Traffic of T and B Lymphocytes in the Normal Spleen

The occurrence of C3b and C3d complement receptors and of Fc and E receptors in normal splenic tissue was studied in cryostat sections incubated with specific marker cells. Thereby the topographic distribution of T and B lymphocytes in the splenic tissue was mapped: T^E lymphocytes are found especially in the periarteriolar sheaths, B lymphocytes in eccentric zones around the arterioles. The cells in the splenic pulp carry especially Fc, but also C3b receptors, whereas C3d receptors appear to be specific of lymphocytes. The basic lymphocyte traffic behind the mapped distribution of the lymphocyte subpopulations in the spleen was elucidated by lymphocyte kinetic studies using autologous ⁵¹Cr-labelled reinfused lymphocytes. These studies were performed partly on normal persons and partly on splenectomized persons without immune defects. The results indicate the presence of an exchangeable pool of T as well as of B lymphocytes in the spleen, the T cell pool being larger and making up in normals about 30 % of the total exchangeable T lymphocyte mass. The matrix for the T lymphocyte pool in the spleen is the periarteriolar sheaths. The difference in structure between the lymph nodes and the lymphoid tissue of the spleen, including differences in the distribution of lymphocyte subpopulations, are explained by the special vascular arrangement in the spleen.     

Semiquantitative determination of Cc and C3 receptors on human lymphocytes by isotope-labelled marker cells.

The number of Fc and C3 receptors on normal B lymphocytes, isolated from blood, was assessed semiquantitatively by a new method in which the marker cells (pretreated sheep erythrocytes) for Fc and C3 receptors are labelled by 99Tc and 51Cr respectively. The mean number of bound marker cells per rosette was assessed simultaneously by measurements of radioactivity. Control studies showed that a measuring system using 1/4 agglutinating titre of antibody on the marker cells is particularly advantageous: (1) Fc and C3 receptors are not demonstrable on T lymphocytes; thus, B lymphocyte fractions with Fc and/or C3 receptors are well-defined. (2) The binding affinity of the marker cells for normal lymphocytes is so weak that the number of bound marker cells per rosette rarely reaches the maximum which was calculated and measured as about 30 marker cells per rosette; thus, the measuring system is sensitive to changes in the number of receptors. (3) By binding of marker cells to lymphocytes bearing Fc as well as C3 receptors it has been demonstrated that interference phenomena between the two marker cells are not operative. Studies of blood lymphocytes from normal human subjects showed B lymphocytes carrying either Fc and C3 receptors as well as a fraction of B lymphocytes having both Fc and C3 receptors. The latter maker up about 6% of the lymphocyte mass. Comparative assessment of the quantitative distribution of these receptors indicates a developmental sequence, B lymphocyte with Fc receptors evidently developing, via B lymphocytes with Fc as well as C3 receptors, into B lymphocytes having C3 receptors only.     

Semiquantitative Determination of Fc and C3 Receptors on Human Lymphocytes by Isotope-Labelled Marker Cells

The number of Fc and C3 receptors on normal B lymphocytes, isolated from blood, was assessed semiquantitatively by a new method in which the marker cells (pre-treated sheep erythrocytes) for Fc and C3 receptors are labelled by ⁹⁹Tc and ⁵¹Cr respectively. The mean number of bound marker cells per rosette was assessed simultaneously by measurements of radioactivity. Control studies showed that a measuring system using 1/4 agglutinating titre of antibody on the marker cells is particularly advantageous: (1) Fc and C3 receptors are not demonstrable on T lymphocytes; thus, B lymphocyte fractions with Fc and/or C3 receptors are well-defined. (2) The binding affinity of the marker cells for normal lymphocytes is so weak that the number of bound marker cells per rosette rarely reaches the maximum which was calculated and measured as about 30 marker cells per rosette; thus, the measuring system is sensitive to changes in the number of receptors. (3) By binding of marker cells to lymphocytes bearing Fc as well as C3 receptors it has been demonstrated that interference phenomena between the two marker cells are not operative. Studies of blood lymphocytes from normal human subjects showed B lymphocytes carrying either Fc or C3 receptors as well as a fraction of B lymphocytes having both Fc and C3 receptors. The latter make up about 6 % of the lymphocyte mass. Comparative assessment of the quantitative distribution of these receptors indicates a developmental sequence, B lymphocyte with Fc receptors evidently developing, via B lymphocytes with Fc as well as C3 receptors, into B lymphocytes having C3 receptors only.     

Distribution of B,T, and O Lymphocytes in Blood and Tissues of Normal Humans Reflecting a Kinetic Model

The occurrence of the lymphocyte subpopulations T^E, B^Fo, B^C3, B^{Fc + C3}, B^Ig, and O^-, percentage as well as absolute, was studied in lymphocyte suspensions from tonsils, abdominal lymph nodes, spleens, bone-marrow aspirates, and at the same time in venous blood. The absolute lymphocyte content (number of lymphocytes per g tissue) was highest in the abdominal lymph nodes, lower in the spleens and tonsils, and lowest in the blood. T^E lymphocytes were found in the significantly highest percentage, 60%, in the blood. B lymphocytes, comprising B^Fc+C3 plus B^Ig, were present in the highest percentage in the bone marrow: 74%. Tonsils, spleens, and abdominal lymph nodes contained fewer B lymphocytes, and the blood fewest: 39%. A significant correlation was found only between the absolute numbers of T and B lymphocytes. A relationship between the absolute number of T lymphocytes and the total number of B lymphocytes as well as fractions thereof was thus demonstrated in the various tissues and in the blood and also between the blood and the tissue. O^- lymphocytes were found in bone marrow, lymph nodes, and spleens, apparently as markerless precursors of other subpopulations. The main conclusion of the study is: In the lymphocytokinetic system the T lymphocytes must play a guiding role as an afferent vector, trigging the B fractions which thus constitute the efferent vector of the system.     

51Cr Labelling of Normal Human T and B Lymphocytes for Kinetic Studies in Vivo

Lymphocyte traffic between blood and tissues was assessed by ⁵¹Cr labelling of lymphocytes and subsequent autologous reinfusion in 10 normal elderly persons. The technique for isolation and platelet depletion of lymphocyte suspensions is described. By the labelling procedure used about 70 μCi ⁵¹Cr may be incubated in about 100 million lymphocytes. This permits measurement of lymphocyte-bound radioactivity on the T and B fractions separately. The blood disappearance curves for labelled lymphocytes indicate the existence of exchangeable pools in the tissues of T as well as of B lymphocytes, that of the T lymphocytes being apparently larger. A characteristic finding in the blood disappearance curves for total lymphocytes is an increase in lymphocyte-bound radioactivity in the blood 4–6 h after reinfusion, designated reappearance. The disappearance curve of the B lymphocytes shows reappearance 4–10 h after reinfusion, whereas that of the T lymphocytes falls exponentially without any recordable reappearance. On the basis of the disappearance curves and a knowledge of the topographic distribution of T and B lymphocytes in the lymphoid tissues, a model of T and B lymphocyte traffic in the lymph nodes is discussed. This model operates with T and B lymphocyte passage by way of postcapillary venules and describes the migration in and around the germinal centres. The T lymphocytes in the periphery of the germinal centres are assumed to derive mainly from the afferent lymph, whereas the B lymphocytes in the centres are exchanged with lymphocytes in the blood in an exchangeable pool. The functional implications are discussed.     

Enhancement of antibody responses by IgD-binding factors induced by anti-IgD treatment of spleen cells.

Treatment of normal BALB/c splenocytes with anti-mouse IgD antibodies at 0 degrees C followed by incubation of the cells at 37 degrees C resulted in the formation of soluble factors that selectively inhibit rosette formation of lymphocytes bearing Fc delta receptors with IgD-coated erythrocytes (i.e., IgD-binding factors). Treatment of the same cells with anti-IgM antibodies failed to induce the formation of the factors. Analysis of cellular mechanisms indicated that polymerized surface IgD on B cells, as well as surface IgD-anti-IgD complexes shed from the B cells, induced T cells bearing Fc delta receptors to form IgD-binding factors. The factors formed by the anti-IgD treatment of splenic lymphocytes are composed of molecular mass species of 70 and 34 kDa, as estimated by gel filtration. Both the 70- and 34-kDa IgD-binding factors enhanced IgM and IgG1 plaque-forming cell responses of sheep erythrocyte-primed mouse spleen cells to the antigen.     

Studies of Lymphocyte Proliferation in Hairy Cell Leukaemia: Activity in Mixed Lymphocyte Reaction and Responses to Mitogens

Subpopulations of splenic lymphocytes from patients with hair cell leukaemia (HCL) were compared with similar subpopulations of lymphocytes from reference individuals for their ability to respond to mitogens and to participate in allogenic and autologous mixed lymphocyte reactions. T cell enriched subpopulations were obtained by double passage of mononuclear cells through mylon wool columns. Non-T cell subpopulations were collected by eluting adherent cells from nylon wool columns and by incubating them with sheep erythrocytes followed by density gradient centrifugation. Unfractionated mononuclear cells, T enriched and non-T subpopulations were compared. Enriched T cell subpopulations from HCL and reference patients responded similarly to allogeneic antigens and phytohaemagglutinin. Splenic non-T cells from reference patients produced a stronger stimulus in the allogeneic mixed lymphocyte reaction than did the unfractionated or the T enriched cells. In contrast, the non-T subpopulations from patients with HCL produced a reduced response compared to that of reference splenic cells when mixed with allogeneic lymphocytes. In addition, non-T cells from HCL patients failed to respond to pokeweed mitogen. Neither reference nor HCL splenic cells produced a significant response in the autologous mixed lymphocyte reactions. The data suggest that splenic non-T cells from patients with HCL either suppress the stimulatory capacity of normal B lymphocytes or fail to stimulate allogeneic lymphocytes in the mixed lymphocyte reactions.     

Possibilities of differentiated stimulation of B- and T-cells (author's transl)]

With the aid of the lymphocyte transformation test (LTT) using different T and B cell mitogens it is possible functionally to examine and differentiate various subpopulations of lymphocytes in man. Normal values of clinically healthy persons were evaluated. The T cell type reactive to Phytohaemagglutinin (PHA) has been found most frequently in the blood (mean transformation rate 79%), less frequently in the spleen and very seldom in the thymus. In 3 patients with infectious mononucleosis a B lymphocyte subpopulation stimulated by Lipopolysaccharide (LPS) was demonstrable in their peripheral blood, being nearly absent in normal persons, but clearly evident in the spleen of splenectomized patients and of 57 Bl-mice. Peripheral leucocytes of healthy persons, however, were able to be transformed into either blast cells by anti-isiotypic immunoglobulin sera or plasma-cell-like cells, while chronic lymphocytic leucamia lymphocytes of several patients reacted in a different way--may be, because of a different degree of insufficiency of these cells. For clinical use it should be possible by means of the LTT to evaluate changes in T and B cell transformation caused by disease or by treatment. This could be useful for differential therapy.     

The effects of splenic irradiation on lymphocyte subpopulations in chronic B-lymphocytic leukemia

We describe the effect of splenic irradiation (SI) (0.5-1 Gy weekly) on lymphocyte subpopulations for 7 patients with progressive B chronic B-lymphocytic leukemia (B-CLL). Using specific cellular characteristics we could distinguish normal from abnormal cells. The irradiation resulted in a decrease of lymph node size, reduction in spleen volume and decrease in peripheral blood lymphocytes. The one exception was a patient with a prolymphocytoid transformation of B-CLL. For 3 patients SI had to be interrupted or stopped because of severe cytopenia. Quantitation of malignant B cells and normal T lymphocytes revealed that the total irradiation dose which resulted in a specific decrease of malignant lymphocytes varied from patient to patient. Normal T-cell subpopulations, which were increased before SI, decreased to normal or abnormally low values during SI. In previously untreated patients, natural killer (NK) cell numbers decreased more rapidly than T-cell subpopulations. For 2 patients refractory to chemotherapy an increase of NK cells was observed upon SI.     

Human Lymphocyte Populations in Blood after Tetanus Vaccination

The content of circulating T and B lymphocytes in 9 healthy volunteers was estimated before and after secondary vaccination with tetanus toxoid. E, Fc, and C3 receptors were demonstrated by rosette techniques and surface Ig by fluorescence technique. The absolute number of T and non-T (B) lymphocytes was simultaneously increased during the first 48 h and at 144 h after vaccination. The correlation between paired T and non-T (B) lymphocyte counts was enhanced after vaccination, prolbably reflecting mechanisms governing the traffic of lymphocytes.     

Distribution of B, T, and O lymphocytes in blood and tissues of normal humans reflecting a kinetic model.

The occurrence of the lymphocyte subpopulations TE, BFc, BC3, BFc + C3, BIg, and O-, percentage as well as absolute, was studied in lymphocyte suspensions from tonsils, abdominal lymph nodes, spleens, bone-marrow aspirates, and at the same time in venous blood. The absolute lymphocyte content (number of lymphocytes per g tissue) was highest in the abdominal lymph nodes, lower in the spleens and tonsils, and lowest in the blood. TE lymphocytes were found in the significantly highest percentage, 60%, in the blood. B lymphocytes, comprising BFc + C3 plus BIg, were present in the highest percentage in the bone marrow: 74%. Tonsils, spleens, and abdominal lymph nodes contained fewer B lymphocytes, and the blood fewest: 39%. A significant correlation was found only between the absolute numbers of T and B lymphocytes. A relationship between the absolute number of T lymphocytes and the total number of B lymphocytes as well as fractions thereof was thus demonstrated in the various tissues and in the blood and also between the blood and the tissue. O- lymphocytes were found in bone marrow, lymph nodes, and spleens, apparently as markerless precursors of other subpopulations. The main conclusion of the study is: In the lymphocytokinetic system the T lymphocytes must play a guiding role as an afferent vector, trigging the B fractions which thus constitute the efferent vector of the system.     

Changes in count and function of splenic lymphocytes from patients with portal hypertension

AIM： To investigate changes in numbers and proliferative function of splenic lymphocytes in patients with hypersplenism due to portal hypertension （PH）, to provide evidence for further study of immune status of the spleen during PH. METHODS： Twelve spleens from patients with hypersplenism due to PH served as the PH group, and four spleens from cases of traumatic spleen rupture were regarded as the control group. After weighing the spleen, lymphocytes were separated and counted using a cell counting plate to calculate the lymphocyte count per gram of spleen tissue （relative quantity） and total lymphocyte count in whole spleen （absolute quantity）. The immunohistochemical SP method was used to observe the density and distribution of lymphocytes in the spleen. The MTT method was used to observe changes in lymphocyte proliferative function. RESULTS： As compared to the control group, the splenic lymphocytes in the PH group showed that： （1） There was no difference in distribution but a significant decreasein density; （2） the number of lymphocytes per gram of spleen （relative quantity） decreased significantly （0.822 ± 0.157） × 108 vs （1.174 ± 0.254） × 108, P 〈 0.01 ; （3） with the significant increase in the weight of the PH spleen （832.6 ± 278.2 g vs 211.7 ± 85.6 g, P 〈 0.01）, the total quantity of lymphocytes （absolute quantity） increased significantly （0.685 ± 0.072） × 1011 vs （0.366 ± 0.057） × 1011, P 〈 0.01 ; and （4） the proliferative function of lymphocytes was enhanced： T lymphocytes, （0.022 ± 0.005 vs 0.015 ± 0.003, P 〈 0.05）, and B lymphocytes （0.034 ± 0.006 vs 0.023 ± 0.001, P 〈 0.01）. CONCLUSION： Although lymphocyte density in the spleen decreased in patients with PH, the total quantity of lymphocytes increased because spleen weight increased greatly, along with the proliferating function. With respect to changes in lymphocytes, PH spleens may still have immune function, although it may be disordered. However,     

Lymphocyte subpopulations in adult coeliac disease.

Rosetting techniques were used to estimate T and B cell subpopulations in the peripheral blood in patients with treated and untreated adult coeliac disease and in control subjects. In patients with untreated coeliac disease, T cell numbers were significantly lower than in controls or treated patients, although there was no difference in total lymphocyte counts. There was no significant difference in B cell numbers between treated and untreated patients, and the subpopulation which increased to replace the T cells in untreated patients comprised cells not identified by B or T cell markers. Total lymphocyte counts and lymphocyte subpopulations were affected by splenic atrophy. It is suggested that these effects might be caused by the loss of lymphocytes from the gastrointestinal tract in untreated coeliac disease.     

Combined T and B Lymphocyte Marker Test in Lymphoproliferative Disorders

The combined T and B lymphocyte marker test of Mendes et al (1974) has been developed to provide an easy, reproducible test for clinical practice. It has been used to study subpopulations of peripheral lymphocytes in patients with lymphoid malignancies. Significant increases in the numbers of lymphocytes which bind both sheep erythrocytes (E) and the fixed, third component of complement (C3) have been found using this test, in the peripheral blood of all untreated lymphosarcoma (L/SA) patients and T cell acute lymphoblastic leukaemia (ALL) patients. Such increases were not found in blood from patients with chronic lymphatic leukaemia (CLL), other non-lymphoid malignancies, null-cell ALL or normal individuals. These mixed E and C3 receptor bearing cells may be Fc receptor positive and represent a separate subpopulation of lymphocytes.     

Distribution of Gc protein (vitamin D binding protein) on the surfaces of normal human lymphocytes and leukemic lymphocytes

Distribution of membrane-bound Gc protein (mGc) in normal lymphocyte subpopulations and leukemic lymphocytes was studied using a rabbit antiserum specific to Gc protein. Forty to fifty percent of the normal peripheral blood mononuclear cells were stained with anti-Gc antiserum by membrane immunofluorescence, and almost all of the B cells (B1+) and NK cells (Leu 11+) were mGc-positive. Only a few resting T cells (T3+) reacted with anti-Gc, but mGc appeared in a significant number of the T cells after activation by PHA. The distribution of mGc on leukemic lymphocytes of various types nearly corresponded to that in normal lymphocyte subpopulations.     

Characterization of peripheral blood, spleen and bone marrow cells from a patient with hairy cell leukaemia

The peripheral blood cells, spleen cells and bone marrow cells from a patient with hairy cell leukaemia were studied by means of several immunological methods and by phase contrast and electron microscopy. Both by light and electron microscopy the cells had the morphology of hairy cells. 60 % of all the peripheral blood cells, and 80 % of the spleen cells had membrane-bound IgGk immunoglobulin. 60 % of the peripheral blood lymphocytes and 90 % of the spleen cells were positive for Ia-antigens, and 80 % of the peripheral blood, and 70 % of the spleen cells had receptors for complement factor C3. The percentages of cells with receptor for the Fc part of IgC and receptors for sheep red blood cells (SRBC) were low both in peripheral blood and in spleen. Reduced numbers of peroxidase positive cells and cytotoxic plaque-forming cells were also observed as well as reduced lymphocyte responses after stimulation of peripheral blood lymphocytes with PHA, PWM, ConA, PPD and allogeneic cells. A normal antibody-dependent cell cytotoxicity (ADCC) and PHA-induced cytotoxicity was observed for the peripheral blood lymphocytes of the patients. Our results suggest that the hairy cells in our patient are derived from B lymphocytes and have a monoclonal origin.     

T- and B-lymphocyte differentiation potentials of spleen colony-forming cells

Cells that generate splenic colonies within 8 days (day-8 colony- forming units-spleen [CFU-s]) are generally thought to differentiate only into erythroid/myeloid cells. The T and B lymphocyte differentiation potentials of day-8 CFU-s were evaluated and compared with those of day-12 and 5-fluorouracil (5-FU) CFU-s. This was achieved by analyzing, after intravenous and intrathymic injection, the lymphocyte progeny of cells contained within individual splenic colonies collected at day 8 and day 12 post-bone marrow cell transfer into irradiated congenic recipients. A large majority of day-8 spleen colonies generated T cells when transferred intrathymically. After intravenous (IV) injection of day-8 colonies, donor-type thymocytes emerged in 33% of the animals reconstituted with only 1 day-8 colony, but in 83% of those inoculated with a pool of 5 colonies. All post-5-FU and 75% of day-12 colonies gave rise to thymocytes after IV injection. B cells were generated by a high proportion of day-8 colonies, and by all day-12 and post 5-FU colonies. These results demonstrate that progenitors of T and B lymphocytes are generated within spleen colonies produced by at least some day-8 CFU-s and virtually all day-12 CFU-s. Whether these progenitors are CFU-s themselves or committed precursors remains an open question.     

Lymphocyte subpopulations in rheumatoid synovial tissue.

Synovial tissue obtained at synovectomy of the knee joint in 21 patients with rheumatoid arthritis contained a significantly lower proportion of T lymphocytes as measured by spontaneous rosette formation with nonsensitized sheep red blood cells than did synovial fluid or blood from the same patients. There was on concomitant increase in synovial tissue lymphocytes with B-cell markers such as surface immunoglobulin or Fc fragment receptors. Removal of lymphocyte receptors with trypsin followed by culture to allow new receptors to form, led to an increase in rosette forming cells, suggesting that part of the synovial cells without B- or T-cell markers may be T lymphocytes with blocked receptors.     

Immunoresponsiveness in acute babesiosis in humans

Immunoresponsiveness in patients with acute babesiosis was characterized. Lymphocyte subpopulations, mononuclear cell responses to nonspecific mitogens, and serum immunochemical values were studied in patients during acute babesiosis and after recovery (four to 40 months), controls, and seropositive individuals. Serum studies were also carried out retrospectively on stored sera from patients with babesiosis. Mean and differential lymphocyte counts and percentages of B lymphocytes were higher than in controls in patients with acute babesiosis, and levels of T lymphocytes bearing the IgG Fc receptor (T gamma) were significantly (P less than 0.01) elevated. Responses to nonspecific mitogens were suppressed during acute babesiosis. Levels of serum IgG and IgM and Clq binding were significantly increased whereas C3 and C4 levels and hemolytic activity were suppressed in acute-phase sera. The apparent paradox of an increased T suppressor/cytotoxic population in the presence of evidence of increased in vivo lymphocyte function is discussed in terms of the heterogeneity of the T gamma subpopulation.     

Fc (IgG) receptor distributions in homogeneous and heterogeneous cell populations by flow microfluorometry.

A flow microfluorometric method has been developed for quantitating the numbers of Fc receptors on individual cells. The cells were equilibrated at 0 degrees C with radiolabeled, affinity-crosslinked rabbit IgG dimers, washed, and treated with fluorescent antibodies against rabbit IgG. The stained cells were analyzed for fluorescence emission by using a fluorescence-activated cell sorter and for bound dimer molecules by using a gamma counter. Standard curves relating fluorescence emission to numbers of dimer molecules bound to cells were used to determine Fc receptor distributions on P388D1 cells, human peripheral blood lymphocytes, and normal mouse spleen cells. Essentially all of the P388D1 cells bore Fc receptors, distributed in a skewed Gaussian profile having a peak at 2 X 10(5) receptors per cell. Human peripheral blood lymphocytes and mouse spleen cells contained positive and negative subpopulations. The percentage of positive cells in human lymphocytes from different donors ranged from 50 to 25; the receptor distributions of these cells were symmetrical and similar in all donors in shape and average receptor density (4.2 X 10(4) receptors per cell). Mouse spleen cells contained 55% positive cells with nonsymmetrical heterogeneous distributions of receptor densities. These cells peaked at 1 to 2 X 10(4) receptors per cell, but significant numbers of cells had receptor densities 10- to 20-fold greater.