A comparison of two fluorescence-activated cell sorters, the FACSIV (laser) and the FACSTm (mercury lamp), as research analyzers for the quantification of T and B cell subsets in human peripheral blood.

T cell subset determinations were performed on 146 peripheral blood samples from healthy volunteers, and on 112 samples from immune deficient patients using two fluorescence-activated cell sorters (the FACSIV laser, and the FACSTm mercury lamp analyzer). The procedures necessary for the use and calibration of the FACSTm analyzer are discussed, and detailed. Using the FACSTm analyzer, counts were made of T and B cell subsets in 28 patients with multiple infections, 9 patients suffering from the acquired immune deficiency syndrome (AIDS) and 16 patients with a primary immunodeficiency disease. These results were compared with data obtained from 47 healthy volunteers, as control references. Results from the two instruments proved closely comparable, both qualitatively and quantitatively.

Comparison of complement-dependent cytotoxicity and indirect immunofluorescence for enumeration of T-cell subpopulations in human peripheral blood.

The monitoring of T-lymphocyte subsets of recipients of organ grafts enables studies on immune reconstitution (after bone-marrow transplantation) and may predict graft rejection (after kidney transplantation). Quantitation of human peripheral T-lymphocyte subsets from healthy volunteers and from recipients of a bone-marrow graft by a complement dependent cytotoxicity (CDC) assay, based on the use of propidium iodide, and by an indirect immunofluorescence (IIF) technique has been compared using the monoclonal antibodies OKT3, OKT4 and OKT8. Except for OKT3 in healthy individuals--for which no significant difference was found between CDC and IIF--CDC detected significantly more cells of each subset than IIF. Furthermore, the CDC results indicated the presence of low numbers of OKT4+8+ cells in the peripheral blood of healthy individuals and--with higher numbers--following marrow transplantation. Results of depletion experiments, obtained by fluorescence activated cell sorting (FACS) for either OKT4 or OKT8, supported this conclusion. OKT4/OKT8 ratios were calculated from enumerations by the CDC assay and by the IIF assay and found to be linearly related, both in healthy persons and in marrow-graft recipients. Thus, the CDC assay is a reliable method for monitoring T-cell subsets, allowing detection of lymphocytes carrying low densities of membrane determinants.

The thymus in "bare lymphocyte" syndrome: significance of expression of major histocompatibility complex antigens on thymic epithelial cells in intrathymic T-cell maturation.

Thymic biopsies from two patients with combined immunodeficiency and defective expression of HLA class I and class II antigens on blood mononuclear cells ("bare lymphocyte" syndrome) were investigated. This made possible an evaluation of the significance of HLA antigen expression in a detailed (immuno)histologic study. Both thymuses showed a normal lobular architecture with distinct cortex-medulla areas, well-differentiated epithelium, including ultrastructurally defined subtypes, and Hassall's corpuscles. Normal numbers of lymphoid cells were present and normal T-cell phenotype was found. Using anti-HLA-A,B,C antisera, confluent staining of the medulla (stroma and lymphocytes) was observed. One of the thymuses was found to be negative for HLA class II antigen expression: the other revealed only HLA-DR positivity of nonlymphoid cells in the medulla. These cells were not of epithelial nature as judged from double staining with anti-keratin antibody. There was no expression of HLA-DC/DS. These observations differ from findings in the normal thymus, wherein epithelial cells in the cortex carry HLA class I and class II antigens, and epithelial cells in the medulla express HLA class I, and for a minor part class II antigens. The results indicate a normal sequential acquisition of T-cell differentiation antigens in the thymus of both cases. It is suggested that the expression of HLA class I and class II antigens on epithelial cells in the normal thymus cortex does not play a significant role in the sequential acquisition of differentiation antigens on T lymphocytes.

HLA and GM in insulin-dependent diabetes in the Netherlands: report on a combined multiplex family and population study.

This report deals with the genetic factors involved in insulin-dependent diabetes mellitus (IDD) in The Netherlands. Twenty-two Dutch multiplex families with IDD were typed for HLA-A, -B, -C, and -DR antigens, for BF, C2, C4, and GLO polymorphisms, as well as for GM allotypes of immunoglobulins. In addition, 53 unrelated IDD children and 31 unrelated patients with adult onset IDD were typed for HLA-A, -B, -C, and -DR antigens. A significant heterogeneity for the frequency of HLA-DR4 related to age of onset was observed. A significant deviation of the Hardy-Weinberg equilibrium was observed for the HLA-DR locus with an excess in patients of heterozygotes HLA-DR3, -DR4.HLA-B8, and HLA-B15 were not only secondary associated, but constituted with HLA-DR3 and -DR4, respectively, a haplotype in association with IDD. Nonrandom segregation of HLA-haplotypes was observed in multiplex families exemplified by an excess of HLA-identical affected sibpairs . Cross- overs between HLA-DR and GLO identified the HLA-DR segment as mainly involved in the association with IDD. Three diabetic haplotypes were confirmed to occur frequently among affected sibs: (a) A1, B8, BFS, C2.1, C4AQO , C4B1 ,DR3, GLO2 ; (b) Aw30, Cw5 ,B18,BFF1,C2.1, C4A3 , C4BQO ,DR3, GLO2 ; (c) A2,Cw3, B15,BFS, C2.1, C4A3 , C4B3 , DR4,GLO1. The segregation of GM allotypes to affected sibpairs was not significantly different from random segregation. The main conclusions from this study are that significant heterogeneity for age of onset exists and that the data are not compatible with simple genetic models including dominant, recessive, and intermediate models of inheritance. The data do require more complex models, involving two different HLA-linked (sets of) susceptibility genes.

Campylobacter jejuni bacteraemia as a cause of recurrent fever in a patient with hypogammaglobulinaemia.

In a hypogammaglobulinaemic patient with faecal cultures persistently positive for Campylobacter jejuni it was shown that C. jejuni bacteraemia was responsible for a long history of self-limiting attacks of fever. A focus of infection outside the gastro-intestinal tract was not found. Antimicrobial treatment failed to eradicate C. jejuni. Antibodies against C. jejuni were not detectable and there was also a defect in serum bactericidal activity. In contrast with normal serum, it was shown that, when patient's serum was used in tests, IgG and the components of complement C3 and C4 did not bind to C. jejuni.

[Genetic carrier detection for the Wiskott-Aldrich syndrome using restriction fragment length polymorphism analysis]. / Draagsterschaponderzoek voor het Wiskott-Aldrich-syndroom met behulp van restrictiefragmentlengte-polymorfisme-analyse.

The gene for the Wiskott-Aldrich syndrome, an X-linked immunodeficiency disease, has been mapped between the RFLP markers DXS7 and DXS14 on the short arm of the X-chromosome. Close linkage to these markers permits accurate carrier detection and prenatal diagnosis. In one family with WAS patients in two generations, RFLP analysis was applied to three women at risk. It could be determined with more than 98.5% accuracy that these women were not carriers.

Blood transfusion suppresses cutaneous cell-mediated immunity.

Cutaneous cell-mediated immunity (CMI) evoked by dinitrochlorobenzene (DNCB) was evaluated in end-stage renal disease patients on regular haemodialysis and before renal transplantation. Twenty-seven per cent of the patients had suppression of cutaneous CMI as shown by a negative response upon DNCB challenge. We analysed seven factors known or postulated to have an influence on renal allograft rejection for their effects on cutaneous CMI. Age, sex, red blood cell groups, pathogenesis of the underlying kidney disease, and HLA-DRw6 status had no direct effect on the DNCB response. The number of blood transfusions and the duration of haemodialysis were related to a decrease of the DNCB response but were at the same time correlated. By multiple regression analysis it was shown that the number of blood transfusions had a major suppressive effect on the DNCB response, whereas the duration of haemodialysis had a minor suppressive effect if any. Thus the cutaneous CMI evoked by DNCB partly reflects a general CMI response involved in allograft rejection as well. At the same time the effect of blood transfusion on cutaneous CMI restricts the application of the DNCB test for prediction of future renal allograft rejection.

Patterns of X chromosome inactivation in haematopoietic cells of female carriers of X linked severe combined immunodeficiency.

We evaluated the use of methylation analysis at the DXS255 locus as a method for carrier detection in X-linked severe combined immunodeficiency (XSCID). We also investigated the variations of X inactivation patterns in several haematopoietic cell lineages of XSCID carriers, both within and between XSCID pedigrees.

X chromosome inactivation patterns in haematopoietic cells of female carriers of X-linked severe combined immunodeficiency determined by methylation analysis at the hypervariable DXS255 locus.

The patterns of X chromosome inactivation were determined in 14 females from three unrelated X-linked severe combined immunodeficiency (XSCID) pedigrees. All the females were found to be heterozygous for the hypervariable DXS255 locus, enabling analysis of differential methylation of this locus in peripheral blood haematopoietic cells. All six obligate carriers manifested a unilateral X chromosome inactivation in the T lymphocyte population. Differential methylation analysis of T lymphocytes was subsequently applied to establish the carrier status of females at risk in the XSCID pedigrees. In the B lymphocyte population of four XSCID carriers a unilateral X chromosome inactivation was observed. Four other carriers had minor fractions and one carrier had a substantial fraction of B lymphocytes with the XSCID gene defect on the active X chromosome. Within single XSCID pedigrees the carriers manifested different patterns. In two pedigrees the granulocyte populations of all carriers showed a random distribution of X chromosome inactivation. In the third pedigree the granulocytes of the three carriers analyzed manifested complete inactivation of the X chromosome that carried the XSCID mutation, exposing a selective disadvantage of granulocytes that express the XSCID defect. The pedigree-dependent differences in the involvement of the granulocyte population suggest the existence of two distinct XSCID defects.

Polyclonal activation of human lymphocytes in vitro. I. Characterization of the lymphocyte response to a T cell-independent B cell mitogen.

The staphylococcal cell wall component protein A (SpA) and formalinized, Cowan I strain Staphylococcal organisms (STA) were compared with the lectins phytohemagglutinin, concanavalin A, and pokeweed mitogen for their ability to trigger proliferation of normal human lymphocytes, lymphocyte subpopulations, and cells from patients with primary immune deficiency diseases. SpA was found to be a potent T cell mitogen, very similar to the other lectins tested. It failed to stimulate purified non-T cells and peripheral blood lymphocytes from patients with different forms of severe combined immunodeficiency disease (SCID). STA, treated to prevent the leakage of soluble SpA during culture, exclusively stimulated non-T cells: the responding cell population was characterized to be E-rosette negative but positive for C3 receptors, surface Ia, a receptor for STA itself, and likely carried surface immunoglobulin. Normal responses to STA were found in patients with the adenosine deaminase-positive form of SCID. In 18 patients with humoral immune deficiency syndromes, the presence of STA responses was correlated with the presence of circulating, surface immunoglobulin-bearing cells. A commercial STA preparation was rendered B cell specific after reformalinization, a procedure that eliminated the shedding of soluble SpA under culture conditions.

Polyclonal activation of human lymphocytes in vitro-II. Reappraisal of T and B cell-specific mitogens.

The capacity of the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Staphylococcus protein A (SpA) to induce B cell proliferation and differentiation was compared with the B cell mitogen, formalinized Staphylococcus aureus (STA). Lymphocyte subpopulations from normal donors and patients with various immunodeficiency diseases were studied. In the presence of the T cell mitogens, irradiated T cells were capable of providing a helper cell activity that enabled co-cultured B lymphocytes to proliferate in response to these mitogens and to differentiate into IgM-secreting (direct) hemolytic plaque-forming cells (PFC). In the PFC response, radioresistant T-helper and radiosensitive T-suppressor cell activities could be demonstrated. T-suppressor cell activity outweighed helper activity only in nonirradiated co-cultures stimulated with Con A. Patients with severe combined immunodeficiency lacked mitogen-induced helper T cells, whereas patients with various forms of humoral immune deficiency were normal in this respect. These findings and the tissue distribution of the helper activity is aquired early in post-thymic T cell differentiation. The data suggest that experiments with cell lineage-specific lymphocyte mitogens should be considered in the context of more complex cell-cell interactions.

Diversity of immunoglobulin kappa light chain gene rearrangements and evidence for somatic mutation in V kappa IV family gene segments in X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency disease in man, characterized by an arrest in B lymphocyte differentiation at the precursor B cell stage. The structure of expressed immunoglobulin (Ig) kappa light (L) chain rearrangements of nine B lymphoblastoid cell lines from one XLA patient was investigated by amplification of cDNA by the polymerase chain reaction using 5' V kappa family-specific primers and a 3' kappa constant region primer. Members of all four V kappa gene families were found to be utilized in Ig kappa L chain rearrangements at frequencies that were consistent with random V kappa family usage. There was no preference for usage of any particular kappa joining segment. Additional diversity was generated by deletions and random nucleotide insertions at the site of juxtaposition. Particular V kappa members seemed to be overrepresented in the sample. The observed homology of the V kappa I, V kappa II and V kappa III region sequences, both to each other and to known germ-line V kappa sequence indicated the absence of somatic mutations in the majority of these expressed Ig genes. In contrast of the single-member V kappa IV family four different sequences were found to be expressed. That these sequences were mutated derivatives of a germ-line V kappa IV element was substantiated both by sequence analysis and oligonucleotide hybridization. This finding shows that the mutation process can occur in early stages of B cell development i.e. before H chain class switch has occurred. The presence of these mutations is probably independent of clonal expansion since XLA patients are unable to respond to antigen. We conclude that the differentiation arrest in XLA does not preclude early onset of somatic mutation events in V kappa gene segments.

Immunoglobulin heavy chain germ-line JH-C mu transcription in human precursor B lymphocytes initiates in a unique region upstream of DQ52.

From human precursor B cells which had both immunoglobulin (Ig) heavy (H) chain loci in germ-line configuration, various IgH chain germ-line transcripts were isolated and sequenced. These transcripts were shown to contain sequences derived from the JH region, the IgH chain enhancer element or the Ig switch region. A number of isolated cDNA clones contained sequences at their 5' end that were derived from a single exon located just upstream of DQ52, designated the mu o' element. Sequence analysis of a 920-bp genomic DNA segment, containing the mu o' exon and its 5' flanking region, revealed the presence of various conserved motifs for DNA-binding proteins, such as E2A, Ets, NF-kappa B and AP-2, which have previously been found in the IgH and L chain enhancers. We propose that the activity of the mu o' element, resulting in germ-line transcription of the DQ52-JH gene segment, is required to generate full accessibility for the V(D)J recombinase.