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Ross River virus (RRV) and other alphaviruses cause chronic musculoskeletal syndromes that are associated with viral persistence, which suggests deficits in immune clearance mechanisms, including CD8 + T-cell responses. Here, we used a recombinant RRV-gp33 that expresses the immunodominant CD8 + T-cell epitope of lymphocytic choriomeningitis virus (LCMV) to directly compare responses with a virus, LCMV, that strongly induces antiviral CD8 + T cells. After footpad injection, we detected fewer gp33-specific CD8 + T cells in the draining lymph node (DLN) after RRV-gp33 than LCMV infection, despite similar viral RNA levels in the foot. However, less RRV RNA was detected in the DLN compared to LCMV, with RRV localizing principally to the subcapsular region and LCMV to the paracortical T-cell zones. Single-cell RNA-sequencing analysis of adoptively transferred gp33-specific transgenic CD8 + T cells showed rapid differentiation into effector cells after LCMV but not RRV infection. This defect in RRV-specific CD8 + T effector cell maturation was corrected by local blockade of type I interferon (IFN) signaling, which also resulted in increased RRV infection in the DLN. Studies in Wdfy4 -/- , CD11c-Cre B2m mice that respectively lack cross-presenting capacity, MHC-I antigen presentation by dendritic cells (DCs), or type I IFN signaling in the DC1 subset show that RRV-specific CD8 fl/fl , or Xcr1-Cre Ifnar1 fl/fl mice that respectively lack cross-presenting capacity, MHC-I antigen presentation by dendritic cells (DCs), or type I IFN signaling in the DC1 subset show that RRV-specific CD8 + T-cell responses can be improved by enhanced direct antigen presentation by DCs. Overall, our experiments suggest that antiviral type I IFN signaling in DCs limits direct alphavirus infection and antigen presentation, which likely delays CD8 + T-cell responses.IMPORTANCEChronic arthritis and musculoskeletal disease are common outcomes of infections caused by arthritogenic alphaviruses, including Ross River virus (RRV), due to incomplete virus clearance. Unlike other viral infections that are efficiently cleared by cytotoxic CD8 + T cells, RRV infection is surprisingly unaffected by CD8 + T cells as mice lacking or having these cells show similar viral persistence in joint and lymphoid tissues. To elucidate the basis for this deficient response, we measured the RRV-specific CD8 + T-cell population size and activation state relative to another virus known to elicit a strong T-cell response. Our findings reveal that RRV induces fewer CD8 + T cells due to limited infection of immune cells in the draining lymph node. By increasing RRV susceptibility in antigen-presenting cells, we elicited a robust CD8 + T-cell response. These results highlight antigen availability and virus tropism as possible targets for intervention against RRV immune evasion and persistence., Competing Interests: M.S.D. is a consultant or advisor for Inbios, Vir Biotechnology, IntegerBio, Moderna, Merck, Bavarian Nordic, GlaxoSmithKline, and Akagera Medicines. The Diamond laboratory has received unrelated funding support in sponsored research agreements from Vir Biotechnology, Moderna, IntegerBio, Bavarian Nordic, and Emergent BioSolutions. more...

Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.) more...

The divergence of the common dendritic cell progenitor 1-3 (CDP) into the conventional type 1 and type 2 dendritic cell (cDC1 and cDC2, respectively) lineages >4,5 is poorly understood. Some transcription factors act in the commitment of already specified progenitors-such as BATF3, which stabilizes Irf8 autoactivation at the +32 kb Irf8 enhancer 4,6 -but the mechanisms controlling the initial divergence of CDPs remain unknown. Here we report the transcriptional basis of CDP divergence and describe the first requirements for pre-cDC2 specification. Genetic epistasis analysis 7 suggested that Nfil3 acts upstream of Id2, Batf3 and Zeb2 in cDC1 development but did not reveal its mechanism or targets. Analysis of newly generated NFIL3 reporter mice showed extremely transient NFIL3 expression during cDC1 specification. CUT&RUN and chromatin immunoprecipitation followed by sequencing identified endogenous NFIL3 binding in the -165 kb Zeb2 enhancer 8 at three sites that also bind the CCAAT-enhancer-binding proteins C/EBPα and C/EBPβ. In vivo mutational analysis using CRISPR-Cas9 targeting showed that these NFIL3-C/EBP sites are functionally redundant, with C/EBPs supporting and NFIL3 repressing Zeb2 expression at these sites. A triple mutation of all three NFIL3-C/EBP sites ablated Zeb2 expression in myeloid, but not lymphoid progenitors, causing the complete loss of pre-cDC2 specification and mature cDC2 development in vivo. These mice did not generate T helper 2 (T H 2) cell responses against Heligmosomoides polygyrus infection, consistent with cDC2 supporting T H 2 responses to helminths 9-11 . Thus, CDP divergence into cDC1 or cDC2 is controlled by competition between NFIL3 and C/EBPs at the -165 kb Zeb2 enhancer., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.) more...

Respiratory infection with vaccinia virus (VacV) elicits robust CD8 + T cell responses that play an important role in host resistance. In the lung, VacV encounters multiple tissue-resident antigen-presenting cell (APC) populations, but which cell plays a dominant role in priming of virus-specific CD8 + effector T cell responses remains poorly defined. We used Batf3 -/- mice to investigate the impact of CD103 + and CD8α + dendritic cell (DC) deficiency on anti-VacV CD8 + T cell responses. We found that Batf3 -/- mice were more susceptible to VacV infection, exhibiting profound weight loss, which correlated with impaired accumulation of gamma interferon (IFN-γ)-producing CD8 + T cells in the lungs. This was largely due to defective priming since early in the response, antigen-specific CD8 + T cells in the draining lymph nodes of Batf3 -/- mice expressed significantly reduced levels of Ki67, CD25, and T-bet. These results underscore a specific role for Batf3-dependent DCs in regulating priming and expansion of effector CD8 + T cells necessary for host resistance against acute respiratory VacV infection. IMPORTANCE During respiratory infection with vaccinia virus (VacV), a member of Poxviridae family, CD8 + T cells play important role in resolving the primary infection. Effector CD8 + T cells clear the virus by accumulating in the infected lungs in large numbers and secreting molecules such as IFN-γ that kill virally infected cells. However, precise cell types that regulate the generation of effector CD8 + T cells in the lungs are not well defined. Dendritic cells (DCs) are a heterogeneous population of immune cells that are recognized as key initiators and regulators of T-cell-mediated immunity. In this study, we reveal that a specific subset of DCs that are dependent on the transcription factor Batf3 for their development regulate the magnitude of CD8 + T cell effector responses in the lungs, thereby providing protection during pulmonary VacV infection., (Copyright © 2018 American Society for Microbiology.) more...

C-type lectin domain family 4, member a4 (Clec4a4) is a C-type lectin inhibitory receptor specific for glycans thought to be exclusively expressed on murine CD8α- conventional dendritic cells. Using newly generated Clec4a4-mCherry knock-in mice, we identify a subset of Clec4a4- expressing eosinophils uniquely localized in the small intestine lamina propria. Clec4a4+ eosinophils evinced an immunomodulatory signature, whereas Clec4a4- eosinophils manifested a proinflammatory profile. Clec4a4+ eosinophils expressed high levels of aryl hydrocarbon receptor (Ahr), which drove the expression of Clec4a4 as well as other immunomodulatory features, such as PD-L1. The abundance of Clec4a4+ eosinophils was dependent on dietary AHR ligands, increased with aging, and declined in inflammatory conditions. Mice lacking AHR in eosinophils expanded innate lymphoid cells of type 2 and cleared Nippostrongylus brasiliensis infection more effectively than did wild-type mice. These results highlight the heterogeneity of eosinophils in response to tissue cues and identify a unique AHR-dependent subset of eosinophils in the small intestine with an immunomodulatory profile. [ABSTRACT FROM AUTHOR] more...