Immune Senescence: Relative Contributions of Age and Cytomegalovirus Infection

Immune senescence, defined as the age-associated dysregulation and dysfunction of the immune system, is characterised by impaired protective immunity and decreased efficacy of vaccines. Recent clinical, epidemiological and immunological studies suggest that Cytomegalovirus (CMV) infection may be associated with accelerated immune senescence, possibly by restricting the naïve T cell repertoire. However, direct evidence whether and how CMV-infection is implicated in immune senescence is still lacking. In this study, we have investigated whether latent mouse CMV (MCMV) infection with or without thymectomy (Tx) alters antiviral immunity of young and aged mice. After infection with lymphocytic choriomeningitis virus (LCMV) or Vaccinia virus, specific antiviral T cell responses were significantly reduced in old, old MCMV-infected and/or Tx mice compared to young mice. Importantly, control of LCMV replication was more profoundly impaired in aged MCMV-infected mice compared to age-matched MCMV-naïve or young mice. In addition, latent MCMV infection was associated with slightly reduced vaccination efficacy in old Tx mice. In contrast to the prevailing hypothesis of a CMV-mediated restriction of the naïve T cell repertoire, we found similar naïve T cell numbers in MCMV-infected and non-infected mice, whereas ageing and Tx clearly reduced the naïve T cell pool. Instead, MCMV-infection expanded the total CD8⁺ T cell pool by a massive accumulation of effector memory T cells. Based on these results, we propose a new model of increased competition between CMV-specific memory T cells and any ‘de novo’ immune response in aged individuals. In summary, our results directly demonstrate in a mouse model that latent CMV-infection impairs immunity in old age and propagates immune senescence.     

Maturation-dependent licensing of naive T cells for rapid TNF production

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4⁺ and CD8⁺ single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.     

TGF-β Sensitivity Restrains CD8+ T Cell Homeostatic Proliferation by Enforcing Sensitivity to IL-7 and IL-15

The pleiotropic cytokine TGF-β has been implicated in the regulation of numerous aspects of the immune response, including naïve T cell homeostasis. Previous studies found that impairing TGF-β responsiveness (through expression of a dominant-negative TGF-β RII [DNRII] transgene) leads to accumulation of memory phenotype CD8 T cells, and it was proposed that this resulted from enhanced IL-15 sensitivity. Here we show naïve DNRII CD8 T cells exhibit enhanced lymphopenia-driven proliferation and generation of “homeostatic” memory cells. However, this enhanced response occurred in the absence of IL-15 and, unexpectedly, even in the combined absence of IL-7 and IL-15, which were thought essential for CD8 T cell homeostatic expansion. DNRII transgenic CD8 T cells still require access to self Class I MHC for homeostatic proliferation, arguing against generalized dysregulation of homeostatic cues. These findings suggest TGF-β responsiveness is critical for enforcing sensitivity to homeostatic cytokines that limit maintenance and composition of the CD8 T cell pool. (154 words).     

TSC1/2 Signaling Complex Is Essential for Peripheral Na?ve CD8+ T Cell Survival and Homeostasis in Mice

The PI3K-Akt-mTOR pathway plays crucial roles in regulating both innate and adaptive immunity. However, the role of TSC1, a critical negative regulator of mTOR, in peripheral T cell homeostasis remains elusive. With T cell-specific Tsc1 conditional knockout (Tsc1 KO) mice, we found that peripheral naïve CD8⁺ T cells but not CD4⁺ T cells were severely reduced. Tsc1 KO naïve CD8⁺ T cells showed profound survival defect in an adoptive transfer model and in culture with either stimulation of IL-7 or IL-15, despite comparable CD122 and CD127 expression between control and KO CD8⁺ T cells. IL-7 stimulated phosphorylation of Akt(S473) was diminished in Tsc1 KO naïve CD8⁺T cells due to hyperactive mTOR-mediated feedback suppression on PI3K-AKT signaling. Furthermore, impaired Foxo1/Foxo3a phosphorylation and increased pro-apoptotic Bim expression in Tsc1 KO naïve CD8⁺T cells were observed upon stimulation of IL-7. Collectively, our study suggests that TSC1 plays an essential role in regulating peripheral naïve CD8⁺ T cell homeostasis, possible via an mTOR-Akt-FoxO-Bim signaling pathway.     

Helios expression is a marker of T cell activation and proliferation

Foxp3+ T-regulatory cells (Tregs) normally serve to attenuate immune responses and are key to maintenance of immune homeostasis. Over the past decade, Treg cells have become a major focus of research for many groups, and various functional subsets have been characterized. Recently, the Ikaros family member, Helios, was reported as a marker to discriminate naturally occurring, thymic-derived Tregs from those peripherally induced from naïve CD4+ T cells. We investigated Helios expression in murine and human T cells under resting or activating conditions, using well-characterized molecules of naïve/effector/memory phenotypes, as well as a set of Treg-associated markers. We found that Helios-negative T cells are enriched for naïve T cell phenotypes and vice versa. Moreover, Helios can be induced during T cell activation and proliferation, but regresses in the same cells under resting conditions. We demonstrated comparable findings using human and murine CD4+Foxp3+ Tregs, as well as in CD4+ and CD8+ T cells. Since Helios expression is associated with T cell activation and cellular division, regardless of the cell subset involved, it does not appear suitable as a marker to distinguish natural and induced Treg cells.     

Cytomegalovirus Infection Impairs Immune Responses and Accentuates T-cell Pool Changes Observed in Mice with Aging

Prominent immune alterations associated with aging include the loss of naïve T-cell numbers, diversity and function. While genetic contributors and mechanistic details in the aging process have been addressed in multiple studies, the role of environmental agents in immune aging remains incompletely understood. From the standpoint of environmental infectious agents, latent cytomegalovirus (CMV) infection has been associated with an immune risk profile in the elderly humans, yet the cause-effect relationship of this association remains unclear. Here we present direct experimental evidence that mouse CMV (MCMV) infection results in select T-cell subset changes associated with immune aging, namely the increase of relative and absolute counts of CD8 T-cells in the blood, with a decreased representation of the naïve and the increased representation of the effector memory blood CD8 T-cells. Moreover, MCMV infection resulted in significantly weaker CD8 responses to superinfection with Influenza, Human Herpes Virus I or West-Nile-Virus, even 16 months following MCMV infection. These irreversible losses in T-cell function could not be observed in uninfected or in vaccinia virus-infected controls and were not due to the immune-evasive action of MCMV genes. Rather, the CD8 activation in draining lymph nodes upon viral challenge was decreased in MCMV infected mice and the immune response correlated directly to the frequency of the naïve and inversely to that of the effector cells in the blood CD8 pool. Therefore, latent MCMV infection resulted in pronounced changes of the T-cell compartment consistent with impaired naïve T-cell function.     

Tentative T cells: memory cells are quick to respond, but slow to divide

T cell memory is a cornerstone of protective immunity, and is the key element in successful vaccination. Upon encountering the relevant pathogen, memory T cells are thought to initiate cell division much more rapidly than their naïve counterparts, and this is thought to confer a significant biological advantage upon an immune host. Here, we use traceable TCR-transgenic T cells to evaluate this proposed characteristic in CD4⁺ and CD8⁺ memory T cells. We find that, even in the presence of abundant antigen that was sufficient to induce in vivo IFNγ production by memory T cells, both memory and naïve T cells show an extended, and indistinguishable, delay in the onset of proliferation. Although memory cells can detect, and respond to, virus infection within a few hours, their proliferation did not begin until ∼3 days after infection, and occurred simultaneously in all anatomical compartments. Thereafter, cell division was extraordinarily rapid for both naïve and memory cells, with the latter showing a somewhat accelerated accumulation. We propose that, by permitting memory T cells to rapidly exert their effector functions while delaying the onset of their proliferation, evolution has provided a safeguard that balances the risk of infection against the consequences of severe T cell–mediated immunopathology.     

Murine B cell development and antibody responses to model antigens are not impaired in the absence of the TNF receptor GITR

The Glucocorticoid-Induced Tumor necrosis factor Receptor GITR, a member of the tumor necrosis factor receptor superfamily, has been shown to be important in modulating immune responses in the context of T cell immunity. B lymphocytes also express GITR, but a role of GITR in humoral immunity has not been fully explored. To address this question, we performed studies to determine the kinetics of GITR expression on naïve and stimulated B cells and the capacity of B cells to develop and mount antibody responses in GITR^−/− mice. Results of our studies indicate that all mature B cells express GITR on the cell surface, albeit at different levels. Expression of GITR on naïve mature B cells is upregulated by BCR signaling, but is counteracted by helper T cell-related factors and other inflammatory signals in vitro. In line with these findings, expression of GITR on germinal center and memory B cells is lower than that on naïve B cells. However, the expression of GITR is strongly upregulated in plasma cells. Despite these differences in GITR expression, the absence of GITR has no effect on T cell-dependent and T cell-independent antibody responses to model antigens in GITR^−/− mice, or on B cell activation and proliferation in vitro. GITR deficiency manifests only with a slight reduction of mature B cell numbers and increased turnover of naïve B cells, suggesting that GITR slightly contributes to mature B cell homeostasis. Overall, our data indicate that GITR does not play a significant role in B cell development and antibody responses to T-dependent and independent model antigens within the context of a GITR-deficient genetic background.     

Differential susceptibility of na?ve and activated human gammadelta T cells to activation-induced cell death by T-cell receptor cross-linking.

BACKGROUND: T cells undergo activation-induced cell death (AICD) through repeated stimulation of their T cell receptors (TCRs). Activated human gammadelta T cells were found to die by apoptosis when their TCRs were cross-linked by antibodies, whereas naïve gammadelta T cells freshly isolated from blood did not. Therefore, we investigated the factors that could contribute to this differential susceptibility. MATERIALS AND METHODS: Gammadelta T cells were isolated from the peripheral blood of healthy human volunteers and their TCRs were cross-linked either directly (naïve) or after an in vitro incubation of 11 days (activated). Their cell cycle profiles, cytokine, Fas and FasL mRNA messages, and surface expression of Fas and FasL were determined. RESULTS: The naïve cells were cycling while the activated T cells exited from the G1 to subG1 phase upon TCR cross-linking. IL-2 and IL-4 mRNAs and surface expression of FasL were detected only in activated T cells in the time period examined. In addition, cFLIP mRNA expression was found only in naïve gammadelta T cells and activated T cells treated with cyclosporin A (CsA), which inhibited AICD in the activated T cells. CsA also downregulated the surface expression of FasL in activated T cells. CONCLUSIONS: The differential expression of cytokines, apoptotic inducers and inhibitors provide the basis for the differential susceptibility of naïve and activated gammadelta T cells to AICD upon TCR cross-linking. This contributes to our understanding of the regulation and maintenance of gammadelta T-cell homeostasis, which would be important in many infectious as well as autoimmune diseases, where gammadelta T cells have been implicated.     

In vivo expansion of naïve CD4+ CD25(high) FOXP3+ regulatory T cells in patients with colorectal carcinoma after IL-2 administration

Regulatory T cells (T_reg cells) are increased in context of malignancies and their expansion can be correlated with higher disease burden and decreased survival. Initially, interleukin 2 (IL-2) has been used as T-cell growth factor in clinical vaccination trials. In murine models, however, a role of IL-2 in development, differentiation, homeostasis, and function of T_reg cells was established. In IL-2 treated cancer patients a further T_reg-cell expansion was described, yet, the mechanism of expansion is still elusive. Here we report that functional T_reg cells of a naïve phenotype - as determined by CCR7 and CD45RA expression - are significantly expanded in colorectal cancer patients. Treatment of 15 UICC stage IV colorectal cancer patients with IL-2 in a phase I/II peptide vaccination trial further enlarges the already increased naïve T_reg-cell pool. Higher frequencies of T-cell receptor excision circles in naïve T_reg cells indicate IL-2 dependent thymic generation of naïve T_reg cells as a mechanism leading to increased frequencies of T_reg cells post IL-2 treatment in cancer patients. This finding could be confirmed in naïve murine T_reg cells after IL-2 administration. These results point to a more complex regulation of T_reg cells in context of IL-2 administration. Future strategies therefore might aim at combining IL-2 therapy with novel strategies to circumvent expansion and differentiation of naïve T_reg cells.     

Lymphoid Tissue Damage in HIV-1 Infection Depletes Na?ve T Cells and Limits T Cell Reconstitution after Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can suppress HIV-1 replication and normalize the chronic immune activation associated with infection, but restoration of naïve CD4⁺ T cell populations is slow and usually incomplete for reasons that have yet to be determined. We tested the hypothesis that damage to the lymphoid tissue (LT) fibroblastic reticular cell (FRC) network contributes to naïve T cell loss in HIV-1 infection by restricting access to critical factors required for T cell survival. We show that collagen deposition and progressive loss of the FRC network in LTs prior to treatment restrict both access to and a major source of the survival factor interleukin-7 (IL-7). As a consequence, apoptosis within naïve T cell populations increases significantly, resulting in progressive depletion of both naïve CD4⁺ and CD8⁺ T cell populations. We further show that the extent of loss of the FRC network and collagen deposition predict the extent of restoration of the naïve T cell population after 6 month of HAART, and that restoration of FRC networks correlates with the stage of disease at which the therapy is initiated. Because restoration of the FRC network and reconstitution of naïve T cell populations are only optimal when therapy is initiated in the early/acute stage of infection, our findings strongly suggest that HAART should be initiated as soon as possible. Moreover, our findings also point to the potential use of adjunctive anti-fibrotic therapies to avert or moderate the pathological consequences of LT fibrosis, thereby improving immune reconstitution.     

Failure of CD4 T-cells to respond to liver-derived antigen and to provide help to CD8 T-cells

CD4 T-cell help is required for the induction of efficient CD8 T-cells responses and the generation of memory cells. Lack of CD4 T-cell help may contribute to an exhausted CD8 phenotype and viral persistence. Little is known about priming of CD4 T-cells by liver-derived antigen. We used TF-OVA mice expressing ovalbumin in hepatocytes to investigate CD4 T-cell priming by liver-derived antigen and the impact of CD4 T-cell help on CD8 T-cell function. Naïve and effector CD4 T-cells specific for ovalbumin were transferred into TF-OVA mice alone or together with naïve ovalbumin-specific CD8 T-cells. T-cell activation and function were analyzed. CD4 T-cells ignored antigen presented by liver antigen-presenting cells (APCs) in vitro and in vivo but were primed in the liver-draining lymph node and the spleen. No priming occurred in the absence of bone-marrow derived APCs capable of presenting ovalbumin in vivo. CD4 T-cells primed in TF-OVA mice displayed defective Th1-effector function and caused no liver damage. CD4 T-cells were not required for the induction of hepatitis by CD8 T-cells. Th1-effector but not naïve CD4 T-cells augmented the severity of liver injury caused by CD8 T-cells. Our data demonstrate that CD4 T-cells fail to respond to liver-derived antigen presented by liver APCs and develop defective effector function after priming in lymph nodes and spleen. The lack of CD4 T-cell help may be responsible for insufficient CD8 T-cell function against hepatic antigens.     

De novo infection of B cells during murine gammaherpesvirus 68 latency

The mechanisms by which gammaherpesviruses maintain latency are unclear. Here we used a murine gammaherpesvirus model to show that previously uninfected B cells in immunocompetent mice can acquire virus during latency. In vivo depletion of T cells allowed viral reactivation, as measured by increased viral loads, but not enhanced transfer of virus to new cells. In the absence of both immune T cells and antibody following the transfer of latently infected cells into naïve animals, there was robust infection of new B cells. These data confirm that both T cells and antibody contribute to the control of gammaherpesvirus latency, reactivation, and spread.     

Increased memory conversion of naïve CD8 T cells activated during late phases of acute virus infection due to decreased cumulative antigen exposure

Background

Memory CD8 T cells form an essential part of protective immunity against viral infections. Antigenic load, costimulation, CD4-help, cytokines and chemokines fluctuate during the course of an antiviral immune response thus affecting CD8 T cell activation and memory conversion.

Methodology/Principal Findings

In the present study, naïve TCR transgenic LCMV-specific P14 CD8 T cells engaged at a late stage during the acute antiviral LCMV response showed reduced expansion kinetics but greater memory conversion in the spleen. Such late activated cells displayed a memory precursor effector phenotype already at the peak of the systemic antiviral response, suggesting that the environment determined their fate during antigen encounter. In the spleen, the majority of late transferred cells exhibited a central memory phenotype compared to the effector memory displayed by the early transferred cells. Increasing the inflammatory response by exogenous administration of IFNγ, PolyI:C or CpG did not affect memory conversion in the late transferred group, suggesting that the diverging antigen load early versus later during acute infection had determined their fate. In agreement, reduction in the LCMV antigenic load after ribavirin treatment enhanced the contribution of early transferred cells to the long lasting memory pool.

Conclusions/Significance

Our results show that naïve CD8 cells, exposed to reduced duration or concentration of antigen during viral infection convert into memory more efficiently, an observation that could have significant implications for vaccine design.     

Antibody V(h) repertoire differences between resolving and chronically evolving hepatitis C virus infections

Despite the production of neutralizing antibodies to hepatitis C virus (HCV), many patients fail to clear the virus and instead develop chronic infection and long-term complications. To understand how HCV infection perturbs the antibody repertoire and to identify molecular features of antibody genes associated with either viral clearance or chronic infection, we sequenced the V(D)J region of naïve and memory B cells of 6 persons who spontaneously resolved an HCV infection (SR), 9 patients with a newly diagnosed chronically evolving infection (CE), and 7 healthy donors. In both naïve and memory B cells, the frequency of use of particular antibody gene subfamilies and segments varied among the three clinical groups, especially between SR and CE. Compared to CE, SR antibody genes used fewer VH, D and JH gene segments in naïve B cells and fewer VH segments in memory B cells. SR and CE groups significantly differed in the frequency of use of 7 gene segments in naïve B cell clones and 3 gene segments in memory clones. The nucleotide mutation rates were similar among groups, but the pattern of replacement and silent mutations in memory B cell clones indicated greater antigen selection in SR than CE. Greater clonal evolution of SR than CE memory B cells was revealed by analysis of phylogenetic trees and CDR3 lengths. Pauciclonality of the peripheral memory B cell population is a distinguishing feature of persons who spontaneously resolved an HCV infection. This finding, previously considered characteristic only of patients with HCV-associated lymphoproliferative disorders, suggests that the B cell clones potentially involved in clearance of the virus may also be those susceptible to abnormal expansion.     

Preparation of myeloid derived suppressor cells (MDSC) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (AutoMACS)

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological conditions including parasitic infection, inflammation, traumatic stress, graft-versus-host disease, diabetes and cancer^1-7. In mice, MDSC express Mac-1 (CD11b) and Gr-1 (Ly6G and Ly6C) surface antigens⁷. It is important to note that MDSC are well studied in various tumor-bearing hosts where they are significantly expanded and suppress anti-tumor immune responses compared to naïve counterparts^7-10. However, depending on the pathological condition, there are different subpopulations of MDSC with distinct mechanisms and targets of suppression^11,12. Therefore, effective methods to isolate viable MDSC populations are important in elucidating their different molecular mechanisms of suppression in vitro and in vivo.Recently, the Ghansah group has reported the expansion of MDSC in a murine pancreatic cancer model. Our tumor-bearing MDSC display a loss of homeostasis and increased suppressive function compared to naïve MDSC ¹³. MDSC percentages are significantly less in lymphoid compartments of naïve vs. tumor-bearing mice. This is a major caveat, which often hinders accurate comparative analyses of these MDSC. Therefore, enriching Gr-1⁺ leukocytes from naïve mice prior to Fluorescence Activated Cell Sorting (FACS) enhances purity, viability and significantly reduces sort time. However, enrichment of Gr-1⁺ leukocytes from tumor-bearing mice is optional as these are in abundance for quick FACS sorting. Therefore, in this protocol, we describe a highly efficient method of immunophenotyping MDSC and enriching Gr-1⁺ leukocytes from spleens of naïve mice for sorting MDSC in a timely manner. Immunocompetent C57BL/6 mice are inoculated with murine Panc02 cells subcutaneously whereas naïve mice receive 1XPBS. Approximately 30 days post inoculation; spleens are harvested and processed into single-cell suspensions using a cell dissociation sieve. Splenocytes are then Red Blood Cell (RBC) lysed and an aliquot of these leukocytes are stained using fluorochrome-conjugated antibodies against Mac-1 and Gr-1 to immunophenotype MDSC percentages using Flow Cytometry. In a parallel experiment, whole leukocytes from naïve mice are stained with fluorescent-conjugated Gr-1 antibodies, incubated with PE-MicroBeads and positively selected using an automated Magnetic Activated Cell Sorting (autoMACS) Pro Separator. Next, an aliquot of Gr-1⁺ leukocytes are stained with Mac-1 antibodies to identify the increase in MDSC percentages using Flow Cytometry. Now, these Gr1⁺ enriched leukocytes are ready for FACS sorting of MDSC to be used in comparative analyses (naïve vs. tumor- bearing) in in vivo and in vitro assays.     

Apoptosis of purified CD4+ T cell subsets is dominated by cytokine deprivation and absence of other cells in new onset diabetic NOD mice

Background

Regulatory T cells (Treg) play a significant role in immune homeostasis and self-tolerance. Excessive sensitivity of isolated Treg to apoptosis has been demonstrated in NOD mice and humans suffering of type 1 diabetes, suggesting a possible role in the immune dysfunction that underlies autoimmune insulitis. In this study the sensitivity to apoptosis was measured in T cells from new onset diabetic NOD females, comparing purified subsets to mixed cultures.

Principal Findings

Apoptotic cells are short lived in vivo and death occurs primarily during isolation, manipulation and culture. Excessive susceptibility of CD25⁺ T cells to spontaneous apoptosis is characteristic of isolated subsets, however disappears when death is measured in mixed splenocyte cultures. In variance, CD25⁻ T cells display balanced sensitivity to apoptosis under both conditions. The isolation procedure removes soluble factors, IL-2 playing a significant role in sustaining Treg viability. In addition, pro- and anti-apoptotic signals are transduced by cell-to-cell interactions: CD3 and CD28 protect CD25⁺ T cells from apoptosis, and in parallel sensitize naïve effector cells to apoptosis. Treg viability is modulated both by other T cells and other subsets within mixed splenocyte cultures. Variations in sensitivity to apoptosis are often hindered by fast proliferation of viable cells, therefore cycling rates are mandatory to adequate interpretation of cell death assays.

Conclusions

The sensitivity of purified Treg to apoptosis is dominated by cytokine deprivation and absence of cell-to-cell interactions, and deviate significantly from measurements in mixed populations. Balanced sensitivity of naïve/effector and regulatory T cells to apoptosis in NOD mice argues against the concept that differential susceptibility affects disease evolution and progression.