Mitogenic response of human thymocytes: identification of functional Ca2+-dependent and independent signals.

In contrast to peripheral blood mononuclear cells (PBMC), human thymocytes do not exhibit a proliferative response to the T cell mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), or Staphylococcal protein A (SPA). In thymocytes and PBMC, Con A and PHA induce increases in free cytosolic calcium concentrations [( Ca2+]i). Since both Con A and PHA induce similar increases in [Ca2+]i in thymocytes and PBMC, the absence of thymocyte proliferation was not due to an inability to induce an increase in [Ca2+]i. The lack of proliferative response was secondary to the failure of the mitogens to induce interleukin 2 (IL-2) production. Incubation of mitogen-treated thymocytes with phorbol esters reconstituted IL-2 production and the proliferative response indicating that the cells were indeed activated by the mitogens. Similarly, addition of exogenous recombinant IL-2 also induced mitogen-treated thymocytes to proliferate. This IL-2-dependent proliferation established that SPA, Con A, and PHA triggered the expression of biologically active IL-2 receptors. Since an increase in [Ca2+]i is a prerequisite, and possibly a trigger, for IL-2 production, the failure of PHA, Con A, or SPA to result in thymocyte proliferation may be due to an inability of thymocytes to respond to increases in [Ca2+]i with subsequent IL-2 production.     

Chromosomes and cell surface markers of marmoset lymphocytes and Epstein-Barr virus-transformed marmoset cell lines

The G-banded karyotypes of both normal lymphocytes and Epstein-Barr virus (EBV)-transformed lymphocytes of cotton-topped marmosets (Saguinus oedipus) were examined. The marmoset lymphocytes and EBV-transformed lymphoblastoid cells had normal diploid chromosomes (2n = 46) with no specific cytogenic change associated with transformation in vitro. EBV-transformed marmoset lymphocytes expressed the cell surface markers of B lymphocytes and EB viral antigens.     

D-penicillamine: Mitogenic effect on mouse,rat and human spleen lymphocytes

The effect of D-penicillamine (D-Pen) on the proliferation of cultures of normal mouse, rat, and human spleen lymphocytes and peripheral blood lymphocytes was examined. D-Pen in concentrations of 2×10^–3 M to 8×10^–3 M in serum-free and in serum-containing medium resulted in a highly significant incorporation of³H-TdR by normal mouse and rat spleen cells. Enhanced incorporation of³H-TdR by normal human spleen cells only occurred in serum-containing medium. D-Pen in concentrations of 10^–4 M to 10^–3 M in serum-free and serum-containing medium resulted in significant inhibition of³H-TdR incorporation by normal and mitogen-stimulated mouse and rat spleen cells. Doses of D-Pen greater than 2×10^–2 M strongly inhibited³H-TdR incorporation by both normal and mitogen-stimulated mouse, rat, and human spleen cells and peripheral blood cells. The latter cells were not stimulated or inhibited at lower concentrations of D-Pen.Results from cell depletion and enriching procedures (specific antibody + C' cell killing, employment of athymic, nude spleen cells, adherent and phagocytic cell removal, E rosette cell separation procedures) suggested that target cells in the mouse spleen for D-Pen activation are non-adherent B cells whereas the D-Pen responsive cells in the human spleen probably are T cells.     

Mitogenic effect of prolactin on chicken lymphocytes in vitro

Lymphocytes obtained from thymus and spleens of 1-6 week old White Leghorn cockerels, untreated or immunized twice with sheep red blood cells (SRBC), were cultured with common T-cell mitogens in serial dilutions and/or different concentrations of bovine prolactin (PRL). [3H]Thymidine incorporation in newly synthesized DNA was used as a measure of lymphocyte mitogenic stimulation. Lymphocytes were stimulated by mitogens, as well as by PRL alone, in a dose-dependent way. Cell cultures prepared from immunized and non-immunized donors differed in their response to mitogens or PRL. The present results demonstrate direct PRL action on avian lymphoid cells and resemble those found in the mammalian immune system.     

T and B lymphocytes in the marmoset: a natural haemopoietic chimera.

The thymus-derived (T) lymphocyte and bone marrow-derived (B) lymphocyte populations of the marmoset were characterized using specific cell surface markers Approximately 85% of the thymocyates formed rosettes with neuraminidase-treated sheep erythrocytes (En). The percentage (approximately 69%) of peripheral blood lymphocytes (PBL) forming rosettes with En was the same as that which stained with fluorescently labelled goat anti-marmoset thymocyte serum (ATS). These two assays identified the same cell population since treatment of cells with ATS and complement resulted in a concomitant decrease in En rosette formation. Marmoset PBL also formed rosettes with human erythrocytes sensitized with antibody and complement (HEAC); since the percentage (approximately 20%) HEAC rosette was the same as that of cells stained with fluorescently labelled goat anti-marmoset IgG, these cells were considered to be B cells. A small percentage of cells (approximately 1-5%) possessed both types of receptors. The mean percentages of T and B cells present in PBL of single-born, presumably non-chimeric animals, were the same as that of iso-sexual and heterosexual chimeras.     

Mitogenic effect of beryllium sulfate on mouse B lymphocytes but not T lymphocytes in vitro

Beryllium metal and its salts can produce disease in man and in animal models. Beryllium disease is thought to involve cell-mediated immunity and an antigen-dependent response by beryllium-specific T cells. Beryllium salts have been shown to stimulate lymphocyte proliferation and release of lymphokines, and to induce granuloma formation and delayed-type hypersensitivity reactions in mice, guinea pigs and man. The studies described here were designed to test the hypothesis that a second lymphocyte population, B cells, may be responding nonspecifically to beryllium. Different populations of BDF1 mouse lymphocytes were cultured in the presence of varying concentrations of beryllium sulfate (BeSO4), and the increase in 125-iodouracildeoxyriboside uptake after 72 h in culture was determined. The data show that BeSO4 is weakly mitogenic for normal mouse spleen cells. Furthermore, BeSO4 is mitogenic for normal and nude mouse spleen B cells and not for spleen T cells or thymocytes in vitro. These findings suggest that BeSO4 can stimulate B cells nonspecifically, and support the hypothesis that polyclonal activation of B cells by beryllium may occur.     

Mitogenic responses of splenic B and T lymphocytes in neonatally capsaicin-treated mice

The proliferative response of spleen cells from neonatally capsaicin-treated mice to 5 mitogens (LPS, PWM, dextran sulfate, ConA and PHA) were tested. Both B- and T-lymphocyte responses were essentially unaffected by the capsaicin treatment, which is known to destroy certain small-sized neuropeptide containing primary sensory neurons. However, the capsaicin-treated mice displayed a shift in the dose response to PHA so that the maximal response was obtained with a lower dose of the mitogen. The results exclude major effects of the affected sensory neurons on the development or expression of immune competence, but point to a possible modulatory effect on some step in the response to PHA.     

Characterization of the antibody response of the marmoset to sheep red blood cells.

The immune competence of two species of marmosets, S. fusciollis and S. oedipus, was evaluated by the intravenous (i.v.) and intramuscular (i.m.) injection of sheep red blood cells (SRBC). In S. fusciollis marmosets, 1 ml of a 50% suspension yielded titres of haemolysin and agglutinating antibodies equal to or greater than 1 ml of a 10% dose of antigen. In both species, the i.v. route, while resulting in formation of 19S and 7S agglutinins, yielded only 19S haemolysins, even after multiple antigen injections. Repeated i.v. injections resulted in a progressive decrease in peak titres, in contrast to the i.m. route, where booster inoculations gave a typical anamnestic response. Jerne plaque-forming cells (PFC) in the spleens of S. oedipus marmosets showed predominately 19S plaques after a primary i.v. challenge; only 19S PFC were detected in the spleen of an animal that had been given multiple inoculations, the type of antibody produced reflecting that found in the serum. 19S but not 7S haemolysins of both species were sensitive to heating at 56 degrees C for 1/2 hr. The serum titres and splenic PFC data from the marmosets suggest these animals, particularly S. oedipus, respond poorly to SRBC when a comparison is made to similar studies in mice and rats.     

Mitogenic effect of IL 2 on non-thymic and thymic lymphocytes of the mouse

Interleukin 2 (IL 2) was obtained from culture fluids of Con A-stimulated rat spleen cells and purified by repeated Sephacryl S-200 column chromatography. The purified IL 2 showed a direct mitogenic effect on C57BL/6 mouse spleen cells and lymph node cells but not on unfractionated thymocytes. Contamination of Con A in the IL 2 was ruled out by the finding that the mitogenic effect of the IL 2 was not diminished by addition of alpha-methyl-D-mannoside in the reaction mixture. The treatment of the spleen cells with anti-Thy 1.2 and complement abolished the response to the IL 2, indicating that the IL 2-responding cells bear a T cell marker. Although unfractionated thymocytes did not blastogenically respond to the IL 2, fractionation of thymocytes by means of discontinuous gradient centrifugation with Percoll resulted in a minor cell population which strongly responded to the IL 2 stimulation. The cells were distributed in lower density fractions. The cells distributed in higher density fractions did not respond to the IL 2.     

Mitogenic activity of two Brucella fractions for murine B lymphocytes.

Two lipid A-free fractions extracted from Brucella melitensis (fractions PI and SF) were shown to behave as B-cell non-specific mitogens for murine lymphocytes: they stimulated the uptake of tritiated thymidine by normal unsensitized murine splenic lymphocytes, by spleen cells from nude mice and by T-cell depleted but not by T-cell enriched and B-cell-deprived splenic populations. Since depletion of adherent cells leads to a two- to three-fold depression of PI- or SF-induced mitogenic responses these two fractions were shown to require accessory adherent cell participation for an optimal mitogenicity. Moreover they behaved as polyclonal activators for murine spleen cells. These results are discussed in terms of a possible classification of PI and SF amongst other B-cell mitogens and of the respective role of the peptidoglycan and lipoprotein moieties in B-cell activation by Brucella fractions.     

Mitogenic responses of canine peripheral blood lymphocytes to staphylococcal protein A

In order to produce long term lymphoid cell cultures from canine lymphocytes of known histocompatibility antigen specificities, mitogenic responses to staphylococcal protein A (SpA) were examined and compared with those of phytohaemagglutinin (PHA) and concanavalin A (Con A). SpA was found to be the strongest mitogen tested with significant responses to concentrations as low as 31 ng/ml. There was a decrease in responsiveness above optimal mitogen concentrations with SpA and PHA. Peak responses were observed at lower concentrations for longer incubation times. PHA showed a rapid fall off in thymidine uptake below optimal concentrations whereas the SpA dose-response curve was less steep and a shoulder or secondary peak of activity was observed at low SpA concentrations in some cases.Continuous SpA stimulation of lymphocyte cultures resulted in an initial period of cell proliferation followed usually by a second period of cell proliferation around week 7 of culture. To date, viable cell cultures have been maintained for up to 12 weeks in vitro.SpA lymphoblast cultures behave normally in microcytotoxicity tests for serologically defined DLA histocompatibility antigens and remain functional in natural killer (NK) and PHA induced cell mediated cytotoxic reactions against ⁵¹Cr-labelled tumour target cells but were not themselves susceptible as target cells for NK activity.     

Blastogenic response of normal lymphocytes to cultured lymphoid cells and non-lymphoid neoplastic cells

Irradiated cultured lymphoid cells (normal and leukaemic) unequivocally stimulated the peripheral lymphocytes from twelve normal individuals. However, irradiated cultured non-lymphoid neoplastic cells (KB cells, melanoma cells and osteogenic sarcoma cells) exerted little or no stimulating effect on lymphocytes of the same donors. These findings indicate that something more than a simple HL-A incompatability is involved in this mixed cell interaction.     

Area 18 corticotectal cells: response properties and identification of sustaining geniculate inputs

1. We examined the response properties and geniculate inputs of 35 antidromically identified corticotectal (CT) cells within area 18 of the paralyzed, anesthetized cat. Twenty-three were either standard complex or hypercomplex, 11 were special complex, and 1 was simple. 2. The response properties of CT cells in area 18 were in general quite similar to those examined in a previous study of area 17 CT cells, including similar proportions of standard and special complex CT cells, virtually identical length-response functions, and similar orientation and direction tuning. 3. Area 18 CT cells are rapidly conducting. They are considerably faster than area 17 CT cells. 4. We investigated the composition of thalamic inputs to CT cells by reversibly inactivating a portion of layer A and/or the C layers of the dorsal lateral geniculate nucleus with injections of cobaltous chloride. Blocking layer A strongly attenuated the visual responsiveness of about half of the cells tested. Blocking the C layers alone generally had only moderate effects, but simultaneous blockade of layer A and the C layers demonstrated a substantial C-layer input to many cells. Unlike area 17 in which there is a strong correlation between CT cell class and dependence on layer A, no single receptive-field parameter nor set of parameters was correlated with dependence on layer A. However, cells least affected by simultaneous blockade of layer A and the C layers were special complex, suggesting that, as in area 17, area 18 special complex CT cells integrate more geniculate inputs than standard complex CT cells. 5. We propose that the similarities of response properties of area 17 and area 18 CT cells results from their participation in similar interlaminar columnar circuits and that differences in the patterns of geniculate control reflect differences in the global patterns of geniculate inputs to these two areas.     

Mitogenic actions of orthoclone OKT3 on human peripheral blood lymphocytes: Effects of monocytes and serum components

The Orthoclone monoclonal antihuman T lymphocyte antibody, OKT3, induced maximal DNA, RNA and protein synthesis in peripheral mononuclear blood cells (PMBC) at concentrations as low as 10 ng ml⁻¹. This pronounced mitogenic activity was highly dependent on the presence of monocytes: removal of these cells from PMBC suspensions by complement (C)-dependent lysis with the antimonocyte antibody OKM1, completely abrogated the proliferative responsiveness of the remaining lymphocytes. The addition of adherent cells to OKM1-treated PMBC demonstrated the strict monocyte requirement for the mitogenic activity of OKT3. Mitogenic responses to OKT3 were most marked when PMBC were cultured in media containing heat-inactivated fetal calf serum (FCS) but they were considerably weaker in cultures supplemented with heat-inactivated human serum (HS). Moreover, aggregated human IgG and its Fc fragments (but not monomeric IgG and its Fab fragments) inhibited the mitogenicity of OKT3: their inhibition could be explained by stimulation of monocytes, resulting in increased prostaglandin E release, since (a) prostaglandin E₂ itself strongly suppressed OKT3 activity and (b) indomethacin blocked the inhibitory effects of aggregated HuIgG.The present data demonstrate that OKT3 shows a particular pattern of mitogenicity: the strict monocyte requirement, the inhibitory effects of HS, aggregated human IgG and prostaglandin E₂ were not observed for the phytomitogen PHA.     

Immunoregulation in the common marmoset, Calithrix jaccus: functional properties of T and B lymphocytes and their response to human interleukins 2 and 4.

Non-human primates have been used to study immune function to a much lesser extent than readily available strains of inbred rodents. Nevertheless, in situations where it might be desirable, but impossible, to study human immune responses in vivo, lower primates could provide an acceptable alternative. In order to extent the knowledge of T- and B-lymphocyte function in lower primates, the common marmoset Callithrix jaccus was used as an experimental model. The functional similarities between this species and humans at the level of T-B co-operation in the antibody response were examined, and xenoreactive T-lymphocyte clones were obtained from marmoset spleen cells using Epstein-Barr virus (EBV)-transformed human B cells as stimulators. These clones could act as helper cells when co-cultured with human B lymphocytes, inducing the secretion of both IgM and IgG. Lymphokine production by mitogen-stimulated marmoset T-cell clones was also examined. Interleukins (IL) 2 and 4 activities were detected in clone supernatants using bioassays and interferon-gamma (IFN-gamma) was detected using a solid-phase ELISA system. However, SDS-PAGE analysis of biosynthetically labelled marmoset and human T-cell clone supernatant proteins revealed major differences between the soluble T-cell products of the two species. The proliferative responses of marmoset T and B cells to recombinant human IL-2 and IL-4 were also examined. Stimulation of [3H]thymidine uptake was detected in both T cell- and anti-IgM-stimulated B-cell cultures with both of the lymphokines. These results suggests that the key components of the antibody response are functionally conserved between lower primates and man and that the common marmoset may be useful as an in vivo model of immune function, particularly with regard to the role of interleukins such as IL-2 and IL-4.     

Ontogeny of mitogen responsive lymphocytes in the bovine fetus

Bovine fetal lymphocytes were examined for their ability to respond to the mitogens phytohemagglutinin (PHA), Concanavalin A (ConA) and pokeweed mitogen (PWM) in the lymphocyte blastogenesis test (LBT). PHA-, Con A- and PWM-responsive lymphocytes appeared simultaneously at 75-80 days of gestation. The response increased with age of the fetus until, by 120 days of gestation, the response to PHA, Con A and PWM of many fetuses was in the range of values obtained with lymphocytes from normal adult cattle.     

Further characterization of lymphocytes from human colonic lamina propria: identification of TG cells.

For the first time, TG cells have been identified in human colon using EDTA-collagenase-prepared, macrophage-depleted isolates of lamina proprial lymphocytes (LPL). Specimens of human colon were obtained from patients undergoing surgery for idiopathic inflammatory bowel disease (IBD), colorectal cancer (Dukes' B or C), other colonic inflammations or benign polyps. Of additional interest were quantitative findings which showed lower TG values in LPL from patients with IBD, regardless of disease activity or steroid therapy, and in Dukes' Group C cancers, compared to the other groups. However, these differences of TG values were not reflected in the peripheral blood lymphocytes (PBL) in which, compared to healthy controls, the numbers of circulating TG cells were greater in patients with Dukes' B or C cancers and in those with moderately or severely active IBD receiving steroids. These quantitative differences re-emphasize the need for concurrent observations on PBL and LPL in these diseases, particularly in experiments to determine the functional properties of their TG subsets, including mediation of natural killing, antibody-dependent cellular cytotoxicity and their immunoregulatory properties. The identification of TG cells per se in colonic LPL provides a basis for such studies.     

Mitogenic response of peripheral blood lymphocytes from patients with Graves' disease incubated with solubilized thyroid cell membranes containing TSH receptor and with thyroglobulin

Transformation of peripheral blood lymphocytes (PBL) in response to solubilized TSH receptor and to human thyroglobulin (hTG) was carried out in patients with Graves' disease (GD). Mean stimulation index (SI) in response to solubilized TSH receptor was significantly increased only in hyperthyroid GD patients (SI = 3.0 +/- 1.8 SD, n = 13, p less than 0.025) when compared to normal subjects (SI = 1.5 +/- 0.8 SD,n = 14). All other subgroups of GD patients (i.e., euthyroid GD patients after treatment as well as those with/without ophthalmopathy or TSH-displacing antibody [TDA]) showed a tendency to elevated mean SI, which, however, was never significantly increased. In contrast, patients who were suspected of having a "disseminated autonomy" showed a tendency to decreased mean SI compared to GD patients. The mitogenic response of PBLs induced by solubilized TSH receptor could be suppressed by incubation of antigen and PBLs with bTSH. Control experiments with purified protein derivate of tuberculin(PPD) as stimulating antigen revealed, however, that bTSH itself suppresses the mitogenic response of PBLs and that the decrease of SI in this experiment is not due to blockage of the antigenic determinants of the TSH receptor protein. Human thyroglobulin (hTG) produced no significant increase in the SI of PBLs. Only 9 out of 47 GD patients showed an SI higher than the mean of normals + 2 SD. All those patients, however, who showed an elevated SI ( greater than 2.0) also revealed an increased SI with solubilized TSH receptor.