A. Arnaiz-Villena

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.

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.

Impaired T cell signal transduction through CD28 in a patient with idiopathic thrombocytopenia

We describe an infant whose peripheral blood mononuclear cells were unable to proliferate or synthesize IL‐2 in response to a mitogenic combination of antibodies directed against CD2 and CD28. This peculiar defect, which has been stable to dale, was attributed to an impairment in CD28‐mediated T cell activation, because further comitogenic combinations containing anti‐CD28 monoclonals also failed to induce normal proliferation of the patients T cells. In contrast, proliferation after membrane stimulation (with anti‐CD2, recombinant IL‐2, or certain lectins) or transmembrane activation (with phorbol ester and calcium ionophore) was normal, suggesting that his lymphocytes did not have a general membrane or intracellular signalling impairment. A T cell line derived from the patient confirmed the existence of a severe defect in CD28‐mediatcd T cell proliferation, but also showed a profound impairment in CD3‐induced T cell proliferation. Other cell surface molecules like CD2 and CD25 were, in contrast, capable of transducing normal proliferation signals. As all relevant molecules were detectable by cytofluorography and immunoprecipilalion, we conclude that the patients lymphocytes had an intrinsic defect in the delivery of CD28‐mediated signals which, in the absence of monocytes, also affected CD3‐mediated proliferation. The study of this novel kind of immunodeficiency may help to unravel the complex interactions that take place among CD2. CD3 and CD28 during T cell activation. The presence of an idiopathic thrombocytopenia in the patient suggests the intriguing possibility of a role for CD28 in the maintenance of peripheral blood platelets levels, although alternative interpretations are not ruled out.

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.

An in vivo functional immune system lacking polyclonal T-cell surface expression of the CD3/Ti(WT31) complex.

A polyclonal T‐cell receptor complex (TCR) expression defect (as detected with monoclonal antibody WT31) has been found in two children belonging to an otherwise healthy Spanish family. One of the sibs (V, who had been vaccinated with attenuated poliomyelitis virus) showed clinical signs of immunodeficiency with an autoimmune syndrome, but the other (older) sib (D, vaccinated with attenuated rubella, measles, mumps, and poliomyelitis viruses) has been symptomless throughout life. In contrast to both sibs normal expression of other peripheral leucocyte markers, as measured by flow cytometry (including CDl, CD2, CD4, CD8, and CD16), only about 6% of CD2+ polyclonal T cells expressed surface antigen‐specific T‐cell receptor (Ti/WT31), and only about 23% weakly expressed surface CD3 determinants. On the remaining CD2+ T cells in each sib the expression of Ti and CD3 was undetectable; the defect in CD3 expression is very likely secondary to the defect in Ti expression. Natural killer (NK) activity was not increased in any of the sibs, ruling out a high content of NK cells among their CD2+ lymphocytes. Functional data indicate that CD3‐mediated T‐cell activation with anti‐CD3 monoclonals and Ti‐mediated responses to allogeneic and tetanus toxoid antigens were severely depressed, whereas activation via CD2 was normal in the T lymphocytes of both sibs. Genes encoding for Ti alpha, beta, and gamma chains did not show major alterations by southern blot analysis, and polyclonal beta chain genes rearrangements were detected in both childrens T‐cell blasts. Family clustering suggests a genetic pathogenesis, but linkage to HLA or other blood group markers has not been found. Sib V had a concomitant autoimmune disease and died after a severe autoimmune haemolytic anaemia, indicating a relationship between the TCR and generation of autoimmune clones. However, the resistance of both individuals to infection and to vaccination with attenuated viruses, and the fact that sib D has been symptomless to date questions the relative importance of the TCR in the immune response against infection, and suggests that alternative T‐cell activation pathways and non‐specific defence mechanisms (external surfaces–bound and/or cellular) may suffice under certain circumstances.

C3 polymorphism, HLA and chronic renal failure in Spaniards

SummaryC3 allele frequencies were studied in 196 unrelated normal Spaniards. The results fit the Hardy-Weinberg equilibrium. No rare variants were detected. The C3 frequency was close but slightly higher than that found in other Caucasoid populations, and higher than that found in Negroids and Orientals. Spanish Basques also showed a high C3F frequency. A North-South decreasing C3F gradient was recorded and compared to other gradients (HLA-D/DR, height, etc.) thought to be due to natural selection. Lod scores in 28 Spanish families excluded C3 gene assignment at less than 45cM of HLA/GLO linkage group; no significant linkage disequilibrium was found between C3 and HLA. C3F was also significantly increased in 20 chronic renal failure (CRF) patients as compared to 196 controls; this would support the existence of functional differences between C3F and C3S alleles.

EXCLUSIVE HLA-DQ FACTORS DO NOT EXPLAIN SUSCEPTIBILITY TO INSULIN-DEPENDENT DIABETES

DQA1, DQA2, DQB1, and DRB1 alleles have been determined and the DQA1 and DQB1 DNA gene sequences assigned by using restriction fragment length polymorphisms in 67 diabetic individuals and 72 controls. It has been found that: 1) DQA2 (U allele) is not a susceptibility factor, 2) non-aspartic acid homozygosity in residue 57 (Asp 57 negative) of the DQ beta chains is positively correlated with insulin-dependent diabetes mellitus (IDDM), and 3) DQ beta Asp-57-negative and DQ alpha arginine-52-positive (Arg-52-positive) individuals are increased among diabetic patients; this latter analysis shows a higher etiologic fraction (delta) value than the one obtained when considering only homozygous DQ beta Asp-57-negative individuals. However, if only non-DR3 or DR4 individuals were considered (both in DQ beta Asp-57-negative homozygous and in DQ beta Asp-57-negative/DQ alpha Arg-52-positive individuals) the correlation with disease disappears. In addition, the postulated risk DQ beta Asp 57-negative and DQ alpha Arg 52 positive is absent in six patients. These data do not discard the possibility that DR3/DR4 may contain the primary susceptibility factors. It is concluded that it is not possible to assign the susceptibility to IDDM to a specific HLA locus and that several loci within the same or the trans haplotype may be involved.

Immunofixation for C2 typing: C2 allotypes in Spaniards in relation to HLA, Bf and C4.

SummaryC2 typing is performed by immunofixation with anti-C2 antiserum instead of by a hemolytic overlay. This method gives sharp band definition, is less cumbersome than the hemolytic overlay, gel files are easily made, and it also enables one to describe putative new nonhemolytic variants. C2 allele frequencies were studied in a sample of the normal Spanish population and were found to be similar to other Caucasoids. HLA-Bw62,-Cw3, and-DR4 were significantly associated with C2 B. Concordantly, the only C2*B extended HLA haplotype found in family material was Bw62-Cw3-Bw6-(DR4)-Bf*S-C2*B-C4A*3 B*2-(GLO*1). C4A*4 B*2 and C4A*4 B*4 are not found within the same haplotype together with C2*B and Bw62 or Bw22 respectively, nor do other C2*B haplotypes occur with common HLA-B alleles. These results may favour the hypothesis that the Bw62-C2*B haplotype is produced by one mutation arising in the Bw62-C2*C haplotype and that subsequent crossovers can explain other C2*B haplotypes (including Bw22-C2*B).

HLA-D determinants are expressed on human seminal cells other than spermatozoa but not on purified spermatozoa

Purified human spermatozoa do not regularly stimulate lymphocytes in spermlymphocyte cultures (SLC). This lack of consistent stimulation was found not to be dependent on sperm/lymphocyte ratio or on culture peak response time. A few weak stimulations obtained are not HLA-D or sex dependent. Contaminating seminal cells other than spermatozoa (SC non-SZ) may be responsible for the high stimulations found in SLC by others, since, in our hands, purified suspensions of SC non-SZ stimulate allogeneic lymphocytes in an HLA-D-dependent fashion, and such responses are abrogated by anti-HLA-DR monoclonal antibodies. These functional data confirm our previous finding of HLA-DR molecules on SC non-SZ by absorptions (and a lack of expression on spermatozoa) and suggest the concomitant expression of HLA-DQ and -DP products.

Antigen Society #15 Report (Bw4 and Bw6)

During the Tenth Workshop, 27 anti-sera in the Antigen Society and 22 anti-sera in the Core serology serum set were provided for the serologic analysis of the supertypic HLA-B locus specificities Bw4 and Bw6. The serum sets included 12 monoclonal antibodies with Bw4 or Bw6 specificity. For each of the submitted sera, Q score, R values, % extra, and missed reactions, as well as “tail” antigens derived by 2x2 comparison of their reactivity on Caucasians, Negroes, and Orientals, are shown in Tables 1 and 2.