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ging is associated with cognitive decline, a heightened tively present in the epithelial layers of the choroid plexus where, at
risk of neurodegenerative disease, and poorer recovery this interface between brain and blood, they are poised to act as
after injury. People aged 80 and over are now the fastest immunomodulators of aging and senescence (6, 10). T cells have
growing segment of the population, projected to reach 19.4 million many diverse functions that influence both immune and nonimmune
in 2030 (1). These statistics highlight growing concerns over the pathways. For example, Scid mice, which lack functional T cells,
burden to the health services. The aging process exerts profound have cognitive deficits compared with wild-type mice, implying that
changes on the nervous and immune systems, which extend into T cells can influence CNS function (1113). However, little is known
nearly all organ systems (24). Indeed, the relationship between these regarding the role and identity of the lymphocytes that traffic
two systems is bidirectional, and the concept of the CNS as an throughout the CNS under normal conditions and with aging.
immune-privileged organ is evolving (5). It is now recognized that the Surveillance is the process whereby the immune system identifies
brain is under constant immune surveillance by resident microglia and eliminates transformed or infected cells before they become
and trafficking systemic T lymphocytes recruited by the choroid cancerous or cause harm (14). Although little is known concerning
plexus (68). These generally serve a protective role as a rapid re- the role of aging on immune surveillance in the CNS, the increase
sponse system to detect damaging agents that disrupt homeostasis. in basal inflammatory levels indicative of the phenomenon known
Although T lymphocytes transit the CNS at low numbers in healthy as inflammaging supports the notion that the necessity for sur-
brain, the role of these cells and how they change with age is not well veillance may be heightened (15). The low-grade inflammatory
understood (9). Recent work has shown that CD4 effector memory
status of the aged CNS may favor immune cell recruitment, but the
(TEM) cells with a TCR repertoire specific to CNS Ag are constitu-
critical mediators of this phenomenon and the functional role of the
specific cell subtypes entering the aged brain in steady state remain
to be established. Stichel and Luebbert (16) showed a significant
*Neuroscience Department, University of Connecticut Health Center, Farmington,
CT 06030; Immunology Department, University of Connecticut Health Center, increase in both CD11c+ dendritic cells and CD3+ T cells with wide
Farmington, CT 06030; and Department of Neurology, McGovern Medical School, distribution throughout the brain beginning at middle age and in-
University of Texas Health Science Center, Houston, TX 77370
creasing with advanced age. Studies assessing donor human brain
Received for publication September 16, 2015. Accepted for publication February 5, tissue have identified the presence of resident CD8+ T cells enriched
2016.
in white matter and regions of blood-brain barrier leakiness (17,
This work was supported by National Institutes of Health Grants R21 NS076293-
01A1 (to L.D.M.) and F31 NS083244-01A1 (to R.M.R.). 18). These findings indicate that the observed changes in leukocyte
Address correspondence and reprint requests to Dr. Louise D. McCullough, De- recruitment in the aging mouse brain most likely have translational
partment of Neurology, McGovern Medical School, University of Texas Health relevance as immune surveillance also occurs in the human CNS.
Science Center, 6431 Fannin Street, Houston, TX 77370. E-mail address: Louise.
D.McCullough@uth.tmc.edu
In the current study, the presence of peripheral leukocytes in the
aging CNS was assessed to determine the functional relevance of these
The online version of this article contains supplemental material.
cells to age-related inflammatory signaling. We evaluated lymphocyte
Abbreviation used in this article: TEM, effector memory.
recruitment guidance and response cues and performed a compre-
Copyright 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 hensive phenotypic analysis of cell subsets found in normal CNS.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502021
2 AGE-RELATED CNS-RESIDENT CD8 T CELLS ARE REACTIVE TO STROKE
Lastly, to determine whether there was a functional consequence of after collecting blood and tissue leukocytes, each sample was divided into
these CNS-resident leukocytes, we assessed production of inflam- 15 separate FACS tubes and stained for the Ab mixture, including 1 of 15
respective TCRvb FITC-conjugated mAbs.
matory mediators in an age-relevant model of brain injury, experi- Phagocytic activity of microglia was performed, as described by Ritzel
mental stroke. et al. (19). Briefly, fluorescent latex beads (Fluoresbrite Yellow Green
carboxylate microspheres; 1 mm diameter; Polysciences) were added to
Materials and Methods sorted microglia in a final dilution of 1:100, as described (19). After 1-h
incubation at 37C, the cells were washed three times with FACS buffer,
Mice/Animals resuspended in FACS buffer, stained for surface markers, and fixed in
C57BL/6J mice of 812 wk (young adult) and 1822 mo (aged) of age paraformaldehyde.
were pair-housed on sawdust bedding in a specific pathogen-free facility
(light cycle 12/12-h light/dark). The average weight of the naive, young ELISA cytokine measurement
mice was 29.6 6 2.3 g and that of aging mice was 35.7 6 3.2 g before Plasma and whole tissue brain chemokine levels were determined by ELISA
sacrifice. All experiments were performed using male C57BL/6J mice, (Milliplex Cytokine/Chemokine Assay; EMD Millipore). In brief, mice
unless otherwise stated. Several experiments were performed using BALB/ were euthanized by avertin injection, and blood was collected by cardiac
cByJ mice of 11 wk (young adult) and 21 mo (aged) of age to determine puncture into heparin-coated syringes. Samples were centrifuged (13,000 3
strain dependence. A cohort of aged C57BL/6J and BALB/cByJ females g for 10 min at 4C), and plasma was collected and stored frozen (280C)
was also included. All animals had access to chow and water ad libitum. until assaying. Brain hemispheres were collected and homogenized in ice-
Animal procedures were performed in accordance with National Institutes cold lysis buffer containing a protease inhibitor mixture (Roche Diag-
of Health guidelines for the care and use of laboratory animals and ap- nostics). Homogenates were sonicated and centrifuged at 4C for 15 min at
proved by the Animal Care Committee of the University of Connecticut 14,000 rpm, and supernatants were assayed for total protein using a
Health Center. Ex vivo studies were performed by an investigator blinded bicinchoninic acid protein assay kit (Pierce, Thermo Scientific). Using a
to age. standard curve, protein concentration was determined and 100 mg each
sample was loaded into each well in duplicate. Samples were assayed
production was measured as above following 2 h of PMA/ionomycin older females exhibiting significantly higher numbers compared
stimulation at 37C. with their age-matched male counterparts (Supplemental Figs.
Statistical analyses 1C, 2AD). The results show that the CNS is more permissive
to CD8 T lymphocyte trafficking with age.
Data represent averages of cell counts obtained from one hemisphere of
individual mice within a group. Data from individual experiments are Age-related chemokine production is associated with enhanced
presented as mean 6 SEM and compared with Student t test where ap- recruitment of CD8 T cells into the aging brain
propriate (GraphPad Prism Software, San Diego, CA). Multiple compari-
sons were assessed by one-way or two-way ANOVA analysis with Tukey To determine whether CD8 T cell recruitment to the aging brain
post hoc correction. Significant differences between paired comparisons could result from a directed, homeostatic process rather than a
were conducted with the Holm-Sidak test. Correlation analyses were random dispersion into tissues, we examined whole brain protein
performed using Pearsons correlation. The p value ,0.05 was considered
statistically significant. expression of chemokines known to be involved in T cell migration.
CCL5 (RANTES), CCL11 (Eotaxin), CXCL9 (monokine induced
by IFN-g), and CXCL10 (IFN-ginducible protein) concentrations
Results
were all significantly upregulated in the brain with age by ELISA
Aging increases the number of peripheral leukocytes in the CNS
(Fig. 2AD). Levels of these chemokines were similarly increased
To confirm that aging increases leukocyte trafficking in the CNS, in aged plasma (Fig. 2EH), suggesting that these gradient levels
we quantified the absolute number of bone marrowderived cells are accessible to circulating T cells. Surface expression of che-
present in each tissue using the gating strategy shown in mokine receptors to these respective chemokine ligands was then
Supplemental Fig. 1A. The number of CD45high leukocytes was assessed on both blood and brain CD8 T cells. An increase in
significantly increased in the aged brain (Fig. 1A). Absolute cell expression of these chemokine receptors was found on blood CD8
FIGURE 1. CNS recruitment of CD8 T cells increases with age. A representative dot plot depicts an increase in CD45high peripheral leukocytes in one
aged brain hemisphere compared with young (A). The absolute number of CD45high peripheral leukocytes present in the young and aged brain is quantified
[(B) n = 13 per group]. The number of cells within the CD45high population is displayed by subset [(C) n = 13 per group]. A representative dot plot shows T
cell subsets in the aged brain (D). The CD4:CD8 ratio of T cells is quantified for blood, spleen, and brain in young and aged mice [(E) n = 1026 per group].
Data are representative of the average of individual mice within a group. Error bars show mean SEM. *p , 0.05, **p , 0.01, ***p , 0.001.
4 AGE-RELATED CNS-RESIDENT CD8 T CELLS ARE REACTIVE TO STROKE
Because ICAM-1 expression is reported to be increased in aged brain injection-labeling experiment, as previously described (23, 24).
vasculature (21, 22), we examined CD8 T cell expression of CD11a All circulating and vessel-associated cells bound to transcardially
(LFA1) and CD49d (VLA4), adhesion molecules required for T cell injected PE-conjugated CD3e Ab were processed for leukocyte
entry into peripheral tissues. Fig. 3AD shows that CD11a and collection and subsequently stained with allophycocyanin eF780-
CD49d expression was significantly increased on circulating CD8 conjugated CD3e Ab. Compared with an injection-labeling effi-
T cells with age (p , 0.05 and p , 0.01, respectively), and further ciency of 98% for all blood CD3e-injected+CD8 T cells, only
elevated on those present in the brain at either age (p , 0.001 and 7.7 6 5% of aged brain CD8 T cells were positive for CD3e-
p , 0.05, respectively). In both tissues, CD49d-positive CD8 T cells injected Ab, suggesting these cells are located outside of the
were CD11ahigh. The vessel capture of T cells implies subsequent vascular network and in the parenchyma or perivascular spaces
extravasation into perivascular and parenchymal CNS tissue. Us- (Fig. 3F, 3G). Immunohistology revealed the presence of CD3+
ing tomato-lectin immunohistochemistry, we identified the putative CD8+ T cells in circumventricular organs (choroid plexus and
presence of CD8 T cells outside of lectin-positive lumen of vessels meninges) and most gray and white matter regions (cortex,
(Fig. 3E). To confirm that T cells resided in the parenchymal tissue, striatum, corpus collosum) throughout the anteriorposterior axis
and were not adherent to the brain endothelium, we performed an of the brain (Fig. 3H, 3I). Furthermore, we found that these cells
The Journal of Immunology 5
were often associated with astrocytes, although the precise nature the aging brain, we found that the vast majority (87 6 4%) of all
of this association is unknown (Fig. 3J). CNS-resident CD8 T cells have an effector memory phenotype,
compared with just 9 6 4% of CD8 T cells in blood (Fig. 4A, 4B).
Age-related, CNS-resident CD8 T cells have memory/effector A similar percentage of T cells with effector memory phenotype
phenotype and expression markers of TCR activation was observed in young brains, despite the fact that T cells were
The trafficking of T cells into CNS tissues is thought to occur found in significantly lower abundance. To determine the general
following antigenic presentation in draining lymph nodes by pro- activation state of CNS-resident CD8 TEM cells, we probed these
fessional APCs. Ag-experienced memory T cell subsets are defined cells with known markers of T cell activation. The early activation
by CD44+CD62L+ (central memory) and CD44+CD62L2 (TEM) and tissue-retention marker, CD69, was highly upregulated on
expression, whereas naive T cells are largely negative for the acti- CNS-resident CD8 T cells compared with those in the blood
vation marker CD44 (25). Applying this paradigm to CD8 T cells in [(Fig. 4C) n = 5 per group, p , 0.001]. Expression of programmed
6 AGE-RELATED CNS-RESIDENT CD8 T CELLS ARE REACTIVE TO STROKE
death-1 receptor, which is upregulated on activated T cells, was also suggest that the increased trafficking of these cells into the CNS is a
dramatically increased on nearly all CNS-resident T cells compared stochastic process, whereby a pool of activated, Ag-experienced
with CD8 T cells in aged blood [(Fig. 4D) n = 5 per group, p , memory effector CD8 T cells is constitutively present throughout
0.0001], implying the presence of widespread T cell activation in adult life in the CNS.
the aged brain. Expression of the resident memory marker aE
integrin CD103 was also found on a significant fraction of these Increased immune surveillance by CD8 T cells in the aging
cells (Fig. 4E). To determine whether CNS-resident memory CD8 brain is associated with microglial function
T cells were responding to antigenic signals present in the aging We investigated the functional role of CNS-resident CD8 T cells by
CNS, CD8 T cells in the blood and brain were screened for TCRvb correlating the absolute number in each brain with the activation
usage to determine T cell clonality in tertiary tissues. No differences status of microglia in that brain. Correlation analyses revealed that
in TCRvb usage between CNS-resident CD8 T cells and their aged brains with greater numbers of CNS-resident CD8 T cells had
blood counterparts were found for any of the 15 TCRvb families significantly more microglia (Fig. 5A, p , 0.05), indicating that
(Fig. 4F). Based on the absence of major clonal expansions in the these cells may either promote microglia survival or, alternatively,
CNS, these results suggest that CD8 T cell entry into the aged brain increase activation-induced proliferation. Brains that contained
is largely stochastic, rather than Ag driven. The following data large numbers of CNS-resident CD8 T cells, however, were also
The Journal of Immunology 7
associated with smaller sized microglia relative to brains with compared with those in the control contralateral hemisphere, as
fewer CD8 T cells (Fig. 5D, p , 0.05), indicative of a more evidenced by 5-(and-6)-chloromethyl-29,79-dichlorodihydrofluorescein
ramified, resting morphology. Functionally, higher numbers of diacetate, acetyl ester staining (Fig. 6G, 6H, p , 0.05). The per-
T cells were associated with increased microglia phagocytosis centage of TNF- and CCL2-positive CD8 T cells was also in-
(Fig. 5E, 5F, p , 0.001) and decreased TNF production (Fig. 5B, creased in ischemic hemisphere compared with control (Fig. 6IL,
5C, p , 0.05). These data suggest that the increased presence of p , 0.05 and p , 0.01, respectively). These data suggest that
CD8 T cells in the aged CNS is positively associated with an anti- activated CNS-resident CD8 T cells contribute to the age-related
inflammatory microglia phenotype in the aged brain. exacerbation of acute ischemic brain injury by amplifying pro-
duction of proinflammatory cytokines and promoting recruitment
Basal and stimulus-driven production of cytokines and of peripheral leukocytes.
chemokines following ischemic stroke
T cells are polarized to effector memory cells following TCR Discussion
reactivation to selected antigenic stimuli. The effector function of This work has identified a novel population of immune surveillant
these cells is largely specified by cytokine production. An in vivo CD8 T cells in the aging CNS that may have opposing roles in
brefeldin protocol was used to measure cytokine production in health and disease. This adds to previous studies demonstrating that
CNS-specific CD8 T cells as in (26). Cytokines commonly asso- immune privilege in the aging CNS is compromised. The aged
ciated with CD8 T cell proinflammatory responses (TNF and brain is more permissive to T cell entry, which has functional
IFN-g) were basally produced at significant levels (Fig. 6AC), consequences, especially after injury. We base this observation on
whereas granzyme B or perforin was not expressed under any several criteria, as follows: 1) increased brain production of T cell
conditions (data not shown). Both the number and relative ex- chemokine cues, 2) enhanced expression of adhesion molecules, 3)
pression level of TNF and IFN-g were significantly increased after higher levels of extravasation into perivascular and parenchymal
stimulation in CNS-resident CD8 T cells compared with those in regions, 4) activated tissue-resident and effector memory pheno-
blood (Fig. 6D, 6E, p , 0.001). CCL2 production by CNS- types, 5) presence alters microglia function, and 6) primed to elicit
resident CD8 T cells was significantly higher after stimulation strong cytokine and chemokine responses after sterile brain injury.
compared with those in the blood (Fig. 6F, p , 0.001). These data These cells have a phenotype indicative of resident memory cells
suggest that CNS-resident T cells in the aged CNS are a rich (CD44highCD62L2CD11a+CD69+CD103+/2). In summary, this
source of proinflammatory cytokines, most likely contributing to work has identified a specific population of age-associated CNS-
the steady increase in overall levels of inflammation that occurs resident memory CD8 T cells and has highlighted their influence
with advanced age. Next we determined whether these cells were on the neuroimmunological landscape.
similarly responsive to stimuli in an age-relevant model of brain For an organ that has long been described as immune privileged
injury. We tested this in an experimental model of ischemic stroke, and devoid of any blood-borne leukocytes, the brains relationship
as this is a disease that mainly affects the aging population. Cy- to the immune system is now being revised with the advent of a
tokine production was assessed at 4 h postreperfusion following meningeallymphatic system and growing recognition that T cells
90 min of occlusion. Reactive oxygen species generation was constitutively traffic throughout the CNS (27). Compared with
significantly increased in CD8 T cells subjected to ischemia many widely used experimental models of CNS injury, the number
8 AGE-RELATED CNS-RESIDENT CD8 T CELLS ARE REACTIVE TO STROKE
FIGURE 6. Acute stimulation of CD8 T cells by PMA/ionomycin and in an age-relevant model of cerebral ischemia. Representative histograms show
CD8 T cell expression of TNF (A), IFN-g (B), and CCL2 (C) in blood (solid black) and brain (solid gray) and after stimulation (dotted). The respective
mean fluorescence intensities (DF) were quantified (n = 410 per group). Aged mice were subject to 90 min of occlusion, followed by 2 h of reperfusion
and 1-h ex vivo stimulation by PMA/ionomycin. CD8 T cells from the ischemic hemisphere (ipsilateral) were compared with those in the contralateral
hemisphere for internal control (n = 46 per group). A representative zebra plot shows reactive oxygen species levels in CD8 T cells after stroke, as
measured by CM-H2DCFDA (G) and quantification of mean fluorescence intensity (H). A representative dot plot shows intracellular production of TNF (I)
and the percentages quantified (J). A representative dot plot depicts a stroke-induced increase in CCL2 production by CD8 T cells in the aged brain (K). The
percentages were quantified (L). Cell-specific fluorescence minus one controls were used to determine positive gating (shaded gray). Error bars show mean
SEM. *p , 0.05, **p , 0.01, ***p , 0.001. a.u.i., arbitrary units of intensity; CM-H2DCFDA, 5-(and-6)-chloromethyl-29,79-dichlorodihydrofluorescein
diacetate, acetyl ester; MFI, mean fluorescence intensity; SSC, side scatter intensity.
The Journal of Immunology 9
of the T cells in the normal aging brain is relatively small, yet Our observation that brain-related chemokine production is in-
emerging data suggests that interfering with this steady-state creased with age suggests possible involvement in facilitating T cell
trafficking of T cells results in significant cognitive impairment migration to the CNS. Moreover, the age-related increase in che-
(12). Moreover, recent findings suggest that the recovery of resi- mokine receptor expression on circulating CD8 T cells as noted in
dent memory CD8 T cells from nonlymphoid tissues may be this study may further enhance this migration. Although speculative,
significantly underestimated, as many cells are either killed or this thinking is premised on several studies in which blocking these
discarded during the lengthy tissue-processing procedure (28). particular chemokines (i.e., CCL5, CCL11, CXCL9, and CXCL10)
The nearly 3-fold increase in CNS-resident CD8 T cells of old interfered with the normal migration of T cells into the injured CNS
mice highlights the role of aging on neuroinflammatory activity (4655). The cellular source of these chemokines was not assessed
and the greater need for immune surveillance. in this study, although glial cells are known to be major producers
Despite the fact that the other CD45high leukocyte populations do of these signals. For example, astrocyte-secreted CXCL10 promotes
not prominently increase in the brain with age, the question arises as the recruitment and entry of lymphocytes into the CNS and worsens
to whether T cell recruitment is gradually increased over time, or acute spinal cord demyelination in experimental allergic encepha-
whether there is a critical period in which the brain is more per- litis (56, 57). Indeed, there is a growing appreciation for astrocyte
missive to these lymphocytes. Although the recent identification of a T cell interactions (5860). The impact of CNS-resident CD8
CNS lymphatic route implies that T cell trafficking is a constitutive, T cells on astrocyte functions in aged brains remains to be shown.
homeostatic process, the presence of CD8 T cells in the young brain Although aged astrocytes exhibit signs of a senescence-associated
suggests that this process begins early. Although our study was phenotype with age (6163) and produce chemokines in response to
limited to two time points, earlier work by Stichel and Luebbert (16) amyloid-b peptides (64), the role of astrocytes as mediators of
demonstrated that CD3+ T cells accumulate in several CNS regions T cell recruitment in the aging brain is not understood.
restricted hemagglutinin (67). OVA-expressing oligodendrocytes works support the notion of an immune surveillant pathway in the
elicit the proliferation and effector memory conversion of CNS that maintains not only the acute steady state trafficking of
responding OT-1 T cells in healthy mice without infiltrating the CD8 T cells, but their long-term maintenance as well. Surveil-
CNS, indicating that Ag sampling is performed by CD8 T cells lance also occurs at CNS drainage sites, as intracranial injection of
under steady state conditions (68). Interestingly, the authors found OVA peptide results in T cell priming in peripheral lymphoid
that neuroinflammation augmented the level of Ag sampling and organs and the subsequent infiltration and persistence of OT-1
led to the accumulation of OT-1 T cells in the inflamed brain. T cells into the brain (36, 80, 81). Because the age-related accu-
Thus, it is increasingly evident that CD8 T cells have the capacity mulation of CD8 memory cells described in our study represents a
to mediate responses against CNS Ags (69). Although the age- naturally occurring population of CNS-resident T cells, it may
related increase in neuroinflammation most likely promotes the stand as a useful model for understanding the biology of tissue-
recruitment of immune surveillant CD8 T cells, how they impact resident memory and immune surveillance. Taken together, the
CNS function is poorly understood. Tissue-resident memory CD8 migration of effector memory CD8 T cells into the aged CNS does
T cells provide superior protection against viral reactivation not require stimulation by CNS Ag, although deeper sequencing
compared with the circulating memory T cell pool, but become of the TCR repertoire might be a useful approach in determining
dependent on the local milieu for function and survival (36, 42, the antigenic specificity of these cells.
70). The increased presence of these cells in the aged CNS may We demonstrated that, upon activation by either PMA/ionomycin
reflect an evolutionary need to generate a rapid response to control stimulation or ischemic brain injury, CNS-resident CD8 T cells in
senescent transformation of cells and/or prevent reactivation of aged brains are prone to elicit highly volatile responses that include
latent neurotropic viruses. the enhanced production of reactive oxygen species, TNF, IFN-g,
The role of immunosurveillance as a defense mechanism that and CCL2. These cells may therefore predispose the aged brain to
biological consequences of these sex differences remains an area vation accompanies brain aging, increasing vulnerability to cognitive decline
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