Secondary Influenza Virus Infections Research Articles

Influenza virus infection selectively triggers the accumulation and persistence of more potent Helios-expressing Foxp3+ regulatory T cells in the lungs.

Foxp3+ regulatory T cells (Tregs) represent a special lineage of CD4+ T cells. Analysis of Treg response during primary and secondary influenza virus infection clearly demonstrates a robust accumulation of Tregs into the infected lungs and the existence of a population of long-lived antigen-specific memory Tregs in the same tissues after resolution of the infection. However, it remains unknown whether these Tregs co-express Helios, a member of the Ikaros transcription factor family. In this study, Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the lungs, mLNs and spleens of influenza virus-infected and uninfected control mice were tracked. The data show that while there is a co-existence of Foxp3+ Helios+ and Foxp3+ Helios- Tregs in the tissues, the accumulated Tregs in the lungs and lung-draining mediastinal lymph nodes (mLNs) of the infected mice are highly enriched for Foxp3+ Helios+ cells. It was further demonstrated that, after the clearance of primary infection, Foxp3+ Helios+ cells have the ability to persist in the tissues over their Helios- counterparts. More importantly, Foxp3+ Helios+ Tregs accumulated in an accelerated kinetics during recall response to reinfection. In vitro analysis of Treg suppressive function revealed that Foxp3+ Helios+ Tregs are more capable of suppressing influenza virus-specific CD8+ T cell activation, cytokine production and proliferation. Together, the data provide new insights into Treg responses during primary and secondary influenza virus infection and suggest that Foxp3+ Helios+ Tregs predominantly drive the Treg responses.

The adjuvant effects of MF59 on antigen-specific regulatory and effector T cells

Abstract Peptide vaccines are an ideal strategy to induce cross-protective T cell immunity against a broad range of influenza virus strains, but they are subimmunogenic and tend to induce regulatory T (Treg) cells. Our previous study has shown that adjuvanted peptide vaccines can suppress the development of antigen-specific Treg cells. MF59 is the first oil-in-water adjuvant approved for protein-based influenza vaccine, which enhances humoral and Th2 cellular immune responses. However, the role of MF59 in peptide-based vaccines regarding the induction of T cell immunity is unknown. We thus investigated the effects of MF59-adjuvanted peptide vaccines on antigen-specific T cell immunity against influenza virus infection. Using OT-I and OT-II TCR transgenic T cells, we found that the MF59-adjuvanted cognate OVA peptide vaccines induced a significant portion of OT-II Treg cells in primary immunization. However, MF59-adjuvanted peptide vaccine could prevent the expansion of pre-existing antigen-specific Treg cells. Of note, primary influenza virus infection induced a negligible portion of antigen-specific Treg cells, but secondary influenza virus infection drove the expansion of pre-existing Treg cells. Interestingly, swapping of the sequential immunizations with MF59-peptide vaccination and influenza virus infection resulted in very different levels of antigen-specific Treg cells, suggesting that the first immunization experience affects the development of antigen-specific Treg cells. Finally, MF59-peptide vaccines stimulated the production of IFN-γ and TNF-α of CD4 and CD8 T cells. In conclusion, MF59-peptide vaccine exhibits a unique feature in stimulation of antigen-specific T cell immunity.

Vaccine-induced antigen-specific regulatory T cells attenuate the antiviral immunity against acute influenza virus infection.

Peptide-based T cell vaccines targeting the conserved epitopes of influenza virus can provide cross-protection against distantly related strains, but they are generally not immunogenic. Foreign antigen-specific regulatory T (Treg) cells are induced under subimmunogenic conditions peripherally, although their development and role in vaccine-mediated antiviral immunity is unclear. Here, we demonstrated primary vaccination with peptides alone significantly induced antigen-specific Foxp3+ Treg cells, which were further expanded by repeated vaccination with unadjuvanted peptides. Certain adjuvants, including CpG, suppressed the induction and expansion of antigen-specific Treg cells by peptide vaccination. Interestingly, secondary influenza virus infection significantly increased the frequency of preexisting antigen-specific Treg cells, although primary infection barely induced them. Importantly, specific depletion of vaccine-induced antigen-specific Treg cells promoted influenza viral clearance, indicating their inhibitory role in vivo. Immunization with CpG-adjuvanted peptides by the subcutaneous prime-intranasal-boost strategy restricted the recruitment and accumulation of antigen-specific Treg cells in lung, and stimulated robust T cell immunity. Finally, subcutaneous prime-intranasal-boost immunization with CpG-adjuvanted peptides or whole-inactivated influenza vaccines protected mice from heterosubtypic influenza virus infection. In conclusion, antigen-specific Treg cells induced by peptide vaccines attenuate the antiviral immunity against influenza virus infection. CpG-adjuvanted peptide vaccines provide heterosubtypic influenza protection probably by inhibiting Treg development and enhancing T cell immunity.

Respiratory Influenza Virus Infection Induces Memory-like Liver NK Cells in Mice.

Although NK cells are classified as innate immune cells, recent studies have demonstrated the transformation of NK cells into long-lived memory cells that contribute to secondary immune responses in certain mouse models. However, whether NK cells mount an Ag-specific memory response to acute influenza virus infection has not yet been examined. Here, we show that, consistent with previous studies, lung NK cells play an important role in controlling viral proliferation after primary influenza virus infection. However, although lung NK cells display a memory phenotype at the late stage of infection, these cells do not protect mice against secondary influenza virus infection. Interestingly, liver NK cells from influenza virus-infected mice possess a memory phenotype and protect mice against secondary influenza virus infection. Memory-like liver NK cells display a CD49a+DX5- phenotype, and the adoptive transfer of purified liver CD49a+DX5- NK cells into naive mice followed by viral infection results in protective immunity and decreased viral titer. Moreover, we demonstrate that primary inactivated influenza virus induces memory NK cells residing in the liver of Rag1-/- mice. Collectively, these data suggest that liver CD49a+DX5- NK cells remember encountered Ag from influenza virus after primary infection and are more protective upon subsequent infection.

Innate Immunity and the Inter-exposure Interval Determine the Dynamics of Secondary Influenza Virus Infection and Explain Observed Viral Hierarchies.

Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus–innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.

Antigen-Specific Memory Regulatory CD4+Foxp3+ T Cells Control Memory Responses to Influenza Virus Infection

Regulatory CD4(+)Foxp3(+) T cells (Tregs) are key regulators of inflammatory responses and control the magnitude of cellular immune responses to viral infections. However, little is known about how Tregs contribute to immune regulation during memory responses to previously encountered pathogens. In this study, we used MHC class II tetramers specific for the 311-325 peptide from influenza nucleoprotein (NP311-325/IA(b)) to track the Ag-specific Treg response to primary and secondary influenza virus infections. During secondary infections, Ag-specific memory Tregs showed accelerated accumulation in the lung-draining lymph node and lung parenchyma relative to a primary infection. Memory Tregs effectively controlled the in vitro proliferation of memory CD8(+) cells in an Ag-specific fashion that was MHC class II dependent. When memory Tregs were depleted before secondary infection, the magnitude of the Ag-specific memory CD8(+) T cell response was increased, as was pulmonary inflammation and airway cytokine/chemokine expression. Replacement of memory Tregs with naive Tregs failed to restore the regulation of the memory CD8 T cell response during secondary infection. Together, these data demonstrate the existence of a previously undescribed population of Ag-specific memory Tregs that shape the cellular immune response to secondary influenza virus challenges and offer an additional parameter to consider when determining the efficacy of vaccinations.

TGF-β dramatically reduces the numbers of short-lived effector CD8 T cells during primary and secondary influenza virus infections. (44.4)

Abstract Highly differentiated CTLs make large quantities of proinflammatory cytokines and contribute to high mortality rates during influenza virus infections. Some pathogens trigger robust responses by short lived effector CD8 T cells (SLECs) which express KLRG1 but not CD127. Other less differentiated CD8 T cells which lack KLRG1 and quickly regain CD127 expression are better equipped to avoid deletion which led to the designation memory precursor cells (MPECs). Recent studies have shown that influenza virus-specific CTLs do not adhere to this pattern, since very small numbers of virus-specific CTLs acquired the KLRG1+CD127- phenotype during the peak of the effector response. We show that exposure to TGF-β during antigen stimulation greatly increases the size of the CTL response and changes the balance between the numbers of MPECs and SLECs during primary and secondary influenza virus infections. We reveal a dichotomy in the response to TGF-β which develops as the virus-specific CTLs become more differentiated. Our data suggest that TGF-β protects the lungs from immune damage through widespread depletion of SLECs, while simultaneously helping less pathogenic virus-specific CTLs colonize the mucosa where they can enhance to response to repeated viral infection.

Memory Treg cells regulate the immune response to influenza virus infection (170.10)

Abstract Regulatory CD4+FoxP3+ T cells (Treg) are key regulators of inflammation and control the magnitude of cellular immune responses to viral infections. However, little is known about Treg regulation of recall responses to previously-encountered pathogens. Here we investigated the influence of Treg on recall responses to influenza virus infection. Utilizing influenza NP311-325/I-Ab Class II tetramers, we tracked the antigen (Ag)-specific Treg during primary and secondary influenza virus infections in C57BL/6 mice. Ag-specific Treg accumulated in the lung-draining lymph node and lung parenchyma during secondary virus infection with accelerated kinetics relative to primary influenza infection, suggesting the existence of a “memory” Treg pool. These Treg contributed to the control of in vivo responses to secondary influenza infection by limiting the magnitude of the Ag-specific memory CD8+ T cell recall response and by contributing to the control of pulmonary inflammation as well as airway chemokine/cytokine expression. In vitro studies verified that memory Treg cells can effectively control the proliferation of memory CD8+ T cells in an Ag-specific fashion, and that this regulatory function was MHC class II dependent. Together, these data demonstrate a previously unappreciated role for Treg in shaping the cellular immune response to secondary influenza virus challenges and need to be considered during vaccine development.

Pre-existing viral antigens change the dynamics of CTL response to secondary influenza virus infection (105.24)

Abstract Cytotoxic T lymphocytes (CTL) can provide immunity between different strains of influenza virus, but the immunity declines over the course of a few months and does not correlate with the number of surviving virus-specific memory CD8 T cells. We previously found that processed peptide antigens were presented to naïve CD8 T cells in the draining lymph nodes of the respiratory tract until ~2 months after primary influenza virus infection, which helped some activated virus-specific CTL colonize the lung airways between repeated infections. Similar results were obtained with multiple strains of influenza virus, which indicates that antigen-persistence is a common characteristic of this pathogen. Confocal imaging has been used to examine the distribution of virus-specific CTL in vivo as they response to reinfection in the presence and absence of the pre-existing viral peptides. Our data show that residual peptides alter the local inflammatory environment and augment the response to reinfection. The gradual disappearance of the viral peptides may account for the early loss of heterosubtypic immunity.

Properties of murine CD8+CD27- T cells

In humans, loss of CD27 expression is associated with the stable acquisition of effector functions by CD8+ T cells. We found that murine (CD8+)CD27- T cells were confined to the primed CD62L(dull/-)CD44(bright)CCR7- T cell population. (CD8+)CD27- T cells were absent from lymph nodes but could be found in blood, spleen and in non-lymphoid organs such as lung and liver. Late after primary influenza virus infection, low percentages of antigen-specific CD27- cells emerged in the lung and spleen. After recovery from secondary influenza virus infection, high percentages of influenza-specific CD27- T cells were found in the lung and the loss of CD27 on lung CD8+ T cells coincided with high granzyme B expression. After murine cytomegalovirus infection, loss of CD27 expression on virus-specific CD8+ T cell populations was sustained and especially marked in liver and lung. We suggest that in mice, CD27 is lost from CD8+ T cells only after repetitive antigenic stimulation. Moreover, the high expression of both granzyme B and perforin in the CD27- T cells suggests that the lack of CD27 on murine CD8+ T cells can be used to identify memory T cells with expression of cytotoxic effector molecules.

Differential antigen presentation regulates the changing patterns of CD8+ T cell immunodominance in primary and secondary influenza virus infections.

The specificity of CD8+ T cell responses can vary dramatically between primary and secondary infections. For example, NP366–374/Db- and PA224–233/Db-specific CD8+ T cells respond in approximately equal numbers to a primary influenza virus infection in C57BL/6 mice, whereas NP366–374/Db-specific CD8+ T cells dominate the secondary response. To investigate the mechanisms underlying this changing pattern of immunodominance, we analyzed the role of antigen presentation in regulating the specificity of the T cell response. The data show that both dendritic and nondendritic cells are able to present the NP366–374/Db epitope, whereas only dendritic cells effectively present the PA224–233/Db epitope after influenza virus infection, both in vitro and in vivo. This difference in epitope expression favored the activation and expansion of NP366–374/Db-specific CD8+ memory T cells during secondary infection. The data also show that the immune response to influenza virus infection may involve T cells specific for epitopes, such as PA224–233/Db, that are poorly expressed at the site of infection. In this regard, vaccination with the PA224–233 peptide actually had a detrimental effect on the clearance of a subsequent influenza virus infection. Thus, differential antigen presentation impacts both the specificity of the T cell response and the efficacy of peptide-based vaccination strategies.