The transmembrane form of TNF-alpha drives autoantibody production in the absence of CD154: studies using MRL/Mp-Fas(lpr) mice

Abstract

It is generally accepted that the interaction between CD40 and its ligand (CD154) plays a decisive role in contact-dependent help for T and B cells. In CD154-deficient MRL/Mp-Fas(lpr) (MRL/lpr) mice, however, high titres of IgG2a-type autoantibodies against small nuclear ribonucleoproteins (snRNPs) are observed. We successfully isolated two CD154-deficient MRL/lpr Th1 lines, which could provide B cell help for anti-snRNP antibody production. The proliferative responses of the Th1 cell lines were MHC class II (I-Ek)-restricted. Although syngeneic B cell proliferation was induced by Th1 lines in both a contact-dependent and -independent manner, the soluble form of TNF-alpha (sTNF-alpha) was not involved in contact-independent B cell proliferation. On the other hand, both anti-TNF-alpha and TNF-receptor 2 (TNF-R2, p75) monoclonal antibody (MoAb) blocked contact-dependent B cell proliferation, suggesting that the transmembrane form of TNF-alpha (mTNF-alpha)-TNF-R2 co-stimulation participates in B cell activation. Similarly, anti-TNF-alpha and TNF-R2 MoAb inhibited anti-snRNP antibody production in vitro, but anti-CD154 or TNF-R1 MoAb did not. These results indicate that the interaction of mTNF-alpha on activated Th1 cells with TNF-R2 on B cells may be involved in the autoimmunity seen in MRL mice, and that the blockade of CD40-CD154 co-stimulation may not always be able to suppress some Th1-related manifestations of lupus.

Fig. 1

CD154 and the transmembrane form of TNF-α (mTNF-α) on activated MRL/lpr T cell lines. CD154-intact autoreactive Th1 (5-1), and Th2 (4-1) lines and CD154-deficient Th1 (P5) were stimulated with plate-bound anti-CD3ɛ MoAb (5 µg/ml) for the indicated time. CD154 was not expressed on P5 during 0–48 h stimulation with anti-CD3ɛ MoAb. Bold lines show staining with anti-CD154 or TNF-α MoAb, and regular lines show isotype control.

Fig. 2

B cell proliferative responses to autoreactive Th1 lines. (a) Anti-CD154 MoAb (MR1) dramatically inhibited B cell proliferation induced by the CD154-intact line (5-1), but not that by the CD154-deficient line (G1 or P5). (b) A membrane insert (\_/) was used to prevent contact by T cell line (P5) with B cells. Irradiated (1500 rad) T cells, which do not proliferate themselves, enhanced purified B cell proliferation. Irradiated (3000 rad) B cells are thought to promote cytokine production from the T cell line as an APC. CD154-deficient lines induce B cell proliferation both in a contact-dependent and -independent manner; however, contact-independent B cell proliferation (black bar) was not inhibited at all by anti-TNF-α MoAb, but was inhibited by anti-IFN-γ or IL-2 MoAb (hatched bar). Concentration of MoAb or isotype control: 25 µg/ml. This figure shows the representative results of three experiments. iT; irradiated T cells (P5), pB; purified B cells, iB; irradiated B cells. **P <0·01 (versus isotype control). (a) □, 5-1 (with hamster IgG); •, G1; ▵, P5; ▪, 5-1; *B cells alone.

Fig. 3

Inhibition by anti-TNF-α/TNF-R MoAb of B cell proliferative responses to CD154-intact (5-1) or -deficient (G1 and P5) Th1 cell lines. Anti-TNF-α MoAb and anti-TNF-R2 MoAb partially inhibit B cell proliferative responses, and are therefore thought to be mediated via contact-dependent T cell help. Anti-TNF-R1 MoAb also seems to block proliferation by G1 and P5, although a high concentration (>25 µg/ml) was required. Rat IgG1 and hamster IgG constitute isotype controls of anti-TNF-α and anti-TNF-R1 or R2 MoAb, respectively. % Inhibition = (1-{[³H]-thymidine uptake with blocking MoAb (cpm)/[³H]-thymidine uptake with isotype control(cpm)}) × 100. Mean cpm of triplicate cultures was used for calculations; s.d. for each experiment was less than 10%. ▪, 5-1; •, G1; ▴, P5.

Fig. 4

In vitro B cell helper assay. (a) A membrane insert was used to prevent contact by the T cell line with B cells (white bar). In the absence of the membrane, G1 and P5 could induce more than the standard level of SI. Stimulation index = (concentration in culture supernatant of experimental wells)/(concentration in culture supernatant of B cells alone). The figure shows representative results of three experiments. (b) Comparison of levels of anti-snRNP antibody in the supernatant of T cell line (P5)–B cell co-culture with the blocking antibody and isotype control determined by ELISA. Anti-TNF-α, IFN-γ, and TNF-R2 MoAb inhibited anti-snRNP antibody production, while anti-TNF-R1 MoAb did not. % Inhibition = (1-{titre of anti-snRNP antibody with blocking MoAb (O.D._405nm)/anti-snRNP antibody with isotype control (O.D._405nm)}) × 100. Concentrations of blocking antibody and isotype control: 25 µg/ml. This figure shows the representative results of three experiments. □, With membrane insert; ▪, without membrane insert.