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.     

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.     

B cells promote inflammation in obesity and type 2 diabetes through regulation of T-cell function and an inflammatory cytokine profile

Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.     

In vitro regulation of immunoglobulin synthesis after human marrow transplantation. II. Deficient T and non-T lymphocyte function within 3-4 months of allogeneic, syngeneic, or autologous marrow grafting for hematologic malignancy

Immunoglobulin secretion was studied in 37 patients between 19 and 106 days after allogeneic HLA-identical (30 patients), allogeneic one HLA- haplotype-identical (three patients), syngeneic (three patients), or autologous (one patient) marrow grafting. E rosette-positive (T) and E rosette-negative (non-T) peripheral blood mononuclear cells were cocultured with pokeweed mitogen for 6 days. Polyvalent immunoglobulin secretion was determined by counting plaque forming cells in a reverse hemolytic plaque assay. The number of antibody secreting cells in cocultures of autologous T and non-T lymphocytes was low in 40 of 44 tests conducted on samples from the 37 patients. Mononuclear or non-T cells from 38 of 40 tests failed to produce antibody when cultured with normal helper T cells. T cells from 23 of 37 tests failed to help normal non-T cells secrete antibody. T lymphocytes from 23 of 41 tests suppressed antibody production greater than 80% by normal T and non-T cells. The suppressor cells were radiosensitive in 17 of the 25 tests. The abnormal function of lymphocyte subpopulations in patients during the first 3 mo after syngeneic, allogeneic or autologous marrow grafting was similar regardless of the type of graft or the presence of acute graft versus host disease.     

支气管哮喘患者记忆性T淋巴细胞对B淋巴细胞产生IgE作用的研究

目的 探讨记忆性（ＣＤ４＾＋ＣＤ４５ＲＯ＾＋）Ｔ淋巴细胞在支气管哮喘的发病中的作用。方法 分别分离出支气管哮喘病人及健康对照的ＣＤ４＾＋ＣＤ４５ＲＯ＾＋Ｔ淋巴细胞亚群,并将其与各自的Ｂ淋巴细胞共同培养,设定刺激组（美洲商陆有丝分裂原）及非刺激组,测定培养上清液中ＩｇＥ的含量。结果 哮喘病人自然状态的ＣＤ４＾＋ＣＤ４５ＲＯ＾＋Ｔ淋巴细胞对Ｂ淋巴细胞产生ＩｇＥ有正向促进作用,但有接受美洲商陆有丝分裂原     

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.     

DNA-synthesizing T and non-T cells in chronic lymphocytic leukemia

Summary In 21 patients with chronic lymphocytic leukemia (CLL) and in 8 hematologically normal persons the number of DNA-synthesizing peripheral blood lymphocytes was investigated by autoradiographic techniques. The lymphocytes were differentiated by En-rosette tests into T and non-T lymphoid cells. The results show a normal number of proliferating T lymphoid cells and an increased number of proliferating non-T lymphoid cells in clinical stages O-I. Stages III–IV demonstrate a significant increase of the proliferation rate of both T and non-T lymphoid cells. The possible pathogenetic factors and the prognostic value of these results are discussed.This work was supported by the Deutsche Forschungsgemeinschaft (Be 79/15) and by the Austrian Funds Zur Förderung der wissenschaftlichen Forschung and Kampf dem Krebs     

Diminished autologous mixed lymphocyte reaction in patients with Hodgkin disease: evidence for non-T cell dysfunction

In the autologous mixed lymphocyte reaction (AMLR), T lymphocytes are stimulated to proliferate by autologous non-T mononuclear cells. In five untreated patients with Hodgkin disease, the AMLR was diminished. In addition, in the same five patients, T cell response PHA was inhibited by a cell in the non-T cell fraction, the response of non-T cells to PWM was diminished, and there was a diminished ability of the non-T cell population to stimulate in allogeneic MLR. However, the response of T cells from patients with Hodgkin disease to allogeneic antigen was normal. The AMLR and allogeneic MLR were then studied in an additional five untreated patients before and after monocyte depletion of the stimulating non-T mononuclear cell population. In this second group of Hodgkin disease patients, the AMLR was again diminished when T cells were incubated either with non-T cells or non-T cells depleted of monocytes. In the Hodgkin patients, monocyte depletion did not alter the T cell response in the AMLR. In the controls, monocyte depletion greatly diminished the proliferative response. The diminished AMLR in untreated Hodgkin disease patients may be the result of a failure of adequate monocyte stimulation of autologous T cells.     

Role of autologous lymph node T cells in membrane expression of mu- or gamma-heavy chain isotype by malignant B NHL cells

We investigated the possibility that T cells observed in lymph nodes involved by B-non-Hodgkin's lymphomas (B-NHL) may have a direct role in the expression of Mu- or Gamma- heavy chain isotype by autologous malignant B cells. T cells were separated from lymph nodes involved by B-NHL cells expressing either surface IgM (19 cases) or surface IgG (4 cases) and compared to peripheral blood T lymphocytes of healthy subjects (19 cases) for their ability to promote both IgG and IgM secretion in Cowan-activated normal B lymphocytes. The mean values of IgG/IgM ratios obtained under the influence of T cells associated with malignant B cells expressing either surface IgM or surface IgG were not statistically different to that obtained with the help of control T cells (0.60 and 0.62 versus 0.47, respectively). These results do not account for the hypothesis that autologous lymph node T cells may directly affect the expression of the heavy chain isotype by malignant B-NHL cells.     

Human intraepithelial lymphocytes

This study evaluates the immunomodulation and receptor binding of vasoactive intestinal peptide on human peripheral blood lymphocytes and intraepithelial lymphocytes. Vasoactive intestinal peptide (VIP, 10^–8 and 10^–12 M) had no effect on the concanavalin A-induced proliferation or the spontaneous cytotoxicity against K-562 targets by either lymphocyte type. Human peripheral blood lymphocytes had a mean of 927 vasoactive intestinal peptide receptors per cell with a K_d of 1.12×10^–10 M, as demonstrated by the competitive displacement of [¹²⁵I]peptide by unlabeled peptide using Scatchard analysis. In contrast, intraepithelial lymphocytes had no high-affinity receptors as shown by the negligible binding of 50 pM [¹²⁵I]VIP. Peptide binding by peripheral blood lymphocytes, although reduced by exposure to dithiothreitol and ethylenediamine tetraacetic acid, was still greater than binding by intraepithelial lymphocytes. As intraepithelial lymphocytes are mainly CD8⁺ T cells, the possibility that this phenotype may not bind VIP at all was tested by specifically depleting peripheral blood lymphocytes by antibody and complement lysis. Peripheral blood lymphocytes expressing CD8, CD4, and/or CD2 were responsible for most of the binding, indicating that CD8⁺ T lymphocytes in the peripheral blood and in the intestinal epithelium differ in their capacity to bind VIP.This work was supported by grants from the National Foundation for Ileitis and Colitis and from the National Institutes of Health (RO1DK42166).     

T and B lymphocytes in leukemia therapy.

In order to evaluate the effect of radio- and chemotherapy on immunity, T and B lymphocyte surface receptors were studies sequentially in the blood from 28 previously untreated leukemic children. Following the initiation of chemotherapy an increase in the percent T and B cells was noted in the peripheral blood. In association with sanctuary therapy and chemotherapy there was a decrease in the total number of circulating T and B cells and a relative increase in lymphocytes lacking markers. Based on total numbers at remission the reduction in B cells was greater than in T cells, and the most marked changes occurred during sanctuary therapy. A reduction in the mean serum immunoglobulin was associated with decreasing B cell numbers.     

Impaired synthesis of immunoglobulin in patients with chronic lymphocytic leukemia

The cause of hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) is unknown. Experiments were performed to determine if sera, monocytes, or non-T cells from patients with CLL suppress the proliferative response and synthesis of immunoglobulin (Ig) following incubation with pokeweed mitogen (PWM) in cocultures with lymphocytes from normal individuals. The data indicate that sera and monocytes from patients with CLL did not suppress the proliferative response or synthesis of Ig normal non-T cells. When various numbers of normal non-T cells and CLL non-T cells were cocultured with a constant number of normal T cells, the proliferative response and the concentration of supernatant Ig decreased as the proportion of CLL non-T cells increased. Since similar results were obtained when irradiated non-T cells from normal individuals were substituted for non-T cells from patients with CLL, we believe that the decrease in proliferative response and diminished synthesis of Ig is not the result of the suppressor non-T cells but is related to the dilution of normal B cells by inert non-T cells. We conclude that these experiments serve as as in vitro model for patients with CLL and suggest that the hypogammaglobulinemia observed in this disease is related to the diluting out of normal B cells by the accumulation of neoplastic B cells in the peripheral blood, bone marrow, and lymphoid tissue of these patients.     

Rosette-formation with mouse erythrocytes: VI. T, B, and third population lymphoid cells in mycosis fungoides and effect of leukopheresis.

Peripheral blood from 16 patients with mycosis fungoides and two patients with Sézary syndrome was examined for T, B, and third-population (K) cells, using a battery of surface markers. T lymphocytes as determined by spontaneous rosette formation with sheep erythrocytes and third-population cells as determined by the Ripley rosette test were present in normal proportions. Surprisingly, B lymphocytes, as determined by surface immunoglobulin and receptors for mouse erythrocytes, were either lacking or were present in low proportions in some patients. Normal proportions were present in others. Repeat studies of two of three patients lacking B lymphocytes, following treatment, revealed normal or low proportions of B cells. Two patients with mycosis fungoides had increased proportions of "null" cells. Study of lymphoid cell subpopulations before and after leukopheresis in a single patient demonstrated a decrease in T cell proportions associated with a concomitant increase in the proportions of "null" cells following this therapy.     

The immune response to influenza vaccination in diabetic patients

Summary The immune response of diabetic patients to influenza vaccination was examined in 31 patients, 10 with Type 1 (insulin-dependent) diabetes and 21 with Type 2 (non-insulin-dependent diabetes), and in 19 normal subjects. Each received a single intramuscular injection of the 3 virus strains (A/Chile,A/Philippines,B/USSR) anti-influenza vaccine recommended by WHO. The antibody titre and the cell-mediated immune response to the 3 virus strains, as evaluated by the generation of activated lymphocytes and enumeration of B lymphocytes, were studied before and 18 h, 72 h and 1, 2, 3 and 6 weeks after vaccination. Overall, the humoral and cell-mediated immune responses were normal in both groups of patients. However, patients with Type 1 diabetes showed a statistically significant increase (p< 0.01) of antibody titre of the A/Chile and an increased percentage of B lymphocytes one week after vaccination compared to age-matched control subjects. Four out of 21 patients with Type 2 diabetes had no antibody response to all 3 virus strains. A significant reduction (p< 0.01) of the percentage of activated cells possessing receptors for interleukin-2 was observed 72 h after vaccination in patients with Type 2 diabetes compared to age-matched control subjects. None of the patients who received the vaccine developed influenza in the course of the following year. These results suggest that valid protection against the influenza virus can be obtained in patients with Type 1 and Type 2 diabetes.     

Acid phosphatase and acid esterase activity in neoplastic and non-neoplastic lymphoid cells A semiquantitative evaluation related to immunological markers in 112 cases

Acid phosphatase (AcP) and acid α-naphthylacetate esterase (ANAE) were examined in lymphoid cells from 104 patients suffering from various lymphoproliferative disorders and from 8 healthy controls. Enzyme activities were evaluated by means of a scoring system. Scores of AcP and ANAE were higher in normal T cells than non-T cells. In comparison, the activated T cells in infectious mononucleosis showed increased AcP and decreased ANAE reaction. Malignant T lymphoblasts had a distinct granular AcP positivity in contrast to the faint reactivity observed in cALL blasts, whereas ANAE showed negative or weak reaction in both subsets. High scores and distinct staining patterns for both enzymes were found in T CLL and T prolymphocytic leukaemia, clearly different from the weak activities seen in B CLL, B PLL and some B cell lymphomas. The latter, too, could be distinguished by mutual differences in enzyme reactions. High AcP and ANAE scores were also found in hairy cell leukaemia, and the staining patterns together with the tartrate resistance firmly established the diagnosis. Thus, simultaneous determinations of AcP and ANAE can be of great value in the diagnosis of lymphoid malignancies.     

Isolation and partial chemical characterization of the IgG Fc receptor of human T lymphocytes and production of an antiserum.

We report here the isolation of an IgG Fc receptor from normal human T lymphocytes. The purified receptor has a nonreduced and a reduced component of molecular weights 120,000 and 60,000, respectively, and it was functionally active in the in vitro blocking of rosette formation between T lymphocytes and IgG-coated ox erythrocytes. An antiserum raised to the Fc receptor and an isolated F(ab')2 fragment of this antiserum, also blocked rosette formation between T cells and IgG-coated ox erythrocytes. In contrast, rosette formation between T lymphocytes and IgM-coated ox or sheep erythrocytes was not blocked by the F(ab')2 fragment, demonstrating the marked specificity of this antiserum for the IgG Fc receptor. In addition, this antiserum did not block the Fc receptors of non-T cells, indicating that the T-cell IgG Fc receptor has unique antigenic determinants not shared with B cells.     

Dissection of distinct human immunoregulatory T-cell subsets by a monoclonal antibody recognizing a cell surface antigen with wide tissue distribution.

A monoclonal antibody, PVR-11, was obtained after hybridization of X63Ag8.653 murine myeloma cells with spleen cells from a mouse immunized with human lymphocytes. It recognizes a 175,000- to 185,000-dalton surface antigen present on approximately 80% of normal human peripheral T lymphocytes, 50% of non-T non-B cells, and less than 10% of B cells as determined by complement-dependent microcytotoxicity. It is also present on various leukemia T cells, on some but not all T lymphoblastoid cell lines, and on a small fraction of some B lymphoblastoid cell lines. Some B-cell chronic lymphocytic leukemia cells also express the PVR-11 antigen. Functional analysis of normal human T lymphocytes demonstrated that the PVR-11-depleted T-cell subset contains the precursors of both cytotoxic and suppressor cells but lacks helper cells. On the other hand, cytotoxic effector T cells express the PVR-11 antigen. These results demonstrate that antigenic determinants with relatively wide tissue distribution can dissect functionally distinct human immunoregulatory T-cell subsets.     

Electronmicroscopical and immunoelectronmicroscopical examination of the lymphocytes of young and old people after influenza vaccination.

Authors examined the lymphocytes of young and old individuals before, 6 days and 6 weeks after influenza vaccination. Vaccination was carried out with killed Influenza B/Hong-Kong virus. Electronmicroscopic structure of the lymphocytes and membrane bound surface IgG and IgM were studied. Membrane bound surface IgG and IgM were seen on the small lymphocytes and on the membrane of the medium-sized lymphocytes in young and aged individuals before and after vaccination. In both age groups intracytoplasmic tubuloreticular structures were seen in the lymphocytes. These findings were not age dependent alterations. Age dependent changes could be detected on the mitochondria of the lymphoid cells. The mitochondrial crysts in aged individuals disappeared and were replaced by myelin-like forms or by faint density substance. The mitochondrial changes were present before vaccination and after vaccination in non labelled, presumably T lymphocytes. The injury of the mitochondrium can influence the function and the proliferative capacity of the cells.     

Defective self-recognition in subjects of far-advanced age

The immunocompetence of 22 subjects aged 85-104 years (mean 90 +/- 1 years) was studied and compared to 21 young subjects aged 19-37 years (mean 30 +/- 1 years). The absolute lymphocyte number and the percentage of T and B lymphocytes in the peripheral blood was similar in the two groups. A marked decrease in phytohemagglutinin response of T-enriched lymphocytes from old subjects was observed. Autologous mixed lymphocyte reaction (MLR) was also profoundly reduced in old subjects. No difference between male and female subjects was observed. The responsiveness of enriched T lymphocytes to allogeneic irradiated non-T cells was only slightly impaired in the old individuals. Non-T cells from old and young subjects functioned equally well as stimulatory cells in allogeneic MLR. The data suggest that an alteration of T lymphocytes with regulatory function and of self-recognition is present in aged humans.     

DNA-synthesizing T and non-T cells in bacterial infections

Summary The results of autoradiographic determination of DNA-synthesizing lymphocytes (³H-thymidine) in 10 patients with bacterial infections were compared with results in 10 normal patients and contrasted with 23 CLL patients in different stages [12]. In patients with infectious diseases the absolute number of T cells was lower and the mean values of S-phase T cells and S-phase non-T cells was higher than in normal persons. In contrast to the patients with infections, CLL patients in stage 0–III have lower S-phase T cell values and higher S-phase non-T cell values. In stage IV, on the other hand, all DNA-synthesizing lymphocytes are increased.Guest of the Deutscher Akademischer Austauschdienst