpha or DR beta chains of human class II major histocompatibility complex antigens were inserted into these vectors and high levels of cell surface HLA-DR antigen were obtained after cotransfection into mouse and human fibroblasts. These vectors were also successfully used to correct the inability of a class II-negative B cell line, derived from a patient with a congenital immunodeficiency, to present peptide antigen to DR-restricted T cells.

Recombinant CD4-Pseudomonas exotoxin hybrid protein displays HIV-specific cytotoxicity without affecting MHC class II-dependent functions.

The present study describes several in vitro activities of CD4(178)-PE40, a recombinant protein containing a portion of human CD4 linked to active regions of Pseudomonas aeruginosa exotoxin A. Using assays for cell viability, we demonstrate that the hybrid toxin displays highly selective cytotoxicity for HIV-infected T lymphocytes. In a latently infected human T-cell line which is inducible for HIV expression, toxin sensitivity is observed only upon virus induction. At concentrations which readily kill HIV-infected T cells, CD4(178)-PE40 has no observable cytotoxic effects on uninfected human cell lines expressing surface major histocompatibility complex (MHC) Class II molecules, and does not interfere with cellular responses known to be dependent on functional association between CD4 and MHC Class II molecules.

HLA and rheumatoid arthritis: susceptibility or severity?

An analysis of data collected on 440 British Caucasoid rheumatoid arthritis patients has confirmed positive association with HLA-DR4, Dw4, DRw53, and A2 and negative associations with HLA-DR2, 3, and 7. HLA-DR4 is more associated with RA 'severity' than with RA 'susceptibility', when measured by the parameters of ARA classification, seropositivity, severity of erosions and extra-articular manifestations. The association between HLA-A2, Cw3, Bw62, DR4, DRw53, and Dw4 and extra-articular disease has been confirmed in this study. The analysis of HLA and RA severity with respect to sex showed high frequencies of DR4, Dw4, and DRw53 in females, which increased in those with severe erosions, seropositivity or extra-articular disease. In males with RA, the disease appears to be associated not only with DR4, Dw4 and DRw53, but also with A2, Cw3 and Bw62. However, no significant differences in these antigen frequencies were found between male patients with severe RA and those without. Despite a significant decrease in the frequencies of DR3, B8 and A1 in most RA patient subsets, RA patients with Sjogren's syndrome showed a marked increase of A1 and B8 and patients with auto-antibodies had a significant increase in HLA-DR3 frequency when compared with patients without these features.

Intra HLA-D region recombinant maps HLA-DR between HLA-B and HLA-D.

A consanguineous family has been typed for HLA-A, B, C, D, DR and GLO, Bf, C2 and C4 and other red cell markers. The results indicate that an expected Dw7 homozygous sibling is a "Dw1"/Dw7 recombinant, probably derived from a crossing over between DR and D on the paternal haplotypes. The anomalous typings by Dw1 HTCs of the recombinant haplotype are best explained by the likely presence of an additional Lad polymorphism which could be a serologically detectable second epitope on the same molecule as the HLA-D determinant or in the form of two linked chains, one encoded by HLA-D and one by another Lad gene in the HLA haplotype.