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The SCID mouse was investigated as a potential animal model for human multiple myeloma (MM). Duplicate samples of bone marrow mononuclear cells (BMMC) and/or peripheral blood mononuclear cells (PBMC) of six MM patients in different clinical phases and one patient with monoclonal gammapathy of undetermined significance (MGUS) were injected intraperitoneally into SCID mice. Human immunoglobulins (Ig) in the SCID sera were quantified with a light-chain isotype-specific ELISA, and their monoclonality biochemically characterized, using a sensitive immunoblotting technique after agar gel electrophoresis. Successful transplantation of bone marrow derived-tumour cells in SCID mice was obtained with BMCC of two MM patients with progressive disease. Human plasma cells were detected in the mesenteric fat tissue around the pancreas and the spleen. This model in SCID mice may facilitate studies on processes involved in tumour progression and provides a new tool for therapeutic approaches in MM.

We present data from a 10-year follow-up study of 11 children who had been infected in the neonatal period by small aliquots of plasma from a single donation. Three of the children died within the first 2.5 years of life, 5 other children died between 6.2 and 11 years after infection and 3 are alive at present. The latter children are classified as P1B (asymptomatic), P2A (non-specific findings) and P2B (neurological changes). All infected children showed progressive decline of cellular immunity. Immunoglobulin levels in serum were increased in the majority of children for prolonged periods and homogeneous immunoglobulin components were present. The severity of the disease was related neither to the clinical condition of the infants in the neonatal period nor to the volume of transfused plasma, the interval between freezing and thawing of the plasma, gestational age at birth and age at transfusion. Coinciding infections with other viruses had no impact on disease progression during the follow-up period of 10 years.

We have investigated fetal and adult T-cell receptor (TCR) A and B V-gene repertoires both by fluorescence-activated cell sorter (FACS) analysis with the available TCR V region-specific mAbs and by the polymerase chain reaction (PCR) with TCR V gene family-specific oligonucleotides. Among the low number of CD3+ T cells, most of the TCR V regions tested for could be detected by FACS analysis in liver, bone marrow, and spleen derived from a 14-week-old fetus and two 15-week-old fetuses. Similarly, the PCR analysis showed that the majority of the TCRAV and TCRBV families were expressed in the peripheral organs of the 13-week-old fetus, although an apparent absence of particular TCR V families was found in liver and bone marrow. This was most probably the consequence of the low number of CD3+ T cells in these organs. In 17-week-old fetal thymi the level of expression of some TCRAV and TCRBV gene families, in particular those that contain a single member, was lower compared to post-partum thymi and adult peripheral blood mononuclear cells. The combined data of FACS and PCR analysis demonstrate that TCR V genes belonging to the majority of TCR V gene families can be used in TCR alpha and beta chain rearrangements during early human fetal life. Our data also suggest that the expression levels of some of the single member TCR V gene families may be influenced by the developmental stage.

We have studied the usage of V beta 6, D beta, and J beta elements, and the composition of the CDR3 regions of human fetal TCR beta chain rearrangements in a 17 week old fetal thymus and in fetal liver, bone marrow, spleen, and cord blood at 11 and 13 weeks of gestation. These fetal sequences were compared with TCR beta chain rearrangements obtained from post-partum thymus, adult spleen, and adult peripheral blood mononuclear cells. Both fetal and adult TCR V beta 6 rearrangements exhibited a non-random usage of V beta and J beta elements. Up to 90% of the sequences obtained at 11 weeks of gestation used J beta 2 elements, most notably J beta 2.1. In the 13 and 17 week old fetal and in the adult tissues, J beta 1 elements were used in approximately 30% of the rearrangements while, within the J beta 2 rearrangements, J beta 2.1 and J beta 2.7 were used most frequently. Both fetal and adult TCR beta chain CDR3 regions showed non-random usage of amino acids. However, the early fetal repertoire was further limited due to the relative absence of N-regions in up to 60% of the 11 and 13 week old TCR beta chain rearrangements, resulting in smaller antigen binding sites. In fetal and adult TCR beta chain rearrangements the distribution patterns of the length of N-regions and the usage profiles of J beta elements were similar in hematopoietic and peripheral organs, suggesting no apparent preference for particular TCR beta chain rearrangements.(ABSTRACT TRUNCATED AT 250 WORDS)

Screening tests for coeliac disease may be useful to select the patients who should undergo a small intestine biopsy. It has been reported that the determination of serum levels of anti-gliadin antibodies is a good screening method for small intestine damage in coeliac disease. We have retrospectively assessed the value of the anti-gliadin antibody test in detecting villous atrophy of the small bowel. Antigliadin antibodies were measured with an ELISA technique in sera of 44 children seen at the Department of Paediatrics, Leiden University Hospital, who underwent 53 small intestine biopsies because of suspected coeliac disease. The relation between the histopathological findings of the small intestine and the results of the anti-gliadin antibody quantifications in serum were studied. In 9 of the 10 children with villous atrophy high Ig total and IgG-titers were found. Five children with villous atrophy, 2 of them with a selective IgA-deficiency, had normal IgA-AGA titers in serum. 17 Children with normal biopsies had high Ig total and IgG-AGA titers, but no child with normal small intestine biopsy had high IgA-AGA titers in serum. We conclude that the determination of anti-gliadin antibodies as screening for mucosal damage must be based on the measurement of antibodies within the IgG as well as the IgA class.

We have developed a mAb (JE-6) that recognizes an Id encoded by the most JH-proximal human VH gene segment (VH6) in or near germ-line configuration. This mAb was used to determine the frequency of Id JE6+ B cells in large collections of monoclonal EBV-transformed and short term B cell lines derived from fetal, neonatal, and adult lymphoid tissues. Moreover, we investigated the presence of Id JE-6+ Ig in sera from neonates and adults and determined the (auto)antigen binding properties of VH6-encoded IgM mAb. We detected a fivefold overrepresentation of VH6-expressing IgM producing B cells in fetal tissues, cord blood, and adult bone marrow relative to adult blood. In cord blood, but not in adult blood sera, germ-line VH6-encoded IgM molecules were readily detectable. IgM secreted by VH6-expressing B cell clones displayed highly conserved and virtually identical autoantigen binding properties, independent of the length and composition of the IgH chain CDR3 region and L chain isotype. Collectively, these results suggest that the VH6 gene and the antibodies it encodes play an important role in early human ontogeny.

The in vitro colony formation of hematopoietic progenitor cells of bone marrow samples, taken before and early after allogeneic bone marrow transplantation (BMT), was investigated prospectively. In order to circumvent culture-related and sample-related variations, a serum-free recombinant growth factor-replenished culture system was developed using T cell- and monocyte-depleted bone marrow samples. Samples of healthy bone marrow donors were used to validate the technique. The standardized culturing technique gave reproducible results, with numbers of colonies above those in conventional conditioned-medium technique. Colony formation in vitro of myelomonocytic precursor cells was found decreased in graft recipients, also after addition of growth factors, in comparison with healthy donors. The growth-promoting effect of the combination of IL-3 + GM-CSF was superior to that of either growth factor alone or conditioned medium. No effect was observed of T lymphocytes and monocytes on in vitro colony formation after bone marrow transplantation, probably as a result of functional impairment of these cells at that period after transplantation.

A rapid recovery of specific humoral immunity in the recipient of an allogeneic bone marrow transplantation (BMT) can be observed after immunization of the donor before graft sampling. This has been attributed to transfer of specific immunity from donor to recipient. However, to maintain the concept of transfer the origin of the antibody producing cells in the recipient after BMT must be demonstrated. To this end, donor-recipient pairs with differences in Gm-allotypes were selected and immunized before BMT with the neo-antigen Helix pomatia haemocyanin (HPH) according to three immunization protocols. Additionally, the recipients were immunized at day 42 after BMT. Serum samples were weekly obtained from the recipients in the first 100 d after BMT. The origin of HPH-specific antibody producing cells was assessed by two approaches: (1) determination of the Gm-allotypes of anti-HPH antibodies within a distinct IgG subclass, (2) analysis of anti-HPH antibody spectrotypes by isoelectric focusing combined with immunoblotting. The results obtained with these two approaches show concordance in most instances and led to the conclusion that the antibody producing cells are of donor origin.

Transfer of specific immunity was investigated in a group of 28 paediatric and adult leukaemia patients during the first 100 d after allogeneic bone marrow transplantation (BMT). These patients and/or their donors were immunized 7-13 d before transplantation with the recall antigen tetanus toxoid (TT) and the neo-antigen Helix pomatia haemocyanin (HPH). The recipients were booster immunized with both antigens at day 42 after transplantation. Transfer of a primary IgM and IgG response to HPH was successful in most paediatric and adult patients, but transfer of a secondary response to TT was established in only a few paediatric recipients. After booster immunization at day 42 most paediatric recipients responded with a rise in serum antibody titre to HPH as opposed to only two of 18 adult recipients. This incapability of the adult recipients to mount a secondary immune response may be related to their conditioning regimen which included Campath-IG in vivo. The results from this study indicate that transfer of immunity against recall- and neo-antigens is possible. However, the establishment of long-term memory may be affected by the regimen used to condition the graft recipient.

Growth characteristics of stromal cells, assessed as adherent cells in long-term bone marrow cultures, and of hematopoietic progenitor cells, prior to and shortly after allogeneic bone marrow transplantation (BMT), were investigated, more specifically with regard to their possible correlations. The main constituent cells of the bone marrow stroma, that is, endothelial cells, reticular cells/fibroblasts, and monocytes/macrophages, showed an as yet inexplicable increased growth in samples taken from recipients prior to BMT, as compared with the growth in samples from their healthy donors and those taken after BMT. In the third week after BMT the in vitro outgrowth of hematopoietic precursors was severely depressed, but cell numbers in the adherent layer were normal. No relationship between in vitro growth of hematopoietic precursor cells and stromal cells was observed at that time. Probably the precursor cells growing in vitro are committed progenitor cells, relatively independent of stromal influences. In the eight week after grafting, endothelial cell outgrowth in vitro was highly correlated with granulocyte-macrophage colony-forming unit (CFU-GM) colony formation and to a lesser extent with mixed-lineage colony-forming unit (CFU-Mix) colony formation. This may indicate the reappearance of cytokine-mediated influences or the reappearance of a direct interaction, for example, by cell-cell contact between stromal cells and hematopoietic progenitor cells at that time.

The in vivo antibody response to the primary T-cell dependent antigen Helix pomatia Haemocyanin (HPH) was studied, in order to detect the possible presence of a humoral immune deficiency in ageing. The IgG subclass distribution of the specific antibodies was also determined. In order to define a dose of HPH which could be used to discriminate between the responsiveness of healthy and immunocompromised individuals, we first established a dose-response curve for this antigen in 60 healthy young volunteers. Their responses were compared with the responses of a group of patients suffering from end stage renal failure. The patients who were treated with haemodialysis showed a significantly lower IgM, IgG and IgA anti-HPH antibody response after immunisation with a dose of 30 micrograms HPH, which could be restored by increasing the antigen dose. Patients treated with continuous ambulant peritoneal dialysis and a group of elderly persons, selected according to the Senieur protocol, showed no impairment of antibody formation after immunisation with 30 micrograms HPH, but in the non-Senieur elderly the anti-HPH antibody response was significantly lower. Furthermore, Senieur and non-Senieur elderly persons showed a diminished IgG2 anti-HPH antibody formation, whereas in the elderly non-Senieur individuals and in the patients with renal insufficiency, IgG1 and IgG3 anti-HPH antibodies were also diminished. This study clearly shows that the so-called age-associated immune deficiency can be the result of disease and is not necessarily due to the ageing process itself.

Twenty-one independent immunoglobulin heavy chain VH3DJH rearrangements were cloned and sequenced from livers of human fetuses at 7, 13 and 18 weeks of gestation. The VH elements expressed were not somatically mutated. Eight out of the estimated 30 VH3 elements were utilized with a preference for five of them. One of these VH3 sequences, designated FL13-28, represented a thus-far unknown VH3 gene segment. From the six functional JH elements the JH3 and JH4 segments were utilized preferentially and from the estimated 30 D segments the DQ52 element and the Dxp family were found to rearrange frequently. D elements were utilized both in normal and inverted orientation, as single copies or in D to D fusions. Addition of N nucleotides, removal of nucleotides from the coding sequences and utilization of DIR elements (D genes with irregular recombination signals) further expanded the third complementarity-determining region (CDR3) diversity. One fourth of the fetal CDR3 regions lacked N regions. Due to utilization of DQ52, the relative absence of N regions and extensive exonuclease activity operating on the D elements, the fetal CDR3 regions were significantly shorter than those found in adult B lymphocytes.

Serum levels of IgM and IgA classes and of IgG subclasses were determined and related to the presence of homogeneous immunoglobulin components (H-Ig) in volunteers equally distributed in age groups from 25 to 98 years, who all met the Senieur admission criteria for immunogerontological studies. In addition, sera of non-Senieur volunteers aged 75 years and older were included. Furthermore, the amount of IgD was determined in sera of Senieur individuals equally distributed in age groups from 15 to 98 years. In the Senieur persons, the contribution of the IgG subclasses and the IgM and IgA classes to the pool of serum immunoglobulins remained relatively unchanged during the course of ageing. In comparison with Senieur individuals aged 25-34 years, a slight increase in IgM and IgA levels was observed from the age 35 to 44 onwards and in IgG1 from the age 55 to 64 onwards. The variability of the immunoglobulin concentrations increased during ageing. The most prominent observation was the continuous decline of serum IgD starting in young adults. The non-Senieur persons differed from their Senieur age-matched counterparts mainly by the elevated IgG2 and IgA levels. During the course of ageing, H-Ig mainly of low concentration were detected at an increasing frequency in the Senieur persons and even more frequently in the elderly non-Senieur volunteers. Although in some individuals the elevation of immunoglobulin levels correlated with the appearance of H-Ig within the corresponding isotype, this relationship was not conclusive for all sera investigated. These results suggest that the rise of serum levels of individual immunoglobulin isotypes associated with ageing is usually the consequence of a polyclonal B cell activation. The occurrence of H-Ig and the decline of serum IgD in aged Senieur persons indicate that these are, at least partly, true phenomena of ageing and not always the consequence of disease.

The antigen-specific antibody secretion in vitro after immunisation with the primary T-cell dependent antigen Helix pomatia Haemocyanin (HPH) was investigated in both young and elderly individuals, who all met the health admission criteria for immunogerontological studies as detailed in the SENIEUR protocol. In addition, elderly non-Senieur persons were incorporated in this study. Young and elderly Senieur volunteers were fully comparable in terms of the occurrence of anti-HPH antibody secreting cells after in vitro simulation of peripheral blood mononuclear cells with variable doses of the antigen. In contrast, the non-Senieur elderly showed a lower number of anti-HPH antibody secreting cells in vitro. PHA-conditioned medium did enhance this in vitro response, whereas the addition of IL-2 remained ineffective. The PHA-induced T-cell proliferation was found to be somewhat impaired in elderly Senieur individuals and significantly lower in elderly non-Senieur individuals compared to young healthy persons. Using an immunofluorescence double staining technique after BrdU incorporation, the phenotype of the proliferating cells was determined. Again the total number of proliferating cells was impaired in the non-Senieur elderly. No changes in the relative contribution of CD4+ or CD8+ cells to the number of proliferating cells were found in the different age groups. On the other hand, a significantly lower number of proliferating cells with IL-2 receptor expression were detected in the non-Senieur individuals, which could account for the lack of response to IL-2 in this group. Our study clearly shows that so-called age-associated immune deficiency can be the result of disease and not necessarily of the ageing process itself.

The amplification of Variable Number of Tandem Repeats (VNTR) by the polymerase chain reaction (PCR) was used to determine the extent of chimaerism in flow sorted lymphoid and myeloid cell populations following allogeneic bone marrow transplantation (BMT). Pre-BMT screening with a set of five VNTR revealed that at least one marker was maximally informative in 95% of donor-recipient pairs. Mixing reconstruction experiments indicated that detection of 1-5% of the minor cell population in a sample of 5 x 10(3) nucleated cells is feasible. Flow sorted post-transplant peripheral blood B- and T-lymphocyte, natural killer and monocyte cell populations were subjected to PCR-VNTR marker analysis. It was shown that this procedure can be used for the early detection of engraftment and the identification of mixed chimaerism in various haematopoietic cell lineages in patients with leukaemia or severe combined immune deficiency, treated with allogeneic BMT.

Discrimination between donor and recipient peripheral blood cells through detection of the Y chromosome can be useful to document chimerism and engraftment after sex-mismatched bone marrow transplantation. Currently applied methods are hampered by the selection of cells (e.g., karyotyping of cells in metaphase) or by the fact that the detection of Y chromosome by in situ hybridization with specific probes does not allow further characterization of the cells. Although quinacrine staining of Y chromosomes can be performed on cells previously marked for membrane antigens, this staining is not fully discriminative between male and female cells. To circumvent this, a technique has been developed, in which mononuclear cells in suspension were stained for membrane antigens by the consecutive use of monoclonal antibodies and tetramethylrhodamine isothiocyanate conjugates. After the cells were spun down on slides and fixed with methanol/acetic acid and formaldehyde, in situ hybridization with a biotinylated Y-chromosome-specific DNA probe was performed. The probe was detected with avidin-fluorescein isothiocyanate and the signal was amplified by consecutive incubation with biotinylated anti-avidin and avidin-fluorescein isothiocyanate. The membrane staining for various antigens remained undisturbed during the hybridization procedure and the Y probe discriminated almost completely between male and female cells. Therefore, this approach allowed us to determine the chimerism within different subpopulations of unseparated mononuclear cells after sex-mismatched bone marrow transplantation with a sensitivity of 98% and a specificity of 100%.

Because of the increasing demand for simple and reliable techniques for the detection of low concentrations of paraproteins against a highly heterogeneous serum background, two techniques were investigated for their sensitivity: isoelectric focusing (IEF) and Wieme high resolution electrophoresis, each with subsequent blotting by diffusion. The techniques were compared using isolated mouse monoclonal antibodies (mAb) of known concentration and specificity. Wieme electrophoresis in combination with immunoblotting (IBL) or antigen-specific immunoblotting (ABL) has a detection limit of 100 ng/ml and 10 ng/ml, respectively. For IEF in combination with IBL or ABL these limits were 1000 and 30 ng/ml, respectively. For ABL, polyvinylidene difluoride (PVDF) and nylon-supported nitrocellulose (NSNC) membranes gave similar detection limits, although for IBL, PVDF is preferred to NSNC. While IEF is essential for investigating the spectrum of the antibody repertoire. Wieme electrophoresis is the most powerful technique for the detection of homogeneous immunoglobulin components (H-Ig). After separation of the proteins. IBL is fast, simple and sensitive enough for routine detection and characterization of H-Ig. However, when the antibody specificity is known, ABL should be chosen for its superior sensitivity.

Two types of T lymphocytes can be discriminated on the basis of expression of either the classical T-cell receptor (TCR) alpha beta or the more recently identified TCR gamma delta. Whereas TCR alpha beta + lymphocytes are known to respond to recognition of antigen in the context of major histocompatibility complex molecules by proliferation, lymphokine secretion, and/or cytotoxicity, the potential ligand specificities and functions of TCR gamma delta + cells have not been completely unraveled. Antibodies specific for either receptor type are important tools to elucidate the role TCR gamma delta + cells play in the immune system. They can be used to quantify TCR gamma delta + cells and TCR alpha beta + cells in normal and disease states, to isolate both T-cell subsets, and to perform in vitro functional assays. Only few antibodies reactive with common determinants on either TCR alpha beta or TCR gamma delta are available. Generally, the monoclonal antibody (mAb) WT31 is used for definition of viable human TCR alpha beta + cells. However, WT31 has recently been shown to cross-react with TCR gamma delta. We describe an mAb, BMA031, that combines the unique features of reactivity with intact viable cells and true specificity for a common determinant on the TCR alpha beta/CD3 complex. Its performance in immunofluorescence staining and immunochemistry has been compared with that of WT31 and anti-TCR gamma delta mAbs, using TCR alpha beta and TCR gamma delta expressing cells isolated from blood and bone marrow of healthy individuals and immunodeficient patients.

A deletion of the immunoglobulin heavy chain (IgH) pseudo-gamma, gamma-2, gamma-4, epsilon, and alpha-2 constant region gene segments was found to segregate unchanged in three generations of a family. The IgG1 locus on the IgH allele carrying the deletion was expressed to the same extent as its normal counterpart. One individual who was heterozygous for the deletion had an IgG2 deficiency, whereas the four other heterozygous individuals had serum levels of IgG2 and IgG4 within the normal ranges. IgA2 levels were low or below the normal range in all heterozygous individuals. The data indicate that the expression of some Ig isotypes can be decreased by hemizygous deletions, possibly due to a lower probability for switching.

Human cord blood mononuclear cells (CBMC) were stimulated in vitro with a number of T cell-dependent antigens. Antigen-induced B cell activation was measured applying a plaque-forming cell assay for the detection of antigen-specific IgM-secreting B cells. With the exception of diphtheria toxoid, the antigens ovalbumin, sheep red blood cells, Helix pomatia hemocyanin, burro red blood cells, and tetanus toxoid elicited an IgM-plaque-forming cell response in cultures of CBMC to levels obtained for peripheral blood mononuclear cells (PBMC) from adult controls. However, for each antigen used, the antigen dose optimal for the induction of a response was consistently found to be a hundred to a thousand times lower than the concentration of the corresponding antigen optimal for adult PBMC. Longitudinal studies on PBMC obtained from infants between 2 and 30 months of age revealed that a shift of the antigen dose toward concentrations needed to induce plaque-forming cells in cultures of adult PBMC occurs at approximately age 8 months. Our data indicate that various antigens can be used for the in vitro analysis of antigen-specific B cell activation and regulatory T cell functions in studies concerning the ontogeny of the humoral immune response in humans.

Rabbit sera contain little IgM. This is a major obstacle for its isolation. However, levels of IgM up to 10 mg/ml are found in rabbits infected with living trypanosomes. A simple procedure, involving gel filtration, standing in the cold, and agarose block electrophoresis is described which allows the isolation of IgM from these sera in 60% yield, with a purity of better than 90%.

In the last 20 years 32 patients with severe combined immunodeficiency (SCID) were treated at our department. The clinical and immunological findings in these patients are presented. In connection with these patients, the recent WHO classification on combined immunodeficiency diseases and the pathogenesis in several forms of SCID, as far as is known, are discussed. SCID appears to represent a very heterogeneous group of disorders, which is affirmed by the findings in several of our patients. At this moment bone marrow transplantation (BMT) is the only way to cure patients with SCID. Without this treatment the prognosis of these patients is very poor. After BMT complete recovery is achieved for the majority of patients, even in the absence of a genotypically HLA identical donor. Besides the therapeutic aspects of SCID the recent developments with regard to carrier-detection and antenatal diagnosis are briefly discussed.

Mononuclear cells from human cord blood (CBMC) are able to mount an antigen-specific IgM plaque-forming cell (PFC) response after primary in vitro stimulation with the T cell-dependent antigen ovalbumin (OA). The antigen dose-response relationship for the induction of PFC in cultures of CBMC is represented by a bell-shaped curve comparable to that found for mononuclear cells from adult peripheral blood (adult PBMC). The dose of OA optimal for the induction of a response in cultures of CBMC consistently, however, is 100-fold lower than the antigen dose optimal for adult PBMC (0.03 microgram OA/ml vs 3.0 micrograms OA/ml). Results obtained from co-culture experiments in which semiallogeneic combinations of parental/neonatal lymphocytes and monocytes were stimulated with a variable dose of OA indicate that the adherent cell (AC) plays a pivotal role in the establishment of the optimum antigen dose. From experiments using antigen-pulsed AC, it was concluded that neonatal and adult AC differ in their antigen handling capacity. In the presence of the prostaglandin synthetase inhibitor indomethacin the antigen dose-response relationship for the induction of PFC in cultures of CBMC shifts to an "adult type" of curve. From pulsing experiments it emerges that indomethacin affects the interaction between antigen and monocytes. Indomethacin causes an enhancement of the expression of HLA-DR at the surface of neonatal as well as adult AC; this can be down regulated by the addition of prostaglandin E2 (PGE2). The addition of PGE2 to cultures of adult PBMC leads to a shift of the optimal antigen dose for induction of PFC toward lower concentrations. Although higher levels of PGE2 were measured in the supernatant of cultured neonatal AC compared with adult AC, it seems unlikely that this observation can explain the distinct antigen dose-response relationship for the induction of a PFC response in cultures of CBMC.

Mononuclear cells isolated from human cord blood (CBL) of full-term neonates were stimulated in vitro with a dose range of T cell-dependent antigens, i.e. ovalbumin or sheep erythrocytes, and tested for the capacity to mount an antigen-specific plaque-forming cell (PFC) response. Both of the antigens used induced in CBL a PFC response with the same kinetics of PFC formation and of the same magnitude as found in cultures of adult peripheral blood lymphocytes (PBL). However, optimal PFC responses in CBL were obtained at a hundredfold lower concentration of the antigens compared with the optimal antigen doses for the induction of a PFC response in adult PBL. This phenomenon was further investigated with respect to the antigen dose dependency of the activation of neonatal B cells and neonatal regulatory T cells. The induction of a PFC response in CBL at antigen concentrations that were suboptimal for adult PBL showed a correlation with the particular antigen dose requirements for the activation of B cells and T helper cells in neonates. Furthermore, the findings suggest that the decrease of the PFC response in CBL stimulated with supraoptimal doses of antigen was not caused by the induction of unresponsiveness at the B cell level or by interference of pregnancy-associated substances with the PFC response, but was rather the result of the activation of antigen-specific T suppressor cells. Neonatal T suppressor cells were activated at antigen concentrations that generated T helper activity in the adult. Thus, although neonatal B cells possess the intrinsic capacity to mature into antigen-specific PFC, the conditions for effective activation of neonatal T cells regulating the B cell response differ from those for the activation of adult regulatory T cells.

A dot-immunobinding assay on nitrocellulose membranes has been developed for the quantification of human IgG subclasses using subclass-specific monoclonal antibodies. The advantages of this technique can be summarized as follows: (1) possibility of rapid semi-quantitative evaluation and/or precise quantitation from the same dot-pattern; (2) simple procedure with very good reproducibility; (3) sensitivity for nanogram concentrations of individual subclasses, therefore applicable not only to serum but also to other body fluids with a low content of IgG; (4) very small amounts of test material needed; (5) very good correlation of results with other techniques (ELISA, radial immunodiffusion) but without some of the inherent problems of the latter methods.