Costimulatory pathways in lymphocyte proliferation induced by the simian immunodeficiency virus SIVsmmPBj14

Abstract

The PBj14 isolate of the simian immunodeficiency virus SIVsmmPBj14 is unique among primate lentiviruses in its ability to induce lymphocyte proliferation and acutely lethal disease. The studies reported here show that viral induction of T-cell proliferation requires accessory cells, such as primary monocytes or Raji B-lymphoma cells, as well as the presence of a putative immunoreceptor tyrosine-based activation motif within the viral Nef protein. Addition of CTLA4-immunoglobulin fusion protein or anti-B7 antibodies to virally infected T cells led to substantial, but not complete, inhibition of monocyte-costimulated T-cell proliferation-suggesting that both CD28/B7-dependent and non-CD28-dependent pathways may contribute to the costimulation of virally induced lymphoproliferation. Finally, cyclosporin A, a specific inhibitor of the calcium-calmodulin-regulated phosphatase activity of calcineurin, which influences activation of the transcription factor nuclear factor of activated T cells, was shown to block virally mediated T-cell proliferation. Taken together, these findings suggest that the effect of SIVsmmPBj14 on T-cell activation may be functionally analogous, at least in part, to the effect of engagement of the T-cell receptor.

FIG. 1

T cells were infected with PBj6.6 virus in the presence or absence of the indicated accessory cells. To prevent their proliferation, accessory cells were X- irradiated with 10,000 rads prior to plating. A total of 2.5 × 10⁴ Raji cells or 5 × 10⁴ autologous macrophages (cultured for 1 week prior to the experiment) were plated per well. The incorporation of [³H]thymidine by accessory cells alone was at background levels (not shown). Results shown are the mean values of three independent observations and are representative of three separate experiments yielding similar conclusions; error bars indicate the standard errors of mean values.

FIG. 2

T cells were plated with irradiated Raji cells in the presence or absence of 20 μg/ml of either CTLA4-Ig, anti-CD40, or both. Cells were mock-infected (+ Raji alone), stimulated with 10 ng of PMA per ml, infected with PBj6.6 virus, or infected with an ITAM-deleted mutant of PBj6.6 virus (PBj6.6YERQ; this virus is isogenic to PBj6.6, except for the substitution of RQ, the corresponding residues in SIVmac239, for YE at amino acids 17 and 18 of Nef [9, 30]). Results shown are the mean values of three independent observations; error bars indicate the standard errors of mean values. Data shown are representative of three separate experiments yielding similar conclusions (note that the experiment using the PBj6.6YERQ virus was performed only once, since this virus consistently fails to trigger lymphoproliferation in PBMC [30]).

FIG. 3

T cells were plated with 5 × 10⁴ homologous macrophages (Mø) per well, in the presence of CTLA4-Ig (5 μg/ml) or of the indicated antibodies (5 μg/ml). Antibodies used in this experiment were as follows: anti-human B7-1 (clone 37711.11), anti-human B7-2 (clone 37301.11), and an isotype-matched normal mouse IgG1 (control IgG1); all antibodies were obtained from R&D Systems. Cells were mock-infected (+Mø alone), stimulated with 10 ng of PMA per ml, or infected with PBj6.6 virus. Results shown are the mean values of three independent observations and are representative of three separate experiments yielding similar conclusions; error bars indicate the standard errors of mean values.

FIG. 4

(A) PBMCs were infected with PBj6.6 virus, or mock-infected, and incubated in the presence of the indicated concentrations of cyclosporin A. (B) PBMCs were infected with PBj6.6 virus, or mock-infected, and CsA (0.1 μg/ml) was then added to the cultures at the indicated times after virus infection (days); proliferation was measured 7 days after initial exposure of the cells to PBj6.6 virus. Values shown represent the means from a single experiment that was performed in triplicate; the standard errors of mean values are marked by bars. The results shown are representative of two experiments that yielded similar results; in addition, the experiment whose results are shown in Panel A was repeated on three occasions, with similar results, with a different molecular clone of SIVsmmPBj14—PBj 1.9 (7, 12).