Cytotoxic activity against maedi-visna virus-infected macrophages.

The cell type predominantly infected by maedi-visna virus (MVV) is the macrophage, and we have looked at the ability of MVV-infected macrophages to interact with cytotoxic T lymphocytes (CTL), important effector cells against virus infections. MVV-specific CTL precursors were detected, after in vitro culture with MVV antigen and recombinant human interleukin-2, in peripheral blood lymphocytes of all MVV-infected sheep. MVV-infected monocyte-derived macrophages and alveolar macrophages were able to stimulate CTL activity in vitro and were targets for these activated CTL. The major effector cell population using MVV-infected macrophage targets was CD8+ lymphocytes, although another population, lymphokine-activated killer cells, may also have been involved. There was no direct cytotoxic activity found in alveolar lymphocytes from MVV-infected sheep without lung lesions.     

Reversal of cell surface abnormalities of T lymphocytes in hodgkin's disease after in vitro incubation in fetal sera.

The capacity of periphal blood lymphocytes from patients with untreated Hodgkin's disease to form E rosettes with sheep erythrocytes and to respond in vitro to PHA stimulation were found to be profoundly impaired. In 49% of the patients, the percentage of E rosette-forming cells (E-RFC) was more than two standard deviations below the mean for normal donors. Overnight incubation of the peripheral blood lymhocytes from these patients in culture media containing 20% fetal calf serum was followed by restoration of the percentage of E-RFC up to normal levels. Similar results have been observed after incubation in fetal human serum, but not in adult human AB serum or adult bovine serum. Incubation of peripheral blood lymphocytes from untreated patients in20% fetal calf serum also resulted in a remarkable restoration of their capacity to respond normally to PHA. Possible mechanisms involved in these reversible cell surface and in vitro lymphocyte function abnormalities in Hodgkin's disease are discussed.     

Activated T lymphocytes within human solid tumors

Summary The majority of lymphocytes separated from tumor cell suspensions were T cells. Conjugates of T lymphocytes and tumor cells were often seen. Variable numbers of T cells exhibited signs of activation such as the ability to form stable E rosettes and attachment to normal and malignant cells (a phenomenon designated natural attachment: NA). A proportion of T cells activated in vitro by allogeneic stimulation regularly exhibit these properties. The T cell-tumor conjugates in the suspensions may represent the NA phenomenon, but they could also be the product of T cells that adhere on the basis of specific recognition of cell surface antigens.Abbreviations BBS balanced salt solution - E rosettes rosettes formed with sheep erythrocytes - EA rosettes rosettes formed with ox erythrocytes coated with anti-ox IgG - FCS fetal calf serum - MLC mixed lymphocyte cultures - NA natural attachment - PBL peripheral blood lymphocytes - SRBC sheep erythrocytes - T lymphocytes thymusderived lymphocytes     

Optimization of an ovine antibody-secreting cell assay for detection of antigen-specific immunoglobulin production in peripheral blood leukocytes

Enumeration of antibody-secreting cells in peripheral blood by enzyme-linked immunospot (ELISPOT) has been used in human studies to detect antigen-specific antibody production at mucosal tissue sites. An alternative assay for detecting and quantitating antigen-specific antibody responses involves culturing circulating peripheral blood antibody-secreting cells and quantitating specific antibody production in culture supernatant by ELISA. In the present study, antigen-specific peripheral blood lymphocytes were isolated from subcutaneously immunized sheep and the parameters for maximizing in vitro antibody production by in vivo-induced antibody-secreting cells optimized for this species. Maximum antibody-secreting cell responses were observed in peripheral blood collected four days after antigen challenge. The addition of lipopolysaccharide and antisheep immunoglobulin had no effect on in vitro antibody secretion by blood antibody-secreting cells, while the effects of pokeweed mitogen were highly variable. However, the combination of anti-Ig and recombinant ovine interleukin-6 to peripheral blood lymphocyte cultures was found to markedly and consistently enhance specific antibody production. In unstimulated cultures, the optimal peripheral blood lymphocyte concentration for generating the greatest antibody responses was 5.0 x 107 cells per mL, but in cultures stimulated with recombinant ovine interleukin-6/antisheep immunoglobulin, the optimal cell concentration was lowered to approximately 1.0 x 107 cells per mL. In vitro, peak immunoglobulin production was usually achieved by day one in unstimulated cultures. In recombinant ovine interleukin-6/antisheep immunoglobulin-stimulated cultures, antibody levels were similar to unstimulated cultures by day one, however, the levels continued to rise during incubation to reach a maximum between days four and five of incubation. This optimized antibody-secreting cell culture assay is amenable for increasing the sensitivity and reducing the cell numbers required for quantitating antigen-specific antibody induction in large-scale immunization trials in sheep and other large animal species.     

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.     

Human recombinant interleukin 2-activated sheep lymphocytes lyse sheep pulmonary microvascular endothelial cells

Administration of lymphokine-activated killer (LAK) cells in combination with interleukin 2 (IL-2) has been effective in reducing tumor mass in humans, but has been accompanied by significant toxicity. We used a chronic awake sheep model to investigate the cause of the vascular leak syndrome associated with IL-2 administration. Sheep repeatedly infused with human recombinant IL-2 (hrIL-2) developed mild pulmonary hypertension, systemic hypotension, acidemia, hypoxemia, and increased flow of protein rich lung lymph. We hypothesized that LAK cells may damage lung endothelium in vivo and cause increased lung vascular permeability. Sheep peripheral blood and lung lymph lymphocytes incubated in vitro with hrIL-2 generated cytotoxic activity for human K-562 cells and sheep pulmonary microvascular endothelial cells. In addition, cytotoxic effector cells were isolated from the peripheral blood of a sheep which had received hrIL-2. These observations suggest that LAK cells possess the ability to damage endothelial cells and may contribute to an increased pulmonary vascular permeability observed following hrIL-2 infusion in sheep.     

Increased T lymphocytes and IgMEA-receptor lymphocytes in Hodgkin's disease spleens.

Splenic lymphocytes from 11 patients with Hodgkin's disease were compared to lymphocytes of six spleens from patients with nonlymphoproliferative diseases. T lymphocytes were increased in patients with histological involvement by Hodgkin's disease. Likewise, lymphocytes from spleens with histological involvement showed increased rosette formation with immunologlobulin M-coated sheep red blood cells (IgMEA). A similar increase in T lymphocytes and in IgMEA rosette formation was not observed with normal peripheral blood lymphocytes, control spleens, or with Hodgkin's disease spleens without evidence of histological involvement.     

In vitro induction of lymphocyte responsiveness by a Strongylus vulgaris-derived mitogen

Proliferation in vitro of peripheral blood lymphocytes both from horses infected with Strongylus vulgaris and from helminth-free ponies was observed in the presence of extracts of the fourth and fifth stage larvae and adults of S. vulgaris. In addition, S. vulgaris extracts induced transformation in cultures of peripheral blood lymphocytes from sheep and dogs and in mouse spleen cell cultures. Nylon wool non-adherent, T cell enriched fractions of lymphocytes from both mice and horses were stimulated by the S. vulgaris larval mitogen while no proliferation was observed in cultures containing nylon wool adherent, B cell enriched fractions. Macrophage co-operation appeared not to be necessary for S. vulgaris mitogen-induced transformation of spleen cells. The S. vulgaris mitogen stimulated a subpopulation of mouse spleen cells different from those responsive to PHA, Con A and LPS. These cells might be T helper cells since B cells were stimulated to proliferate in the presence of both T cells and S. vulgaris larval mitogen. In addition, the supernatant of in vitro cultured larvae of S. vulgaris induced slight, but significant transformation of equine peripheral blood lymphocytes. Therefore, it is possible that the S. vulgaris mitogen released by both viable parasites and degenerating larvae might induce T cell dependent production of immunoglobulin in vivo and account for the beta-globulinaemia, of which IgG(T) is a major component, in S vulgaris infected horses.     

Cytogenetic study of some tissues and age-related changes in cell proportions in a goat-sheep chimera.

Different percentages of cells with a female sheep or male goat karyotype were found in kidney (12.0% vs. 88.0%) and lung (42.6% vs. 57.4%) cell cultures from a 10-year-old chimera. Skin biopsies from patches with goat hair or sheep wool showed different, age-related goat-to-sheep fibroblast ratios. Karyotypic analysis of lymphocytes obtained from peripheral blood of the chimera at 6 and 10 yr of age showed no chimerism. Two weeks after birth, however, lymphocytes with both sheep (54,XX) and goat (60,XY) karyotypes were apparent in the blood of this chimera. Twenty percent of the blood cells examined at 2 wk had a caprine karyotype; this proportion declined with time, until it was totally eliminated at age 6.     

Functional differentiation of B lymphocytes in congenital agammaglobulinemia. I. Generation of hemolytic plaque-forming cells.

Despite the absence of B lymphocytes, peripheral blood lymphocytes (PBL) from four of five patients with congenital agammaglobulinemia (cAgamma) generated a specific hemolytic plaque-forming cell (HcPFC) response in vitro to sheep red blood cells and ovalbumin. The kinetics, antigenic, and cellular requirements were similar to normals, but significantly less HcPFC were found in patient cultures. Normal but not patient HcPFC-precursor cells were inactivated by treatment with anti-mu antisera whereas generated HcPFC in both controls and patients were sensitive to treatment with anti-mu. Pokeweed mitogen (PWM) and dextran sulfate (DXS) enhanced the HcPFC-response of normal PBL; cAgamma-cells were unresponsive to DxS and, in the presence of PWM, the development of HcPFC was inhibited. These findings indicate the presence of B lymphocyte precursors in the majority of patients with cAgamma investigated.     

Cellular changes in the intestinal lymph of sheep infected with the enteric nematode, Trichostrongylus colubriformis

Some changes produced in the cell populations of intestinal lymph by infection with the enteric nematode, Trichostrongylus colubriformis, were studied in sheep regularly re-infused with all discharged lymph. Lymphocyte traffic through the intestinal lymphatic duct was reduced until day 35 of primary infection, mainly due to the absence of lymphocytes with smaller cell volumes, but was increased two-fold after day 70 and in immune sheep. Antigen-reactive lymphocytes in blood and lymph were assayed by the uptake of 3H-thymidine in cell culture stimulated by extracts from the larvae of T. colubriformis. Cells from the blood and lymph of immune sheep were highly reactive to worm antigen. A relatively smaller reactivity was present in the blood of worm-free sheep and was abolished during the first 12 days of primary infection. Antigen reactive cells were not detected in intestinal lymph until 12 days after primary infection, and in vitro antigen reactivity in intestinal lymph of immune sheep was increased after challenge with infective larvae. Responses to the mitogens, concanvalin A and phytohaemagglutinin, in cultures of cells from both intestinal lymph and blood were depressed on days 7 and 12 of primary infection. It is proposed that the diminished traffic of lymphocytes in intestinal lymph and the reduced numbers of mitogen and nematode antigen-reactive lymphocytes in both blood and intestinal lymph during the early stages of infection with T. colubriformis is closely related to the slow development of protective immunity to this parasite.     

Simultaneous detection of T-, B- and D-rosette forming lymphocytes and null cells in humans]

For the identification of T-, B-, and D-rosette-forming lymphocytes and the "null" cells in the human peripheral blood a simultaneous reaction of rosette-forming cells with the use of zymosan-complement, sheep red blood cells, fixation and staining of smears in extraction of the lymphocytes tested with the aid of verographin may be recommended.     

Influence of human thymocytes on B-lymphocyte differentiation in man.

Human thymocytes from children less than 6 years of age were tested for their influence on differentiation of normal B cells. The addition of either thymocytes or a culture supernatant from thymocytes to normal peripheral blood lymphocytes (PBL) enhanced pokeweed mitogen-induced B-cell differentiation as tested in a plaque-forming assay for antibody to sheep red blood cells. The thymocytes, however, could not substitute for T lymphocytes in cultures of PBL which had been previously depleted of T lymphocytes. Further, prior treatment of thymocytes with concanavalin A did not result in generation of suppressor cells for either B-cell differentiation or for the responses of PBL to mitogens. Thus, although thymocytes were functionally immature by these assays as compared to mature T lymphocytes they exerted an influence on B-cell differentiation in cultures of normal peripheral blood lymphocytes.     

Immunoglobulin-positive mononuclear cells in human peripheral blood: detection by mixed anti-globulin and direct anti-globulin-rosetting reactions.

Ig-bearing mononuclear cells were identified in Ficoll-Hypaque preparations of human peripheral blood by using mixed anti-globulin (MAG) and direct anti-globulin rosettes; indicator cells consisted of sheep erythrocytes coated with human F(ab')2 or anti-F(ab')2 antibody, respectively. Of the cell population isolated from 10 normal subjects, a mean of 68% was lymphocytes. However, fewer than 50% of the cells with detectable surface Ig were lymphocytes. On viable cell preparations using chromic chloride-treated sheep erythrocytes (CrCl3SRBC) coated with anti-F(ab')2 antibody, a mean of 20.1% of the lymphocytes formed rosettes, i.e., were B. Up to 6% of peripheral blood lymphocytes formed mixed Ig-rosettes and E-rosettes. On viable lymphocytes using F(ab')2-coated CrCl3SRBC, MAG rosettes were insensitive in detection of B lymphocytes. Formaldehyde treatment of lymphocytes increased the number of B cells detectable to 25.5% of the lymphocyte population. Study of T-enriched and B-enriched populations showed that the observed increase in B cell reactivity was real and not due to MAG-rosetting T cells. A one-stage procedure for T and B lymphocyte separation is described.     

Changes in the spontaneous DNA-synthesizing activity in the peripheral blood lymphocytes of irradiated animals

In lymphocytes of sheep exposed to 52 and 103 mC/kg radiation revealed was an increase in spontaneous incorporation of 3H-thymidine into DNA. A change in spontaneous DNA-synthesizing activity in lymphocytes of exposed animals was accompanied by the increase in the total protein content of the peripheral blood lymphocytes.     

Immunological analysis of human lymphocytes bearing different surface receptors]

Cell surface receptors on human lymphocytes being studied, essential differences were revealed in a relative content of E-, EA- and EAC-rosette-forming cells in peripheral blood and tonsils. In tonsils, part of lymphocytes carrying receptors for sheep erythrocytes have been shown to possess C'3-receptors for sheep erythrocytes. Apparently, C'-receptor,--being B-lymphocyte surface marker,--may also appear at definite stages of T-cell differentiation.     

E1Lymphocytopenia in pseudo-lupus-erythmetaosus syndrome]

Seven female patients with pseudo-lupus erythematosus (LE)-syndrome had markedly reduced lymphocyte counts in their peripheral blood during the active phase of the disease. One patient, we were able to study during the active phase of her disease, had a diminuation of spontaneous rosettes-forming (T-)lymphocytes, using neuraminidase-treated sheep red blood cells for the test. In this case the percentage of surface-immunoglobulin-bearing (B-)lymphocytes determined by an indirect immunofluorescence technique was augmented. In comparison with 20 normal controls 6 other patients did not show any alterations in the relation of B- and T-lymphocytes in the peripheral blood. 5 patients had circulating lymphocytotoxic antibodies in their serum. A specificity of these antibodies for T-lymphocytes could not be realized.     

Detection of plaque-forming cells in the peripheral blood of actively immunized humans.

Ten adult human volunteers were immunized with Salmonella typhi and their peripheral blood leukocytes were collected for 14 days after immunization. These peripheral blood leukocyted, rich in lymphocytes, were plaqued in a modified Jerne assay against sheep erythrocytes coated with either Salmonella or Escherichia lipopolysaccharide. A specific direct and indirect PFC response developed in immunized individuals by day 7 and peaked at day 10. This vigorous PFC response rapidly declined to normal levels by day 14. This marked and specific PFC response of human peripheral blood leukocytes may be developed as a useful tool for monitoring the humoral immune response of patients with Gram-negative bacterial infections.     

Identification of Ia on a subpopulation of human T lymphocytes that stimulate in a mixed lymphocyte reaction

The delineation of discrete subpopulations of human T lymphocytes has permitted preliminary analyses of the complex cellular network regulating the immune response in man. We previously showed that a subset of T lymphocytes, designated as theophylline-sensitive because of their inability to bind sheep red blood cells in the presence of the drug, are responsible for antigen-specific suppression or regulation in an in vitro plaque-forming cell assay. We now show that 25 to 45% of these theophylline-sensitive T cells were Ia-positive by immunofluorescence with a rabbit antiserum raised against purified B lymphoblast surface antigenic material. These data suggested that 4 to 7% of peripheral blood T cells carry Ia determinants. The presence of Ia determinants on this T cell subset was confirmed by gel analysis of radioiodinated surface material. Furthermore, in mixed lymphocyte culture, the theophylline-sensitive cells demonstrated HLA-D determinants and were 10-fold more potent stimulators than equal numbers of B lymphocytes. The presence of Ia determinants on these T cells indicates the expression of major histocompatibility complex-related regulatory gene products on a specific human T lymphocyte subpopulation.     

Peripheral blood lymphocyte subpopulations in sarcoidosis.

We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sarcoidosis and 15 healthy controls. T cells were estimated from the number of lymphocytes forming rosettes in vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreased cellular immunity and increased reactivity of the antibody-forming system as commonly seen in sarcoidosis.