Brigitte Jenewein

Long-Term Cytomegalovirus Infection Leads to Significant Changes in the Composition of the CD8+ T-Cell Repertoire, Which May Be the Basis for an Imbalance in the Cytokine Production Profile in Elderly Persons

ABSTRACT In spite of the present belief that latent cytomegalovirus (CMV) infection drives CD8+ T-cell differentiation and induces premature immune senescence, no systematic studies have so far been performed to compare phenotypical and functional changes in the CD8+ T-cell repertoire in CMV-infected and noninfected persons of different age groups. In the present study, number, cytokine production, and growth potential of naïve (CD45RA+ CD28+), memory (CD45RA− CD28+), and effector (CD45RA+ CD28− or CD45RA− CD28−) CD8+ T cells were analyzed in young, middle-aged, and elderly clinically healthy persons with a positive or negative CMV antibody serology. Numbers and functional properties of CMVpp65495-503-specific CD8+ T cells were also studied. We demonstrate that aging as well as CMV infection lead to a decrease in the size of the naïve CD8+ T-cell pool but to an increase in the number of CD8+ effector T cells, which produce gamma interferon but lack substantial growth potential. The size of the CD8+ memory T-cell population, which grows well and produces interleukin-2 (IL-2) and IL-4, also increases with aging, but this increase is missing in CMV carriers. Life-long latent CMV infection seems thus to diminish the size of the naïve and the early memory T-cell pool and to drive a Th1 polarization within the immune system. This can lead to a reduced diversity of CD8 responses and to chronic inflammatory processes which may be the basis of severe health problems in elderly persons.

Age‐related loss of naïve T cells and dysregulation of T‐cell/B‐cell interactions in human lymph nodes

In this study we analysed the effects of age on T and B lymphocytes in human lymph nodes by comparing lymphocyte subsets in paraffin sections from lymph node tissue taken from healthy young and elderly people. We demonstrate that the relative number of CD8+ T cells decreases with age but that the relative number of CD4+ T cells does not. There is also a very pronounced age‐dependent loss of CD45RA+ naïve T cells. The number and size of follicles and the relative number of CD20+ B cells are similar in young and elderly donors. For polymerase chain reaction analysis of the T‐cell receptor (TCR) repertoire the TCR‐γ gene rearrangements were used as a marker of clonality. This is a reliable tool to detect not only clonal TCR‐γδ populations but also TCR‐αβ populations. Young donors with clonal T‐cell expansions in their lymph node tissue do, however, have a higher number of CD20+ B cells, a higher relative size of germinal centres compared to the follicle mantles and a higher number of immunoglobulin M‐expressing cells than young donors without evidence of clonal T‐cell expansions. Corresponding changes are not observed in elderly donors with clonal T‐cell expansions in their lymph node tissue. In summary our findings demonstrate characteristic effects of aging on human lymph node tissue, the most striking feature being the depletion of naïve T cells and the apparent dysregulation of T‐cell/B‐cell interactions in old age.

Human Bone Marrow Hosts Polyfunctional Memory CD4+ and CD8+ T Cells with Close Contact to IL-15–Producing Cells

Recently, a key role in memory T cell homing and survival has been attributed to the bone marrow (BM) in mice. In the human BM, the repertoire, function, and survival niches of CD4+ and CD8+ T cells have not yet been elucidated. In this study, we demonstrate that CD4+ and CD8+ effector memory T cells accumulate in the human BM and are in a heightened activation state as revealed by CD69 expression. BM-resident memory T cells produce more IFN-γ and are frequently polyfunctional. Immunofluorescence analysis revealed that CD4+ and CD8+ T cells are in the immediate vicinity of IL-15–producing BM cells, suggesting a close interaction between these two cell types and a regulatory role of IL-15 on T cells. Accordingly, IL-15 induced an identical pattern of CD69 expression in peripheral blood CD4+ and CD8+ T cell subsets. Moreover, the IL-15–inducible molecules Bcl-xL, MIP-1α, MIP-1β, and CCR5 were upregulated in the human BM. In summary, our results indicate that the human BM microenvironment, in particular IL-15–producing cells, is important for the maintenance of a polyfunctional memory CD4+ and CD8+ T cell pool.

IL-4-Producing CD8+ T Cells with a CD62L++(bright) Phenotype Accumulate in a Subgroup of Older Adults and Are Associated with the Maintenance of Intact Humoral Immunity in Old Age

An increased production of proinflammatory cytokines occurs in a high percentage of elderly persons and is associated with an impaired humoral immune response. However, high IL-4 production has also been observed in old age. We now demonstrate an IL-4-producing subpopulation of CD8+ T cells in a subgroup of healthy older adults. This T cell subset is substantial in size and has a characteristic phenotype expressing CD45RO, CD28, CD62L, and CD25. IL-4-producing CD8+ T cells produce large amounts of IL-2 but not IFN-γ or perforin, and these cells do not have a regulatory suppressive effect on other T cells. In vivo IL-4-producing CD8+ T cells can be stably detected over a year. When put into culture they also have a stable cytokine production pattern but fail to produce perforin even in the presence of IL-12. This special T cell type does not occur in persons under the age of 40, but is present in 36% of the persons >60 years of age. In this age group, IL-4-producing CD8+ T cells are more frequent in persons who are still capable of raising a humoral immune response following immunization than in others who fail to produce protective Abs after vaccination. Our results suggest that CD8+ T cells with a CD62L++(bright) phenotype accumulate in a subgroup of older adults. Due to their phenotype that enables them to migrate into lymphoid tissues and to their capacity to produce IL-4, these cells may counterbalance the overproduction of proinflammatory cytokines in old age.

The impact of aging on memory T cell phenotype and function in the human bone marrow.

Recently, the BM has been shown to play a key role in regulating the survival and function of memory T cells. However, the impact of aging on these processes has not yet been studied. We demonstrate that the number of CD4+ and CD8+ T cells in the BM is maintained during aging. However, the composition of the T cell pool in the aged BM is altered with a decline of naïve and an increase in TEM cells. In contrast to the PB, a highly activated CD8+CD28– T cell population, which lacks the late differentiation marker CD57, accumulates in the BM of elderly persons. IL‐6 and IL‐15, which are both increased in the aged BM, efficiently induce the activation, proliferation, and differentiation of CD8+ T cells in vitro, highlighting a role of these cytokines in the age‐dependent accumulation of highly activated CD8+CD28– T cells in the BM. Yet, these age‐related changes do not impair the maintenance of a high number of polyfunctional memory CD4+ and CD8+ T cells in the BM of elderly persons. In summary, aging leads to the accumulation of a highly activated CD8+CD28– T cell population in the BM, which is driven by the age‐related increase of IL‐6 and IL‐15. Despite these changes, the aged BM is a rich source of polyfunctional memory T cells and may thus represent an important line of defense to fight recurrent infections in old age.

Association of increased neopterin production with decreased humoral immunity in the elderly

Tetanus toxoid (TT) antibodies of 447 adult persons aged 27-69 years were investigated and analyzed in relationship with the time span since the last vaccination against tetanus as well as the serum concentration of neopterin. Neopterin is a pteridine, which is produced by monocytes/macrophages upon stimulation with the type 1 T cell-derived cytokine interferon-gamma. There was an inverse correlation between serum neopterin and TT antibody concentrations (Spearmans rank correlation coefficient: r(s)=-0.259; p<0.0001) which was even stronger when persons with neopterin concentrations and TT antibodies below the third quartile of the study population were excluded (residual group: n=210; r(s)=-0.718; p<0.0001). The study demonstrates that an immunoregulatory shift towards type 1 immunity as indicated by higher neopterin concentrations coincides with lower TT antibody concentrations in the elderly.

Changes in the expression of CD31 and CXCR3 in CD4+ naı̈ve T cells in elderly persons

So far, very few studies exist on the naïve T cell population of elderly persons. Only recently an increase in the percentage of long lived CD4(+)CD31(-) naïve T cells has been claimed to occur with aging. We, therefore, characterised CD31(+) and CD31(-) CD45RA(+) CD4(+) T cells in young and healthy elderly persons. The production of IL-2 and IFN-gamma by the different subpopulations was studied following stimulation with PMA and Ionomycin. The expression of CD28, CD11a, CD62L, CXCR3 and CCR7 was also analysed. The results of this study demonstrate a pronounced increase in the percentage of CD31(-) CD45RA(+) T cells within the CD4 subpopulation of elderly persons. Both, CD31(-) and CD31(+) CD45RA(+) cells expressed CD28, CD62L, were CD11a (dim) and produced IL-2 but no IFN-gamma. This phenotype confirms that they were naïve T cells. IL-2 production by naïve T cells was not impaired in elderly persons. Interestingly, CD31(+) as well as CD31(-) naïve T cells contained a subpopulation of CXCR3(+) cells in elderly individuals, but not in young ones. In spite of expressing this chemokine receptor that enables the cells to migrate into inflammatory tissues, they were still CCR7(+) and CD62L(+). We speculate that due to previous contact with local environmental factors, this subset of naïve T cells acquires a different chemokine receptor phenotype, resulting in an altered migratory capacity in old age. Aberrant contact with antigen and effector cell differentiation in unorthodox locations may be the consequence. This could also affect Th1/Th2 polarisation, which is known to be impaired in elderly persons.

UVB-Induced Senescence of Human Dermal Fibroblasts Involves Impairment of Proteasome and Enhanced Autophagic Activity

In the current study, we have extended previous findings aiming at a better understanding of molecular mechanisms underlying UVB-induced senescence of diploid human dermal fibroblasts (HDFs), an experimental model to study the process of photoaging in the skin. We provide evidence that the inhibition of proteasomal degradation of damaged proteins and the activation of autophagosome formation are early events in UVB-induced senescence of HDFs, dependent on UVB-induced accumulation of reactive oxygen species. Our data suggest that autophagy is required for the establishment of the senescent phenotype in UVB-treated HDFs and that inhibition of autophagy is sufficient to change the cell fate from senescence to cell death by apoptosis. Studies in reconstructed skin equivalents revealed that UVB irradiation triggers hallmarks of autophagy induction in the dermal layer. These findings have potential implications for fundamental as well as translational research into skin aging, in particular photoaging.

Immunodominant peptides from conserved influenza proteins--a tool for more efficient vaccination in the elderly?

ZusammenfassungInfluenza-spezifische zytotoxische CD8+ T-Zellen stellen einen wesentlichen Bestandteil bei der Überwindung einer Infektion dar, insbesondere in Hochrisikogruppen wie älteren Personen. Eine Aktivierung dieser Zellen durch eine Impfung wäre deshalb eine wichtige Voraussetzung um einen besseren Schutz für diese Altersgruppe zu erreichen. Deshalb haben wir die Häufigkeit, den Phänotyp und die Funktion von Influenza M158–66 Peptid-spezifischen CD8+ T-Zellen in drei verschiedenen Altersgruppen (27 ± 1 Jahre; 46 ± 3 Jahre und 72 ± 2 Jahre) ex vivo und nach in vitro Stimulation untersucht. Bei Personen ab einem Alter von 38 Jahren stellten wir eine geringere Anzahl von M158–66-spezifischen CD8+ T-Zellen fest. Diese Zellen exprimierten CD28, CD62L und waren entweder CD45RAlowCD45ROlow oder CD45RA-CD45RO+, produzierten aber kein Perforin. Kein Unterschied zeigte sich beim Phänotyp von Influenza-spezifischen CD8+ T-Zellen zwischen den drei Altersgruppen. Trotz der zu Beginn geringen Anzahl von M158–66-spezifischen CD8+ T-Zellen in älteren Personen, konnte diese Population nach in vitro Stimulation mit dem M158–66-Peptid signifikant vergrößert werden. Diese M158–66-spezifische CD8+ T-Zellen produzierten Perforin und hatten einen CD45RO+CD28+CD62L+/− Phänotyp. Unsere Ergebnisse zeigen, dass trotz einer geringen Anzahl M158–66-spezifischer CD8+ T-Zellen bei älteren Personen, diese Zellen nach in vitro Stimulation mit einem Peptid gut expandieren und in Perforin-produzierende Effektor-T-Zellen differenzierbar sind. M158–66-Peptid oder andere immundominante Peptide aus konservierten Influenzaproteinen könnten somit einen wichtigen Bestandteil zukünftiger Influenzaimpfstoffe darstellen um älteren Personen einen verbesserten Schutz vor Influenza zu bieten, insbesondere beim Auftreten einer Influenza Pandemie.SummaryInfluenza-specific CD8+ T cells are important for the clearance of infection especially in high risk groups such as elderly persons. Activation of these cells by immunization might therefore be a useful tool for a better protection of this specific age group. We therefore analyzed the frequency, phenotype and function of CD8+ T cells with specificity to the influenza M158–66 peptide in young, middle-aged and elderly persons ex vivo and after in vitro stimulation. Significantly lower numbers of M158–66-specific CD8+ T cells were detected in the middle-aged and elderly compared to young donors. M158–66-specific CD8+ T cells were either CD45RAlowCD45ROlow or CD45RA-CD45RO+, expressed CD28 and CD62L and did not produce perforin. There was no difference in the phenotype of influenza-specific CD8+ T cells between the three age groups. Despite the initially low numbers of M158–66-specific CD8+ T cells in the older age groups, these cells could be expanded in vitro following peptide stimulation. They also acquired a CD45RO+CD28+ CD62L+/− phenotype and produced perforin. Our results demonstrate that although initially low in number, M158–66-specific CD8+ T cells from elderly persons can be propagated and differentiated into perforin producing effector cells upon appropriate stimulation. M158–66 peptide or other immunodominant peptides derived from conserved influenza proteins could therefore be useful in future influenza vaccines in order to render elderly persons better protected against disease, in particular in the case of an influenza pandemia.

Increased Neopterin Concentration in Older Age Coincides with Decline of CD28 + CD45RA + T-cells

Abstract Neopterin is produced from human monocyte-derived macrophages and dendritic cells upon Stimulation with interferon-y, and increased neopterin concentrations thus indicate cell-mediated immune activation. In healthy individuals increasing neopterin concentrations are found with increasing age, which is also evident from the agedependency of reference values of serum neopterin concentrations. Increase of neopterin concentrations in the elderly may relate to, e.g., altered T lymphocyte differentiation with age. In this study, serum neopterin concentrations of 138 persons (median age: 34 years; interquartile range 29.0 - 67.8 years) were investigated and compared to age and to the percentage of CD28+CD45RA+ or CD28+CD45RO+ subsets among CD8+ T cells. With increasing age, the percentage of CD28+CD45RA+ in CD8+ T-cells decreased (Spearmans rank correlation coefficient: rs = -0.561; p < 0.0001) accompanied by an increase of the percentage of CD28+CD45RO+ in CD8+ T-cells (rs = +0.221; p < 0.01). Serum neopterin concentrations increased with age (rs = -+0.541; p < 0.0001). This increase of neopterin concentration was accompanied by a decreased percentage of CD28+CD45RA+ in CD8+ T-cells (r+ = -0.287; p < 0.001). Multivariate analyses revealed that the inverse relationship between the percentage of CD28+CD45RA+ in CD8+ T-cells and neopterin concentrations was at least partly independent from age. Thus, investigation allows to conclude that an increase of neopterin concentrations with older age is accompanied by a loss of naive CD28+CD45RA+ CD8+ T-cells. Data suggest that the subset of CD28+CD8+ T-cells, which is developing in states of sustained immune activation, is important for a chronic production of interferon-γ which in turn gives rise to increased neopterin concentrations.