Immune Senescence, Immunosenescence and Aging.

With aging, there is increased dysfunction of both innate and adaptive immune responses, which contributes to impaired immune responses to pathogens and greater mortality and morbidity. This age-related immune dysfunction is defined in general as immunosenescence and includes an increase in the number of memory T cells, loss of ability to respond to antigen and a lingering level of low-grade inflammation. However, certain features of immunosenescence are similar to cellular senescence, which is defined as the irreversible loss of proliferation in response to damage and stress. Importantly, senescence cells can develop an inflammatory senescence-associated secretory phenotype (SASP), that also drives non-autonomous cellular senescence and immune dysfunction. Interestingly, viral infection can increase the extent of immune senescence both directly and indirectly, leading to increased immune dysfunction and inflammation, especially in the elderly. This review focuses on age-related immune dysfunction, cellular senescence and the impaired immune response to pathogens.

An aged immune system drives senescence and ageing of solid organs.

Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.

Intersection of immunometabolism and immunosenescence during aging.

Aging is associated with the highest risk for morbidity and mortality to chronic or metabolic diseases, which are present in 50% of the elderly. Improving metabolic and immune function of the elderly would improve quality of life and reduce the risk for all other diseases. Tissue-resident macrophages and the NLRP3 inflammasome are established drivers of inflammaging and metabolic dysfunction. Energy-sensing signaling pathways connect sterile and metabolic inflammation with cellular senescence and tissue dysfunction. We discuss recent advances in the immunometabolism field. Common themes revealed by recent publications include the alterations in metabolic signaling (SIRTUIN, AMPK, or mTOR pathways) in aged immune cells, the impact of senescence on inflammaging and tissue dysfunction, and the age-related changes in metabolic tissues, especially adipose tissue, as an immunological organ. Promising gerotherapeutics are candidates to broadly target nutrient and energy sensing, inflammatory and senescence pathways, and have potential to improve healthspan and treat age-related diseases.

Characterization of age-associated exhausted CD8⁺ T cells defined by increased expression of Tim-3 and PD-1.

Aging is accompanied by altered T-cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD-1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T-cell exhaustion and aging-associated immune dysfunction. In this study, we demonstrate that T-cell immunoglobulin mucin domain-3 (Tim-3), another exhaustion marker, is up-regulated on aged T cells, especially CD8(+) T cells. Tim-3-expressing cells also produced PD-1, but Tim-3(+) PD-1(+) CD8(+) T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim-3(-) PD-1(+) cells. Tim-3(+) PD-1(+) CD8(+) T cells showed more evident properties associated with exhaustion than Tim-3(-) PD-1(+) CD8(+) T cells: an exhaustion-related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL-10 and induced normal CD8(+) T cells to produce IL-10, which might contribute to immune dysregulation in aged mice. The generation of Tim-3-expressing CD8(+) T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vß usage. In conclusion, we found that a CD8(+) T-cell population with age-associated exhaustion was distinguishable by its expression of Tim-3. These results provide clues for understanding the alterations that occur in T-cell populations with age and for improving dysfunctions related to the aging of the immune system.

A multimeric carcinoembryonic antigen signal inhibits the activation of human T cells by a SHP-independent mechanism: a potential mechanism for tumor-mediated suppression of T-cell immunity.

Carcinoembryonic antigen (CEA) is a well-known tumor antigen that is found in the serum of patients with various cancers and is correlated with an increased risk of cancer recurrence and metastasis. To understand the tumor environment and to develop antitumor therapies, CEA has been studied as an antigen to activate/tolerate specific T cells. In this study, we show that CEA can function as a coinhibitory molecule and can inhibit the activation of human peripheral blood mononucleated cell-derived T cells. The addition of CEA-overexpressing tumor cells or immobilized CEA dampened both cell proliferation and the expression of IL-2 and CD69 expression in T cells after TCR stimulation. The phosphorylation of ERK and translocation of NFAT were hampered in these cells, whereas the phosphorylation of proximal TCR signaling molecules such as ZAP70 and phospholipase Cγ was not affected by immobilized CEA. To determine the relevance of carcinoembryonic antigen-related cell adhesion molecule-1 and Src homology region 2 domain-containing phosphatase (SHP) molecules to CEA-mediated suppression, we tested the effect of the SHP inhibitor, NSC-87877, on CEA-mediated suppression of T cells; however, it did not reverse the effect of CEA. Collectively, these results indicate that CEA can function as a modulator of T-cell responses suggesting a novel mechanism of tumor evasion.

Detection of constant domain of human T cell antigen receptor alpha-chain via novel monoclonal antibody 7F18.

The αß T cell antigen receptor (TCR) endows T lymphocytes with immune specificity and controls their effector functions. Each person possesses a vast repertoire of TCRs that is generated by the well-studied processes of somatic recombination and thymic selection. While many antibodies specific for TCRß variable domains are available, antibodies specific for human TCRα are rare. We now report a novel monoclonal antibody, 7F18, which binds to human TCRα constant region, with specificity for a denatured epitope that can be visualized by SDS-PAGE followed by Western blot. Both immature and mature TCR α-chain products can be visualized, making 7F18 potentially applicable to various biochemical assays of multiprotein complex assembly and maturation. This new monoclonal antibody provides a tool that can potentially facilitate the biochemical analysis of comprehensive populations of human αß TCR complexes that need not be limited to small subsets of the repertoire.

Tumor-derived osteopontin suppresses antitumor immunity by promoting extramedullary myelopoiesis.

Extramedullary myelopoiesis occurs commonly in tumor-bearing animals and is known to lead to accumulation of peripheral myeloid-derived suppressor cells (MDSC), which play an important role in immune escape. However, the cellular and molecular mechanisms by which tumors induce extramedullary myelopoiesis are poorly understood. In this study, we found that osteopontin expressed by tumor cells enhances extramedullary myelopoiesis in a CD44-dependent manner through the Erk1/2-MAPK pathway. Osteopontin-mediated extramedullary myelopoiesis was directly associated with increased MDSCs in tumor-bearing hosts. More importantly, osteopontin silencing in tumor cells delayed both tumor growth and extramedullary myelopoiesis, while the same treatment did not affect tumor growth in vitro. Finally, treatment with an antibody against osteopontin inhibited tumor growth and synergized with cell-based immunotherapeutic vaccines in mediating antitumor immunity. Our findings unveil a novel immunosuppressive role for tumor-derived osteopontin and offer a rationale for its therapeutic targeting in cancer treatment.

The Wnt inhibitor secreted Frizzled-Related Protein 1 (sFRP1) promotes human Th17 differentiation.

Wnt/ß-catenin signaling plays a crucial role during embryogenesis and tumorigenesis, and in T cells, promotes the differentiation of Th2 cells. However, the role of Wnt signals in the differentiation and maintenance of human Th17 cells remains poorly understood. We found that the higher levels of IL-17 in the synovial fluid of rheumatoid arthritis (RA) patients compared with that of osteoarthritis (OA) patients were associated with a higher concentration of sFRP1 (secreted Frizzled-Related Protein 1), an inhibitor of the Wnt/ß-catenin pathway. The addition of sFRP1 during TCR-mediated stimulation induced a significant increase in IL-17 production by both naïve and memory CD4(+) T cells. Moreover, under Th17-differentiation conditions, the addition of sFRP1 significantly reduced the requirement for TGF-ß. Mechanistically, we observed that sFRP1 significantly enhanced the phosphorylation of Smad2/3 in CD4(+) T cells upon TGF-ß stimulation and that blocking TGF-ß signaling abolished the Th17-promoting activity of sFRP1. Our findings reveal a novel function for sFRP1 as a potent inducer of human Th17-cell differentiation. Consequently, sFRP1 may represent a promising target for the treatment of Th17-mediated disease in humans.

Retinoic acid alleviates Con A-induced hepatitis and differentially regulates effector production in NKT cells.

Retinoic acid (RA) is a diverse regulator of immune responses. Although RA promotes natural killer T (NKT) cell activation in vitro by increasing CD1d expression on antigen-presenting cells (APCs), the direct effects of RA on NKT-cell responses in vivo are not known. In the present study, we demonstrated the effect of RA on the severity of Con A-induced hepatitis and molecular changes of NKT cells. First, we demonstrated that Con A-induced liver damage was ameliorated by RA. In correlation with cytokine levels in serum, RA regulated the production of IFN-γ and IL-4 but not TNF-α by NKT cells without influencing the NKT-cell activation status. However, RA did not alleviate α-GalCer-induced liver injury, even though it reduced IFN-γ and IL-4 but not TNF-α levels in serum. This regulation was also detected when liver mononuclear cells (MNCs) or NKT hybridoma cells were treated with RA in vitro. The regulatory effect of RA on NKT cells was mediated by RAR-α, and RA reduced the phosphorylation of MAPK. These results suggest that RA differentially modulates the production of effector cytokines by NKT cells in hepatitis, and the suppressive effect of RA on hepatitis varies with the pathogenic mechanism of liver injury.

An α-GalCer analogue with branched acyl chain enhances protective immune responses in a nasal influenza vaccine.

α-Galactosylceramide (α-GalCer) is a safe and effective adjuvant for nasal vaccines and induces protective immune responses against tumors and viral infections. In our previous study, the fatty acyl chains of α-GalCer were modified based on the CD1d/glycolipid structure to generate α-GalCer analogues with branched acyl chains. In this study, two α-GalCer analogues, KBC-007 and KBC-009, that have different branched chain lengths were prepared and evaluated for their efficacy as nasal influenza vaccine adjuvants. These analogues displayed improved solubility over α-GalCer and potently stimulated NKT cells in both murine and in vitro human systems. Examination of serum cytokines in vivo revealed that these analogues elicited different cytokine release profiles compared to α-GalCer. KBC-009 induced both Th1/Th2 cytokines, whereas KBC-007 induced a more Th2-polarized cytokine response with diminished IFN-γ production. We found that a single immunization of inactivated influenza virus A/PR/8/34 (PR8) combined with α-GalCer analogues enhanced PR8-specific humoral and cellular immune responses in both systemic and mucosal compartments. Notably, KBC-009 exhibited potent adjuvant effects, inducing significantly higher systemic IgG and mucosal IgA antibody titers and enhancing cytotoxic T lymphocyte generation when compared to immunization with inactivated PR8 alone. In contrast, addition of KBC-007 to inactivated PR8 only marginally increased PR8-specific immune responses. The protective effect of KBC-009 against challenge infection was comparable to the effect produced by α-GalCer. These results suggest that an α-GalCer analogue with a branched acyl chain could be used as an effective mucosal adjuvant for the induction of protective immune responses against influenza virus infection.

Immunosuppressive myeloid-derived suppressor cells can be converted into immunogenic APCs with the help of activated NKT cells: an alternative cell-based antitumor vaccine.

Myeloid-derived suppressor cells (MDSCs), which are known to be accumulated in the blood, spleen, and bone marrow of tumor-bearing mice and cancer patients, were tested as APCs for a cellular vaccine because they have phenotypical similarity with inflammatory monocytes and may be differentiated from the same precursors as monocytes. Although MDSCs have immunosuppressive properties, in vivo transferred MDSCs, which present tumor Ag and NKT cell ligand (alpha-galactosylceramide), significantly prolonged survival time in metastatic tumor-bearing mice in a CD8(+) cell-, NK cell-, and NKT cell-dependent manner vs a CD4(+) T cell- and host dendritic cell-independent manner. Major concerns about using MDSCs as APCs in a vaccine are their suppression of CTLs and their induction of Foxp3(+) regulatory T cells. However, alpha-galactosylceramide-loaded MDSCs did not suppress CD4(+) and CD8(+) T cells and allowed for the generation of Ag-specific CTL immunity without increasing the generation of regulatory T cells. Furthermore, stimulation with activated NKT cells induced changes on MDSCs in phenotypical or maturation markers, including CD11b, CD11c, and CD86. Taken together, these findings suggest that NKT cells facilitate the conversion of immunosuppressive MDSCs into immunogenic APCs, eliciting successful antitumor immunity and providing the basis for alternative cell-based vaccines.

Functional maturation of lamina propria dendritic cells by activation of NKT cells mediates the abrogation of oral tolerance.

We previously showed that although systemic administration of alpha-galactosylceramide (alphaGalCer) or agonistic anti-CD40 induced functional maturation of dendritic cells (DC) in mesenteric lymph nodes, only the former treatment succeeded in breaking the induction of oral tolerance. In this study, we looked for the essential factor responsible for the disruption of oral tolerance. We found that lamina propria (LP)-DC was responsible for the oral OVA presentation and that Peyer's patch was not essential for the induction of oral tolerance. Therefore, we investigated the role of LP-DC. Treatment with alphaGalCer but not with anti-CD40 induced the full maturation of LP-DC at an early time point. This functional activation of LP-DC was mediated by strong activation of NKT cells that reside abundantly in the small intestinal lamina propria (SI-LP) and interferon-gamma partially contributed to the LP-DC activation. LP-DC isolated from alphaGalCer-treated OVA-fed mice induced the differentiation of naïve CD4+ T cells into Th1 and Th2 and was associated with the reduced Foxp3+ population. In contrast, LP-DC isolated from anti-CD40-treated OVA-fed mice failed to generate Th cell differentiation but induced more Foxp3+ CD4+ T cells. Our results demonstrate that triggered by NKT cells in SI-LP, functional maturation of Ag-capturing DC from SI-LP is necessary for the abrogation of oral tolerance induction.

A distinct subset of natural killer T cells produces IL-17, contributing to airway infiltration of neutrophils but not to airway hyperreactivity.

Activated natural killer T (NKT) cells produce a broad range of cytokines, including IL-4 and IFN-gamma, that determine immunomodulatory functions in various animal models. In this report, we show that a well-known proinflammatory cytokine, IL-17 is also produced by a distinct population of NKT cells upon TCR stimulation. Administration of alpha-galactosylceramide (alpha-GalCer), a strong agonist of NKT cells, induces rapid IL-17 production by a small population of NKT cells, mostly belonging to a population different from that of IL-4- and IFN-gamma-producing NKT cells. IL-17-producing NKT cells showed unresponsiveness after stimulation of alpha-GalCer as conventional NKT cells. During airway inflammation induced by pulmonary activation of NKT cells with alpha-GalCer, IL-17 contributes to the infiltration of neutrophils into the airway but has no effect on airway hyperreactivity (AHR). These results indicate that TCR stimulation induces IL-17 expression by a novel population of NKT cells and may help to explain diverse NKT cell functions.

4-1BB engagement costimulates NKT cell activation and exacerbates NKT cell ligand-induced airway hyperresponsiveness and inflammation.

Multiple studies have demonstrated that 4-1BB (CD137), a member of the TNF receptor superfamily, is expressed on several immune cells including activated T cells. However, the expression and the role of 4-1BB on natural killer T (NKT) cells have not been fully characterized. In this study, it was shown that 4-1BB was not expressed on naive NKT cells but was rapidly induced on activated NKT cells by TCR engagement with alpha-galactosylceramide (alpha-GalCer). Also, 4-1BB signaling provided by 3H3, an agonistic anti-4-1BB mAb, promoted NKT cell activation resulting in enhanced cytokine production of NKT cells driven by alpha-GalCer. When NKT cell-driven airway immune responses were evaluated by intranasal administration of alpha-GalCer, airway hyperresponsiveness (AHR) and lung inflammation were significantly more aggravated in mice treated with 3H3 and alpha-GalCer than in mice treated with alpha-GalCer alone. These aggravations were accompanied by up-regulation of IL-4, IL-13, and IFN-gamma production. Interestingly, AHR was not developed in IL-4Ralpha-deficient mice treated with alpha-GalCer with or without 3H3 but was exacerbated in IFN-gamma-deficient mice. Our study suggests that 4-1BB on NKT cells functions as a costimulatory molecule and exacerbates the induction of NKT cell-mediated AHR, which is dependent on the IL-4Ralpha-mediated pathway.

Phenotypic changes induced by IL-12 priming regulate effector and memory CD8 T cell differentiation.

In addition to TCR and co-stimulatory signals, inflammatory cytokines such as IL-12 provide important signals for differentiation and survival of activated CD8 T cells. In the present study, to investigate the mechanisms by which IL-12 priming contributes to activation and enhanced survival of CD8 T cells, we searched the differentially regulated genes and markers by IL-12 during antigenic stimulation. Here, we show that IL-12 priming results in the increased subpopulation of CD127(hi) cells, which differentiates into long-lived memory cells. We also found that IL-12 priming induces IL-10 expression from activated CD8 T cells, which is distinct from CD127 up-regulation. Direct IL-10 priming of CD8 T cells results in the significant increase of effector and memory CD8 T cell population after adoptive transfer, and this priming effect is closely associated with less susceptibility to apoptosis. Although IL-10 is known as a cytokine with anti-inflammatory and immunosuppressive properties, our results have shown that IL-10 has a direct and positive effect on the survival of CD8 T cells. Together, we suggest that IL-10-dependent and independent effects of IL-12 play important roles in regulating differentiation and survival of activated CD8 T cells into effector and memory cells.

Mediastinal lymph node CD8alpha- DC initiate antigen presentation following intranasal coadministration of alpha-GalCer.

Our previous study revealed that alpha-galactosylceramide (alpha-GalCer) is a potent nasal vaccine adjuvant inducing both potent humoral and cellular immune responses and affording complete protection against viral infections and tumors. However, the antigen-presenting cells (APC) that are activated by NKT cells and thereby initiate the immune responses following intranasal coadministration of protein antigen and alpha-GalCer are poorly understood. We assessed here where antigen presentation occurs and which APC subset mediates the early stages of immune responses when protein antigen and alpha-GalCer are intranasally administered. We show that dendritic cells (DC), but not B cells, initiated the mucosal immune responses at mediastinal lymph nodes. Of the DC subsets, the CD8alpha-B220-CD11c+ DC subset played the most prominent role in the direct and cross-presentation of protein antigen to naive T cells and in triggering the naive T cells to differentiate into effector T cells. This might be mainly caused by a relatively larger population of CD1dhigh cells of CD8alpha-B220-CD11c+ DC subset than those of other DC subsets. These results indicate that CD8alpha-B220-CD11c+ DC is the principal subset becoming immunogenic after interaction with NKT cells and abrogating tolerance to intranasally administered protein antigen when alpha-GalCer is coadministered as a nasal vaccine adjuvant.

A single intranasal immunization with inactivated influenza virus and alpha-galactosylceramide induces long-term protective immunity without redirecting antigen to the central nervous system.

alpha-Galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, was known to activate natural killer T (NKT) cells through CD1d-mediated Ag presentation and induce Th1 and/or Th2 immunity. In this study, we evaluated the nasal adjuvanticity of alpha-GalCer when co-administered with formalin-inactivated influenza virus A/PR/8/34 (PR8) in BALB/c mice. A single nasal immunization of inactivated PR8 and alpha-GalCer induced brisk levels of PR8-specific IgG and IgA Abs in serum and lung washes. Antigen-specific Ab responses lasted for 3 months, providing protective immunity against challenge with live PR8. In addition, mice given alpha-GalCer also exhibited cellular immune responses including cytotoxic T lymphocyte (CTL) generation. Because it did not redirect Ags into brain, alpha-GalCer would likely pose no risk if administered as a nasal adjuvant. These results suggest for the first time that a single nasal immunization of inactivated virus and alpha-GalCer is a safe and effective means of preventing influenza infection.