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.

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.

Promotion of a subdominant CD8 T cell response during murine gammaherpesvirus 68 infection in the absence of CD4 T cell help.

CD8 and CD4 T cells are each critically important for immune control of murine gammaherpesvirus 68 (γHV68) infection. In immunocompetent mice, acute γHV68 infection results in lifelong latency, but in the absence of CD4 T cell help, mice succumb to viral recrudescence and disease. However, the requirements for CD4 T cell help in the generation and maintenance of antiviral CD8 T cell responses are incompletely understood, and it is unclear whether there are epitope-specific differences in the requirement of CD8 T cells for CD4 help. In this report, we characterized the CD8 T cell response to γHV68 in major histocompatibility complex (MHC) class II(-/-) mice, which lack CD4 T cells, or after antibody-mediated depletion of CD4 T cells. All antiviral CD8 T cells exhibited marked upregulation of surface expression of the inhibitory receptor programmed death-1 (PD-1), but surprisingly, while the immunodominant memory response appeared to be functionally impaired, helpless CD8 T cells of a subdominant specificity had increased numbers and enhanced functionality. Thus, we demonstrate differential requirements for CD4 help in the antiviral CD8 T cell response to a latent gammaherpesvirus. Importance: γHV68 is a mouse pathogen closely related to the oncogenic human γHVs, which infect a majority of the world's population. Reactivation of these viruses from latency can lead to complications, disease, and even death. CD4 T cells are required for complete immune control of long-term infection, in part by providing key signals to dendritic cells that in turn instruct optimal antiviral CD8 T cell responses. We have investigated multiple virus-specific CD8 T cell responses during infection and identified a subdominant CD8 T cell response that is numerically and functionally enhanced in the absence of CD4 T cell help. This occurs in spite of high surface expression of an inhibitory receptor and in contrast to the immunodominant response, which is impaired. Our data suggest that signals from CD4 T cells are important in maintaining the CD8 T cell hierarchy during γHV infections.

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.

Activation phenotype, rather than central- or effector-memory phenotype, predicts the recall efficacy of memory CD8+ T cells.

The contributions of different subsets of memory CD8+ T cells to recall responses at mucosal sites of infection are poorly understood. Here, we analyzed the CD8+ T cell recall responses to respiratory virus infection in mice and demonstrate that activation markers, such as CD27 and CD43, define three distinct subpopulations of memory CD8+ T cells that differ in their capacities to mount recall responses. These subpopulations are distinct from effector- and central-memory subsets, coordinately express other markers associated with activation status, including CXCR3, CD127, and killer cell lectin-like receptor G1, and are superior to CD62L in predicting the capacity of memory T cells to mediate recall responses. Furthermore, the capacity of vaccines to elicit these memory T cell subpopulations predicted the efficacy of the recall response. These findings extend our understanding of how recall responses are generated and suggest that activation and migration markers define distinct, and unrelated, characteristics of memory T cells.

Gammaherpesvirus latency induces antibody-associated thrombocytopenia in mice.

Human herpesviruses establish lifelong latency. Viral recrudescence can lead to the development of cancers, immunoproliferative disorders, transplantation complications, and thrombocytopenia. Although platelet-specific autoantibodies have been reported in patients infected with the Epstein-Barr virus (EBV), the mechanisms by which thrombocytopenia is induced remain unclear, as do the relative contributions of lytic viral replication and latent viral gene expression. The human gammaherpesviruses are tightly restricted in their ability to infect other mammals, so they are difficult to study in live animal models. Here we show that infection of mice with murine gammaherpesvirus-68 (γHV68), a rodent-specific pathogen closely related to EBV, induces the production of platelet-binding antibodies and causes thrombocytopenia. Infection of antibody-deficient mice does not lead to thrombocytopenia, indicating the platelet decrease is mediated by antibody. Additionally, infection with a latency-null recombinant γHV68 does not induce thrombocytopenia, suggesting factors associated with viral latency drive the infection-induced antibody-mediated thrombocytopenia. These studies describe an important animal model of gammaherpesvirus-induced autoimmune thrombocytopenia and demonstrate that this pathology is mediated by antibody and dependent on viral latency. This model will allow studies of the underlying mechanisms of disease progression and the testing of therapeutic strategies for the alleviation of virus-induced thrombocytopenia.

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.

Differential contributions of central and effector memory T cells to recall responses.

Although the absolute number of memory CD8+ T cells established in the spleen following antigen encounter remains stable for many years, the relative capacity of these cells to mediate recall responses is not known. Here we used a dual adoptive transfer approach to demonstrate a progressive increase in the quality of memory T cell pools in terms of their ability to proliferate and accumulate at effector sites in response to secondary pathogen challenge. This temporal increase in efficacy occurred in CD62L lo (effector memory) and CD62L hi (central memory) subpopulations, but was most prominent in the CD62L hi subpopulation. These data indicate that the contribution of effector memory and central memory T cells to the recall response changes substantially over time.

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.

ESAT-6-specific CD4 T cell responses to aerosol Mycobacterium tuberculosis infection are initiated in the mediastinal lymph nodes.

CD4(+) T cell responses to aerosol Mycobacterium tuberculosis (Mtb) infection are characterized by the relatively delayed appearance of effector T cells in the lungs. This delay in the adaptive response is likely critical in allowing the bacteria to establish persistent infection. Because of limitations associated with the detection of low frequencies of naïve T cells, it had not been possible to precisely determine when and where naïve antigen-specific T cells are first activated. We have addressed this problem by using early secreted antigenic target 6 (ESAT-6)-specific transgenic CD4 T cells to monitor early T cell activation in vivo. By using an adoptive transfer approach, we directly show that T cell priming to ESAT-6 occurs only after 10 days of infection, is initially restricted to the mediastinal lymph nodes, and does not involve other lymph nodes or the lungs. Primed CD4 T cells rapidly differentiated into proliferating effector cells and ultimately acquired the ability to produce IFN-gamma and TNF-alpha ex vivo. Initiation of T cell priming was enhanced by two full days depending on the magnitude of the challenge inoculum, which suggests that antigen availability is a factor limiting the early CD4 T cell response. These data define a key period in the adaptive immune response to Mtb infection.

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.

Aging and CD8+ T cell immunity to respiratory virus infections.

The capacity of the immune system to mediate effective immune responses to pathogens declines with age. In the case of immune responses to newly encountered antigens, several studies have demonstrated that this decline reflects both a loss of naïve T cells and changes in the repertoire and function of these cells over time. However, comparatively little is known about the impact of age on established memory T cells pools. Here we discuss age-related changes in memory CD8(+) T cell pools elicited by influenza and parainfluenza viruses and the impact of these changes on immunity in general.

Dose-rate-dependent damage of cerium dioxide in the scanning transmission electron microscope.

Beam damage caused by energetic electrons in the transmission electron microscope is a fundamental constraint limiting the collection of artifact-free information. Through understanding the influence of the electron beam, experimental routines may be adjusted to improve the data collection process. Investigations of CeO2 indicate that there is not a critical dose required for the accumulation of electron beam damage. Instead, measurements using annular dark field scanning transmission electron microscopy and electron energy loss spectroscopy demonstrate that the onset of measurable damage occurs when a critical dose rate is exceeded. The mechanism behind this phenomenon is that oxygen vacancies created by exposure to a 300keV electron beam are actively annihilated as the sample re-oxidizes in the microscope environment. As a result, only when the rate of vacancy creation exceeds the recovery rate will beam damage begin to accumulate. This observation suggests that dose-intensive experiments can be accomplished without disrupting the native structure of the sample when executed using dose rates below the appropriate threshold. Furthermore, the presence of an encapsulating carbonaceous layer inhibits processes that cause beam damage, markedly increasing the dose rate threshold for the accumulation of damage.

Oxidation-state sensitive imaging of cerium dioxide by atomic-resolution low-angle annular dark field scanning transmission electron microscopy.

Low-angle annular dark field (LAADF) scanning transmission electron microscopy (STEM) imaging is presented as a method that is sensitive to the oxidation state of cerium ions in CeO2 nanoparticles. This relationship was validated through electron energy loss spectroscopy (EELS), in situ measurements, as well as multislice image simulations. Static displacements caused by the increased ionic radius of Ce(3+) influence the electron channeling process and increase electron scattering to low angles while reducing scatter to high angles. This process manifests itself by reducing the high-angle annular dark field (HAADF) signal intensity while increasing the LAADF signal intensity in close proximity to Ce(3+) ions. This technique can supplement STEM-EELS and in so doing, relax the experimental challenges associated with acquiring oxidation state information at high spatial resolutions.

Inflammatory chemokine receptors regulate CD8(+) T cell contraction and memory generation following infection.

The development of T cell memory from naive precursors is influenced by molecular cues received during T cell activation and differentiation. In this study, we describe a novel role for the chemokine receptors CCR5 and CXCR3 in regulating effector CD8(+) T cell contraction and memory generation after influenza virus infection. We find that Ccr5(-/-) Cxcr3(-/-) cells show markedly decreased contraction after viral clearance, leading to the establishment of massive numbers of memory CD8(+) T cells. Ccr5(-/-) Cxcr3(-/-) cells show reduced expression of CD69 in the lung during the peak of infection, which coincides with differential localization and the rapid appearance of memory precursor cells. Analysis of single chemokine receptor-deficient cells revealed that CXCR3 is primarily responsible for this phenotype, although there is also a role for CCR5 in the enhancement of T cell memory. The phenotype could be reversed by adding exogenous antigen, resulting in the activation and contraction of Ccr5(-/-) Cxcr3(-/-) cells. Similar results were observed during chronic Mycobacterium tuberculosis infection. Together, the data support a model of memory CD8(+) T cell generation in which the chemokine-directed localization of T cells within infected tissues regulates antigen encounter and controls the extent of CD8(+) T cell activation and differentiation, which ultimately regulates effector versus memory cell fate decisions.

The route of priming influences the ability of respiratory virus-specific memory CD8+ T cells to be activated by residual antigen.

After respiratory virus infections, memory CD8+ T cells are maintained in the lung airways by a process of continual recruitment. Previous studies have suggested that this process is controlled, at least in the initial weeks after virus clearance, by residual antigen in the lung-draining mediastinal lymph nodes (MLNs). We used mouse models of influenza and parainfluenza virus infection to show that intranasally (i.n.) primed memory CD8+ T cells possess a unique ability to be reactivated by residual antigen in the MLN compared with intraperitoneally (i.p.) primed CD8+ T cells, resulting in the preferential recruitment of i.n.-primed memory CD8+ T cells to the lung airways. Furthermore, we demonstrate that the inability of i.p.-primed memory CD8+ T cells to access residual antigen can be corrected by a subsequent i.n. virus infection. Thus, two independent factors, initial CD8+ T cell priming in the MLN and prolonged presentation of residual antigen in the MLN, are required to maintain large numbers of antigen-specific memory CD8+ T cells in the lung airways.

Antigen-specific CD8+ T cell clonal expansions develop from memory T cell pools established by acute respiratory virus infections.

Increasing age is associated with the development of CD8+ T cell clonal expansions (TCE) that can dominate the peripheral T cell repertoire and interfere with immune responses to infection and vaccination. Some TCE are driven by chronic infections, consistent with dysregulated outgrowth of T cell clones in response to persistent antigenic stimulation. However, a second class of TCE develops with age in the absence of chronic infections and is poorly understood in terms of origin or Ag dependence. In this study, we present evidence that Ag-specific TCE develop at high frequencies from conventional memory CD8+ T cell pools elicited by nonpersistent influenza and parainfluenza virus infections. Putative TCE occurred in both the central- and effector-memory CD8+ T cell populations and did not require Ag for their maintenance. In addition, they were similar to normal memory T cells in terms of phenotype and function, suggesting that they develop stochastically from the memory T cell pool. These data suggest that memory T cell pools become progressively dysregulated over time and this may have a significant impact on immune responsiveness in the aged.