Effect of antibodies against immunoglobulins and the theta antigen on the specific and non-specific stimulation of mouse spleen cells in vitro

Normal mouse spleen cells were stimulated in culture by phytohaemagglutinin (PHA), pokeweed mitogen (PWM), allogeneic cells; keyhole limpet haemocyanin (KLH) sensitized cells by the specific antigen. The stimulation of the cells was measured by [³H]thymidine incorporation into the TCA precipitable fraction of the cultures. In this system, the effect of treating the cells with an antibody against the theta antigen and an antibody against immunoglobulin, with or without complement, was investigated. Treatment of the cells with antisera and complement or without complement gave similar results. The secondary in vitro stimulation with soluble antigen KLH could be markedly reduced with both anti-θ and anti-immunoglobulin serum. The response to allogeneic cells in the mixed lymphocyte reaction was only reduced by anti-θ serum and not by anti-immunoglobulin serum. No definite effect could be demonstrated by either antibody on the non-specific stimulation by PHA or PWM.     

The transformation of human lymphocytes by monkey antisera to human immunoglobulins

Cultures of human peripheral leucocytes were stimulated to incorporate tritiated thymidine when incubated with monkey antisera to human immunoglobulins. Twenty-five of forty-four monkey antisera were active and stimulated 90 per cent of leucocyte (WBC) cultures to incorporate a small but significantly greater amount of tritiated thymidine (TdR³H) than that incorporated by controls. This stimulation of TdR³H uptake correlated with an increase from 2 to 8 per cent lymphoblasts in the cultures. Leucocytes washed free of serum immunoglobulins responded to a greater degree to the anti-immunoglobulin sera than when they were cultured in the presence of human serum. Prior absorption of antisera with either whole serum or homologous immunoglobulin blocked antiserum stimulation completely. The anti-IgG and anti-IgM antisera were consistently more effective than anti-IgA, anti-κ and anti-λ chain antisera. Sequential stimulation by antisera against two different immunoglobulins was not significantly different from those stimulated by only one of the two. Lymphocytes from three asymptomatic subjects with low or absent serum IgA levels transformed as well with anti-IgA as did lymphocytes from subjects with normal serum IgA levels. Antisera were cytotoxic to the lymphocytes only in the presence of complement. Presumably the transformation of human lymphocytes was due to a reaction of anti-immunoglobulin antisera with specific immunoglobulin antigenic determinants present on or in the circulating lymphocytes.     

The cytotoxic activity of lymphocytes from human lymph in vitro

The in vitro cytotoxicity and DNA synthesis of thoracic duct and blood lymphocytes from four patients have been studied on the 1st day of drainage. Three patients were being drained as a pretreatment for kidney transplantation and one had myasthenia gravis. In one patient lymphocytes were obtained from a lymphatic fistula in the groin and from the blood 5 weeks after drainage began. Lysis of tissue culture cells (Chang cells) in the presence of PHA or antiserum to target cell antigens was quantitated by [⁵¹Cr]chromate release.

Lymphocytes from lymph were at best poorly cytotoxic to antibody-treated target cells under conditions where purified blood lymphocytes from the same donors had normal lytic activity. PHA-induced cytotoxicity by lymph-borne lymphocytes was noted but was considerably weaker than that of blood lymphocytes. In contrast, incorporation of [¹⁴C]thymidine into DNA of thoracic duct lymphocytes after stimulation with PHA was about 60% of that of the patients' blood lymphocytes. The DNA synthesis of thoracic duct lymphocytes induced by PPD or allogeneic lymphocytes was as good as that of blood lymphocytes. The mitotic response to PHA by lymphocytes from the lymph was reduced after two weeks drainage.

It is assumed that the number of effector cells and/or supporting cells in antibody-induced cytotoxicity in thoracic duct lymph is too small to induce target cell lysis under the present experimental conditions. Moreover, our data indicate that PHA-induced and antibody-mediated cytotoxicity are at least partly mediated by different lymphocyte subpopulations.

     

Function and distribution pattern of human T lymphocytes. I. Production of anti-T lymphocyte specific sera as estimated by cytotoxicity and elimination of function lymphocytes

Antisera were prepared in rabbits against lymphoid cells from peripheral blood of a patient with Bruton-type agammaglobulinaemia. Such antisera could be shown to display a two plateau level of cytotoxicity against human peripheral lymphocytes in the presence of complement. This suggested the presence of species- as well as subgroup-specific antibodies in these antisera.

The subgroup-activity of the antisera could be shown to be directed against human T lymphocytes on the basis of the following results. When lymphocytes are filtered through columns coated with anti-immunoglobulin antibodies lymphocytes with high surface concentrations of immunoglobulin are retained. The filtered cells are highly enriched in cells sensitive to the subgroup-specific antibodies. A close to complete inactivation of mixed leucocyte reactivity or stimulability with soluble PHA was induced by preincubating with the antisera and complement. Using identical conditions only marginal inhibition of immunoglobulin production of peripheral lymphocytes in vitro was induced.

In conclusion, we believe these antisera to selectively kill human T lymphocytes at serum concentrations, which will not kill or inhibit the function of human B lymphocytes.

     

Lymphokine Production in Ty Lymphoproliferative Disorders

We have studied five patients with chronic lymphocytosis consisting of large granular lymphocytes (LGL). The increased numbers of LGL in these patients had little or no natural killer activity, mediated antibody-dependent cellular cytotoxicity, and were induced to kill tumour lines after culture for 3 days with interleukin 2 (IL-2). Patients' LGL showed considerable reactivity with HNK-1 and AB8.28 monoclonal antibodies (MoAb), whereas positivity for OKM1 and N901 was found in only two subjects, and only one patient reacted with B73.1. No appreciable reactivity has been found with anti-Tac MoAb in the four patients tested. In the absence of stimulation, the patients' LGL produced no IL-2 and only minimal amounts of IL-1 and interferon (IFN). On stimulation with lipopolysaccharides (for IL-1) or phytohaemagglutinin A (PHA) (for IL-2 and IFN), they produced IL-1 and IFN in amounts similar to those produced by normal lymphocytes, but only modest levels of IL-2. These results indicated that proliferating LGL, like normal LGL, have a secretory capacity. The lack of constitutive lymphokine production, the lack of Tac receptor expression, and the defect in IL-2 production after PHA stimulation do not support the hypothesis of an autocrine proliferation sustained by a known growth factor.     

Nonspecific suppression of T lymphocyte responses in mice carrying progressively growing tumors

Unfractionated spleen cells from mice with four different types of progressively growing tumors were unable to show enhanced DNA or protein synthesis upon stimulation with phytohemagglutinin (PHA) in vitro. After separation by velocity sedimentation, or passage over an anti-immunoglobulin column, cells were obtained which could respond to this mitogen. An auxiliary population of cells obtained by velocity sedimentation, sedimenting with the characteristics of activated B lymphocytes and sensitive to the cytotoxic effects of anti-immunoglobulin serum and complement, could depress the response of normal spleen cells challenged with PHA. A role for this cell type in the tumor progression seen in the host animals is discussed.     

Structure and biological functions of human IgD. XII. Anti-IgD enhancement of PHA responsiveness in patients with chronic lymphocytic leukaemia.

Specifically purifed anti-human delta stimulated the in vitro incorporation of [3H]thymidine by human peripheral lymphocytes from patients with chronic lymphocytic leukaemia (CLL). The peak response varied between individuals; those with 5--52% IgD-bearing lymphocytes exhibited maximum stimulation at 3 days, whereas a patient with only 1% IgD-bearing cells showed optimal activation at 6 days. In agreement with others, our data indicated that, in most instances, lymphocytes from patients with CLL respond poorly to PHA. One of the most important findings in this study is the enhancement of PHA responsiveness by anti-delta. Lymphocytes that exhibited reduced responsiveness to PHA alone, when pre-treated with anti-delta, showed transformation greater than the sum of the anti-delta plus PHA responses.     

Age-related refractoriness of PHA-induced lymphocyte transformation. I. Comparable sensitivity of spleen cells from young and old mice to culture conditions.

The decline in phytohemagglutinin (PHA) responsiveness of spleen cells from aged mice was studied in an effort to determine the nature of the depressed PHA reactivity. The reduced response of DNA synthesis (thymidine incorporation) to PHA stimulation could not be attributed to a decreased viability of spleen lymphocytes from old mice in in vitro culture conditions, to different PHA dose requirements, or to longer periods of culture time for old mouse spleen cells to reach maximal activity. Culture of cells for 24-48 h prior to addition of PHA gave patterns of thymidine incorporation similar to those of freshly prepared spleen cells. The elimination of red blood cells by a brief hypotonic shock significantly increased thymidine incorporation in spleen lymphocytes from both yound and old mice.     

In vitro cytotoxicity of lymphocytes from patients with `acquired'' and sex-linked agammaglobulinaemia

The in vitro cytotoxicity for fowl erythrocytes of circulating lymphocytes from nine patients with `acquired' and three patients with sex-linked agammaglobulinaemia was tested by release of sodium chromate (<sup>51</sup>Cr) after 42 hr of culture in the presence of phytohaemagglutinin (PHA). The lymphocyte cytotoxicity of three patients with `acquired' agammaglobulinaemia was low, but that of the other six and of the three patients with sex-linked agammaglobulinaemia were normal. All the `acquired' agammaglobulinaemic patients had markedly diminished thymidine uptake in response to PHA at 3 days, but a normal response at 7–10 days, but the three boys with sex-linked agammaglobulinaemia showed a normal response. The findings further demonstrate the heterogeneity of lymphocyte abnormalities in patients with agammaglobulinaemia.     

Lymphocyte surface components. I. Stimulation of enzyme-treated rabbit lymphocytes by non-specific mitogens

Rabbit lymphocytes from various locations were treated briefly with proteases and the rate of incorporation of thymidine following stimulation by `non-specific' stimulants (Staphylococcus filtrate, SF, and Phaseolus vulgaris phytohaemagglutinin, PHA) measured. The proteases themselves, especially at concentrations exceeding 0·005%, sometimes caused cells to incorporate more thymidine than untreated cells. Stimulation with SF or PHA was much greater when lymphocytes were first treated with protease at low concentrations (when enzyme alone failed to stimulate). This effect was shown particularly by lymphocytes from gut-associated lymphoid organs. Peripheral blood lymphocytes, while unaffected by proteases at a concentration of 0·005%, incorporated increased amounts of thymidine after treatment with proteases at higher concentrations: pronase is especially potent. Further evidence is presented, distinguishing between the cytoagglutinating and mitogenic characters of PHA.     

Lymphocyte transformation and production of a human mononuclear leucocyte chemotactic factor in patients with Hodgkin''s disease

Human peripheral lymphocytes stimulated in vitro with phytohaemagglutinin (PHA) produce a factor with chemotactic activity for homologous monocytes. The production of this factor and lymphocyte transformation was investigated in patients with Hodgkin's disease. It has been found that the PHA response as measured by incorporation of [³H]thymidine was markedly depressed in patients when compared to the response of normal lymphocytes. In contrast, the production of the chemotactic factor was not significantly depressed in patients with Hodgkin's disease.     

Activation and proliferation of normal resting human T lymphocytes in serum-free culture: role of IL-4 and IL-6

Purified human T lymphocytes, completely depleted of accessory cells [i.e. monocytes, large granular lymphocytes (LGL) and B lymphocytes], have been grown in serum-free culture in presence of a mitogenic lectin (phytohaemagglutinin, PHA) and different recombinant cytokines. Only IL-2 and IL-4 induced a marked stimulation of [3H] thymidine ([3H]TdR) uptake, cell proliferation and expression of activation markers [transferrin receptor (TrfR), IL-2R]. The other cytokines (IL-1 alpha, IL-1 beta, IFN-gamma, GM-CSF, TNF-alpha) had no significant effect, except for a moderate, but significant, stimulation of [3H]TdR uptake induced by IL-3. Simultaneous addition of IL-4 and anti-IL-2 neutralizing monoclonal antibodies (mAb) did not modify the effects induced by IL-4 alone. Furthermore, IL-2 was not detected in the supernatant of T cells grown in the presence of PHA and IL-4. Thus, our results indicate that IL-4 acts on T lymphocytes independently of IL-2. We also observed that IL-6 moderately activates DNA synthesis in PHA-stimulated T lymphocytes, but markedly potentiates the proliferative effect of suboptimal amounts of IL-2. In conclusion, the present study suggests that B-cell growth factors, in addition to IL-2, control the proliferation of normal circulating T lymphocytes.     

Mitogen-induced membrane changes and cell proliferation in T lymphocyte subpopulations.

Rabbit thymus-dependent lymphocytes were exposed to phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) or anti-immunoglobulin at various stages of maturation. Proliferation (induction of DNA synthesis) and early membrane events (turnover of membrane phospholipids) were measured in neonatal thymocytes, normal adult thymocytes, prednisolone-resistant thymocytes and lymph node lymphocytes. In immature thymocytes PHA induced only a marginal increase in DNA synthesis. The mitotic response increased with maturation, but only peripheral T lymphocytes exhibited maximum stimulation. Con A and PWM were able to induce DNA synthesis in immature thymocytes and the degree of stimulation was shown to increase with maturation. In contrast to the different degree of proliferation of thymocytes induced by PHA or Con A the incorporation of [14C]oleate, [14C]choline or [14C]acetate into phospholipids was stimulated to the same degree by these lectins. Reactivity of T lymphocytes, as measured by early membrane changes at different stages of maturation, to different T cell mitogens appears to be identical. Differences in degree of cell proliferation therefore may be secondary phenomena due, in part, to tissue culture conditions. Reactivity to mitogens as measured by phospholipid turnover appears to be an early acquired function in the maturation of lymphocytes of the T cell line.     

A new ultra-microculture system. I. Stimulation of human T lymphocytes by phytohemagglutinin (PHA)

We have developed an ultra-microtechnique for culturing lymphocytes in glass capillary tubes at a final culture volume of 1 microliter or 2 microliter. The advantage of the method is that a substantially lower number of cells and minute amounts of culture medium are required. The cultures are premixed in microtubes, sucked into glass capillary tubes and incubated for an appropriate culture period. For determination of [3H]thymidine ([3H]TdR) incorporation, the cells are transferred into the wells of microtiter plates. Some special accessories have been developed which allow routine use of this system for large numbers of cultures. Optimal culture conditions for stimulation of human T lymphocytes by PHA are described.     

Cellular immunity in newborn infants and children: stimulation of lymphocyte protein synthesis as a measure of immune competence.

An assay based on the early stimulation of protein synthesis in lymphocytes has been used as an in vitro measure of cellular immune competence. 3H-labelled leucine incorporation into human peripheral lymphocytes (PBL) stimulated by the mitogens phytohaemagglutinin (PHA), wax bean agglutinin (WBA) and Concanavalin A (Con A) was measured after one day in culture. This assay offers a technical advantage over the analogous 3H-labelled thymidine incorporation assay, because of the short incubation time required and the absence of homologous serum in the assay system. Newborn infants and patients with Down's syndrome as a group had normal responses, whereas those suffering from recurrent infections demonstrated normal or hyper-reactive responses. Patients with lymphoproliferative disorders, ataxia telangiectasia, and some patients under steroid therapy had diminished immune proliferative reactions. These results are in agreement with most previously reported studies using other assay systems.     

Reactivity of RA and SLE lymphocytes stimulated by anti-Ig antibodies

In vitro human lymphocyte stimulation response was studied using phytohaemagglutinin and immunoabsorbent-column purified rabbit antibodies to γA, γM and γG with peripheral blood lymphocytes from normal subjects as well as patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). In addition, lymphocytes from a group of patients receiving immunosuppressive therapy in connection with renal transplantation or metastatic carcinoma were studied as controls. No essential differences in degree of response were noted when normal subjects were compared to RA patients, however, most SLE patients showed a decreased response with the various purified anti-Ig antibodies. This hyporesponsiveness was most marked using anti-IgM and anti-IgG antibody (P<0·01). Patients receiving immunosuppressive drugs for maintenance of renal transplants or in the course of treatment for metastatic carcinoma also showed significant depression of responses in lymphocyte culture (P<0·01) after stimulation with anti-IgG, IgA, or IgM antibody. In addition, baseline unstimulated cellular incorporation of labelled thymidine, as well as degree of stimulation with phytohaemagglutinin, was significantly reduced (P<0·01) among the immunosuppressed control group when compared to normals or the groups of SLE or RA patients. Diminished culture response in SLE patients and subjects receiving immunosuppressive therapy may be related to concurrent therapy or to other alterations intrinsic to the disease process itself.     

Methyl inosine monophosphate (MIMP) augments T-lymphocyte mitogen responses and reverses various immunosuppressants

Methyl inosine monophosphate (MIMP) augments preferentially the in vitro responses of human and murine lymphocytes to a T-cell mitogen such as phytohemagglutinin (PHA) and inconsistently to a B-cell mitogen such as pokeweed or lipopolysaccharide (LPS). In a normal interleukin-2-dependent cell line (CTLL), MIMP showed little or no effect on IL-2 action; however, in a murine CTLL line exhibiting impaired responses to IL-2, MIMP stimulated thymidine incorporation and restored the response to IL-2. MIMP augments the PHA responses of both CD4⁺ and CD8⁺ human peripheral blood T-cells. The effect of MIMP to augment the PHA response of human lymphocytes is paralleled by the parent molecule, IMP. MIMP, but not IMP, is resistant to hydrolysis by 5'nucleotidase; thus, MIMP appears to be a protected analogue of IMP which is capable of in vivo action. MIMP (100 μg/ml) augments the PHA responses of 15 of 24 elderly humans. MIMP also augments the PHA responses of eight HIV-infected pre-AIDS patients but not of eight AIDS patients. When PHA responses of human lymphocytes are suppressed in vitro by an HIV-derived immunosuppressive peptide, interferon α, or prostaglandin PGE₂, MIMP (0.1–100 μg/ml) progressively restores the depressed response; however, when the suppression is severe (greater than 50%), MIMP cannot restore the response. These data indicate that MIMP potentiates normal T-lymphocyte mitogen responses and restores those impaired by a variety of inflammatory and immunosuppressive influences.     

Somatomedin production and response to mitogen by lymphocytes in children with growth hormone deficiency

Studies on the lymphocyte proliferative activity of the sera from growth hormone (GH) deficient patients have resulted in contradictory observations. The ability of lymphocytes to synthesize somatomedin-C (Sm-C) and thus account for a normal proliferative activity (previously observed by us) of the GH-deficient sera was studied, by measuring Sm-C concentration in the culture medium using a standard radioimmunoassay for Sm-C. The response of lymphocytes to the mitogen phytohemagglutinin (PHA), as determined by the incorporation of 3H thymidine into DNA, was also studied. The Sm-C concentrations in the cultures reflected the Sm-C concentrations of the respective serum added and did not alter with significant increases in the cell number induced by PHA. The lymphocytes from GH-deficient children and normal children were indistinguishable in their ability to respond to PHA. We conclude that lymphocyte proliferation in short-term culture, was not associated with an increase in Sm-C and that in the lymphocyte proliferation assay the sera and the lymphocytes from GH-deficient children respond similarly to the sera and lymphocytes from normal children.     

Cell cycle-specific effects of glucocorticoids on phytohaemagglutinin-stimulated lymphocytes.

Effects of steroid hormones and colchicine on the response of pig lymphocytes to phytohaemagglutinin (PHA) were assessed by measurement of [6-3H]thymidine incorporation. At steroid concentrations of 1 microM and below, only glucocorticoids and progesterone inhibited PHA-stimulated [6-3H]thymidine incorporation but at 100 microM inhibition was also produced by oestrogens, androgens and physiologically inactive steroids. Measurement of [6-3H]thymidine incorporation 18-24 hr, 6-12 hr or 0-6 hr after the delayed addition of the synthetic glucocorticoid, dexamethasone, to PHA-stimulated lymphocytes revealed a succession of alternating phases of sensitivity and insensitivity to the effects of the steroid which suggested that it was acting, perhaps indirectly, in a cell cycle stage-specific manner to arrest the progression of activated lymphocytes from G1 to S. Similar effects were observed with colchicine, but 100 microM 11-epicortisol inhibited [6-3H]thymidine incorporation in a non-cycle-specific manner. Glucocorticoid receptor levels in pig lymphocytes were increased 2-5-fold within 24 hr of PHA stimulation.     

The influence of the serum/PHA ratio,microplate well shape and 2-mercaptoethanol on the stimulation of different numbers of cells in lymphocyte cultures from the chinese hamster

Conditions for microculture of Chinese hamster lymphocytes are described which allow measurement of thymidine uptake with 6000 to 1000 lymphocytes per culture.The relationship between degree of cell stimulation, PHA concentration, culture surface and cell concentration is described, as well as the influence of addition of 2-mercapto-ethanol to the cultures.