CD40-associated TRAF 6 signaling is required for disease induction in a retrovirus-induced murine immunodeficiency

Abstract

LP-BM5 retrovirus-infected C57BL/6 mice develop splenomegaly, lymphadenopathy, hypergammaglobulinemia, and immunodeficiency; thus, this disease has been named mouse AIDS. In this syndrome, CD154/CD40 interactions are required for but do not mediate disease by upregulation of CD80 or CD86. We report here that there is nonetheless a necessity for CD40 signaling competence, specifically an intact tumor necrosis factor receptor-associated factor 6 (TRAF 6) binding site.

FIG. 1.

wt chimeric hu/mouse CD40 transgenic mice (TRAF 2,3/5⁺, 6⁺) develop MAIDS-associated disease after LP-BM5 infection (3 × 10⁴ PFU of B ecotropic helper virus). In contrast, infected transgenic mice with CD40 cytoplasmic tail mutations that inhibit TRAF protein binding to the TRAF 2,3/5, 6 sites (TRAF 2,3/5⁻, 6⁻) do not develop MAIDS. B- or T-cell mitogen responsiveness was determined, respectively, by a 72-h in vitro stimulation with either 10 μg of LPS/ml or 4 μg of ConA/ml, followed by a terminal 6-h pulse with [³H]thymidine. These transgenic mice were all on the CD40 k.o. background. LP-BM5-defective viral load (bottom panel) for spleen tissue was determined by quantitative real-time reverse transcriptase PCR, as members of our laboratory previously reported (9). Briefly, total RNA was isolated and was DNase I treated. One microgram of DNA-free RNA was reverse transcribed to cDNA by using random hexamer priming. Reverse transcriptase PCR was performed with BM5-defective gag (9, 12), ecotropic gag (9), and β-actin (9) primers by amplifying resulting cDNA in the presence of SYBR Green stain on an iCycler iQ instrument (Bio-Rad) by using previously reported temperatures, cycle numbers, and normalization to β-actin expression (9). By this assay, levels of the BM5-defective and BM5 ecotropic viruses were not detectable (ND) in uninfected mice. This experiment is representative of two additional experiments for the TRAF 2,3/5⁻, 6⁻ transgenic mice, including one in which there was no significant loss of ConA response for the infected and uninfected mice of this group and one additional experiment for the wt transgenic mice. Mean values and standard deviations were derived from four mice per group. *, P < 0.05 by the Student t test, compared to uninfected control mice of the same strain. NS, not significant; rel. exp., relative expression.

FIG. 2.

LP-BM5-infected (+) CD40 transgenic mice with mutated TRAF 2,3/5 and intact TRAF 6 binding sites (TRAF 2,3/5⁻, 6⁺) are MAIDS susceptible, whereas TRAF 2,3/5⁺, 6⁻ transgenic mice are relatively insusceptible as assessed by readouts for spleen size and B- and T-cell immunodeficiency. Allo-CTL responses were generated after stimulation with major histocompatibility complex-mismatched irradiated stimulator cells, followed by a standard ⁵¹Cr release assay (13, 14). Mitogen responses and viral load assessment were performed as indicated in the legend to Fig. 1. Mean values and standard deviations represent a composite of two experiments of four to seven mice per group. *, P < 0.05 by the Student t test compared to uninfected control mice of the same strain. E:T, effector to target cell.

FIG. 3.

MAIDS-associated spleen cell phenotypic changes are clearly evident in LP-BM5-infected CD40 transgenic TRAF 2,3/5⁻, 6⁺ and B6 wt mice. Increased numbers of spleen cells that are CD19⁺ CD23⁻ and CD4⁺ Thy1.2⁻ have been reported as a consequence of MAIDS pathogenesis (25). Histograms are for spleen cell populations from uninfected (−) or LP-BM5-infected (+) CD40 transgenic and B6 control mice. Cells were doubly stained with either the appropriate isotype controls or with either fluorescein isothiocyanate (x axis)- or phycoerythrin (y axis)-labeled αCD23 and αCD19 or αThy1.2 and αCD4 monoclonal antibodies (BD Pharmingen, San Diego, Calif.). The percentages of double-positive cells are found in the upper right quadrant and those of single-positive cells are in the upper left. Cells were analyzed on a FACScan, and histograms are for one representative mouse per group. See Table 1 for the entire data set.