Tetherin/BST-2 promotes dendritic cell activation and function during acute retrovirus infection

Tetherin/BST-2 is a host restriction factor that inhibits retrovirus release from infected cells in vitro by tethering nascent virions to the plasma membrane. However, contradictory data exists on whether Tetherin inhibits acute retrovirus infection in vivo. Previously, we reported that Tetherin-mediated inhibition of Friend retrovirus (FV) replication at 2 weeks post-infection correlated with stronger natural killer, CD4+ T and CD8+ T cell responses. Here, we further investigated the role of Tetherin in counteracting retrovirus replication in vivo. FV infection levels were similar between wild-type (WT) and Tetherin KO mice at 3 to 7 days post-infection despite removal of a potent restriction factor, Apobec3/Rfv3. However, during this phase of acute infection, Tetherin enhanced myeloid dendritic cell (DC) function. DCs from infected, but not uninfected, WT mice expressed significantly higher MHC class II and the co-stimulatory molecule CD80 compared to Tetherin KO DCs. Tetherin-associated DC activation during acute FV infection correlated with stronger NK cell responses. Furthermore, Tetherin+ DCs from FV-infected mice more strongly stimulated FV-specific CD4+ T cells ex vivo compared to Tetherin KO DCs. The results link the antiretroviral and immunomodulatory activity of Tetherin in vivo to improved DC activation and MHC class II antigen presentation.

We recently provided evidence that Tetherin could promote innate and adaptive cell-mediated immune responses against FV infection 16 . FV is a complex of a replication-competent but non-pathogenic helper Friend MuLV (F-MuLV), and a replication-defective but pathogenic spleen focus forming virus (SFFV). FV infects adult immunocompetent mice and causes splenomegaly and erythroleukemia 17 . Classical restriction genes such as Fv2 and mA3/Rfv3 strongly influence the susceptibility of mice to FV disease 17 . C57BL/6 (B6) mice encode resistant forms of Fv2 and mA3/Rfv3, which significantly inhibit splenomegaly induction 18 and promote neutralizing antibody responses 14,15 , respectively. However, B6 mice remain susceptible to infection and erythroleukemia especially at high FV inoculum dose, older age 19 and compromised CD8+ T cell responses 20 . In addition to CD8+ T cell responses, NK cell and CD4+ T cell responses are also required for effective control of FV infection in B6 mice [21][22][23][24][25][26] .
During peak T cell responses to FV, Tetherin KO mice had weaker IFNγ expression in NK cells, CD4+ T cells, and CD8+ T cells, and weaker cytotoxic responses in NK and CD8+ T cells 16 . In addition, Tetherin KO mice had reduced numbers of virus-specific CD8+ T cells. These cell-mediated immune responses correlated with lower plasma viral loads and cellular infection levels. These results demonstrated a role for Tetherin in promoting the cell-mediated immune response to retroviral infection. However, it remained unclear whether Tetherin had a direct effect on acute FV replication. Higher FV replication in Tetherin KO versus WT mice during early stages of the infection may result in weaker cell-mediated immune responses in Tetherin KO mice due to higher FV-induced immune dysfunction.
Dendritic cells (DCs) play key roles in priming both NK and T cell responses 27,28 and are susceptible to FV infection in vivo 29 . Thus, an effect of Tetherin on DC function may govern its ability to stimulate cell-mediated immune responses. In the current study, we explored Tetherin's role in early retroviral infection, when DCs were at the crucial stage of priming cell-mediated immune responses. Utilizing B6 WT and Tetherin KO mice, we show that Tetherin had no effect on early retrovirus replication, but promoted DC function. Our results highlight Tetherin-mediated DC activation as a critical mechanism for how Tetherin influenced retrovirus cell-mediated immune responses that subsequently inhibited retrovirus replication in vivo.

Results
Tetherin does not influence acute FV infection levels. Innate restriction factors are expected to counteract virus infection prior to the onset of adaptive immune responses. Tetherin is widely considered as an innate restriction factor and its expression is significantly induced in bone marrow and spleen 24 h after FV infection 16 . We therefore determined if Tetherin could inhibit acute FV infection levels in vivo. B6 WT and Tetherin KO mice were infected with FV and analyzed at 7 days post infection (dpi). Splenocytes from these mice were stained for surface expression levels of F-MuLV env and analyzed by flow cytometry. F-MuLV env+ cells were quantified using gates set with a stained uninfected sample as < 1% as previously described 30 . The percentage of F-MuLV env+ splenocytes was not significantly different between WT and Tetherin KO mice (Fig. 1a). Viral RNA copies (a) Comparison of % F-MuLV env+ splenocytes from WT and Tetherin KO mice. (b) Plasma viral load comparison between WT and Tetherin KO mice. Plasma samples at 7 dpi were evaluated for F-MuLV RNA copies by qPCR. Absolute viral copies were determined using an F-MuLV RNA standard. Log-transformed values are shown. In both panels, lines represent means, with each dot representing data from an individual mouse. Statistical analyses were performed using a 2-tailed Student's t test. Data for each group were combined from 2 independent experiments. *p < 0.05; ns, not significant (p > 0.05).
Scientific RepoRts | 6:20425 | DOI: 10.1038/srep20425 in plasma were also found to be similar between WT and Tetherin KO mice (Fig. 1b). Thus, Tetherin did not significantly influence acute FV infection levels.
Tetherin does not activate NF-κB in mouse NIH3T3 cells. We previously showed that Tetherin inhibited FV replication at 14 dpi 16 . Since Tetherin had no effect on FV infection levels at 7 dpi (Fig. 1), the effect of Tetherin at a later time point may not be due to its direct restriction properties. Notably, Tetherin-mediated inhibition of virus replication at 14 dpi correlated with stronger NK cell and virus-specific CD8+ T cell responses 16 . The underlying mechanism for how Tetherin influenced cell-mediated immunity remains unclear. Recently, human Tetherin was found to activate NF-κ B, which controls many immunity-related genes [31][32][33][34] . Mouse Tetherin could not activate NF-κ B, but the assays were performed in human cells 31 . We therefore tested if mouse Tetherin could activate NF-κ B in murine NIH3T3 cells. Similar to findings in human 293T cells, human Tetherin induced NF-κ B in murine NIH3T3 cells, but mouse Tetherin did not ( Supplementary Fig. 1 online).

Tetherin does not alter DC phenotypes in the absence of infection. Conventional myeloid DCs
(referred simply as DCs in this study) could modulate both NK and T cell responses 27 . Thus, we next tested whether Tetherin could influence the activity of DCs. We initially determined if Tetherin had an intrinsic effect on DC activation in the absence of infection. Splenocytes from naïve WT and Tetherin KO mice were stained for DC markers and surface proteins indicative of DC maturation. Major histocompatibility complex II (MHC-II) binds antigenic peptide for presentation to CD4+ T cells and its expression on DCs usually increase following infection 35,36 . DC activation also results in the induction of costimulatory molecules CD80 and CD86, which work in conjunction with peptide-bound MHC to prime T cell responses 37 . Splenocytes were analyzed by flow cytometry and DCs (CD11c+ CD11b+ CD19-F4/80-) were evaluated for MHC-II, CD80, and CD86 expression based on median fluorescence intensity (MFI) (Fig. 2a). Expression of MHC-II, CD80, and CD86 were not significantly different between DCs from WT and Tetherin KO mice (Fig. 2b).
Although Tetherin did not affect MHC-II, CD80, and CD86 expression in DCs in uninfected mice, Tetherin may still influence DC function. Thus, splenic DCs from uninfected mice were enriched by negative magnetic selection, pulsed with F-MuLV env 122-141 peptide and cocultured with H5, an FV env 122−141 -specific CD4+ T cell hybridoma derived from infected B6 mice 38 . IL2 levels in the supernatant after 2 d of DC:H5 co-culture were determined by ELISA. Naïve, peptide-loaded WT and Tetherin KO DCs did not differ in their ability to stimulate IL2 production by the H5 CD4+ T cells (Fig. 2c). Thus, Tetherin did not impact the ability of naïve DCs to stimulate virus-specific CD4+ T cells.

Tetherin promotes DC activation during acute FV infection.
Since Tetherin had no effect on DC activation in the absence of infection, we next tested if Tetherin influenced DC activation following FV infection. Splenocytes were collected from FV-infected mice at 5 dpi and gated DCs were analyzed for MHC-II, CD80, and CD86 MFI levels. DCs from infected WT mice showed significantly higher expression of MHC-II and CD80 compared with DCs from infected Tetherin KO mice (Fig. 3a). CD86 expression in DCs was higher in WT versus Tetherin KO mice, but this difference did not quite reach statistical significance (p = 0.06). By contrast, MHC-II expression in B cells, which could also serve as antigen presenting cells 39 were not significantly different between WT and Tetherin KO mice (Fig. 3b). Thus, Tetherin promoted the expression of select proteins involved in antigen presentation in DCs but not in B cells during acute FV infection.
Early Tetherin-mediated DC activation correlates with NK cell activity . We previously showed that Tetherin improved NK cell responses to FV at 14 dpi 16 . However, NK cell responses should already be induced by 1 week post-FV infection 25 . We therefore determined if Tetherin influenced NK cell responses at an earlier time point (5 dpi). Splenocytes from FV-infected mice were stimulated with PMA and ionomycin, stained for NK cell markers (CD3-NK1.1+ DX5+ ), and then analyzed by flow cytometry for expression of IFNγ and CD107a, a marker of NK cell degranulation. A significantly higher percentage of splenic IFNγ + NK cells were found in WT mice compared to Tetherin KO mice (Fig. 4a). The percentage of IFNγ + NK cells correlated with DC MHC-II, CD80 and CD86 expression (Fig. 4b). WT mice exhibited higher percentage of CD107a+ cells compared to Tetherin KO mice, but this did not quite reach statistical significance ( Fig. 4c; p = 0.058). Nevertheless, the percentage of CD107a+ NK cells correlated with DC MHC-II, CD80 and CD86 MFI (Fig. 4d). These data revealed that Tetherin influenced NK cell responses at an early time point following FV infection that correlated with 3 markers of DC activation.
Tetherin promotes BM IL15 expression. One mechanism by which DCs stimulate NK cells is the production of IL15 40 . IL15 is particularly critical for NK cell development in the BM 41 . We therefore analyzed the levels of IL15 transcripts in BM cells from FV-infected mice by qPCR. IL15 mRNA was expressed at higher levels in the BM of WT mice than in Tetherin KO mice at 3 dpi (Fig. 5), but this difference was lost by 5 dpi (p > 0.05). Thus, Tetherin transiently enhanced BM IL15 mRNA expression during early retroviral infection.

Stimulation of FV-specific CD4+ T cells by Tetherin+ versus Tetherin KO DCs. Tetherin influ-
enced CD4+ T cell responses at 14 dpi 16 . Our results above demonstrated that Tetherin influenced DC activation and function. Thus, we hypothesized that the impact of Tetherin on CD4+ T cell responses may be preceded by enhanced antigen presentation qualities of Tetherin+ DCs obtained at an earlier time point. WT and Tetherin KO mice were infected with 10 4 SFFU of FV and at 3 dpi, untouched splenic DCs were isolated by negative selection using magnetic beads. Enriched DCs were cocultured with H5 cells as in Fig. 2d. IL2 release in the DC:H5 cell coculture supernatants were determined by ELISA after 2 days. Isolated DCs from 3 dpi infected WT mice did not stimulate detectable IL2 release from the H5 CD4+ T cells. We therefore pulsed DCs with the F-MuLV env 122-141 peptide prior to co-culture with H5 cells (Fig. 6a, left panel). Notably, WT DCs stimulated IL2 production by H5 cells to a greater degree than Tetherin KO DCs (Fig. 6a, right panel). To confirm that the DCs isolated in the mice were exposed to similar amounts of virus, F-MuLV proviral DNA loads in the isolated DCs were quantified. As expected, Tetherin+ and Tetherin KO DCs had similar proviral F-MuLV DNA loads (Fig. 6b). Altogether, the data demonstrate that Tetherin enhanced the antigen presentation capability of DCs to virus-specific CD4+ T cells independent of FV infection levels.

Tetherin promotes DC activation and function in an Apobec3 KO background. The resistance
genes that influence FV infection may exhibit epistasis, such that the presence of one resistance gene may mask the impact of another. In a previous B 1 test cross of Fv2-susceptible mice, we found that a Tetherin SNP influenced acute FV infection in mice in the mA3/Rfv3 susceptible but not the mA3/Rfv3 resistant background 30 . Thus, Tetherin may have an impact on acute FV replication in an mA3-null background. To test this hypothesis, B6 mA3 KO mice were crossed with Tetherin KO mice to generate mA3/Tetherin double KO (dKO) mice. We then compared FV infection levels, DC activation and antigen presentation capability between B6 mA3 KO (which express Tetherin) and mA3/Tetherin dKO mice.
We first compared FV infection levels between mA3 KO and mA3/Tetherin dKO mice at early time points by flow cytometry. As shown in Fig. 7a, both strains of mice had similar FV infection levels at 5 and 7 dpi. At 5 dpi, DCs of mA3 KO mice expressed higher MHC-II and CD80 MFI levels compared to mA3/Tetherin dKO mice (Fig. 7b). Interestingly, BM cells from mA3 KO mice exhibited higher IL15 expression at 5 dpi compared to mA3/ Tetherin dKO mice ( Supplementary Fig. 2), in contrast to B6 mice which transiently induced BM IL15 at 3 dpi (Fig. 5). Isolated DCs from 3 dpi mA3 KO mice also more efficiently stimulated IL2 release from virus-specific CD4+ T cells compared to DCs from mA3/Tetherin KO mice (Fig. 7c), despite similar proviral FV DNA loads (Fig. 7d). In contrast to Fig. 6a, a prior peptide pulse was not required to reveal the differences in IL2 release, possibly because of higher FV infection levels in mA3 KO compared to WT mice 42,43 . Thus, we confirmed in a second genetic background lacking mA3/Rfv3 that Tetherin significantly promoted DC activation and function during acute retrovirus infection.

Discussion
Tetherin is a potent retrovirus restriction factor in vitro but its impact in vivo is still being determined. Using the FV infection model, we previously provided evidence that Tetherin promoted NK cell, CD4+ T cell and CD8+ T cell responses 16 . These stronger cell-mediated immune responses correlated with lower infection levels suggesting that Tetherin-mediated retrovirus control operated by modulating adaptive immunity. Direct inhibition of FV by Tetherin at earlier time points could explain Tetherin's subsequent immunological effects. Therefore, in the current study, we examined FV infection levels in WT and Tetherin KO mice at earlier acute infection time points. Tetherin had no effect on FV infection levels in the spleen, plasma or isolated DCs from 3 to 7 dpi. Removal of mA3 resulted in increased FV infection, but FV infection levels were still not different between mice with and without Tetherin in this mA3-null background. Our results concur with previous findings using the Mo-MuLV and LP-BM5 infection models that Tetherin did not restrict acute retroviral infection in vivo 10,11 . Thus, the impact of Tetherin on retrovirus replication was not immediate, as would be expected for an innate restriction factor.
Tetherin's effects on different arms of cell-mediated immunity, which rely on antigen presenting cells for activation, led us to investigate the possible role of DCs, which serve as critical bridges between innate and adaptive immunity. After confirming that Tetherin did not alter DC phenotypes in uninfected mice, we evaluated DC activation and function during acute FV infection. The antigen presenting function of DCs is dependent on their activation, which is characterized by the upregulation of MHC-II molecules and costimulatory molecules such as CD80 and CD86. Here, we found that DCs from FV+ Tetherin WT mice displayed higher expression of MHC-II and CD80 than DCs from FV+ KO mice. Tetherin+ DCs also more potently stimulated virus-specific CD4+ T cells compared to Tetherin KO DCs ex vivo despite similar virus infection levels. The enhanced MHC-II antigen presentation capability of Tetherin+ DCs could explain why WT mice had stronger CD4+ T cell responses compared to Tetherin KO mice 16 . CD4+ T cells play critical roles in the maturation, expansion and function of CD8+ T cells 44,45 . Thus, improved MHC-II antigen presentation due to Tetherin activity could indirectly impact CD8+ T cell responses by enhancing CD4+ T cell help. Moreover, NK cells could regulate CD8+ T cell responses against pathogens 46,47 . Tetherin influenced NK cell responses as early as 5 dpi, and this phenomenon could also contribute to enhanced CD8+ T cell responses by 14 dpi. Tetherin-mediated virion aggregation on the cell surface was recently proposed as a mechanism to augment NK cell mediated killing [48][49][50] . This current model stems from in vitro studies showing that cells infected with HIV-1Δ Vpu, which could not counteract Tetherin and whose virions aggregate at higher densities on the cell surface, were more susceptible to NK cell mediated antibody-dependent cellular cytotoxicity (ADCC). However, our results from a test cross to evaluate a unique Tetherin single nucleotide polymorphism (SNP) in mice suggest otherwise 16 . Relative to B6 mice, NZW/LacJ (NZW) mice harbor a Tetherin SNP that results in a truncation of the cytoplasmic tail that encodes the endocytosis motif. Endocytosis-defective NZW Tetherin more strongly restricted FV release in vitro, consistent with higher retention of virions on the cell surface 30 . Thus, if the virion retention/ADCC model was true, then mice encoding NZW Tetherin should have better NK cell responses than those encoding B6 Tetherin. Surprisingly, we observed the opposite result: mice encoding endocytosis-competent B6 Tetherin demonstrated stronger NK cell-associated immune control of FV in vivo 16 . Thus, endocytosis appeared to be critical for Tetherin's impact on cell-mediated immunity. Interestingly, transfection studies linked Tetherin with HIV-1 Gag accumulation in intracellular compartments 2,51,52 . Internalization of tethered virions could promote viral sensing by endosomal sensors, resulting in the upregulation of cytokines required for NK cell function such as IL15. Consistent with this theory, we observed higher BM IL15 mRNA levels in WT versus Tetherin KO mice. A recent study found an association between the endosomal sensor TLR3 and cytotoxic NK and CD8+ T cell responses against FV infection 53 . BM-derived DCs from FV+ TLR3 KO mice were less able to stimulate proliferation of CD8+ T cells in culture, whereas splenic DCs from FV+ TLR3 KO mice had lower expression of activation markers. Thus, we hypothesize that Tetherin-mediated virion endocytosis may enhance TLR3 sensing in DCs to promote NK cell responses.
Tetherin-mediated endocytosis of virions could explain the connection between Tetherin and MHC-II antigen presentation found in the current study. First, endosomal recycling of virions could promote sensing to induce MHC-II and co-stimulatory molecule expression. Second, endosomal reuptake may significantly promote the presentation of virion-derived peptides to MHC-II. In contrast to soluble protein antigens that could readily be cleaved by endosomal proteases, virions have macromolecular structures that may render them more protease-resistant 54 . A mechanism that would retain virions in MHC-II antigen-processing compartments, which resemble multivesicular bodies 55 , could therefore increase the amount of viral peptides loaded to MHC-II. Notably, at least 9 H-2 b -restricted F-MuLV CD4+ T cell epitopes were identified in infected B6 mice 56 . It remains to be determined if this broad epitope diversity could be linked Tetherin. Overall, the results of the current study justify further investigations on Tetherin as a modulator of viral antigen presentation to MHC-II.
Initial models proposed that virus tethering functioned to prevent the release of viruses from infected cells 57 . However, viral tethering may serve a different purpose than just restricting virus release. Tetherin induced NF-κ B when tethering HIV-1 [31][32][33][34] , suggesting that Tetherin could act as a viral sensor. As mouse Tetherin could not activate NF-κ B, additional immunological phenotypes may be altered by human Tetherin in vivo. In this study, we found that human Tetherin activated NF-κ B in mouse cells, suggesting that transgenic mice encoding human Tetherin may be useful for probing these additional immunological effects in vivo. Interestingly, mouse Tetherin enhanced type I IFN production by a specialized subset of cells known as plasmacytoid dendritic cells (pDC),  (a) DCs were isolated from splenocytes of mice infected with 10 4 SFFU of FV at 3 dpi by negative magnetic selection. The DCs were co-cultured with an FV-specific CD4+ T cell hybridoma line, H5, and after 2 d, IL2 in the supernatant was quantified by ELISA. DCs from WT and Tetherin KO mice were pulsed with 5 μ g/ml F-MuLV env peptide prior to H5 coculture. One Tetherin KO mouse had unusually high splenomegaly (2-sided Grubb's test, p < 0.05) and was excluded. Lines correspond to means, and differences were evaluated using a 2-tailed Mann-Whitney U test. (b) Viral infection of DCs. Proviral DNA was quantified from the isolated 3 dpi DCs by qPCR, normalizing to the number of cell equivalents. The lines correspond to geometric means. The dots correspond to individual mice combined from 2 independent experiments. Statistical analyses were performed using a 2-tailed Student's t test; ns, not significant (p > 0.05). (d) Proviral DNA load in isolated 3 dpi DCs were determined by qPCR, normalizing to the number of cell equivalents. For all panels, the lines correspond to means and dots correspond to individual mice combined from 2 independent cohorts. The data were analyzed using a 2-tailed unpaired Student's t test. P values > 0.05 were considered not significant (ns). Exact P values are shown for significant differences. when stimulated with virus in vitro 11 . This appears to contradict data revealing that the interaction between human Tetherin and ILT7 lowers pDC activation 58 . However, a recent report revealed that HIV-1 Vpu may facilitate Tetherin-mediated suppression of pDC activation by targeting Tetherin outside viral assembly sites to interact with ILT7 59 . Studies are now underway to determine if Tetherin influences pDC function and type I IFN production during retrovirus infection in vivo.
In conclusion, we provide evidence that enhanced myeloid DC activation and function underlie the observed connection between Tetherin and retrovirus cell-mediated immune responses. Furthermore, the data raise the possibility that Tetherin's primary role during retrovirus infection may be immunological, as Tetherin-mediated inhibition of acute FV infection was not observed. Data from the current study and previous work on an endocytosis-defective Tetherin allele 16,30 support a model whereby viral tethering triggers the endocytic recycling of tethered virus for enhanced TLR sensing and antigen presentation. Specific molecular details of this proposed pathway remain to be unraveled. Answering these emerging mechanistic questions on Tetherin immunobiology could have important implications in boosting cell-mediated immune responses against pathogenic retrovirus infections including HIV-1.

Methods
Mice. B6 mice (Fv1 b/b Fv2 r/r H-2 b/b mA3/Rfv3 r/r ) were purchased from The Jackson Laboratory. mA3 KO mice were generated from the XN450 gene-trap embryonic stem cell line (BayGenomics) and backcrossed for nine generations into B6 60 . Tetherin KO mice were directly generated in the B6 genetic background 10 . mA3 KO mice were crossed with Tetherin KO mice and resulting F 1 progeny were crossed to each other to generate mA3/ Tetherin double KO (dKO) mice. Mice used in this study ranged from 8 to 12 weeks of age. Mice were handled in accordance with the regulations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The procedures include tail clipping at < 3 weeks of age for genotyping, breeding, infections with FV via the intravenous route and terminal euthanasia involving carbon dioxide inhalation followed by cervical dislocation. Infections were performed under isoflurane anesthesia with regular monitoring and all efforts were made to minimize suffering of the animals. This study was approved by the University of Colorado Institutional Animal Care and Use Committee Permit Number B-89712(08)1E.   61,62 . All virological assays used in this study were specific for the F-MuLV helper virus, which is critical for FV replication. The virus stock was prepared and titered in BALB/c mice 63 .
Plasma Viral Load analysis. Plasma viral load was measured by quantitative real-time PCR (qPCR) 64 .
DC and CD4+ T cell coculture. Splenocytes were collected from uninfected or FV-infected mice at 3 dpi and passed through a 100 μ m nylon filter. Cells were then treated with RBC lysis buffer (eBioscience). A pan-dendritic cell negative selection kit (Miltenyi 130-100-875) was used for Magnetic-Activated Cell Sorting (MACS) of DCs from the splenocytes. MACS separation was performed according to the manufacturer's instructions. 10 5 DCs from each mouse were aliquoted into wells of a 96-well tissue culture plate. DCs were pulsed with 0, 5, 10, 20, or 50 μ g/ml of F-MuLV env 122−141 peptide (DEPLTSLTPRCNTAWNRLKL) (Genscript) diluted in water for 1 h. Excess peptide was washed from the cells. 4 × 10 5 H5 cells 38 were added to each DC culture for a final volume of 200 μ l. After 2 d of coculture at 37 °C and 5% CO 2 , supernatants were collected and used for IL2 ELISA.
IL2 ELISA. IL2 in supernatants collected from coculture experiments were quantified using an IL2 ELISA kit (eBioscience 88-7024-22). Experiments were performed according to the manufacturers' instructions. IL15 expression. RNA was isolated from BM using the Qiagen RNAeasy kit. cDNA synthesis was performed using the QuantiTect Reverse Transcription Kit (Qiagen) and the product was diluted 1:10 in water for a final volume of 200 μ l. Diluted cDNA (10 μ l) was added to 1× QuantiTect SYBR Green PCR master mix (Qiagen) and 5 pmol of the following primers: IL15.for, CATTTTGGGCTGTGTCAGTG; IL15.rev, CATTTTGGGCTGTGTCAGTG. The following thermocycling conditions were used: 95 °C for 10 min, FV proviral load. FV proviral loads in enriched DCs were determined by qPCR 68 . DNA was extracted from enriched DCs from the spleen using the Qiagen DNAeasy Blood and Tissue kit, and 100 ng DNA was added to 1× Taqman Gene Expression Master Mix (Qiagen) along with 10 pmol F-MuLV-specific primers and probe described above. Absolute F-MuLV DNA copy numbers were interpolated using F-MuLV DNA standards. FV DNA copies per cell was determined by also measuring mA3 DNA copies, with 2 mA3 copies = 1 cell. 10 pmol of the following primers and probe were added to the same master mix for duplex PCR: mA3.sense, 5′ -ATCCTCTTCCTTGATAAGATTCGGTCCATG-3′ ; mA3.antisense, 5′ -GATCCC TGATTGCCACAGAGAACAC-3′ ; mA3.probe, HEX-ATC TACACCTCCCGCCTGTATTTCCACT-TAMRA. mA3 DNA standards were used to interpolate absolute mA3 DNA copy numbers. The reaction was performed under these thermocycling conditions: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 1 min. Statistical analysis. Statistical analyses were performed using Prism 5.0 (Graphpad). Two-tailed Student's t test was used for 2-group comparisons. For datasets with skewed distribution based on the Kolmogorov-Smirnov normality test (p < 0.05), a nonparametric 2-tailed Mann-Whitney U test was performed. Correlations were computed using Pearson's r. P values < 0.05 were considered statistically significant.