Immunoglobulin turnover in B lymphocyte subpopulations.

"In vitro" turnover of leucine-labeled and of radioiodinated IgM has been studied with cells from various lymphoid organs of nude mice, i.e. lymph nodes, thoracic duct, spleen and bone marrow, as well as with subpopulations of B cells from spleen and bone marrow separated by free flow electrophoresis. Three types of IgM-producing lymphocytes could be distinguished by their turnover rates of IgM, by the size of the released IgM and by the capacity of the IgM molecules to be labeled by the lactoperoxidase-catalyzed radioiodination reaction and/or by incorporation of radioactive leucine. Type I cells release 7-8 S IgM rapidly (t1/2 = 1-3h); the released IGM is leucine-labeled and radioiodinated. Type II cells release 7-8 S IgM slowly (t1/2 =10-30); the released IgM is leucine labeled and radioiodinated. Type III cells release 19 S IgM rapidly (t1/2 =2-4 h); the released IgM is leucine labeled, but not radioiodinated. Lymph nodes and thoracic duct contain predominantly type II cells, bone marrow contains type I and II cells, spleen contains type I,II and III cells. It is suggested that type III cells are Ig-secreting plaque-forming plasma cells, type II cells are small, resting "memory" B cells, and type I cells may be newly formed antigen-inexperienced B cells.     

A phenotypic study of B lymphocyte subpopulations in human bone marrow.

The regulatory mechanisms that monitor the size of the peripheral B cell pool and determine cell death or survival are poorly understood. In rodents B lymphopoiesis is maintained at a high rate throughout adult life, and under resting conditions there is little recruitment into the long-lived peripheral pool; it therefore follows that most newly formed B lymphocytes have a very short lifespan. The maturation stages of B lymphopoiesis in humans and in experimental mammals appear to be similar. We have determined the phenotype of sIgM- and sIgD-expressing cells from normal adult human bone-marrow and peripheral blood by dual immunofluorescence with an extensive panel of monoclonal antibodies representative of major B cell clusters, in order to identify antigenic differences that may play a regulatory role. Antibodies of the CD21, CD22 and CD9 clusters, the unclustered restricted B antibody 7-F-7 and anti-IgD were reactive with different proportions of sIgM+ cells in blood and bone marrow; 29.5% (range 5-60%) of sIgM+ cells in marrow were sIgD- and most of these cells were also CD21- and CD22-, thus defining a unique marrow population. However, newly formed and mature re-circulating cells comprising the sIgM+sIgD+ population could not be distinguished by the panel of antibodies.     

T lymphocyte subpopulations and B lymphocyte cells in caecum and spleen of chicks infected with Salmonella enteritidis

The effect of low and high doses of Salmonella enteritidis PT4 (SE) on immunocompetent cells in caecum and spleen of one-day-old chicks was investigated. Subsets of T lymphocytes positive for CD3, CD4, CD8 and B lymphocytes (Bu1b-positive cells) were counted in the caecum after immunohistochemical staining and the relative percentage of these cells in the spleen was analysed using a FACScan cytometer on days 7, 10, 14, 21, and 27 post-inoculation (pi). In the low dose group, the number of CD3+ and CD4+ cells in the caecum had significantly increased at day 10 pi. Both CD8+ and Bu1b+ cells were significantly higher on day 14 pi in this group. In the high-dose group, the number of CD4+ cells had significantly increased at day 7 pi. CD3+, CD8+, and Bu1b+ cells showed prolonged proliferation at days 7 up to 21 pi. Splenic lymphocytes demonstrated significant changes only in the high dose group. The percentage of splenic CD4+ cells was decreased at day 7 pi. A decrease in CD3+ and CD8+ cells was found at day 14 pi in this group.     

Beta adrenergic receptors in lymphocyte subpopulations

To further evaluate the potential utility of lymphocyte beta adrenergic receptor assays in the study of receptor alterations in human disease, we studied highly purified populations of B and T lymphocytes in peripheral blood to see if differences existed in the concentration or affinity of beta adrenergic receptors and catecholamine-responsive cAMP levels. The mean number of receptors present in particulate fractions of B cells did not differ significantly from the number found in T cells. Similarly, no significant difference in the dissociation constant for (-)[3H]dihydroalprenolol was found. Cyclic adenosine monophosphate (cAMP) accumulation in whole lymphocytes as measured by radioimmunoassay was comparable, although a tendency toward lower basal and stimulated levels in the T cells was evident. The data suggest that differences observed in concentrations of beta adrenergic receptors or catecholamine-responsive cAMP accumulation in lymphocytes from patients with varying illnesses are not likely to be due to differences in the proportions of circulating B and T lymphocytes.     

Circulating lymphocyte subpopulations in Hashimoto thyroiditis

Peripheral blood and T and B lymphocytes and [¹²⁵I]thyroglobulin-binding lymphocytes were investigated in twenty-two euthyroid Hashimoto thyroiditis patients and in twenty-two age- and sex-matched normal subjects. Although the total lymphocyte count in Hashimoto patients (mean±SEM = 1226±187/mm³) was lower than in normal subjects (1603±156/mm³) this difference was not statistically significant. There was, however, a statistically significant reduction in the proportion of circulating T lymphocytes in the Hashimoto patients (mean±SEM = 57·4±2·5%) as assessed by the sheep red-cell rosette method when compared with the normal controls (mean±SEM = 66·7±1·8%). The proportion of B lymphocytes in the peripheral blood as assessed by indirect immunofluorescence, was not significantly different being 21·6±2·1% in the Hashimoto patients and 20·2±1·1% in normal subjects.

[¹²⁵I]thyroglobulin-binding lymphocytes, as assessed by autoradiography were present in the circulation of nineteen Hashimoto patients with a mean frequency of 8·37±1·15/10⁴ lymphocytes and in thirteen normal subjects with a mean of 8·84±0·93/10⁴ lymphocytes. There was no difference in the degree of [¹²⁵I]thyroglobulin binding between the two groups as determined by grain count analysis. There was no apparent correlation between age or thyroglobulin antibody titres and the frequency of [¹²⁵I]thyroglobulin-binding lymphocytes. Thyroglobulin-binding lymphocytes were increased 100-fold in a Hashimoto thyroid biopsy in comparison to the patient's peripheral blood.

     

Reference values for T and B lymphocyte subpopulations in Turkish children and adults

Background/aim Established reference values are critical for the interpretation of immunologic assessments. In particular, the proportion and absolute counts of T- and B- cell subpopulations are subject to change with age and ethnicity. We aimed to establish age-specific reference values for lymphocyte subsets using updated immunophenotyping panels.Materials and methods We studied a total of 297 healthy Turkish subjects aged 0 to 50 years, stratified into major age brackets in a cluster factor of 10 per age-group. The predetermined age intervals contained randomly allocated participants enrolled over a period of 6 months, who were homogenously distributed by sex. We analyzed a complete blood count test and simultaneously with detailed immunophenotyping enumerated the percent and absolute cell counts of lymphocyte subsets. Results The percentage and absolute counts of lymphocyte subsets show a marked surge across the age-span. T helper, T cytotoxic, and the natural killer cell numbers were increasing from birth until 6 months, followed by a gradual decrease thereafter. B cell numbers were rising until 2 years, followed by a gradual decrease for the upcoming years, accompanied by a steady expansion of unclass-switched- and class-switched- B cells.Conclusion We provide updated extensive reference intervals for lymphocyte subpopulations in Turkish people.     

Development of lymphocyte subpopulations in preterm infants

In preterm neonates the immune system is thought to be less developed at birth, but very little is known about the actual size of lymphocyte subpopulations, and even less about the maturation of these subpopulations during the first months after a premature birth. To evaluate the development of lymphocyte subpopulations in preterm infants during the first 3 months after birth, we performed a prospective longitudinal study in two hospitals in the Netherlands. Preterm neonates (n = 38) of all post-menstrual ages were included and blood samples were taken from cord blood, and at 1 week, 6 weeks, and 3 months. Lymphocyte subpopulations were measured by four-colour flow cytometry. The data were compared with follow-up data obtained in healthy term neonates (n = 8), and with single samples from school age children (n = 5) and adults (n = 5). Overall, we found a similar pattern of post-natal development of lymphocyte subpopulations in the term and preterm infants. Both B lymphocytes and helper and cytotoxic T lymphocytes mainly consist of naive cells at birth and during the 3 months of follow-up in all neonatal age groups. So, the preterm immune system seems to be able to generate an outburst of naive T and B lymphocytes from the thymus and bone marrow within the same time span after the start of post-natal antigenic stimulation from the environment as the term immune system, but, with lower post-menstrual age, the absolute counts of naive helper T lymphocytes are lower.     

Differentiation of human B lymphocyte subpopulations induced by an alloreactive helper T-cell clone

We have used cloned alloreactive helper T cells to determine if direct T cell-B cell interaction can induce differentiation of human peripheral blood B cells which do not respond to pokeweed mitogen (PWM). T-cell clone 2F8 was derived from a one-way mixed lymphocyte reaction. 2F8 cells are T3⁺T4⁺T8^–IL-2R⁺ and proliferate in response to irradiated stimulator cells, but not autologous cells, in the absence of exogenous interleukin-2. 2F8 cells provide allospecific help for polyclonal proliferation and differentiation of B cells in the absence of any other stimulus. The magnitude of this response is comparable to that of the response of the same B cells to PWM and fresh autologous T cells. 2F8 cells could also provide nonspecific help for unrelated donor B cells in the presence of PWM, with no requirement for costimulation by irradiated stimulator cells. Allospecific stimulation of B cells was completely inhibited by antibodies to class II major histocompatibility complex (MHC) framework determinants and was abrogated by 1000-rad irradiation. Cloned 2F8 T cells stimulated differentiation of both small, high-density B cells and larger B cells, generating up to 30% plasma cells with either fraction. B cells forming rosettes with mouse erythrocytes were also induced to differentiate by the helper T cell clone. As found previously, neither small, high-density B cells nor mouse rosette⁺ B cells responded well to PWM. Direct interaction with allospecific T cells induces differentiation of a broader spectrum of B cells than soluble growth and differentiation factors in conjunction with polyclonal activators such as PWM and protein A containing staphylococci.     

Sephadex G-10 columns do not retain selectively T or B lymphocyte subpopulations

The passage through Sephadex G-10 columns of lymphocytes prepared from different lymphoid compartments of man, guinea pig and rabbit, did not result in a selective loss of lymphocyte subsets, as assessed by various rosetting assays. This lack of T and B lymphocyte loss was seen irrespective of whether glass beads or a fine mesh net was used as a support for the Sephadex G-10 column.     

Atopic asthma: T lymphocyte subpopulations

Atopy is associated with diminished cell-mediated immunity and increased amounts of IgE, both of which may be caused by imbalances of T lymphocyte subsets. We compared the composition of highly purified peripheral-blood T cells of fifteen atopic asthmatics with ten non-atopic control subjects. Each subject was examined on five separate occasions. Indirect immunofluorescence using monoclonal antibodies was used to define T cell subsets. We examined the proportion of T cells with T3 (most T cells), T4 (helper/inducer), T8 (suppressor/cytotoxic), M1 (natural killer), and Ia (activated T cells) surface antigens. Blood was obtained at the same time of day to eliminate the effects of circadian rhythm. Subjects were taking no medications. We found no difference between the groups of the percentage of T cells with T4, T8, M1, and Ia antigens, nor the ratio of T4+ (helper) to T8+ (suppressor) cells. T3 percent was slightly (94.3 vs 92.5%) higher in the atopic group. We conclude that atopic asthma is not associated with imbalances of peripheral-blood T cell subsets.     

Alterations in lymphocyte subpopulations in copper-deficient mice.

Analyses of cell surface determinants of splenocytes from copper-deficient C58 mice indicate alterations in lymphocyte subpopulation characteristics. Both the absolute number and the relative percentage of surface immunoglobulin-bearing (B) cells from copper-deficient mice were significantly greater than those from copper-supplemented controls. The relative percentage of Thy 1.2-positive (T) cells was decreased, and the decrease was most prominent within the Lyt 1-positive (helper) T-cell subset. The functional responsiveness of both B cells and T cells was decreased in copper deficiency.     

Immunoglobulin production by lymphocyte hybridomas.

We have investigated immunoglobulin production by cloned hybrid cells. gamma2b heavy chains, but not gamma1 or gamma21, exist in two forms, differing in molecular weight, galactose labeling and charge. Hybrid molecules containing gamma2b and gamma1 heavy chains were secreted by hybridoma cells synthesizing Ig molecules of both subclasses. However, cells synthesizing both IgM and IgG chains did not secrete mu-gamma hybrid molecules. We have surveyed many new hybridomas derived from different mouse strains: 10% of those secreting IgM produce a mu chain which is about 5000 daltons larger than normal mu. Two IgM-secreting lines were studied in detail and were found to bear mu but not delta in their membrane. Intra- and extracellular mu differ in molecular size; pentameric IgM-containing molecules of both sizes were observed in the membrane fraction.     

Blood lymphocyte subpopulations in extrinsic and intrinsic asthmatics

Blood leukocyte numbers and proportions of T lymphocyte subsets were studied in extrinsic asthmatics (EA), intrinsic asthmatics (IA), IA systemically treated with corticosteroids (IA + C), and in age-matched control subjects. The EA and IA showed an increased number of eosinophils. During corticosteroid therapy of IA, the eosinophil number remained elevated, whereas there was a slight decrease in the number of circulating lymphocytes. The proportion of T cells of the suppressor/cytotoxic phenotype carrying the Leu-2a antigen was significantly lower in the IA than in all other groups. In the IA + C group, the proportions of Leu-3a/3b and Leu-2a positive T lymphocytes returned to normal, although the patients still exhibited asthmatic symptoms. These findings suggest that cellular immunologic factors might be involved in the pathogenesis of intrinsic asthma.     

Peripheral lymphocyte subpopulations in human falciparum malaria.

The concentration of circulating T, B, and 'null' lymphocytes was determined in thirty children and three adults with Plasmodium falciparum infections in West Africa. During infection, both percentage as well as concentration of T cells were decreased as compared to levels following treatment. The percentage but not concentration of B cells was increased. Both percentage and concentration of 'null' cells were increased in malaria. Patients with splenomegaly had the most severe alterations in T-cell number; no other historic or clinical parameter correlated with the degree or pattern or change in circulating lymphocyte subpopulations. These alterations were rapidly reversible after antimalarial treatment and presumably represent the sequestration of T cells in the spleen or other organs.     

T lymphocyte subpopulations in congenital cytomegalovirus infection.

T lymphocyte subpopulations in the peripheral blood of children with congenital cytomegalovirus infections and in controls were enumerated with monoclonal antibodies. Infants of less than 1 year of age with symptomatic infections showed significant increases in the proportion of suppressor cells and decreases in the ratio of helper to suppressor cells, whereas T lymphocyte populations in older symptomatic patients and asymptomatic infants and children did not differ from those in controls.     

Changes in T and B lymphocyte subpopulations before, during and after chemotherapy for malignant melanoma

The behaviour of T and B lymphocyte subpopulations before, during and after chemotherapy with DTIC or CCNU was studied in 87 melanoma patients. The effect on immune status of DTIC administered as adjuvant therapy was also reported. The immunosuppressive effect was found to be higher in patients with normal pretreatment T-cell values than in patients with low pretreatment values, and it was higher in metastatic than in nonmetastatic patients. Where T and B lymphocyte subpopulations were assessed in 10 metastatic patients after each course of chemotherapy, a progressive reduction of T-lymphocytes was observed, indicating a cumulative effect of the drugs with advancing course. The reduction of T lymphocytes was mainly due to the decrease of OKT4+ cells. A normalization of T lymphocyte subpopulations was observed after the suspension of therapy. No significant decrease in the number of B cells was observed in the DTIC treated metastatic patients, whereas a significative reduction in the absolute number was found in those treated with CCNU.