Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help

Abstract

In general, humoral immune responses depend critically upon T cell help. In transplantation, prevention or treatment of humoral rejection therefore require drugs that ideally inhibit both B cell and T helper cell activity. Here, we studied the effects of commonly used immunosuppressive drugs [tacrolimus, cyclosporin, mycophenolic acid (MPA) and rapamycin] on T cell helper activity and on T cell-dependent B cell responses. T cells were activated polyclonally in the presence of immunosuppressive drugs in order to analyse the effect of these drugs on T cell proliferation, co-stimulatory ligand expression and cytokines. The impact of immunosuppressive drugs on T cell-dependent immunoglobulin production by B cells was addressed in T-B cell co-cultures. All drugs affected T cell proliferation and attenuated T cell co-stimulatory ligand (CD154 and CD278) expression when T cells were activated polyclonally. Tacrolimus, cyclosporin and rapamycin also attenuated B cell stimulatory cytokine mRNA levels in T cells. As a consequence, a decrease in immunoglobulin levels was observed in autologous T-B cell co-cultures, where T cell help is essential for immunoglobulin production. In contrast, when pre-activated T cells were used to stimulate autologous B cells, calcineurin inhibitors failed to inhibit B cell immunoglobulin production, whereas MPA and rapamycin did show inhibition. From these studies, it is evident that calcineurin inhibitors affect the humoral immune response by interfering with T helper signals, but not by targeting B cells directly. Furthermore, our studies support the necessity of intervening in T cell helper function to attenuate humoral responses.

Fig. 1

Immunosuppressive drugs (ISD) did not alter T cell activation but did inhibit T cell proliferation. T cell activation with anti-CD3 monoclonal antibody (mAb)/anti-CD28 mAb for 24 h resulted in up-regulation of CD25 and CD69 (a). None of the ISD affected CD25 levels, whereas cyclosporin inhibited the expression of CD69. Drug concentrations were 1·0 ng/ml tacrolimus, 100 ng/ml cyclosporin, 100 ng/ml mycophenolic acid (MPA) and 8·0 ng/ml rapamycin. Data are expressed as percentage of cells expressing CD25 or CD69 compared to medium controls (dotted lines), n = 4 (b). Carboxyfluorescein succinimidyl ester (CFSE)-labelled T cells were activated with anti-CD3 mAb/anti-CD28 mAb for 3 days in the presence of ISD. Data from a representative experiment are shown. Similar results were obtained in three independent experiments (c). Percentage inhibition of the T cell proliferation index by ISD compared to medium controls (dotted lines) are depicted, n = 3 (d). Drug concentrations were: 0·3 ng/ml tacrolimus, 50 ng/ml cyclosporin, 100 ng/ml MPA and 4 ng/ml rapamycin. **P < 0·01.

Fig. 2

Effect of immunosuppressive drugs (ISD) on T cell stimulatory signals. T cell stimulation with anti-CD3 monoclonal antibody (mAb)/anti-CD28 mAb for 24 h resulted in up-regulation of CD154 and CD278 (a). All ISD inhibited the expression of CD154 and CD278. Drug concentrations were 1·0 ng/ml tacrolimus, 100 ng/ml cyclosporin, 100 ng/ml mycophenolic acid (MPA) and 8·0 ng/ml rapamycin. Data are expressed as percentage of cells expressing CD154 or CD278 as compared to medium controls (dotted lines), n = 4 (b). ISD inhibit the mRNA levels of B cell stimulatory cytokines produced by T cells to various extents. Data are expressed as percentage of cytokine mRNA inhibition compared to mediums controls, n = 3. Drug concentrations are identical to those of Fig. 2b (c). *P < 0·05; **P < 0·01; ***P < 0·001.

Fig. 3

T cells were necessary for B cell activation in a cell–cell contact-dependent fashion. B cells were cultured in anti-CD3 monoclonal antibody (mAb)-coated wells with soluble anti-CD28 mAb in the absence or presence of autologous T cells. After 9 days supernatants were harvested for immunoglobulin assessment (a). Co-cultures of B cells and prestimulated T cells were performed in either regular or transwell plates (B cells in lower compartment). Supernatants were harvested at day 6 for detection of immunoglobulins (b). **P < 0·01; ****P < 0·0001.

Fig. 4

All immunosuppressive drugs (ISD) were capable of inhibiting immunoglobulin production when B cells are cultured with non-pre-activated T cells, but calcineurin inhibitors failed to inhibit immunoglobulins levels when pre-activated T cells were used to stimulate B cells. B cells were cultured with fresh, autologous T cells and anti-CD3 monoclonal antibody (mAb)/anti-CD28 mAb with cytosine-guanine dinucleotide (CpG) in the presence of graded concentrations of ISD for 9 days, whereupon supernatants were tested for immunoglobulin M (IgM) (a) and IgG levels (b). B cells were cultured with ISD for 6 days with CpG in the presence of prestimulated T cells. Depicted are the percentages of IgM (c) and IgG (d) levels compared to medium controls (dotted lines). Horizontal bars indicate mean values (n = 5). *P < 0·05; **P < 0·01; ***P < 0·001; ****P < 0·0001.