CD19 Signaling Pathways Play a Major Role for Murine AIDS Induction and Progression

Mice

C57BL/6 (B6) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6.CD19^−/− (CD19^−/−) mice were originally generated by R. Rickert (University of Cologne, Cologne, Germany) (14). B6.Vav-1^−/− (Vav^−/−) mice (27) were a gift from J. Rivera (National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD). B6.Cr2^−/− (hereafter referred to as CR2^−/−) mice (16) were obtained from M. Carroll (Harvard Medical School, Boston, MA). The murine Cr2 locus encodes both mCR1 and mCR2 (complement receptor 1 and 2); disruption of this locus renders mice deficient in both mCR1 and mCR2 (CD35 and CD21). B6.CD22^−/− mice (26) were generated by T. Tedder (Duke University Medical Center, Durham, NC). Mice were inoculated i.p. with 0.1 ml of LP-BM5 mixed virus pools at 6–10 wk of age.

Virus and virus assays

BM5eco and LP-BM5 virus stocks were prepared from the G6 clone of chronically infected SC-1 cells as described previously (6). LP-BM5 virus stocks contain a mixture of nonpathogenic ecotropic and MCF MuLV and a disease-causing defective genome BM5def. At selected time points after infection, mice were killed and bled; serum was stored at −20°C until use. Spleen weight, degree of lymphadenopathy, and histopathologic evaluations of selected tissues obtained at autopsy were used to stage the progression of MAIDS by criteria detailed previously (28, 29, 30). The frequencies of spleen cells expressing infectious ecotropic or MCF MuLV were determined by infectious center tests of mitomycin C-treated cells as described elsewhere (28, 31, 32).

Flow cytometry

All staining was done at 4°C. Cells were stained with FITC- or PE-conjugated Ab directed against Ig κ L chain, CD23, B220, FcγRII/III (CD16/CD32), and CD11b (all from BD PharMingen, San Diego, CA). Propidium iodide was added to allow gating on live cells. Flow cytometry was performed on a FACScan (BD Biosciences, San Jose, CA), and data were analyzed with CellQuest software (BD Biosciences).

Spleen cell response to mitogens

Spleen cells (1 × 10⁵) from control and experimental mice were plated in triplicate into 96-well plates with medium containing 10% FCS, l-glutamine, 2-ME, antibiotics, and a final concentration of 20 μg/ml LPS and 2 μg/ml Con A or of 20 ng/ml PMA and 250 ng/ml ionomycin. After 48 h, all wells were pulsed with 1 μCi [³H]thymidine (PerkinElmer, NEN, Boston, MA) and harvested 6 h later for assessment of thymidine incorporation by scintillation counting (Betaplate system; PerkinElmer Wallac, Boston, MA).

Measurement of serum Ig levels

Serum IgE levels were measured by ELISA using Ab and standards purchased from BD PharMingen according to their recommendations. Briefly, 96-well microtiter plates (Corning, Corning, NY) were coated with 2 μg/ml of the 02111D anti-IgE Ab overnight. After blocking the plates with BSA and incubation with the samples, biotinylated anti-mouse IgE Ab (02122D) was added, followed by incubation with streptavidin-HRP. Plates were developed with ABTS peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

For ELISA determinations of serum IgG1, IgG3, and IgM, affinity-purified goat anti-mouse IgG1, IgG3, or IgM Ab (Southern Biotechnology Associates, Birmingham, AL) were diluted to 5 μg/ml to coat 96-well microtiter plates overnight at 4°C. The plates were then blocked with BSA, and sera were plated and allowed to incubate for 2 h at 37°C. The plates were washed, and HRP-labeled goat anti-mouse IgG1, IgG3, or IgM (Southern Biotechnology Associates) was then added. After 2 h at 37°C, plates were developed with ABTS peroxidase substrate.

RNA purification, reverse transcription, Southern blotting, and real-time PCR

Mouse spleen samples were stored at −70°C in TRIzol Reagent (Invitrogen, Carlsbad, CA) until further processing. RNA was extracted according to the manufacturer’s directions. RNA (1 μg) was reverse transcribed using MuLV reverse transcriptase (Invitrogen) according to the recommendations of the manufacturer. The cDNA solution was diluted to 200 μl, and 10 μl were used for specific amplification by PCR. The primers and probe used for BM5def were 5′-CCTTTTCCTTTATCGACACT-3′ (sense), 5′-ACCAGGGGGGGAATACCTCG-3′ (antisense), and 5′-CTCTGCCAAAGGGACCAGTT-3′ (probe). The primers and probe for hypoxanthine phosphoribosyltransferase (HPRT) were 5′-GTTGGATACAGGCCAAGACTTTGT-3′ (sense), 5′-GATTCAAACTTGCGCTCATCTTAGGC-3′ (antisense), and 5′-GTTGTTGGATTGCCCTTGAC-3′ (probe). Amplifications were done for 22 cycles (BM5def) and 24 cycles (HPRT). The PCR products were separated on 1.5% agarose gels, blotted onto a nylon membrane (Hybond-N⁺; Amersham Pharmacia Biotech, Buckinghamshire, U.K.), and hybridized with the fluorescein-labeled probe using the Gene Images 3′-oligolabeling and ECF signal amplification system (Amersham Pharmacia Biotech).

For real-time PCR, 1 μl of diluted cDNA was amplified using Sybr Green PCR Master Mix (PE Applied Biosystems, Foster City, CA) and 0.33 μM each of sense and antisense primers. The primers used for BM5def were 5′-CCTCCTAAGTCCCGGGTTCTC-3′ (sense) and 5′-CGGCCGCCTCTTCTTAACT-3′ (antisense); the primers for HPRT were 5′-GGGAGGCCATCACATTGTG-3′ (sense) and 5′-TCCAGCAGGTCAGCAAAGAAC-3′ (antisense). All PCR were performed on an ABI PRISM 7700 sequence detector system (PE Applied Biosystems). Serial logarithmic dilutions of a pool of cDNA obtained from three B6 mice 12 wk postinfection (p.i.) with LP-BM5 were prepared in triplicate to generate standard curves. The fluorescence signals of each well were collected every 7 s, and threshold cycles were set by determining the point at which the fluorescence intensity increased in a linear manner. The ratio between the BM5def and HPRT content in the samples was defined as the normalization factor. Relative BM5def mRNA quantities were determined by dividing the interpolation-derived values from the standard curve by the normalization factor.

Virus-binding assays

Virus binding was detected as previously described (33, 34) with modifications. To reduce virus interference with non-B cells, spleen cells were treated with ACK lysis buffer (Biofluids, Rockville, MD) to lyse RBC and subsequently depleted of CD43⁺ and CD4⁺ cells using anti-CD43 and anti-CD4 paramagnetic beads (Miltenyi Biotech, Auburn, CA) and column purification according to the manufacturer’s protocol (VarioMACS; Miltenyi Biotech). Cells (1 × 10⁶) were pelleted and resuspended in 100 μl of BM5eco virus supernatant plus 8 μg/ml polybrene. Cells were incubated at 37°C for 40 min with gentle agitation to maintain cell suspension. Following incubation with virus, cells were washed twice with 2 ml of ice-cold PBS-5% FBS. Cells were then incubated for 30 min on ice with PE-conjugated anti-B220 (BD PharMingen) and biotinylated mAb 83A25, which recognizes ecotropic viral envelope gp70. Cells were then washed and incubated with FITC-conjugated avidin (Zymed Laboratories, San Francisco, CA) for 30 min at 4°C. After another wash, cells were analyzed by flow cytometry. The human lymphoblastoid cell line WIL2-NS (American Type Culture Collection, Rockville, MD), which lacks ecotropic virus receptor was assayed for nonspecific viral binding using the same methods as described without B220 staining.

In vitro infection and integration

Spleen cells (10⁶/ml) were cocultured with ecotropic MuLV-green fluorescent protein (GFP)-producing NIH 3T3 cells (35) in the presence of 2 μg/ml polybrene in RPMI 1640 supplemented with 10% FCS and 50 mM 2-ME. For B cell stimulation, LPS (Sigma-Aldrich, St. Louis, MO) at a concentration of 20 μg/ml, anti-IgM (Jackson ImmunoResearch Laboratories, West Grove, PA) at a concentration of 15 μg/ml, or PMA (20 ng/ml; Sigma-Aldrich) and ionomycin (250 ng/ml; Sigma-Aldrich) were added. Cells were harvested after 48 or 72 h, after plating, and infection was analyzed by flow cytometry using determination of green fluorescence. To exclude contaminating producer cells from the analysis, cells were stained with B220-PE Ab as a B cell marker.

In vivo BrdU labeling

BrdU (Sigma-Aldrich) was added to the drinking water of wild-type and CD19- and Vav-1-deficient mice at a concentration of 1 mg/ml 6 days before harvesting spleens. The incorporation of BrdU was determined by flow cytometry using a BrdU Flow kit (BD PharMingen). Splenocytes were labeled with PE-conjugated anti-B220 to detect B cells, washed, fixed, and stained for incorporated BrdU using FITC-conjugated anti-BrdU Ab according to the manufacturer’s instruction.

MAIDS-associated lymphoproliferative disease and histologic changes in wild-type and CR2-, CD22-, CD19- and Vav-1-deficient mice

At 4, 8, and 12 wk p.i., mice were screened for MAIDS-associated splenomegaly, lymphadenopathy, and histopathologic changes by criteria previously described. Spleens and lymph nodes of infected B6 and CR2-deficient mice were found severalfold enlarged compared with those of uninfected mice (Table I⇓). In contrast, the CD19-deficient mice displayed only slight splenomegaly or lymphadenopathy after infection with LP-BM5 (Table I⇓). The histopathological picture in splenic sections ranged from no lesions to appearance of follicular hyperplasia and development of a few small active GC. To determine whether MAIDS induction is impaired because of the reduced number of B cells in CD19-deficient mice (14) or as a result of the disrupted signaling pathway initiated by CD19 ligation, we infected Vav-1-deficient mice with the disease-inducing virus mixture. Signaling through CD19 induces formation of a CD19-Vav-phosphatidylinositol 3 kinase complex that activates the Vav-mitogen-activated protein kinase pathway. Vav-1-deficient mice have normal numbers of conventional B cells (36). When infected with the MAIDS-inducing virus mixture, Vav-1-deficient mice showed somewhat increased spleen and lymph node sizes with spleen weights doubling at 12 wk p.i. This was accompanied by mild immunopathologic changes characterized by the appearance of proliferating immunoblasts and plasma cells in splenic follicles (Table I⇓).

CD22 is another critical cell surface molecule capable of modulating signals through the BCR as well as BCR-independent signals (26). Notably, infected CD22-deficient mice displayed a rapid onset of splenomegaly and lymphadenopathy; at 4 wk p.i., spleen and lymph node weights were ∼2-fold higher than those of infected B6 mice (Table I⇑). However, this hyperreactivity was not observed at later times, indicating an acceleration in disease progression rather than an increased susceptibility.

Phenotypic changes in spleen cells from wild-type and CR2-, CD19-, Vav-1-, and CD22-deficient mice

FACS analysis of spleen cells from B6 and CR2^−/− mice obtained 4 and 12 wk p.i. revealed that these mice had comparable phenotypic changes typical for MAIDS at these time points (data not shown). At 4 wk p.i., spleen cells obtained from infected CD22^−/− mice showed FACS profiles indicative of a more advanced disease state than that revealed by studies of B6 spleen cells (Fig. 1⇓A). Blast populations, indicated by increased forward angle scatter, were seen along with reduced expression of Ig κ L chain as signs of B cell activation. Surface labeling of CD23 was reduced in infected CD22^−/− mice. Expression of IgG FcR was greatly increased, and the size of the CD11b⁺ population was expanded. Furthermore, an expanded subpopulation of CD4⁺Thy-1⁻ cells characteristic of MAIDS could be detected (37). At 12 wk p.i., spleen cells from infected B6, CR2^−/−, and CD22^−/− mice showed identical profiles for all markers tested (Fig. 1⇓B and data not shown). In contrast to the marked changes in B6, CR2^−/−, and CD22^−/− mice induced by LP-BM5 infection, the phenotype of spleen cells from infected CD19^−/− and Vav-1^−/− mice was very similar to that of uninfected mice at 4 and 12 wk p.i., with no evidence of B cell activation or differentiation (Fig. 1⇓B and data not shown). To evaluate further B cell activation markers, we screened B6 and CD19^−/− B cells for CD80 and CD86 as well as for MHC class II expression at 8 wk p.i. As expected, B cells from B6 mice carried increased levels of these molecules, whereas B cells from CD19^−/− mice showed no significant changes in expression (data not shown).

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FIGURE 1.

Phenotype of spleen cells from mice infected with LP-BM5 viruses for 4 (A) or 12 (B) wk. Histograms and dot plots show expression of the indicated molecules and are gated on propidium iodide-negative cells. FSC, Forward angle scatter. MFI, Mean fluorescence intensity.

Immunodeficiency in CR2- and CD22-deficient mice but not in CD19- and Vav-1-KO animals

Immunodeficiency of increasing severity is a second prominent feature of MAIDS (38, 39). At 4, 8, and 12 wk p.i., spleen cells were prepared from wild-type and KO mice and stimulated for 48 h with LPS, Con A, and PMA/ionomycin. As soon as 4 wk p.i., cells from infected B6, CR2^−/−, and CD22^−/− mice showed decreased proliferative response to mitogenic stimulation compared with cells from uninfected control mice (Fig. 2⇓A). It was notable that cells from infected CD22-deficient mice were significantly less responsive to all mitogens tested at this early time point (p < 0.001; Fig. 2⇓A). In contrast, mitogenic responses of infected Vav-1- and CD19-deficient mice were not affected. At 12 wk p.i., spleen cells from infected B6 and CR2- and CD22-deficient mice were almost totally nonresponsive to both B and T cell mitogens (Fig. 2⇓B). In contrast, cells from infected CD19- and Vav-1-deficient mice showed no or moderate proliferative defects (p < 0.05; Fig. 2⇓B). It should be noted that the intrinsic ability of CD19^−/− spleen cells to proliferate in response to LPS was reduced in all experiments (two experiments per time point); the same was true for the ability of Vav-1-deficient spleen cells to respond to Con A.

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FIGURE 2.

Proliferative responses of spleen cells to stimulation with mitogens or PMA plus ionomycin. Cells (1 × 10⁵/well) from spleens of uninfected mice (□) or mice infected for 4 and 12 wk (▪) were cultured for 48 h in triplicate in 96-well plates with Con A (2 μg/ml), LPS (20 μg/ml), or PMA (20 ng/ml) plus inonomycin (250 ng/ml). Cultures were pulsed with [³H]thymidine for the last 6 h of culture. Data are average values ± 1 SEM for three mice per time point. The p values were derived by Student’s t test. ∗, p < 0.05; ∗∗, p < 0.01; and ∗∗∗, p < 0.001.

LP-BM5 infection induces polyclonal Ig production, including switch to IgE in CR2- and CD22-deficient mice

Elevated levels of polyclonal Ab in serum are a well-known feature of MAIDS; however, the absence of CR2, CD19, or Vav results in severely impaired humoral responses to T cell-dependent Ag (14, 16, 20). Therefore, we measured serum Ab levels by ELISA in groups of control and BM5-infected B6 and KO mice at 8 wk p.i. (Fig. 3⇓). Increased levels of IgE, IgM, IgG1, and IgG3 were detected in LP-BM5-infected B6 and CR2- and CD22-deficient mice. When comparing Ig levels in infected CD19^−/− and Vav-1^−/− mice vs uninfected controls, only Vav^−/− mice showed slightly increased IgE and IgM levels. Thus, the polyclonal B cell response that leads to accumulation of serum Ig does not occur in CD19-deficient mice and only marginally in Vav-1-deficient mice. Furthermore, these results suggest that MAIDS infection can induce class switching in the absence of CR2.

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FIGURE 3.

Serum Ig levels in control mice (○) and mice infected with LP-BM5 8 wk p.i. (•). Sera were assayed for the indicated Ig by isotype-specific ELISA. Each circle represents a different animal, and data shown are the mean of triplicate values within a typical experiment.

Suppression of viral replication in CD19- and Vav-1-deficient mice

To examine whether the mild disease course in CD19- and Vav-1-deficient mice is due to impaired virus replication in these mice, we used RT-PCR followed by Southern blotting of amplified transcripts to detect levels of BM5def mRNA in spleen samples at 12 wk p.i. Of interest, BM5def transcripts in spleen cells from CD19-deficient mice were below the limits of detection, and Vav-1^−/− mice showed clearly reduced levels compared with infected B6 mice (Fig. 4⇓A). Real-time PCR confirmed these results; no BM5def transcripts were detected in spleen cells from CD19^−/− mice, and BM5def levels in Vav-1-deficient animals ranged from 30 to 64% of those found in infected wild-type mice 12 wk p.i. (data not shown).

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FIGURE 4.

Viral load and virus binding in wild-type and KO mice. A, Semiquantitative RT-PCR analyses of BM5def and HPRT transcripts in spleens of B6 and CD19- and Vav-1-deficient mice either left uninfected (−) or infected (+) for 12 wk. The length of the amplified products for BM5def and HPRT were 245 and 163 bp, respectively. The electrophoresed amplified products were transferred to a nylon membrane and detected by ECL as described in Materials and Methods. B, Virus binding to wild-type and CD19^−/− B cells. Spleen cells were assayed for ecotropic virus binding using mAb 83A25 as described in Materials and Methods. The human lymphoblastoid cell line WIL2-NS was used to test for specificity of the assay. Histogram overlays are gated on propidium iodide-negative, B220⁺ cells and show cells incubated with ecotropic virus (filled) over noninfected control cells (unfilled).

The frequencies of spleen cells producing ecotropic and MCF viruses also differed among the strains (Table II⇓). Cells from B6 mice expressed both classes of MuLV, whereas cells from CD19^−/− mice did not harbor any ecotropic MuLV, and Vav-1-deficient mice expressed ecotropic MuLv at much lower levels (Table II⇓). Infectious MCF viruses were also not detected in the spleens of CD19-deficient mice. However, after passage of infected Mus dunni cells, low to moderate amounts of MCF could be noticed in blind-passaged cultures. Levels of MCF-producing cells were ∼10-fold lower in Vav-1-KO mice compared with B6 mice. In summary, replication of all LP-BM5 virus types was clearly suppressed in CD19- and Vav-1-deficient mice.

Virus binding is not affected by CD19 deficiency

To further investigate the nature of viral inhibition in CD19-deficient B cells, we examined the capacity of virus to bind to these cells. B cell-enriched spleen cells obtained from wild-type as well as CD19 KO mice were incubated with ecotropic virus for 40 min and subsequently labeled with mAb 83A25, which recognizes ecotropic viral envelope gp70. A human lymphoblastoid cell line that lacks receptors for ecotropic virus was used as a control for specificity of the assay. As shown in Fig. 4⇑B, virus binding was equivalent in both wild-type and CD19^−/− B cell cultures as opposed to minimal binding of ecotropic virus to the human cell line. Thus, absence of CD19 does not influence virus binding to B cells.

Viral integration is not impaired in CD19- and Vav-1-deficient B cells

Another critical step in viral replication is integration of newly synthesized viral DNA into the host genome. To test whether CD19 or Vav-1 deficiency interferes with this process, we cocultured spleen cells from wild-type and CD19- and Vav-1-deficient animals with an ecotropic MuLV-GFP-producing cell line (35). Viral integration was assessed by flow cytometry. Wild-type and CD19- and Vav-1-deficient spleen cells were infectable in culture and expressed GFP after stimulation with anti-IgM, LPS, and PMA/ionomycin (Fig. 5⇓ and data not shown). Although reduced GFP expression could be noted only in Vav-1-deficient B cells after anti-IgM treatment (Fig. 5⇓B), this was due to an impaired proliferative response to anti-IgM as determined by [³H]thymidine uptake (data not shown), which had been shown previously (20). These findings indicate that the reduced viral load in CD19- and Vav-1-deficient animals is not due to defective integration.

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FIGURE 5.

In vitro infection and integration. Spleen cells from B6 and CD19- and Vav-1-deficient animals were cocultured with ecotropic MuLV-GFP-producing NIH 3T3 cells as described in Materials and Methods. Cells were harvested 48 h after plating, and infection was analyzed flow cytometrically by determination of green fluorescence in B220⁺ B cells. Histogram overlays show B cells cocultured with virus producer cells (filled) over noninfected control cells (unfilled). For B cell stimulation, LPS (20 μg/ml; A) or anti-IgM (15 μg/ml; B) was added to the cultures.

In vivo proliferation

Integration was reduced in Vav-1-deficient B cells in vitro, most likely due to impaired proliferation after BCR engagement. This is in accordance with previous studies that suggested that retroviral integration might depend on cell division because viral integration complexes enter the nucleus only after the nuclear envelope is transiently disassembled during mitosis (40). Therefore, impaired in vivo B cell turnover might be a limiting factor for LP-BM5 replication in CD19- and Vav-1-deficient mice. To analyze in vivo B cell proliferation in B6 and CD19- and Vav-1-KO animals, BrdU was added to the drinking water. We then surface labeled the splenic B cell pool with PE-anti-B220 and counterstained with FITC-anti-BrdU to detect cells that had divided during the 6-day in vivo labeling. On average, 12.9% of wild-type B cells, 15.6% of CD19^−/− B cells, and 11.3% of B cells from Vav-1-deficient mice were positive for BrdU (Fig. 6⇓). Thus, in vivo B cell proliferation is not impaired in CD19- or Vav-1-deficient animals.

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FIGURE 6.

In vivo B cell proliferation in wild-type and CD19- and Vav-1-deficient mice. BrdU was added to the drinking water 6 days before sacrificing mice (n = 3 per group). Spleens were harvested, and cells were surface labeled with PE-anti-B220, fixed, permeabilized, and stained with FITC-labeled anti-BrdU Ab. Numbers indicate percentage of positive cells ± SD.