Interaction of measles virus glycoproteins with the surface of uninfected peripheral blood lymphocytes induces immunosuppression in vitro

Abstract

A marked suppression of immune function has long been recognized as a major cause of the high morbidity and mortality rate associated with acute measles. As a hallmark of measles virus (MV)-induced immunosuppression, peripheral blood lymphocytes (PBLs) isolated from patients exhibit a significantly reduced capacity to proliferate in response to mitogens, allogens, or recall antigens. In an in vitro system we show that proliferation of naive PBLs [responder cells (RCs)] in response to a variety of stimuli was significantly impaired after cocultivation with MV-infected, UV-irradiated autologous PBLs [presenter cells (PCs]. We further observed that a 50% reduction in proliferation of RCs could still be observed when the ratio of PC to RC was 1:100. The effect was completely abolished after physical separation of the two populations, which suggests that soluble factors were not involved. Proliferative inhibition of the RCs was observed after short cocultivation with MV-infected cells, which indicates that surface contact between one or more viral proteins and the RC population was required. We identified that the complex of both MV glycoproteins, F and H, is critically involved in triggering MV-induced suppression of mitogen-dependent proliferation, since the effect was not observed (i) using a recombinant MV in which F and H were replaced with vesicular stomatitis virus G or (ii) when either of these proteins was expressed alone. Coexpression of F and H, however, lead to a significant proliferative inhibition in the RC population. Our data indicate that a small number of MV-infected PBLs can induce a general nonresponsiveness in uninfected PBLs by surface contact, which may, in turn, account for the general suppression of immune responses observed in patients with acute measles.

Figure 1

Induction of proliferative inhibition of PBLs after MV infection or cocultivation with MV-infected PBLs. (A) PHA-stimulated human PBLs were infected with MV-ED (MOI of 1) for 48 hr and stained for the expression of MV-H protein (MV-PBLs). The negative control was performed using a coronavirus-specific antibody. (B) MV-PBLs or mock-infected PBLs, respectively, were UV-irradiated and added in decreasing proportions as indicated in the axis legend (PCs) together with PHA (2.5 μg/ml) to naive autologous PBLs (RCs). Proliferative activity of the RCs was determined as described after a 72-hr incubation period followed by a further 16-hr labeling time. Each value represents the result of three independent experiments, and each was performed with triplicate assays. Open circles indicate the values obtained with MV-ED-infected PCs, solid circles indicate those with MV-WTF-infected PCs. (C) PBLs were infected with MV-ED at the MOIs indicated in the axis legend, stimulated with PHA (2.5 μg/ml), and their proliferative activity was determined after 72 hr by a labeling with [³H]thymidine for 16 hr. Proliferative inhibition is indicated in relation to the proliferation of uninfected, PHA-stimulated PBLs.

Figure 2

Inactivated MV, but not VSV, induces inhibition of mitogen-dependent proliferation of naive PBLs. RCs (naive PBLs) were incubated (in the presence of PHA) with amounts of MV-ED stock virus corresponding to MOIs of 5 (lane 1), 3 (lane 2), 2 (lane 3), 1 (lane 4), and 0.1 (lane 5) or VSV corresponding to an MOI of 3 (lane 6), which were previously inactivated by UV-irradiation (5 J/cm²). Proliferative inhibition was determined in relation to PHA-stimulated untreated controls after 72 hr and a 16-hr labeling period.

Figure 3

Physical separation of RCs from PCs abolishes the induction of proliferative inhibition. RCs were seeded into clusters of a 96-well plate and separated by filters with a pore size of 0.2 μm from a mixture of PCs/RCs (1:1, lane 1; 1:10, lane 3; 1:100, lane 5) in the presence of PHA. Filters were removed from the wells after 72 hr and the proliferative activity of the seeded RCs was determined after a further 16 hr of labeling. Control values indicate the results obtained in the standard proliferation assay using PC/RC ratios of 1:1 (lane 2), 1:10 (lane 4), and 1:100 (lane 6) in the absence of the filter membranes.

Figure 4

Short contact with PCs leads to proliferative inhibition of RCs. Primary macrophages were separated by adhesion from PBMCs, infected with MV (MOI 3 for 2 days until they were 80% positive for MV antigens) (MV-M) and UV-irradiated. For each value, 1 × 10⁶ MV-M were mixed with 2 × 10⁶ RCs in the presence of PHA and the PCs were separated by anti-CD14 magnetic cell sorting selection after 10 min (lane 1), 30 min (lane 2), or 60 min (lane 3) from the RCs. RCs were further cultivated in the absence of PCs for 72 hr and subsequently labeled for 16 hr.

Figure 5

The MV glycoproteins are required for induction on nonresponsiveness of PBLs. PCs were infected with either MV-ED (lanes 2, 4, and 6) or MGV (schematically shown in B) (lanes 1, 3, and 5), UV-irradiated, and cocultivated at a PC/RC ratio of 1:1 (lanes 1 and 2), 1:10 (lanes 3 and 4), or 1:100 (lanes 5 and 6) for a total of 96 hr (72 hr plus 16 hr labeling), and proliferative inhibition was determined in relation to the control culture with mock-infected PCs. Infection levels in both PC populations were confirmed prior to the cocultivation by FACScan using MV-N- and MV-H-specific antibodies (for MV-ED) and MV-N and VSV-G-specific antibodies (for MGV).

Figure 6

Both MV-F and MV-H proteins are required for the induction of nonresponsiveness of PBLs. (A) The expression levels of MV-F in 293-F cells, of MV-H in 293-H cells (B), and MV-F and MV-H in 293 cells productively infected with MV (C) were determined using specific antibodies. The negative controls were performed using a coronavirus-specific antibody. (D) Standard proliferation assays were performed using PBLs as RCs and 293-T7 (lane 1), 293-T7 supertransfected with pCG-H (lane 2), 293-F (lane 3), 293-F supertransfected with pCG-H (lane 4), 293-F supertransfected with pCG-F (lane 5), 293-H (lane 6), 293-H supertransfected with pCG-H (lane 7), 293-H cells supertransfected with pCG-F (lane 8), a 1:1 mixture of 293-F and 293-H (lane 9), or 293-MV infected (lane 10) cells, each at a PC/RC ratio of 1:2. Each value represents three independent experiments each performed as triplicate assay.