All animal experiments were conformed to the guidelines of the animal ethics committee at National Institute of Immunology (NII) (IAEC approval# 536/19) and biosafety committee guidelines (IBSC approval# 413/20). IRF8^−/− and IRF8^R294C mice were procured from Jackson Laboratory. IRF8^R294C mice were crossed with C57BL/6 wild-type female for at least three generations to eliminate the vertical transmission of Murine Leukemia Virus (MuLV). Progenies were crossed to generate the mice carrying homozygous alleles for the mutation, and all the mice were used at the age of 8–12 weeks. Bone marrow mononuclear cells were isolated from the femur and tibia and cultured in the presence of FLT3L (100 ng/ml, PeproTech) as described previously (51). After day 8, loosely adherent cells were harvested and used for different treatments, stimulations, and experiments. For stimulation with CpG (1826 ODN, Merck), as employed earlier (23, 27, 43), cells were stimulated with 1 μg/ml CpG for the mentioned time period and were analyzed for transcript or proceeded to flow cytometry. For the 7-AAD experiment, cells were harvested after mentioned time point and stained with different fluorochrome conjugated antibodies and finally with 7-AAD before analyzing through flow cytometry. NDV (LaSota strain) was a kind gift from Dr. Himanshu Kumar (IISER Bhopal). NDV infections were set up in serum-free media for 90 min at MOI 1 followed by replenishment with fresh culture media for the mentioned time period after washing with PBS. DCs were stained for surface markers with the following immunophenotyping antibodies: anti-B220 FITC (RA3-6B2), anti-CD11c APC (N418), anti-CD24 PE (M1/69), anti-CD80 V450 (16-10A1), anti-CD8α PE (53-6.7), anti-CD4 APC Cy7 (GK1.5), anti-Sirpα BV421 (P84) (all from BD biosciences); anti-CD40 FITC (HM40-3) (from eBioscience); anti-CD86 PE (GL-1) (from BioLegend), and biotin-labeled anti-SiglecH (551.3D3) (Miltenyi Biotech). Events were captured in FACS CantoII using FACS Diva software, and data were analyzed using Flowjo software (version 9.3.2).

HEK293T and U2OS cells were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 50 μg/ml streptomycin. Retroviral vectors expressing murine IRF8^WT (wild type) and IRF8^R294C were generated, and HEK293T cells were seeded in six-well dish for retroviral transduction with viral supernatants and 4 μg/ml polybrene by spinoculation as described previously (52). Transduced cells were then used for further experiments.

Spleens were harvested from 8- to 12-week-old mice, and splenocytes were isolated. Briefly, dendritic cell-enriched, low-density cell fraction from spleen digested by Liberase (Roche laboratory) was isolated using 30% BSA in PBS as described previously (25). The isolated cells were stained using anti-CD11c, anti-B220, anti-CD8α, anti-CD4 (all from BD biosciences), and biotin-labeled anti-SiglecH (Miltenyi Biotech) antibodies; and CD11c⁺B220⁺SiglecH⁺ pDCs were sorted using FACS Aria III cell sorter. Sorted cells were lysed in RNAiso Plus (Takara), and RNA was isolated using RNeasy mini kit (Qiagen). After the QC procedures, mRNA was enriched using oligo(dT) beads. Then, the mRNA was fragmented randomly by adding fragmentation buffer, and cDNA was synthesized using mRNA template and random hexamer primers, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNaseH, and DNA polymerase I were added to initiate the second-strand synthesis. Followed by terminal repair, A ligation, and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment. Finally, RNA sequencing was carried out in Illumina platform (150 bp paired end), and generated paired end reads were used for downstream analysis. Raw data QC was carried out to check data quality. The quality trimmed reads were aligned using HISAT2 aligner (v.2.1.0) and mapped to GRCm39 as reference genome. HTSeq-count (v.0.12.4) was utilized to generate read count, and DESeq2 (v.1.30.1) R-package was employed for differential gene expression analysis.

RNA was isolated using Nucleospin RNA isolation kit (Machery-Nagel), and cDNA was prepared using high-capacity reverse transcription kit (Applied Biosystems) according to the manufacturer’s protocol. cDNA was diluted appropriately, and qRT-PCR was performed using SYBR Green PCR master mix and 7500 Fast RT PCR machine (Applied Biosystems). Relative mRNA expression was calculated using delta Ct method, and to normalize the variability in expression level, Gapdh was used as internal control. Primers used for qRT-PCR are listed in Supplementary Table 1 .

U2OS cells seeded in 24-well plates were co-transfected with 100 ng of reporter plasmid pGL4.20-Luc (Blank), pGL4.20-Ifnβ-Luc containing promoter region of murine Ifnb promoter alone (control), pGL4.20-Ifnα6-Luc containing promoter region of murine Ifna6 promoter alone (control) or along with pcDNA-IRF8^WT or IRF8^R294C (300ng) expressing plasmids. Transfection was carried out using Lipofectamine 2000 according to the manufacturer’s protocol and as transfection control Renilla luciferase plasmid pRL-TK (Promega, 50 ng) was used. Cells were harvested 24 h post-transfection, and protein activity of firefly and Renilla luciferase was quantified using dual-luciferase reagents (Promega) and Sirius Luminometer V3.2. Renilla luciferase was used to normalize firefly luciferase activity.

NDV stock was prepared in embryonated chicken eggs and titrated by hemagglutination assay as described previously (53) in Dr. Himanshu Kumar’s laboratory in IISER Bhopal. Then 1,200 hemagglutination assay (HA) unit of NDV was used for the infection where HA unit denotes the HA titer of the virus calculated as reciprocal of highest dilution at which complete agglutination of RBC was observed in hemagglutination assay. Eight- to 12-week-old mice were injected with PBS (control) or 1,200 HA NDV intravenously and kept for 8 h. Then blood was collected and spleens were harvested. DC-enriched low-density cell fraction from spleen was isolated as mentioned above. Blood was centrifuged at 2,000 g for 15 min, and serum was collected and stored for further analysis.

FLDC culture supernatants after 24 h treatment or serum from in vivo experiment were subjected to IFN-α1 and IFN-β ELISA using Abcam IFN-α1 (ab252352) and Abcam IFN-β (ab252363) ELISA kits respectively according to the manufacturer’s protocol.

Statistical analysis was performed using unpaired Student’s t test, and significance is represented as *p < 0.05, **p < 0.01, ***p < 0.001, and ns means non-significant.

Numerous studies established that Irf8 is a key transcription factor for cDC1 and pDC development (1, 25, 30, 54). The characterization of splenic DCs and FLDC cultures from Irf8^−/− mice exhibited complete absence of both cDC1 and pDC populations; however, Irf8^R294C mutation selectively abrogated cDC1 development, without affecting that of pDC ( Supplementary Figures 1A, B ) in coherence with previous studies (30, 34, 54). This intriguing phenomenon of selective loss of cDC1 population in IRF8^R294C but retention of pDC population led us to investigate whether Irf8 plays a differential role in cDC1 and pDC development and function. We found that transcript level of Irf8 is elevated in pDCs from IRF8^R294C mice in comparison with IRF8^WT (wild type) mice ( Supplementary Figure 1C ). The increased level of Irf8 transcript in IRF8^R294C pDCs led us to further explore the role of IRF8 in pDC biology.

To investigate this, we sorted pDCs (CD11c⁺ B220⁺ SiglecH⁺) from IRF8^WT and IRF8^R294C mice spleen, carried out transcriptome analysis, and studied differentially expressed genes ( Supplementary Figure 2A ). Principal component analysis (PCA) of RNA-seq data showed differential segregation of clusters of IRF8^WT and IRF8^R294C pDCs ( Figure 1A ). Additional analysis demonstrated a total of 3,494 genes were differentially expressed between these two groups after adjusting the p value cutoff <0.05. Further setting the next cutoff parameter of Log₂FC value implemented at <=−1 or >=1, the number of differentially regulated genes were reduced to 2,542. Out of 2,542 genes, 898 genes were found to be upregulated in IRF8^WT and the rest of the genes (1,644) were upregulated in IRF8^R294C. To visualize gene expression data sets, the two-dimensional (2D) scatter MA plot (Bland Altman plot, “M” denotes log ratio and “A” denotes mean average) was constructed ( Figure 1B ). Global gene expression values, which were obtained from analysis of IRF8^WT and IRF8^R294C transcriptomic data, were represented in volcano plot ( Figure 1C ).

To further understand the ontological features of differentially regulated genes, we implemented KEGG pathway analysis on divergently upregulated ( Figure 1D ) and downregulated ( Supplementary Figure 2B ) genes in IRF8^WT pDCs. Genes significantly enriched in KEGG pathway analysis included those involved in different pathways related to immune response and viral infections such as antigen processing and presentation, human cytomegalovirus infection, human immunodeficiency virus 1 infection, etc., and genes regulating essential signaling mechanisms such as MAPK signaling pathway, calcium signaling pathway, Ras signaling pathway, etc. Genes that showed differential expression in IRF8^WT and IRF8^R294C pDCs were represented in heatmaps ( Supplementary Figures 2C, D and Figure 1E ). Intriguingly, among various genes, the gene Ifnb1 (also known as Ifnb), which encodes interferon β (IFN-β), was found to be significantly downregulated in pDCs from IRF8^R294C than IRF8^WT mice ( Figures 1C, E ). IFN-β, a key cytokine belonging to type I IFN family, plays pivotal role in pDC biology and is well established for its indispensability in mediating antiviral immune response (12, 13). Also, Ifnl2, which encodes interferon lambda 2 (also known as IL28A), was found to be downregulated in IRF8^R294C ( Figure 1E ). IFNL2, distantly related to type I IFN and IL10 family, is important for host defense from antiviral challenge and is involved in antitumor activity (55–58). From these analyses, we can clearly infer that although the point mutation Irf8^R294C does not affect development of pDC subset, it results in significant changes at the transcriptome level.

Since Irf8 is a fundamental transcription factor involved in DC development and the fact that pDCs, which are the key producers of type I IFN, are showing decreased level of Ifnb in IRF8^R294C mice strongly suggest the probable impact of this specific point mutation in changing the pDC transcriptome and immune response. So, in IRF8^R294C pDCs, the decreased expression of Ifnb further prompted us to investigate the effect of Irf8^R294C mutation in immune response to gain mechanistic insight of the expression and regulation of downstream signaling molecules.

First, to confirm observed reduction of Ifnb expression in RNA-seq data, we determined the transcript level of Ifnb from FLDC cultures derived from IRF8^WT and IRF8^R294C mice by qRT-PCR and found that indeed Ifnb was downregulated in IRF8^R294C compared to its wild-type counterpart ( Figure 2A ). Next, we wanted to examine the impact of Irf8^R294C mutation on type I IFN production upon CpG stimulation (TLR9 agonist) in FLDC cultures as pDCs induce type I IFN transcription upon encountering CpG by TLR9. It was found that induction of both Ifnb and Ifna (type I IFN) transcripts were greatly reduced in IRF8^R294C DCs post-stimulation ( Figures 2B, C ). As pDCs play a major role in initiating antiviral response through type I IFN production, we wanted to determine the type I IFN production upon viral infection. To address this, FLDC cultures from IRF8^WT and IRF8^R294C mice were infected by a single-stranded RNA virus—Newcastle disease virus (NDV). NDV infection in IRF8^WT led to marked augmentation of type I IFN transcript level, whereas in IRF8^R294C there was abrogation of type I IFN expression ( Figures 2D, E ), which was consistent with the result from CpG stimulation.

Further, defective induction of type I IFN transcripts in IRF8^R294C FLDCs was concomitant with increased NDV burden in comparison with IRF8^WT ( Figure 2F ). This observation was further confirmed in the protein level by ELISA as we observed IFN-β and IFN-α1 were greatly declined in FLDC culture supernatants from IRF8^R294C mice compared to IRF8^WT upon CpG stimulation as well as NDV infection ( Figures 2G, H ). So, these experiments infer that the pDCs from IRF8^R294C mice are defective in type I IFN production. Further, we wanted to ascertain the effect of this mutation on the time kinetics of type I IFN production upon immune challenge. FLDCs from IRF8^R294C mice stimulated with CpG were found to be defective in type I IFN production starting from early time points to late time points ( Figures 2I, J ). Next, we examined time kinetics of type I IFN production in FLDCs infected with NDV and found that in this scenario also, type I IFN production was greatly hindered in IRF8^R294C in comparison with IRF8^WT ( Figures 2K, L ). In summary, these data conclude that IRF8^R294C pDCs are defective in type I IFN production from very early time points to late time points.

We speculated that the defect of type I IFN production in IRF8^R294C FLDCs may be explained by either increased cell death in IRF8^R294C or due to defect in pDC transcriptional program regulated by IRF8 or any other plausible mechanism mediated by the point mutation Irf8^R294C . To examine if the decreased production of type I IFN was due to increased cell death in IRF8^R294C, we stimulated FLDCs derived from IRF8^WT and IRF8^R294C mice with CpG for 6 h and 24 h and stained the treated as well as untreated cells with viability dye 7-AAD. Flow cytometric analysis revealed that the live pDC percentages were similar in IRF8^R294C with that of IRF8^WT mice under untreated as well as CpG stimulated condition for both 6 h and 24 h time points ( Figures 3A–D ). This experiment clearly refuted the hypothesis that reduced type I IFN production in IRF8^R294C was due to increased cell death.

Next, we wanted to decipher if this impairment of type I IFN production in IRF8^R294C pDCs was an effect of indirect regulation of other pDC-specific factors or due to any mechanism mediated by mutation Irf8^R294C itself. RIG-I like receptor (RLR) is a family of cytosolic pathogen recognition receptor (PRR) which recognizes viral RNA and induces type I IFN production (59). Fibroblasts are known to mediate antiviral response through RLR signaling (60), and mechanism for sensing NDV in HEK293T by RLR differs from that of pDCs mediated by TLR. HEK293T cell line was employed previously for studying interferon induction in response to viral infection (61). Hence, to further investigate, we infected HEK293T cells stably expressing IRF8^WT, IRF8^R294C separately with NDV, studied time kinetics of type I IFN transcript level ( Supplementary Figures 3A, B ) and found that type I IFN induction was curtailed in IRF8^R294C-expressing cells. This experiment indicated that point mutation Irf8^R294C directly affects the type I IFN production.

Next, we wanted to investigate the mechanism and the downstream effect of impaired type I IFN production. We hypothesized that the inability of type I IFN production in IRF8^R294C could be due to ineffective type I IFN promoter activation. Through promoter assay we demonstrated that, IRF8^WT induced Ifnb and Ifna6 promoters, but induction of Ifnb and Ifna6 promoters was defective in IRF8^R294C-expressing cells ( Figures 3E, F ). This observation suggested that inefficient promoter activation by IRF8^R294C translated to abrogated type I IFN production. Upon generation, type I IFN further promotes transcription of other effector molecules of several downstream signaling cascades which carry out different aspects of immune response (14). So, we speculated the defective type I IFN production may result in ineffective downstream signaling subsequently. We observed that, indeed, the production of ISGs was declined significantly in FLDCs from IRF8^R294C mice stimulated with CpG compared to IRF8^WT ( Figure 4A ). Similar observations were made in FLDCs from IRF8^R294C mice infected with NDV ( Figure 4B ). IRF7 plays an important role in type I IFN-mediated signaling pathway and is known to be one of the major regulators of the pathway (59). IFIT3 and other ISGs—OAS3, OAS2, OAS1a, OAS1g, OASL2—which are 2’-5’ oligoadenylate synthetases, are involved in mounting antiviral innate immune response by restricting viral replication (62–64). Overall, these results suggest that the reduced expression of ISGs reflects the inability in induction of type I IFNs, and reduced production of these ISGs due to impaired type I IFN production in IRF8^R294C may further compromise the response to CpG stimulation and NDV infection.

pDCs in resting state express the costimulatory molecules in low level, but once pDCs are activated, they upregulate several costimulatory molecules (CD40, CD86, CD80) and initiate a cascade of activities leading to instigating adaptive immune response (13, 65, 66). So, to characterize pDC activation, we next assessed the upregulation of costimulatory molecules in pDCs from IRF8^WT and IRF8^R294C mice stimulated with CpG or infected with NDV. In comparison to IRF8^WT, the IRF8^R294C pDCs displayed significant partial but not complete reduction in upregulation of CD40, CD86, and CD80 expression upon CpG stimulation as well as NDV infection ( Figures 5A–F ). Taken together, these experiments suggest that Irf8^R294C point mutation renders pDCs defective in activation as the consequence of impaired type I IFN induction, leading to deficient expression of costimulatory molecules.

To corroborate our in vitro studies, we infected both IRF8^WT and IRF8^R294C mice with NDV ( Figure 6A ) and investigated type I IFN induction, NDV transcript, and status of costimulatory molecules in spleen, as spleen is the major site to elicit successful antiviral immune response against systemic infection (67, 68). There was significant impairment in production of type I IFN transcripts in NDV-infected IRF8^R294C mice splenocytes compared to IRF8^WT counterpart ( Figures 6B, C ). Further, the absence of type I IFN transcript augmentation was associated with increased viral burden in IRF8^R294C mice splenocytes ( Figure 6D ). Next, we examined the concentration of IFN-α1 and IFN-β in sera by ELISA. Consistent with increased transcript levels, the upregulation of serum IFN-α1 and IFN-β in IRF8^WT mice infected with NDV but not IRF8^R294C counterpart bolstered that our observation from in vitro system indeed translated to in vivo infection condition ( Figures 6E, F ). Next, we examined the activation status of splenic pDCs from infected and uninfected mice. Flow cytometric analysis of splenic pDCs further revealed that IRF8^WT mice infected with NDV showed significant upregulation of all the costimulatory molecules—CD40, CD86, CD80 ( Figures 7A–F ). In IRF8^R294C mice, upregulation of CD40, CD86, and CD80 in pDCs was compromised upon NDV infection. Also, IRF8^R294C pDCs, in unstimulated condition, showed lower expression of CD80 than wild-type counterpart in contrast to previous study in Irf8^fl/flItgax-cre mice (43), suggesting the difference in IRF8^R294C pDCs carrying mutant IRF8 as against its deletion in Irf8^fl/flItgax-cre counterpart. We also compared the MFI of these costimulatory molecules to study the average expression of these markers on each cell ( Supplementary Figures 4A–C ). Expression of CD40, CD80, and CD86 displayed clear shift in histogram of respective markers in IRF8^WT mice infected with NDV than all the other experimental groups ( Figure 7G ). Collectively, these experiments indicate that decreased type I IFN production in IRF8^R294C mice further leads to immune suppression.

Conclusively, these results infer that the inability of mutant IRF8^R294C to induce type I IFN promoters translates to impaired type I IFN production, which further abrogates production of ISGs and impairs pDC activation, ultimately leading to increased viral burden and futile immune response ( Figure 8 ). Taken together, our study unravels the important role of IRF8 in mediating a successful immune response by controlling pDC function and the differential role played by IRF8 with respect to the cDC1 and pDC subsets. While in cDC1 subset, IRF8 controls the development and differentiation, in pDC subset, IRF8 controls the functionality, and Irf8^{R294 C} mutation greatly changes the functions mediated by IRF8.