Terutaka Kakiuchi

The effect of cardiopulmonary bypass on T cells and their subpopulations.

To investigate the effect of cardiopulmonary bypass (CPB) on T cells, lymphocyte subsets of peripheral blood and lymphoid organs were monitored during and after open-heart surgery (Group 1). As a control, lymphocyte subsets of peripheral blood were measured in patients undergoing thoracovascular operations without CPB (Group 2). In Group 1, analysis of each subset-to-total lymphocyte ratio revealed that observed lymphocytopenia in the early postoperative days was mainly the result of T cell reduction, and that the decrease of helper/inducer T cells contributed to this decrease. In contrast, no significant fluctuation of any lymphocyte subpopulation ratio was observed in Group 2. Analysis of lymphocyte subpopulation ratios in lymphoid organs showed that reciprocal changes of T cells and their subsets were observed in the bone marrow, thus indicating that the redistribution of T cells (especially of helper/inducer cells) seems to occur between peripheral blood and bone marrow in Group 1. Furthermore, there was no relationship between serum cortisol levels and the changes in lymphocyte subset ratios in Group 1 patients.

IL-2 production by B cells stimulated with a specific antigen☆

The ability of a specific antigen (Ag) to stimulate B cells to produce IL-2 was examined with a murine B lymphoma line, A20-HL, which expressed surface IgM specific for trinitrophenyl (TNP). The culture supernatant of A20-HL cells stimulated with TNP3.9-ovalbumin (-OVA) or anti-IgM goat IgG contained an activity which supported the proliferation of an IL-2-dependent T cell line, CTLL-2. Neither TNP3.9-OVA nor anti-IgM antibody stimulated the parent line, A20.2J, which did not bear TNP-specific sIg, whereas anti-mouse Ig rabbit IgG F(ab)2 did stimulate both A20-HL cells and A20.2J cells. The active material in the culture supernatant was identified as IL-2 based on the experiments in which the activity was inhibited by anti-IL-2 mAb, and IL-2 mRNA was expressed in A20-HL cells stimulated with TNP3.9-OVA or anti-IgM antibody. These results support the conclusion that a specific Ag can stimulate A20-HL cells to produce IL-2. For IL-2 production, TNP receptors on A20-HL cells have to be appropriately cross-linked, inasmuch as either TNP3.9-OVA or TNP6.7-OVA was much more effective than TNP1.2-OVA and TNP22.9-OVA in the induction of IL-2 production by A20-HL cells.

B cells as antigen-presenting cells: Antibody production in vitro against a T-dependent antigen

It was examined whether B cells can serve as antigen-presenting cells (APC) in the antibody response to a T-dependent antigen, trinitrophenyl-ovalbumin (TNP-OVA). B cells purified from mice primed with TNP (TNP-B cells) responded to TNP-OVA in the presence of purified T cells sensitized with OVA (OVA-T cells). OVA-T cells required the addition of APC to proliferate in response to TNP-OVA. APC activity of TNP-B cells in the T-cell proliferation was abolished by 4000 R irradiation. Our experiments also revealed that an antibody response requires more adherent cells than the T-cell proliferation. These results indicate that adherent cells possibly accompanying the T- and B-cell preparations were at a less than functional level. There was genetic restriction between T and B cells for the antibody response. B cells in the pellet fraction of 70% Percoll density sedimentation behaved similarly to the unfractionated TNP-B cells in the antibody response. A T-cell clone specific for human gamma-globulin (HGG) also induced an anti-TNP antibody response in B cells from unprimed mice in the presence of TNP-HGG. These results suggest that B cells are able to elicit an antibody response to a T-dependent antigen in the presence of carrier-primed T cells without the participation of macrophages.

Role of Newly Synthesized MHC Class II Molecules in Antigen-Specific Antigen Presentation by B cells

Using a B lymphoma, A20-HL, bearing IgM receptors for TNP, we have shown that presentation of an Ag taken up through the receptors is highly sensitive, whereas that of an Ag taken up nonspecifically is resistant to inhibition of protein synthesis or protein transport from the endoplasmic reticulum. To analyze the difference, we have examined the effect of protein synthesis inhibition on A20-HL cells in terms of internalization and fragmentation of a specific Ag, TNP-OVA, and distribution of MHC class II molecules. Inhibition of protein synthesis in A20-HL cells with emetine, an irreversible protein synthesis inhibitor, did not decrease the surface expression of anti-TNP receptors, or the kinetics of internalization of 125I-TNP-OVA. To detect fragmentation of TNP-OVA, A20-HL cells were incubated at 37 degrees C in the presence of 125I-TNP-OVA, and the cell lysate was analyzed in SDS-PAGE. The number of detectable fragments increased with the incubation period, and inhibition of protein synthesis did not change the electrophoretic pattern. Expression of MHC class II molecules on the surface of A20-HL cells was not affected by inhibition of protein synthesis. However, intracellular MHC class II molecules markedly decreased in amount in the emetine-treated cells. Thus, presentation of an Ag taken up through Ag receptors seems to be dependent on intracellular MHC class II molecules, whereas that of an Ag taken up nonspecifically does not, suggesting that the Ag-processing pathway in B cells for a specific Ag is different from that for a nonspecific one, at least partly.

Accessory cell function in a Con A response: Role of Ia and interleukin 1☆

Accessory cell (A-cell) function in a Con A response was analyzed. Irradiated P388D1 cells efficiently induced a proliferative response to Con A of T cells purified from spleen cells, whereas paraformaldehyde-fixed P388D1 cells failed to serve as A cells. Although IL-1 containing culture supernatant (SN) of a macrophage hybridoma induced the Con A response of the T-cell preparations, the depletion of Ia+ cells by the treatment with anti-Ia antibody and complement abrogated the response in the presence of IL-1. Fixed P388D1 cells and the hybridoma SN synergized in the reconstitution of the response. A 15,000-Da fraction of the hybridoma SN or human recombinant IL-1 alpha was able to substitute the hybridoma SN for the response. The reconstitution of the response by IL-1 and fixed P388D1 cells was inhibited by the addition of monoclonal anti-Ia antibody. These results indicate that IL-1 or fixed P388D1 cell does not exert a sufficient signal by itself and both of them are required for the reconstitution of a Con A response of highly purified T cells, and that Ia on fixed P388D1 cells play an important role.

Responses of spleen cells from mice with X-linked B-cell defect to polyclonal B-cell activators, purified protein derivative of tuberculin, and dextran sulfate

Abstract Polyclonal responses to LPS, PPD, and DxS of spleen cells from mice expressing a X-linked B-cell defect were examined. Spleen cells from young (CBA/N × BALB/c)F1 male mice responded slightly lower to LPS, significantly lower to PPD than the cells from age-matched F1 female mice, and showed no response to DxS stimulation. This hypo- or unresponsiveness of F1 male cells to PPD or DxS could not be explained by a shift in the dose-response or time kinetics of the responding cells, and also could not be due to the defect in the function of T cells or macrophages. Suppressor T cells to polyclonal response to PPD or DxS could not be shown in F1 male spleen cells. The response of F1 male cells to PPD was dramatically improved with age but not to DxS. These results suggest that B cells responsive to DxS may belong to a distinct subpopulation from the cells responsive to LPS or PPD.

B cells as antigen-presenting cells: Antigen-specific IL-2 production by cloned T cells without expression of IL-2 receptors

A murine T cell clone, 24-2C, responds specifically to human IgG (HGG) in the context of I-Ab. B cells purified from mouse spleen cells were examined for their function as antigen-presenting cells (APC) in the response of 24-2C cells to HGG. B cells functioned as APC for IL-2 production but not for proliferation, whereas spleen cells or spleen-adherent cells functioned as APC for both IL-2 production and proliferation. LPS-activated B cells also failed to induce the proliferative response. The addition of the culture supernatant of 24-2C cells stimulated with HGG presented by irradiated spleen cells to the culture of 24-2C cells, irradiated B cells, and HGG induced the proliferative response of 24-2C cells, whereas IL-1, IL-3, and/or interferon-gamma did not reconstitute the proliferation. The expression of IL-2 receptors (IL-2R) on 24-2C cells was examined using a monoclonal anti-mouse IL-2R antibody AMT 13 or 7D4. 24-2C cells cultured with spleen cells as APC expressed IL-2R. Those cultured alone or with B cells as APC did not express IL-2R. Enlargement of 24-2C cells in response to HGG was also examined, and the relative cell size of those cultured with B cells or spleen cells as APC was larger than that of those cultured alone. These results demonstrate that B cells as APC induce IL-2 production and cell size enlargement in the response of 24-2C cloned T cells to HGG, but not IL-2R expression nor proliferation.

Antibody response of murine spleen cells with or without receptors for c3 to antigenic or mitogenic stimulation.

Abstract Bone marrow-derived lymphocytes (B cells) with or without receptors for third component of complement (CR) were studies in their responsiveness to T-independent antigens and B-cell mitogens in terms of anti-DNP PFC response. Spleen cells were fractionated by centrifugation over a Ficoll-Hypaque density gradient after they were rosetted with antigen-antibody-complement complexes. The cells in interface fraction could not respond to any T-independent antigen tested, while they responded well to polyclonal stimulations by lipopolysaccharide and polymerized flagellin but not by keyhole limpet hemocyanin. Reduced response to T-independent antigen of the cells in interface fraction could not be explained by a shift of the kinetics, lack of number of B cells, or by the depletion of macrophages. Significance of CR in B-cell differentiation is discussed.

Fc receptors on IgM and IgG antibody-forming cells in an early immune response

Abstract Antibody-forming cells were studied to determine whether they had receptors for the Fc portion of IgG(FcR). Spleen cells from mice immunized with horse red blood cells(HRBC) were assayed for anti-HRBC plaque-forming cells (PFC) after they were depleted by Ficoll-Hypaque density sedimentation of cells forming rosettes with sheep red blood cells(SRBC) coated with IgG anti-SRBC. Direct PFC were shown to bear FcR but the receptor was not on indirect PFC. FcR on direct PFC were indicated as being occupied by antigen-antibody complexes at a certain stage of the immune response. Dose of antigen and immunization route were of crucial importance for the occupation of FcR on PFC.

B cells as accessory cells in a Con A response of a T cell clone

Accessory cell (AC) function of B cells was examined in Con A response of a cloned T cell line, 22-9D, which is Thy 1+,L3T4+,Lyt2-,H-2KbDb+ and I-Ab-.22-9D cells produced IL 2 in the presence of Con A without participation of AC. For the initiation of a proliferative response to Con A, the addition of spleen cells or spleen adherent cells was required. B cells as AC were unable to induce the proliferative response. In the presence of culture supernatant of spleen cells stimulated with Con A (CAS), 22-9D cells showed proliferative response to Con A with B cell AC. The response was inhibited by a relevant monoclonal anti-I-A antibody. Although irradiated spleen cells as AC induced IL 2 receptor expression of 22-9D cells in the presence of Con A, B cells were shown to require the addition of unknown factor(s) in CAS, which was suggested to be different from IL 1, IL 2, IL 3, or IFN-gamma, for the induction of the receptor expression on 22-9D cells.