CD8ß Depletion Does Not Prevent Control of Viral Replication or Protection from Challenge in Macaques Chronically Infected with a Live Attenuated Simian Immunodeficiency Virus.

We evaluated the contribution of CD8αß+ T cells to control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8ß-specific mAb CD8ß255R1 selectively depleted CD8αß+ T cells without also depleting non-CD8+ T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8ß255R1, plasma viremia transiently increased coincident with declining peripheral CD8αß+ T cells. Interestingly, plasma viremia returned to predepletion levels even when peripheral CD8αß+ T cells did not. Although depletion of CD8αß+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were 3-fold lower (P = 0.006) in animals that received CD8ß255R1 than in those that received control IgG. It is possible that these residual SIV-specific CD8αß+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8ß255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8ß255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8ß255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8ß255R1 infusion on the immunological composition in cynomolgus macaques, compared to an isotype-matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8ß255R1 administration.IMPORTANCE Studies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αß+) and minor (CD8αα+) populations of CD8+ T cells but additionally deplete non-CD8+ T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8ß-specific depleting mAb CD8ß255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αß+ T cells without removing CD8αα+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8ß255R1 for studying the direct contribution of CD8αß+ T cells in various disease states.

Pulmonary Dendritic Cell Subsets Shape the Respiratory Syncytial Virus-Specific CD8+ T Cell Immunodominance Hierarchy in Neonates.

Young infants are generally more susceptible to viral infections and experience more severe disease than do adults. CD8+ T cells are important for viral clearance, and although often ineffective in neonates they can be protective when adequately stimulated. Using a murine CB6F1/J hybrid model of respiratory syncytial virus (RSV) infection, we previously demonstrated that the CD8+ T cell immunodominance hierarchy to two RSV-derived epitopes, KdM282-90 and DbM187-195, was determined by the age at infection. To determine whether age-dependent RSV-specific CD8+ T cell responses could be modified through enhanced innate signaling, we used TLR4 or TLR9 agonist treatment at the time of infection, which remarkably changed the neonatal codominant response to an adult-like KdM282-90 CD8+ T cell immunodominant response. This shift was associated with an increase in the number of conventional dendritic cells, CD11b+ and CD103+ dendritic cells, in the lung-draining lymph node, as well as increased expression of the costimulatory molecule CD86. The magnitude of the KdM282-90 CD8+ T cell response in TLR agonist-treated neonates could be blocked with Abs against CD80 and CD86. These studies demonstrate the age-dependent function of conventional dendritic cells, their role in determining immunodominance hierarchy, and epitope-specific CD8+ T cell requirements for costimulation, all of which influence the immune response magnitude. The unique impact of TLR agonists on neonatal T cell responses is important to consider for RSV vaccines designed for young infants.

Interleukin-2 inhibits FMS-like tyrosine kinase 3 receptor ligand (flt3L)-dependent development and function of conventional and plasmacytoid dendritic cells.

Steady-state development of plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) requires the ligand for FMS-like tyrosine kinase 3 receptor (flt3L), but little is known about how other cytokines may also control this process. In this study, we show that IL-2 inhibits the development of both pDCs and cDCs from bone marrow cells under flt3L stimulation, by acting on lineage(-) flt3(+) precursors. This inhibition of DC development by IL-2 requires IL-2Rα and IL2Rß. IL-2Rα is specifically expressed in one stage of the DC precursor: the monocyte and DC progenitors (MDPs). Furthermore, more MDPs are found in flt3L-stimulated bone marrow cultures when IL-2 is present, suggesting that IL-2 may be inhibiting DC development at the MDP stage. Consistent with our in vitro findings, we observe that nonobese diabetic (NOD) mice, which express less IL-2 compared with diabetes-resistant NOD.Idd3/5 mice, have more splenic pDCs. Additionally, DCs developed in vitro in the presence of flt3L and IL-2 display reduced ability to stimulate T-cell proliferation compared with DCs developed in the presence of flt3L alone. Although the addition of IL-2 does not increase the apoptosis of DCs during their development, DCs developed in the presence of IL-2 are more prone to apoptosis upon interaction with T cells. Together our data show that IL-2 can inhibit both the development and the function of DCs. This pathway may have implications for the loss of immune tolerance: Reduced IL-2 signaling may lead to increased DC number and T-cell stimulatory capacity.

Increased innate immune activation induces protective RSV-specific lung-resident memory T cells in neonatal mice.

Young infants frequently experience respiratory tract infections, yet vaccines designed to provide mucosal protection are lacking. Localizing pathogen-specific cellular and humoral immune responses to the lung could provide improved immune protection. We used a well-characterized murine model of respiratory syncytial virus (RSV) to study the development of lung-resident memory T cells (TRM) in neonatal compared to adult mice. We demonstrated that priming with RSV during the neonatal period failed to retain RSV-specific clusters of differentiation (CD8) TRM 6 weeks post infection, in contrast to priming during adulthood. The reduced development of RSV-specific TRM was associated with poor acquisition of two key markers of tissue residence: CD69 and CD103. However, by augmenting both innate immune activation and antigen exposure, neonatal RSV-specific CD8 T cells increased expression of tissue-residence markers and were maintained in the lung at memory time points. Establishment of TRM correlated with more rapid control of the virus in the lungs upon reinfection. This is the first strategy to effectively establish RSV-specific TRM in neonates providing new insight into neonatal memory T cell development and vaccine strategies.

Type I IFN ineffectively activates neonatal dendritic cells limiting respiratory antiviral T-cell responses.

Insufficient T-cell responses contribute to the increased burden of viral respiratory disease in infancy. Neonatal dendritic cells (DCs) often provide defective activation of pathogen-specific T cells through mechanisms that are incompletely understood, which hinders vaccine design for this vulnerable age group. Enhancing our characterization of neonatal DC sub-specialization and function is therefore critical to developing their potential for immunomodulation of T-cell responses. In this study, we engineered respiratory syncytial virus (RSV) to express a model protein, ovalbumin, to track antigen-presenting DCs in vivo. We found that murine neonatal conventional DC1s (cDC1s) efficiently migrated and presented RSV-derived antigen, challenging the paradigm that neonatal DCs are globally immature. In a key observation, however, we discovered that during infection neonatal cDC1s presenting viral antigen were unable to upregulate costimulatory molecules in response to type I interferons (IFN-I), contributing to poor antiviral T-cell responses. Importantly, we showed that the deficient response to IFN-I was also exhibited by human neonatal cDC1s, independent of infection. These findings reveal a functionally distinct response to IFN-I by neonatal cDC1s that may leave young infants susceptible to viral infections, and provide a new target for exploration, in light of failed efforts to design neonatal RSV vaccines.

Restoration of the type I IFN-IL-1 balance through targeted blockade of PTGER4 inhibits autoimmunity in NOD mice.

Type I IFN (IFN-I) dysregulation contributes to type 1 diabetes (T1D) development, and although increased IFN-I signals are pathogenic at the initiation of autoimmune diabetes, IFN-I dysregulation at later pathogenic stages more relevant for therapeutic intervention is not well understood. We discovered that 5 key antigen-presenting cell subsets from adult prediabetic NOD mice have reduced responsiveness to IFN-I that is dominated by a decrease in the tonic-sensitive subset of IFN-I response genes. Blockade of IFNAR1 in prediabetic NOD mice accelerated diabetes and increased Th1 responses. Therefore, IFN-I responses shift from pathogenic to protective as autoimmunity progresses, consistent with chronic IFN-I exposure. In contrast, IL-1-associated inflammatory pathways were elevated in prediabetic mice. These changes correlated with human T1D onset-associated gene expression. Prostaglandin E2 (PGE2) and prostaglandin receptor 4 (PTGER4), a receptor for PGE2 that mediates both inflammatory and regulatory eicosanoid signaling, were higher in NOD mice and drive innate immune dysregulation. Treating prediabetic NOD mice with a PTGER4 antagonist restored IFNAR signaling, decreased IL-1 signaling, and decreased infiltration of leukocytes into the islets. Therefore, innate cytokine alterations contribute to both T1D-associated inflammation and autoimmune pathogenesis. Modulating innate immune balance via signals such as PTGER4 may contribute to treatments for autoimmunity.

CD8+ dendritic cell-mediated tolerance of autoreactive CD4+ T cells is deficient in NOD mice and can be corrected by blocking CD40L.

DCs are important mediators of peripheral tolerance for the prevention of autoimmunity. Chimeric αDEC-205 antibodies with attached antigens allow in vivo antigen-specific stimulation of T cells by CD8(+) DCs, resulting in tolerance in nonautoimmune mice. However, it is not clear whether DC-mediated tolerance induction occurs in the context of ongoing autoimmunity. We assessed the role of CD8(+) DCs in stimulation of autoreactive CD4(+) T cells in the NOD mouse model of type 1 diabetes. Targeting of antigen to CD8(+) DCs via αDEC-205 led to proliferation and expansion of ß-cell specific BDC2.5 T cells. These T cells also produced IL-2 and IFN-γ and did not up-regulate FoxP3, consistent with an activated rather than tolerant phenotype. Similarly, endogenous BDC peptide-reactive T cells, identified with I-A(g7) tetramers, did not become tolerant after antigen delivery via αDEC-205: no deletion or Treg induction was observed. We observed that CD8(+) DCs from NOD mice expressed higher surface levels of CD40 than CD8(+) DCs from C57BL/6 mice. Blockade of CD40-CD40L interactions reduced the number of BDC2.5 T cells remaining in mice, 10 days after antigen targeting to CD8 DCs, and blocked IFN-γ production by BDC2.5 T cells. These data indicate that the ability of autoreactive CD4(+) T cells to undergo tolerance mediated by CD8(+) DCs is defective in NOD mice and that blocking CD40-CD40L interactions can restore tolerance induction.

Chimpanzee adenovirus vaccine generates acute and durable protective immunity against ebolavirus challenge.

Ebolavirus disease causes high mortality, and the current outbreak has spread unabated through West Africa. Human adenovirus type 5 vectors (rAd5) encoding ebolavirus glycoprotein (GP) generate protective immunity against acute lethal Zaire ebolavirus (EBOV) challenge in macaques, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans. Here we show that a chimpanzee-derived replication-defective adenovirus (ChAd) vaccine also rapidly induced uniform protection against acute lethal EBOV challenge in macaques. Because protection waned over several months, we boosted ChAd3 with modified vaccinia Ankara (MVA) and generated, for the first time, durable protection against lethal EBOV challenge.