Lymphocyte Rosette Research Articles

Spontaneous rosette formation and rosette inhibition assays in patients with squamous cell carcinoma of the head and neck

Peripheral blood lymphocytes from patients with squamous cell carcinoma (SCC) of the head and neck were studied by spontaneous lymphocyte rosette and rosette inhibition (RI) assays prior to treatment. The patients were clinically staged and the results of the assays compared with the clinical stage of the disease. The percentage of T-lymphocytes as determined by the spontaneous lymphocyte rosette test was significantly lower (p less than .01) for the patient group when compared with a normal population. Patients with stage I and II disease did not differ significantly from controls. Individuals with stage III or IV disease, however, had significantly lower T-lymphocyte counts. The tumors were histologically graded as well, moderately well, or poorly differentiated SCC. Patients with poorly differentiated neoplasms had significantly lower T-cell counts. The RI assay (using horse anti-human thymocyte globulin to inhibit rosette formation) was abnormal in many of the patients but did not appear to be a more sensitive in vitro measure of cell mediated immunity in these patients. Performing both tests detected more patients with cellular immunologic incompetence than either one alone.

Characterization of immunocompetent cells recovered from the respiratory tract and tracheobronchial lymph node of normal guinea pigs.

To gain insight into the structure of the lung's immune system, the identity and function of immunocompetent cells in the broncho-alveolar air spaces and tracheobronchial lymph nodes of normal guinea pigs were examined. Guinea pig thymus-derived (T) lymphocytes (mediators of cellular immunity) were identified by their ability to form rosettes with rabbit erythrocytes (E rosettes). Bone marrow-derived (B) lymphocytes (mediators of humoral immunity) were identified by their ability to form rosettes with sheep erythrocytes sensitized with antibody and complement (EAC rosettes) and by the presence of surface immunoglobulin, detected by direct immunofluorescence. Total lung lavage yielded 14+/-5.0 X 10(6) (mean +/- SD) cells. There were 68+/-5 per cent cells of the monocyte-macrophage series as judged by morphology, the ingestion of latex particles, and the uptake of neutral red; 20+/-9 per cent were eosinophils, and 12+/-5 per cent were lymphocytes. Stable populations of T and B lymphocytes were recovered from guinea pig airways and tracheobronchial lymph nodes. T cells represented 76 per cent of lymphocytes from the airways and 64 per cent of lymphocytes in tracheobronchial lymph node; B cells equalled 14 per cent and 30 per cent, respectively. The functional potential of T cells present in the lung and tracheobronchial lymph node was demonstrated by their proliferative response to phytohemagglutinin. These results indicated that, although macrophages are the predominant cell type, viable T and B lymphocytes are present in the guinea pig broncho-alveolar air space. The composition of these cells is comparable to what has been observed in normal human lungs and provides preliminary evidence that the guinea pig may be a useful model for humans. This report also describes a method using 2 vital stains whereby macrophages, lymphocytes, and rosetted lymphocytes can be distinguished in unseparated cell suspensions prepared from bronchial aspirates.

The use of frozen-stored sheep erythrocytes for demonstrating lymphocytic membrane markers

The usefulness of cryopreserved sheep erythrocytes (unsensitized or coated with fluorescein, IgG-type antibody or complement) for marker tests on human blood lymphocytes was studied. It appears that the rosetting properties of freshly prepared or frozen-stored erythrocytes are not significantly different. The method may be used to provide homogeneous stocks of sensitized erythrocytes for lymphocyte rosetting techniques.

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 T and B lymphocyte rosette tests: Effect of enzymatic modification of sheep erythrocytes (E) and the specificity of neuraminidase-treated E

Sheep erythrocytes (E) were treated with papain or neuraminidase to evaluate what effect these enzymes would have on the E rosette test for human T lymphocytes. Few or no rosettes were detected with sheep erythrocytes treated with papain (E P). Sensitization of E P with rabbit antibody and mouse complement resulted in a rosette indicator (E PAC) which could be used to detect peripheral blood complement receptor lymphocytes (CRL) and thymocyte CRL without having to perform the rosette assay at 37 °C. Neuraminidase treatment of E (E N) enabled the detection of approximately 20% more rosette-forming cells (RFC) in human peripheral blood lymphocyte (PBL) suspensions compared to untreated E. Rosette studies on a patient with severe combined immunodeficiency disease and on human lymphoblastoid cell lines showed that the additional rosettes detected with E N were T lymphocytes.

Marijuana and T lymphocyte rosettes.

The effect of marijuana smoking on sheep cell rosetting properties of both early (active) and total T lymphocytes was studied in vitro. Significantly fewer active rosettes were formed by T cells from a population of 35 who appeared to be chronic marijuana smokers than the 34 controls. The late, or cold-enhanced rosettes formed by smokers and nonsmokers were similar, suggesting that similar numbers of rosette-forming T cells were present in the peripheral blood of smokers. These data suggest that marijuana smoking may affect the function of a subpopulation of T lymphocytes, identified by their capacity to form early rosettes, possibly by affecting these cell membranes.

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.

Unification and technical aspects of total t, active t and b lymphocyte rosette assays.

Conditions for rosette formation of human adult T and B lymphocytes with unsensitized and complement coated SRBC respectively, have been examined in this collaborative study. Modified procedures for identifying, by rosette formation, the total T, active T and B-RFC lymphocytes are described. The procedures reported here yielded results for the percent of peripheral blood lymphocytes forming total T, active T and B-RFC for control individuals that are comparable with those previously reported. The advantage of the procedures described here are that they are 1) easy to perform; 2) highly reproducible; 3) allow for the preparation of a single lymphocyte concentration and common reaction mixture volume to be used in all three rosette assays and 4) allow for the removal of non-lymphocytic cells which would interfere with the determination of B-RFC. The use of these procedures would allow other laboratories to obtain similar values so results for controls and various types of patients could be compared.

Ultrastructure of cell-mediated immunity. I. Comparison between E and EA rosettes.

The ultrastructural comparison between E and EA rosettes showed that, in the former, the rosetting lymphocytes are mostly round in shape and their interaction with sheep erythrocytes only consists of limited areas of membrane contact, in the latter, rosetting lymphocytes are mostly in the shape of uropods and surrounding ox red cells show pseudopods protruding towards the lymphocyte and coming into contact with it.

Effect of cholinesterase inhibition by eserine and phospholipase D on human T lymphocyte Rosetting.

La formation de rosettes entre les lymphocytes T humains et les globules rouges du mouton est inhibée par l'ésérine et la phospholipase D. Cela suggère que l'acétylcholinesterase liée aux membranes influence les récepteurs impliqués dans la formation des rosettes.

Surface characteristics of synovial fluid and peripheral blood lymphocytes in inflammatory arthritis.

AbstractIn order to characterize the T‐ and B‐cell populations of inflammatory arthritides, synovial fluid and peripheral blood lymphocytes from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), juvenile rheumatoid arthritis (JRA), and rheumatoid variant diseases (RV) were studied. Normal peripheral blood lymphocytes were examined as controls. T cells were identified by spontaneous sheep red blood cell (E) rosette formation. B cells were determined by complement receptor lymphocyte (EAC) rosette formation and by the presence of surface immunoglobulins (SIg) utilizing fluoresceinated polyvalent antiglobulin. Synovial fluids were analyzed in terms of duration, mucin quality, white cell count, and differential. Synovial fluid and peripheral blood lymphocytes from patients with RA and RV were distributed in T‐ and B‐cell percentages similar to those found in normal peripheral blood. In contrast, significant T‐cell depression was observed in the percentage of synovial fluid and peripheral blood lymphocytes of SLE and JRA patients. This depression was apparent in comparison with normal peripheral blood and with the synovial fluid and peripheral blood from RA patients. B‐cell percentages were similar in all patient groups and in comparison to normal peripheral blood lymphocytes. No differences were noted in B‐cell percentages when the EAC and SIg techniques of identification were compared. The percentage of cells bearing neither T‐ nor B‐cell markers (null cells) was enumerated for each patient group and found to be significantly elevated in the synovial fluid and peripheral blood of SLE and JRA patients. Though the mean synovial fluid and peripheral blood null cell percentages in RA patients were similar to those in controls, a definite bimodal distribution was found in the synovial fluids. These data suggest that evaluation of T‐, B‐, and null‐cell populations may be clinically useful in differentiating patients with SLE and JRA from those with rheumatoid arthritis and variant diseases.

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.

Lymphocyte Rosette Formation and Lung Lesions

Lymphocyte Rosette Formation and Lung Lesions

T and B Lymphocyte Rosetting in Undernourished Children

T and B lymphocyte rosetting values were obtained for 18 children with kwashiorkor, marasmus, or nutritional edema. T cell values were subnormal in all malnutrition classes, but were lowest in children with kwashiorkor. Four of five malnourished children who were sensitized with 2,4-dinitrochlorobenzene (DNCB) before refeeding failed to respond to repeated subsequent challenges; five of six children who were sensitized after refeeding responded strongly to the first challenge.

Inhibition of Fc receptors on a murine lymphoid cell line by cholera exotoxin

AFTER binding to its receptor, Gml ganglioside1,2, cholera exotoxin has been demonstrated to be a potent activator of the adenyl cyclase system in several cell systems. The response to exotoxin is related to an increase in intracellular cyclic 3′, 5′ adenosine monophosphate (cyclic AMP). Agents that increase intracellular cyclic AMP have been shown to suppress certain immunological responses, including histamine release3, lysosome degranulation4, mitogenic stimulation5, T lymphocyte rosette formation6 and antibody-dependent cellular cytotoxicity7. We now report that cholera exotoxin can inhibit the expression of plasma membrane receptors for the Fc portion of IgG in a murine lymphoid cell system.

Thymus-derived lymphocytes in patients with solid malignancies

A spontaneous lymphocyte rosette assay revealed subnormal peripheral blood levels of T cells in 94 patients with clinically operable solid malignancies when compared with 240 normal controls. Subnormal levels occurred in patients with melanomas, sarcomas, and squamous carcinomas, and even lower levels were found in patients with inoperable or recurrent malignancies. Forty-six patients clinically cured of adenocarcinomas, melanomas, and sarcomas had normal levels, but 79 patients cured of squamous carcinomas had subnormal levels. These findings in tumor-bearing and cured cancer patients, including the persisting defects in cured squamous cancer patients, parallel the findings with other assays and indicate that the spontaneous lymphocyte rosette assay is a clinically useful in vitro correlate of cell-mediated immunity in cancer patients.

Thymus-dependent lymphocyte levels in bronchogenic carcinoma: Correlations with histology, clinical stage, and clinical course after surgical treatment

The in vitro spontaneous lymphocyte rosette (T cell) assay was used to determine cellular immunologic competence in 112 patients with bronchogenic carcinoma. Among preoperative patients with localized tumors. T cell levels were significantly lower than in 237 normal controls. With advanced stages of disease, T cell levels declined progressively among patients with squamous cell carcinoma, oat cell carcinoma, and undifferentiated carcinoma, but not among patients with adenocarcinoma. Squamous carcinoma patients considered cured had persisting low T cell levels, but cured adenocarcinoma patients had normal levels. Serial determinations that showed a fall in T cell levels preceded the development of clinically evident metastases by an average of 2.5 months. Postoperative patients with rising T cell levels have remained clinically free of disease. The data indicate that T cell levels correlate with extent of tumor and clinical course of patients with bronchogenic carcinoma. The assay may, therefore, provide a rational basis for the selection of patients who are at high risk for the development of recurrence after surgical resection and who may benefit from the early institution of adjunctive therapy.

Studies on human T lymphocyte rosette formation with human and sheep erythrocytes

A large proportion of normal human blood lymphocytes forms rosettes with neuraminidase-treated autologous and allogeneic human erythrocytes (nHRBC) or sheep red blood cells (SRBC). In contrast, the percentage of rosette-forming cells is extremely reduced in the peripheral blood of patients with chronic lymphocytic leukemia. The formation of mixed rosettes after addition of nHRBC and SRBC suggests that most normal rosette-forming lymphocytes have receptor sites for both types of erythrocytes. Rosette formation with neuraminidase-untreated HRBC occurs only with a small fraction of normal lymphocytes, but can be increased by preincubating the lymphocytes with neuraminidase. Similar to rosette formation with SRBC. the binding of nHRBC is temperature-dependent, is prevented by pretreatment of the lymphocytes with proteolytic enzymes, but is not inhibited by EDTA. Rosette dissociation during incubation at 37 degree C is accompanied by conversion of ring- or morula-like rosettes into horseshoes or caps.

β-Adrenergic Inhibition of Human T Lymphocyte Rosettes

Abstract The cyclic nucleotides adenosine and guanosine monophosphate (CAMP and CGMP)1 have been reported to affect significantly the magnitude of cellular and humoral in vitro immune responses. Mitogenic lymphocyte stimulation (1, 2), lymphocyte-mediated cytotoxicity (LMC) (3, 4) and the effect of migration inhibitory factor on target cells (5) are inhibited by β adrenergic stimulants of intracellular CAMP, and dibutyryl CAMP. CGMP has been shown to enhance LMC (4). In vitro antibody synthesis has recently been shown to be inhibited by CAMP and enhanced by CGMP (6). Furthermore, CAMP has been reported to alter significantly mammalian cell morphology in vitro (7). Since the cyclic nucleotides have profound effects on the morphology and function of cells, we chose to study the effects of dibutyryl CAMP and the β adrenergic stimulant isoproterenol on the human T lymphocyte's binding affinity to sheep red cells (E rosettes).

Enhancement of sheep red blood cell human lymphocyte rosette formation by the sulfhydryl compound 2-amino ethylisothiouronium bromide

In vitro treatment of sheep red blood cells with the sulfhydryl compound 2-aminoethylisothiouronium bromide (AET) under suitable experimental conditions greatly enhances their ability to form rosettes with human lymphoid cells. The effect of AET depends on the concentration, pH of the solution and time of incubation. AET treated sheep red blood cells lose their ability to form rosettes after varying time periods depending on the temperature of storage and on the concentration of the AET solution. Treatment of human peripheral lymphocytes with AET abolishes their capability to form rosettes with both normal and AET treated sheep red blood cells. Lymphocytes from some patients with chronic lymphocytic leukemia react differently with AET-treated sheep red blood cells than with normal sheep red blood cells.