RNA-Sensing Pattern Recognition Receptors and Their Effects on T-Cell Immune Responses: A Dissertation

Virus infection is sensed by the innate immune system through germline encoded pattern recognition receptors (PRRs). Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs) serve as PRRs that recognize different viral components. Microbial nucleic acids such as Ribonucleic acid (RNA) are important virus-derived pathogen-associated molecular patterns (PAMPs) to be recognized by PRRs. Virus recognition may occur at multiple stages of the viral life cycle. Replication intermediates such as single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) are detected by the RNA-sensing PRRs that initiate innate and adaptive immune responses. Triggering of the innate immune system is a critical event that can shape the adaptive immune response to virus infection. Better vaccination strategies that lead to improved T-cell and antibody responses are needed for protection against pathogens. We sought to delineate the RNA-sensing PRR pathways that are activated during infection with an RNA virus, the signaling mediators involved and the influence on subsequent virus-specific adaptive immune responses. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vitro, we performed direct co-stimulation experiments on CD4+ T-cells of high purity. We utilized synthetic RNA-like immune response modifiers (IRMs) R-848 (MyD88-dependent) and poly I:C (MyD88-independent) as RNA PAMPs to determine the direct effects of RNA-sensing PRR activation on CD4+ T-cells. RNA PAMPs can act directly on CD4+ T-cells and modulate their function and phenotype. Maximal direct co-stimulatory effects were observed in CD4+ T-cells cultured with poly I:C compared to R-848. The cytoplasmic dsRNA-dependent protein kinase R (PKR) was also involved in poly I:C-mediated signaling in CD4+ T-cells. We found differences in the RNA-sensing PRRs activated by R-848 between mouse and human CD4+ T-cells. We observed minimal direct co-stimulatory effects by R-848 in mouse CD4+ T-cells. In contrast, augmentation of Th1 responses by R-848 was observed in human CD4+ T-cells. TLR8 activation in human CD4+ T-cells may explain the observed differences. We next explored the signaling pathways activated by RNA PAMPs in conventional dendritic cells (cDCs) and CD4+ T-cells that drive Th1 CD4 T-cell responses in isolated cDC/CD4 T-cell interactions. Allogeneic cDCs and CD4+ T-cells of high purity were cultured together with R-848 and poly I:C in MHC congenic mixed leukocyte reactions (MLRs). R-848 and poly I:C stimulation of type I IFN production and signaling was essential but not sufficient for driving CD4+ Th1 responses. The early production of IL-1α and IL-1β was equally critical. To analyze the role of RNA-sensing PRRs in T-cell immune responses in vivo, we utilized a mouse model of heterosubtypic influenza A virus (IAV) infections. Using MyD88-/-, TLR7-/- and IL-1-deficient mice, we explored the role of MyD88-signaling in the generation of heterosubtypic memory CD4+ T-cell, CD8+ T-cell and antibody responses. We found that MyD88 signaling played an important role in anti-IAV spleen and lung CD4+ T-cell, spleen CD8+ T-cell and Th1 antibody immune responses. Anti-IAV lung heterosubtypic CD8+ T-cell responses were not dependent on MyD88 signaling. Our in vitro and in vivo results show the pivotal role of RNA-sensing PRR pathway activation in T-cell immune responses. Understanding the complexity of the PRR pathways involved during viral infections and defining the subsequent immune response would have important implications for the generation of more effective vaccine strategies

The combination of early and rapid type I IFN, IL-1alpha, and IL-1beta production are essential mediators of RNA-like adjuvant driven CD4+ Th1 responses

There is a growing need for novel vaccine adjuvants that can provide safe and potent T-helper type 1 (Th1) activity. RNA-like immune response modifiers (IRMs) are candidate T-cell adjuvants that skew acquired immune responses towards a Th1 phenotype. We set out to delineate the essential signaling pathways by which the RNA-like IRMs, resiquimod (R-848) and polyinosinic:polycytidylic acid (poly I:C), augment CD4+ T-helper 1 (Th1) responses. Highly purified murine conventional dendritic cells (cDCs) and conventional CD4+ T-cells were co-cultured in allogeneic and MHC congenic mixed leukocyte reactions. The activation of CD4+ Th1 cells was examined utilizing cells from mice deficient in specific RNA-sensing pattern recognition receptors and signaling mediators. R-848 and poly I:C stimulation of Type I interferon production and signaling in cDCs was essential but not sufficient for driving CD4+ Th1 responses. The early and rapid production of IL-1alpha and IL-1beta was equally critical for the optimal activation of Th1 CD4+ T-cells. R-848 activation of Toll-like receptor 7/MyD88-dependent signaling in cDCs led to a rapid upregulation of pro-IL-1alpha and pro-IL-1beta production compared to poly I:C activation of MyD88-independent signaling pathways. The in vitro data show that CD4+ T-cell adjuvant activity of RNA-like IRMs is mediated by a critical combination of early and rapid Type I interferon, IL-1alpha and IL-1beta production. These results provide important insights into the key signaling pathways responsible for RNA-like IRM CD4+ Th1 activation. A better understanding of the critical signaling pathways by which RNA-like IRMs stimulate CD4+ Th1 responses is relevant to the rational design of improved vaccine adjuvants

Plasmacytoid dendritic cell interferon-alpha production to R-848 stimulation is decreased in male infants

BACKGROUND: Sex differences in response to microbial infections, especially viral ones, may be associated with Toll-like receptor (TLR)-mediated responses by plasmacytoid dendritic cells (pDCs). RESULTS: In this study, we identified sex differences in human infant pDC interferon-alpha production following challenge with the TLR7/8 agonist R-848. Male pDC responses were significantly lower than those of females during early infancy. This difference may be attributed to the androgen surge experienced by males during the early infancy period. Pretreatment of human pDCs with dihydrotestosterone produced a significant reduction in interferon-alpha production following R-848 challenge. CONCLUSIONS: Androgen-mediated regulation of pDC TLR7-driven innate immune responses may contribute to the observed sex differences in response to infections during early infancy

The role of MyD88 signaling in heterosubtypic influenza A virus infections

A mouse model of heterosubtypic influenza A virus infections was used to determine the role of MyD88 signaling in CD4+ T-cell, CD8+ T-cell, and IgG immune responses. We found that MyD88 signaling played an important role in anti-influenza A virus heterosubtypic lung and spleen CD4+ T-cell, and spleen CD8+ T-cell, immune responses. MyD88 dependent signaling was important for T-helper 1 cytokine production in anti-influenza A virus lung and spleen heterosubtypic CD4+ T-cells, but not for their frequencies. Toll-like receptor 7 dependent signaling played a partial role in anti-influenza A virus lung heterosubtypic CD4+ T-helper 1 responses and anti-influenza A virus heterosubtypic IgG2c antibody levels. Our results have important implications for the generation of effective universal influenza vaccines

The combination of early and rapid type I IFN, IL-1α, and IL-1β production are essential mediators of RNA-like adjuvant driven CD4+ Th1 responses.

There is a growing need for novel vaccine adjuvants that can provide safe and potent T-helper type 1 (Th1) activity. RNA-like immune response modifiers (IRMs) are candidate T-cell adjuvants that skew acquired immune responses towards a Th1 phenotype. We set out to delineate the essential signaling pathways by which the RNA-like IRMs, resiquimod (R-848) and polyinosinic:polycytidylic acid (poly I:C), augment CD4+ T-helper 1 (Th1) responses. Highly purified murine conventional dendritic cells (cDCs) and conventional CD4+ T-cells were co-cultured in allogeneic and MHC congenic mixed leukocyte reactions. The activation of CD4+ Th1 cells was examined utilizing cells from mice deficient in specific RNA-sensing pattern recognition receptors and signaling mediators. R-848 and poly I:C stimulation of Type I interferon production and signaling in cDCs was essential but not sufficient for driving CD4+ Th1 responses. The early and rapid production of IL-1α and IL-1β was equally critical for the optimal activation of Th1 CD4+ T-cells. R-848 activation of Toll-like receptor 7/MyD88-dependent signaling in cDCs led to a rapid upregulation of pro-IL-1α and pro-IL-1β production compared to poly I:C activation of MyD88-independent signaling pathways. The in vitro data show that CD4+ T-cell adjuvant activity of RNA-like IRMs is mediated by a critical combination of early and rapid Type I interferon, IL-1α and IL-1β production. These results provide important insights into the key signaling pathways responsible for RNA-like IRM CD4+ Th1 activation. A better understanding of the critical signaling pathways by which RNA-like IRMs stimulate CD4+ Th1 responses is relevant to the rational design of improved vaccine adjuvants

Plasmacytoid dendritic cell interferon-α production to R-848 stimulation is decreased in male infants

Abstract Background Sex differences in response to microbial infections, especially viral ones, may be associated with Toll-like receptor (TLR)-mediated responses by plasmacytoid dendritic cells (pDCs). Results In this study, we identified sex differences in human infant pDC interferon-α production following challenge with the TLR7/8 agonist R-848. Male pDC responses were significantly lower than those of females during early infancy. This difference may be attributed to the androgen surge experienced by males during the early infancy period. Pretreatment of human pDCs with dihydrotestosterone produced a significant reduction in interferon-α production following R-848 challenge. Conclusions Androgen-mediated regulation of pDC TLR7-driven innate immune responses may contribute to the observed sex differences in response to infections during early infancy.</p