Prognostic irrelevance of HLA-G in B-cell chronic lymphocytic leukemia.

In the last few years, it has been suggested that the involvement of human leukocyte antigen-G (HLA-G) in several tumoral processes and its likely participation as a factor of immune tolerance in malignant cells. Recently, positive HLA-G surface expression has been associated with a poor prognosis in a small group of patients with B-cell chronic lymphocytic leukemia (B-CLL), a lymphoproliferative disorder characterized by a heterogeneous clinical course. In the present work, 169 patients suffering from B-CLL were analyzed for the expression of HLA-G by flow cytometry in order to verify its prognostic value in a larger cohort. We observed a low expression of this molecule on leukemic B cells and no significant relation to clinical data or progression-free survival time, indicating that this molecule is not as good immunologic prognostic marker for B-CLL as suggested.

Interleukin-10 promoter polymorphisms in patients with systemic lupus erythematosus from the Canary Islands.

The purpose of this study was to examine whether several allelic variants in the polymorphic interleukin (IL)-10 promoter region were related with an increased risk of developing systemic lupus erythematosus (SLE) in Spanish patients from Canary Islands. Microsatellites (MS) at positions -4000 and -1200 (IL10R and IL10G, respectively) and single nucleotide polymorphisms (SNPs) (MS) at positions -1082G/A, -819C/T and -592C/A of the IL-10 promoter were analysed in patients with SLE and healthy controls from Canary Islands (Spain). We found that SNPs but not MS were associated with SLE. The GCC haplotype frequency was significantly higher in SLE patients (0.43) than in healthy donors (0.33) [P = 0.02; OR = 1.50 (95% CI = 1.06-2.14)], whereas the ACC haplotype was less represented in patients (0.28 vs. 0.37) [P = 0.02; OR = 0.64 (95% CI = 0.44-0.92)]. To assess the functional role of genotypes, serum IL-10 levels from patients and controls were quantified by ELISA. Also, the lipopolysaccharide-induced IL-10 secretion by monocytes from healthy controls was evaluated in vitro. Serum IL-10 levels were higher in patients [median (interquartile range) = 2.8 pg/mL (1.8-4.2)] than in controls [0.9 pg/mL (0-3.5)] (P = 0.02), but no association was observed between serum IL-10 levels or lipopolysaccharide-induced IL-10 secretion and the IL-10 promoter haplotypes. These data suggest that the IL-10 promoter haplotype that produces higher levels of cytokine is associated with SLE in patients from Canary Islands.

A sustained activation of PI3K/NF-kappaB pathway is critical for the survival of chronic lymphocytic leukemia B cells.

The progressive rise of mature CD5+ B lymphocytes, despite the low proportion of proliferating cells, has led to the notion that B cell chronic lymphocytic leukemia (B-CLL) is primarily related to defective apoptosis. The microenvironment likely plays a prominent role because the malignant cells progressively accumulate in vivo, whereas they rapidly undergo spontaneous apoptosis when cultured in vitro. To assess microenvironment-mediated survival signals, B-CLL cells were cultured with a murine fibroblast cell line, Ltk-, with and without an agonistic antibody to CD40. Spontaneous apoptosis was associated with the loss of Akt and NF-kappaB activities. Interactions with fibroblasts sustained a basal level of Akt and NF-kappaB activities, which was dependent on phosphatidylinositol-3 kinase (PI3K). Constitutive activity of the PI3K pathway in B-CLL cells when cultured with fibroblasts prevented the downregulation of the prosurvival Bcl-2 family protein Bcl-xL and the caspase inhibitor proteins FLIPL and XIAP, and consequently caspase-3 activation and apoptosis. CD40 crosslinking in B-CLL cells did not further prevent murine fibroblasts-mediated apoptosis but induced cell proliferation, which was associated with an increase of Akt and NF-kappaB activation compared with cells cultured with fibroblasts alone. The PI3K pathway seems to play a pivotal role in B-CLL cell survival and growth.

The dinucleotide repeat polymorphism in the 3'UTR of the CD154 gene has a functional role on protein expression and is associated with systemic lupus erythematosus.

OBJECTIVE: To investigate the association of the (CA)n dinucleotide repeat in the 3' untranslated region (3'UTR) of the CD154 gene with systemic lupus erythematosus (SLE), and its functional role in protein expression. METHODS: The allelic and genotypic distributions of the polymorphism were compared in 80 patients with SLE and 80 controls. A complete clinical and analytical database was recorded in each patient in order to correlate the clinical manifestations in SLE with different alleles. To investigate the functional role of the polymorphism, the CD154 protein expression on activated lymphocytes from healthy homozygous controls was evaluated by flow cytometry. RESULTS: The 24 CA allele was the most represented in controls (p = 0.029), whereas the alleles containing >24 CA repeats were found in patients (p = 0.0043). Furthermore, when only homozygous women were considered, most controls carried two 24 CA alleles (p = 0.041), whereas most patients carried two alleles containing >24 CA repeats (p = 0.032). Also, patients carrying at least one 24 CA allele had less neurological involvement (p = 0.034), and carriers of at least one allele with fewer than 24 CA repeats presented more livedo reticularis (p = 0.006) and anti-Sm (p = 0.01) and anti-RNP (p = 0.038) autoantibodies. CD154 maximum expression in activated lymphocytes from all controls was reached after 54 hours, but it was more prolonged in controls carrying two alleles with >24 CA repeats (p = 0.0068). CONCLUSION: The CD154 3'UTR microsatellite is associated with SLE, and the most represented alleles in patients were accompanied by a more prolonged protein expression in activated lymphocytes from controls.

IL4-R1 (5q31-q33) and FcepsilonRI-betaca (11q13) markers and atopy: a case/control study in a spanish population.

BACKGROUND: Rhinoconjunctivitis and bronchial asthma are atopic diseases with a high prevalence in the Canary Islands (Spain). Given that the most prevalent allergen is the house-dust mite Dermatophagoides pteronyssinus, early detection of genetically susceptible subjects would allow the application of preventive measures. The objective was to investigate the possible association of IL4-R1 (chromosome 5q31-q33) and FcepsilonRI-betaca (chromosome 11q13) markers with the atopic disease in our population. METHODS: We performed a case/control study in which patients were recruited on the basis of diagnosis of rhinoconjunctivitis and/or bronchial asthma, and positive skin prick test to D. pteronyssinus. Analysis of IL4-R1 and FcepsilonRI-betaca microsatellites was carried out by PCR and electrophoresis in acrylamide gels. RESULTS: We have not found evidence of association between IL4-R1 and FcepsilonRI-betaca markers and atopic disease in our population. In addition, these markers have shown a high percentage of homozygosis. CONCLUSIONS: IL4-R1 and FcepsilonRI-betaca markers have not proved to be useful genetic markers for linkage or association studies in our population.

CD154 polymorphism in Spanish populations. Differences in the allelic distribution between Canary islanders and Peninsulars.

The CD154 gene contains a dinucleotide repeat (CA)n in the 3' untranslated region. Allelic distribution in Spanish populations from two areas with different genetic background, the Canary Islands and Peninsula, are described. Seven alleles with different allelic distribution between the two groups, were found. This represents a highly polymorphic marker, useful for genetic studies on a critical molecule in immunity.

Pocket 4 in the HLA-DRB1 antigen-binding groove: an association with atopy.

BACKGROUND: Many studies have attempted to identify an association between HLA genes and atopy, given the role of HLA molecules in the regulation of the immune response. In the case of house-dust mites, it is difficult to find an association with a particular HLA allele, due to the complexity of the allergen. The objective was to investigate whether HLA-DRB1 functional groups are better correlated with the atopic disease in our population than DRB1 alleles. METHODS: The method was reanalysis of the HLA-DRB1 data of a previous case/ control study. RESULTS: The "Dr" group was found to be associated with the atopic disease in our population. CONCLUSIONS: Grouping HLA-DRB1 alleles into functional categories may assist in the search for predictive factors in relation to atopic disease.

Association of HLA-DR11 with the anaphylactoid reaction caused by nonsteroidal anti-inflammatory drugs.

BACKGROUND: Several HLA alleles have been associated with asthma induced by nonsteroidal anti-inflammatory drugs (NSAIDs). The existence of HLA markers linked to other NSAID-induced reactions, such as cutaneous and anaphylactoid reactions, has not been established. OBJECTIVE: The purpose of our work was to study the HLA-DRB1 and HLA-DQB1 alleles in patients with cutaneous and anaphylactoid reactions caused by NSAIDs. METHODS: We have analyzed 114 HLA DRB1 and 26 HLA-DQB1 alleles in 21 patients with anaphylactoid reactions caused by NSAIDs, 47 patients who had exclusively cutaneous reactions during single-blind, placebo-controlled oral challenges with NSAIDs, and 167 tolerant control subjects (29 of whom had also had an IgE-mediated anaphylaxis to different agents). HLA-DRB1 and HLA-DQB1 alleles were typed by the polymerase chain reaction sequence-specific primers method with genomic DNA. RESULTS: The frequency of HLA-DR11 alleles was 58.8% in the anaphylactoid reaction group, compared with 15.9% in the NSAID-tolerant healthy control subjects (OR, 7:3; 95% confidence interval, 2.8-19.0; P <.02) and 6.3% in the group of the patients with a tolerance for NSAIDs and with IgE-mediated anaphylaxis (OR, 18.75; 95% confidence interval, 4.3-81.1; P <.004). No differences were observed among HLA-DR11 alleles analyzed. There were no significant HLA-DQB1 associations with NSAID-induced anaphylactoid reactions. Patients with cutaneous reactions had HLA frequencies that did not differ significantly from the tolerant control subjects. CONCLUSION: The HLA-DRB1*11 alleles showed a positive association with NSAID-induced anaphylactoid reactions.

Linkage of house dust mite allergy with the HLA region.

BACKGROUND: Atopy is a multifactorial disease, the pathogenesis of which is influenced by both genetic and environmental conditions. Genes in the HLA region have been involved in the control of the IgE response. OBJECTIVE: In order to investigate whether allergy to house dust mite is associated with HLA in our population, we performed sib-pair analysis in 18 families and a case/control study of 161 non-related individuals. METHODS: Levels of total and specific IgE were determined, skin-prick tests were carried out and clinical history was reviewed for every subject in the study. HLA class II typing was performed by the polymerase chain reaction with sequence specific primers. RESULTS: We observed a significant difference from expected values in haplotypes shared by affected sibs; however, the case/control study did not reveal any association with any particular allele. CONCLUSION: These results suggest that any particular HLA-DRB1/DQA1/DQB1 allele is responsible for the development of allergy to house dust mite in the Spanish population. Some other locus in or close to the HLA region might be involved, e.g., the tumour necrosis factor gene, a possibility that would explain the significant difference from expected values in the segregation of HLA haplotypes.

Clinical and immune aspects of idiopathic acute tubulointerstitial nephritis and uveitis syndrome.

Five patients with idiopathic interstitial nephritis and uveitis without bone marrow granulomas were followed-up for 1 year. Ophthalmological examination revealed bilateral anterior uveitis. Light microscopy of the renal tissue revealed predominant lymphocyte infiltration of the interstitium. Immunohistochemical analysis revealed a clear predominance of memory T lymphocytes (CD45RO+) in the interstitial and tubular infiltration. HLA typing, and immunophenotypic studies of peripheral blood mononuclear cells including absolute lymphocyte and monocyte counts were assessed. The patients' peripheral T-cell subpopulation did not significantly differ from control studies. With steroid treatment maintained during a period of 6-9 months renal function and uveitis responded dramatically in all patients. After 1-year follow-up, only 1 patient showed a relapse of uveitis, but there was complete clinical recovery of the nephritis in all 5 patients. The aim of this study was to describe the 1-year follow-up of 5 new cases of acute tubulointerstitial nephritis and uveitis syndrome, and assess some aspects of their cellular immunity.

Selective disbalances of peripheral blood T lymphocyte subsets in human CD3 gamma deficiency.

The selection of T lymphocytes in the thymus and their activation upon the encounter with foreign antigens in the periphery require the aggregation and signals of the T cell receptor (TcR)/CD3 complex and several surface molecules termed coreceptors (notably CD4 or CD8 and CD45). The spatial arrangement and interactions of the different molecules in the resulting multimolecular recognition structure are mostly unknown. Here we report, from studies on a healthy human CD3 gamma deficiency, that the lack of the CD3 gamma component of the TcR/CD3 complex is associated with a long-term severe defect of peripheral blood CD4+ CD45RA+ and CD8+ lymphocytes, whereas CD4+CD45RO+, B and natural killer lymphocytes are unaffected. These results suggest that the CD3 gamma site of the TcR/CD3 complex is required for the peripheral representation of certain T cell types.

From pathology to physiology of the human T-lymphocyte receptor.

The recent description of a selective human CD3 gamma deficiency and other T-cell receptor (TCR)/CD3 structural and functional defects, together with previous biochemical data on the structure and interactions of the TCR/CD3 complex, may aid in elucidating the physiology of this multi-subunit membrane ensemble. CD3 gamma seemed to be required for the commitment and thymic maturation of an important fraction of T lymphocytes to the CD8 (but not CD4) lineage, perhaps by participating with the CD8 co-receptor in the instructive signal delivered through the alpha beta TCR during intrathymic positive selection by HLA class I molecules. The homologous CD3 delta component would, in contrast, be necessary for the selection of CD4 lymphocytes by HLA class II molecules. The interaction of CD4 and CD8 with the TCR/CD3 complex during antigen recognition may thus be asymmetrical, taking place through CD3 delta and gamma, respectively. Also, the existence of in vivo functional TCR/CD3 hemireceptors (lacking either CD3 gamma or CD3 delta) is suggested, and defects in their relative amount on the T-cell surface may disrupt unresponsiveness to self antigens and generate autoimmunity.

High frequency of the HLA-DRB1*0405-(Dw15)-DQw8 haplotype in Spaniards and its relationship to diabetes susceptibility.

A study of DR4 subtypes has been done in Spanish unrelated controls and insulin-dependent diabetics by using dot blot hybridization with specific DR4B1 exon-2 oligonucleotides and automated dideoxy DNA sequencing. Dw15-DQw8 is the predominant DR4 subtype present in our normal population (37%); this DR4 frequency characteristic singles out our population from all other Caucasoids tested so far and may also be a marker of the original Iberian paleo-North African population. Dw15-DQw8 is not significantly increased in our insulin-dependent diabetics sample and despite its relative high frequency in the control population it does not have a bearing in lowering insulin-dependent diabetes mellitus frequency of DR4-positive Spaniards. In addition, no particular DR4 split is by itself significantly increased in Spanish diabetics; this may indicate that selective diabetogenic environmental factors may be working upon DR4-positive individuals, but on genes (or gene products) other than DR or at least not upon the polymorphic sites of DRB1 exon-2 products.

Immunogenetics of systemic lupus erythematosus in Spanish patients: differential HLA markers.

HLA-DR3 antigen included in the compound phenotype B18BfF1 (but not the one linked to the B8BfS compound phenotype) was found to be significantly increased in our SLE patients. It is remarkable that in our Southern-Mediterranean population, B18BfF1DR3 individuals (but not B8BfSDR3) are prone to SLE with renal disease, in contrast with other Northern European and Caucasoid populations. Also, patients with autoantibodies to Ro/La have a significant increase of the B8DR3 compound phenotype. Production of autoantibodies against Ro alone was associated to DR2 and production of anti-Sm/nRNP to DR3 (either B18BfF1 or B8BfS associated) only in the subgroup without renal disease. The distinctive HLA and autoimmune associations to SLE with and without renal disease suggests that both clinical forms may not share a common identical pathogenesis.

T-lymphocyte dysfunctions occurring together with apical gut epithelial cell autoantibodies.

Gut epithelial cell autoantibodies have been considered a hallmark of autoimmune enteropathy, a disorder occurring in children with protracted diarrhea of unknown etiology. Four patients (two male and two female) with such autoantibodies were studied. Immunofluorescence analysis showed two different disjunctive staining patterns: complement-fixing apical (three of four) and cytoplasmic (the remaining fourth one), which are shown to be directed against different structures. All three patients positive for complement-fixing apical gut epithelial cell autoantibodies had abnormal T-cell responses in vitro, one of them with an immunoglobulin G2 immunoglobulin deficiency and another with an immunoglobulin A deficiency. An immunoglobulin A deficiency without T-cell alterations was also diagnosed in the cytoplasmic gut epithelial cell autoantibody-positive patient. These findings suggest that different immunologic alterations (either a T-cell abnormality or immunoglobulin deficiency) may favor the appearance of gut epithelial cell autoantibodies (complement-fixing apical or cytoplasmic, respectively). Furthermore, these autoantibodies should not be considered a specific marker of autoimmune enteropathy, because they may not always be associated with such a disease: two patients with apical gut epithelial cell autoantibodies showed no signs of intestinal lesion or diarrhea.

Expression and function of a variant T cell receptor complex lacking CD3-gamma.

A T cell line termed DIL2 has been derived from an infant with a polyclonal T cell receptor (TCR)/CD3 cell surface expression defect. Indirect immunofluorescence showed that the expression of certain TCR/CD3 epitopes (like those detected by WT31 and BMA031 monoclonals) was strongly reduced (around five-fold) on DIL2, whereas other epitopes (like those detected by SP34 and Leu4) were only around two-fold lower than in normal T cell lines. Specific immunoprecipitates of surface-radioiodinated DIL2 cells contained TCR-alpha, TCR-beta, CD3-delta, CD3-epsilon and TCR-zeta chains, but lacked CD3-gamma. This structural TCR/CD3 variant was, however, capable of transducing certain activation signals, since normal proliferation and a low but significant calcium flux was observed in DIL2 cells after engagement with specific antibodies. Our data suggest that a functional TCR/CD3 complex can be expressed on the surface of T cells in the absence of CD3-gamma.

Biochemical basis of a novel T lymphocyte receptor immunodeficiency by immunohistochemistry. A possible CD3 gamma abnormality.

Necropsic lymphoid tissues obtained from an infant with a novel type of immunodeficiency consisting of a peripheral blood T lymphocyte antigen receptor (TCR) surface expression defect, were analyzed by immunohistochemistry for the expression of various TCR-associated epitopes. The work was aimed to characterize the biochemical basis of this kind of disorder and confirm the defect in different lymphoid tissues. Within an assessed lymphoid depletion, the patient's tissues showed a normal expression of several TCR epitopes (those associated to CD3 epsilon, CD3 delta and the clonotypic -Ti- alpha and beta chains). In contrast, the expression of the epitopes recognized by the monoclonals OKT3, WT31, and BMA031 was severely diminished. Our results therefore support that CD3 epsilon, CD3 delta, Ti alpha and Ti beta are probably not involved in this type of immunodeficiency, and strongly suggest that CD3 gamma (forming part of the epitope recognized by OKT3) may rather be the affected chain giving rise to the defective surface T cell phenotype; however, alternative interpretations are not ruled out. The disrupted TCR thus formed, containing Ti alpha beta heterodimers and CD3 epsilon and CD3 delta subunits, but lacking normal CD3 gamma, would in this scheme lack the conformational framework determinants recognized by WT31 and BMA031.

A diallelic RFLP of the CD3-epsilon chain of the clonotypic T-lymphocyte receptor is not associated with certain autoimmune diseases.

A diallelic restriction fragment length polymorphism of the CD3-epsilon (epsilon) gene, which encodes for an invariant component of the human T-lymphocyte receptor, is observed when using genomic DNA TaqI digests probed with a CD3-epsilon chain cDNA probe. This combination shows two alleles of 9.1 kb and 8.4 kb with a frequency of 0.66 and 0.34, respectively, in the Spanish population. None of these alleles is associated with susceptibility to juvenile rheumatoid arthritis (JRA) or insulin-dependent diabetes mellitus (IDDM).

Congenital T-cell receptor immunodeficiencies in man.

The T-cell receptor (TCR) for antigen is a complex consisting of at least seven polypeptides chains. Two chains form the clonotypic heterodimer, which determines antigen-specificity. The other five constitute the monomorphic CD3 components, which are thought to be involved in signal transduction. We have reported a familial defect in the surface expression of the TCR/CD3 complex on otherwise phenotypically normal T lymphocytes. As a consequence of the surface defect, an impaired T-lymphocyte activation through the TCR and CD3 complex by both antigens and mitogens is observed in this type of immunodeficiency (ID) for which we propose the name TCR ID. In contrast, normal proliferative responses were recorded to TCR-independent activation or proliferation signals (i.e: anti-CD2, phorbol esters and IL-2). We believe that other ID with similar clinical and immunological characteristics, described before the general availability of monoclonal antibodies, may have also been TCR ID. Severe and mild clinical phenotypes of the disease exist which correlate with differences in the levels of TCR surface expression and of T-cell function in vitro. The biochemical basis of the defect in one of the cases thus far studied is an impaired association of CD3-zeta chain with the other chains of the complex. This defect prevented the maturation and transport of the incomplete complex to the cell surface. Due to the multi-subunit nature of the TCR/CD3 complex it is possible that other biochemical defects in TCR assembly may also give rise to TCR ID. The description of mild as well as a severe phenotype in TCR ID points to a threshold effect of TCR/CD3 expression necessary for normal T cell function in vivo since mild TCR ID is clinically asymptomatic. In contrast, severe TCR ID behaves as a clinical SCID, with clear autoimmune features and profound lymphoid tissue depletion. TCR ID and other naturally occurring SCID variants may shed light on fundamental questions of the development, function and biochemistry of the in vivo immune system. Their study and characterization also opens the door to somatic gene therapy in this kind of disorder, which is an obvious goal in ID research.