Description of the effect of polyclonal mitogens on a population of splenic lymphocytes in the presence of serum against isologous aggregated mouse immunoglobulins

Antiserum against isologous aggregated mouse immunoglobulins was shown to be mitogenic for spleen cells during the first 48 hours of culture. The mitogenic effect of the serum factor is mediated through T lymphocytes and may be macrophage-dependent. Whe incubated with spleen cells the serum was demonstrated to inhibit the DNA synthesis in B cells in response to sulfate dextran and lipopolysaccharide. Experiments with B cells and the thymidine "suicide" test suggest that the target cells for the serum factor may reside in the population of radiosensitive and highly proliferating B lymphocytes. The degree of suppressing by the antiserum factor depends on the differentiation stage and the presence of antigen-binding receptors on the membrane of B cells.     

Patients treated for amebic liver abscess develop cell-mediated immune responses effective in vitro against Entamoeba histolytica

We studied the afferent and efferent cell-mediated immune response in 15 patients treated for amebic liver abscess. Patients had a lower T4 to T8 ratio (1.25 +/- 0.65) compared with age- and sex-matched controls (1.89 +/- 0.44, p less than 0.01) due to a decrease in T4-"helper" cells and an increase in T8-"suppressor" cells (p less than 0.01). The in vitro proliferative response of patient T lymphocytes to the plant mitogen concanavalin A (Con A) was depressed; responses to phytohemagglutinin were not. The proliferative response of patient lymphocytes to an amebic soluble protein preparation (SPP) was greater than the mitogenic response seen in control lymphocytes (mean of 68,300 delta cpm and 22,300 delta cpm, respectively, p less than 0.001), correlated with the T4 to T8 ratio (p less than 0.05) and the duration of time from initiation of antiamebic therapy (p less than 0.01). Supernatants from patient lymphocytes exposed to the amebic SPP activated normal monocyte-derived macrophages to kill virulent axenic E. histolytica trophozoites (p less than 0.001); patient monocyte-derived macrophages activated by Con A-elicited lymphokine could also kill amebae. Finally, when incubated with the amebic SPP for 5 days, T lymphocytes from patients were able to kill virulent amebae (p less than 0.005); patient T lymphocytes not exposed to the amebic SPP or control T lymphocytes incubated for 5 days with the amebic SPP were not cytotoxic to E. histolytica trophozoites. In summary, after cure of amebic liver abscess, specific cell-mediated immune mechanisms develop that are effective in vitro against the parasite.     

Regulatory mechanisms of cutaneous delayed-type hypersensitivity: I. Suppression of cutaneous delayed-type hypersensitivity by migration inhibitory factor

The macrophage migration inhibitory factor (MIF) fraction was prepared from the immunoadsorbent column by using anti-guinea pig MIF antiserum. Suppression of cutaneous delayedtype hypersensitivity was achieved by intraperitoneal injection of the MIF fraction into the animals bearing macrophage-rich peritoneal exudates. Skin reactions induced by phytohemagglutinin (PHA) were also suppressed in these animals. Reactivity to skin reactive factor (SRF) was suppressed in these animals as well. The sera obtained from these animals exhibited the inhibitory activity against production of lymphokines from sensitized lymphocytes.     

Production and in vivo effect of antibodies against guinea pig lymphokines

Supernatants from guinea pig lymph node lymphocytes stimulated with insoluble Concanavalin A in serum-free medium were fractionated by Sephadex chromatography and preparative electrophoresis. The isolated fraction possessed migration inhibition, mitogenic, and skin reactive activities. Associated with these were apparently two newly synthesized haemoproteins of unknown function. Antibodies were prepared against this partially purified lymphokine fraction. MIF produced by sensitized lymphocytes activated with an antigen (PPD tuberculin) could be completely absorbed from whole supernatants by immunoadsorbent columns prepared with that antibody whereas mitogenic factor and skin reactive factor were not retained. The anti-lymphokine antiserum totally inhibited the delayed skin response of sensitized guinea pigs challenged with PPD.     

Clinical studies on cell-mediated immunity in patients with malignant disease

Summary In vitro mitogenic responses of lymphocytes of patients with advanced cancer of the stomach or lung were determined and the cells involved in the depressed responses of patients characterized. Proliferative responses to phytohemagglutinin and concanavalin A of lymphocyte rich mononuclear cells of the patients were impaired, but increased after in vitro unstimulated culture for seven days. Mitogenic responses were enhanced by depletion of monocytes using a Sephadex G-10 column, and further enhanced by removal of nylon wool nonadherent cells. Nylon wool nonadherent cells of the patients suppressed the mitogenic responses of autologous and allogeneic lymphocytes, but lost the suppressive activity during seven days' in vitro culture. Nonadherent cells of normal donors did not inhibit mitogenic responses. The results suggest that peripheral blood mononuclear cells of cancer patients may contain at least two populations of suppressor cells for mitogenic responses, monocytes and nylon wool nonadherent cells, which could be one of the causes of impaired mitogenic responses in these cancer patients.     

Inhibition of interleukin 1 activity by a factor in submandibular glands of rats

With the sequential use of ammonium sulfate precipitation, gel filtration and chromatofocusing, we have partially purified from extracts of the submandibular glands of rats a factor (referred to as submandibular gland's immunosuppressive factor or SMG-ISF) capable of inhibiting the in vitro proliferation of mitogen- and antigen-stimulated murine lymphocytes. The semi-purified suppressor fractions had an isoelectric point of 4.4 to 4.5 and consisted of at least three molecular species. These active fractions suppressed the mitogenic effects of Concanavalin A phytohemagglutinin, and lipopolysaccharide. In vitro immune reactions such as the mixed lymphocyte culture MLC reaction and the production of cytotoxic T lymphocytes (CTL) across major histocompatibility barriers in mice were also suppressed. These in vitro immunosuppressive effects required the addition of the suppressor fractions early after the initiation of the cultures and were reversed if the factor was removed from the cultures at least 48 to 72 hr before the completion of the assays. The active fractions did not affect the proliferation of CTLL 2 cells induced by interleukin 2 (IL 2), but inhibited the mitogenic and co-stimulatory effects of IL 1 on mouse thymocytes, and in this effect showed a dose-response relation suggestive of a competitive mechanism. These characteristics of SMG-ISF indicate a specific inhibition of the activity of IL 1.     

Inhibition of human lymphocyte transformation by human alpha-fetoprotein (HAFP); HAFP monomers and multimers and a resistant lymphocyte subpopulation.

We have previously shown that human alpha-fetoprotein (HAFP) inhibits the response in vitro of human lymphocytes to a variety of mitogenic stimuli. In this report we present evidence that HAFP preparations containing dimeric and trimeric forms are no more inhibitory than HAFP monomeric isolates. HAFP isolates of low inhibitory potency cannot impede the capacity of high-potency HAFP to inhibit lymphocyte transformation. Since lymphocyte responses to phytomitogens or anti-human thymocyte antiserum cannot be totally suppressed by increasing doses of HAFP, we conclude that human lymphocyte preparations contain a population of lymphocytes resistant to the inhibitory action of HAFP.     

Lymph node, spleen and peripheral blood lymphocytes as stimulators of alloreactivity

Before and after kidney transplantations, in vitro tests that measure the level of reactivity between donor and recipient lymphocytes are performed for better organ selection and as indicator of possible organ rejection. In these tests, donor's and recipient's lymphocytes are stimulated for proliferation, which intensity is measured and accordingly organ recipient reactivity towards graft is determined. Lymph node, spleen and peripheral blood lymphocytes are used for those purposes. For better interpretation of these in vitro tests it should be important to determine mitogenic ability of lymphocytes of different origin and to choose the most adequate cells. To compare mitogenic ability of deceased donor lymph node, spleen and peripheral blood lymphocytes one-way mixed lymphocyte culture (MLC) was used. As stimulators irradiated lymphocytes from spleen, lymph node and peripheral blood samples of 12 deceased donors were used while as responders lymphocytes from peripheral blood of healthy individuals, chosen according HLA-DRB1 alleles (stimulators and responders were HLA-DRB1 identical, semi-identical or different), were used. Spleen lymphocyte activity was the best with different cells and the weakest with identical cells. Impact of polyclonal mitogens (PHA - phytohemagglutinin, Con A - concanavalin A and PWM - pokeweed mitogen) on lymphocyte proliferation was tested on lymphocytes from spleen and lymph node of deceased donors. Results obtained in culture in vitro showed that spleen cells had exerted the best mitogenic potential and PHA had the greatest impact upon lymphocyte proliferation. This investigation is of importance for establishing the best model to reflect in vivo situation in transplanted patient.     

Direct and cooperative mechanisms of lymphocyte triggering in liquid and solid cultures.

The role of cell interactions in lymphocyte stimulation was analyzed by studying the kinetics of lymphocyte proliferation at different cell concentrations, and also by a lymphocyte microculture technique in solid medium. An absolute requirement for cell interactions was found in lymphocyte responses to concanavalin A, pokeweed mitogen, sodium periodate, purified protein derivative from Mycobacterium tuberculosis, and zinc chloride. No requirement for cell interactions was found in lymphocyte responses to calcium ionophore A23187. The existence of lymphocyte subpopulations with different requirements for cell interactions was observed in lymphocyte responses to phytohemagglutinin P, phytohemagglutinin HA 17, tetradecanoyl-phorbolacetate, antiserum to MOLT-4 lymphoblasts, antiserum to B411-4 lymphoblasts, antiserum to human embryo lung fibroblasts, and antiserum to HeLa cells infected with Herpes simplex virus. Lymphocyte responses to phytohemagglutinin P were potentiated by incorporation into the solid cultures of red blood cells of their membrane preparations suggesting that membrane-membrane interactions, either directly, or through soluble mediators are likely to be the basis of cell cooperation in this system. In solid cultures, phytohemagglutinin P, phytohemagglutinin P plus red blood cells, phytohemagglutinin HA 17, tetradecanoyl-phorbol-acetate and antiserum to MOLT-4 lymphoblasts were found to stimulate mainly thymus-dependent lymphocytes, whereas antiserum to Hela cells infected with Herpes simplex virus stimulated mainly non-thymus-dependent lymphocytes. Antiserum to B411-4 lymphoblasts stimulated both thymus-dependent and non-thymus dependent lymphocytes.     

Proliferation of spleen cells in response to Bordetella pertussis

Vaccine strain 305 of B. pertussis in a dose of 10(8)-10(11) cells was shown to be mitogenic for splenocytes of BALB/c mice and nude mice. When added in a dose of 10(10) B. pertussis exerted a more pronounced mitogenic effect than phytohemagglutinin P, which was less powerful, however, than that of Con A, B. pertussis caused a greater stimulation of DNA synthesis in lymphocytes than B mitogens whose action depended on the differentiation stages of B lymphocytes. This is likely to hint towards a possible action of B. pertussis on immature B cells and/or their precursors. The cells of T lineage (T1 cells and/or T precursors) can also be involved.     

The interaction of lymphocyte surface-bound immune complexes and neutrophils

Neutrophils can be stimulated directly by a variety of stimulants resulting in the production of highly reactive oxygen derivatives. Included in these stimulants are peripheral blood lymphocytes with bovine serum albumin-anti-bovine serum albumin immune complexes (BSA-IC) or aggregated gamma-globulin (AHG) bound to their surface receptors. Through the use of chemiluminescence (CL) studies, we found that B lymphocytes preincubated with AHG stimulated neutrophils to a much greater extent than similarly preincubated T lymphocytes. Preincubated Raji cells (a B lymphoblastoid cell line) were also capable of stimulating neutrophils. We further demonstrated that after periods of mixed incubation with neutrophils, lymphocytes with surface-bound AHG did show an abnormal proliferative response to pokeweed mitogen (PWM), but not to phytohemagglutinin (PHA). This was not due to loss of cell viability, as judged by chromium release cytotoxicity assays and trypan blue exclusion, or to neutrophil enzyme release. The data suggest that neutrophils, stimulated with lymphocyte surface-bound immune complexes, are capable of producing an environment of in vitro oxidant stress. This stress, although not great enough to cause a significant decrease in lymphocyte viability, can cause impaired lymphocyte function. Physiologically, this may relate in the long term to immunologic malfunction observed in patients with high levels of circulating immune complexes.     

Effect of mouse antiserum against isologous aggregated immunoglobulins on the transplantation and antitumor immunity]

It was shown that mouse antiserum against isologous thermically aggregated immunoglobulins MAAS injected repeatedly to the animals prolonged the survival of the skin allotransplant, induced Moloney's sarcoma diminished the latent period of the tumour development sharply increased the formation of the tumours which in the majority of cases, led to the death of mice, deranged the normal age barrier in fibrosarcoma induction, and sharply inhibited the intensity of Rauscher's leukemia development. This action of MAAS on the transplantation and antitumour immunity was of immunological nature.     

Concanavalin A-inducible suppressor cells in pleural effusions and peripheral blood of cancer patients

Summary Carcinomatous pleural effusions of 18 of 20 patients with lung cancer contained suppressor cell precursors that could be activated by concanavalin A (Con A) to suppress the proliferative responses of autologous and allogeneic lymphocytes to phytohemagglutinin and Con A. However, pleural effusion cells showed no suppressor function without prior activation by Con A. In contrast, the peripheral blood of the cancer patients exhibiting impaired mitogenic response contained nonspecific spontaneous suppressor cells capable of inhibiting the lymphoproliferative response to mitogens without prior activation by Con A, but these cells were not able to show further suppressor function even after activation by Con A. The maximum suppression was observed after 48-h treatment of lymphocytes with optimally mitogenic doses of Con A. The Con A-inducible suppressor cells of the pleural effusion and spontaneous suppressor cells of the peripheral blood of cancer patients had the same characteristics with regard to the capacity to suppress the mitogenic responses of autologous and allogeneic lymphocytes, belonging to the group of nylon wool-nonadherent T cells and being sensitive to in vitro culture and resistant to treatment with mitomycin C.     

Activation of murine lymphocytes by cyclic guanosine 3′,5′-monophosphate: Specificity and role in mitogen action

Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) stimulates nucleic acid synthesis in lymphocytes, and has been implicated as the intracellular effector of the actions of mitogenic agents on these cells. In the present study, we examined the specificity of the mitogenic activity of cyclic GMP and of its 8-bromo (Br) derivatives, and the effects of the T cell mitogens, concanavalin A, phytohemagglutinin, and staphylococcal entertoxin B (SEB) on the cyclic GMP content and guanylate cyclase activity of mouse splenic lymphocytes. Cyclic GMP and guanosine modestly increased the incorporation of [³H]thymidine into DNA by cultured lymphocytes, but were far less effective than their 8-Br-derivatives. However, on a molar basis the mitogenic activity of both 8-Br-guanosine and 8-Br-5′-GMP exceeded that of 8-Br-cyclic GMP, when tested in the presence and absence of serum in the culture media. Combined addition of maximal doses of these nucleotides did not give additive stimulatory effects, suggesting an action on a common subpopulation of cells, and possibly a common mechanism. By contrast, cyclic AMP, 8-Br-cyclic AMP, 8-Br-adenosine, cholera toxin and prostaglandin E₁ suppressed both basal [³]thymidine incorporation and stimulation of this parameter by T-cell line mitogens and the guanosine nucleotides. Rapid effects of concanavalin A, phytohemagglutinin, SEB, guanosine, 5′-GMP, 8-Br-guanosine, and 8-Br-5′-GMP on the cyclic GMP content of murine lymphocytes could not be demonstrated. Similarly, concanalin A, phytohemagglutinin and SEB failed to alter guanylate cyclase activity when added directly to cellular homogenates or pre-incubated with intact cels. Conversely, carbamylcholine rapidly increased lymphocyte cyclic GMP but was not mitogenic.These results are consistent with the hypothesis that cyclic GMP and cyclic AMP are antagonistic in their influence on lymphocyte mitogenesis. However, they also demonstrate that related nucleotides are more potent mitogens than cyclic GMP and suggest that activation of murine lymphocytes by concanavalin A, phytohemagglutinin and SEB may not be mediated by rapid increases in cellular cyclic GMP content. Since high concentrations of exogenous cyclic GMP and related nucleotides must be used to influence DNA synthesis, the biologic significance of this effect remains uncertain.     

Impaired T- and B-cell functions in patients with Hodgkin's disease

Summary The mitogenic response of T-cell subsets, the production of interleukin-1 (Il-1) and interleukin-2 (Il-2) and in vitro immunoglobulin production was investigated in patients with Hodgkin's disease (HD). The mitogenic response of mononuclear cells (MNC) and the OKT4⁺ and OKT8⁺ subsets was greatly reduced in advanced disease stages and could only partially be restored with exogeneous Il-2. In untreated patients with HD — except those with highly advanced disease — the OKT4⁺ lymphocytes showed normal response to phytohemagglutinin in contrast to the MNC suggesting inhibiting agents or cells within the MNC. These findings corresponded to reduced Il-2 synthesis of MNC, whereas isolated OKT4⁺ — cells produced normal or elevated amounts of Il-2. MNC or monocytes produced normal or even higher amounts of lipopolysaccharide-induced Il-1 than controls. The results do not confirm a defect in this component of the interleukin system in HD. The immunological impairment was not limited to the T-cell system but involved B-cell activation and differentiation as well. The pokeweed mitogen-induced IgM, IgG and IgG production was highly suppressed in untreated HD, whereas the MNC of previously treated patients produced subnormal amounts of immunoglobulin in vitro. It is not yet clear whether this defect is T-cell-mediated or primarily a B-cell deficiency.     

Immunosuppression of experimental allergic encephalomyelitis. III. In vitro evidence for induction of suppressor T lymphocytes in draining lymph node cells of animals immunized with myelin basic protein complexed to lipopolysaccharides

We recently demonstrated that Lewis rats immunized with bacterial lipopolysaccharides (LPS) precomplexed to guinea pig myelin basic protein (BP) in complete Freund's adjuvant were less effective in inducing experimental allergic encephalomyelitis (EAE) than BP-immunized controls. When tested in vitro both lymph node cells (LNC) and spleen cells (SpC) of animals immunized with BP-LPS were less effective in proliferative responses to various mitogens, which included phytohemagglutinin, concanavalin A, purified protein derivative of tuberculin, LPS, and BP. Of importance immunization of rats with BP complexed to LPS results in the generation of cells in lymph nodes of these animals that suppress the mitogenic response of BP-immunized LNC and also SpC in mixed lymphocyte cultures. The suppressive effect of these cells in mixed lymphocyte culture reaction was found specifically in response to BP and to a lesser extent to LPS in LNC. SpC of BP-LPS immunized animals did not suppress the proliferative response to SpC of BP-immunized animals. Treatment of these LNC with antithymocyte serum and complement abolished this suppressive effect of LNC, suggesting that the immunoregulatory cells in LNC of BP-LPS immunized animals are suppressor T lymphocytes. The parallel between the in vitro induction of suppressor T lymphocytes in the draining LNC and the function of LPS in the development of EAE in Lewis rats suggests a possible immunologic significance of the effect.     

Interaction of mitogenic lectins with lymphocyte plasma membranes]

It is shown that mitogenic stimuli from concanavalin A and phytohemagglutinin are summed up in the time. Under given experimental conditions each of the mitogens used did not induce mitogenesis when the interaction with lymphocytes continued for 14 and/or 20 hours. The results obtained are discussed from the point of view of the cell-to-cell interaction between the lymphocytes. It is assumed that the ability of the stimuli to sum up in the time underlies the nonspecific mechanism of defense from tolerance.     

Cellular regulation of human lymphocyte mitogenic factor release in vitro]

Production of the mitogenic facotr by human lymphocytes stimulated with phytohemagglutinin (PHA) was investigated. Anti-PHA antibodies were used for studying the culture medium mitogenic activity. The mitogenic factor production markedly increased after lymphocyte irradiation. When macrophages were eliminated, using iron powder, mitogenic factor generation was also increased. It was demonstrated that lymphocyte irradiation and macrophage elimination stimulated the mitogenic factor production throgh different mechanisms.     

Analysis of the cyclase enzyme activity of the cell membrane following lymphocyte stimulation with a mitogenic polyanion

Stimulant action of the mitogenic polyanion, polyacrylic acid (PAA) was investigated in mouse lymphocyte culture in vitro. B cell division was induced by "impulsive" PAA treatment. Shortly after PAA treatment the activity of the membrane enzymes, adenylate and guanylate cyclases, was assayed according to the changes in the concentration of cAMP and cGMP. The effect of PAA on the time course of cAMP and cGMP in lymphocytes was compared to the effect of B cell mitogen of other chemical nature--bacterial lipopolysaccharide (LPS). PAA was demonstrated to produce no effect on the activity of membrane cyclase enzymes. On the contrary, following LPS addition guanylate cyclase in the lymphocyte membrane was activated within the first 5-10 minutes. Later on (after 2h) the cells activated with LPS showed an increase in adenylate cyclase activity. By the 12th-24th hour the concentration of cAMP in the LPS-stimulated cells reached 250% of the control level. The differences are discussed between the mitogenic polyanion (PAA) and the lipid-modifying mitogen (LPS) in the molecular mechanisms by which the lymphocyte responses are activated.