Crossreaction of antilymphocyte globulin with human granulocyte colony-forming cells.

Clinical preparations of horse antilymphocyteglobulin (ALG) were found to inhibit human bone marrow granulocyte colony growth. This effect was enhanced by complement and was dose dependent, being almost complete at ALG concentrations of 100 microgram/ml. Inhibition was a property of ALG but not of normal horse globulin. However, short incubation of ALG with bone marrow cells occasionally stimulated colony growth and normal horse globulin regularly stimulated it. Three hours' incubation of bone marrow cells with ALG was needed to produce consistent colony inhibition, which was measurable as a reduction in the expected number of colonies and as a fall in the colony: cluster ratio of surviving cell aggregates. Absorption of ALG on acute myeloid leukaemia blast cells removed the inhibiting property of the ALG while preserving its lymphocytotoxic action. Serum from two patients receiving ALG treatment inhibited colony growth for up to 48 hours after ALG administration. The results suggest the presence in ALG of antibodies specifically cytotoxic to myeloid stem cells which may relate to its myleosuppressive properties in vivo, and also indicate that it should be possible to remove antimyeloid antibodies from ALG by absorption. The use of such purified ALG would have advantages in clinical bone marrow transplantation.     

The mediating effect of anti-rat lymphocytic globulin on the uptake of homologous lymphocytes by rat macrophages

The uptake of homologous lymphocytes by rat macrophages was observed in tissue culture chambers. This uptake was quantified as an opsonization index. The effect on this index of treatment of either lymphocytes or macrophages by horse anti-lymphocytic globulin (ALG) or normal horse IgG (N.IgG) in the presence or absence of complement was measured. ALG-treated lymphocytes were shown to attach to immune adherence receptors on human erythrocytes in the presence of complement. A complement attachment of ALG-treated lymphocytes to macrophages was also observed following blocking of cytophilic receptors by N.IgG.     

Bovine lymphocytes: recognition of cells forming spontaneous (E) rosettes.

About 20% of thymus lymphocytes from neonatal calves formed spontaneous (E) rosettes with SRBCs in medium consisting of 50-100% foetal calf serum (FCS); other media were less satisfactory. FCS was necessary both to allow rosette formation to occur and to maintain stability of the rosettes once formed. Rosettes were stable at 0 degrees C but unstable at 18 degrees C and 37 degrees C. Dead thymus cell (sodium azide treated) did not form rosettes. Treatment of thymus cells with antiserum to bovine Ig-inhibited rosette formation, but this inhibition was considered non-specific since it also occurred with normal rabbit serum. Treatment of SRBCs with neuraminidase slightly enhanced rosette formation by thymus cells, but did not induce peripheral blood lymphocytes to form rosettes. Rosette formation did not occur under a variety of conditions with normal or neuraminidase-treated human, horse, pig, rabbit, guinea-pig, chicken or autologous RBCs. SRBC rosette forming cells were also found in lymph nodes (2-14%) and spleen (less than 5%), but rarely or never in peripheral blood and bone marrow of calves and adults. In foetuses at 80 days of gestation, 49% of thymus cells formed E rosettes. Foetal lymph node cells formed E rosettes at 160 days and spleen at 180 days. Cells with membrane-bound Ig were observed by IFT; their distribution did not coincide with the occurrence of E rosettes. E-rosette formation might be a marker for a subpopulation of bovine T cells.     

Distribution of IgM-, IgG-, and IgA-positive cells in lymphatic organs of normo- and dysgammaglobulinemic UM-B19 chickens

The cytotoxic activities of lupus sera were measured against IgG Fc-receptor-bearing T lymphocytes (T gamma cells) and T lymphocytes lacking this receptor [T gamma (-) cells], and the activities were compared with the inhibitory activities of the sera for the formation of rosettes (T gamma rosettes) between T lymphocytes and ox erythrocytes sensitized with IgG antibody. The cytotoxic activities of the sera against both T gamma and T gamma (-) cells well correlated with their T gamma rosette inhibitory rates. Also, the cytotoxic activities after the removal of IgM antibodies strongly correlated with the inhibitory rates. Among them, the highest correlation was observed between IgG T gamma-specific cytotoxic antibodies and T gamma rosette inhibitory rates of the sera. Gel filtration, ultracentrifugation, pepsin digestion, and reduction and alkylation of the sera revealed that main inhibitory activities were contained in IgG fractions. These results suggested that IgG T gamma-specific antibody suppressed T gamma rosette formation and might contribute to the reduction of T gamma cell number in patients with systemic lupus erythematosus.     

Autoantibodies Against Ganglioside GM3 Represent a Portion of Anti-Lymphocyte Antibodies in AIDS Patients

In this study we analysed the relationship between anti-lymphocytic ganglioside antibodies and anti-lymphocyte antibodies in AIDS patients. Anti-lymphocytic ganglioside antibodies were detected by thin layer chromatography (TLC) immunostaining; three colour flow cytometry was used to analyse circulating antibodies against different lymphocyte subsets.
Anti-lymphocytic ganglioside antibodies were detected in 23 out of 49 AIDS patient sera (46.9%). All positive sera reacted selectively with the GM3 comigrating band from AIDS lymphocytes. Twenty two out of the 23 anti-lymphocytic GM3 positive sera also had antibodies against CD4⁺ T cells, versus 17/26 anti-GM3 negative. Furthermore, patients with lymphocytic GM3 antibodies showed a significantly higher antibody reactivity against CD4⁺ T cells than patients in which these antibodies were not detected. The absorption tests revealed that preincubation of positive sera with GM3 was followed by a decrease in the reaction with target lymphocytes.
These findings suggest that anti-GM3 antibodies are a portion, but not the majority, of antibodies reacting with CD4⁺ T cells.     

Erythrocyte-rosette receptor expression by chronic lymphocytic leukemia B lymphocytes

Erythrocyte (E) rosette-forming cells have been investigated in three patients with B chronic lymphocytic leukemia whose leukemic lymphocytes were easily identifiable. A small percentage of fresh neoplastic cells formed E rosettes in two patients. In every patient, most unstimulated, cultured leukemic lymphocytes became E+ and this was further enhanced by phytohemagglutinin and pokeweed mitogen stimulation or neuraminidase treatment. These E+ B cells lacked detectable T cell antigens (except for a weak expression of an antigen associated with the helper T cell subpopulation in one case). They were unreactive or weakly reactive by immunofluorescence with a monoclonal antibody to the E receptor. However, this antibody completely inhibited E-rosette formation. The enhanced expression of E-rosette receptors by in vitro cultured cells appeared to be dependent upon the presence of a small number of E-rosetting cells at the beginning of the culture. E-rosette receptor expression by leukemic lymphocytes was most likely in a fourth case (out of 9 patients studied). This finding may account for some of the discrepancies in the study of so-called T cells in B chronic lymphocytic leukemia.     

Mechanisms of E-rosette formation by mitogen-stimulated canine lymphocytes.

This study examined the mechanisms of E-rosette formation of mitogen-stimulated canine peripheral blood lymphocytes with human and guinea-pig erythrocytes (E). In vitro culture with phytohaemagglutinin-P (PHA-P) increased the number of E-rosette forming cells two- to three-fold within 1 h and ten-fold by 48 h. Mitogen-enhancement of 1 h rosettes occurred via cross-linking of E to lymphocytes by PHA-P. Forty-eight hour rosettes, mediated by natural cytophilic antibodies in serum, resulted from PHA-P-induced increases in the number of cellular Fc receptors for this immunoglobulin. The data obtained in this study indicate that E-rosette formation by resting or mitogen-stimulated canine lymphocytes is neither a reliable nor a specific T-cell marker in this species.     

Study of human T and B lymphocytes with heterologous antisera. I Preparation, specificity and properties of antisera.

Heterologous antisera have been raised in the horse and rabbit against human lymphocytes. Appropriate absorptions on either B or T cells were performed to make antisera specific for human T (anti-HTLA) or B (serum 789) lymphocytes respectively. In addition serum 789 was found to react with circulating monocytes. The percentages of T and B cells detected by anti-HTLA and 789 sera in the different lymphoid organs averaged respectively: 78-7 per cent and 14-7 per cent in peripheral blood, 91-4 per cent and 4-0 per cent in thymus, 73-0 per cent and 14-5 per cent in lymph nodes, 53-6 per cent and 30-0 per cent in spleen, 47-1 per cent and 47-6 per cent in tonsils and 17-5 per cent and 13-5 per cent in bone marrow. Anti-HTLA serum appeared to supress E-rosette formation but did not affect binding of C3-coated erythrocytes. Serum 789 did not prevent E-rosette formation and reduced the number of EAC rosettes by 50 per cent. Anti-HTLA serum was found able to suti-lymphocyte serum, and PWM in the presence of complement; it was found highly mitogenic by itself. Serum 789 decreased the proliferative response to phytomitogens in about the proportion of cells killed by the antiserum. These results indicate that the presence of T cells is necessary for the mitogen-induced proliferation to occur, and that B lymphocytes are induced to proliferate in the presence of T cells and phytomitogens. Anti-HTLA serum was found not to inhibit K-cell activity of lymphocytes against antibody-coated chicken erythrocytes. These antisera appear very useful tools for the study of the role of human B and T lymphocytes involved in in vitro immune reactions.     

Tuberculin-specific transfer factor in dogs.

The present study examined the specificity of guinea pig erythrocyte (E) and erythrocyte-antibody-complement (EAC) rosette formation assays with suspensions of canine peripheral blood lymphocytes. Neutrophils, monocytes, and lymphocytes bound EAC but not erythrocyte-antibody (EA) controls. Similarly, all three cell types formed rosettes with guinea pig E. Adherence of guinea pig E to these cells was apparently mediated by natural cytophilic antibodies present in the serum used in the suspension medium. The nonspecificity of the guinea pig E-rosette formation assay with canine lymphocytes renders the technique unreliable for the identification of thymus-derived lymphocytes in dogs.     

Rosette formation by peripheral lymphocytes

In preparations of human peripheral lymphocytes suspended in serum absorbed with sheep red cells, up to 30% of the lymphocytes may make rosettes with sheep erythrocytes.

Washed lymphocytes suspended in Hanks' solution make many rosettes if tested without delay. Such lymphocytes rapidly lose their capacity to make rosettes, but it can be restored by adding the serum of man or of the horse, rabbit or guinea-pig. The lymphocytes of three newborn babies, and of one adult who had no detectable antisheep agglutinin in the serum, made rosettes with sheep cells.

Rosette formation is uncorrelated with serum agglutinin levels. Many normal adults have far higher titres of agglutinins against the red cells of other animals than against sheep cells; yet their lymphocytes do not make rosettes with the cells of these other animals.

Sodium cyanide (0·01 M) abolished rosette formation, and horse antihuman lymphocyte globulin inhibits it.

It is concluded that sheep cell rosette formation by human peripheral lymphocytes is not due to humoral antibody or delayed hypersensitivity, because of the great proportion of lymphocytes that are capable of it. Its nature is obscure, but it is suggested that it may be due to a substance, not primarily an antibody, that is elaborated by a large proportion of circulating lymphocytes and cross-reacts with some red cell antigens as plant lectins do.

Caution is advised in using the system to test antihuman lymphocyte serum until more is known about it.

     

A methodological study of E-rosette formation using AET-treated sheep red blood cells.

The influence of some of the well knwon technical variables on the E-rosette technique was examined using sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (AET). With AET treatment, E-rosette formation becomes less dependent on time and temperature and on the presence of serum. The mechanical stability of the rosettes is enhanced, and the number of SRBC attached to each rosette-forming lymphocyte (RFC) is markedly increased, leading to a sharper distinction between RFC and non-RFC. Ultimately, significantly more E-receptor carrying lymphocytes become detectable. Evidence is provided that the specificity of the E-rosette technique is unchanged after AET treatment of SRBC, in spite of the enhanced binding. A simple and reliable method for E-RFC identification is documented.     

Immune complexes in Takayasu''s arteritis.

We examined sera and Fc receptor-bearing lymphocytes from peripheral blood of patients with Takayasu's arteritis for the purpose of investigating the presence of immune complexes (IC). IC in sera were assayed by solid-phase conglutinin-binding test. Seven of 29 patients exceeded the normal range of circulating IC. IC combined with Fc receptors were estimated by enumerating EA-RFC. EA-RFC of lymphocytes from patients with Takayasu's arteritis were 13.0% and those of normal controls were 29.1%. Low EA-RFC in the patient group increased significantly when lymphocytes were incubated with EA after rising lymphocytes with medium at 37 degrees C. On the contrary, EA-RFC from healthy subjects did not increase after rinsing cells. These findings indicate that IC combined with Fc receptors and hindered EA rosette formation and that rinsing cells with medium at 37 degrees C removed IC from Fc receptors. Comparable results were obtained by a membrane immunofluorescence method using FITC-conjugated anti-human immunoglobulin. In order to confirm that EA rosette formation was really blocked by IC, lymphocytes from a healthy donor were incubated with heat-aggregated human IgG. Incubating cells with IgG aggregates caused reduction of EA-RFC and these lymphocytes restored their ability to form rosettes with EA by rinsing cells with medium at 37 degrees C. In conclusion, we could confirm the presence of IC both in sera and on lymphocyte Fc receptors in some cases of Takayasu's arteritis.     

The immunomodulating effect of theophylline on lymphocytes from chronic lymphocytic leukemia patients

The in vitro immunomodulating effects of theophylline on E-rosette formation, phytohemagglutinin (PHA) response, and Ig surface receptors of B lymphocytes were studied on fresh as well as on preincubated lymphocytes from patients with B cell chronic lymphocytic leukemia (CLL). In 11 out of 14 CLL patients, 24 hours preincubation at 37 degrees C significantly enhanced E-rosette formation. Subsequent treatment of preincubated cells with appropriate concentrations of theophylline further enhanced E-rosette formation in 11 cases. On fresh lymphocytes the enhancing effect of theophylline on E-rosette formation was not significant. The same was true for PHA stimulation; in 5 out of 7 cases the mitogen enhanced the stimulating effect of preincubation and had no significant effect on fresh lymphocytes from CLL patients. Preincubation significantly reduced the percentage of surface immunoglobulin positive B cells from CLL patients in all cases studied, and theophylline treatment had an additional effect on this phenomenon. No such effect of theophylline on fresh B cells from CLL patients could be observed. Preincubation had no significant effect on control lymphocytes. The effect of theophylline on control lymphocytes as compared to lymphocytes from CLL patients was completely different for T as well as for B lymphocytes. E-rosette formation from control lymphocytes (fresh and preincubated) was significantly inhibited in the presence of theophylline. No significantly enhanced responsiveness to PHA could be observed after treatment of fresh or preincubated lymphocytes with theophylline. Preincubation and theophylline treatment had no significant effect on the percentage of Ig positive B cells from control lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies of lymphocytotoxins in infectious mononucleosis and systemic lupus erythematosus: evidence for immune complex-mediated cytotoxicity.

The hypothesis that serum lymphocytotoxins are antigen-antibody complexes was examined. High molecular weight fractions from the sera of eighteen patients with infectious mononucleosis (IM), thirteen patients with systemic lupus erythematosus (SLE) and six healthy controls, were prepared by precipitation with polyethylene glycol 6000 (PEG). The lymphocytotoxic activity (LCA) of these PEG precipitates was significantly greater (P less than 0.01) than that of the corresponding sera and a significant correlation (r = 0.66, P less than 0.01) was observed between the LCA of sera and the PEG precipitates. In contrast to the concentration of LCA in the PEG precipitates, the heterophil antibody titres of the precipitates from IM sera were significantly less (P less than 0 05) than serum titres. Antisera raised against PEG precipitates from sera from nine patients with IM contained significant LCA. The nature of this LCA differed from that of the LCA in the original sera in temperature dependence and the molecular size. Antigen-antibody complexes in seven sera (four IM, three SLE) were dissociated at low pH (3.0) and fractionated by gel filtration at pH 3.9. The LCA of these fractions was compared with the LCA of equivalent fractions obtained by gel filtration at pH 7.2. The heterophil antibody present in sera from patients with IM and the cytotoxicity of anti-lymphocyte globulin (ALG) were used as 'antibody controls'. In this way it was shown that the LCA in patient sera, but not heterophil antibody or ALG cytotoxicity was significantly reduced (P less than 0.001) by low pH gel filtration.     

E and EAC rosetting lymphocytes in patients with carcinoma of bronchus I. Some parameters of the test and of its prognostic significance.

Using peripheral blood lymphocytes separated by a Ficoll method and suspended in saline, means of 77-1% (s.d. 5-2) E rosettes (T lymphocytes) and 20-1% (s.d. 6-7) EAC rosettes (B lymphocytes) have been obtained with normal healthy donors. Poorer E-rosette formation resulted from higher centrifugation speeds during the washing of lymphocytes or erythrocytes, insufficient chilling, or rough handling. The presence of 5% albumin in the final mixture stabilized the rosettes and brought a constant subpopulation of B lymphocytes into rosetting. In patients with bronchial carcinoma who, at the time of diagnosis, had E-rosette percentages below 1 s.d. of the mean for normal donors, the length of survival was significantly shorter than in those with normal or high values. The same was true for those in whom null cells were detected. In each case the correlation effect was mainly found in the group of patients with squamous carcinoma.     

Induction and suppression of the cytotoxic activity of human lymphocytes in vitro by heterologous anti-lymphocyte serum

Heterologous anti-lymphocyte sera were demonstrated to induce a cytotoxic potential in normal non-immunized human lymphocytes against allogeneic fibroblast target cells. The cytotoxicity-inducing capacity was restricted to certain dilutions of anti-lymphocytic serum above and below which no cytotoxic effect was obtained. This optimal concentration shifted towards higher dilutions in sera taken late during the immunization course. The antisera were shown to stimulate the DNA-synthesis in lymphocytes and to aggregate the lymphocytes to the target cells. The DNA-synthesis and the aggregation as well were maximal at the same dilution of anti-lymphocytic serum which induced cytotoxicity. No cytotoxic effect was demonstrable on sheep fibroblasts. It is, therefore, suggested that the anti-lymphocytic serum antibody induces lymphocyte-mediated cytotoxicity against allogeneic fibroblasts in a two step manner: it stimulates the lymphocytes into a cytotoxic state; it aggregates the human lymphocytes to the human fibroblasts by virtue of its bivalent structure.

Anti-lymphocytic serum was also found to suppress the cytotoxic activity of lymphocytes induced by various non-specific agents, such as phytohaemagglutinin, streptolysin O and anti-lymphocytic serum itself. The mechanism for this inhibition is extensively discussed and it is suggested that anti-lymphocytic serum suppresses the reaction by coating the lymphocytes, thereby preventing the intimate contact between effector and target cell. A similar mechanism may operate in vivo and could be a partial explanation of the in vivo immunosuppressive effect of anti-lymphocytic serum. Purified 7S γ-globulin possessed all activities of the whole antiserum.

     

Characterisation and separation of human lymphocytes forming mouse red cell rosettes.

Rosette formation between human lymphocytes and mouse red blood cells (MRBC) was examined as a marker for B lymphocytes and as a method of B lymphocyte separation. A small proportion of lymphocytes formed spontaneous rosettes with MRBC (mean value 6%) and the number was considerably increased by pretreating the lymphocytes with neuraminidase (mean value 16%). Double marker tests demonstrated that lymphocytes forming MRBC rosette were immunoglobulin (Ig) bearing cells, with a high proportion of IgM bearing cells, but not all Ig bearing cells formed MRBC rosettes. Lymphocyte populations enriched with T or B lymphocytes, by SRBC rosette sedimentation and nylon column filtration, gave values for MRBC rosettes consistent with a subpopulation of Ig bearing cells. Separation of MRBC rosette-forming cells gave a relatively poor degree of separation and rerosetting with MRBC produced variable results.     

Peripheral blood B-lymphocyte abnormalities associated with hyperthyroidism of Graves' disease.

Proportions of peripheral blood thymus-derived T lymphocytes (T cells) and bone-marrow-derived B-lymphocytes (B cells) were studied in twelve hyperthyroid patients and ninety-nine non-hyperthyroid control subjects including thirty-nine healthy individuals and sixty patients with various disorders. All hyperthyroid patients had Graves' disease and eight were untreated. The sheep erythrocyte (E)-rosetting technique was employed for enumeration of T cells and the immunofluorescent technique was used for identification of lymphocytes with surface immunoglobulins (SIg), a marker for B cells. The results showed that hyperthyroid patients had higher percentages of lymphocytes stainable for SIg, whereas their T-cell proportions were the same as our control values. In addition, approximately half or more of the fluorescein stainable lymphocytes reacted with each of the five antisera against individual heavy chain determinants, including the epsilon chain, indicating the presence of more than three Ig determinants on the same cell. The fluorescein-stainable cells did not form E rosettes. Blocking of the Fc receptor on lymphocytes by incubating the patients' cells with heat-aggregated human IgG or heated goat anti-bovine serum albumin (anti-BSA) failed to abolish the subsequent fluorescent staining of the cells. Incubation of patients' lymphocytes with non-fluorescent anti-epsilon inhibited the subsequent staining of cells with fluoresceinated anti-epsilon but not staining with fluoresceinated anti-mu or anti-gamma. Thus, the study revealed B-cell abnormalities associated with hyperthyroidism, manifested by the simultaneous presence of multiple Ig classes, including IgE, on a single B cell. Results of studies of incubation of the patients' plasmas with lymphocytes from health individuals and studies of SIg by overnight culture of the patients' lymphocytes with or without prior trypsinization suggested that the SIg was generated endogenously by the cell on which it resided.     

A comparison of in vitro, immuno-suppressive and clinical effects of goat and horse anti-human thymocyte globulin in macaca monkeys

Anti-human thymocyte globulin was raised in four goats and two horses. The in vitro properties were defined, and clinical effects and immunosuppressive efficacy were tested in Macaca monkeys bearing skin xenografts or allografts. All goat and one horse ALG preparation produced significant graft prolongation; one horse ALG showed no in vivo immune suppression. Graft survival correlated well with the rosette-inhibiting activities of the various ALG batches. Goat ALG was well tolerated and produced no local, systemic or anaphylactic reactions; both horse ALG preparations consistently produced local reactions, serum sickness, and, occasionally severe anaphylactic reactions in sensitized and non-sensitized monkeys. Prolonged treatment with goat or horse ALG for up to 10 months caused lymphocyte depletion and plasmacytosis in lymph nodes and spleen but no other apparent pathological changes in a wide variety of tissues studied; infective complications did not occur. Goat IgG was more immunogenic than horse but circulating antibody titres to xenogenic IgG could not be related to clinical responses to ALG, to deposition of xenogenic IgG in kidneys, or to immunosuppressive potency of ALG batches. It was concluded that goat ALG was consistently more potent and less toxic than horse ALG.     

In vitro radiosensitivity of human T and B lymphocytes evaluated using lymphocyte transformation tests and rosette formation tests.

The influence of irradiation upon human lymphocytes was studied using lymphocyte transformation tests and formation of E and HEAC rosettes. Irradiation was given in vitro using doses between 0 and 50,000 rad. It was shown that blast transformation after stimulation with T-cell stimulating agents (as PHA, PWM, Con.A and PPD) was suppressed by irradiation. The effect of irradiation upon T lymphocytes was also shown in different kinds of MLC experiments. Both the effect of irradiation upon rosetts formation and the influence of irradiation upon already formed rosettes were analyzed. The ability of lymphocytes to form E rosettes was affected after irradiation with 500 rad: there were fewer E rosettes with 3 SRBCs, decrease in total number of E rosettes and more null cells, with no depression of the number of HEAC rosettes formed. Already formed E and HEAC rosettes were totally unaffected of irradiation, and this radioresistance was also observed for 18-hour-old rosettes. The ability to form spontaneous E rosettes was decreased after irradiation of the lymphocytes with 100 rad; increasing doses did not cause further depression, and already formed spontaneous E rosettes were radioresistant. The mechanisms involved in E-rosette formation thus seem to be radiosensitive.