Anti-cytomegalovirus antibody levels stratify human immune profiles across the lifespan.

Human cytomegalovirus (hCMV) is a ubiquitous latent persistent herpesvirus infecting 60-90% of the population worldwide. hCMV carriage in immunocompetent people is asymptomatic; thus, hCMV can be considered a component of normative aging. However, hCMV powerfully modulates many features of the immune, and likely other, systems and organs. Questions remain as to how hCMV carriage affects the human host. We used anti-CMV antibody titers as a stratifying criterion to examine the impact of "intensity" of hCMV infection as a potential biomarker of aging, inflammation, and immune homeostasis in a cohort of 247 participants stratified into younger (21-40 years) and older (> 65 years of age) groups. We showed that anti-CMV antibody titers increased with age and directly correlated to increased levels of soluble tumor necrosis factor (sTNFR) I in younger but not older participants. CD8 + cell numbers were reduced in the older group due to the loss in CD8 + T naïve (Tn) cells. In CMV carriers and, in particular, in anti-CMV Ab-high participants, this loss was mitigated or reversed by an increase in the numbers of CD8 + T effector memory (Tem) and T effector memory reexpressing CD45RA (Temra) cells. Analysis of CD38, HLA-DR, and CD57 expression revealed subset (CD4 or CD8)-specific changes that correlated with anti-CMV Ab levels. In addition, anti-CMV Ab levels predicted anti-CMV CD8 T cell responsiveness to different CMV open reading frames (ORFs) selectively in older participants, which correlated to the transcriptional order of expression of specific CMV ORFs. Implications of these results for the potential predictive value of anti-CMV Ab titers during aging are discussed.

Inflammatory and immune markers in HIV-infected older adults on long-term antiretroviral therapy: Persistent elevation of sCD14 and of proinflammatory effector memory T cells.

HIV-positive patients whose viral loads are successfully controlled by active antiretroviral therapy (ART) show no clinical signs of AIDS. However, their lifespan is shorter compared with individuals with no HIV infection and they prematurely exhibit a multitude of chronic diseases typically associated with advanced age. It was hypothesized that immune system aging may correlate with, and provide useful biomarkers for, this premature loss of healthspan in HIV-positive subjects. Here, we tested whether the immune correlates of aging, including cell numbers and phenotypes, inflammatory status, and control of human cytomegalovirus (hCMV) in HIV-positive subjects on long-term successful ART (HIV+) may reveal increased "immunological age" compared with HIV-negative, age-matched cohort (HIV-) in participants between 50 and 69 years of age. Specifically, we expected that younger HIV+ subjects may immunologically resemble older individuals without HIV. We found no evidence to support this hypothesis. While T cells from HIV+ participants displayed differential expression in several differentiation and/or inhibitory/exhaustion markers in different T cell subpopulations, aging by a decade did not pronounce these changes. Similarly, while the HIV+ participants exhibited higher T cell responses and elevated inflammatory marker levels in plasma, indicative of chronic inflammation, this trait was not age-sensitive. We did find differences in immune control of hCMV, and, more importantly, a sustained elevation of sCD14 and of proinflammatory CD4 and CD8 T cell responses across age groups, pointing towards uncontrolled inflammation as a factor in reduced healthspan in successfully treated older HIV+ patients.

Antibody therapy reverses biological signatures of COVID-19 progression.

Understanding who is at risk of progression to severe coronavirus disease 2019 (COVID-19) is key to clinical decision making and effective treatment. We study correlates of disease severity in the COMET-ICE clinical trial that randomized 1:1 to placebo or to sotrovimab, a monoclonal antibody for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (ClinicalTrials.gov04545060). Laboratory parameters identify study participants at greater risk of severe disease, including a high neutrophil-to-lymphocyte ratio (NLR), a negative SARS-CoV-2 serologic test, and whole-blood transcriptome profiles. Sotrovimab treatment is associated with normalization of NLR and the transcriptomic profile and with a decrease of viral RNA in nasopharyngeal samples. Transcriptomics provides the most sensitive detection of participants who would go on to be hospitalized or die. To facilitate timely measurement, we identify a 10-gene signature with similar predictive accuracy. We identify markers of risk for disease progression and demonstrate that normalization of these parameters occurs with antibody treatment of established infection.

Infection-induced type I interferons critically modulate the homeostasis and function of CD8+ naïve T cells.

Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide-MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C+ CD8+ Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5hiCD8+ cells, which in the case of polyinfection translates into a numerical CD8+ Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C+ Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C+CD8+ Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy.

Corrigendum: Defective Transcriptional Programming of Effector CD8 T Cells in Aged Mice Is Cell-Extrinsic and Can Be Corrected by Administration of IL-12 and IL-18.

[This corrects the article DOI: 10.3389/fimmu.2019.02206.].

Defects in Antiviral T Cell Responses Inflicted by Aging-Associated miR-181a Deficiency.

Generation of protective immunity to infections and vaccinations declines with age. Studies in healthy individuals have implicated reduced miR-181a expression in T cells as contributing to this defect. To understand the impact of miR-181a expression on antiviral responses, we examined LCMV infection in mice with miR-181ab1-deficient T cells. We found that miR-181a deficiency delays viral clearance, thereby biasing the immune response in favor of CD4 over CD8 T cells. Antigen-specific CD4 T cells in mice with miR-181a-deficient T cells expand more and have a broader TCR repertoire with preferential expansion of high-affinity T cells than in wild-type mice. Importantly, generation of antigen-specific miR-181a-deficient CD8 effector T cells is particularly impaired, resulting in lower frequencies of CD8 T cells in the liver even at time points when the infection has been cleared. Consistent with the mouse model, CD4 memory T cells in individuals infected with West Nile virus at older ages tend to be more frequent and of higher affinity.

Defective Transcriptional Programming of Effector CD8 T Cells in Aged Mice Is Cell-Extrinsic and Can Be Corrected by Administration of IL-12 and IL-18.

In response to infection with intracellular microorganisms, old mice mobilize decreased numbers of antigen-specific CD8 T cells with reduced expression of effector molecules and impaired cytolytic activity. Molecular mechanisms behind these defects and the cell-intrinsic (affecting naïve CD8 T cells themselves) vs. extrinsic, microenvironmental origin of such defects remain unclear. Using reciprocal transfer experiments of highly purified naïve T cells from adult and old transgenic OT-1 mice, we decisively show that the dominant effect is cell-extrinsic. Naïve adult OT-1 T cells failed to expand and terminally differentiate in the old organism infected with Listeria-OVA. This defect was preceded by blunted expression of the master transcription factor T-bet and impaired glycolytic switch when T cells are primed in the old organism. However, both old and adult naïve CD8 T cells proliferated and produced effector molecules to a similar extent when stimulated in vitro with polyclonal stimuli, as well as when transferred into adult recipients. Multiple inflammatory cytokines with direct effects on T cell effector differentiation were decreased in spleens of old animals, particularly IL-12 and IL-18. Of note, in vivo treatment of mice with IL-12 and IL-18 on days 4-6 of Listeria infection reconstituted cytotoxic T cell response of aged mice to the level of adult. Therefore, critical cytokine signals which are underproduced in the old priming environment can restore proper transcriptional programming of old naïve CD8 T cells and improve immune defense against intracellular microorganisms.

Lymph nodes as barriers to T-cell rejuvenation in aging mice and nonhuman primates.

In youth, thymic involution curtails production of new naïve T cells, placing the onus of T-cell maintenance upon secondary lymphoid organs (SLO). This peripheral maintenance preserves the size of the T-cell pool for much of the lifespan, but wanes in the last third of life, leading to a dearth of naïve T cells in blood and SLO, and contributing to suboptimal immune defense. Both keratinocyte growth factor (KGF) and sex steroid ablation (SSA) have been shown to transiently increase the size and cellularity of the old thymus. It is less clear whether this increase can improve protection of old animals from infectious challenge. Here, we directly measured the extent to which thymic rejuvenation benefits the peripheral T-cell compartment of old mice and nonhuman primates. Following treatment of old animals with either KGF or SSA, we observed robust rejuvenation of thymic size and cellularity, and, in a reporter mouse model, an increase in recent thymic emigrants (RTE) in the blood. However, few RTE were found in the spleen and even fewer in the lymph nodes, and SSA-treated mice showed no improvement in immune defense against West Nile virus. In parallel, we found increased disorganization and fibrosis in old LN of both mice and nonhuman primates. These results suggest that SLO defects with aging can negate the effects of successful thymic rejuvenation in immune defense.

Lifelong CMV infection improves immune defense in old mice by broadening the mobilized TCR repertoire against third-party infection.

Lifelong interactions between host and the ubiquitous and persistent cytomegalovirus (CMV) have been proposed to contribute to the age-related decline in immunity. Prior work from us and others found some support for that idea, yet evidence that this led to increased vulnerability to other infections was not obtained. Moreover, evidence has accumulated that CMV infection can be beneficial to immune defense in young/adult mice and humans, dominantly via enhanced innate immunity. Here, we describe an unexpected impact of murine CMV (MCMV) upon the T cell response of old mice to Listeria monocytogenes expressing the model antigen, OVA (Lm-OVA). Single-cell sequencing of the OVA-specific CD8 T cell receptor ß (TCRß) repertoire of old mice demonstrated that old MCMV-infected mice recruited many diverse clonotypes that afforded broad and often more efficient recognition of antigenic peptide variants. This stood in contrast to old control mice, which exhibited strong narrowing and homogenization of the elicited repertoire. High-throughput sequencing of the total naïve CD8 TCRß repertoire showed that many of these diverse OVA-specific clonotypes were present in the naïve CD8 repertoire of mice in all groups (adult, old control, and old MCMV+) yet were only recruited into the Lm-OVA response in MCMV+ old mice. These results have profound implications for our understanding of T cell immunity over a life span and suggest that our coevolution with CMV may include surprising, potentially positive impacts on adaptive heterologous immunity in late life.

Diminished antibody response to influenza vaccination is characterized by expansion of an age-associated B-cell population with low PAX5.

Individuals over the age of 65 comprise a substantial portion of the world population and become more susceptible to vaccine-preventable infections with age as vaccination response diminishes. The underlying reason for this impaired vaccine response in older individuals is not entirely clear. We evaluated potential differences in phenotypic and functional responses of B cells from healthy younger (22-45years) and older (64-95years) individuals that may associate with a diminished antibody response to influenza vaccination. We report that age is associated with expansion of atypical memory B cells (CD10-CD20+CD21-CD27-) and an age-associated B cell (ABC, CD21-T-bet+CD11c+) phenotype. Reduced expression of PAX5 was also seen in older individuals. Poor influenza-specific antibody production following vaccination was associated with low PAX5 expression and a distinct composition of the ABC compartment. Collectively, these findings demonstrate that the characteristics of the ABC populations of older individuals are associated with antibody production following influenza vaccination.

Intrinsic and extrinsic contributors to defective CD8+ T cell responses with aging.

Aging has a profound effect on the immune system, and both innate and adaptive arms of the immune system show functional decline with age. In response to infection with intracellular microorganisms, old animals mobilize decreased numbers of antigen-specific CD8+ T cells with reduced production of effector molecules and impaired cytolytic activity. However, the CD8+ T cell-intrinsic contribution to, and molecular mechanisms behind, these defects remain unclear. In this review we will discuss the mechanistic contributions of age related changes in the CD8+ T cell pool and the relative roles of intrinsic functional defects in aged CD8+ T cells vs. defects in the aged environment initiating the CD8+ T cell response.

Known unknowns: how might the persistent herpesvirome shape immunity and aging?

The microbial community that colonizes all living organisms is gaining appreciation for its contributions to both physiologic and pathogenic processes. The virome, a subset of the overall microbiome, large and diverse, including viruses that persistently inhabit host cells, endogenous viral elements genomically or epigenomically integrated into cells, and viruses that infect the other (bacterial, protozoan, fungal, and archaeal) microbiome phylla. These viruses live in the organism for its life, and therefore are to be considered part of the aging process experienced by the organism. This review considers the impact of the persistent latent virome on immune aging. Specific attention will be devoted to the role of herpesviruses, and within them, the cytomegalovirus, as the key modulators of immune aging.

Cutting Edge: The Aging Immune System Reveals the Biological Impact of Direct Antigen Presentation on CD8 T Cell Responses.

The vertebrate immune system uses multiple, sometimes redundant, mechanisms to contain pathogenic microorganisms that are always evolving to evade host defenses. Thus, the cowpox virus (CPXV) uses genes encoding CPXV12 and CPXV203 to prevent direct MHC class I presentation of viral peptides by infected cells. However, CD8 T cells are effectively primed against CPXV by cross-presentation of viral Ags in young mice. Old mice accumulate defects in both CD8 T cell activation and cross-presentation. Using a double-deletion mutant (∆12∆203) of CPXV, we show that direct priming of CD8 T cells in old mice yields superior recall responses, establishing a key contribution of this mechanism to host antipoxvirus responses and enhancing our fundamental understanding of how viral manipulation of direct presentation impacts pathogenesis. This also provides a proof of principle that suboptimal CD8 T cell in old organisms can be optimized by manipulating Ag presentation, with implications for vaccine design.

Functional and Homeostatic Impact of Age-Related Changes in Lymph Node Stroma.

Adults over 65 years of age are more vulnerable to infectious disease and show poor responses to vaccination relative to those under 50. A complex set of age-related changes in the immune system is believed to be largely responsible for these defects. These changes, collectively termed immune senescence, encompass alterations in both the innate and adaptive immune systems, in the microenvironments where immune cells develop or reside, and in soluble factors that guide immune homeostasis and function. While age-related changes in primary lymphoid organs (bone marrow, and, in particular, the thymus, which involutes in the first third of life) have been long appreciated, changes affecting aging secondary lymphoid organs, and, in particular, aging lymph nodes (LNs) have been less well characterized. Over the last 20 years, LN stromal cells have emerged as key players in maintaining LN morphology and immune homeostasis, as well as in coordinating immune responses to pathogens. Here, we review recent progress in understanding the contributions of LN stromal cells to immune senescence. We discuss approaches to understand the mechanisms behind the decline in LN stromal cells and conclude by considering potential strategies to rejuvenate aging LN stroma to improve immune homeostasis, immune responses, and vaccine efficacy in the elderly.

Acute systemic DNA damage in youth does not impair immune defense with aging.

Aging-related decline in immunity is believed to be the main driver behind decreased vaccine efficacy and reduced resistance to infections in older adults. Unrepaired DNA damage is known to precipitate cellular senescence, which was hypothesized to be the underlying cause of certain age-related phenotypes. Consistent with this, some hallmarks of immune aging were more prevalent in individuals exposed to whole-body irradiation (WBI), which leaves no anatomical repository of undamaged hematopoietic cells. To decisively test whether and to what extent WBI in youth will leave a mark on the immune system as it ages, we exposed young male C57BL/6 mice to sublethal WBI (0.5-4 Gy), mimicking human survivor exposure during nuclear catastrophe. We followed lymphocyte homeostasis thorough the lifespan, response to vaccination, and ability to resist lethal viral challenge in the old age. None of the irradiated groups showed significant differences compared with mock-irradiated (0 Gy) animals for the parameters measured. Even the mice that received the highest dose of sublethal WBI in youth (4 Gy) exhibited equilibrated lymphocyte homeostasis, robust T- and B-cell responses to live attenuated West Nile virus (WNV) vaccine and full survival following vaccination upon lethal WNV challenge. Therefore, a single dose of nonlethal WBI in youth, resulting in widespread DNA damage and repopulation stress in hematopoietic cells, leaves no significant trace of increased immune aging in a lethal vaccine challenge model.

Lifespan-extending caloric restriction or mTOR inhibition impair adaptive immunity of old mice by distinct mechanisms.

Aging of the world population and a concomitant increase in age-related diseases and disabilities mandates the search for strategies to increase healthspan, the length of time an individual lives healthy and productively. Due to the age-related decline of the immune system, infectious diseases remain among the top 5-10 causes of mortality and morbidity in the elderly, and improving immune function during aging remains an important aspect of healthspan extension. Calorie restriction (CR) and more recently rapamycin (rapa) feeding have both been used to extend lifespan in mice. Preciously few studies have actually investigated the impact of each of these interventions upon in vivo immune defense against relevant microbial challenge in old organisms. We tested how rapa and CR each impacted the immune system in adult and old mice. We report that each intervention differentially altered T-cell development in the thymus, peripheral T-cell maintenance, T-cell function and host survival after West Nile virus infection, inducing distinct but deleterious consequences to the aging immune system. We conclude that neither rapa feeding nor CR, in the current form/administration regimen, may be optimal strategies for extending healthy immune function and, with it, lifespan.

Lost in translation: mice, men and cutaneous immunity in old age.

Translational research programs offer incredible opportunities to bring cutting edge science into clinical practice. To facilitate these medical advances, funding agencies are increasingly focusing on a translational "payoff" within grant applications and larger programs. As this is the underlying promise of biomedical research-delivering advances to public health to improve the quality of life-such strategic initiatives are paramount. However, the process of taking experimental observations between model systems and human subjects can be extraordinarily frustrating. We brought together the collective expertise of our mouse and human immunology research programs to reverse engineer a clinical observation into a mouse model system. Our goal was to model (in mice) the age-related impaired delayed-type hypersensitivity response observed in humans, and then evaluate the efficacy of interventions to improve cutaneous immunity. We report here on what worked, what didn't, and what we learned along the way.

Histone deacetylation critically determines T cell subset radiosensitivity.

Lymphocytes are sensitive to ionizing radiation and naive lymphocytes are more radiosensitive than their memory counterparts. Less is known about radiosensitivity of memory cell subsets. We examined the radiosensitivity of naive (TN), effector memory (TEM), and central memory (TCM) T cell subsets in C57BL/6 mice and found TEM to be more resistant to radiation-induced apoptosis than either TN or TCM. Surprisingly, we found no correlation between the extent of radiation-induced apoptosis in T cell subsets and 1) levels of pro- and antiapoptotic Bcl-2 family members or 2) the H2AX content and maximal γH2AX fold change. Rather, TEM cell survival correlated with higher levels of immediate γH2AX marking, immediate break binding and genome-wide open chromatin structure. T cells were able to mark DNA damage seemingly instantly (30 s), even if kept on ice. Relaxing chromatin with the histone deacetylase inhibitor valproic acid following radiation or etoposide treatment improved the survival of TCM and TN cells up to levels seen in the resistant TEM cells but did not improve survival from caspase-mediated apoptosis. We conclude that an open genome-wide chromatin state is the key determinant of efficient immediate repair of DNA damage in T cells, explaining the observed T cell subset radiosensitivity differences.

Immune memory-boosting dose of rapamycin impairs macrophage vesicle acidification and curtails glycolysis in effector CD8 cells, impairing defense against acute infections.

Direct mammalian target of rapamycin (Rapa) complex 1 inhibition by short-term low-dose Rapa treatment has recently been shown to improve CD8 T cell immunological memory. Whereas these studies focused on memory development, the impact of low-dose Rapa on the primary immune response, particularly as it relates to functional effector immunity, is far less clear. In this study, we investigated the impact of acute Rapa treatment on immune effector cell function during the primary immune response to several acute infections. We found that functional CD8 T cell and macrophage responses to both viral and intracellular bacterial pathogens were depressed in mice in vivo and in humans to phorbol ester and calcium ionophore stimulation in vitro in the face of low-dose Rapa treatment. Mechanistically, the CD8 defect was linked to impaired glycolytic switch in stimulated naive cells and the reduced formation of short-lived effector cells. Therefore, more than one cell type required for a protective effector immune response is impaired by Rapa in both mice and humans, at the dose shown to improve immune memory and extend lifespan. This urges caution with regard to the relative therapeutic costs and benefits of Rapa treatment as means to improve immune memory.