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F U L L T I T L E : Functional recreation of age-related CD8 T cells in young mice identifies drivers of aging- and human-specific tissue pathology.
O P E N A C C E S S S O U R C E : Mechanisms of Aging and Development
Highlights
• Expansion of CD8 T cells in nude mice recreates age-related phenotype, function.
• Expanded CD8 T cells are reactive to self antigen and accumulate in tissue.
• Persistence of expanded CD8 T cells in brain requires T cell lytic function.
• Expanded CD8 T cells elicit tissue pathology of aging in skin, brain in nude hosts.
• Expanded CD8 T cells humanize pathological response to brain injury in wild-type hosts.
Abstract
Mitigating effects of aging on human health remains elusive because aging impacts multiple systems simultaneously, and because experimental animals exhibit critical aging differences relative to humans. Separation of aging into discrete processes may identify targetable drivers of pathology, particularly when applied to human-specific features. Gradual homeostatic expansion of CD8 T cells dominantly alters their function in aging humans but not in mice. Injecting T cells into athymic mice induces rapid homeostatic expansion, but its relevance to aging remains uncertain. We hypothesized that homeostatic expansion of T cells injected into T-deficient hosts models physiologically relevant CD8 T cell aging in young mice, and aimed to analyze age-related T cell phenotype and tissue pathology in such animals. Indeed, we found that such injection conferred uniform age-related phenotype, genotype, and function to mouse CD8 T cells, heightened age-associated tissue pathology in young athymic hosts, and humanized amyloidosis after brain injury in secondary wild-type recipients. This validates a model conferring a human-specific aging feature to mice that identifies targetable drivers of tissue pathology. Similar examination of independent aging features should promote systematic understanding of aging and identify additional targets to mitigate its effects on human health.
1. Introduction
One of the central unanswered questions in biology is how aging leads to coordinated dysfunction in multiple tissues throughout the body. Related to this is how aging increases susceptibility to tissue pathology. The complexity and pervasiveness of aging across multiple bodily systems underlies this conundrum. In addition, critical aspects of aging differ substantially between the experimental rodent systems widely used to study them, and humans. To identify discrete targetable features of age-related tissue pathology, and relevance to human health, there is thus a critical need to deconvolute aging into separable components, as well as render it more human-like in experimental rodent systems. Current health-related aging research focuses on cellular senescence, yet some aspects of aging are initiated upon sexual maturity, and as such predate the onset of cellular senescence by years or decades. Examining these earliest age-related processes may reap the most substantial benefits in any effort to deconvolute aging.
While accumulation of senescent cells is thought to contribute to age-related pathologies, immune dysfunction, particularly within the cytolytic (CD8) T cell compartment, can occur completely separate from aging or time-dependent cellular senescence [Zlamy et al., 2016]. Shortly after puberty, an aberrant subpopulation of phenotypically and functionally distinct memory CD8 T cells begins to progressively accumulate in the circulation, partially in response to thymic involution [Zlamy et al., 2016; LeMaoult et al., 2000; Messaoudi et al., 2006]. This subpopulation exhibits age-related changes in homing to tissues including brain in both humans and mice, and expresses markers of resident-memory CD8 T cells (CD8 TRM) [Park and Kupper, 2015; Wakim et al., 2012; Smolders et al., 2013; Ritzel et al., 2016; Rodriguez-Garcia et al., 2018]. In the circulation, age-related CD8 TRM become prominent by middle age in most individual people, and can promote immune-mediated tissue damage upon re-stimulation [Clambey et al., 2005; Clambey et al., 2008; den Braber et al., 2012; Schwab et al., 1997]. As such, CD8 TRM accumulation is a prime candidate to coordinate the breakdown of multiple tissues during aging. Nevertheless, the impact of this phenomenon on tissue pathology outside the immune system itself is poorly understood, in large part because it is not easily studied in experimental rodents.
In contrast to humans where they dominantly overtake the T cell pool in aging, the impact of CD8 TRM expansion in mice is offset by compensatory factors including continual production of new T cells by the thymus [Clambey et al., 2005; den Braber et al., 2012]. Hence, expanded CD8 TRM exhibit a relatively minor influence on the mouse T cell pool even at advanced ages [den Braber et al., 2012]. Moreover, and as with most age-related phenomena, CD8 TRM accumulation also occurs simultaneously with all other age-related changes in humans and in mice. Thus, determining its impact on aging pathology is problematic without models to examine it in isolation. Such models would ideally mimic the impact of CD8 TRM expansion in humans to be most informative for human health. To achieve this, we considered the physiological causes of age-related CD8 TRM expansion.
Age-related accumulation of CD8 TRM is dependent on homeostatic expansion. This ordinarily occurs gradually as production of new CD8 T cells progressively wanes over time, and existing CD8 T cells begin to expand in response to available antigens, refilling the depleted T cell niche. Rapid homeostatic expansion of T cells also occurs when T cells are introduced into hosts that intrinsically lack a full T cell niche (athymic or lymphocyte-deficient strains), but the relevance of this inducible age-independent phenomena to age-related CD8 TRM expansion and age-related disorders has not been examined. We hypothesized that homeostatic expansion in T-deficient hosts models physiologically relevant CD8 T cell aging in young mice, and conducted phenotypic and physiological studies to test this. Specifically, we examined age-related T cell phenotype in nude hosts injected with CD8 T cells, as well as examine age-related tissue pathology in such mice, most notably in brain with and without accompanying injury.
We established that inducible homeostatic expansion of CD8 T cells by injection into athymic mouse hosts (nude; B6.Foxn1) mimics the molecular, phenotypic, and functional properties of aberrant age-related CD8 TRM with 100% penetrance. We therefore examined the resulting homeostatically-induced- or hiTRM-bearing mice for evidence of tissue pathology in skin and brain, and their role in “humanizing” distinct pathological responses in mice.
Our findings strongly suggest that age-dependent accumulation of CD8 TRM is accurately modeled by inducible homeostatic expansion of CD8 T cells in young mice. Additionally, CD8 TRM in these mice mediated coordinated damage to multiple tissues including skin and brain, that were reminiscent of aging. Finally, CD8 TRM elictied human-specific molecular pathology in response to brain trauma, essentially eliminating a clinical disparity between mice and humans. Our model represents a reductionist model to study the role of a discrete component of aging separate from others, that promises to illuminate not only aspects of age-related immune dysfunction, but targetable drivers of age-related pathology in distinct tissues such as skin and brain.
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4. Results
4.1. Uniform induction of “hiT” cells in nude mice
CD8 T cells from young (<9 wk) C57BL/B6 (B6) donors, CD45.1-congenic B6, or B6 donors with effector gene knockout (PrfKO or IfngKO) were injected into B6.Foxn1 recipients to examine the role of CD8 T cell pro-inflammatory and lytic function on homeostatic expansion and related sequelae. Where warranted (i.e., in wild-type B6 recipients injected with CD45.1-congenic CD8 T cells), lymphocytes were gated for donor surface marker CD45.2 by flow cytometry where appropriate, and evaluated for peripheral expansion (Fig. 1A, B). B6 CD8 T cells were also injected into young nude hosts lacking the Amyloid Precursor Protein gene (B6.Foxn1/AppKO) to examine the possible role of App as a promoters of tissue-specific inflammation in modulating donor cell expansion [Puig et al., 2017; Puig et al., 2012]. Donor CD8 T cells injected in young B6.Foxn1 recipients (CD8→B6.Foxn1 respectively) expanded within 8 days regardless of donor effector gene knockout and remained in circulation. Donor CD8 T cells injected into young B6.Foxn1/AppKO also expanded within 8 days (Fig. 1C). By contrast, CD8 T cells serially transferred from nude recipients back into wild-type B6 or B6.CD45.2-congenic hosts (CD8→B6.Foxn1→B6) did not expand (Fig. 1C). As expected, CD8 T cells labeled with the cytoplasmic dye, CFSE, prior to injection into B6.Foxn1 hosts exhibited the laddered dye dilution and population enlargement typical of homeostatic expansion within 4 days (Fig. 1D). This early expansion was delayed, however, in B6.Foxn1/AppKO recipients (Fig. 1D), suggesting that the earliest phase of homeostatic expansion may be dependent on reactivity to a self App epitope. Unfortunately, B6.Foxn1/AppKO hosts were about a third the size of either parental strain and did not survive more than a few weeks, and so did not contribute to further analyses.
Fig. 1. Expansion of donor CD8 T cells in mouse recipients. Purified CD8 T cells from female C57BL/6, B6.CD45.1-congenic (B6 recipients only; CD8→B6), or congenic effector knockout hosts (Perforin-1- or Ifnγ-deficient), or from previously injected B6.Foxn1 donors, were injected into 8-10 week-old female C57BL/6 J, B6.Foxn1, or B6.Foxn1/AppKO, recipients as indicated (A). Blood was analyzed by flow cytometry 5-8 days later using the gating and antibodies to T cell markers as shown (B), with % CD3ε+CD8+ in gated donor cells compiled in ©, where “% donor T cells” refers to the percent donor T cells within gated recipient lymphocytes. Note that both B6.Foxn1 mice and C57BL/6 are CD45.2. Fig. 1B depicts lymphocytes from CD45.2+ donors in CD45.2+ B6.Foxn1, and thus contains plentiful non-T lymphocytes. B6.Foxn1 mice were crossed to B6.App-knockout mice, homozygous double-mutants (B6.Foxn1/AppKO) verified by PCR and phenotype at Jackson Laboratories (Bar Harbor, MN). Purified CD8 T cells were labeled with CFSE, and their expansion assessed by CFSE dilution in B6.Foxn1 and B6.Foxn1/AppKO female recipients (D; n = 3 B6.Foxn1 & n = 5 B6.Foxn1/AppKO; *P < 0.04, ***P < 0.00001 by 2-tailed T-test in >3 independent tests for all markers).
4.2. hiT and aged CD8 T cell phenotype, genotype, function
Analysis of homeostatically-induced donor CD8 T cells (“hiT” cells) in B6.Foxn1 hosts by flow cytometry revealed a surface marker profile identical to CD8 T cells undergoing clonal expansion in aged mice (CD122hi, CD127hi, CD44hi, KLRG1hi, PNAhi, CD8lo, CD103+; Fig. 2A–D) [LeMaoult et al., 2000; Messaoudi et al., 2006; Clambey et al., 2005; Clambey et al., 2008; Messaoudi et al., 2004]. A similar phenotype is found on CD8 T cell clonal expansions in aging humans [Clambey et al., 2005]. The T cell markers CD44 and KLRG1 establish that these homeostatically induced, or “hiT” cells belong to a memory T cell subset associated with aging [Clambey et al., 2008], while CD103 expression is further characteristic of resident-memory CD8 T cells, or TRM [Mackay et al., 2012; Gebhardt and Mackay, 2012]. Nevertheless, resident-memory T cells normally reside in peripheral non-immune tissues, although they can migrate into the general circulation as “ex-TRM” [Fonseca et al., 2020]. Since in our model, these cells appear to arise within the circulation, we thus regard them as “pre-TRM” cells when observed in the general circulation/spleen, but more generally refer to them as “homeostatically-induced” or “hiT cells” in recipient mice, unless they are within non-immune tissues.
Fig. 2. Age-related hiT cell phenotype in young mice. Representative flow cytometry analysis of age-related markers on splenic CD8 T cells from young (< 10 weeks) and old (> 12 months) C57BL/6 (B6), and young (6 weeks) B6.Foxn1 recipients of i.v. CD8 T cells (CD8→B6.Foxn1) 3-5 weeks after injection (A). Antibody combinations used were: CD3 PEcy5, CD8 PE, CD4 FITC (control, not shown); CD8 PECy5, CD122 FITC, CD127 PE, CD45.2 PacBlue (top panel); CD8 FITC, CD44 PE, KLRG1 Biotin/SACy, CD45.2 PacBlue (2nd panel); CD8 FITC, PNA APC (3rd panel); CD8 PacBlue, CD103 FITC (4th panel). Percentage of lymphocytes (B) and mean fluorescence intensity (C, D) from flow cytometry compiled from n > 6 mice/group. T cell receptor (TCR) gene segment usage and diversity in nude mice harboring hiTRM. Proportions of mice with “diverse” TCRVβ D→J gene segment usage (> 3 segments/brain) and specific D→J segments within brains of young (<10 weeks), middle-aged (6 months), and old (> 12 months) B6 mice, reveals an age-dependent pattern of progressively decreased diversity and increased usage of particular D→J segments (i.e., clonality; E, F). D→J diversity and segment usage was significantly correlated only between old B6 and young CD8→B6.Foxn1 brain; colors for specific D-J joints are derived from E & F (G). Schematic of forward (right-facing arowhead) and reverse (left-facing arrowhead) TCRβ locus D1-J1 and D2-J2 primers is depicted beneath E-F. Additional detail and representative gels are provided in Supplemental Fig. S1.*P < 0.05, **P < 0.01, ***P < 0.005 by 2-sided T-test relative to B6 for flow cytometric markers, and by Pearson’s correlations in n > 10 mice/group for PCR compilations.
Age-related expansion decreases clonal diversity of CD8 T cells [LeMaoult et al., 2000; Schwab et al., 1997; Messaoudi et al., 2004; Ahmed et al., 2009; Degauque et al., 2011; Morley et al., 1995; Posnett et al., 1994; Posnett et al., 2003; Ricalton et al., 1998; Buchholz et al., 2011]. We thus sought to quantify hiT clonality. To do this, we analyzed variable region D→J rearrangements in T Cell Receptor beta gene segments by PCR from brain, as previously described [Aifantis et al., 1997; Gärtner et al., 1999]. This methodology provides a measure of overall clonal diversity T cells without extensive sequencing data from each individual TCR V region/joint as in TCR spectratyping, by detecting productive TCRβ D-J joints. These DNA recombination events are required for the generation of a productive TCR and for T cell maturation. The methodology is additionally insensitive to the propensity of tissue-resident CD8 T cells to undergo apoptosis upon tissue dissociation [Wakim et al., 2010], as well as to brain autofluorescence that can complicate flow cytometric analysis [Duong and Han, 2013]. We thus determined the proportion of biological replicate brain samples using each of the detectable 12 D-J joints. Consistent with previous reports, splenic T cells in wild-type mice aged 12 months showed minimal clonal skewing in TCRVβ, whereas those injected into nude recipients exhibited both D1→J1 and D2→J2 clonal skewing after just 10 weeks (Supplemental Fig. S1). Increasing D1→J1 and D2→J2 clonal skewing was most evident, however, in brains of middle-aged (6 months) and aged (>12 months) wild-type mice (Fig. 2E–G, Supplemental Fig. S1). These cohorts exhibited progressive reduction of D→J diversity with age in brain, whereas D→J usage in young wild-type mouse brain exhibited substantial diversity (Fig. 1E, F). D→J joints in brains of young hiT-bearing nude mice also exhibited reduced diversity similar to that of aged mice (Fig. 2E), and D→J segment usage in these as well as aged mice were both significantly different from that in young mice (Fig. 2F, Supplemental Fig. S1). Indeed, D→J segment usage in brains of young hiT-bearing nude hosts was most similar to that in aged wild-type mice (Fig. 1G).
TCR Vβ D→J joint analysis strongly suggested that hiT reside in brain. We thus examined presence, phenotype, and antigen reactivity of hiT cells in brain more directly by flow cytometry and Western blot analysis. CFSE-labeled donor CD8 T cells injected into B6.Foxn1 hosts were found increased in brain parenchyma three days later, confirming rapid localization of hiT cells to brain (Fig. 3A, B). CD8 protein on brain Western blots was also increased in both nude hiT recipients 10 weeks after injection, and in aged relative to young wild-type mice (Fig. 2C), suggesting similar functional localization to brain in hiT-bearing nudes and affected aged mice [Ritzel et al., 2016]. Although overall levels of CD8 T cells in brain appeared similar to wild-type by flow cytometry, this likely reflected sensitivity to tissue dissociation [Wakim et al., 2010]. Qualitatively, however, flow cytometry revealed that IFNγ+ and KLRG1+ CD8 T cells that retained CD103 expression were significantly increased in hiTRM-bearing nude brains (Fig. 3D). Unexpectedly high apoptosis of lymphocytes derived from brains of aging mice prohibited direct comparison by flow cytometry. Nevertheless, multiple studies have documented that resident memory CD8 T cells in aging tissues including brain and others, express CD103, and up-regulate both KLRG1 and pro-inflammatory cytokines including IFNγ [Ritzel et al., 2016; Clambey et al., 2008; Onyema et al., 2012; Schenkel et al., 2013]. Thus, hiT cells in nude brain phenotypically correspond to typical CD8 TRM within aging tissues. This altered T cell population was also evident in the periphery, as KLRG1+ CD8 T cells reactive to MHC I-restricted antigens including Tyrosinase-related Protein-2/Dopachrome Tautamerase (Trp-2/DCT[180-188]) and APP[470-478] were expanded in blood, corroborating host T cell expansion dynamics in B6.Foxn1/AppKO hosts (Fig. 1D; 3E, F). Nevertheless, only APP-reactive CD8 TRM were significantly increased in brain (Fig. 3E, F). Thus, hiT cells reactive to two distinct self antigens expanded peripherally in nude hosts, whereas those reactive to an APP epitope selectively accumulated in brain.
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