Erythrocyte-rosetting properties of feline blood lymphocytes and their relationship to monoclonal antibodies to T lymphocytes

Rosette formation of feline peripheral blood leukocytes with guinea pig (GP) and gerbil (G) erythrocytes (E) has been shown in an earlier study to identify T lymphocytes expressing helper and suppressor cell activity, respectively. This T lymphocyte distinction was based on the removal of the E-rosetting populations from peripheral blood leukocytes (PBL) and the subsequent functional evaluation of the remaining cells in a pokeweed mitogen (PWM)-induced synthesis of immunoglobulin (Ig). In the present study, we demonstrate a direct helper and suppressor function of GPE- and GE-rosetted cells, respectively, wherein the induction of Ig synthesis is altered in a positive or negative way by the addition of the cells to a control target population. A pan-T monoclonal antibody (mAb), CT843, and mAbs to the CD4 (CT248) and CD8 (CT87) subsets are also described; their specificities are established in functional assays, the PWM-induced Ig synthesis and the production of interleukin-2 following Concanavalin A stimulation of PBL, and a biochemical analysis of the surface membrane antigens detected by the mAbs. Immunoprecipitation and SDS-PAGE analyses showed CT248 to react with a approximately 60-kDa protein under both reducing and nonreducing conditions. Under reducing conditions, CT87 reacted with one subunit at approximately 35 kDa; a second faint band at approximately 39 kDa was poorly resolved. mAb CT843 detected a heterodimer of approximately 70 and approximately 60 kDa under both reducing and nonreducing conditions. The relationship of the mAbs to E-rosetting was examined in FACScan analyses and rosette inhibition studies. The percentage of GE-rosetting cells agreed with the percentage of cells stained with the CD8 mAb, whereas a comparison of GPE-rosetting and staining with the CD4 mAb showed variability. The binding of GE to PBL was blocked by pretreatment of PBL with the CD8 mAb, whereas no inhibition of GPE rosettes was observed with any of the mAbs. In a previous study, we had shown that an overnight culture of feline PBL at 37 degrees C leads to the development of a second population of GPE-rosetting cells, also having a helper function. The relationship of the two GPE-rosetting populations to the CD4 mAb, CT248, was examined in rosette depletion studies and FACScan analyses. It was found that depletion of the GPE-rosetting cells from fresh, i.e., Day 0 cells, removed only a small percentage of cells reactive with the CD4 mAb, whereas GPE-rosette depletions performed on Day 1 PBL, which contained both populations of GPE-rosetting cells, removed almost all cells reactive with this antibody. The latter study suggests that the GPE-rosetting phenomenon is detecting two subsets of CD4 cells with T helper function, those present in fresh blood and those acquiring the GPE receptor after an overnight culture.     

Two populations of guinea pig erythrocyte-rosetting cells in the cat: evidence for their T-helper function in mitogen-induced synthesis of Ig and interleukin-2.

Studies in our laboratory have shown that T-helper (T-H) and T-suppressor (T-S) cells in cat peripheral blood leukocytes (PBL) rosette with guinea pig (GP) and gerbil (G) erythrocytes (E), respectively. Removal of GE-rosetting cells leads to an enhanced (two- to threefold) synthesis of Ig in a pokeweed mitogen (PWM)-driven system as measured by plaque-forming cells (PFC) to protein A-coated sheep RBC, while depletion of GPE-rosetting cells yields a PFC response only 10-15% of the control. Surprisingly, removal of both GE- and GPE-rosetting cells gave a response equivalent to 40-100% of the control PBL. Analysis of the mixed GE/GPE rosette depleted cultures revealed the reappearance of GPE- but not GE-rosetting cells, reaching maximum values within 12-18 hr after in vitro culture. Cultures of control PBL and those following the mixed rosette depletion showed two populations of GPE-rosetting cells; the GPE-1 cells, present on Day 0 before culture, and the GPE-2 cells, those appearing on Day 1. Addition of cycloheximide prevented development of the GPE receptor while colchicine and mitomycin C were without effect. The development of PFC after the mixed GE/GPE rosette depletion was interpreted as being due to the GPE-2 cells functioning as T-H cells in the absence of any T-S (GE-rosetting) cells. This thesis was supported by showing a marked decrease in the PWM-induced Ig response when both the GPE-1 and GPE-2 populations were removed on Day 1. Additional evidence for functional T-H cells in the GPE-rosetting population was obtained by analyzing interleukin-2 (IL-2) production. Removal of the GPE-rosetting cells (GPE-1 and/or GPE-2) from PBL led to a marked decrease in Con A-induced IL-2 synthesis while removal of the GE-rosetting cells yielded a normal or slightly greater than normal response.     

Guinea pig and gerbil erythrocytes rosette with different cells in the blood, bone marrow, and thymus of the cat

Cat thymocytes, bone marrow cells, and peripheral blood leukocytes (PBL) formed rosettes with guinea pig (GP) and gerbil (G) erythrocytes (E). In PBL from adult cats the frequency of rosettes was 27% with GPE and GE, while an average of 33% bone marrow cells formed rosettes with GPE and only 4% with GE. Thymocytes from kittens showed a high percentage of rosettes with both GPE and GE (35 to 81%), with the frequency of each type varying with the thymus tested. Fluorescein isothiocyanate labeling of one of the erythrocyte species revealed these cells to be rosetting with different nucleated cells; i.e., a low percentage (3-5%) of the rosettes formed with PBL and bone marrow had both labeled and unlabeled erythrocytes. In contrast, "mixed" rosettes were observed with a significant number of thymocytes, averaging 33% of thymocytes from six animals. A further distinction between the GE- and GPE-rosetting cells was revealed by a monoclonal antibody which blocked GE rosette formation without interfering with the binding of GPE to PBL and thymocytes. PBL could be depleted of either GPE- or GE-rosetting cells, with retention of IgG+ cells and cells capable of rosetting with the second erythrocyte species in the nonrosetting fractions. Stimulation of the latter nonrosetting fractions with pokeweed mitogen for induction of Ig synthesis revealed a T-lymphocyte specificity of the GE- and GPE-rosetting cells. PBL depleted of GE-rosetting cells yielded an increased Ig production, two- to threefold above the control; in contrast, depletion of GPE-rosetting cells from PBL resulted in a failure of the remaining cells to respond. These results suggest that T-suppressor cells of the cat are contained in the GE-rosetting fraction and T-helper cells are rosetted with GPE.     

The role of interleukin 1 and interleukin 2 in human T colony formation

We investigated the roles of interleukin 1 (IL1) and interleukin 2 (IL2) on T colony formation by PHA-stimulated peripheral blood lymphocytes (PBL). Purified T cells stimulated by PHA could not generate T colonies as did PBL. Media conditioned by PHA-stimulated PBL (PHA-LCM) contained IL2 and a T colony-promoting activity (TCPA) which induced T colony formation in PHA-stimulated purified T cells. IL2 and TCPA are coeluted in the same peak of 18,000 molecular weight after gel filtration chromatography. Moreover, TCPA present in the PHA-LCM could be absorbed on IL2-sensitive cells which possessed specific receptors for IL2. These results suggest that TCPA and IL2 are related entities. Monocytes or IL1 (a monokine released by activated monocytes) also induced T colony formation in purified T cells. Phorbol myristate acetate (PMA) could replace monocytes in the induction of T colony. Monocytes, IL1, or PMA are known to be crucial requirements for IL2 production by PHA-stimulated T cells. This combined with the fact that IL2 participates in T colony formation suggests that monocytes induce T colony formation through IL2 production.     

Isolation and characteristics of IKO-GM-1 monoclonal antibodies

The hybridoma clone IKO-GM-1 was obtained as a result of fusion of P3X63Ag8.653 cells with splenocytes of BALB/c mice with the aid of 50% polyethyleneglycol. Antigen demonstrated by antibodies IKO-GM-1 expressed on 100% polymorphonuclear neutrophils, 54.4 +/- 3.1% monocytes, 25.9 +/- 2.4% mononuclears of healthy subjects' blood, on 11.1 +/- 1.0% T lymphocytes, on 14.6 +/- 1.6% T lymphocytes bearing Fc-receptor for IgG, on 49.3 +/- 8.2% enriched population of B lymphocytes and O cells. The treatment of healthy donors' mononuclears with antibodies IKO-GM-1 and complement blocked EK cellular activity against Molt-2 cells but not against K-562 cells. Antigen demonstrated by MAT IKO-GM-1 did not express on the colony-forming granulocyte or macrophagal cells. Antigen expressed on blast cells of patients with AMonoL, on those in part of patients with AML and AMML, on leukocytes of patients with chronic ML, on monocytes of a patient with chronic MonoL. Antigen was absent from blast cells of patients with ALL, LSA, on lymphocytes of patients with ChLL.     

Phagocytosis by B-cells and neutrophils in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.)

Phagocytosis by fish cells has mostly been studied using adherent leucocytes, excluding suspended cells such as the majority of B-cells and neutrophils, but a recent study describes professional phagocytosis of latex beads and bacteria by B-cells from rainbow trout. In the present study, phagocytosis by B-cells and neutrophils from salmon and cod was studied. Leucocytes were isolated from peripheral blood (PBL) and head kidney (HKL). By flow cytometry analyses, proportions of MAb labelled cell populations with internalized fluorescent beads, as well as the number of beads within each cell, could be determined. Phagocytic capacity and ability were demonstrated in B-cells and neutrophils from salmon and cod. In salmon, B-cells had higher phagocytic ability than neutrophils in HKL, but not in PBL. For cod the phagocytic ability of B-cells were lower than for neutrophils in both HKL and PBL, but the phagocytic capacity of cod B-cells were higher than for neutrophils in both HKL and PBL. For salmon B-cells the phagocytic capacity was lower than or similar to neutrophils in HKL and PBL. The total phagocytic ability of leucocytes was different in the species studied. The highest phagocytic ability was observed in cod, showing similar values for PBL and HKL. Salmon PBL displayed about twice the phagocytic ability of cod PBL. There seemed to be some major differences between the two fish species concerning phagocytosis. In salmon, a rather large proportion of phagocytic leucocytes were phagocytic B-cells, indicating that B-cells may have an important function in particle clearance in this species. In cod, phagocytic leucocytes in HKL and PBL were mostly neutrophils, and only a small proportion of B-cells were phagocytic, supporting the more prominent role of innate immune functions in cod neutrophils.     

Target cells for interferon production in human leukocytes stimulated by sendai virus.

Whole leukocytes, mononuclear cells, polymorphonuclear cells (PMN), MONOCYTES, PURIFIED LYMPHOCYTES, AND T (rosette-forming cells, RFC) and non-T (nonrosette-forming cells, nonRFC) lymphocytes isolated from the human peripheral blood were stimulated by Sendai virus, respectively, and examined for interferon production in their culture fluids. High levels of interferon were produced by mononuclear cells, but not by PMN. Removal of monocytes from the mononuclear cell population did not affect at all the levels of interferon produced, although it strongly suppressed interferon induction by polyinosinic-polycytidylic acid (poly IC) and mitogenic response to phytohemagglutinin (PHA) of the lymphocytes. Purified monocytes and T lymphocytes were unresponsive to the virus. In contrast, a population of purified non-T lymphocytes produced high levels of interferon. Addition of monocytes to the interferon-producing non-T lymphocytes did not affect the levels of interferon produced. No detectable levels of interferon were produced in the mixture of T lymphocytes and monocytes. It is concluded that non-T lymphocytes may be a major target for interferon induction of human leukocytes by Sendai virus.     

Mitogen responsiveness and inhibitory activity of mesenteric lymph node cells

Summary Blood-, lymph node-, and tumour-infiltrating lymphocytes (PBL, LNC, and TIL, respectively) from patients with colonic neoplasms were tested for responsiveness to phytohaemagglutinin (PHA). All populations responded, with LNC and PBL showing comparable reactivities while TIL were less reactive as assessed by incorporation of ³H-thymidine. Increased mitogen responsiveness was observed for T cells enriched by SRBC rosette formation or passage through nylon columns. Mitomycin C-treated LNC and TIL inhibited PHA induced ³H-thymidine incorporation of admixed autologous PBL, suggesting the presence of suppressor cells. Suppressor activity resided primarily in the SRBC rosetting population and was dose-dependent, with increasing numbers of LNC giving greater diminution of PHA response. Suppression by LNC was apparent only when they were added to PBL responders within 6 h of the initiation of stimulation assays, in common with the effects of Concanavalin A (Con A)-induced suppressors on PBL phytomitogen responsiveness. Con A-induced and LNC-suppressor activity could be reversed by addition of lymphocyte-conditioned medium (CM) containing T cell growth factor (TCGF; interleukin IL-2). These data provide further evidence that the suppressor phenomena observed in this system are a function of activated T cells present both in drainage lymph nodes and at the tumour site.     

Strong suppression by monocytes of T cell mitogenesis in chicken peripheral blood leukocytes

Peripheral blood leukocytes (PBL) isolated from chicken blood by flotation on Ficoll-Hypaque (FH) have much lower proliferative responses to Con A and PHA than PBL isolated by slow-speed (SS) centrifuging at 60 X G. FH preparations contain all categories of blood cells except erythrocytes, whereas SS are almost devoid of nonlymphoid cells. FH responses approach SS levels after filtration through Sephadex G-10, which removes almost all monocytes detectable by neutral red and nonspecific esterase methods, while only partially depleting granulocytes and thrombocytes. Thus, the low responses of FH are probably associated with suppressor activity of monocytes in these preparations, numbering 6 to 8% of mononuclear cells. G-10 filtration decreases responses of SS preparations, indicating that the few (less than 0.1%) monocytes in SS function mainly as helper cells. In co-cultures, irradiated FH cells (FHX) but not SSX produce as much as 90% suppression of SS or FH responses. Suppression by FHX is totally inhibited by heat killing (56 degrees C for 45 min) or monocyte inactivation by preincubation with Trypan blue, and is removed by G-10 filtration, although not completely. Adherent cells isolated from unirradiated FH on Cytodex beads (ADC-CY) produce up to 99% suppression in co-culture with SS or nonadherent cells derived from FH. Soluble suppressor factors can be detected in conditioned media from supernatants of Con A-stimulated cocultures containing suppressor monocytes, but their suppressor activity is partially opposed by stimulatory factors, possibly interleukin 2, also present in supernatants. It is concluded that in FH preparations and therefore in blood, but not in SS, monocytes that have not been activated exert strong suppressor activity on mitogen-induced T cell proliferation. Occasional chickens of one inbred line, RPRL 72, had exceptionally high suppressor activity of monocytes, as shown by very low FH responses and very high suppressor effects of FHX well outside the normal range of variation found in this line or in line RPRL 63. This abnormal suppressor activity may have resulted from activation of suppressor monocytes as a response to subclinical infection.     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

Interleukin 1 can replace monocytes for the specific human B-cell response to a particulate antigen

It is shown that the anti-trinitrophenyl (TNP) response of human B cells to trinitrophenyl polyacrylamide beads (TNP-PAA) is monocyte dependent. This response is abolished by extensive adherent cell depletion and restored by the addition of monocytes. The optimal response is obtained with 3% monocytes, higher numbers being suppressive. Supernatants from muramyl dipeptide (MDP)-activated monocytes can restore the response of monocyte-depleted preparations even when cells are cultured at suboptimal concentration. A partially purified preparation of interleukin (IL-1) has a comparable restorative ability. The following arguments suggest that monocytes do not function as antigen-presenting cells for this particulate antigen: (i) anti-genpulsed monocytes induce neither an anti-TNP response nor a specific T-cell proliferative response; (ii) allogeneic monocytes function as well as autologous monocytes to restore the response of nonadherent cells; (iii) HLA-DR-negative cells from the human leukemia cell line K562 can replace monocytes for this response. Monocyte supernatants do not replace T cells for the response of B-enriched lymphocytes, showing that T cells are directly involved in B-cell activation.     

Comparative analysis of the heterogeneity of mononuclear cells present in adult and cord blood by simultaneous examination of multiple phenotypic characteristics

In an attempt to better relate specific membrane characteristics of human adult and cord blood lymphocytes to specific functional activities, the phenotypic differences that exist in these two populations have been examined. Cord blood cells have considerably more spontaneous suppressor cell activity than adult cells. A technique that allows cells to be examined simultaneously for their ability to ingest latex beads, react with specific monoclonal antisera, bind sheep erythrocytes, or react with the Fc portion of IgG was used. As well as assessing fresh populations, phenotypic changes that occur when such cells are held in culture or stimulated with phytohemagglutinin for 3 days were sought. Many differences were found when comparing these mononuclear populations. These included the observations that 12% of adult and 9% of cord blood E-rosette-forming cells ingest latex beads and that 9% of OKT3 reactive cells in both populations did not form E rosettes. In cord blood 58% of T cells that bind OKT8 do not form E rosettes. A similar percentage of cord blood T8-positive cells express a receptor for Fc gamma, such cells being very uncommon in adult blood. Four "monocyte" subpopulations were identified in both samples. One such population (an OKM1- and Fc gamma-positive, nonphagocytic cell) was three times more common in cord blood. In cord blood some OKM1-positive cells also appeared to be simultaneously OKT8 positive. These phenotypic variations forward populations that may be candidates responsible for the functional differences noted in vitro.     

New antigenic determinant (Sa) on human lymphocytes and phagocytes

Presence of antigenic determinants reacting with homogeneous IgM/kappa cold agglutinin (CA) of a new specificity, tentatively called Sa, was investigated by bithermic cytotoxicity assay and by immunofluorescence. CA Sa killed on average 38% allogeneic peripheral blood lymphocytes (PBL) and up to 74% of autologous PBL. There was preferential kill of B-PBL compared to T-PBL. Some preference toward B cells was also noted using tonsillary B and T lymphocytes. Cytotoxic activity of CA Sa against chronic lymphocytic leukemia cells of B-type was almost equal to that of potent anti-I CA and much stronger than anti-i CA. Presence of additional B-cytotoxic factor in the serum was excluded by the use of red blood cell eluate composed solely of homogeneous CA. Thymocytes and helper-type T cells from a patient with T cell chronic lymphocytic leukemia were very susceptible to the cytotoxic action of Sa. CA Sa killed 39% of monocytes, but there was almost no kill of polymorphonuclear leukocytes. Lymphocytotoxicity of CA Sa was abolished by sialyllactose and was not influenced by I-active glycoproteins. Comparison of CA Sa to CA of other specificities showed marked differences, supporting the view that Sa has new, previously unrecognized specificity.     

Inhibition of human T lymphocyte E rosette formation by neutrophils and hydrogen peroxide. Differential sensitivity between helper and suppressor T lymphocytes

Co-incubation of human mononuclear cells with small numbers of autologous polymorphonuclear leukocytes resulted in a ratio-dependent inhibition of T lymphocyte rosette formation with sheep erythrocytes (SRBC). Inhibition by polymorphonuclear leukocytes was reversed with catalase, implicating H2O2 or associated products as the suppressive agent(s). Exposing T lymphocytes directly to micromolar concentrations of H2O2 also inhibited subsequent rosette formation. Inhibition was dose-dependent between 40 and 320 microM H2O2 and was not due to cytotoxicity at those H2O2 concentrations. Kinetic studies demonstrated that after exposing T lymphocytes to H2O2 a proportion of cells appeared to be relatively resistant in that they retained their ability to form E rosettes. T cell phenotype analysis of these cells showed that, in contrast to untreated T cells, H2O2-treated T rosette-forming cells were almost exclusively of the helper/inducer phenotype. Analysis with the monoclonal antibody 4B4 revealed that H2O2-resistant T rosette-forming cells contained significantly fewer 4B4-positive cells than predicted for the T helper population, indicating that H2O2-resistant T cells might be a subset of helper/inducer T lymphocytes. The interaction between H2O2 and T cells was rapid but did not lead to loss of Leu-5b binding sites. Preliminary functional analysis indicates that interleukin 2 production and phytohemagglutinin-induced proliferation by the relatively H2O2-resistant T cells is similar to untreated T cells. In contrast, H2O2-treated T cells which lost their ability to form E rosettes produced undetectable levels of interleukin 2 and proliferated poorly in response to phytohemagglutinin. Finally, mononuclear cells pretreated with H2O2 and subsequently stimulated with mitogens demonstrated a preferential expansion of the T helper population with concomitant loss of T suppressors. Our results show that, although neutrophil-released H2O2 inhibits effective interactions between SRBC and T lymphocyte sheep erythrocyte receptors, there appears to be a population of T helper cells which is relatively resistant to H2O2 both in terms of SRBC rosette formation and response to mitogens. These data suggest that at sites of inflammation phagocyte-released H2O2 could exert immunoregulatory effects on T cells by altering T cell subset survival and allowing the function of a particular lymphocyte population to predominate.     

Feline cytotoxic large granular lymphocytes induced by recombinant human IL-2

Large granular lymphocytes (LGL) have been characterized phenotypically and functionally as cytotoxic T lymphocytes, NK cells or lymphokine-activated killer cells. The most prominent morphologic feature of LGL is large cytoplasmic granules that are thought to contain the molecules responsible for cell lysis. In this study, we describe the morphologic and functional characteristics of IL-2-dependent cytotoxic lymphocytes derived from feline PBL. Stimulation of feline PBL with Con A followed by culturing in 50 U of gibbon monkey IL-2 human rIL-2 induced long term lymphocyte cultures. These lymphocytes are cytotoxic for the feline leukemia virus-induced T cell lymphoma (FL74), in a 4-h 51Cr release assay. All cell lines are either constitutively cytotoxic for FL74 cells, or cytotoxic in a lectin-dependent cell cytotoxic assay, the latter being a characteristic of low passage cultures. In contrast, no cell lines express self lysis or lysis for other lines. [3H]TdR uptake showed that 1 U of human rIL-2 produces a 50% maximal proliferative response by feline lymphocytes suggesting a high degree of homology between the ligand binding sites of feline and human IL-2R. Feline cytotoxic lymphocytes possess abundant cytoplasm containing large azurophilic granules characteristic of LGL. These granules are bound by a bilipid membrane and contain numerous smaller membrane-bound vesicles 50 to 60 nm in diameter. A model is proposed, whereby subsequent to binding of LGL to target cell the large granules fuse to the LGL plasma membrane and release the small vesicles into the binding pocket. The vesicles then transport the lytic molecules directly and selectively to the target cell membrane.     

Neutrophils and B cells express XCR1 receptor and chemotactically respond to lymphotactin

The C chemokine lymphotactin (Lptn) has been reported to act specifically on CD4(+) and CD8(+) T lymphocytes and natural killer (NK) cells, but not monocytes. However, the chemotactic effect of Lptn on other types of hematopoietic cells has not been well studied. In this study we investigated (i) the chemotactic influences of Lptn on T and B lymphocytes, neutrophils, monocytes, and dendritic cells, and (ii) the expression of the Lptn receptor (XCR1) by these cells, using RT-PCR. Our data showed that Lptn is chemotactic for B lymphocytes and neutrophils as well as T lymphocytes, but not for monocytes or dendritic cells, and that XCR1 expression is found only in association with T and B lymphocytes and neutrophils, but not monocytes or dendritic cells. Thus, this study is the first demonstration of a chemotactic effect of Lptn on neutrophils and confirms the association of this effect with expression of the XCR1 receptor on these cells. These data suggest that Lptn could potentially be an important protein in the regulation of T and B lymphocytes and neutrophil trafficking, and thereby also their roles in inflammatory and immunological responses.     

Cytokinetic basis for the impaired activation of lymphocytes from patients with primary intracranial tumors

Patients with malignant brain tumors have a variety of immunologic abnormalities, including the impaired responsiveness of peripheral blood lymphocytes (PBL) to mitogens and alloantigens. We further investigated this impairment of lymphocyte reactivity by employing the techniques of limiting dilution analysis and cytokinetic analysis. PBL preparations from patients have approximately six times fewer phytohemagglutinin (PHA)-responsive cells than PBL from normal subjects. Similar results were obtained with purified T cell preparations. Cytokinetic analysis of PHA-induced [3H]thymidine incorporation employing colchicine blocking of mitosis demonstrated that the number of first generation cells entering the S-phase of mitosis for each 24-hr period was less for PBL from patients than for PBL from normal individuals. First generation responding cells from patients and normal subjects entered DNA synthesis at the same time (48 to 72 hr). Cytokinetic analysis over a period of 168 hr demonstrated that whereas PBL from normal individuals demonstrated second generation responding cells, PBL from the majority of patients did not, thus indicating a defect in their ability to undergo clonal expansion. Measurement of interleukin 2 (IL 2) activity in culture fluids from PHA-activated PBL from normal subjects and patients revealed significantly lower IL 2 levels in culture fluids from PBL from patients. The addition of various concentrations of lectin-free IL 2 to PBL from patients stimulated with PHA did not restore responsiveness to normal values. There was no difference between the levels of interleukin 1 (IL 1) produced by lipopolysaccharide-activated monocytes from normal subjects and patients. Overall, these results suggest that an intrinsic defect exists in T cells obtained from brain tumor patients that renders them unable to enter into normal mitogen-induced blastogenesis.     

Studies on the reticulo-endothelial system of the dogfish,Scyliorhinus canicula

Summary Clearance and subsequent localisation of a range of materials, including colloidal carbon, latex beads, sheep erythrocytes, bacteria and dextran were followed in the lesser spotted dogfish, Scyliorhinus canicula. It was found that two populations of peripheral blood leucocytes — monocytes and thrombocytes, but not granulocytes — were involved in clearance of the circulation. In the case of carbon, this material was cleared from the plasma after 12 h, and both the colloid-containing thrombocytes and monocytes disappeared from circulation by 8 weeks post injection. Upon injection of some of the materials, and particularly bacteria, a settling out of monocytes containing phagocytosed material was seen in the secondary lamellae and cavernous bodies of the gills. Large clumps of monocytes were found in the gills as early as 30 min post injection and these increased in size for up to one week, after which they gradually dispersed. The lining cells of the cavernous body, known as CB cells, were also responsible for the sequestration of carbon, latex beads and probably erythrocytes, but dextran and bacteria were not internalised. The origin, functions and phylogenetic significance of the CB cells are discussed.     

Specific Role of Each Human Leukocyte Type in Viral Infections I. Monocyte as Host Cell for Vesicular Stomatitis Virus Replication In Vitro

Each major leukocyte type of the peripheral blood of healthy donors was studied in vitro for its ability to support vesicular stomatitis virus (VSV) replication. Purified cultures of each white blood cell type were prepared by the selective adsorption and elution of cells from silicone-treated glass beads. It was found that monocytes and macrophages (derived from the rapid transformation of monocytes in vitro) were the principal host cells for VSV replication. Interferon added to mixed leukocyte cultures, prior to virus inoculation, reduced virus yields and prevented destruction of macrophages. Cultures of small lymphocytes, containing no detectable monocytes or macrophages, produced amounts of virus equivalent to 1% of that produced in leukocyte cultures which contained 7% monocytes. Small lymphocytes did not undergo demonstrable cytopathic alterations in virus-infected cultures. VSV neither replicated nor produced cytopathic effects in polymorphonuclear leukocytes.     

TGF-beta inhibits proliferation of and promotes differentiation of human promonocytic leukemia cells.

Transforming growth factor-beta 1 (TGF-beta 1) has been implicated in a variety of responses associated with wound healing and inflammation. Thus, TGF-beta 1 enhances production of several extracellular matrix proteins both in vitro and in vivo, is chemotactic for monocytes, and alters the functioning of lymphocytes. We have examined the ability of TGF-beta 1 to affect the behavior of human THP-1 promonocytic leukemia cells, a cell line with the capacity to differentiate into macrophage-like cells. TGF-beta 1 reduces the growth rate of these cells, induces morphologic changes, and promotes adherence to culture surfaces. In addition, the adherent cell population expresses high levels of esterase activity, acquires the ability to ingest latex beads, and releases elevated levels of interleukin 1. TGF-beta 1-treated cells also express elevated levels of the beta 2 family of integrins. Taken together, these results suggest that TGF-beta 1 is capable of promoting the maturation of promonocytic cells into macrophages. This outcome has implications at wound sites where TGF-beta 1 and a myriad of other factors interact with many cell types to facilitate healing.