Clinical Immunology (2008) 127, 107–118
`
`a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
`
`w w w. e l s e v i e r. c o m / l o c a t e / y c l i m
`
`T cell subset-specific susceptibility to aging
`Marta Czesnikiewicz-Guzik a, Won-Woo Lee a, Dapeng Cui a, Yuko Hiruma a,
`David L. Lamar a, Zhi-Zhang Yang b, Joseph G. Ouslander c,
`Cornelia M. Weyand a, Jörg J. Goronzy a,⁎
`
`a Kathleen B. and Mason I. Lowance Center for Human Immunology, Emory University School of Medicine, Atlanta, GA, USA
`b Division of Hematology, Mayo Graduate School, Rochester, MN, USA
`c Division of Geriatric Medicine and Gerontology, Emory University School of Medicine, Atlanta, GA, USA
`
`Received 18 October 2007; accepted with revision 7 December 2007
`Available online 28 January 2008
`
`KEYWORDS
`Immunosenescence;
`Aging;
`T-cell subset;
`T-cell homeostasis;
`CD4;
`CD8;
`Killer
`immunoglobulin-like
`receptors;
`CD85
`
`With increasing age, the competence of the immune system to fight infections and
`Abstract
`tumors declines. Age-dependent changes have been mostly described for human CD8 Tcells, raising
`the question of whether the response patterns for CD4 Tcells are different. Gene expression arrays of
`memory CD4 Tcells yielded a similar age-induced fingerprint as has been described for CD8 Tcells. In
`cross-sectional studies, the phenotypic changes were not qualitatively different for CD4 and CD8 T
`cells, but occurred much more frequently in CD8 Tcells. Homeostatic stability partially explained this
`lesser age sensitivity of CD4 Tcells. With aging, naïve and central memory CD8 Tcells were lost at the
`expense of phenotypically distinct CD8 effector Tcells, while effector CD4 Tcells did not accumulate.
`However, phenotypic shifts on central memory Tcells were also more pronounced in CD8 Tcells. This
`distinct stability in cell surface marker expression can be reproduced in vitro. The data show that CD8
`T cells are age sensitive by at least two partially independent mechanisms: fragile homeostatic
`control and gene expression instability in a large set of regulatory cell surface molecules.
`© 2007 Elsevier Inc. All rights reserved.
`
`Introduction
`
`Failure of adaptive immunity with age is a major cause for
`morbidity and mortality in the elderly [1,2]. As a highly
`dynamic organ, the immune system is in constant turnover,
`even in the absence of infections or obvious challenges with
`exogenous antigens [3]. Naïve T cells have a half-life of 6 to
`12 months and memory T cells of 15 to 45 days [4,5]. Thymic
`
`⁎ Corresponding author. Lowance Center for Human Immunology,
`Emory University School of Medicine, 101 Woodruff Circle #1003,
`Atlanta, GA 30322, USA. Fax: +1 404 727 7371.
`E-mail address: jgoronz@emory.edu (J.J. Goronzy).
`
`production of new Tcells dwindles with age and does not meet
`the replenishment demand during adulthood [6–8]. After the
`ages of 40 to 50 years old, virtually the entire T-cell supply is
`generated from existing naïve and memory T cells [9]. In this
`setting, insufficient homeostatic mechanisms may lead to a
`progressive loss of naïve and memory Tcells and contraction of
`T-cell receptor diversity [10–14]. In addition, the replicative
`stress associated with continuous turnover can induce cellular
`senescence and lead to phenotypic changes that impinge on
`the competence of the adaptive immune system [15–18].
`Both mechanisms contribute to failure to respond to new
`antigenic challenges, poor vaccine responses [19–22] and in-
`creased morbidity with newly arising infections, such as is seen
`with antigenic shift or drift of the influenza virus [23,24].
`
`1521-6616/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
`doi:10.1016/j.clim.2007.12.002
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`108
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`M. Czesnikiewicz-Guzik et al.
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`Moreover, memory T-cell responses to some persisting viruses
`wane—a prime example is the increased incidence of herpes
`zoster with age [25]. Epidemiological evidence suggests that
`signs of decreased immune competence first occur after the
`age of 50 and accelerate after the ages of 65 to 70 [20,23,25].
`It is currently unclear which functional and phenotypic chan-
`ges in the immune system occur at what ages and, in particular,
`whether different cell types are affected to distinct extents,
`possibly providing a model for the patterns of age-dependent
`susceptibilities for different viral infections.
`Circumstantial evidence suggests that CD4 and CD8 T cells
`behave differently in response to aging. Oligoclonal expansions
`in the CD8 T-cell compartment can be readily detected with
`age and appear to be induced by chronic persisting viruses, in
`particular CMV, but also arise from uneven homeostatic pro-
`liferation [12,15,26,27]. In contrast, oligoclonal expansions
`within the CD4 compartment are rare and preferentially found
`in patients with autoimmune diseases [28]; they do not reach
`the clonal size seen with CD8 Tcells [18]. Even at the age of 65,
`decades after the involution of the thymus, both naïve and
`memory CD4 Tcells are highly diverse and show no contraction
`in T-cell receptor diversity compared to young adults [10].
`Also, the classical phenotypic change of CD28 loss, frequently
`encountered in CD8 Tcells with age [17], is only inconsistently
`seen for CD4 T cells [11]. However, the molecular mechanisms
`regulating CD28 expression and loss appear to be identical in
`both T-cell subsets [29,30], suggesting a fundamental differ-
`ence in CD4 and CD8 T cells in response to aging, possibly in
`addition to distinct cell-specific transcriptional regulation.
`The objective of this study was to compare CD4 and CD8 Tcells
`and to determine whether differences in their phenotypic
`response pattern to aging are explained by differences in T-cell
`subset homeostasis or whether CD4 and CD8 T cells are in-
`trinsically different in controlling gene expression.
`
`Materials and methods
`
`Study population
`
`Peripheral blood was obtained from 140 individuals aged
`20–90 years and immediately processed. The study cohort
`included 68 individuals age 20 to 39 years, 31 age 40 to 59,
`and 41 age 60 to 90 years. Exclusion criteria included the
`presence or a history of cancer, uncontrolled hypertension,
`diabetes mellitus, any chronic inflammatory or autoimmune
`disease, or any acute disease. Appropriate written informed
`consent was obtained, and the study was approved by the
`Emory Institutional Review Board.
`
`Gene expression microarray studies
`
`CD4 T cells were negatively enriched with human CD4+ T-cell
`enrichment cocktail (RosetteSep; StemCell Technologies,
`Vancouver, Canada) from PBMC of five 20- to 30- and five
`70- to 75-year-old healthy volunteers. CD45RA−CD4+ memory
`T cells were isolated by depletion of CD45RA+ subsets with
`anti-CD45RA magnetic beads (Miltenyi Biotec, Auburn, CA).
`Purity was between 95% and 98%. Total RNA was extracted
`using an RNeasy Mini Kit (Qiagen, Valencia, CA), amplified,
`and labeled using a modification of the technique described
`by Baugh et al. [31]. A first round of in vitro transcription (IVT)
`
`was followed by a second round of reverse transcription,
`second strand synthesis, and IVT. aRNA was hybridized with
`Affymetrix U133A GeneChips by the Institute of System Bio-
`logy (Seattle, WA). Gene expression signal was summarized by
`robust multi-array average RMA [32]. Genes that showed a
`1.25-fold difference between young and old samples were
`identified.
`In a second microarray series, CD4+CD28+ and CD4+CD28− T
`cells were purified from three donors by cell sorting using FACS
`vantage (BD Biosciences, San Jose, CA). Cells were expanded
`by stimulation with anti-CD3 (OKT3; Ortho Diagnostics, Ro-
`chester, NY) immobilized at 1 μg/ml in IL-2-containing me-
`dium. RNA was extracted 2 weeks after stimulation. This time
`point was chosen because gene expression in T cells cultured
`under these conditions is stable and kinetic changes induced by
`the stimulation are no longer observed (data not shown).
`Labeled aRNA was hybridized with Affymetrix Hu-95Av2
`GeneChips (Mayo Cancer Microarray Core Facility, Mayo
`Foundation; Rochester, MN). Data were analyzed with Micro-
`array Analysis Software version 5.0 (Affymetrix). A gene was
`called differentially expressed if comparisons for all three
`individuals yielded a consistent change call, or if two com-
`parisons yielded consistent change calls and the third yielded a
`“no change” call.
`
`Immunophenotyping and flow cytometry analysis
`
`To confirm age-dependent differential expression of cell
`surface markers identified in the gene arrays, PBMC were
`stained with the following monoclonal antibodies in 5-color
`panels: FITC-conjugated anti-CD158b/j (GLI83; CH-L), PE-anti-
`CD85j (HP-F1), PerCP-Cy5.5-anti-CD28 (L293), APC-anti-CD8
`(RPA-T8), APC-Cy7-anti-CD3 (SK7), FITC-anti-CD57 (HNK-1),
`PE-anti-CD26 (M-A261), PerCP-anti-CD4 (L200), APC-anti-CD3
`(HIT3a); FITC-conjugated anti-HLA-DR (G46-6), PE-anti-CD3
`(HIT3a), PerCP-anti-CD4 (L200), APC-anti-CD45R0 (4CHL1), PE-
`Cy7-anti-CD69 (FN 50). FITC- or APC-anti-CD45RA (RA5H9,
`HI100), PE- or APC-anti-CCR7 (150503), and PerCP-anti-CD8
`(SK1) were used to define the functional subset distribution.
`To determine the frequency of cytomegalovirus (CMV)-
`specific CD8 Tcells in different age groups, PBMC from HLA-A2
`individuals were stained with APC-conjugated HLA-A⁎0201/
`CMV pp65495–504 (NLVPMVATV) pentamer (Proimmune Inc,
`Springfield, VA) at room temperature for 10 min. Following
`washing, the cells were incubated with PE-anti-CD85j, PerCP-,
`or PE-Cy7-anti-CD8; FITC- or PerCP-Cy5.5-anti-CD28; and APC-
`Cy7-anti-CD3 antibodies for 20 min.
`All samples were acquired with FACSort or LSRII (BD Bio-
`sciences), and data were analyzed by using CellQuest, FACS
`DIVA software (both BD Biosciences), or FlowJo (Tree Star, Inc.,
`Ashland, OR). All antibodies were from BD Biosciences, except
`PE-, APC-anti-CCR7 (R&D Systems, Minneapolis, MN, USA), and
`anti-CD85j (Beckman Coulter, Fullerton, CA, USA).
`
`In vitro cultures
`
`CD28 positive cells were sorted from PBMC with biotin-labeled
`anti-CD28 antibody (BD Biosciences), and anti-biotin labeled
`microbeads (Miltenyi). Isolated cells were labeled with CFSE
`and stimulated with anti-CD3 (OKT3, 30 ng/ml) in the presence
`of irradiated EBV transformed cells, rhIl-2 (50 U/ml) and rhIl-
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`T cell subset-specific susceptibility to aging
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`109
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`15 (100 ng/ml). Cultures were split every 4 days. Cells were
`analyzed at 6- to 8-day intervals for the expression of CD85j,
`CD28 and HLA-DR on CD4 and CD8 T cells that had undergone
`the same number of divisions as determined by CSFE dilution.
`
`Statistical analysis
`
`Results are expressed as medians, 25th/75th percentiles as
`boxes and 10th/90th percentiles as whiskers. Groups were
`compared using ANOVA or non-parametric Mann–Whitney U
`test. p values b 0.05 were considered statistically significant.
`
`Results
`
`Differential loss of CD28 in CD8 versus CD4 T cells
`
`Loss of CD28 expression, the dominant phenotypic change of
`T cells with aging, is seen much more frequently in CD8 than
`in CD4 T cells. Figure 1 shows a cross-sectional study of CD28
`expression in different age groups. In the vast majority of
`individuals, lack of CD28 is a rare event in CD4 T cells,
`irrespective of age. There was a small increase in CD4+CD28−
`T-cell frequencies (p b 0.001) and increasing variance in
`healthy individuals older than 60 years; however, even in
`this age group only a median of 7.9% of CD4 T cells lost CD28
`expression, and individuals with more than 20% CD4+CD28− T
`cells were the rare exception. In contrast, 21% of CD8 T cells
`were already negative for CD28 expression in individuals
`between the age of 20 and 39 years. The frequency increased
`to 25.5% in 40- to 59-year-old healthy individuals (p b 0.001).
`After that age, CD28 loss accelerated to a median frequency
`of 53.9% in the 60–90-year cohort (p b 0.001). Again the
`variance increased with age, indicating higher heterogeneity
`in the older population.
`
`Age-dependent changes in the transcriptional profile
`of CD4 memory T cells
`
`Regulation of CD28 may be cell specific, and CD8 T cells may
`lose CD28 more easily. Indeed, the DNA-binding proteins con-
`trolling the initiation of CD28 transcription differ in composi-
`tion between CD4 and CD8 T cells [30]. However, the basic
`mechanism of CD28 repression is the same in both T-cell sub-
`sets, suggesting that factors not related to CD28 transcription
`
`play a role [29]. The CD4 memory T cells may be a more stable
`cell type, with lesser susceptibility to age-dependent pheno-
`typic and functional changes. To determine whether CD4 T
`cells principally undergo the same changes in gene expression
`with age as CD8 T cells but do so at a substantially reduced or
`delayed rate, we performed gene expression arrays on (a)
`CD45RA−CD4+ cells purified from PBMC of five 20- to 30-year-
`old and five 70- to 75-year-old healthy donors and (b)
`CD28+CD4+ and CD28−CD4+ T cells from the same donor and
`compared them to published gene profiles for CD8 Tcells [33];
`The gene expression profiles in young and old memory CD4 T
`cells highly correlated indicating that age-dependent changes
`in CD4 T-cell transcription were small or only involved a minor
`subpopulation. Because even small changes in gene expression
`or emergence of a low-frequent subpopulation could have
`physiological consequences, we used a low cut-off of a 1.25-
`fold difference in expression levels between the two age
`groups to screen for genes that warrant further studies. 536
`genes were identified, including the cell surface molecules
`listed in Table 1. Several of the genes over-expressed in the
`elderly mapped to the HLA region. CD28 was among the few
`genes that were less expressed in elderly memory CD4 cells.
`Hypothesizing that the differences were small because only a
`minor subset of CD4 T cells is affected by aging and these
`senescent cells are characterized by the loss of CD28, in ana-
`logy to CD8+CD28− T cells, we compared the gene expression
`profiles of CD4+CD28− to CD4+CD28+ T cells. In general,
`identified differences in this subset were of larger magnitude
`than the age-dependent differences observed in the global
`memory CD4 T-cell pool. Cell surface molecules identified as
`differentially expressed partially overlapped with the genes
`identified in the global memory cell populations (Table 1). MHC
`class II genes again were among those that were overexpressed
`in the cells with the senescent CD4+CD28− phenotype. Also,
`the KLRC2 genes were underrepresented in both the elderly
`CD4+ memory cells and CD4+CD28− T cells. As expected, CD27
`transcripts that are known to correlate with CD28 loss in CD4
`and CD8 T cells were reduced in CD4+CD28− T cells. The most
`striking feature of these cells was the overexpression of
`several genes encoded in the leukocyte regulatory cluster,
`including several KIR genes.
`In comparing our results with those of Fann et al., it is
`evident that aging CD4 and CD8 T cells undergo similar
`transcriptional changes. In agreement with their findings for
`senescent CD8 Tcells [33], we observed a significant decrease
`
`Influence of age on CD28 loss within CD4+ and CD8+ T cells. The frequency of CD28 loss in CD4 (left panel) and CD8 (right
`Figure 1
`panel) T-cell subpopulations was determined by FACS in healthy individuals of different ages. Results are shown as box plots with
`medians, 25th and 75th percentiles as boxes and 10th and 90th percentiles as whiskers for different age strata. Although decline was
`significant for both CD4 (p b 0.001) and CD8 T cells (p b 0.001), loss was much more dramatic and earlier in life in the CD8 population
`and was already significant for middle-aged individuals (p b 0.001).
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`Table 1
`
`Age of donor
`
`Cell type
`Differentially overexpressed cell surface molecules
`
`Influence of age on gene expression in CD4 T cells
`20–30 years
`CD4+CD45RA−
`KLRB1
`CD28
`KLRC2
`CCR6
`
`M. Czesnikiewicz-Guzik et al.
`
`65–75 years
`CD4+CD45RA−CD28+
`TCRβ-chain
`ICAM-2
`CD7
`KLRC2
`IL9R
`IL7R
`CD27
`
`70–75 years
`CD4+CD45RA−
`HLA-DRA
`CCR4
`CD26
`HLA-DPB1
`HLA-DPB5
`HLA-DPB4
`HLA-F
`HLA-DPA1
`CCR8
`CD58
`IL17RB
`LAIT1
`CD3E
`
`CD4+CD45RA−CD28−
`KIR2DL3
`KIR2DL4
`KIR3DL2
`CD70
`HLA-DRA
`HLA-DQA
`CD74
`HLA-DRB1
`HLA-DPB1
`HLA-DMA
`HLA-DPA
`CCR5
`HLA-DQB1
`CD2
`
`in CD28 expression in memory CD4 T cells from elderly donors.
`We found that expression of the costimulatory molecules
`CTLA4 and PD-1 did not differ with respect to age in CD4
`memory cells, a finding which mirrored Fann et al.'s results
`with respect to senescence phenotype in CD8 T cells. While
`CD40L is significantly downregulated in CD8+CD28− Tcells [33],
`we did not detect an age-dependent decline in memory CD4
`cells; however, we have previously shown that CD28−CD4+ T
`cells lack the ability to induce CD40L [34]. Furthermore, there
`are many similarities in the expression of NK cell receptors on
`CD28−CD8 and CD4 T cells. For example, our results showed
`significant downregulation of KLRB1 (CD161) expression in old
`memory CD4 cells, as Fann et al. demonstrated for CD8+CD28−
`cells. Similarly, the expression of KIR2DL2, KIR2DL3, and
`KIR2DL4 increased in CD4+CD28− cells, although these differ-
`ences did not reach the significance found in CD8+CD28− T
`cells. We did not detect any changes in expression of KLRD1,
`KLRG1, and KLRK1 between old and young memory CD4 cells,
`in contrast to the significant upregulation of these genes in
`CD8+CD28− cells. Several chemokine receptors exhibited ex-
`pression patterns in CD4 memory cells that are analogous to
`those found in CD8 cells. CCR2, CCR6, and CCR7, which are
`all significantly downregulated in CD8+CD28− cells [33],
`decrease with age in memory CD4 T cells, with CCR6 reaching
`significance.
`
`Preferential age-dependent expression of negative
`regulatory receptors on CD8 T cells
`
`Candidate cell surface molecules implicated in the gene arrays
`were examined for their age-dependent expression in a cohort
`of 140 healthy individuals. One of the most striking findings of
`the gene array studies was the overexpression of several KIR
`genes in CD4+CD28− T cells. KIR genes have allelic polymorph-
`isms, and we selected the antibody to CD158b/j that re-
`cognizes the most frequent genetic variants KIR2DL2, KIR2DL3,
`and KIR2DS2 for further analysis [35]. In addition, we included
`CD85j, which is encoded in the leukocyte cluster near the KIR
`genes, in our analysis. Like most KIR genes, CD85j is a negative
`regulatory receptor that is irregularly expressed on T cells.
`Cross-sectional studies showed preferential expression of
`CD85j and CD158b/j in CD8 T cells, while they were infre-
`
`quently found in CD4 T cells (Fig. 2A). Expression of both
`molecules, but particularly CD85j, increased with age; the in-
`crease was already apparent in the age group of 40- to 59-year
`olds, but clearly accelerated after that age. Compared to the
`20–39-year-old individuals, CD158b/j (p b 0.001) and CD85j
`(p b 0.001) expression was significantly more frequent in CD8
`T cells from individuals older than 60 years. In many of these
`individuals, the majority of CD8 Tcells were positive for CD85j.
`The age-dependent increase was much less striking in CD4 T
`cells and did not reach significance for either molecule. How-
`ever, in both T-cell subsets, the expression of these receptors
`was closely related to the loss of CD28. In both the CD4 and CD8
`populations, the majority of CD28− T cells expressed CD85j. In
`contrast, CD4+CD28+ Tcells expressed almost no CD85j. A small
`fraction of CD8+CD28+ Tcells, however, was consistently found
`to express CD85j (Fig. 2B). CD158b/j showed the same dis-
`tribution pattern but was much less frequent and exceeded
`10% expression in only a few individuals, even on CD28− Tcells.
`KIR and CD85j expression patterns resembled those of CD57,
`a generally accepted T-cell senescence marker [36]. Gain in
`CD57 expression showed a similar age dependence (Fig. 2A),
`with a median of 5.1% of CD4 T cells and 33.35% of CD8 T cells
`being CD57-positive in 60- to 90-year olds. CD57 expression was
`mainly restricted to the CD28− cell population and occurred
`only infrequently in CD28+ cells (Fig. 2B). Thus, in both CD4 and
`CD8 Tcells, the gains in CD57, CD85j, and CD158b/j, and loss of
`CD28 with age were highly correlated, suggesting that the
`changes are qualitatively similar. However, they occurred much
`more frequently in CD8 T cells. Table 2 shows the slopes of the
`linear regression curves examining the relationship between
`age and frequency of cell surface expression. For each
`biomarker shown, the slope is significantly steeper for CD8 T
`cells, documenting a fundamental difference in CD4 and CD8 T
`cells in their responses to aging.
`
`Defective T-cell homeostasis in CD8 Tcells contributes
`to the preferential acquisition of negative regulatory
`receptors
`
`Since CD28 loss is mostly seen on effector T cells, we
`addressed the question of whether the higher susceptibility
`of CD8 T cells to undergo age-dependent phenotypic changes
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`T cell subset-specific susceptibility to aging
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`111
`
`Age-dependent gain of CD57, CD85j, and CD158b/j expression in CD8+ T cells. (A) Frequencies of CD85j, CD158b/j, and
`Figure 2
`CD57 cells within CD4 (left panel) and CD8 (right panel) T cells were determined by FACS. Results are shown as box plots with medians,
`25th and 75th percentiles as boxes, and 10th and 90th percentiles as whiskers for healthy individuals representing different age strata.
`(B) In both CD4 and CD8 T cells, CD57 and CD85j were preferentially expressed on CD28− (shaded areas) and less on CD28+ (dark lines).
`
`reflected different stability in homeostatic control mechan-
`isms. Quantification of naïve, central, and effector memory
`cells using the CD45RA and CCR7 markers in different age
`groups indeed showed considerable differences between CD4
`and CD8 T cells. In both compartments, naïve T cells were
`reduced in elderly individuals ( p b 0.001); however, the loss
`was much more dramatic for CD8 (Fig. 3B) than for CD4 Tcells
`(Fig. 3A). The functional subset distribution within the CD4
`memory compartment was stable with age (Fig. 3C). In
`contrast, the subset of central CD8 memory T cells was
`
`Linear regression analysis of the age dependency
`Table 2
`of cell surface molecule expression in CD4 and CD8 T cells
`
`CD8 T cells
`CD4 T cells
`Δ frequency
`Δ frequency
`(%/year)
`(%/year)
`−0.03 ± 0.04 −0.6 ± 0.07
`CD28
`0.1 ± 0.07
`0.7 ± 0.07
`CD85j
`0.2 ± 0.07
`0.4 ± 0.07
`CD57
`CD158b/j −0.02 ± 0.01
`0.1 ± 0.03
`0.09 ± 0.07 −0.5 ± 0.1
`CD26
`HLA-DR
`0.2 ± 0.03
`0.6 ± 0.8
`
`F
`
`p
`
`F = 36.798 p b 0.0001
`p b 0.001
`F = 25.75
`F = 4.942
`p = 0.03
`F = 12.483 p = 0.0005
`F = 12.46
`p = 0.0006
`p b 0.0001
`F = 26.02
`
`diminished (p = 0.01), and the CD8 effector memory T cells
`increasingly acquired a CD45RA phenotype ( p b 0.001, Fig.
`3D) The CD4/CD8 ratio was not affected by age, contrary to
`the hypothesis that expansion of CD8 effector T cells
`outcompetes CD4 T cells.
`The altered subset distribution in part accounted for the
`preferential phenotypic changes in CD8 Tcells. CD158b/j was
`exclusively expressed on CD45RA terminal effector T cells,
`suggesting that the accumulation of this subset was
`responsible for the emergence of CD158b/j cells with age.
`CD57 and CD85j were also preferentially expressed on
`CD45RA effector T cells; however, expression was not subset
`specific, and all three memory subsets gained expression of
`these molecules with age (Fig. 3E).
`
`Expansion of CMV-specific T cells does not account
`for the preference of age-dependent changes for
`the CD8 compartment
`
`Several recent studies have attributed disordered CD8 T-cell
`homeostasis to chronic persistent anti-CMV responses and
`the accumulation of oligoclonal CMV-specific effector T cells
`[14,15,27]. To examine the relationship between CMV
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`112
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`M. Czesnikiewicz-Guzik et al.
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`Influence of age on functional T-cell subset distribution. PBMC were isolated from healthy young (n = 31; 27.9 ± 5.5 years)
`Figure 3
`and elderly (n = 26; 73.1 ± 4.2 years) individuals and stained with anti-CD3, -CD4, -CD8, -CD45RA, and -CCR7 mAbs to determine
`frequencies of T-cell subsets. Results are shown for CD45RA+CCR7+ naïve CD4 (A) and CD8 (B), CD45RA−CCR7+ central memory (CM),
`CD45RA−CCR7− effector memory (EM) and CD45RA+CCR7− effector (CD45RA effector) CD4 (C) and CD8 cells (D). Expression of CD57,
`CD85j and CD158b/j in the elderly was highest in effector CD8 Tcells, but CD57 and CD85j was also present in a central memory Tcells.
`Representative histograms are shown (E).
`
`responses and age-dependent phenotypic changes, we
`selected HLA-A2+ individuals and determined the frequency
`and phenotype of HLA-A2-restricted T cells specific for the
`CMV peptide NLVPMVATV (A2/NLV). While the majority of
`individuals in our population did not have any detectable
`(b0.1%) CMV pp65495–504 peptide-specific clones irrespective
`of age, clonal expansions of more than 1% were seen in about
`
`one half of the middle-aged and elderly populations
`(Fig. 4A). In contrast to the global CD8 T-cell compartment,
`CMV-specific clones did not undergo phenotypic changes with
`age; approximately 60% had lost CD28 expression and 50%
`had gained CD85j expression in young and elderly individuals
`(Fig. 4B). Individuals with CMV-specific clones had overall
`higher frequencies of CD8+CD28−CD85j+ T cells than
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`Expansion of CMV-specific T cells does not account for the preference of age-dependent phenotypic changes for the CD8
`Figure 4
`compartment. PBMC were isolated from HLA-A2+ individuals and stained with HLA-A2-CMV peptide (NLVPMVATV) (A2/NLV) pentamer,
`anti-CD3, -CD8, -CD28, and -CD85j mAbs. (A) Frequencies of CMV peptide-specific CD8 T cells are shown in correlation to age. (B) CMV-
`peptide-specific (solid boxes) and total CD8 T cells (open boxes) from young and elderly individuals who had N0.1% of CMV-peptide
`specific cells, were compared. (C) Individuals who had (solid bars) or did not have expanded CMV-peptide specific T cells (open bars)
`were compared for the frequency of CD85j and CD28 expression on CD8 T cells.
`
`CD4+ and CD8+ T cells differ in CD26 expression with age. Results of CD26 expression within CD4 (left panel) and CD8 (right
`Figure 5
`panel) T-cell populations in relation to age in cross-sectional studies are shown as box plots (A). Representative histograms of CD26
`expression in CD28+ and CD28−CD4 and CD8 cells from a 27 (top row) and 73-year-old (bottom row) individual are shown in (B).
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`individuals without detectable CMV T-cell responses; how-
`ever, both subject populations exhibited the same age-
`dependent phenotypic shift in their CD8 compartment
`(Fig. 4C). These data suggest that CMV-specific responses
`are not the sole and, in most individuals, not a major cause
`for the observed age-dependent phenotypic conversions and
`the disequilibrium in functional T-cell subset distribution
`that mostly affect CD8 T cells.
`
`Preference for phenotypic conversions in CD8 T cells
`independent of homeostatic imbalance
`
`Several genes including CD26 and MHC class II molecules were
`found to be overexpressed in the gene array of elderly
`memory T cells independent of CD28 loss, raising the issue of
`whether their expression was independent of homeostatic
`dysregulation. Flow cytometric studies were performed to
`compare the effect of age on CD4 and CD8 T cells for these
`
`markers. A complex CD26 expression pattern was found,
`particularly on CD8 T cells. Three separate populations of
`CD26-expressing CD8 T cells could be distinguished: high,
`medium, and low expression. Most CD8 cells that had lost
`CD28 expression were also negative for CD26 (Fig. 5B, right
`panel). In addition, CD26high CD8 Tcells virtually disappeared
`with age. Both phenomena contributed to an age-dependent
`decrease in CD26 expression in CD8 Tcells ( p b 0.001, Fig. 5A).
`CD4 T cells were mostly CD26intermediate (Fig. 5B, left panel)
`and, contrary to the gene array, expression did not change
`with age (Fig. 5A, left panel). Thus, again there were striking
`differences between CD4 and CD8 T cells in age-dependent
`phenotypic changes.
`Genes of the MHC class II region were found to be dif-
`ferentially expressed in both series of gene arrays. Flow
`cytometry studies confirmed that the expression of HLA-DR is
`age dependent (Fig. 6A). A small increase in the frequencies of
`HLA-DR+ cells was already seen between the ages of 20 and
`59 years and then accelerated in the age group of 60 years and
`
`Increased expression of HLA-DR on CD4 and CD8 T cells with age. (A) The frequencies of HLA-DR+ cells within CD4 (left
`Figure 6
`panel) and CD8 (right panel) T cells are shown as box plots for different age groups. HLA-DR in elderly individuals is expressed on CD28+
`as well as CD28− T cells (B) and on central as well as effector memory CD8 T cells (C). Representative histograms from a 27- (top row)
`and 70-year-old (bottom row) individual are shown.
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`Phenotypic changes of the surface molecules: CD28, CD85j, HLA-DR in CD8 and CD4 subsets caused by long-term culture of
`Figure 7
`CD28 positive T cells. CD28 positive cells were sorted, labeled with CFSE, and stimulated with irradiated, EBV transformed PBMC and
`OKT3. FACS analysis was carried out every seventh day beginning from the first stimulation. CD28, HLA-DR, and CD85j molecules
`surface expression changes over time and is much more pronounced in CD8 T cells compared to the CD4 subset, suggesting that the
`phenotypic instability is intrinsic to CD8 T cells.
`
`older who had significantly higher frequencies of HLA-DR-
`expressing CD4 and CD8 T cells compared to young adults
`( p b 0.001). In contrast to KIRs, CD57 and CD85j, HLA-DR ex-
`pression did not coincide with CD28 loss. Representative
`histograms of CD4 and CD8 T cells are shown in Figure 6B. In
`both CD4 and CD8 subsets, very few Tcells expressed HLA-DR in
`a 27-year-old individual. Only CD8+CD28− T cells had a slightly
`higher expression of 6%. In contrast, 30% of CD8+CD28+ and 44%
`of CD8+CD28− T cells in a 70-year-old individual were positive
`for HLA-DR. 10 and 13%, respectively, of CD4+CD28+ and CD28−
`T cells in the same individual expressed the molecule. HLA-DR
`is known to be expressed on activated T cells, raising the
`possibility that elderly individuals have a larger fraction of
`activated cells. However, HLA-DR-positive cells had morpho-
`logical characteristics similar to HLA-DR-negative cells in the
`forward-side scatter, and there was no correlation with other
`activation markers, such as CD69, which does not increase with
`age (data not shown). Increased HLA-DR expression can
`therefore not be explained as a result of constitutive
`activation of T cells in the elderly. Although the pattern of
`HLA-DR expression was strikingly different from those of CD57
`and CD85j, the influence of age on HLA-DR expression was
`again more pronounced in CD8 than in CD4 T cells (Table 2).
`These findings suggest that CD8 Tcells are more susceptible to
`phenotypic conversions with age independent of their pre-
`ferred differentiation into effector cells.
`
`Intrinsic increased susceptibility of CD8 T cells to
`undergo phenotypic conversions
`
`To examine whether the phenotypic instability is intrinsic to
`CD8 Tcells, we activated CD4 and CD8 Tcells in vitro, expanded
`them in the presence of IL-2 and IL-15 and determined the
`expression of CD28, HLA-DR, and CD85j over time. To control
`for differences in population doublings, cells were labeled
`with CFSE, and CD4 and CD8 Tcells with equal division numbers
`were compared. Representative histograms are shown in
`Figure 7. CD28 was more rapidly lost in CD8 than in CD4 T
`cells. On day 31, most CD8, but few CD4, Tcells had lost CD28.
`Expression of CD85j emerged in a subset of CD8 Tcells after 3 to
`4 weeks of culture, but was nearly absent in CD4 Tcells. Both T-
`cell subsets gained HLA-DR expression early during culture,
`and expression in CD8 Tcells again preceded that in CD4 Tcells.
`In general, phenotypic shifts were more pronounced and
`earlier in older individuals (data not shown).
`
`Discussion
`
`In this manuscript, we describe that CD4 T cells are more
`resistant to phenotypic and functional changes with aging
`than CD8 T cells. CD4 and CD8 T cells undergo the same
`principal phenotypic shifts; however, the rate at which they
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`occur or accumulate with age is vastly different. The
`increased susceptibility of CD8 T