Type I interferons regulate cytolytic activity of memory CD8(+) T cells in the lung airways during respiratory virus challenge.

Memory CD8(+) T cells in the lung airways provide protection from secondary respiratory virus challenge by limiting early viral replication. Here, we demonstrate that although airway-resident memory CD8(+) T cells were poorly cytolytic, memory CD8(+) T cells recruited to the airways early during a recall response showed markedly enhanced cytolytic ability. This enhanced lytic activity did not require cognate antigen stimulation, but rather was dependent on STAT1 transcription factor signaling through the interferon-alpha receptor (Ifnar1), resulting in the antigen-independent expression of granzyme B protein in both murine and human virus-specific T cells. Signaling through Ifnar1 was required for the enhanced lytic activity and control of early viral replication by memory CD8(+) T cells in the lung airways. These findings demonstrate that innate inflammatory signals act directly on memory T cells, enabling them to rapidly destroy infected host cells once they enter infected tissues.

Correction to: Virtual memory cells make a major contribution to the response of aged influenza-naïve mice to influenza virus infection.

[This corrects the article DOI: 10.1186/s12979-018-0122-y.].

Virtual memory cells make a major contribution to the response of aged influenza-naïve mice to influenza virus infection.

BACKGROUND: A diverse repertoire of naïve T cells is thought to be essential for a robust response to new infections. However, a key aspect of aging of the T cell compartment is a decline in numbers and diversity of peripheral naïve T cells. We have hypothesized that the age-related decline in naïve T cells forces the immune system to respond to new infections using cross-reactive memory T cells generated to previous infections that dominate the aged peripheral T cell repertoire. RESULTS: Here we confirm that the CD8 T cell response of aged, influenza-naïve mice to primary infection with influenza virus is dominated by T cells that derive from the memory T cell pool. These cells exhibit the phenotypic characteristics of virtual memory cells rather than true memory cells. Furthermore, we find that the repertoire of responding CD8 T cells is constrained compared with that of young mice, and differs significantly between individual aged mice. After infection, these virtual memory CD8 T cells effectively develop into granzyme-producing effector cells, and clear virus with kinetics comparable to naïve CD8 T cells from young mice. CONCLUSIONS: The response of aged, influenza-naive mice to a new influenza infection is mediated largely by memory CD8 T cells. However, unexpectedly, they have the phenotype of VM cells. In response to de novo influenza virus infection, the VM cells develop into granzyme-producing effector cells and clear virus with comparable kinetics to young CD8 T cells.

The chemokine receptor CCR5 plays a key role in the early memory CD8+ T cell response to respiratory virus infections.

Innate recognition of invading pathogens in peripheral tissues results in the recruitment of circulating memory CD8(+) T cells to sites of localized inflammation during the early phase of a recall response. However, the mechanisms that control the rapid recruitment of these cells to peripheral sites are poorly understood, particularly in relation to influenza and parainfluenza infections of the respiratory tract. In this study, we demonstrate a crucial role for C-C chemokine receptor 5 (CCR5) in the accelerated recruitment of memory CD8(+) T cells to the lung airways during virus challenge. Most importantly, CCR5 deficiency resulted in decreased recruitment of memory T cells expressing key effector molecules and impaired control of virus replication during the initial stages of a secondary response. These data highlight the critical importance of early memory T cell recruitment for the efficacy of cellular immunity in the lung.

CD4 T cells specific for a latency-associated γ-herpesvirus epitope are polyfunctional and cytotoxic.

The oncogenic γ-herpesviruses EBV and Kaposi sarcoma-associated herpesvirus are ubiquitous human pathogens that establish lifelong latent infections maintained by intermittent viral reactivation and reinfection. Effector CD4 T cells are critical for control of viral latency and in immune therapies for virus-associated tumors. In this study, we exploited γHV68 infection of mice to enhance our understanding of the CD4 T cell response during γ-herpesvirus infection. Using a consensus prediction approach, we identified 16 new CD4 epitope-specific responses that arise during lytic infection. An additional epitope encoded by the M2 protein induced uniquely latency-associated CD4 T cells, which were not detected at the peak of lytic infection but only during latency and were not induced postinfection with a latency-deficient virus. M2-specific CD4 T cells were selectively cytotoxic, produced multiple antiviral cytokines, and sustained IL-2 production. Identification of latency-associated cytolytic CD4 T cells will aid in dissecting mechanisms of CD4 immune control of γ-herpesvirus latency and the development of therapeutic approaches to control viral reactivation and pathology.

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.

Persistent loss of IL-27 responsiveness in CD8+ memory T cells abrogates IL-10 expression in a recall response.

CD8+ T cells are central to the eradication of intracellular pathogens, but they can also act to limit inflammation and immunopathology. During primary respiratory viral infection CD8+ effector T cells release the immunosuppressive cytokine IL-10, which is essential for host survival. Here we report that CD8+ T-cell-derived IL-10 is absent in a recall response. We show in mice that the lack of IL-10 is due to a persistent loss of IL-27 responsiveness in CD8+ memory T cells, caused by down-regulation of the common cytokine receptor, glycoprotein 130. CD8+ memory T cells secreted less IL-10 when activated in the presence of IL-27 than did naïve controls, and retroviral expression of glycoprotein 130 restored IL-10 and reduced IFN-γ production upon restimulation. We demonstrate that human CD8+ memory cells are also characterized by impaired IL-27 responsiveness. Our data suggest that CD8+ T-cell activation involves a persistent loss of specific cytokine receptors that determines the functional potential of these cells during rechallenge infection.

Zika purified inactivated virus (ZPIV) vaccine reduced vertical transmission in pregnant immunocompetent mice.

Zika virus (ZIKV) is a significant threat to pregnant women and their fetuses as it can cause severe birth defects and congenital neurodevelopmental disorders, referred to as congenital Zika syndrome (CZS). Thus, a safe and effective ZIKV vaccine for pregnant women to prevent in utero ZIKV infection is of utmost importance. Murine models of ZIKV infection are limited by the fact that immunocompetent mice are resistant to ZIKV infection. As such, interferon-deficient mice have been used in some preclinical studies to test the efficacy of ZIKV vaccine candidates against lethal virus challenge. However, interferon-deficient mouse models have limitations in assessing the immunogenicity of vaccines, necessitating the use of immunocompetent mouse pregnancy models. Using the human stat2 knock-in (hSTAT2KI) mouse pregnancy model, we show that vaccination with a purified formalin-inactivated Zika virus (ZPIV) vaccine prior to pregnancy successfully prevented vertical transmission. In addition, maternal immunity protected offspring against postnatal challenge for up to 28 days. Furthermore, passive transfer of human IgG purified from hyper-immune sera of ZPIV vaccinees prevented maternal and fetal ZIKV infection, providing strong evidence that the neutralizing antibody response may serve as a meaningful correlate of protection.

Sequential Early-Life Infections Alter Peripheral Blood Transcriptomics in Aging Female Mice but Not the Response to De Novo Infection with Influenza Virus or M. tuberculosis.

To determine the impact of accumulating Ag exposure on immunity in the aging mouse, and to develop a model more relevant to humans who are exposed to multiple pathogens during life, we sequentially infected young female mice with four distinct pathogens at 8-wk intervals: murine γ-herpesvirus 68, Sendai virus, murine CMV, and Heligmosomoides polygyrus. Mock-infected mice received PBS. After aging the sequentially infected and mock-infected mice to 18-25 mo under specific pathogen-free conditions, we analyzed multiple immune parameters. We assessed transcriptional activity in peripheral blood, T cell phenotype, the diversity of influenza epitopes recognized by CD8 T cells, and the response of the animals to infection with influenza virus and Mycobacterium tuberculosis. Our data show enhanced transcriptional activation in sequentially infected aged mice, with changes in some CD8 T cell subsets. However, there was no measurable difference in the response of mock-infected and sequentially infected aged mice to de novo infection with either influenza virus or M. tuberculosis at 18-21 mo. Unexpectedly, a single experiment in which 25-mo-old female mice were challenged with influenza virus revealed a significantly higher survival rate for sequentially infected (80%) versus mock-infected (20%) mice. These data suggest that although exposure to a variety of pathogen challenges in the mouse model does not overtly impact cellular markers of immunity in aged female mice following de novo respiratory infection, subtle changes may emerge in other compartments or with increasing age.

Impact of prior dengue virus infection on Zika virus infection during pregnancy in marmosets.

Zika virus (ZIKV) infection during pregnancy causes severe developmental defects in newborns, termed congenital Zika syndrome (CZS). Factors contributing to a surge in ZIKV-associated CZS are poorly understood. One possibility is that ZIKV may exploit the antibody-dependent enhancement of infection mechanism, mediated by cross-reactive antibodies from prior dengue virus (DENV) infection, which may exacerbate ZIKV infection during pregnancy. In this study, we investigated the impact of prior DENV infection or no DENV infection on ZIKV pathogenesis during pregnancy in a total of four female common marmosets with five or six fetuses per group. The results showed that negative-sense viral RNA copies increased in the placental and fetal tissues of DENV-immune dams but not in DENV-naïve dams. In addition, viral proteins were prevalent in endothelial cells, macrophages, and neonatal Fc receptor-expressing cells in the placental trabeculae and in neuronal cells in the brains of fetuses from DENV-immune dams. DENV-immune marmosets maintained high titers of cross-reactive ZIKV-binding antibodies that were poorly neutralizing, raising the possibility that these antibodies might be involved in the exacerbation of ZIKV infection. These findings need to be verified in a larger study, and the mechanism involved in the exacerbation of ZIKV infection in DENV-immune marmosets needs further investigation. However, the results suggest a potential negative impact of preexisting DENV immunity on subsequent ZIKV infection during pregnancy in vivo.

Nonmalignant clonal expansions of memory CD8+ T cells that arise with age vary in their capacity to mount recall responses to infection.

Immune responsiveness declines with age in part due to the development of CD8(+) T cell clonal expansions (TCEs) that can dominate the peripheral T cell pool. Although some TCEs arise due to persistent Ag stimulation from chronic infections, others arise in the apparent absence of chronic infection. We have recently shown that this latter class of TCEs can arise over time from the memory CD8(+) T cell pool established by an acute viral infection. Unlike TCEs driven by chronic infections, these age-related TCEs do not display the phenotypic and in vitro functional characteristics of exhausted cells. However, the rate at which these age-related TCEs develop from the memory CD8(+) T cell pool, as well as their ability to mount a recall response to secondary pathogen challenge in vivo, is not known. In this study, we analyzed large cohorts of mice over time for the development of TCE following Sendai virus infection and found a progressive increase in the appearance of TCEs, such that most mice showed evidence of TCE within the memory T cell pool by 2 y postinfection. Using a dual adoptive transfer approach to address the recall potential of virus-specific TCEs, we also demonstrate that most TCEs examined are poorly responsive to a secondary infection. Therefore, we provide evidence that the development of TCE is a common occurrence due to the progressive dysregulation of the virus-specific memory T cell pool with age, but many TCEs are profoundly defective in their ability to mediate recall responses.

Early dysregulation of the memory CD8+ T cell repertoire leads to compromised immune responses to secondary viral infection in the aged.

BACKGROUND: Virus-specific memory CD8+ T cells persist long after infection is resolved and are important for mediating recall responses to secondary infection. Although the number of memory T cells remains relatively constant over time, little is known about the overall stability of the memory T cell pool, particularly with respect to T cell clonal diversity. In this study we developed a novel assay to measure the composition of the memory T cell pool in large cohorts of mice over time following respiratory virus infection. RESULTS: We find that the clonal composition of the virus-specific memory CD8+ T cell pool begins to change within months of the initial infection. These early clonal perturbations eventually result in large clonal expansions that have been associated with ageing. CONCLUSIONS: Maintenance of clonal diversity is important for effective long-term memory responses and dysregulation of the memory response begins early after infection.

Efficacy of an inactivated Zika vaccine against virus infection during pregnancy in mice and marmosets.

Zika virus (ZIKV) is a mosquito-borne arbovirus that can cause severe congenital birth defects. The utmost goal of ZIKV vaccines is to prevent both maternal-fetal infection and congenital Zika syndrome. A Zika purified inactivated virus (ZPIV) was previously shown to be protective in non-pregnant mice and rhesus macaques. In this study, we further examined the efficacy of ZPIV against ZIKV infection during pregnancy in immunocompetent C57BL6 mice and common marmoset monkeys (Callithrix jacchus). We showed that, in C57BL/6 mice, ZPIV significantly reduced ZIKV-induced fetal malformations. Protection of fetuses was positively correlated with virus-neutralizing antibody levels. In marmosets, the vaccine prevented vertical transmission of ZIKV and elicited neutralizing antibodies that remained above a previously determined threshold of protection for up to 18 months. These proof-of-concept studies demonstrate ZPIV's protective efficacy is both potent and durable and has the potential to prevent the harmful consequence of ZIKV infection during pregnancy.

Two kinetic patterns of epitope-specific CD8 T-cell responses following murine gammaherpesvirus 68 infection.

Murine gammaherpesvirus 68 (gammaHV68) provides an important experimental model for understanding mechanisms of immune control of the latent human gammaherpesviruses. Antiviral CD8 T cells play a key role throughout three separate phases of the infection: clearance of lytic virus, control of the latency amplification stage, and prevention of reactivation of latently infected cells. Previous analyses have shown that T-cell responses to two well-characterized epitopes derived from ORF6 and ORF61 progress with distinct kinetics. ORF6(487)-specific cells predominate early in infection and then decline rapidly, whereas ORF61(524)-specific cells continue to expand through early latency, due to sustained epitope expression. However, the paucity of identified epitopes to this virus has limited our understanding of the overall complexities of CD8 T-cell immune control throughout infection. Here we screened 1,383 predicted H-2(b)-restricted peptides and identified 33 responses, of which 21 have not previously been reported. Kinetic analysis revealed a spectrum of T-cell responses based on the rapidity of their decline after the peak acute response that generally corresponded to the expression patterns of the two previously characterized epitopes. The slowly declining responses that were maintained during latency amplification proliferated more rapidly and underwent maturation of functional avidity over time. Furthermore, the kinetics of decline was accelerated following infection with a latency-null mutant virus. Overall, the data show that gammaHV68 infection elicits a highly heterogeneous CD8 T-cell response that segregates into two distinctive kinetic patterns controlled by differential epitope expression during the lytic and latency amplification stages of infection.

CXCR3 directs antigen-specific effector CD4+ T cell migration to the lung during parainfluenza virus infection.

Effector T cells are a crucial component of the adaptive immune response to respiratory virus infections. Although it was previously reported that the chemokine receptors CCR5 and CXCR3 affect trafficking of respiratory virus-specific CD8+ T cells, it is unclear whether these receptors govern effector CD4+ T cell migration to the lungs. To assess the role of CCR5 and CXCR3 in vivo, we directly compared the migration of Ag-specific wild-type and chemokine receptor-deficient effector T cells in mixed bone marrow chimeric mice during a parainfluenza virus infection. CXCR3-deficient effector CD4+ T cells were 5- to 10-fold less efficient at migrating to the lung compared with wild-type cells, whereas CCR5-deficient effector T cells were not impaired in their migration to the lung. In contrast to its role in trafficking, CXCR3 had no impact on effector CD4+ T cell proliferation, phenotype, or function in any of the tissues examined. These findings demonstrate that CXCR3 controls virus-specific effector CD4+ T cell migration in vivo, and suggest that blocking CXCR3-mediated recruitment may limit T cell-induced immunopathology during respiratory virus infections.

Visualization of Resident Memory CD8 T Cells in the Lungs of Young and Aged Influenza Memory Mice and after Heterosubtypic Challenge.

Memory T cells that are resident in the tissues (T resident memory [Trm]) serve as frontline responders to prevent reinfection by pathogens. Trm in the lung protect against respiratory viruses. Although these cells have been well characterized, little is known about the impact of immune aging on the establishment, maintenance, function and recall of lung-resident Trm in the context of an influenza virus infection. Aging is associated with a progressive decline in immune function and a generalized inflammatory syndrome, referred to as inflammaging. In this study, we analyzed inflammation in the lung and assessed numbers and function of lung Trm after primary influenza infection and heterosubtypic challenge of young and aged mice. Our analysis showed that aged mice had more severe and sustained lung inflammation than young mice. Analysis of Trm numbers by flow cytometry and direct imaging showed comparable or higher numbers of Trm in aged compared with young mice, with a similar rate of decline over time in both groups of mice. Furthermore, influenza virus-specific Trm from young and aged memory mice were both functional in vitro, and the mice were protected from heterosubtypic challenge. Finally, there were enhanced numbers of T cells resident in the lungs of aged compared with young mice after heterosubtypic viral challenge. The data suggest that the generation, maintenance, and function of Trm in aged mice are not severely impaired and the increased numbers in aged compared with young mice after heterosubtypic challenge may be associated with enhanced lung inflammation in the aged mice.

Differential impact of ageing on cellular and humoral immunity to a persistent murine gamma-herpesvirus.

BACKGROUND: Oncogenic gamma-herpesviruses establish life-long infections in their hosts and control of these latent infections is dependent on continual immune surveillance. Immune function declines with age, raising the possibility that immune control of gamma-herpesvirus infection becomes compromised with increasing age, allowing viral reactivation and/or increased latent load, both of which are associated with the development of malignancies. RESULTS: In this study, we use the experimental mouse gamma-herpesvirus model, gammaHV68, to investigate viral immunity in aged mice. We found no evidence of viral recrudescence or increased latent load in aged latently-infected mice, suggesting that effective immune control of gamma-herpesvirus infection remains intact with ageing. As both cellular and humoral immunity have been implicated in host control of gammaHV68 latency, we independently examined the impact of ageing on gammaHV68-specific CD8 T cell function and antibody responses. Virus-specific CD8 T cell numbers and cytolytic function were not profoundly diminished with age. In contrast, whereas ELISA titers of virus-specific IgG were maintained over time, there was a progressive decline in neutralizing activity. In addition, although aged mice were able to control de novo acute infection with only slightly delayed viral clearance, serum titers of neutralizing antibody were reduced in aged mice as compared to young mice. CONCLUSION: Although there is no obvious loss of immune control of latent virus, these data indicate that ageing has differential impacts on anti-viral cellular and humoral immune protection during persistent gammaHV68 infection. This observation has potential relevance for understanding gamma-herpesvirus immune control during disease-associated or therapeutic immunosuppression.

Vaccination of aged mice with adjuvanted recombinant influenza nucleoprotein enhances protective immunity.

Elderly individuals are highly susceptible to influenza virus (IAV) infection and respond poorly to influenza vaccines. Although the generally accepted correlate of protection following influenza vaccination is neutralizing antibody titers, cytotoxic T cell activity has been found to be a better correlate in the elderly. This suggests that vaccines designed to protect against influenza in the elderly should induce both humoral and cellular immunity. The co-induction of T cell immunity is additionally advantageous, as virus-specific T cells are frequently cross-reactive against different strains of IAV. Here, we tested the capacity of a synthetic TLR-4 adjuvant, SLA-SE (second-generation lipid adjuvant formulated in a squalene-based oil-in-water emulsion) to elicit T cell immunity to a recombinant influenza nucleoprotein (rNP), in both young and aged mice. IAV challenge of vaccinated mice resulted in a modest increase in the numbers of NP-specific CD4 and CD8 effector T cells in the spleen, but did not increase numbers of memory phenotype CD8 T cells generated following viral clearance (compared to control vaccinated mice). Cytotoxic activity of CD8, but not CD4 T cells was increased. In addition, SLA-SE adjuvanted vaccination specifically enhanced the production of NP-specific IgG2c antibodies in both young and aged mice. Although NP-specific antibodies are not neutralizing, they can cooperate with CD8 T cells and antigen-presenting cells to enhance protective immunity. Importantly, SLA-SE adjuvanted rNP-vaccination of aged mice resulted in significantly enhanced viral clearance. In addition, vaccination of aged mice resulted in enhanced survival after lethal challenge compared to control vaccination, that approached statistical significance. These data demonstrate the potential of SLA-SE adjuvanted rNP vaccines to (i) generate both cellular and humoral immunity to relatively conserved IAV proteins and (ii) elicit protective immunity to IAV in aged mice.