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.

T lymphocyte receptor deficiencies.

Signalling through the TCR is mediated by the cytoplasmic tails of the CD3 complex. Deficiencies in the expression of different CD3 components have lead to dramatic, yet dissimilar, effects on T-cell development and to selective deficits in peripheral T-cell subsets. Recent studies of human patients and animal models with CD3 deficiencies are providing insights into the redundant and unique roles of these molecules.

An Eco RI polymorphic site in the human complement C4 gene distinguishes juvenile rheumatoid arthritis (JRA) susceptibility-bearing haplotypes.

Susceptibility to acquire Juvenile Rheumatoid Arthritis (JRA) is linked to HLA-DR5 and DRw8 antigens in Caucasoid populations. However, the frequency of HLA-DR5 is too high in the normal Spanish population and JRA cannot thus be found to be associated with this antigen. It has been found a 14.3 kb-C4-Eco RI restriction fragment length polymorphism which correlates significantly with JRA and may be used as a marker for this disorder in Spaniards.

Construction of retroviral vectors carrying human CD3 gamma cDNA and reconstitution of CD3 gamma expression and T cell receptor surface expression and function in a CD3 gamma-deficient mutant T cell line.

CD3 gamma, a subunit of the T cell receptor-CD3 (TCR/CD3) complex, helps to support surface TCR/CD3 expression and participates in signal transduction for gene induction after antigen recognition by T lymphocytes, and in TCR/CD3 down-modulation. Humans with primary immunodeficiencies caused by inherited mutations in the CD3 gamma gene or in the gene encoding epsilon CD3é, another subunit of TCR/CD3 complex, have been previously reported. To develop a gene therapy protocol for CD3-deficient patients, CD3 gamma cDNA was orientationally inserted into two retroviral vectors (LNCX and LXSN), which resulted in recombinant vectors LNCG and LGSN, respectively. Two vector producer cell lines Am12/LNCG and Am12/LGSN were established from packaging cells GP+envAm12. Their mean viral titers were 6.5 x 10(6) and 2.0 x 10(7) cfu/ml, respectively, as shown by an improved retroviral vector production and transduction method that increases titers around five-fold over conventional methods. The presence of helper virus in vector stocks was tested by marker rescue assay and found to be < 1 cfu/ml. Southern blot analysis showed that multiple copies of the vectors were present in the genome of high-titer producers and that both vectors could transfer CD3 gamma cDNA into the genome of 3T3 cells. The vectors were used to correct in vitro a CD3 gamma-deficient Jurkat mutant cell line lacking TCR/CD3 expression and termed JGN (for Jurkat gamma negative). Both vectors increased TCR/CD3 expression in JGN (normally 2% using WT31 monoclonal antibody) to 34% and 37%, respectively, in G418-selected 3-week bulk cultures. Two clones from transduced JGN cells termed JGN/LNCG13 and JGN/LNCG15, with high TCR/CD3 expression (88% and 79%, respectively), were selected for further analyses. First, CD3 gamma protein reconstitution was demonstrated by immunoprecipitation. Second, interleukin-2 production after TCR/CD3 engagement and TCR/CD3 down-modulation in response to phorbol myristate acetate were shown to be comparable to wild-type Jurkat cells. We conclude that LNCG and LGSN may be useful for gene therapy purposes.

Toward gene therapy for human CD3 deficiencies.

The CD3 subunits of the T cell receptor-CD3 complex (TCR-CD3) help to regulate surface TCR-CD3 expression, and participate in signal transduction leading to intrathymic selection and peripheral antigen recognition by T lymphocytes. Humans who lack individual CD3 chains show impairments in the expression and activation-induced downregulation of TCR-CD3, and the defective immune responses that result may be lethal. We have investigated delivery of a normal CD3 chain to treat disorders of this type. Retroviral transduction of CD3gamma into CD3gamma-deficient peripheral blood T lymphocytes from two unrelated patients selectively corrected the observed TCR-CD3 expression and downregulation defects, but unexpectedly seemed to cause adverse effects that can be explained by an autoreactive recognition mechanism. These data support the feasibility of gene therapy for human CD3 deficiencies, but also suggest that gene transfer into postthymic lymphocytes carrying mutations on T cell recognition or activation pathways may disrupt their intrathymic calibration and become harmful to the host.

Lack of preferential transmission of diabetic HLA alleles by healthy parents to offspring in Spanish diabetic families.

HLA-DR3 or -DR4 segregation distortion to normal or insulin-dependent (ID) diabetic offspring of 108 Spanish families whose parents were healthy was not observed; however, DR3 or DR4 ID offspring is significantly increased in the present study, since parents were chosen after tracing ID children. These results are discrepant with those found by others in families with diabetic parents in other ethnic groups. These conflicting data could be due to sampling errors or segregation distortion. Thus, ethnic group differences in a genetic (T/t-like) or metabolic mechanism might confer advantages to DR3- or DR4-bearing gametes from ID diabetic parents, but segregation distortion might only affect certain HLA DR3 or DR4 extended haplotypes which are frequent and characteristic for certain ethnic groups (i.e. B8-DR3-BfS-C4AQOB1 and Bw62-DR3-BfS-C4A383 in most caucasians) but not for other haplotypes in other ethnic groups (Spaniards; B18-DR3-BfF1-C4A3BQO and BwX-DR4-BfX-C4AXBX).

Herpes virus saimiri transformation of T cells in CD3 gamma immunodeficiency: phenotypic and functional characterization.

The characterization of T cell immunodeficiencies could in part be supported by using stable cell lines in which biochemical and molecular studies of the defect could be carried out thereby omitting frequent bleeding of patients. First attempts to obtain such cell lines included HTLV-I transformation and exogenous IL-2 administration, but both models have important disadvantages. Recently, a virus isolated from the squirrel monkey, Herpes virus saimiri (HVS), has been reported to have the ability to transform T cells. A stable IL-2-dependent HVS-transformed T cell line from a CD3 gamma deficient patient has been obtained; and this cell line displays both the phenotypic and the functional characteristics of the patient's lymphocytes. Moreover, the line down-modulates TCR/CD3 surface expression upon CD3 engagement, as do the patient's lymphocytes, showing that CD3 gamma and its phosphorylation are not necessary for TCR/CD3 internalization. In addition, the abnormal staining pattern of different anti-TCR/CD3 monoclonal antibodies is preserved in the HVS-patient line. Since HVS is capable of transforming CD3 gamma- T cells, the CD3 gamma chain does not seem to be involved in the HVS receptor process. The fact that it is not possible to obtain a CD8+ HVS line from the CD3 gamma- patient supports the existence of a functional anomaly in his scanty CD8+ peripheral lymphocytes. Thus, HVS transformation is a suitable model for T cell immunodeficiency studies and characterization. It may also be used in the future in cellular models for in vitro gene therapy trials.

Peripheral blood reduction of memory (CD29+, CD45RO+, and "bright" CD2+ and LFA-1+) T lymphocytes in Papillon-Lefèvre syndrome.

A Papillon-Lefèvre patient with characteristic chronic periodontal disease and palmoplantar keratoderma was studied over a 4-year period. An abnormal T-cell phenotype was steadily observed in peripheral blood; both low numbers of CD29+ and CD45RO+ cells and a low density surface expression of CD2 and LFA-1 molecules were found. T-cell activation through CD3, CD2 and ConA, PWM and IL-2 receptors was normal; however, there was impairment in the activation via CD28. CD2, LFA-1 and CD45 molecules were normal in charge and molecular weight. There was no tissue sequestering of T lymphocytes in periodontal lesions, but rather a relative T-cell reduction. It is suggested that an important decrease of the so-called "memory/hyperreactive" (CD45RO-positive) T cells does exist; therefore, hyperreactive T cells would not be available in sufficient numbers to leave the bloodstream through blood vessel endothelium, and the periodontium would be left without these important defenses and thus exposed to chronic infections. A disregulated factor affecting the transition from "naive" to "memory" T cells and the increase in certain surface molecules expression (i.e., CD2, LFA-1, CD29, and CD45RO) or the reversion from memory to naive T cells may be responsible for the disease pathogenesis. CD2 and LFA-1 molecule synthesis might be conjointly regulated on T lymphocytes.

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.

HLA typing of dried sperm.

HLA human histocompatibility leukocyte antigens -A and -B antigens are expressed on human spermatozoa. A micro- and a macro-technique are described to HLA-type dried sperm coming from unknown subjects. The high HLA genetic polymorphism allows a high degree of sperm individualization which may be of value to blame or discard suspects in forensic science cases.

Human MHC class III (Bf, C2, C4) genes and GLO: their association with other HLA antigens and extended haplotypes in the Spanish population.

C4 allotype frequencies and their combination with factor B and C2 alleles (complotypes) were studied in a sample of the Spanish population in relation to MHC class I, class II and GLO alleles. The shorter genetic distances found for C4 between Spaniards and North Africans and the high frequency of extended HLA haplotypes (GLO 2) HLA-DR3 F1C30 HLA-B18 HLA-Cw5 (HLA-A30) and HLA-DR7 S1C21 HLA-Bw50 HLA-Cw6 are consistent with a paleo-North African ethnic origin (about 20,000 years B.C.) of a part of present Spaniards (Iberians), and with the effect of racial admixture during late Moslem invasions (from the 8th to the 15th century). The complotype null alleles C4A QO and C4B QO may be under natural selection pressure when found in cis position, since they are never in the same haplotype in families. The underestimation of these C4 null alleles' frequencies in unrelated individuals as compared to genotyped families is shown to be a very likely event and a serious hindrance for C4-disease association studies. We have not found any C4 duplications in the Spanish population; this may be due to sample size limitations or to the degree of admixture of our population. Strikingly, no positive linkage disequilibrium between C4A and C4B alleles is detected in unrelated individuals nor in families, although strong associations are maintained among Bf, C2, C4, HLA-A, HLA-B, HLA-C and HLA-DR markers. Assuming that all MHC polymorphisms have reached equilibrium, several explanations are proposed, including the possibility of no, different or additional natural selection mechanisms operating on some MHC class III genes (Bf, C2, C4 alleles combinations for most appropriate C3 convertases), as compared to those affecting class I and class II gene clusters (most advantageous immune response genes sets).

C3 polymorphism, HLA and chronic renal failure in Spaniards.

C3 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 45 cM 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.

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

C2 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-A, -B, -C, -Bw4, Bw6 and -DR antigens are expressed on purified seminal cells other than spermatozoa.

HLA-A, HLA-B, HLA-C, HLA-Bw4, Bw6 and HLA-DR antigens have been detected on purified seminal cells than spermatozoa (SCnonSz) using specific absorptions. These results contrast with those obtained on spermatozoa, which only express significant amounts of HLA-A and HLA-B antigens, and which may be relevant to explain immunosuppressive alloimmunization in the acquired immunodeficiency syndrome (AIDS).

CD11b-bearing mononuclear leucocytes and IgA levels in the staging of human immunodeficiency virus infection.

Certain immunological parameters (i.e. low CD4+ T cell numbers, high serum soluble CD8) have been described as prognostic factors for the progression of human immunodeficiency virus (HIV) infection to later clinical stages. In the present study we have found in one hundred HIV-infected Spanish patients (81% drug abusers, 7% homosexuals, 6% heterosexuals, and 6% other or unknown risk groups) that CD11b+ peripheral blood mononuclear cells are increased in those with persistent lymphadenopathy as compared to other clinical stages (asymptomatic, AIDS-related complex and AIDS). Serum IgA was significantly increased in AIDS patients, and in patients at any other clinical stage who had concomitant infections (mainly mycobacterial and fungal). CD11b (an integrin with complement receptor functions) may thus be of clinical interest for the staging of HIV-infected patients, and reflect stage-selective immunological changes in mononuclear cell biology during HIV infection. High IgA on the other hand, would be a marker of concomitant infection as well as of disease progression. The results concern mostly drug addicts (the main risk group in Spain), but may apply to the other risk groups because no significant differences were detected between drug addicts (n = 81) and non-drug addicts (n = 19) for the studied variables (p greater than 0.05).