Influenza- and MCMV-induced memory CD8 T cells control respiratory vaccinia virus infection despite residence in distinct anatomical niches.

Induction of memory CD8 T cells residing in peripheral tissues is of interest for T cell-based vaccines as these cells are located at mucosal and barrier sites and can immediately exert effector functions, thus providing protection in case of local pathogen encounter. Different memory CD8 T cell subsets patrol peripheral tissues, but it is unclear which subset is superior in providing protection upon secondary infections. We used influenza virus to induce predominantly tissue resident memory T cells or cytomegalovirus to elicit a large pool of effector-like memory cells in the lungs and determined their early protective capacity and mechanism of reactivation. Both memory CD8 T cell pools have unique characteristics with respect to their phenotype, localization, and maintenance. However, these distinct features do not translate into different capacities to control a respiratory vaccinia virus challenge in an antigen-specific manner, although differential activation mechanisms are utilized. While influenza-induced memory CD8 T cells respond to antigen by local proliferation, MCMV-induced memory CD8 T cells relocate from the vasculature into the tissue in an antigen-independent and partially chemokine-driven manner. Together these results bear relevance for the development of vaccines aimed at eliciting a protective memory CD8 T cell pool at mucosal sites.

Landscape of Exhausted Virus-Specific CD8 T Cells in Chronic LCMV Infection.

A hallmark of chronic infections is the presence of exhausted CD8 T cells, characterized by a distinct transcriptional program compared with functional effector or memory cells, co-expression of multiple inhibitory receptors, and impaired effector function, mainly driven by recurrent T cell receptor engagement. In the context of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, most studies focused on studying splenic virus-specific CD8 T cells. Here, we provide a detailed characterization of exhausted CD8 T cells isolated from six different tissues during established LCMV infection, using single-cell RNA sequencing. Our data reveal that exhausted cells are heterogeneous, adopt organ-specific transcriptomic profiles, and can be divided into five main functional subpopulations: advanced exhaustion, effector-like, intermediate, proliferating, or memory-like. Adoptive transfer experiments showed that these phenotypes are plastic, suggesting that the tissue microenvironment has a major impact in shaping the phenotype and function of virus-specific CD8 T cells during chronic infection.

Profiling Virus-Specific Tcf1+ T Cell Repertoires During Acute and Chronic Viral Infection.

CD8 T cells play a crucial role in providing protection from viral infections. It has recently been established that a subset of CD8 T cells expressing Tcf1 are responsible for sustaining exhausted T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection. Many of these studies, however, have been performed using T cell receptor (TCR) transgenic mice, in which CD8 T cells express a monoclonal TCR specific for the LCMV glycoprotein. To investigate whether the Tcf1+ and Tcf1- repertoires are naturally composed of similar or different clones in wild-type mice exposed to acute or chronic LCMV infection, we performed TCR repertoire sequencing of virus-specific CD8 T cells, including Tcf1+ and Tcf1- populations. Our analysis revealed that the Tcf1+ TCR repertoire is maintained at an equal or higher degree of clonal diversity despite harboring fewer cells. Additionally, within the same animal, there was extensive clonal overlap between the Tcf1+ and Tcf1- repertoires in both chronic and acute LCMV infection. We could further detect these virus-specific clones in longitudinal blood samples earlier in the infection. With respect to common repertoire parameters (clonal overlap, germline gene usage, and clonal expansion), we found minor differences between the virus-specific TCR repertoire of acute and chronic LCMV infection 40 days post infection. Overall, our results indicate that the Tcf1+ population emerging during chronic LCMV infection is not clonally distinct from the Tcf1- population, supporting the notion that the Tcf1+ pool is indeed a fuel for the more exhausted Tcf1- population within the heterogenous repertoire of LCMV-specific CD8 T cells.

Tcf1+ cells are required to maintain the inflationary T cell pool upon MCMV infection.

Cytomegalovirus-based vaccine vectors offer interesting opportunities for T cell-based vaccination purposes as CMV infection induces large numbers of functional effector-like cells that accumulate in peripheral tissues, a process termed memory inflation. Maintenance of high numbers of peripheral CD8 T cells requires continuous replenishment of the inflationary T cell pool. Here, we show that the inflationary T cell population contains a small subset of cells expressing the transcription factor Tcf1. These Tcf1+ cells resemble central memory T cells and are proliferation competent. Upon sensing viral reactivation events, Tcf1+ cells feed into the pool of peripheral Tcf1- cells and depletion of Tcf1+ cells hampers memory inflation. TCR repertoires of Tcf1+ and Tcf1- populations largely overlap, with the Tcf1+ population showing higher clonal diversity. These data show that Tcf1+ cells are necessary for sustaining the inflationary T cell response, and upholding this subset is likely critical for the success of CMV-based vaccination approaches.

Chronic viral infections impinge on naive bystander CD8 T cells.

INTRODUCTION: Epidemiological data suggest that persistent viral infections impair immune homeostasis and immune responsiveness. Previous studies showed that chronic virus infections negatively impact bystander T-cell differentiation and memory formation but there is limited knowledge of how chronic virus infections impinge on heterologous naive T-cell populations. METHODS: We used adoptive transfer of naive CD8 T cells with defined nonviral specificity into hosts, which were subsequently chronically infected with lymphocytic choriomeningitis virus, followed by analyses of numeric, phenotypic, and functional changes provoked in the chronically infected host. RESULTS: We demonstrate that chronic virus infections have a profound effect on the number and phenotype of naive bystander CD8 T cells. Moreover, primary expansion upon antigen encounter was severely compromised in chronically infected hosts. However, when naive bystander CD8 T cells were transferred from the chronically infected mice into naive hosts, they regained their expansion potential. Conversely, when chronically infected hosts were supplied with additional antigen-presenting cells (APCs), primary expansion of the naive CD8 T cells was restored to levels of the uninfected hosts. CONCLUSIONS: Our results document numeric, phenotypic, and functional adaptation of bystander naive CD8 T cells during nonrelated chronic viral infection. Their functional impairment was only evident in the chronically infected host, indicating that T-cell extrinsic factors, in particular the quality of priming APCs, are responsible for the impaired function of naive bystander T cells in the chronically infected hosts.

Quantitative and Qualitative Analysis of Humoral Immunity Reveals Continued and Personalized Evolution in Chronic Viral Infection.

Control of established chronic lymphocytic choriomeningitis virus (LCMV) infection requires the production of neutralizing antibodies, but it remains unknown how the ensemble of antibodies evolves during ongoing infection. Here, we analyze the evolution of antibody responses during acute or chronic LCMV infection, combining quantitative functional assays and time-resolved antibody repertoire sequencing. We establish that antibody responses initially converge in both infection types on a functional and repertoire level, but diverge later during chronic infection, showing increased clonal diversity, the appearance of mouse-specific persistent clones, and distinct phylogenetic signatures. Chronic infection is characterized by a longer-lasting germinal center reaction and a continuous differentiation of plasma cells, resulting in the emergence of higher-affinity plasma cells exhibiting increased antibody secretion rates. Taken together, our findings reveal the emergence of a personalized antibody response in chronic infection and support the concept that maintaining B cell diversity throughout chronic LCMV infection correlates with the development of infection-resolving antibodies.

Early primed KLRG1- CMV-specific T cells determine the size of the inflationary T cell pool.

Memory T cell inflation is a process in which a subset of cytomegalovirus (CMV) specific CD8 T cells continuously expands mainly during latent infection and establishes a large and stable population of effector memory cells in peripheral tissues. Here we set out to identify in vivo parameters that promote and limit CD8 T cell inflation in the context of MCMV infection. We found that the inflationary T cell pool comprised mainly high avidity CD8 T cells, outcompeting lower avidity CD8 T cells. Furthermore, the size of the inflationary T cell pool was not restricted by the availability of specific tissue niches, but it was directly related to the number of virus-specific CD8 T cells that were activated during priming. In particular, the amount of early-primed KLRG1- cells and the number of inflationary cells with a central memory phenotype were a critical determinant for the overall magnitude of the inflationary T cell pool. Inflationary memory CD8 T cells provided protection from a Vaccinia virus challenge and this protection directly correlated with the size of the inflationary memory T cell pool in peripheral tissues. These results highlight the remarkable protective potential of inflationary CD8 T cells that can be harnessed for CMV-based T cell vaccine approaches.

Fuel and brake of memory T cell inflation.

Memory T cell inflation is a process in which a large number of effector memory T cells accumulates in peripheral tissues. This phenomenon is observed upon certain low level persistent virus infections, but it is most commonly described upon infection with the ß-herpesvirus Cytomegalovirus. Due to the induction of this large pool of functional effector CD8 T cells in peripheral tissues, the interest in using CMV-based vaccine vectors for vaccination purposes is rising. However, the exact mechanisms of memory T cell inflation are not yet fully understood. It is clear that repetitive exposure to antigen is a key determinant for memory inflation, and therefore the viral inoculum dose and the subsequent number of viral reactivation events strongly impact on the magnitude of the inflationary T cell pool. In addition, the number of CMV-specific CD8 T cells that is able to sense these reactivation events affects the size of the inflationary T cell pool. In the following, we will discuss factors that either promote or limit T cell inflation from both the virus and host perspective. These factors mostly operate by influencing the amount of available antigen or by affecting the T cell pool that is able to respond to the antigen. Furthermore, we will discuss the recent use of CMV-based vaccines in pre-clinical experimental settings, where these vectors have shown promising results by inducing prolonged effector memory T cell responses to foreign-introduced epitopes and thereby provided protection from subsequent virus or tumour challenges.

Chronic virus infection compromises memory bystander T cell function in an IL-6/STAT1-dependent manner.

Chronic viral infections are widespread among humans, with ∼8-12 chronic viral infections per individual, and there is epidemiological proof that these impair heterologous immunity. We studied the impact of chronic LCMV infection on the phenotype and function of memory bystander CD8+ T cells. Active chronic LCMV infection had a profound effect on total numbers, phenotype, and function of memory bystander T cells in mice. The phenotypic changes included up-regulation of markers commonly associated with effector and exhausted cells and were induced by IL-6 in a STAT1-dependent manner in the context of chronic virus infection. Furthermore, bystander CD8 T cell functions were reduced with respect to their ability to produce inflammatory cytokines and to undergo secondary expansion upon cognate antigen challenge with major cell-extrinsic contributions responsible for the diminished memory potential of bystander CD8+ T cells. These findings open new perspectives for immunity and vaccination during chronic viral infections.

The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity.

Regulatory factor X 7 (Rfx7) is an uncharacterized transcription factor belonging to a family involved in ciliogenesis and immunity. Here, we found that deletion of Rfx7 leads to a decrease in natural killer (NK) cell maintenance and immunity in vivo. Genomic approaches showed that Rfx7 coordinated a transcriptional network controlling cell metabolism. Rfx7-/- NK lymphocytes presented increased size, granularity, proliferation, and energetic state, whereas genetic reduction of mTOR activity mitigated those defects. Notably, Rfx7-deficient NK lymphocytes were rescued by interleukin 15 through engagement of the Janus kinase (Jak) pathway, thus revealing the importance of this signaling for maintenance of such spontaneously activated NK cells. Rfx7 therefore emerges as a novel transcriptional regulator of NK cell homeostasis and metabolic quiescence.

Memory CD8 T cell inflation vs tissue-resident memory T cells: Same patrollers, same controllers?

The induction of long-lived populations of memory T cells residing in peripheral tissues is of considerable interest for T cell-based vaccines, as they can execute immediate effector functions and thus provide protection in case of pathogen encounter at mucosal and barrier sites. Cytomegalovirus (CMV)-based vaccines support the induction and accumulation of a large population of effector memory CD8 T cells in peripheral tissues, in a process called memory inflation. Tissue-resident memory (TRM ) T cells, induced by various infections and vaccination regimens, constitute another subset of memory cells that take long-term residence in peripheral tissues. Both memory T cell subsets have evoked substantial interest in exploitation for vaccine purposes. However, a direct comparison between these two peripheral tissue-localizing memory T cell subsets with respect to their short- and long-term ability to provide protection against heterologous challenge is pending. Here, we discuss communalities and differences between TRM and inflationary CD8 T cells with respect to their development, maintenance, function, and protective capacity. In addition, we discuss differences and similarities between the transcriptional profiles of TRM and inflationary T cells, supporting the notion that they are distinct memory T cell populations.

Tissue maintenance of CMV-specific inflationary memory T cells by IL-15.

Cytomegalovirus (CMV) infection induces an atypical CD8 T cell response, termed inflationary, that is characterised by accumulation and maintenance of high numbers of effector memory like cells in circulation and peripheral tissues-a feature being successfully harnessed for vaccine purposes. Although stability of this population depends on recurrent antigen encounter, the requirements for prolonged survival in peripheral tissues remain unknown. Here, we reveal that murine CMV-specific inflationary CD8 T cells are maintained in an antigen-independent manner and have a half-life of 12 weeks in the lung tissue. This half-life is drastically longer than the one of phenotypically comparable inflationary effector cells. IL-15 alone, and none of other common γ-cytokines, was crucial for survival of inflationary cells in peripheral organs. IL-15, mainly produced by non-hematopoietic cells in lung tissue and being trans-presented, promoted inflationary T cell survival by increasing expression of Bcl-2. These results indicate that inflationary CD8 T cells are not just simply effector-like cells, rather they share properties of both effector and memory CD8 T cells and they appear to be long-lived cells compared to the effector cells from acute virus infections.

Cytomegalovirus infection exacerbates autoimmune mediated neuroinflammation.

Cytomegalovirus (CMV) is a latent virus which causes chronic activation of the immune system. Here, we demonstrate that cytotoxic and pro-inflammatory CD4+CD28null T cells are only present in CMV seropositive donors and that CMV-specific Immunoglobulin (Ig) G titers correlate with the percentage of these cells. In vitro stimulation of peripheral blood mononuclear cells with CMVpp65 peptide resulted in the expansion of pre-existing CD4+CD28null T cells. In vivo, we observed de novo formation, as well as expansion of CD4+CD28null T cells in two different chronic inflammation models, namely the murine CMV (MCMV) model and the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis (MS). In EAE, the percentage of peripheral CD4+CD28null T cells correlated with disease severity. Pre-exposure to MCMV further aggravated EAE symptoms, which was paralleled by peripheral expansion of CD4+CD28null T cells, increased splenocyte MOG reactivity and higher levels of spinal cord demyelination. Cytotoxic CD4+ T cells were identified in demyelinated spinal cord regions, suggesting that peripherally expanded CD4+CD28null T cells migrate towards the central nervous system to inflict damage. Taken together, we demonstrate that CMV drives the expansion of CD4+CD28null T cells, thereby boosting the activation of disease-specific CD4+ T cells and aggravating autoimmune mediated inflammation and demyelination.

The Contribution of Cytomegalovirus Infection to Immune Senescence Is Set by the Infectious Dose.

The relationship between human cytomegalovirus (HCMV) infections and accelerated immune senescence is controversial. Whereas some studies reported a CMV-associated impaired capacity to control heterologous infections at old age, other studies could not confirm this. We hypothesized that these discrepancies might relate to the variability in the infectious dose of CMV occurring in real life. Here, we investigated the influence of persistent CMV infection on immune perturbations and specifically addressed the role of the infectious dose on the contribution of CMV to accelerated immune senescence. We show in experimental mouse models that the degree of mouse CMV (MCMV)-specific memory CD8+ T cell accumulation and the phenotypic T cell profile are directly influenced by the infectious dose, and data on HCMV-specific T cells indicate a similar connection. Detailed cluster analysis of the memory CD8+ T cell development showed that high-dose infection causes a differentiation pathway that progresses faster throughout the life span of the host, suggesting a virus-host balance that is influenced by aging and infectious dose. Importantly, short-term MCMV infection in adult mice is not disadvantageous for heterologous superinfection with lymphocytic choriomeningitis virus (LCMV). However, following long-term CMV infection the strength of the CD8+ T cell immunity to LCMV superinfection was affected by the initial CMV infectious dose, wherein a high infectious dose was found to be a prerequisite for impaired heterologous immunity. Altogether our results underscore the importance of stratification based on the size and differentiation of the CMV-specific memory T cell pools for the impact on immune senescence, and indicate that reduction of the latent/lytic viral load can be beneficial to diminish CMV-associated immune senescence.

Viral Persistence Induces Antibody Inflation without Altering Antibody Avidity.

UNLABELLED: Antibodies are implicated in long-term immunity against numerous pathogens, and because of this property, antibody induction is the basis for many vaccines. Little is known about the influence of viral persistence on the evolving antibody response. Here, we examined the characteristics of antibody responses to persistent infection by employing the prototypic betaherpesvirus family member cytomegalovirus (CMV) in experimental mouse models. During the course of infection, mouse CMV (MCMV)-specific IgM and IgG responses are elicited; however, IgG levels gradually inflate in the persistent phase of infection while IgM levels are stably maintained. Whereas CD27-CD70 interactions are dispensable, the CD28/B7 costimulatory pathway is critical for the class switching of MCMV-specific IgM-to-IgG B cell responses, which corresponds to the CD28/B7-dependent formation of CD4(+)T follicular helper cells (TFH) and germinal center (GC) B cells. Furthermore, the initial viral inoculum dose dictates the height of the antibody levels during IgG antibody inflation and relates to the induction of long-lived plasma cells and memory B cells. Antibody avidity nonetheless is not altered after the establishment of viral persistence and occurs independently of the inoculum doses. However, repetitive challenge with intact viral particles, accompanied by increased GC reactivity, promotes the development of high-avidity IgG responses with neutralizing capacity. These insights can be used for the rational design of CMV-based vaccines aimed at inducing antibody responses. IMPORTANCE: Antibodies provide long-term protection to different pathogens. However, how antibody responses develop during persistent virus infection is not entirely clear. Here, we characterize factors that influence the virus-specific antibody response to persistent CMV. This study describes that during persistent infection, CMV-specific IgM antibody levels are stably maintained while IgG2b and IgG2c levels gradually inflate over time. In contrast, the IgG avidity remains similar after the establishment of viral persistence. The induction of T follicular helper cells and GC B cells requires CD4(+)T cell help and CD28/B7 costimulation signals and is essential for the development of CMV-specific IgG antibody responses. Furthermore, neutralizing CMV-specific antibodies appear to develop late after infection, yet the neutralizing capacity can be improved upon repetitive viral challenge that is associated with increased GC reactivity. The results described here could inform the use of CMV-based vaccines and may help to understand how our immune system copes with this persistent virus.

The Quantity of Autocrine IL-2 Governs the Expansion Potential of CD8+ T Cells.

Adequate responsiveness of CD8(+) T cell populations is of utmost importance for the efficacy of many vaccines and immunotherapeutic strategies against intracellular pathogens and cancer. In this study, we show in mouse models that the relative number of IL-2-producing cells within Ag-specific CD8(+) T cell populations predicts the population expansion capacity upon challenge. We further demonstrate that IL-2 producers constitute the best responding subset. Notably, we show that elevated production of IL-2 by CD8(+) T cells results in concomitant improved population expansion capacity and immunity. The amount of IL-2 produced on a per-cell basis essentially connects directly to the superior CD8(+) T cell population expansion. Together, our findings identified that autocrine IL-2 production operates in a dose-dependent fashion to facilitate the expansion potential of Ag-specific CD8(+) T cell populations, which may instigate ways to augment therapies depending on fit CD8(+) T cells.

The viral context instructs the redundancy of costimulatory pathways in driving CD8(+) T cell expansion.

Signals delivered by costimulatory molecules are implicated in driving T cell expansion. The requirements for these signals, however, vary from dispensable to essential in different infections. We examined the underlying mechanisms of this differential T cell costimulation dependence and found that the viral context determined the dependence on CD28/B7-mediated costimulation for expansion of naive and memory CD8(+) T cells, indicating that the requirement for costimulatory signals is not imprinted. Notably, related to the high-level costimulatory molecule expression induced by lymphocytic choriomeningitis virus (LCMV), CD28/B7-mediated costimulation was dispensable for accumulation of LCMV-specific CD8(+) T cells because of redundancy with the costimulatory pathways induced by TNF receptor family members (i.e., CD27, OX40, and 4-1BB). Type I IFN signaling in viral-specific CD8(+) T cells is slightly redundant with costimulatory signals. These results highlight that pathogen-specific conditions differentially and uniquely dictate the utilization of costimulatory pathways allowing shaping of effector and memory antigen-specific CD8(+) T cell responses.

Viral inoculum dose impacts memory T-cell inflation.

Memory T-cell inflation develops during certain persistent viral infections and is characterized by the accumulation and maintenance of large numbers of effector-memory T cells, albeit with varying degrees in size and phenotype among infected hosts. The underlying mechanisms that control memory T-cell inflation are not yet fully understood. Here, we dissected CMV-specific memory T-cell formation and its connection to the initial infectious dose by varying the inoculum size. After low dose inoculum with mouse CMV, the accumulation of inflationary memory T cells was severely hampered and correlated with reduced reservoirs of latent virus in nonhematopoietic cells and diminished antigen-driven T-cell proliferation. Moreover, lowering of the initial viral dose turned the characteristic effector memory-like inflationary T cells into more central memory-like cells as evidenced by the cell-surface phenotype of CD27(high) , CD62L(+) , CD127(+) , and KLRG1(-) , and by improved secondary expansion potential. These data show the impact of the viral inoculum on the degree of memory T-cell inflation and provide a rationale for the observed variation of human CMV-specific T-cell responses in terms of magnitude and phenotype.

The distinct role of T cell costimulation in antiviral immunity.

Defining the critical molecular interactions for inducing antiviral T cell responses is important for improvement of vaccines. Recent progress in understanding the role of T cell costimulatory molecules provides the insight that these molecules not only enhance CD4 and CD8 T cell responses in acute infections but also have an impact in latent and chronic viral infections. Intriguingly, the requirements for T cell costimulation seem to be distinct for each virus but nonetheless at least one or more costimulatory pathways are instrumental for development of protective immunity. Remarkably, certain viruses have evolved mechanisms to evade host costimulatory pathways to their advantage. These new insights have important implications for rational vaccine design.

CD27-CD70 costimulation controls T cell immunity during acute and persistent cytomegalovirus infection.

Cytomegaloviruses (CMVs) establish lifelong infections that are controlled in part by CD4(+) and CD8(+) T cells. To promote persistence, CMVs utilize multiple strategies to evade host immunity, including modulation of costimulatory molecules on infected antigen-presenting cells. In humans, CMV-specific memory T cells are characterized by the loss of CD27 expression, which suggests a critical role of the costimulatory receptor-ligand pair CD27-CD70 for the development of CMV-specific T cell immunity. In this study, the in vivo role of CD27-CD70 costimulation during mouse CMV infection was examined. During the acute phase of infection, the magnitudes of CMV-specific CD4(+) and CD8(+) T cell responses were decreased in mice with abrogated CD27-CD70 costimulation. Moreover, the accumulation of inflationary memory T cells during the persistent phase of infection and the ability to undergo secondary expansion required CD27-CD70 interactions. The downmodulation of CD27 expression, however, which occurs gradually and exclusively on inflationary memory T cells, is ligand independent. Furthermore, the IL-2 production in both noninflationary and inflationary CMV-specific T cells was dependent on CD27-CD70 costimulation. Collectively, these results highlight the importance of the CD27-CD70 costimulation pathway for the development of CMV-specific T cell immunity during acute and persistent infection.