Non-responsiveness to hepatitis B surface antigen vaccines is not caused by defective antigen presentation or a lack of B7 co-stimulation

Abstract

The mechanisms causing non-responsiveness to hepatitis B surface antigen (HBsAg) vaccines in man remain elusive. The increased incidence of non-responsiveness in subjects with HLA-DR3(+) or -DR7(+) haplotypes suggests that immune response mechanisms governed by genes of the MHC are involved. Homozygotes for these two haplotypes are found almost exclusively in the non-responder (NR) population. It is conceivable that antigen-presenting cells (APC) of NR are defective in the uptake of HBsAg and that they are unable to present this Ag adequately. Previously, we demonstrated that DR2(+), DR7(+) and DP4(+) NR were able to present HBsAg. In the present paper we demonstrate that six DR0301(+) NR, five of which are homozygous for this marker, were able to take up, process and present HBsAg to HBsAg-specific, DR0301-restricted T cell lines. Non-fractionated peripheral blood mononuclear cells (PBMC) from the DR0301(+) NR did not proliferate to HBsAg in vitro, whereas they proliferated vigorously upon stimulation with tetanus toxoid, thus ruling out the presence of a generalized immunodeficiency. We therefore conclude that HLA-DR0301(+) NR vaccinees are not deficient in their HBsAg-presentation. Because it was demonstrated that recently activated T cells can apparently bypass the requirement for B7, we may have overlooked the role of the B7-co-stimulation in our set-up that used HBsAg-specific T cell lines. Therefore we examined the expression of B7 co-stimulatory molecules on NR-APC. CD86 was normally present on these cells and was not down-regulated after culturing the PBMC in the presence of HBsAg. We conclude that CD86 expression on CD14(+) monocytes of DR0301- and DR07-homozygous poor responders is not deficient and cannot be the mechanism underlying the non-responsiveness of these subjects.

Fig. 1

APC dose titration. Increasing numbers of PBMC from two non-responders (NR60 and NR34) or a high responder (R18) were added to HBsAg-specific T cells (2 × 10⁴) of cell line HBL-50-DO and HBsAg (3 µg /ml). Lymphoproliferation was measured by [³H]-TdR incorporation on day 4. Data show the proliferative response (Δ cpm) of HBL-50-DO in the presence of increasing PBMC numbers of NR60, NR34 or R18. The number of APC required to reach 50% of the maximal prolifertive response can be determined and is shown.

Fig. 2

HBsAg dose titration. Increasing doses of HBsAg were added to PBMC from two non-responders (NR60 and NR34) or a high responder (R18). HBL-50-DO T cells were cultured with HBsAg and allogeneic PBMC (10⁵) as described in Fig. 1. Data show the proliferative response (Δ cpm) of HBL-50-DO in the presence of increasing HBsAg doses. The dose of HBsAg required to reach 50% of the maximal proliferative response can be determined and is shown.

Fig. 3

Dose-dependent inhibition of antigen-specific T cell proliferation by anti-CD86, anti-CD80 MoAbs and a combination of both. Non-fractionated PBMC (4 × 10⁵ cells/well) from R17 were cultured with HBsAg (3 µg/ml) in the presence of variable amounts of anti-CD86 + anti-CD80 (▪), anti-CD80 alone (•) or anti-CD86 alone (□). Proliferation was measured after 6 days of incubation. Mean [³H]-thymidine incorporation in control culture with HBsAg alone was 8181 ± 472 cpm and in medium control was 311 ± 25 cpm. Results are expressed as % inhibition of the proliferative response.

Fig. 4

Expression of CD80 and CD86 on CD14⁺ cells from good and poor responders to the hepatitis B vaccine. PBMC from good (R17, R21, upper panels) and poor responders (NR1, NR46, lower panels) to the HB vaccine were cultured for 96 h in the presence of HBsAg (▪), TT (▵) or medium alone (×). At the initiation of the culture (0 h) and different moments thereafter (24, 48, 72 and 96 h; x-axis) the expression of CD80 and CD86 on the cultured cells was measured and expressed as % of total CD14⁺ cells (y-axis). In the time-curves, the expression of CD80 and CD86 was measured after incubation with 3 µg HBsAg/ml or 5 µg TT/ml. The x-axis of the adjacent figures represents a titration of HBsAg. In these figures, the dose-dependency of the CD80 expression is shown at 72 h post-incubation, and the expression of CD86 is shown at 48 h post-incubation.