Human immunodeficiency virus type 1 infection after sexual abuse: value of nucleic acid sequence analysis in identifying the offender.

Nucleic acid sequence analysis of human immunodeficiency virus type 1-specific sequences allowed the identification of the source of infection in a case of sexual abuse of a 10-year-old girl.

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients.

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4+ and CD8+ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4+ and CD8+ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR+CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4+ lymphocytes following TCR+CD28 stimulation. In addition, stimulation via TCR+CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Th1-type cytokines such as IL-2, and is expressed in both CD4+ and CD8+ T cell subsets.

Inhibition of IL-10 protein synthesis induces major histocompatibility complex class II gene expression in class II-deficient patients.

Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency, characterized by a lack of constitutive expression of the human leukocyte antigen (HLA) class II genes. The patients investigated in this study are histoidentical twin brothers with a new phenotype in MHC class II deficiency. Examination of HLA-D locus genes in their fractionated peripheral mononuclear cells (MNCs) revealed an unusual and uncoordinated mRNA pattern. Here we analyzed the distribution of pro- and anti-inflammatory cytokines expressed in these patients' adherent and nonadherent MNCs. We show that gene expression of IL-1 alpha, IL-1 beta, IL-6, granulocyte-colony-stimulating factor, and IL-10 was induced in both cell fractions, whereas increased mRNA levels of interferon-gamma and the inducible nitric oxide synthase were exclusively detected in the patients' nonadherent MNCs. As IL-10 is known to be able to downregulate transcription of MHC class II and expression of IL-10 in the patients' MNCs was increased, we investigated the regulatory function of this cytokine. Interestingly, inhibition of IL-10 protein synthesis with IL-10-specific antisense oligonucleotide DNA (IL-10-AS-ODN) induced HLA-D locus genes in these MHC class II-deficient patients. Exposure of the nonadherent cell fraction to IL-10-AS-ODN resulted in a profound induction of a previously absent DR beta 1 and DP alpha gene expression. HLA-DQ beta mRNA levels, however, were increased in both the adherent and the nonadherent MNC population. Albeit expression of HLA-D locus genes was inducible via inhibition of IL-10 translation, surface expression of HLA class II antigens on the patients' MNCs was essentially negative. The data presented support the concept of a coordinated network of pro- and anti-inflammatory cytokine regulation and this network obviously has a significant role in the cell-type-specific regulation of MHC class II expression.

Anti-inflammatory properties of human serum IgA: induction of IL-1 receptor antagonist and Fc alpha R (CD89)-mediated down-regulation of tumour necrosis factor-alpha (TNF-alpha) and IL-6 in human monocytes.

A deregulated expression and/or release of large amounts of inflammatory cytokines such as IL-1 and TNF-alpha accounts for most pathophysiological events in a variety of systemic inflammatory diseases, the effect being mediated by the interaction of these cytokines with their respective receptors. IL-1 receptor antagonist (IL-1Ra), mainly produced by monocytes/macrophages, is an inhibitor of IL-1 activity. The present study shows that human serum IgA induces significant IL-1Ra release in human peripheral blood mononuclear cells and adherent monocytes. IgA induced higher levels of IL-1Ra than Haemophilus influenzae type b (Hib) expressing lipopolysaccharide (LPS), purified LPS or phorbol myristate acetate (PMA), without induction of IL-1 beta release, and even inhibited LPS-induced IL-1 beta release. Induction of IL-1Ra by IgA could be detected both at the mRNA and protein levels in resting and activated monocytes. Ligation of Fc alpha R with MoAb My-43 or treatment with human serum IgA induced protein tyrosine phosphorylation in human monocytes, and herbimycin A, a specific inhibitor of protein tyrosine kinase activity, inhibited IgA-induced IL-1Ra production, suggesting that Fc alpha R-mediated induction of tyrosine phosphorylation is required for the IgA-induced stimulation of IL-1Ra release. In addition, triggering of Fc alpha R with MoAb specifically down-regulated TNF-alpha and IL-6 release in human monocytes activated with Hib. By the induction of IL-1Ra and down-regulation of the release of inflammatory cytokines such as IL-1 beta, TNF-alpha and IL-6, interaction of IgA with human monocytes may actively contribute to the regulation of the inflammatory response.

Genetic evidence for a new type of major histocompatibility complex class II combined immunodeficiency characterized by a dyscoordinate regulation of HLA-D alpha and beta chains.

Major histocompatibility complex (MHC) class II combined immunodeficiency (CID), also known as type II bare lymphocyte syndrome, is an autosomal recessive genetic disorder characterized by the complete lack of expression of MHC class II antigens. The defect results from a coordinated lack of transcription of all class II genes. Cell fusion studies using many patient- and experimentally derived class II-negative cell lines have identified four distinct genetic complementation groups. In this report, we present genetic evidence that cell lines derived from two newly described MHC class II-deficient patients, KER and KEN, represent a fifth complementation group. In addition, the KER and KEN cell lines display a unique pattern of dyscoordinate regulation of their MHC class II genes, which is reflected in a new phenotype of in vivo promoter occupancy as revealed by in vivo genomic footprinting. These data point to a new defect that can result in the MHC class II-deficient phenotype.

Activation via the antigen receptor is impaired in T cells, but not in B cells from patients with common variable immunodeficiency.

The patients included in this study belong to a subset of common variable immunodeficiency (CVID) patients whose peripheral blood T cells have a T cell receptor (TCR)-mediated activation defect leading to impaired expression of the interleukin (IL)-2 gene upon stimulation with recall antigens (tetanus toxoid, Escherichia coli) or superantigens (staphylococcal enterotoxins). In the present report we demonstrate that the patients' peripheral blood T cells failed to generate the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) following stimulation with superantigen or mAb specific for the monomorphic region of the TCR beta-chain. Patients' T cell lines were also impaired in generating Ins(1,4,5)P3 when stimulated with tetanus toxoid-pulsed autologous monocytes. Addition of a second or third co-stimulatory signal provided by recombinant IL-2, CD28 or both had no effect on the Ins(1,4,5)P3 formation of the patients' antigen-driven T cell lines. The T cell activation defect, however, was not absolute, as Ins(1,4,5)P3 formation in the patients' T cells after phytohemagglutinin or aluminium fluoride stimulation was normal. The impairment in signal transduction via the T cell antigen receptor was limited to the patients' T cells, as no activation defect after ligation of surface immunoglobulin, the antigen receptor on B cells, could be detected.

Molecular characterization of major histocompatibility complex class II gene expression and demonstration of antigen-specific T cell response indicate a new phenotype in class II-deficient patients.

Major histocompatibility complex (MHC) class II deficiency is an inherited autosomal recessive combined immunodeficiency. The disease is known as bare lymphocyte syndrome (BLS). BLS is characterized by a lack of constitutive MHC class II expression on macrophages and B cells as well as a lack of induced MHC class II expression on cells other than professional antigen-presenting cells (APCs) due to the absence of mRNA and protein of the human leukocyte antigen (HLA) class II molecules, designated HLA-DR, -DQ, and -DP. The defect in gene expression is located at the transcriptional level and affects all class II genes simultaneously. Here we have analyzed transcription and protein expression of class II antigens in Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines and mononuclear cells (MNCs) of twin brothers. Whereas flow cytometric analysis failed to detect class II antigens on the cell surface of the patients' EBV-B cells and MNCs, examination of the genes coding for HLA-DR, -DQ, -DP, and the invariant chain (Ii) by reverse transcriptase-polymerase chain reaction amplification resulted in an unusual mRNA pattern in the B cell lines of the patients (HLA-DR alpha +, -DR beta, -DQ alpha +, -DQ beta -, -DP alpha -; -DP beta +, Ii+). In accordance with these findings no HLA-DR beta-specific protein was detected by immunoblotting, whereas low levels of HLA-DR alpha and normal levels of Ii were present. In contrast to EBV-B cells, the MNCs of both patients displayed a residual HLA-DR beta, -DQ beta, and -DP alpha mRNA signal. Furthermore, HLA-DR beta-specific protein was found in addition to HLA-DR alpha by immunoblotting of cell lysates, even though it was clearly decreased as compared with controls. Our results indicate that the defect in class II antigen expression is not necessarily present to the same extent in B cells and cells of other lineages. mRNA levels of HLA-DR beta were found to be enriched in adherent cells within the MNC fraction. Further investigations indicated that the MHC class II expressed is functional in antigen presentation, as the two boys' CD4+ T cells became activated and expressed interleukin-2R after stimulation of peripheral blood mononuclear cell cultures with recall antigen (tetanus toxoid). Furthermore, T cells tested in one of the two patients responded to both MHC class I and II allostimulation, and this response was inhibited by monoclonal antibodies of the respective specificity.(ABSTRACT TRUNCATED AT 400 WORDS)

Defective TCR surface expression associated with impaired TCR beta-chain assembly in a patient with cutaneous T-cell lymphoma.

We report on a patient with cutaneous T-cell lymphoma (CTCL) of long-standing duration. Phenotypic analysis of his peripheral blood mononuclear cells revealed an increased CD4+ T-helper subset and a decreased CD8+ cytotoxic T-cell population. Eighty-three to ninety-three percent of the patient's CD4+ T cells in the peripheral blood and 70% of the CD4+ T cells in the lesional skin lacked surface expression of the TCR/CD3 complex and showed a clonal rearrangement pattern of the TCR gamma-chain gene (V11-J1/J2). The lack in TCR surface expression correlated with defective assembly of the TCR beta-chain. Although mRNA for the TCR constant region beta 1 was found in the patient's purified CD4+ TCR-CD3- T cells, no intracytoplasmic TCR beta protein was detectable. In contrast, the patient's purified CD4+ TCR-CD3- T cells not only expressed mRNA specific for the TCR alpha-chain and for all CD3 chains, but intracytoplasmic TCR alpha and CD3 epsilon proteins could also be found. The lack of TCR beta protein clearly explains the defective surface expression of the TCR/CD3 complex in the patient's malignant T cells.

Reduced IL-2 expression upon antigen stimulation is accompanied by deficient IL-9 gene expression in T cells of patients with CVID.

Patients with common variable immunodeficiency (CVID) are heterogeneous in the clinical manifestation of the disease as well as in the underlying mechanisms leading to the immunodeficiency. In a previous study we identified a subgroup of patients with a primary immunodeficiency disease affecting IL-2 and IFN-gamma gene expression. The T cells of these patients revealed impaired proliferative response and reduced levels of IL-2 and IFN-gamma-specific mRNA after antigen stimulation in vitro, while cellular and molecular response to phorbol ester and the calcium ionophore ionomycin (PMA+IM) or anti-CD3 monoclonal antibodies (MoAbs) (OKT3) were comparable to those of healthy control individuals. Here we show that stimulation of these patients' T cells with tetanus toxoid (TT) resulted in dramatically reduced levels of IL-2, IL-9 and IFN-gamma mRNA, while IL-3 gene expression in three patients was comparable or even increased to the healthy controls. As expected, addition of exogenous IL-2 to tetanus toxoid pulsed cultures had virtually no effect on IL-2 transcription, but corrected the defect in IL-9 gene expression, while IFN-gamma mRNA levels were still reduced. In conclusion, these data suggest that recombinant IL-2 alone is not able to induce the IL-9 gene adequately in our patients, but clearly increases IL-9 mRNA levels in combination with tetanus toxoid.

A defect in the early phase of T-cell receptor-mediated T-cell activation in patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is characterized by an impairment of specific antibody production and a decrease in all or selected Ig isotypes. Abnormalities at the level of the B cells, T cells, and antigen-presenting cells have been described. In the present study, we have focused our attention on T-cell activation in CVID. T cells from 15 of 24 patients failed to respond to recall antigens (eg, tetanus toxoid, Escherichia coli). Of these 15 patients, 11 were studied in detail and showed significantly decreased T-cell proliferative responses and/or decreased interleukin-2 and interferon-gamma production on T-cell receptor-mediated stimulation with recall antigens and superantigens (staphylococcal enterotoxins [SE]); however, T-cell response to mitogens (anti-CD3 monoclonal antibody, phytohemagglutinin) was normal. The defect in interleukin-2 and interferon-gamma release on tetanus toxoid stimulation could also be documented in purified CD4 T cells of the patients and was present in patients with high and normal CD8 counts alike. Furthermore, patients' T cells failed to mount a significant elevation in free intracellular calcium (Ca++ flux) in response to superantigen, whereas the response to phorbol myristate acetate and ionomycin, bypassing receptor-mediated signaling, was unimpaired. These results indicate a defect in the early phase of T-cell activation after triggering of the T-cell receptor in a significant subgroup of CVID patients.

Impaired TCR signal transduction, but normal antigen presentation, in a patient with common variable immunodeficiency.

We describe a 27-year-old white man with common variable immunodeficiency (CVID) who has two healthy histoidentical brothers and one IgA-deficient sister who shares one HLA haplotype with the patient. T cells from the patient with CVID showed an impaired response to recall antigens (tetanus toxoid, E. coli), whereas his IgA-deficient sister and his two healthy histoidentical brothers responded normally. Cross-mixing experiments using isolated monocytes and T cells from the CVID patient and one histoidentical brother revealed that the patient's monocytes were fully functional in processing and presenting antigen to resting T cells of his brother, and provided normal accessory cell function for superantigen-induced activation of his brother's resting T cells. In contrast, the patient's T cells were unable to respond to antigen presented by the brother's monocytes and failed to respond with an increase in intracellular free Ca++ to stimulation with superantigen, which is known to bind to the TCR V beta-chain outside the antigen-binding groove. However, stimulation with a combination of PMA and IM, directly activating protein kinase C and increasing intracellular free Ca++ by bypassing membrane receptors, induced normal Ca++ flux. These data indicate that the patient with CVID has a defect in TCR-mediated signalling at the level of the T cells which is not present in his histoidentical healthy brothers or in his haploidentical IgA-deficient sister.

T cell differentiation and generation of the antigen-specific T cell repertoire in man: observations in MHC class II deficiency.

The circulating T cell pool of an MHC class II-deficient patient was shown to lack the MHC class II-specific T cell functions. This was demonstrated by the absence of MHC class II-specific alloreactive T cells and a substantially decreased number of circulating CD4+ lymphocytes. The patient's T cells did respond to an allostimulus, although the restriction pattern of this reaction remains speculative. The function and distribution of peripheral T cell subsets from the patient resemble findings in MHC class II-deficient mice, which also lack interaction of T cell precursors with MHC class II-bearing accessory cells during thymic differentiation. Our data support the concept that T cell differentiation in humans is similar, and that the human MHC-restricted T cell repertoire depends on prior interaction of T cell precursors with self MHC.

Defective interleukin-2 and interferon-gamma gene expression in response to antigen in a subgroup of patients with common variable immunodeficiency.

Patients with common variable immunodeficiency (CVID) are heterogeneous in the clinical manifestations of the disease and in the underlying mechanisms leading to the immunodeficiency. The impairment of B-cell function can be due to an intrinsic defect of the B cells, a deficiency in the function of the antigen-presenting cell, or a dysfunction in the course of T-cell activation. In the present report we have focused our attention on T-lymphocyte activation in three patients with CVID. Numbers of T and B cells, as well as CD4 and CD8 T cell subsets, were within the normal range. The patients' B cells secreted IgM but did not secrete IgG and IgA in response to B-cell stimuli in vitro. Addition of allogeneic T cells was followed by an increase in IgM production but had no effect on the other immunoglobulin isotypes. Examination of T-cell function revealed impaired proliferative response, interleukin-2 (IL-2) and interferon-gamma gene expression and IL-2 release after antigenic stimulation, whereas T-cell proliferation, as well as IL-2 gene expression and release in response to stimulation via anti-CD3, were comparable to those of healthy control subjects. Anti-CD3-induced IFN-gamma gene activation in T cells from two patients was comparable to that of control subjects, whereas T cells from the third patient showed reduced expression of IFN-gamma mRNA. In contrast to the decreased IL-2 and IFN-gamma mRNA levels, IL-2R transcripts examined in parallel were normal in CVID T cells on stimulation with antigen. The defect in IL-2 and IFN-gamma mRNA expression after stimulation with antigen, but not with anti-CD3, suggests an abnormality confined to T-cell activation by the T-cell receptor.

Activation of CVID patients' T cells with conventional antigens and superantigens.

Defects in T cell function are known to be present in a subset of patients with CVID, but the true nature of these defects still has to be revealed. In prior studies we described that T cells from these patients show an impaired proliferative response following activation with recall antigens (E. coli, Tet. Tox., TBE and PPD). Gene expression of IL2 and IFN-gamma in patients' T cells following antigenic stimulation was significantly reduced compared to controls, while IL-2R transcripts were normal. To further characterize the defect we examined T cell responses to bacterial enterotoxins, collectively termed superantigens. Following stimulation with optimal (10 ng/ml p < 0.05) as well as suboptimal (1 ng/ml p < 0.0025) concentrations of staphylococcal enterotoxin A (SEA), proliferative response and cytokine release (IL-2 and IFNg) were significantly decreased in patients' T cells as compared to controls'. When patients' T cells were stimulated with staph. enterotox. C3 (SEC3) an even more pronounced difference between patients' and controls' T cells could be observed (10 ng/ml p < 0.002, 1 ng/ml p < 0.0005). Our data indicate that, in addition to the defect in antigen-induced T cell activation, T cells of CVID patients express a broader impairment in the interaction between the antigen presenting cell and the TCR.