Rosette-forming Lymphocytes Research Articles

Rosette Formation of Pig Peripheral Blood Lymphocytes with Sheep Red Blood Cells

Peripheral blood lymphocytes of normal young pigs formed spontaneous and EAC rosettes with sheep red blood cells. Pretreatment of the SRBC with a sulphydryl reagent increased the percentage of spontaneous rosettes and the number of SRBC bound by the lymphocytes. The influence of an incubation period prior to centrifugation and the duration and temperature of the following incubation were tested for spontaneous and EAC rosettes.The importance is stressed to define the number of bound SRBC to call a lymphocyte a «rosette-forming cell». Rosette formation is interpreted as a quantitative rather than a qualitative marker.

Fibrin Monomer Complexes in Pre-Eclampsia

On the assumption that the number of E-rosettable lymphocytes (active T lymphocytes) is an index of cell-mediated immunity, rosette assays were performed at early cycle and at midcycle for 6 women taking oral contraceptives (OCs) for 1-4 years. OC subjects at midcycle had 21.4% active rosette-forming lymphocytes as compared with 14.1% in controls (p less than .05). The 2 youngest subjects had higher values during the early cycle. These results imply the possibility of hormonal regulation of human T-cell activity.

EA-rosette-forming cells in chickens: Presence in spleens of bursectomized chickens and relationships to B-lymphocytes

Spleen cells from 3-month-old chickens which had been surgically and chemically bursectomized were examined for rosette-forming lymphocytes (RFL). Finding no correlation between the degree of agammaglobulinemia in bursectomized chickens and the percentage of RFL in their spleens demonstrated a dissociation between two B-cell dependent functions, namely, immunoglobulin production and rosette-forming ability. In fact, the percentage of RFL in the spleens of agammaglobulinemic chickens was found to be slightly higher than those of normal chickens. The data indicate that RFL in spleens of normal chickens are B cells, and suggest the existence of a population of rosette-forming B lymphocytes in agammaglobulinemic bursectomized chickens.

Immunological Studies in Psoriasis

Quantitative techniques of sensitization to 2,4-dinitrochlorobenzene (DNCB) was used to determine in psoriasis the intensity and frequency of allergic reactions to DNCB following primary challenge with 2,000 microgram allergen and secondary challenge with decreasing doses of DNCB. 56 patients with psoriasis and 23 healthy volunteers were examined. Frequency of positive reactions to DNCB was similar in both groups, since all normal controls were sensitized, whereas only 8 of 56 psoriasis cases failed to develop delayed hypersensitivity to DNCB. However, the intensity of acquired contact allergy was significantly diminished in psoriasis in comparison with controls. The patients with stationary skin lesions resembled the normal population in the intensity of reaction to DNCB. Decreased intensity of DNCB sensitization seemed to be related to the activity of the disease, but not correlated with the extent of the lesions. A relationship was found between reduced reactivity to DNCB and decrease in E rosette-forming lymphocytes. The data suggest that the impaired function of T lymphocytes in active psoriasis could be responsible for both, defective recognition of contact antigens, such as DNCB, and the alteration of secondary response to DNCB.

Proportions of Mouse Erythrocyte Rosette-Forming Lymphocytes, Immunoglobulin-Bearing Cells and E Rosettes in Patients with Lymphoproliferative Diseases

The proportions of mouse erythrocyte rosette-forming (MERF) lymphocytes, immunoglobulin (Ig)-bearing cells and E rosettes were examined in the peripheral blood of patients with lymphoproliferative diseases. The proportion of MERF lymphocytes was considerably increased in chronic lymphocytic leukaemia. The correlation between the proportions of MERF and Ig-bearing cells, characteristic of healthy individuals, is not observed in the lymphoproliferative diseases. It is suggested that in a part of the patients with these diseases, the study of the proportion of MERF cells provides more accurate information on the proportion of lymphocytes of bursal origin than does counting of the Ig-bearing cells.

Modified E-rosette test for detection of total and active rosette-forming lymphocytes

The human peripheral blood T-lymphocyte population can be demonstrated with an economy of time by using bromelin-treated sheep red blood cells (SRBC). Similarity, the lower affinity, active T-cell population can be determined using untreated SRBC. Both tests are done without serum, or prolonged incubation of the cell mixture, using the same procedural steps reflecting changes as readily as longer test methods.

B and T cell surface markers on peripheral lymphocytes from diabetic patients.

A study of T and B cell surface markers was carried out on peripheral blood lymphocytes from diabetic patients, employing immunofluorescent staining for membrane Ig, and the E rosette test. According to the immunofluorescent studies the % of sIg bearing lymphocytes decreases with age; this trend is more evident in healthy individuals. Lymphocytes from diabetic patients posses a higher amount of membrane IgG than age-matched controls. It was only in the younger diabetic patients that a definite decrease was observed in the % of IgM bearing lymphocytes. The evaluation of E rosette-forming lymphocytes revealed a similar age-associated reduction both in diabetics and in controls. Some common features of the pattern of T and B cell markers on lymphocytes from younger diabetic patients and older controls suggest that the former undergo, at least from the immunological standpoint, a more precocious ageing process. It is possible that some metabolic disorders in lymphocyte activity may play a role in this, resulting in a deficiency in the control of immunological mechanisms.

Modulation of human T lymphocyte rosette formation by autonomic agonists and cyclic nucleotides

Early rosette formation by T lymphocytes appears to be modulated by cyclic nucleotides. Dibutyryl cyclic 3′,5′ adenosine monophosphate (cyclic AMP) 10 −2 M inhibited E rosette formation up to 83%, while 10 −6 M 8-bromo-cyclic guanosine monophosphate (cyclic GMP) increased rosette formation maximally to 67.4% with less pronounced effect at increased concentrations. T lymphocyte receptors for beta adrenergic, alpha adrenergic, and muscarinic cholinergic agonists appear to influence rosette formation. Isoproterenol 10 −2 M induced 67.3% inhibition, while phenylephrine 10 −5 M and carbamylcholine 10 −4 M were associated with enhanced rosette formation of 67.2% and 57.8%, respectively. Selective blockade with propranolol, phentolamine, and atropine indicated the presence of separate receptor sites. The lack of effect of histamine at concentrations of 10 −3 M and above suggests the absence of specific receptors on peripheral human T lymphocytes.

Human Lymphocyte Subpopulations: Giant Srbc Rosettes

Human thymus-derived lymphocytes have the ability to form rosettes with sheep red blood cells (SRBC) in vitro. In the investigation of rosettes of peripheral blood lymphocytes of 10 normal subjects, the number of SRBC adhering to the lymphocyte in each of 100 rosettes was assessed. The percentage of rosettes with SRBC greater than or equal to 36 per rosette was only 1.2 +/- 0.5. These were defined as giant SRBC rosettes. Peripheral blood lymphocytes were stimulated in vitro by four mitogens: sodium periodate, neuraminidase plus galactose oxidase, pokeweed mitogen, and concanavalin A. The lymphocytes were then cultured at 37 degrees C. The giant rosette-forming lymphocytes became significantly increased 4 to 24 hr after stimulation, prior to the appearance of lymphoblasts or increased incorporation of tritiated thymidine. The giant rosettes were not caused by the hemagglutinating properties of pokeweed mitogen and concanavalin A that were adsorbed on the lymphocyte surfaces. This was shown by the fact that, on removal of the receptors by trypsinization, they were regenerated on culture in vitro in the absence of the mitogens. It was concluded that giant SRBC rosettes constituted a marker for some of the activated lymphocytes. Their appearance was independent of the increase in size of the cells or of DNA synthesis. These receptors were intrinsic to lymphocytes and not caused by mitogens adsorbed on their surfaces.

Inhibition of the Fc receptor of human lymphoid cells by antisera-recognizing determinants of the hla system.

The inhibitory effect of HLA antisera on Fc receptors of human lymphoid cells was investigated. The ability of lymphoid cells to form rosettes (FcRFC) with antibody-coated sheep red blood cells and to function as effector cells (K cells) in antibody-dependent cell-mediated cytotoxicity were used as assay systems. We found that antisera recognizing determinants of the HLA-A, -B, and -C series had no effect on FcRFC, while a specific inhibitory effect was observed of antisera probably reacting with determinants identical to or closely associated with those of the HLA-D series. This inhibitory effect was retained in the F(ab')2 fragments. Specific inhibition of K cells was observed with all HLA antisera, but this effect was lost in the F(ab')2 fragments. We conclude that the Fc receptor of rosette-forming lymphocytes may be closely associated with products of the HLA-D region. This is analogous to the association between the Fc receptor and the Ia antigens on murine rosette-forming B lymphocytes.

EA rosette forming lymphoid cells: Distribution as to cell types and organs from chickens of different developmental stages

The interaction of chicken spleen cells with sheep erythrocytes coated with chicken antibody (EA complexes) was studied using a rosette assay. The results reported in this paper indicate that subpopulations of chicken lymphocytes, monocytes, and heterophils have a receptor for EA. The formation of rosettes between chicken lymphoid cells and sheep erythrocytes (SRBC) was dependent upon the concentration of antibody used to sensitize the SRBC. In a developmental study of rosette-forming lymphocytes (RFL), the bursa was the first site of appearance of large numbers of RFL. The percentage of RFL in the bursa reached a peak at 17 days of embryonic life, and declined to a low by hatching. The percentage of RFL in the spleen, however, began to increase at the time of hatching and by 6 weeks of age the spleen far surpassed the bursa in percentage RFL. At no age were significant numbers of RFL detected in the thymus.

Decreased T-Lymphocytes in Patients with Mammary Cancer

The levels of thymus-derived lymphocytes (T-cells) in samples of peripheral blood from 47 patients with carcinoma of the breast, 36 patients with benign mammary lesions, and 33 matched normal controls were determined. Results were analyzed with three statistical tests. It was found that the mean percentages of T-lymphocytes differed significantly among the three groups of patients (p less than 0.001). The mean percentages of rosette-forming lymphocytes were 72.8 for the normal group, 60.6 for patients with benign mammary lesions, and 45.1 for the patients with carcinoma. The finding of lower levels of T-lymphocytes in the peripheral blood of individuals with mammary cancer is of particular significance since all the patients in this study were otherwise in apparently good general health and undergoing no treatment.

Ontogeny of lymphocyte subpopulations in human fetal liver.

Lymphocytes were isolated from five fetal livers (13-17 weeks) and examined for different surface markers. Immunoglobulin M was found on 0.5-4.0% of lymphocytes. No membrane-bound surface IgD, IgA. or IgG was detected. Mouse erythrocyte rosette-forming lymphocytes ranged from 0.5 to 6.0%. Thymus-processed (T) lymphocytes, which were defined as those forming spontaneous rosettes with sheep erythrocytes, as well as lymphocytes with IgG Fc receptors, were present in a small proportion. No complement-receptor-bearing lymphocytes were found in the two cell populations of fetal liver studied. It is evident that during the ontogeny of bone-marrow-derived (B) lymphocytes IgM is the first surface immunoglobulin to appear. Receptors for the binding of mouse erythrocytes are present at the same time as surface IgM. The slight excess of mouse erythrocyte rosette-forming cells over cells having surface immunoglobulin M could mean that IgM appears later than do the receptors for the binding of mouse erythrocytes.

Antigen heterogeneity of human B and T lymphocytes.

Rhesus monkeys were immunized with normal human lymphoid cells, cultured lymphoid cells, and chronic leukemic lymphocytes. Antisera were analyzed by cytotoxicity and immunofluorescence techniques to study the antigenic characteristics of human lymphocytes. In an attempt to obtain a reagent specifically reactive with T (thymus-derived) lymphocytes, an antispleen antiserum was absorbed with cellf from five B- (bone marrow-derived) cell lines. After absorption, the antiserum killed 60-75% of peripheral blood lymphocytes and 40-50% of tonsil cells, so that there was a relationship between the percentage of killed cells and the proportion of T lymphocytes. However, when cells after cytotoxic treatment were assayed for rosette formation with sheep erythrocytes (a T-cell marker) 5-20% of viable rosette-forming lymphocytes were found. Therefore, this antiserum was cytotoxic for only 75-90% of T cells. From studies performed with antisera prepared against spleen and B-cell lines, we conclude that lymphoblastoid cells are antigenically different and deficient in comparison to normal B lymphocytes. In addition, cultured B-cell lines appear to be antigenically heterogenous, as shown by the cytotoxic activity remaining in antispleen and anti-B-cell lines sera after absorption with various numbers and types of lymphoid cell lines. After absorption with normal lymphocytes, an antiserum produced against chronic lymphatic leukemia cells had specific activity associated with 12 chronic lymphatic leukemia cells tested. Absorption of the same antiserum with leukemic cells from two patients showed that a certain degree of antigenic heterogeneity also exists among chronic leukemic lymphocytes.

Rosette-forming lymphocytes and measles vaccination

Rosette-forming lymphocytes and measles vaccination

The Effect of Neonatal Rat Graft-vs-Host Disease (GVHD) on Fc Receptor Lymphocytes

The level of Fc receptor rosette-forming lymphocytes (Fc-RFL) was examined in spleen and lymph node cell suspension from neonatal DA and BN rats inoculated within 24 hr of birth with either allogeneic L (experimental) or syngeneic (control) lymphoid cells. In addition, these levels were compared to fetal and neonatal animals that received no injection. The indicator cells (EA) were sheep erythrocytes sensitized with one-half concentration of the highest dilution of rabbit anti-sheep erythrocyte IgG(A) which agglutinated an equal amount of 1% suspension of E. Care was taken to exclude scoring macrophages by injecting colloidal carbon at least 1 hr before killing the test animals. The spleen of 19-day DA fetal rats exhibited a level of 19.3% Fc-RFL, similar to that of animals having received adult syngeneic cells at birth (40.0%) by day 7. Thereafter the level of Fc-RFL did not vary appreciably between these two groups. However, as early as 2 days after inoculation there was a significantly greater number of Fc-RFL in the spleen of experimental DA neonates compared to controls. The lymph nodes of experimental animals did not exhibit a significantly greater number of Fc-RFL until day 6 with both tissue compartments peaking at day 10 and remaining significantly higher than controls until death. In neonatal BN animals significantly higher levels of Fc-RFL in experimental animals were not evident as early and peaked later (day 12) in both tissue compartments but again these differences remained until death. Cytotoxic alloantisera demonstrated that on days 6, 10, and 12 most, if not all, of the Fc-RFL were host in origion in both DA and BN GVHD, with a very significant host plasma cell response in such GVHD animals. One-micron tissue section revealed the presence of a great number of plasma cell especially prominent in the medulla of lymph nodes with the cortex of lymph nodes and white pulp of the spleen markedly depleted of lymphocytes indicative of cytotoxicity.

Electron microscope study on human lymphocyte-sheep erythrocyte rosettes.

Nonimmune rosette formation of human peripheral blood lymphocytes and cultered MOLT 3 and MOLT 4 cells with sheep red blood cells was studied by transmission electron microscopy. At the cell contact area of the rosette, lymphoid and red cell membranes were parallel and 80-100 A apart. The inner leaflet of the lymphoid cell membrane seemed denser, and amorphous substance attached to its cytoplasmic face. The cell contact area was 100-1000 nm wide and frequently on the lymphoiid cell body rather than on microvilli, though some cells extended microvilli near red cells.

T and B lymphocytes in skin changes of cutaneous lymphoma (author's transl)

In 30 patients with benign or malignant lymphoreticular proliferation of the skin (38 skin biopsies) the percentage of immunoglobulin-bearing (B) and of spontaneous rosette-forming (T) lymphocytes was determined in cell suspension from homogenized tissue. Four main classes of cutaneous lymphoma could be differentiated: lymphocyte-rich (more than 50% of the infiltrate cells, predominantly B-lymphocytic, predominantly T-lymphocytic, mixed B and T-lymphocytic), lymphocyte-depleted (less than 50% of infiltrate cells), reticulo-histio-monocytic infiltrates, and reticulo- or lymphosarcoma. A definite statement was not possible (other than in leukaemic processes) if the accumulation of lymphocytic cells represented the primary malignant process rather than a concomitant inflammatory reaction. A clinically useful differentiation was into hyperplasia, paraplasia and neoplasia, as this takes into account different histopathological characteristics of cutaneous lymphomas (reactive hyperplastic/autonomic proliferative: benign/malignant; reversible/irreversible; systemic/nonsystemic; metastasizing/nonmetastasizing).

Cell-Mediated Cytotoxicity in Vitro of Human Lymphocytes against a Tissue Culture Melanoma Cell Line (IGR3)

Abstract Purified peripheral blood lymphocytes from 13 healthy donors, 6 melanoma patients and 1 halo nevus patient were tested for cytotoxic activity against an allogeneic melanoma cell line (IGR3) in, at least, one of the following assays: cell-mediated cytotoxicity (CMC), antibody-dependent cellular cytotoxicity (ADCC) and microcytotoxicity assays (MA). The lymphocytes were isolated by Ficoll-Triosil gradient centrifugation (fraction F) followed by removal of iron-phagocytosing and adherent cells (fraction FFF) and by subsequent passage through anti-IgG columns (fraction FFF-C). Leukocytes of each fraction were identified by different methods including morphology, rosetteformation, phagocytic activity, and membrane fluorescence. CMC activity paralled ADCC activity at a log lower level of sensitivity. In both assays lymphocytes of fractions F and FFF had the highest activity, whereas in fraction FFF-C cytotoxicity was strongly reduced. In all three lymphocyte fractions CMC and ADCC activity could be blocked by preincubation of the effector cells in aggregated IgG. Furthermore, depletion of E rosette-forming lymphocytes slightly increased ADCC and CMC activity, whereas depletion of EA and EAC rosette-forming lymphocytes strongly decreased it. Our results therefore indicate that in both CMC and ADCC assays, non-adherent, non-phagocytic Fc receptor-bearing lymphocytes (“K” cells) were the active cytotoxic cells. In MA, on the other hand, mononuclear phagocytes seemed to be the most active cell population. So far no significant difference was observed in CMC, ADCC, and MA between control persons and melanoma patients.

X-ray enhancement of splenic rosette-forming cells in nonimmune mice.

The formation of rosettes between spleen cells from nonimmune mice and sheep red blood cells (SRBC) has been investigated. The number of splenic rosette-forming cells (RFC) in the intact spleen was very low but greatly increased after sublethal whole body x-irradiation. These cells were inhibited by pretreatment of the cells with AKR anti-C3H- antigen antiserum, in the presence of fresh guinea pig serum, but not with rabbit anti-mouse immunoglobulin antiserum. The splenic RFC also increased after cortisone acetate administration into mice. These results suggest that the RFC are a T cell sub-population having x-ray resistant and cortisone-insensitive characteristics. The x-irradiation of mice in vivo selectively increased the number of splenic RFC in consequence of reduction in the number of cells having non-T cell characteristics. These RFC are presumably antigen-specific sheep red blood cell rosettes, a quite different situation as in the case of human T lymphocyte rosette formation.