Ulla Persson

The Role of Adherent Cells in B and T Lymphocyte Activation

Macrophages have been found to be of importance in several difierent immunological reactions. Thus, macrophages are required for induction of a primary immune response to heterologous red blood cells in vitro (Mosier & Coppleson 1968), and for a proliferative response induced in vitro to alloantigens (Alter & Bach 1970, Rode & Gordon 1970, Twormey et al. 1970), as well as for the developement of cytotoxic cells (Wagner et al. 1972, McDonald et al. 1973). Fxirthermore, macrophage and factors released by macrophages can induce proliferation in spleen cells and cause production of polyclonal immunoglobuiins (Lemke & Opitz 1976, Moller et al. 1976, Opitz et al. 1976). In all these systems, however, fetal calf serum together with 2-mercaptoethanol (2-ME) can substitute for macrophages, indicating a nonspecific function of adherent cells (Chen & Hirsh 1972, Bevan et al. 1974, Lemke & Opitz 1976). In this paper, we report on some effects of adherent cells on B and T cell activation induced by polyclonal cell activators, and we discuss the possible mechanisms behind the synergistic effect observed between B and T cell mitogens and adherent cells. In the experiments shown, Fi hybrids of the inbred mouse strains A/Sn and C57B1 or B10.5M were used, if not otherwise indicated. Cells were cultured senimfree in microcultures (usually triplicate cultures with 5 X 10* cells/0.2 ml of medium) and incorporation of ^H-thymidine was measured on day 2.

Enumeration of IFN-γ-producing cells by flow cytometry: Comparison with fluorescence microscopy

Abstract A new intracytoplasmic immunofluorescence staining to detect and quantify human interferon-γ (IFN-γ)-producing cells by means of flow cytometry is described. Mononuclear leukocytes, stimulated in vitro to produce IFN-γ, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic IFN-γ was demonstrated by indirect immunofluorescence using IFN-γ-specific mouse monoclonal antibodies. The staining exhibited a very characteristic morphology and was localized in the Golgi apparatus. An excellent agreement between the enumeration of cytoplasmic IFN-γ-positive cells by immunofluorescence microscopy and flow cytometry was noted. However, the latter has the advantage of a standardized control, is less labor consuming and is observer independent.

Different Requirements for T Cells Responding to Various Doses of Concanavalin A

Con A is known to activate T cells to proliferation and the development of effector cells. Conflicting reports have been published as to the need for accessory cells in the T‐cell response induced by Con A. We have found that the proliferative response in purified mouse spleen T cells induced by various doses of Con A requires different culture conditions and helper cells. The Con‐A‐induced response induced by optimal and high concentrations of the ligand requires the presence of either serum or adherent cells obtained from the peritoneal cavity or the spleen. For induction of a proliferative response by low doses of Con A both serum and helper cells must be present. The T‐cell response to suboptimal concentrations of Con A is further characterized by the fact (hat the presence of an Ia‐positive cell is required. Removal of Ia‐positive cells from purified T cells results in a loss of the response of remaining cells to low doses of Con A hut has less effect on the response induced by higher concentrations of the activating ligand. The possibility that distinct subsets of T cells are responding to low and high concentrations of Con A will he discussed.

An association between human leucocyte antigen alleles and acute and chronic graft-versus-host disease after allogeneic haematopoietic stem cell transplantation

Summary. The association between various human leucocyte antigen (HLA) alleles and the occurrence of acute and chronic graft‐versus‐host disease (GVHD) was evaluated in 493 haematopoietic stem‐cell transplant (HSCT) patients with HLA identical sibling donors. There were 307 men and 186 women with a median age of 30 years (0·2–77). Most of the patients had a haematological malignancy and received total body irradiation or busulphan combined with cyclophosphamide as conditioning before transplantation. GVHD prophylaxis consisted of monotherapy with methotrexate (MTX) or cyclosporin (CsA) in 118 patients, MTX + CsA in 323, T‐cell depletion in 28 and other combinations in 24. In total, 84 patients (17%) received a peripheral blood stem‐cell graft, whereas the rest received bone marrow. The cumulative incidence of acute GVHD grades II–IV was 20%, and chronic GVHD 46%. In the multivariate analysis, HLA‐A10 (OR 2·14, CI 1·04–4·41, P = 0·03) and HLA‐B7 (OR 1·80, CI 1·04–3·12, P = 0·03) correlated with an increased risk of acute GVHD grades II‐IV. We also found an association between HLA‐B27 (RR 0·60, CI 0·37–0·95, P = 0·04) and a lower incidence of chronic GVHD. These HLA alleles were independent of other known risk factors for acute or chronic GVHD, as shown by multivariate analysis. These results show that major histocompatibility comlex (MHC) alleles may influence the incidence of GVHD in HSCT with HLA identical sibling donors.

Are Increased IgE‐Levels a Signal of an Acute Graft‐Versus‐Host Reaction?

IgE-levels are markedly elevated following bone-marrow transplantation in patients with and without GVHD. In patients with GVHD, there is a significant correlation between the timing of the IgE-increase and the appearance of clinical GVHD (p less than 0.01). The highest IgE-level (8000 kU/l) was noted in a recipient of a syngeneic graft. During the IgE-peak, the serum from this patient contained low concentrations of IgE reacting with several tested allergens as well as for the hapten TNP, which indicated polyclonal activation. In a patient with a known allergy to animal danders, RAST tests were positive against dog and cat both before and six weeks after total body irradiation and transplantation with marrow from a non-allergic donor. A slight increase in the amount of allergen-specific, IgE-antibodies was seen during the increase in total IgE. A non-allergic patient was transplanted with marrow from a donor allergic to timothy. Timothy-specific, IgE-antibodies were detected immediately after transplantation but they disappeared within a few days and could not be detected during the period of increase in total IgE. We believe that the IgE-elevation seen after conditioning with cytotoxic drugs and total body irradiation in BMT-patients is a polyclonal response in host B-cells induced during an acute, GVHR and influenced by disturbed regulatory T-cells. Lymphocytes from patients with acute GVHD had unusually large numbers of IgG/PFC in vitro after stimulation with staph. aureus Cowan 1 (p less than 0.001), which may reflect a clonal expansion of responsive B-cells.

Concanavalin A inhibits the effector phase of specific cytotoxicity.

The effects of concanavalin A (Con A) on the effector phase of specific and nonspecific cytotoxicity were studied. The addition of the lectin to the cytotoxicity assay resulted in inhibition of specific cytotoxicity and induced the lysis of nonspecific targets only when the lectin was added after the target cells. Preincubation of the effector cells with the ligand strongly inhibited specific cytotoxicity and did not induce nonspecific cytotoxicity. However, preincubation of the target cells with Con A before addition to the assay had no effect on the specific lysis and strongly facilitated the lysis of nonspecific targets. The inhibitory effect was not due to the agglutinating property of the lectin, since another agglutinogenic and non‐mitogenic lectin (Helix pomatia) did not inhibit cytotoxicity. Induction of effector‐to‐effector killing seemed unlikely, since the addition of Con A to 51Cr‐labelled effector cells did not significantly enhance the release of isotope. The inhibitory effect could be reversed by a subsequent incubation of the Con‐A‐treated effectors with alpha‐methyln‐mannoside. We suggest that Con A inhibits specific alloreactive cytotoxicity by blocking the antigen‐binding receptors of T cells and induces nonspecific cytotoxocity by already activated cytotoxic T lymphocytes (CTLs) by binding to the major histocompatibility complex (MHC) antigens of the targets and creating structures mimicking allogeneic MHC products that will be recognized by CTLs via the antigen‐binding receptor.

Improved cell depletion in a panning technique using covalent binding of immunoglobulins to surface modified polystyrene dishes.

A method for the chemical modification of plastic surfaces permits covalent binding of proteins and we have used this method in the development of an efficient panning technique. Thus, human peripheral T lymphocytes coated with mouse monoclonal antibodies directed against the CD4 marker may be selectively and reproducibly removed from a lymphocyte population by a short incubation in modified plastic dishes coated with rabbit anti-mouse IgG antibody. Due to the higher protein binding capacity of the dishes the use of the IgG fraction of the coating antibody was sufficient for optimal and reproducible results. In contrast, control dishes with passively adsorbed antibody required an affinity-purified fraction and even then were less efficient.

The effect of lipopolysaccharide on the primary immune response to the hapten NNP.

We have studied the effects of lipopolysaccharide (LPS) on the primary in vivo immune response to the hapten (4‐hydroxy‐3,5‐dinitrophenyl)acetyl (NNP), with special reference to the avidity and affinity of the early appearing 19S and antibodies Comparisons were made of the immune response to NNP in groups of mice given either antigen alone. LPS alone, or antigen plus LPS The avidity of antibodies induced by LPS plus antigen was similar to that found after injection of antigen alone, in spite of the fact that the antibodies were more numerous However, when comparing the avidity of antibodies produced in animals given only LPS with those given LPS plus antigen, the latter group was often found to have fewer low‐avidity 19S‐antibody‐producing cells The affinity of 7S antibodies was also similar in the two groups given antigen or antigen plus LPS Kinetic studies of the effect of LPS on the primary immune response to NNP showed that synergy was observed only before or after the peak response in groups given antigen alone It is concluded that LPS under synergy conditions acts preferentially on specific antigen‐sensitive cells which are distinct from those that are activated to polyclonal antibody synthesis by LPS alone. Possible mechanisms for the adjuvant effect of LPS are discussed

Regulation of in vitro immunocyte activation: origin of and targets for cell-released inhibitors.

Supernatants from incubated normal mouse spleen cells suppressed the DNA synthetic response induced by polyclonal B‐ and T‐cell activators and the primary immune response to sheep erythrocytes in normal spleen cells in vitro. The inhibitory effects of the supernatants were found to be dependent on the culture system used. The main cell population producing or releasing the inhibitory factors was nonadherent spleen cells, but macrophages and bone marrow cells could also give supernatants with inhibitory effects. Thymocytes did nut release any inhibitors under the same conditions. Separation of the supernatants showed that there were it least two factors with inhibitory effects on proliferation in lymphocytes. One of these factors had a molecular weight of less than 10,000 (not degradable by protease) and suppressed activation by all tested polyclonal B‐ and T‐cell activators. Another factor (a protein with a molecular weight of more than 30, 000) inhibited activation by some polyclonal B‐cell activators (lipopolysaccharide, type III pneumococcal polysaccharide) whereas the proliferation induced by dextran sulfate Was less or not at all affected under the same culture conditions. Trivial mechanisms for inhibition by supernatant factors, such as medium depletion, general toxicity, and accumulation of free thymidine in the medium, were excluded.

Alveolar Lymphocytes from Patients with Sarcoidosis Respond Poorly to Activation by Mitogenic Lectins

Attempts have been made to activate alveolar cells obtained by bronchoalveolar lavage from 17 patients with sarcoidosis in different stages with the lectins concanavalin A and leukoagglutinin. The cells obtained from 2 of the patients responded well by proliferation whereas the cells from the other 15 patients responded poorly. Coculture experiments indicate that the low proliferative response was not due to inhibitory effects exerted by alveolar lymphocytes or macrophages, but rather to a lack of responding T cells. The results are discussed in relation to other studies showing increased immune reactivity in alveolar lymphocytes from patients with sarcoidosis.