Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Immunobiology. 2014 Oct 30;220(4):518–524. doi: 10.1016/j.imbio.2014.10.019

Batf3 deficiency is not critical for the generation of CD8α+ dendritic cells

Kevin R Mott 1, Hadi Maazi 2, Sariah J Allen 1, Mandana Zandian 1, Harry Matundan 1, Yasamin N Ghiasi 2, Behrooz G Sharifi 3, David Underhill 4, Omid Akbari 2, Homayon Ghiasi 1,*
PMCID: PMC4355210  NIHMSID: NIHMS639606  PMID: 25468565

Abstract

Recently, we have reported that CD8α+ DCs, rather than CD8+ T cells, are involved in the establishment and maintenance of HSV-1 latency in the trigeminal ganglia (TG) of ocularly infected mice. In the current study, we investigated whether similar results can be obtained using Batf3−/− mice that previously were reported to lack CD8α+ DCs. However, our results demonstrate that Batf3−/− mice, without any known infection, express CD8α+ DCs. Consequently, due to the presence of CD8α+ DCs, no differences were detected in the level of HSV-1 latency between Batf3−/− mice compared with wild type control mice.

Keywords: dendritic cells, HSV-1, latency, knockout, compensation

INTRODUCTION

Dendritic cells (DCs) are highly heterogeneous cells and have been classified into several subsets based on their immunophenotype, resident location, and functional differences (Shortman and Naik, 2007, Belz and Nutt, 2012). In general, DCs are divided into two main groups: conventional (cDCs) and plasmacytoid (pDCs) (Shortman and Naik, 2007, Belz and Nutt, 2012, Heath and Carbone, 2009). In mouse lymph nodes (LNs), conventional DCs include the CD8α+ and CD11b+ subgroups, the latter subdivided based on CD4+ expression (Vremec, et al., 2000, Belz, et al., 2004). Human blood DC antigen 3 (CD141, BDCA3) is reported to be the homologue of mouse CD8α+ DCs (Jongbloed, et al., 2007).

Recently, we reported that DCs isolated from CD8α−/− mice harbor fewer viruses than DCs isolated from wild-type, CD8β−/−, or β2m−/− mice (Mott, et al., 2014). In addition, we demonstrated that CD8α+ DCs are responsible for enhanced viral latency in ocularly infected mice (Mott, et al., 2014, Mott, et al., 2014, Mott and Ghiasi, 2008, Mott, et al., 2008). In those studies, we showed that HSV-1 infected DCs interfered with DC function, such as inhibiting induction of antiviral responses, leading to higher latency and T cell exhaustion. Similarly, it was shown that the absence of CD8α+ DCs in mice enhances resistance to the intracellular bacterium Listeria monocytogenes (Edelson, et al., 2011). These results are also consistent with reports on the negative role of certain DC populations in control of various viral infections (Engelmayer, et al., 1999, Granelli-Piperno, et al., 1998, Geijtenbeek, et al., 2000, Wu, et al., 2000, Paroli, et al., 2000).

Production of CD8α+ DCs is regulated by several transcription factors including interferon regulatory factor 8 (Irf8), nuclear factor interleukin 3 regulated (Nfil3), inhibitor of DNA binding 2 (Id2), and basic leucine zipper transcriptional factor ATF-like 3 (Batf3) (Belz and Nutt, 2012, Edelson, et al., 2010, Kashiwada, et al., 2011, Hildner, et al., 2008). Based on these studies, the absence of IRF8, Nfil3, Id2, or Batf3 blocked production of CD8α+ DCs. However, it was recently shown that CD8α+ DCs can be induced in the absence of the transcription factors Id2, Nfil3, or Batf3, but not IRF8 (Seillet, et al., 2013). BXH2 mice have a spontaneous point mutation in the IRF8 gene similar to that in IRF8−/− mice that blocks the generation of CD8α+ DCs (Mott, et al., 2014, Turcotte, et al., 2004, Tailor, et al., 2008).

In our published studies, we have shown that the levels of both HSV-1 glycoprotein B (gB) DNA and latency associated transcript (LAT) RNA were significantly lower in latently infected mice that did not produce CD8α+ DCs compared with mice that produced CD8α+ DCs (Mott, et al., 2014). In the present study, we examined whether Batf3−/− mice, which were reported to lack CD8α+ DCs (Hildner, et al., 2008), may have lower HSV-1 latency, as we reported for BXH2 mice (Mott, et al., 2014). In contrast to BXH2, Batf3−/− mice produced CD8α+ DCs and had similar levels of HSV-1 latency as WT control mice. Therefore, in the absence of any known infection, our results point to the lack of a key role for Batf3 in generation of CD8α+ DCs. Thus, among the known transcription factors for the development of CD8α+ DCs, only IRF-8 is essential for generation of CD8α+ DCs (Mott, et al., 2014, Turcotte, et al., 2004, Tailor, et al., 2008, Fung-Leung, et al., 1991).

Materials and Methods

Virus and mice

Plaque-purified virulent HSV-1 strain McKrae was grown in rabbit skin (RS) cell monolayers in minimal essential medium (MEM) containing 5% fetal calf serum (FCS) as we described previously (Osorio and Ghiasi, 2003). WT C57BL/6 and C57BL/6-Batf3−/− mice were purchased from The Jackson Laboratories and were bred in-house. Mice were housed in static microisolator cages in negatively pressurized isolation cubicles.

Ocular infection

Mice were infected ocularly with 2 × 105 PFU per eye of HSV-1 strain McKrae in 2 μl of tissue culture medium and administered as an eye drop without prior corneal scarification.

Confocal microscopy and image analysis of BM-derived DCs cultures

Six week-old mice were used as a source of bone marrow (BM) for the generation of mouse dendritic cells (DCs) in culture as we described previously (Mott, et al., 2009). DCs isolated from WT and Batf3−/− strains of mice were grown on Lab-Tex chamber slides (Sigma-Alderich, St. Louise, MO) as we described previously (Mott, et al., 2014). Briefly, cells were fixed by incubating slides in methanol for 10 min followed by acetone for 5 min at −20°C. Afterwards, slides were rinsed three times for 5 min each at ambient temperature in PBS containing 0.05% v/v Tween-20 (PBS-T). Slides were then blocked for 30 min at ambient temperature in PBS-T containing 1% w/v BSA. Rat anti-CD8α (clone 53-6.7, eBioscience, San Diego, CA), rat anti-CD4 (clone Gk1.5, eBioscience), rat anti-CD8α (clone YTS156.7.7, BioLegend), and hamster anti-CD11c (clone HL3, BD Biosciences) were used for IHC. Immunostaining was done using CD11c/CD4, CD11c /CD8α, or CD11c /CD8β antibody combinations and staining for 1 h at 25°C. After three rinses for 5 min each in PBS, slides were incubated for 1 h at 25°C with secondary antibodies labeled with anti-hamster FITC or anti-Rat TRITC (Invitrogen). Slides were washed three times with PBS, air-dried and mounted with Prolong Gold DAPI mounting medium (Invitrogen).

Images were captured at 1024 × 1024 pixels (original magnification = 20×) in independent fluorescence channels using a Nikon C1 eclipse inverted confocal microscope.

FACS analyses

Single cell suspension from wt C57BL/6 or Batf3−/− mouse was made by chopping the spleen into small pieces and incubating in 200 U/ml collagenase D (Worthington Biochemical, Lakewood, NJ) for 45 minutes at 37°C followed by filtering through 40 μm cell strainer (BD biosciences, San Jose, CA) and washing twice in PBS + 1% BSA. 2×106 cells were taken for each test. Cell were then stained with APC-eFluor® 780-anti mouse CD11c, APC-anti mouse CD8β and PE/Cy7-anti mouse CD8α (all from eBioscience, San Diego, CA) in PBS + 1% BSA containing 1μg/ml anti mouse FC-receptor block antibody (BioXCell, West Lebanon, NH). Stained cells were washed twice in PBS + 1% BSA then 5 × 105 cells were acquired on BD FACSCANTO-II (BD biosciences) and results were analyzed using FlowJo software (TreeStar, Ashland, OR).

Genotyping of Batf3−/− mouse

Half cm sections of WT and Batf3−/− mouse tail were processed using the DirectPCR lysis reagent (Cat # 101-T, ViagenBiotech, Los Angeles, CA) as per manufacturer protocol. Briefly, tail pieces were added to approximately 200 ul of lysis reagent containing freshly prepared 0.2-0.4 mg/ml Proteinase K (Cat # 501-PK, ViagenBiotech, Los Angeles, CA) and incubated for 5-6 hours at 55 °C. Following incubation, crude lysates were incubated at 85°C for 45 minutes and 1ul of lysate was used for PCR reactions. Standard PCR was carried out using Ready PCR Mix (Cat # N806, Amresco Corporation, Solon, OH) and the Batf3 Fast MCA-Master Protocol- version 2, September 2011- Stock number 013755 (The Jackson Laboratory, Sacramento, CA). Briefly, 1 ul of crude DNA lysate was amplified at the following conditions: 94°C 3min (1 cycle); 94°C 30sec, 59°C 60sec, 72°C 60sec (35 Cycles); 72°C 2min. Primers used for the analysis were as follows: Primer 11387-Common Forward- 5′-TGC TAT GCA CAA ACC ACA AAC C- 3′; Primer 11388- Wild Type Reverse- 5′-GTT GTG AGT CGA AAC CAC GC-3′; Primer 11390- Mutant Reverse- 5′-GAT ACA GGC TGC TGA TGA TCT GAG-3′. The expected PCR product sizes identified the following mouse genotype: Mutant = 600bp; Heterozygote = 600bp and 288bp; and Wild type = 288bp.

RNA and DNA extraction from TG of latently infected mice

DNA was isolated from homogenized latently infected individual TG using the commercially available DNeasy Blood &Tissue Kit (Qiagen, Stanford, CA) according to the manufacturer’s instructions. PCR analyses were done using gB specific primers (Forward - 5′-AACGCGACGCACATCAAG-3′; Reverse - 5′-CTGGTACGCGATCAGAAAGC-3′; and Probe - 5′-FAM-CAGCCGCAGTACTACC-3′). The amplicon length for this primer set was 72 bp. Relative copy numbers for gB DNA were calculated using standard curves generated from the plasmid pAc-gB1 (Ghiasi, et al., 1992). In all experiments, GAPDH was used for normalization of transcripts.

RNA was extracted from latent TG or infected DCs as we have described previously (Mott, et al., 2009, Mott, et al., 2007, Mott, et al., 2007). Following RNA extraction, 1 μg of total RNA was reverse-transcribed using random hexamer primers and Murine Leukemia Virus (MuLV) Reverse Transcriptase from the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA), in accordance with the manufacturer’s recommendations. The levels of various transcripts were evaluated using commercially available TaqMan Gene Expression Assays (Applied Biosystems) with optimized primer and probe concentrations. Primer-probe sets consisted of two unlabeled PCR primers and the FAM™ dye-labeled TaqMan MGB probe formulated into a single mixture. Additionally, all cellular amplicons included an intron-exon junction to eliminate signal from genomic DNA contamination. PCR analyses were done using LAT specific primers (forward primer 5′-GGGTGGGCTCGTGTTACAG-3′; reverse primer, 5′-GGACGGGTAAGTAACAGAGTCTCTA-3′; and probe, 5′- FAMACACCAGCCCGTTCTTT-3′– Amplicon Length =81 bp, corresponding to LAT nts 119553-119634. As an internal control, a set of GAPDH primers from Applied Biosystems (ASSAY I.D. m999999.15_G1 - Amplicon Length = 107 bp) was used.

Quantitative real-time PCR (qRT-PCR) was performed as we described previously (Mott, et al., 2007, Mott, et al., 2007). Real-time PCR was performed in triplicate for each tissue sample. The threshold cycle (Ct) values, which represents the PCR cycle at which there is a noticeable increase in the reporter fluorescence above baseline, were determined using SDS 2.2 Software. GAPDH transcript was used for normalization purposes.

Statistical analysis

Student’s t tests were performed using the computer program Instat (GraphPad, San Diego). Results were considered statistically significant when the P value was < 0.05.

RESULTS and DISCUSSION

In our published study we have shown that BHX2 and CD8α−/− mice did not produce CD8α+ DCs, while WT, CD8β−/−, and β2m−/− mice produced CD8α+ DCs (Mott, et al., 2014). We have also reported that the absence of CD8α+ DCs was associated with lower HSV-1 latency and reactivation compared with mice that produce CD8α+ DCs (Mott, et al., 2014, Mott, et al., 2014). Therefore, to confirm our recent observation that deficiency of CD8α+ DCs leads to lower latency in ocularly infected mice, we used Batf3−/− mice that were reported previously to lack CD8α+ DCs (Hildner, et al., 2008). We first set out to confirm that Batf3−/− mice, similar to BHX2 and CD8α−/− mice, are unable to produce CD8α+ DCs. BM-derived DCs were isolated from mice that we received upon arrival from the Jackson Laboratory (WT or Batf3−/−JAX) or from their offspring that we bred in-house (Batf3−/−InHouse). Cells were stained with various combinations of CD11c, CD8α, CD8β, or CD4 antibody. Immunohistochemistry (IHC) results for DCs isolated from Jackson mice are shown in Figure 1. CD8α+ CD11c+ or CD8β+ CD11c+ expression was not detected on DCs isolated from Batf3−/−JAX mice, while CD4+CD11c+ cells were present (Figure 1, Batf3−/−JAX DCs). In contrast, BM-derived DCs from our in-house breeding colony expressed both CD8α+CD11c+ and CD8β+CD11c+ cells (Figure 2). Similar to the Batf3−/−JAX result (Figure 1, CD4), expression of CD4+CD11c+ cells in BM-derived DCs from in-house breeding was not affected (Figure 2). The expression level of CD8α+CD11c+ in BM-derived DCs from Batf3−/−InHouse mice was similar to the results for WT mice. Taken together, these results suggest that due to some environmental factor, mice that lack the Batf3 gene reprogram hematopoetic stem cells to allow for production of CD8α+ DCs after we start breeding them in-house.

Figure 1. IHC of DCs isolated from Batf3−/−JAX mice.

Figure 1

Open in a new tab

BM-derived DCs from WT and Batf3−/− mice following arrival from The Jackson Laboratory were isolated and grown on Lab-Tex chamber slides. At 24 hr post culture, DCs were fixed and stained with anti-CD11c/anti-CD4, CD11c/anti-CD8α, or CD11c/anti-CD8β antibodies, followed by incubation with relevant secondary antibody to each primary antibody as described in Materials and Methods. DAPI is shown as a nuclear counter-stain. Upper panels: from left to right DAPI, CD11c (FITC), CD4 (TRITC), Merge; middle panels: from left to right DAPI, CD11c (FITC), CD8α (TRITC), Merge; and bottom panels: from left to right DAPI, CD11c (FITC), CD8β (TRITC), Merge.

Figure 2. IHC of DCs isolated from Batf3−/−in-house mice.

Figure 2

Open in a new tab

BM-derived DCs from WT and Batf3−/− mice bred in-house were isolated and grown on Lab-Tex chamber slides and processed as described in the legend of Figure 1 above. DAPI is shown as a nuclear counter-stain. Upper panels: from left to right DAPI, CD11c (FITC), CD4 (TRITC), Merge; middle panels: from left to right DAPI, CD11c (FITC), CD8α (TRITC), Merge; and bottom panels: from left to right DAPI, CD11c (FITC), CD8β (TRITC), Merge.

The IHC results described above show expression of CD8α+ in in-house-derived DCs compared with Jackson -derived DCs. Therefore, it was of interest to determine if similar changes could be detected in the spleens of Jackson versus in-house mice. Single cell suspensions of spleen cells were prepared as described in Materials and Methods. Cells were stained with anti-CD3, anti-CD11c, and anti-CD8 antibodies. Cells were gated for expression of CD11c+CD8α+ and the FACS data are shown in Figure 3. Similar to our IHC results for BM-derived DCs, 0.09% of DCs in the spleens of Jackson mice were CD11c+CD8α+, while this increased to 0.60% in the spleens of in-house mice (Figure 3). This was similar to the 0.54% level of CD11c+CD8α+ expression in WT mice (Figure 3). Thus, these results further support our in vitro data and suggest that the inability to produce CD8α+ DC in Batf3-deficient mice is overcome by another factor upon in-house breeding, possibly due to changes in animal housing conditions.

Figure 3. CD8α+ expression was altered in Batf3−/− offspring without infection.

Figure 3

Open in a new tab

Spleens from Batf3−/−JAX and their in-house offspring, as well as from WT mice, were isolated, single cell suspensions were prepared, and subsequently stained with anti-CD11c and anti-CD8 antibodies and gated for expression of CD11c and CD8α. A minimum of 105 events were acquired on a gate including viable cells. Numbers in each quadrant indicate the percentage of single or double-positive cells for each treatment condition.

Previously, it was shown that mice of C57BL/6 genetic background at steady-state express low levels of CD8α+ DCs in their spleens and lymph nodes (Edelson, et al., 2011, Jackson, et al., 2011). Similarly, in this study we detected CD8α+ DCs in spleens and BM-derived DCs of mice with the same genetic background. However, the expression level of CD8α+ DCs in the in-house Batf3−/− mice was similar to that of WT mice. It was recently shown that IL-12 administration or infection with T. gondii, L. monocytogenes, and Mycobacterium tuberculosis restored CD8α+ DCs production in Batf3−/− mice (Tussiwand, et al., 2012). The authors concluded that Batf and Batf2 compensate for the absence of Batf3 gene during infection with intracellular pathogens, and this is mediated by IL-12 and IFN-γ. Basic leucine zipper transcription factor ATF-like (Batf, Batf2 and Batf3) comprise the Batf family, which is a subgroup of the larger family of basic leucine zipper (bZIP) transcription factors (Finn, et al., 2010). The expression of Batf and Batf3 is restricted to the haematopoietic system (Hildner, et al., 2008, Williams, et al., 2001), while Batf2 is expressed in both haematopoietic and non-haematopoietic tissues (Su, et al., 2008). Batf is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17 cells) (Schraml, et al., 2009, Ise, et al., 2011), promotes effector CD8 T-cell differentiation (Kuroda, et al., 2011), and antibody production (Betz, et al., 2010). Batf2 exerts cancer-selective growth-inhibitory effects (Su, et al., 2008) and Batf2−/− mice had normal development of NK, T and B cells, pDCs, neutrophils, resting cDCs, and peritoneal, liver and lung macrophages (Tussiwand, et al., 2012). Batf3 was originally reported to be required for development of the CD8α+ lymphoid-resident DC subset and of the related CD103+ peripheral DC subset, and produce IL-12 in response to pathogens (Hildner, et al., 2008, Williams, et al., 2001, Mashayekhi, et al., 2011). Thus, these studies suggest that Batf, Batf2 and Batf3 genes each have their unique and non-overlapping function. However, when using Batf−/−Batf3−/− and Batf2−/−Batf3−/− mice a reduction of CD8α+ cDC expansion was shown following IL-12 treatment compared with similarly treated Batf3−/− mice (Tussiwand, et al., 2012). The authors concluded that expression of either Batf or Batf2 can compensate for Batf3 in the expansion of the CD8α+ cDC population. Although both Batf−/− and Batf2−/− mice had no apparent DCs defect (Tussiwand, et al., 2012). However, the involvement of other factors to compensate for the absence of Batf3 in the absence of any infection or IL-12 injection can not be ruled out. Moreover, our in-house mice are certified pathogen-free and were not exposed to any type of infection. Thus, it is possible that, in contrast to the reported study by Tussiwand et al (Tussiwand, et al., 2012), changes in microflora of in-house mice that are independent of any type of infection contribute to the appearance of CD8α+ DCs in Batf3−/− mice. It is widely understood that intestinal microflora can profoundly influence the homeostasis of the host immune system (Iliev, et al., 2012, Tanoue, et al., 2010, Jarchum and Pamer, 2011, Chinen and Rudensky, 2012). In addition, commensal microbes can affect immune cell progenitors (Gollwitzer, et al., 2014, Trompette, et al., 2014, Khosravi, et al., 2014). Consequently, the changes in microflora without any infection may be the reason that the Jackson mice do not express CD8α+ DCs, while our in-house mice do.

Both IHC and FACS data described above have shown that CD8α+ DCs are produced at WT levels in Batf3−/−InHouse but not in Batf3−/−JAX mice. To confirm the absence of Batf3 gene in both JAX and in-house Batf3−/− mice, qPCR was performed on mice genomic DNA as described in Materials and Methods. As expected, DNA isolated from both Jackson and in-house Batf3−/− mice produced a band of 600 bp, while DNA isolated from WT mice produced a band of 288 bp (Fig. 4). Thus, both JAX and in-house Batf3−/− mice lack the Batf3 gene and the observed differences are not due to genetic reversion.

Fig. 4. Confirmation of the absence of Batf3 in JAX and in-house knockout mice.

Fig. 4

Open in a new tab

Tail DNA from WT and Batf3−/− mice was isolated and PCR was performed using the primers as mentioned in Materials and Methods. Panels: (Left) Shown is a representative gel scan from WT and JAX mice demonstrating a band of 600 bp in Batf3−/−JAX mice and a band of 288 bp in WT mice; and (Right) Shown is a representative gel scan from WT and Batf3−/−in-house mice demonstrating a band of 600 bp in in-house mice and a band of 288 bp in WT mouse.

Finally, to determine if the absence of Batf3 gene, despite the presence of CD8α+ DCs, alters latency in infected mice, WT and Batf3−/−InHouse mice were ocularly infected with HSV-1, as described in Materials and Methods. No differences in virus replication in the eye, eye disease, or survival were detected between WT and Batf3-deficient mice (not shown). Individual TG from surviving mice were isolated on day 28 post-infection (PI), and total DNA was prepared. TaqMan PCR was performed as described in Materials and Methods to quantitate viral gB DNA as a measure of HSV-1 genome copies and hence, latency. The amount of viral DNA during latency in WT mice was similar to that of Batf3-deficient mice (Figure 5A; WT vs. Batf3−/−, p>0.05). This suggests that the lack of Batf3 does not impact viral latency, consistent with the normal presence of CD8α+ DCs in these mice (Mott, et al., 2014). During HSV-1 neuronal latency, only the LAT region is consistently expressed at high levels (Rock, et al., 1987, Dobson, et al., 1990). We next sought to confirm our gB DNA latency results by quantitating LAT expression levels. On day 28, TG from surviving mice were harvested, and expression of LAT was determined by TaqMan RT-PCR from total RNA isolated from individual TG. In agreement with the gB DNA results (Figure 5A), the amount of LAT RNA detected in the TG of Batf3-deficient mice was similar to that of WT mice (Figure 5B, p>0.05). These results demonstrate that the absence of Batf3 did not reduce HSV-1 latency due to the presence of CD8α+ DCs.

Figure 5. Quantitation of gB DNA and LAT RNA in trigeminal ganglia of HSV-1 latently-infected mice.

Figure 5

Open in a new tab

Wild-type (WT) C57BL/6 or Batf3−/− mice were ocularly-infected with HSV-1 strain McKrae. On day 28 PI, TG were harvested from latently infected mice. Quantitative PCR and RT-PCR was performed on each individual mouse TG. In each experiment, an estimated relative copy number of the HSV-1 gB (for viral DNA) and LAT (for viral RNA) were calculated using standard curves generated from pGem-gB1 and pGem5317, respectively. Briefly, DNA template was serially diluted 10-fold such that 5 μl contained from 103 to 1011 copies of gB, then subjected to TaqMan PCR with the same set of primers. By comparing the normalized threshold cycle of each sample to the threshold cycle of the standard, the copy number for each reaction was determined. GAPDH expression was used to normalize the relative expression of viral (gB) DNA and LAT RNA in the TG. Each data point represents the mean ± SEM from 10 TG. Panels: A) gB DNA; and B) LAT RNA.

Acknowledgements

This work was supported by Public Health Service grants AI093941 and EY13615.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Disclosure Statement

The authors declare that there are no competing financial interests.

REFERENCES

  1. Shortman K, Naik SH. Steady-state and inflammatory dendritic-cell development. Nat Rev Immunol. 2007;7:19. doi: 10.1038/nri1996. [DOI] [PubMed] [Google Scholar]
  2. Belz GT, Nutt SL. Transcriptional programming of the dendritic cell network. Nat Rev Immunol. 2012;12:101. doi: 10.1038/nri3149. [DOI] [PubMed] [Google Scholar]
  3. Heath WR, Carbone FR. Dendritic cell subsets in primary and secondary T cell responses at body surfaces. Nat Immunol. 2009;10:1237. doi: 10.1038/ni.1822. [DOI] [PubMed] [Google Scholar]
  4. Vremec D, Pooley J, Hochrein H, Wu L, Shortman K. CD4 and CD8 expression by dendritic cell subtypes in mouse thymus and spleen. J Immunol. 2000;164:2978. doi: 10.4049/jimmunol.164.6.2978. [DOI] [PubMed] [Google Scholar]
  5. Belz GT, Smith CM, Eichner D, Shortman K, Karupiah G, Carbone FR, Heath WR. Cutting edge: conventional CD8alpha(+) dendritic cells are generally involved in priming CTL immunity to viruses. J Immunol. 2004;172:1996. doi: 10.4049/jimmunol.172.4.1996. [DOI] [PubMed] [Google Scholar]
  6. Jongbloed SL, Kassianos AJ, McDonald KJ, Clark GJ, Ju X, Angel CE, Chen CJ, Dunbar PR, Wadley RB, Jeet V, Vulink AJ, Hart DN, Radford KJ. Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens. J Exp Med. 2007;207:1247. doi: 10.1084/jem.20092140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mott KR, Allen SJ, Zandian M, Konda B, Sharifi BG, Jones C, Wechsler SL, Town T, Ghiasi H. CD8a dendritic cells drive establishment of HSV-1 latency. PLoS One. 2014;9:e93444. doi: 10.1371/journal.pone.0093444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Mott KR, Allen SJ, Zandian M, Ghiasi H. Coregulatory Interactions between CD8α DCs, LAT, and PD-1 Contribute to higher HSV-1 Latency. J Virol. 2014;88:6599. doi: 10.1128/JVI.00590-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Mott KR, Ghiasi H. Role of dendritic cells in enhancement of herpes simplex virus type 1 latency and reactivation in vaccinated mice. Clin Vaccine Immunol. 2008;15:1859. doi: 10.1128/CVI.00318-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mott KR, UnderHill D, Wechsler SL, Ghiasi H. Lymphoid-related CD11c+CD8a+ dendritic cells are involved in enhancing HSV-1 latency. J Virol. 2008;82:9870. doi: 10.1128/JVI.00566-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Edelson BT, Bradstreet TR, Hildner K, Carrero JA, Frederick KE, Kc W, Belizaire R, Aoshi T, Schreiber RD, Miller MJ, Murphy TL, Unanue ER, Murphy KM. CD8alpha(+) dendritic cells are an obligate cellular entry point for productive infection by Listeria monocytogenes. Immunity. 2011;35:236. doi: 10.1016/j.immuni.2011.06.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Engelmayer J, Larsson M, Subklewe M, Chahroudi A, Cox WI, Steinman RM, Bhardwaj N. Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion. J Immunol. 1999;163:6762. [PubMed] [Google Scholar]
  13. Granelli-Piperno A, Delgado E, Finkel V, Paxton W, Steinman RM. Immature dendritic cells selectively replicate macrophagetropic (M-tropic) human immunodeficiency virus type 1, while mature cells efficiently transmit both M- and T-tropic virus to T cells. J Virol. 1998;72:2733. doi: 10.1128/jvi.72.4.2733-2737.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geijtenbeek TB, Kwon DS, Torensma R, van Vliet SJ, van Duijnhoven GC, Middel J, Cornelissen IL, Nottet HS, KewalRamani VN, Littman DR, Figdor CG, van Kooyk Y. DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell. 2000;100:587. doi: 10.1016/s0092-8674(00)80694-7. [DOI] [PubMed] [Google Scholar]
  15. Wu SJ, Grouard-Vogel G, Sun W, Mascola JR, Brachtel E, Putvatana R, Louder MK, Filgueira L, Marovich MA, Wong HK, Blauvelt A, Murphy GS, Robb ML, Innes BL, Birx DL, Hayes CG, Frankel SS. Human skin Langerhans cells are targets of dengue virus infection. Nature Med. 2000;6:816. doi: 10.1038/77553. [DOI] [PubMed] [Google Scholar]
  16. Paroli M, Schiaffella E, Di Rosa F, Barnaba V. Persisting viruses and autoimmunity. J Neuroimmunol. 2000;107:201. doi: 10.1016/s0165-5728(00)00228-9. [DOI] [PubMed] [Google Scholar]
  17. Edelson BT, Kc W, Juang R, Kohyama M, Benoit LA, Klekotka PA, Moon C, Albring JC, Ise W, Michael DG, Bhattacharya D, Stappenbeck TS, Holtzman MJ, Sung SS, Murphy TL, Hildner K, Murphy KM. Peripheral CD103+ dendritic cells form a unified subset developmentally related to CD8alpha+ conventional dendritic cells. J Exp Med. 2010;207:823. doi: 10.1084/jem.20091627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kashiwada M, Pham NL, Pewe LL, Harty JT, Rothman PB. NFIL3/E4BP4 is a key transcription factor for CD8alpha(+) dendritic cell development. Blood. 2011;117:6193. doi: 10.1182/blood-2010-07-295873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hildner K, Edelson BT, Purtha WE, Diamond M, Matsushita H, Kohyama M, Calderon B, Schraml BU, Unanue ER, Diamond MS, Schreiber RD, Murphy TL, Murphy KM. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science. 2008;322:1097. doi: 10.1126/science.1164206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seillet C, Jackson JT, Markey KA, Brady HJ, Hill GR, Macdonald KP, Nutt SL, Belz GT. CD8alpha+ DCs can be induced in the absence of transcription factors Id2, Nfil3, and Batf3. Blood. 2013;121:1574. doi: 10.1182/blood-2012-07-445650. [DOI] [PubMed] [Google Scholar]
  21. Turcotte K, Gauthier S, Mitsos LM, Shustik C, Copeland NG, Jenkins NA, Fournet JC, Jolicoeur P, Gros P. Genetic control of myeloproliferation in BXH-2 mice. Blood. 2004;103:2343. doi: 10.1182/blood-2003-06-1852. [DOI] [PubMed] [Google Scholar]
  22. Tailor P, Tamura T, Morse HC, 3rd, Ozato K. The BXH2 mutation in IRF8 differentially impairs dendritic cell subset development in the mouse. Blood. 2008;111:1942. doi: 10.1182/blood-2007-07-100750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Fung-Leung WP, Schilham MW, Rahemtulla A, Kundig TM, Vollenweider M, Potter J, van Ewijk W, Mak TW. CD8 is needed for development of cytotoxic T cells but not helper T cells. Cell. 1991;65:443. doi: 10.1016/0092-8674(91)90462-8. [DOI] [PubMed] [Google Scholar]
  24. Osorio Y, Ghiasi H. Comparison of adjuvant efficacy of herpes simplex virus type 1 recombinant viruses expressing TH1 and TH2 cytokine genes. J Virol. 2003;77:5774. doi: 10.1128/JVI.77.10.5774-5783.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mott KR, Underhill D, Wechsler SL, Town T, Ghiasi H. A role for the JAK-STAT1 pathway in blocking replication of HSV-1 in dendritic cells and macrophages. Virol J. 2009;6:56. doi: 10.1186/1743-422X-6-56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ghiasi H, Kaiwar R, Nesburn AB, Wechsler SL. Expression of herpes simplex virus type 1 glycoprotein B in insect cells. Initial analysis of its biochemical and immunological properties. Virus Res. 1992;22:25. doi: 10.1016/0168-1702(92)90087-p. [DOI] [PubMed] [Google Scholar]
  27. Mott KR, Perng GC, Osorio Y, Kousoulas KG, Ghiasi H. A Recombinant Herpes Simplex Virus Type 1 Expressing Two Additional Copies of gK Is More Pathogenic than Wild-Type Virus in Two Different Strains of Mice. J Virol. 2007;81:12962. doi: 10.1128/JVI.01442-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mott KR, Osorio Y, Brown DJ, Morishige N, Wahlert A, Jester JV, Ghiasi H. The corneas of naive mice contain both CD4+ and CD8+ T cells. Mol Vis. 2007;13:1802. [PubMed] [Google Scholar]
  29. Edelson BT, Bradstreet TR, Kc W, Hildner K, Herzog JW, Sim J, Russell JH, Murphy TL, Unanue ER, Murphy KM. Batf3-dependent CD11b(low/−) peripheral dendritic cells are GM-CSF-independent and are not required for Th cell priming after subcutaneous immunization. PLoS One. 2011;6:e25660. doi: 10.1371/journal.pone.0025660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jackson JT, Hu Y, Liu R, Masson F, D’Amico A, Carotta S, Xin A, Camilleri MJ, Mount AM, Kallies A, Wu L, Smyth GK, Nutt SL, Belz GT. Id2 expression delineates differential checkpoints in the genetic program of CD8alpha+ and CD103+ dendritic cell lineages. EMBO J. 2011;30:2690. doi: 10.1038/emboj.2011.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tussiwand R, Lee WL, Murphy TL, Mashayekhi M, Wumesh KC, Albring JC, Satpathy AT, Rotondo JA, Edelson BT, Kretzer NM, Wu X, Weiss LA, Glasmacher E, Li P, Liao W, Behnke M, Lam SS, Aurthur CT, Leonard WJ, Singh H, Stallings CL, Sibley LD, Schreiber RD, Murphy KM. Compensatory dendritic cell development mediated by BATF-IRF interactions. Nature. 2012;490:502. doi: 10.1038/nature11531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A. The Pfam protein families database. Nucleic Acids Res. 2010;38:D211. doi: 10.1093/nar/gkp985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Williams KL, Nanda I, Lyons GE, Kuo CT, Schmid M, Leiden JM, Kaplan MH, Taparowsky EJ. Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus. Eur J Immunol. 2001;31:1620. doi: 10.1002/1521-4141(200105)31:5<1620::aid-immu1620>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  34. Su ZZ, Lee SG, Emdad L, Lebdeva IV, Gupta P, Valerie K, Sarkar D, Fisher PB. Cloning and characterization of SARI (suppressor of AP-1, regulated by IFN) Proc Natl Acad Sci U S A. 2008;105:20906. doi: 10.1073/pnas.0807975106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schraml BU, Hildner K, Ise W, Lee WL, Smith WA, Solomon B, Sahota G, Sim J, Mukasa R, Cemerski S, Hatton RD, Stormo GD, Weaver CT, Russell JH, Murphy TL, Murphy KM. The AP-1 transcription factor Batf controls T(H)17 differentiation. Nature. 2009;460:405. doi: 10.1038/nature08114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ise W, Kohyama M, Schraml BU, Zhang T, Schwer B, Basu U, Alt FW, Tang J, Oltz EM, Murphy TL, Murphy KM. The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells. Nat Immunol. 2011;12:536. doi: 10.1038/ni.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Kuroda S, Yamazaki M, Abe M, Sakimura K, Takayanagi H, Iwai Y. Basic leucine zipper transcription factor, ATF-like (BATF) regulates epigenetically and energetically effector CD8 T-cell differentiation via Sirt1 expression. Proc Natl Acad Sci U S A. 2011;108:14885. doi: 10.1073/pnas.1105133108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Betz BC, Jordan-Williams KL, Wang C, Kang SG, Liao J, Logan MR, Kim CH, Taparowsky EJ. Batf coordinates multiple aspects of B and T cell function required for normal antibody responses. J Exp Med. 2010;207:933. doi: 10.1084/jem.20091548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mashayekhi M, Sandau MM, Dunay IR, Frickel EM, Khan A, Goldszmid RS, Sher A, Ploegh HL, Murphy TL, Sibley LD, Murphy KM. CD8alpha(+) dendritic cells are the critical source of interleukin-12 that controls acute infection by Toxoplasma gondii tachyzoites. Immunity. 2011;35:249. doi: 10.1016/j.immuni.2011.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Iliev ID, Funari VA, Taylor KD, Nguyen Q, Reyes CN, Strom SP, Brown J, Becker CA, Fleshner PR, Dubinsky M, Rotter JI, Wang HL, McGovern DP, Brown GD, Underhill DM. Interactions between commensal fungi and the C-type lectin receptor Dectin-1 influence colitis. Science. 2012;336:1314. doi: 10.1126/science.1221789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Tanoue T, Umesaki Y, Honda K. Immune responses to gut microbiota-commensals and pathogens. Gut Microbes. 2010;1:224. doi: 10.4161/gmic.1.4.12613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Jarchum I, Pamer EG. Regulation of innate and adaptive immunity by the commensal microbiota. Curr Opin Immunol. 2011;23:353. doi: 10.1016/j.coi.2011.03.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Chinen T, Rudensky AY. The effects of commensal microbiota on immune cell subsets and inflammatory responses. Immunol Rev. 2012;245:45. doi: 10.1111/j.1600-065X.2011.01083.x. [DOI] [PubMed] [Google Scholar]
  44. Gollwitzer ES, Saglani S, Trompette A, Yadava K, Sherburn R, McCoy KD, Nicod LP, Lloyd CM, Marsland BJ. Lung microbiota promotes tolerance to allergens in neonates via PD-L1. Nat Med. 2014;20:642. doi: 10.1038/nm.3568. [DOI] [PubMed] [Google Scholar]
  45. Trompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, Ngom-Bru C, Blanchard C, Junt T, Nicod LP, Harris NL, Marsland BJ. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med. 2014;20:159. doi: 10.1038/nm.3444. [DOI] [PubMed] [Google Scholar]
  46. Khosravi A, Yanez A, Price JG, Chow A, Merad M, Goodridge HS, Mazmanian SK. Gut microbiota promote hematopoiesis to control bacterial infection. Cell Host Microbe. 2014;15:374. doi: 10.1016/j.chom.2014.02.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rock DL, Nesburn AB, Ghiasi H, Ong J, Lewis TL, Lokensgard JR, Wechsler SL. Detection of latency-related viral RNAs in trigeminal ganglia of rabbits latently infected with herpes simplex virus type 1. J Virol. 1987;61:3820. doi: 10.1128/jvi.61.12.3820-3826.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Dobson AT, Margolis TP, Sedarati F, Stevens JG, Feldman LT. A latent, nonpathogenic HSV-1-derived vector stably expresses beta- galactosidase in mouse neurons. Neuron. 1990;5:353. doi: 10.1016/0896-6273(90)90171-b. [DOI] [PubMed] [Google Scholar]

RESOURCES