CNS-Specific Immunity at the Choroid Plexus Shifts Toward Destructive Th2 Inflammation in Brain Aging

CNS-speciﬁc immunity at the choroid plexus shifts toward destructive Th2 inﬂammation in brain aging

Kuti Barucha,1, Noga Ron-Harelb,1, Hilah Galc,1, Aleksandra Deczkowskaa, Eric Shifrutc, Wilfred Ndifonc, Nataly Mirlas-Neisberga, Michal Cardona, Ilan Vaknina, Liora Cahalona, Tamara Berkutzkia, Mark P. Mattsond, Fernando Gomez-Pinillae,f, Nir Friedmanc,2, and Michal Schwartza,2,3

Departments of aNeurobiology and cImmunology, Weizmann Institute of Science, Rehovot 76100, Israel; bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; dLaboratory of Neurosciences, National Institute on Aging Intramural Research Program, National Institutes of Health, Baltimore, MD 21224; and Departments of eIntegrative Biology and Physiology and fNeurosurgery, University of California, Los Angeles, CA 90095

Edited* by Michael Sela, Weizmann Institute of Science, Rehovot, Israel, and approved November 13, 2012 (received for review July 4, 2012) In the present study, we show that the CP in young and aged The adaptive arm of the immune system has been suggested as an + important factor in brain function. However, given the fact that animals retains CD4 effector memory cells with a T-cell receptor fi interactions of neurons or glial cells with T lymphocytes rarely occur (TCR) repertoire speci c to CNS antigens. With aging, the CP mani- fl within the healthy CNS parenchyma, the underlying mechanism is fested signs of T helper type 2 (Th2) in ammation, linking its still a mystery. Here we found that at the interface between the functional plasticity to age-related cognitive decline. Using an immunological manipulation that induced homeostasis-driven brain and blood circulation, the epithelial layers of the choroid + proliferation and partially restored cognitive ability, we demon- plexus (CP) are constitutively populated with CD4 effector memory fi strate that peripheral rejuvenation of the immune system can cells with a T-cell receptor repertoire speci c to CNS antigens. With immunomodulate the CP, shifting its cytokine balance and age, whereas CNS specificity in this compartment was largely main- modifying hippocampal plasticity. tained, the cytokine balance shifted in favor of the T helper type 2 (Th2) response; the Th2-derived cytokine IL-4 was elevated in the CP Results γ + of old mice, relative to IFN- , which decreased. We found this local The Choroid Plexus Is Populated by Effector Memory CD4 T Cells. cytokine shift to critically affect the CP epithelium, triggering it to Using flow cytometry, we found that, similar to the lymph nodes produce the chemokine CCL11 shown to be associated with cognitive (LNs), the majority (62 ± 2.76%) of T cells found in CPs were IMMUNOLOGY + + + dysfunction. Partial restoration of cognitive ability in aged mice, CD4 cells (Fig. 1A). Because CD4 T cells (rather than CD8 by lymphopenia-induced homeostasis-driven proliferation of mem- cells) were previously implicated in supporting CNS plasticity (1, ory T cells, was correlated with restoration of the IL-4:IFN-γ ratio at 2, 8, 12), we further characterized this subpopulation. We focused the CP and modulated the expression of plasticity-related genes at our interest on memory T cells, commonly divided into two subsets (Fig. 1B): CD44high/CD62Lhigh are central memory T cells the hippocampus. Our data indicate that the cytokine milieu at the high −/low CP epithelium is affected by peripheral immunosenescence, with (TCM), whereas CD44 /CD62L are effector memory T detrimental consequences to the aged brain. Amenable to immuno- cells (TEM) that can be locally activated to an immediate effector function upon encountering their cognate antigen (15). In the CP, modulation, this interface is a unique target for arresting age-related unlike the blood and LNs, the vast majority of memory T cells cognitive decline. expressed TEM markers (Fig. 1C). In aged mice (22-mo-old), we observed an increase relative to young mice (3-mo-old) in the fre- blood-cerebrospinal fluid barrier | brain senescence | neuroinflammation quency of TEM cells in both the blood circulation and the LNs, whereas their levels in the CP were unchanged (Fig. 1 C and D). + irculating immune cells have been repeatedly shown to be Thus, unlike the CSF, which is dominated by CD4 TCM cells (16, fi + Cessential for central nervous system (CNS) maintenance (1–3). 17), the CP speci cally retains CD4 TEM cells throughout life. T Specifically, T cells that recognize CNS antigens contribute to the cells in the CP were colocalized with the epithelium (Fig. 1E)and functional integrity of the CNS under both normal and patho- adjacent to MHCII-expressing APCs (Fig. 1F), indicating their logical conditions (2, 4–6), supporting hippocampus-dependent potential functional interactions there. We therefore assumed that learning and memory, adult neurogenesis, and neurotrophic factor the TEM cells that reside in the CP are activated by APCs that present their cognate antigens, and are likely to be specificforbrain production (2). fi Under physiological conditions, T cells are rarely found in the antigens. Thus, we next focused on identifying the speci city of the brain parenchyma and are mainly observed at the borders of the T cells that reside in the CP and whether they undergo changes with CNS: the choroid plexus (CP) of the brain’s ventricles, forming age that could, to some extent, explain brain senescence. – fl the blood cerebrospinal uid barrier (BCSFB), the meningeal + fi spaces, and the cerebrospinal fluid (CSF) (7). T cells were shown The Choroid Plexus CD4 TCR Repertoire Is Enriched with CNS-Speci c to accumulate in these compartments in response to signals from Clones. Sequencing of a TCR by itself cannot identify its antigenic fi specificity. Therefore, we developed a unique approach that the CNS, speci cally in the meninges after performance of cog- fi nitive tasks (8) and in the CP after exposure to mental stress (9). allowed us to identify clonotypic enrichment of CNS-speci cT cells. Mice were immunized with spinal cord homogenate (SCH), In the meningeal spaces, these cells were further characterized as producing the cytokine interleukin 4 (IL-4), known for its beneficial role in CNS maintenance and neuroprotection (8, 10–13). However, the questions of why, where, and how T-cell specificity Author contributions: K.B., N.R.-H., H.G., W.N., N.F., and M.S. designed research; K.B., N.R.-H., H.G., A.D., E.S., W.N., N.M.-N., M.C., I.V., L.C., and T.B. performed research; M.P.M. and F.G.-P. is needed for brain plasticity remained mysterious. contributed new reagents/analytic tools; K.B., N.R.-H., H.G., A.D., E.S., W.N., and N.M.-N. The CP is strategically positioned at the lining between the CNS analyzed data; and K.B. and M.S. wrote the paper. and the immune system and, in addition to its classically known The authors declare no conflict of interest. role in generating the CSF, can enable bidirectional communi- cation between the CNS parenchyma and blood circulation (14). *This Direct Submission article had a prearranged editor. Accordingly, we envisioned that T cells that reside in this com- 1K.B., N.R.-H., and H.G. contributed equally to this work. partment interact with their relevant antigen-presenting cells 2N.F. and M.S. contributed equally to this work. (APCs) and respond, upon stimulation, by secreting cytokines 3To whom correspondence should be addressed. E-mail: [email protected]. that modulate the properties of the CP epithelium, thereby af- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. fecting the CNS parenchyma from afar. 1073/pnas.1211270110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1211270110 PNAS Early Edition | 1of6 Fig. 1. The Choroid plexus is populated by effector + A Lymph nodes Choroid plexus B memory CD4 T cells. (A) Flow cytometry plots of 70% 62% + + Central CD4 /CD8 T-cell frequencies (mean; %) in the in- memory + guinal LNs and the CPs, pregated by CD45 and + Effector β = β memory 4 TCR (n 5 per group). (B) Flow cytometry gating C D 36% S TCR CD44 CD62L S 28% C strategy for inguinal LN, blood, and CP tissues. Cells CD4 + + CD8 FSC CD45 TCRβ CD4 CD4 were stained for markers of effector (CD45 , TCRβ , + high − + CD4 , CD44 , and CD62L ) and central (CD45 , C Young DEFCD3 MHC-II / CD3 Lymph nodes Blood Choroid plexus *** * β+ + high + + 100 TCR , CD4 , CD44 , and CD62L ) memory CD4 T 39.9 51.4 3.1% EM ±0.9% ±4.6% 80 T cells. FCS, forward scatter; SSC, side scatter. (C) TCM

60.1 48.6 96.9% Representative plots for young (3-mo-old) and aged ±0.9% ±0.4% 60 + CD62L (22-mo-old) mice effector and central memory CD4 CD4 40 T-cell frequencies in inguinal LN, blood, and CP tis- Old Lymph nodes Blood Choroid plexus 20

sues. (D) Quantitative analysis of the data shown Population frequency (%) 26.3 28.2 0.7% ±0.4% ±4.4% 0 in C. Data shown are representative of three inde- N L P P B C C 73.6 71.8 gLN dL gBL d g 99.3% n n Ol u Ol Old pendent experiments. Bar graphs throughout the ±0.4% ±4.4% o oun You Y Y

fi ± = – CD62L gure show mean SE of each group (n 4 5 per CD4 group; *P < 0.05; ***P < 0.001; Student’s t test). BL, blood. (E) Localization of T cells (stained for CD3; arrowheads) in the CP of naïve animals. (Scale bar, 50 μm.) (F) Confocal images of CP tissues from naïve animals showing the distribution of APCs (stained for MHCII; green) and their colocalization with T cells (stained for CD3; red) using orthogonal projections of confocal z stacks. (Scale bar, 25 μm.) containing a wide array of CNS proteins, in order to enrich the maintained clonotypic specificity to CNS antigens, though the spleen with CNS-specific T cells. RNA isolated from splenic abundance of CNS-specific clones was somewhat lower relative + CD4 T cells and CP-resident T cells was analyzed using TCR- to young CP (Fig. 2 C and D). Following immunization with seq, a high-throughput sequencing procedure of the TCRβ SCH, the abundance of CNS-specific clones was elevated in the CDR3 region (18–20). Using bioinformatics tools developed in- CP compared with the nonimmunized CP (Fig. 2D), suggesting house (see SI Materials and Methods for a detailed description), that the accumulation of CNS-specific T cells at the CP is subject full characterization of each CDR3 region was achieved from to changes and reflects peripheral immunity. individual sequencing reads, identifying variable (Vβ), diversity (Dβ), and joining (Jβ) gene segments use. Data obtained were Th2-Mediated Inflammation of the CP Epithelium During the Aging further analyzed by a specially designed analysis pipeline en- Process. Based on the above results, we hypothesized that aging abling extraction of reliable quantitative information on TCRβ of the CP might be associated with changes in T-cell effector phe- repertoire composition, providing a list of annotated TCRβ se- notype bias, an established phenomenon outside the CNS during quences (nucleotide and amino acid sequences), and their rela- aging (22, 23). To examine this, we measured mRNA levels of the tive abundance, for each sample. cytokines ifn-γ and il-4 in this compartment, representing the Th1 Once we established the TCRβ repertoire of the spleens of and Th2 effector phenotypes, respectively. We found preferential animals immunized with CNS antigens, we compared it to the elevation of il-4 expression and a decline in ifn-γ expression with repertoire of T cells from the CP of nonimmunized animals. We aging (Fig. 3A). observed a high level of similarity in Vβ use between the TCRβ IL-4–producing cells were recently identified at the meningeal repertoire found in CP of naïve animals and that found in the spaces of the brain and shown to support cognitive function (8). spleens of animals immunized with CNS antigens (SCH) (Fig. S1 Outside the CNS, however, IL-4 was shown to induce expression A and B). In contrast, the naïve CP TCRβ repertoire showed of CCL11 (24), a chemokine associated with age-related cogni- much lower correlation with the repertoire found in spleens of tive decline, and is elevated in the CSF and plasma of aged mice naïve or ovalbumin (OVA)-immunized mice. Jβ use analysis gave and humans (25). This apparent contradiction between the similar results (Fig. S1 C and D). beneficial roles of IL-4 in cognitive performance and its known Next, we applied average-linkage clustering analysis to the potential to induce CCL11 expression outside the CNS led us to different groups, based on the group average relative frequencies consider a link between the two effects in the aged brain; namely, of all Vβ and Jβ segments (36 parameters overall). The naïve CP we envisioned that the age-related CCL11 found in the CSF formed a distinct linkage cluster with the SCH-immunized spleen, during aging (25) may be a product of the CP epithelium, re- whereas the OVA-immunized spleen clustered with the naïve sulting from overwhelming levels of IL-4 that develop in this spleen (Fig. 2A). The repertoire of T cells residing in the naïve compartment with aging. CP showed a high level of similarity in Vβ and Jβ segment use to We therefore examined mRNA and protein levels of CCL11 spleens of SCH-immunized mice, yet not with naïve or OVA- in the aged CP, and found them to be elevated (Fig. 3 B and C) immunized spleens. Finally, we examined the naïve CP repertoire relative to young animals. CCL11 immunoreactivity was barely in terms of specific clones. From our TCR-seq data, we compiled detected in young animals, whereas it was highly abundant in a library of 2,752 TCRβ amino acid sequences that were signifi- aged CP tissue (Fig. 3D). To understand how the local cytokine cantly up-regulated in the SCH-immunized spleens compared balance controls CCL11 induction, we examined its expression with spleens of nonimmunized animals. These clones showed bytheCPinresponsetoIL-4invitro.ccl11 mRNA expression only a small overlapping with OVA-immunized spleens (Fig. S2). levels by young CP cells were significantly up-regulated in a Examining the most frequent clones in the naïve CP revealed that direct relationship to IL-4 concentrations (Fig. 3E). Moreover, these clones were found in similar frequencies in spleens of CNS- whereas treatment of young CP epithelial cells with IFN-γ alone immunized mice but not in naïve spleens (Fig. 2B). Importantly, had no effect on ccl11 expression, addition of IFN-γ together with in some cases, we observe different nucleotide sequences en- IL-4 reversed the effect of IL-4 on ccl11 production (Fig. 3F). coding the same amino acid CDR3 sequence (Fig. 2B), suggesting Intracellular CCL11 immunostaining confirmed that the CP selection based on specificity. epithelium produced CCL11 in response to IL-4, and that We also determined how many of the most highly expressed co-incubation with IFN-γ reversed this induction (Fig. 3G). These clones from each analyzed tissue were found in the CNS-specific results confirmed that although young CP epithelial cells hardly TCRβ library (Fig. 2C). The spleens of aged mice were found to produce CCL11, their exposure to IL-4 up-regulates CCL11 be enriched with CNS-specific clones compared with naïve production and IFN-γ can mitigate this effect. We therefore spleens of young mice (Fig. 2C), possibly reflecting the lifelong tested whether CCL11 production by aged CP is amenable to exposure to autoantigens believed to result in accumulation of down-regulation by IFN-γ. We cultured CP epithelial cells of autoreactive clones with age (21). T cells from CP of aged mice young and old mice and monitored their ccl11 expression in the

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1211270110 Baruch et al. IFN-γR in the knockout animals resulted in significantly higher A ccl11 expression by the CP (Fig. 3I), further demonstrating the role of IFN-γ in balancing IL-4 response in this compartment. Thus, it appears that IL-4–induced CCL11 expression could be balanced by IFN-γ in vivo, further suggesting that this regulation might be applicable to the aged CP in vivo. When applied on astrocytes (8) or microglia (13), IL-4 induces brain-derived neurotropic factor (BDNF), a key molecule in mediating CNS plasticity, learning, and memory. We examined whether IL-4 would have a similar effect on the CP. We found that bdnf was up-regulated in the CP cultures in response to IL-4, up to a certain concentration threshold, beyond which, upon further increase of IL-4 concentration, bdnf levels were reduced (Fig. 3J). Immunostaining the CP cultures showed the localization of BDNF to the epithelial cells (Fig. 3K). Thus, whereas high levels of IL-4 induced the CP to produce CCL11, low levels of IL-4 induced BDNF, suggesting an unexpected dose-dependent opposite role of CP-associated IL-4 in mediating CNS homeostasis. BC To further substantiate the dominance of IL-4 in the aged CP, we measured levels of arginase 1 (arg1), a known marker of epithelial Th2-mediated inflammation; in cases of asthma, chronic obstructive pulmonary disease, and cystic fibrosis, increased local arginase activity was shown to have detrimental effects on the airway epithelium (26). Moreover, chronic Th2-mediated inflammation of the airways induces remodeling of the epithelium and contributes to a progressive decline in lung function (27). We found arg1 mRNA and protein levels to be strongly up-regulated in the aged CP (Fig. 3 L and M) as well as in IL-4–treated CP cultures (Fig. 3N). Another effect of the exposure to IL-4 was

dysregulation of the epithelial tight junctions (Fig. 3N), as ob- IMMUNOLOGY served here by staining for ZO-1, and as previously reported in the case of human lung epithelial cells (28). Histological examination of the CP revealed signs of hypertrophism in the aged CP epithelium (Fig. S3), a phenomenon described in the murine lung epithelium in response to IL-4 (29). Finally, we examined arg1 mRNA expression in the CP of young IFN-γR-KO animals and D found it to be significantly up-regulated (Fig. S4). Together, these data indicate that the changes in the IL-4:IFN-γ ratio in the CP of aged mice critically affect gene expression and morphology of the BCSFB, and may potentially explain the age-related cognitive decline that was observed in correlation with elevated CCL11 levels in the blood and CSF (25). Because brain aging and impaired hippocampal plasticity have been associated with elevation of parenchymal proinflammatory cytokines (30, 31) such as IL-1β,IL-6,andTNF-α, we assumed that the CP of aged mice is exposed to such a proinflammatory milleu. Examining the aged CP for the presence of proinflammatory cytokines revealed the elevated protein levels of IL-1β and IL-6 (Fig. S5), suggesting that the cytokine milieu of the aged parenchyma is signaling to the CP, thereby possibly contributing to its dysfunction.

+ Fig. 2. The choroid plexus CD4 TCR repertoire is enriched with CNS-specific Lymphopenia-Induced Homeostasis-Driven Proliferation of T Cells β β clones. (A) Group hierarchical clustering based on the average V and J use Affects the CP and Hippocampus. The increased levels of IL-4 in β described in Fig. S1.(B) Examples of TCR clones found to be expanded in the the CP of aged mice without proper balance by IFN-γ could re- spleen of SCH-immunized mice and in the naïve CP (n = 3 mice per group). flect the well-characterized alternations in circulating immune Colors indicate different nucleotide sequences that encode the same amino cells during aging (22, 23). One way by which immunose- acid CDR3 sequence. (C) A library of CNS-specific clones was compiled from β nescence can be alleviated and memory T cells can be expanded is measured TCR amino acid sequences from all mice in the spleen SCH-im- the induction of lymphopenia, which is followed by homeostasis- munized group. The library contains clones that were significantly expanded compared with the group of naïve mouse spleen. The 50 most abundant driven proliferation (HDP) of the residual T-cell population. We clones from each tissue were examined for their presence in this library (n = therefore induced HDP by irradiation of the peripheral organs 3–12 per group; *P < 0.05; ***P < 0.001; one-way ANOVA, Newman–Keuls while shielding the head. Following lymphopenia, we restored the post hoc). (D) Quantitative comparison between the fractions of clones hematopoietic cell lineage with syngeneic (pseudoautologous) present in the CNS-specific library using different cutoffs for the most bone marrow (BM) derived from genetically identical age- abundant clones of each tissue (P < 0.05; two-way ANOVA). matched donors. We expected that memory T cells that are presentintheagedBMwouldprovide,inadditiontothere- sidual T cells of the recipients, a source of memory cells for their fi γ expansion under these lymphopenic conditions. We rst con- presence or absence of IFN- . In aged CP cultures the basal level firmed that aged BM contained memory T cells, and determined of ccl11 was higher than those in young CP (Fig. 3H), and the ad- whether their specificity resembles that of cells found in SCH- dition of IFN-γ decreased it to the basal level of young CP culture. immunized spleen, old spleen, and naïve CP. We found that BM To verify this IL-4–IFN-γ interplay at the CP in vivo, we also from aged mice contained significantly higher levels of memory + examined this compartment in IFN-γ receptor knockout ani- CD4 cells relative to young animals (Fig. S6). Testing the mals (IFN-γR-KO). We found that the complete absence of clonotypic repertoire revealed that T cells in the BM were

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Fig. 3. Th2-mediated inflammation of the CP epithelium during the aging process. (A)mRNAlevelsofil-4, ifn-γ, and their ratio in the CP of mice at the ages of 3, 6, 12, and 18 mo (n = 8–10 per group; one-way ANOVA, Newman–Keuls post hoc). (B) ccl11 mRNA levels in the CP of young and old mice (n = 5–6 per group; Student’s t test). (C) CCL11 protein levels (pg/mg tissue) in the CP of young (3-mo-old) and old (20- to 22-mo-old) mice (n = 16 per group; Student’s t test). (D) Representative micrographs showing immunohistochemical stainings for CCL11 in young and old mice. (Scale bars, 100 μm.) (E) ccl11 mRNA levels 24 h after the addition of IL-4 (untreated, and 0.12, 0.37, 1.1, 3.3, 10, and 30 ng/mL) of cultured CP epithelial cells (n = 3 per group; one-way ANOVA, Newman–Keuls post hoc; one representative experiment is shown out of three independently performed). (F) ccl11 mRNA levels of cultured CP epithelial cells from young (3-mo-old) mice, 24 h after the addition of the cytokines IL-4 (10 ng/mL), IFN-γ (100 ng/mL), or their combination (n = 3 per group; one-way ANOVA, Newman–Keuls post hoc; one representative experiment is shown out of three independently performed). (G) Representative micrographs of cultured CP epithelial cells from young mice exposed to IL-4 (10 ng/mL) or the combination of IL-4 (10 ng/mL) and IFN-γ (100 ng/mL). Cells were treated with Brefeldin A 2 h before fixation and stained for the epithelial marker cytokeratin (green), CCL11 (red), and Hoechst nuclear staining (blue). (Scale bars, 25 μm.) (H) ccl11 mRNA levels, measured by qPCR, of cultured CP epithelial cells from young and old mice, 24 h after the addition of the cytokines IL-4 (10 ng/mL) or IFN-γ (100 ng/mL) (n = 3 per group; one-way ANOVA, Newman–Keuls post hoc). (I) ccl11 mRNA levels, measured by qPCR, in the CP of young wild-type (3-mo-old) and age-matched IFN-γR-KO mice (n = 5–6 per group; Student’s t test). (J) bdnf mRNA levels 24 h after the addition of IL-4 (untreated, and 0.12, 0.37, 1.1, 3.3, 10, and 30 ng/mL), measured by qPCR, of cultured CP epithelial cells (n = 3 per group; one-way ANOVA, Newman–Keuls post hoc; one representative experiment is shown out of three independently performed). (K) Representative micrographs of cultured CP epithelial cells from young (3-mo-old) mice, exposed to IL-4 at the concentrations of 1 or 10 ng/mL or untreated. Cells were stained for ZO-1 (green) and for BDNF (red). (Scale bars, 25 μm.) (L) arg1 mRNA levels, measured by qPCR, in the CP of young (3-mo-old) and old (22-mo-old) mice (n = 5–6 per group; Student’s t test). (M)Repre- sentative micrographs showing staining for Claudin 1 (tight junctions; green), arginase 1 (red), and Hoechst nuclear staining (blue). (Scale bars, 25 μm.) (N)Rep- resentative micrographs of cultured CP epithelial cells from young (3-mo-old) mice exposed to IL-4 or left untreated. Cells were stained for ZO-1 (green), arginase 1 (red), and Hoechst nuclear staining (blue). (Scale bars, 25 μm.) Bar graphs throughout the figure show mean ± SE of each group (*P < 0.05; **P < 0.01; ***P < 0.001). significantly enriched for CNS specificity (Fig. 4A). To verifiy improvement in aged animals (32). Because our protocol involved that following BM transplantation, the T cells from the aged aged BM as our source of additional memory T cells beyond the donor BM repopulated the recipient lymphoid organs, donor BM irradiation remnants that undergo proliferation following irradi- cells were labeled with the intracellular fluorescent dye carboxy- ation-induced lymphopenia, we verified here the cognitive im- fluorescein succinimidyl ester (CFSE) before transplantation. provement previously reported is indeed attributable to the BM-derived T cells migrated to the spleen, where they pro- transferred T cells that reside in the donor BM. To this end, HDP liferated over time (Fig. S7); in young and aged recipients, these was induced in cognitively impaired aged mice that were either T cells could be detected in the spleen and cervical LNs, and transplanted with aged-matched BM or with the same BM de- showed dilution of the CFSE label (indicating proliferation). pleted of T cells. The mice were tested again, 8 wk later, in the In previous experiments, using a similar protocol of HDP and Morris water maze (MWM) for their hippocampus-dependent BM transplantation from aged donors, we demonstrated cognitive spatial learning/memory relative to young mice. Old mice

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Fig. 4. Syngeneic homeostasis-driven proliferation effects on the CP and hippocampus. (A) The 100 most abundant TCRβ clones from the spleen and bone marrow of young (3-mo-old) and old (22-mo-old) mice were examined for their presence in the CNS-specific clonal library (n = 8 per group; one-way ANOVA, Newman–Keuls post hoc). (B) Morris water maze test for young mice (3-mo-old), untreated old (22-mo-old), and aged littermates (old-HDP) 3 months following homeostasis-driven proliferation. Mice were divided into intact or impaired memory groups according to their MWM score, where an average latency time of above 60 s on the fourth day of acquisition was classified as “impaired memory” and a latency of less than 60 s was defined as “intact memory” (n = 10–14 per group; χ2 test). (C) il-4 and ifn-γ mRNA levels and ratio in the CP of young, old, and old-HDP mice (one-way ANOVA, Newman–Keuls post hoc). (D) Correlation analysis between the CP mRNA levels of il-4:ifn-γ ratio and ccl11 (n = 17; Pearson r2 = 0.2294). (E) bdnf, ho1,andpgc1α mRNA levels in the hippocampus of young, old, and old-HDP mice (one-way ANOVA, Newman–Keuls post hoc). Bar graphs throughout the figure show mean ± SE of each group (*P < 0.05; **P < 0.01; ***P < 0.001). receiving BM depleted of T cells failed to reestablish learning/ their CNS-specificity (2). Here, we found that the CP, under phys- memory skills (Fig. S8). iological conditions, retains a broad repertoire of CNS-specific + Based on the above results, we adopted here the same HDP CD4 T-cell clones. These T cells, upon recognition and reac- procedure using pseudoautologous aged BM to examine whether tivation by their cognate antigens, potentially presented to them by + such peripheral immunomodulation, which leads to cognitive im- the Flt3 dendritic cells found in the CP (39), either enter the CSF provement, would be associated with changes of the IL-4:IFN-γ or remain in the CP (40, 41). Although TCR-seq of the CP allowed ratio at the CP. Before excising the CP and the hippocampi of the us to identify CNS-specific clonotypic enrichment both in young and animals, we verified using MWM that the treatment was indeed aged mice, other parameters of age-related changes of the TCR fi bene cial for learning/memory 3 mo following transplantation. repertoire should not be ruled out. In addition, the appearance IMMUNOLOGY The proportion of aged animals with impaired spatial learning/ of such clones in other organs, such as the lung (42), should be memory was 74%, as commonly found (33); among the aged further investigated. animals subjected to HDP, the proportion of cognitively impaired One of the most prominent features of aging is a shift toward animals was reduced to 45% (Fig. 4B). Testing the CP of the a dominance of the helper Th2-related response. Accordingly, same mice for IL-4 and IFN-γ expression showed that in the old murine models of aging were shown to manifest age-associated animals following HDP, the IL-4:IFN-γ ratio was similar to that dysregulation in cytokine production, specifically involving found in young animals (Fig. 4C). The causal connection between reduced production of IFN-γ, and increased production of IL-4 the IL-4:IFN-γ ratio and CCL11 levels, and thus the impact on (23, 33). Examining the local effector cytokine balance in the cognitive ability, was further demonstrated by the positive cor- CP revealed a gradual increase in IL-4:IFN-γ ratio during ag- relation found between the IL-4:IFN-γ ratio and CCL11 levels in ing, which may reflect the general condition of the aged im- the CP of old animals (treated and untreated) (Fig. 4D). mune system, further supporting the contention that the Because spatial learning/memory is hippocampus-dependent, immune response within the CP is an integral part of systemic we also tested whether the HDP-induced changes in the CP cy- immunity. Notably, the ongoing T-cell dialogue in this com- + tokine balance were also reflected in the expression of plasticity- partment was also manifested by the CD4 clonotypic enrich- related molecules in the hippocampi of these mice. We found that ment of CNS-specific TCR in the CP of mice immunized with bdnf levels, repeatedly linked with age-associated loss of cognitive CNS antigens, but not of mice immunized with ovalbumin. function (34), were down-regulated with age and restored fol- The age-related change in CP cytokine milieu was found here to lowing HDP (Fig. 4E). In addition, we measured the expression of critically affect this compartment. When not properly balanced by heme oxygenase 1 (ho1), previously linked to aging of the hippo- IFN-γ, high levels of IL-4 induced the CP epithelium to produce campus (35), and found it to be up-regulated in the aged group CCL11, a chemokine associated with age-related cognitive im- compared with the young animals. HDP reduced ho1 in aged pairments that accumulates at the aged CSF (25) and can directly animals to its expression levels in young animals (Fig. 4E). We also interfere with IL-4 signaling (43). Because cognitive tasks lead to examined whether rejuvenation of the CP cytokine balance was accumulation of IL-4–producing T cells in the meningeal spaces of associated with elevation of PPARγ coactivator 1α (pgc1α), a key the brain (8) and IL-4 has a beneficial role in learning and memory, regulator in coping with reactive oxygen species and a hippo- our findings highlight a dual role of IL-4 in the CNS, that may campal neuroprotective mediator (36). Old animals after HDP depend on its spatial localization and dosing. Accordingly, epithe- had significantly higher levels of hippocampal pgc1α (Fig. 4E), lial IL-4–induced CCL11 might impair cognition by its potential possibly correlating with their improved cognitive ability. Taken ability to suppress IL-4 originated from the meninges, as recently together, this peripheral immunomodulation, which improved suggested (44). Importantly, however, it is very likely that not only cognitive ability and affected cytokine balance at the CP, also meningeal IL-4 supports brain plasticity, but also basal/low levels correlated with molecular changes at the hippocampus. of IL-4 at the CP are part of a lifelong role of this compartment in keeping CNS homeostasis, as was manifested here by BDNF ex- Discussion pression by CP epithelial cells. If this is the case, the age-related Here we show that the choroid plexus epithelium, an active interface overwhelming levels of IL-4 at the CP have a negative outcome for between blood circulation and the brain, is populated by CNS-spe- the functioning brain, not only because of induction of CCL11 but + cific effector memory-type CD4 T cells. We further demonstrate also because of dysregulation of BDNF and possibly other CP- that with aging, the overall specificity of CP resident T cells toward derived neurotrophic factors. Notably, the CP produces BDNF CNS antigens was maintained, although the cytokine–chemokine following CNS trauma (45) and chronic stress (46). balance changed toward a Th2-like epithelial inflammation, with The fact that CCL11 was found here to be produced by the CP detrimental consequences to brain function. epithelium, and specifically in response to high levels of IL-4, CNS specificity plays a part in the beneficial effector function of coupled with the negative effects of CCL11 on brain functional T cells in CNS plasticity and repair (2–4, 6, 37, 38). Experimentally, plasticity, supports our hypothesis that the loss of cognitive T cells involvement in brain plasticity was found to be dependent on ability in old mice is a reflection of dysregulation of epithelium-T

Baruch et al. PNAS Early Edition | 5of6 cell cross-talk at the CP. Such Th2-like inflammation is known in the BCSFB, may lead to hitherto unexplored approaches to reverse epithelial pathologies outside the CNS, such as the lung epi- or arrest dementia, other age-related cognitive deficits, or neu- thelium in asthma (47). Intriguingly, examining the aged CP rodegenerative diseases. epithelium in analogy to the asthmatic Th2-inflamed lung epithelium revealed similarities in mechanistic and functional dysreg- Materials and Methods fl ulation that may re ect common disease-like processes amenable Young and aged C57BL/6 and IFN-γR1-KO mice were handled according to to immunomodulation. regulations formulated by The Weizmann Institute’s Animal Care and Use Previous studies carried out by our group have demonstrated Committee and maintained in a pathogen-free environment. A detailed de- that T cell-mediated immunity supports hippocampus-dependent fl cognitive ability, and that age-related memory loss may be re- scription of all experimental procedures, including ow cytometry, active versed, at least to some extent, by peripheral immunomodulation immunizations, TCR-Seq, primary culture of choroid plexus, immunohisto- in the form of lethal irradiation followed by syngeneic bone mar- chemistry, immunocytochemistry, multiplex cytokine analysis, RNA and cDNA row transplantation (2, 34). Using the same paradigm here, we preparation for quantitative PCR, HDP procedure, cognitive tests, and statistical demonstrate that this peripheral immunomodulation could locally analysis can be found in SI Materials and Methods. influence the CP, alleviating age-related overwhelming IL-4 levels, and suggest a mechanism as to how these changes affect ACKNOWLEDGMENTS. We thank Dr. Gilad Kunis for technical assistance, cognitive ability. Dr. Shelley Schwarzbaum for editing the manuscript, Dr. Hillary Voet for In summary, the present study identifies the choroid plexus and statistical consultation, and Margalit Azoulay for animal handling. This fi research was supported by a European Research Council Grant Award, its CNS-speci c immunity as unique players that functionally link a Seventh Framework Programme HEALTH-2011 Grant (to M.S.), and by the brain aging and immunosenescence. We further show that modu- Intramural Research Program of the National Institute on Aging (to M.P.M.). lating the cytokine imbalance in this compartment has the potential N.F. is the incumbent Pauline Recanati Career Development Chair of to partially restore memory function. It is therefore possible that Immunology. M.S. holds The Maurice and Ilse Katz Professorial Chair targeting treatment for brain aging, based on immunomodulating in Neuroimmunology.

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6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1211270110 Baruch et al. Supporting Information

Baruch et al. 10.1073/pnas.1211270110 SI Materials and Methods using the QIAquick PCR Purification Kit (Qiagen), followed by Animals. Inbred male 3-mo-old C57BL/6 mice were supplied by enzymatic digestion, in accordance with the ACUI protocol the Animal Breeding Center of The Weizmann Institute of (New England BioLabs). The ACUI enzyme was used to cleave Science. Inbred male 17- to 24-mo-old C57BL/6 mice were the 14 bp downstream of its binding site, enabling positioning of supplied by the National Institute on Aging (NIA). IFN-γ re- the Illumina sequencing primer in close proximity to the junction ceptor knockout animals (IFN-γR1-KO) mice on the B6 back- region and assuring sequencing of the entire variable CDR3 ground were purchased from The Jackson Laboratory. Aged region. Digestion produced a 2-bp overhang for the ligation of mice were allowed a 1-mo adaptation period following shipment the Illumina 5′ adapter, which was linked to a 3-bp barcode se- from the NIA to our laboratory. The cages were placed in a light- quence at its 3′ end. Overnight ligation was performed using T4 and temperature-controlled room, and all behavioral tests were ligase (Fermentas) at 16 °C in accordance with the manufacturer’s conducted during the dark hours. All animals were handled ac- protocol. A second round of PCR amplification was performed cording to regulations formulated by The Weizmann Institute’s (24 cycles), using primers for the 5′ and 3′ Illumina adapters. Final Animal Care and Use Committee and maintained in a pathogen- PCR products were run on an agarose gel (2%) and purified using free environment. the Wizard SV Gel and PCR Clean-Up System (Promega). Final library concentrations were measured using a NanoDrop spec- Flow Cytometric Cell-Sample Preparation and Analysis. Before tissue trophotometer. The libraries were sequenced using an Illumina collection, blood was collected into heparin-containing tubes, and Genome Analyzer II. mice were intracardially perfused with PBS. Spleens were mashed with the plunger of a syringe and treated with ammonium-chloride- Computational Analysis of TCRβ Sequencing Data. For preprocessing potassium (ACK) lysing buffer to remove erythrocytes. Choroid of the data, we used the Smith–Waterman alignment algorithm (1) plexus (CP) tissues were isolated from the lateral, third, and fourth to assign to each sequencing read its variable (Vβ) and joining (Jβ) ventricles of the brain, incubated at 37 °C for 45 min in PBS (with gene, using germ-line Vβ/Jβ gene segment sequences downloaded 2+ 2+ Ca /Mg ) containing 400 U/mL collagenase type IV (Wor- from the IMGT database (2). Reads that were not assigned either thington Biochemical), and then manually homogenized by pipe- aVβ or Jβ and other erroneous reads were eliminated. We then tation. Lymph nodes were mashed with the plunger of a syringe. clustered the library-derived reads using a version of the quality ’ All samples were stained according to the antibody s protocols. threshold clustering algorithm (3) to correct for nucleotide copy- – Antibodies used included Alexa-700 conjugated anti-CD45.2, ing errors (up to two errors for each read). The clustering pro- – β PercpCy5.5-conjugated anti T-cell receptor (TCR) , PE-conju- cedure identified unique CDR3β clonotypes, defined as the most gated anti-CD4, FITC-conjugated anti-CD44, and antigen-pre- prevalent read found in each cluster. The clonotype sequences – senting cell conjugated anti-CD62L (BD Pharmingen and were then translated, and those clonotypes that lacked a stop co- eBioscience). Cells were analyzed on an LSRII cytometer (BD don in-frame with the V/D/J sequences were considered for fur- Biosciences) using FACSDiva (BD Biosciences) and FlowJo (Tree ther analysis. This analysis computed statistics of V/D/J use, Star) software. In each experiment, relevant negative-control statistical properties of the number of deletions and insertions of groups and single-stained samples for each tissue were used to nucleotides at both VD and DJ junctions, as well as distributions of identify the populations of interest and to exclude others. CDR3 lengths. The analysis was done using the R statistical software package (R Development Core Team; www.r-project.org). Active Immunization and Cell Isolation. Mice were immunized s.c. at β β their flanks with either spinal cord homogenate (SCH) or oval- Frequencies of V and J segment use were measured for all fi samples in each treatment group. Correlation coefficients were bumin (OVA), each emulsi ed in an equal volume of CFA con- β β taining 2 mg/mL Mycobacterium tuberculosis. SCH was prepared calculated, based on the sample mean of combined V and J use by manually homogenizing the spinal cord of young C57BL/6 in each group, using MATLAB (Mathworks). Hierarchical clus- β β mice in PBS. Mice were intracardially perfused with PBS 7 d tering was performed, based on combined V and J use, in all after immunization, and their spleens, bone marrow, and CPs were groups (clustergram; MATLAB). extracted and processed to single-cell suspensions as described + + above. CD4 cells were isolated by magnetic depletion of CD4 Primary Culture of Choroid Plexus Cells. Mice were perfused from the left ventricle of the heart with PBS, and their CPs were re- T cells on a MACS column (Miltenyi Biotec) according to the manufacturer’s protocol. moved under a dissecting microscope (Stemi DV4; Zeiss) in PBS into tubes containing 0.25% trypsin and kept on ice. The tubes cDNA Library Preparation for TCRβ Sequencing. Total RNA was were shaken for 20 min at 37 °C and the tissue was dissociated by + extracted from CD4 T cells derived from naïve spleen and CP pipetting. The cell suspension was washed in culture medium for tissues of C57BL/6 mice as well as from spleens of OVA-im- epithelial cells (DMEM/HAM’s F12; Invitrogen) supplemented munized and SCH-immunized mice. RNA was extracted using with 10% (vol/vol) FCS (Sigma-Aldrich), 1 mM l-glutamine, 1 mM an RNeasy Mini Kit (Qiagen) and reverse-transcribed with Su- sodium pyruvate, 100 U/mL penicillin, 100 mg/mL streptomycin, perScript II reverse transcriptase (Invitrogen) using a primer 5 μg/mL insulin, 20 μM Ara-C, 5 ng/mL sodium selenite, and specific for the TCRβ constant region linked to the Solexa 3′ 10 ng/mL EGF, and cultured (∼250,000 cells per well) at 37 °C, adapter (Cβ-3′adp). cDNA was then used as a template for high- 5% CO2 in 24-well plates (Corning) coated with poly-L-lysine fidelity PCR amplification (Phusion; Finnzymes) using a pool of (PLL; Sigma-Aldrich). After 24 h, the medium was changed and 23 Vβ-specific primers, divided into five primer groups, to min- the cells were either left untreated or treated with either 100 ng/mL imize potential for cross-hybridization. Each Vβ primer was IFN-γ (Peprotec), serial dilution or 10 ng/mL IL-4 (Peprotec), or linked to a restriction-site sequence for the ACUI restriction a combination of 100 ng/mL IFN-γ and 10 ng/mL IL-4 for 24 h. enzyme (New England BioLabs). PCR reactions were performed RNA isolation was done with an RNA MicroPrep Kit (Zymo in duplicate, and PCR products were then pooled and cleaned Research) according to the manufacturer’s protocol.

Baruch et al. www.pnas.org/cgi/content/short/1211270110 1of7 Immunohistochemistry and Immunocytochemistry. For whole-mount in triplicate for each sample using the standard curve method. staining of the choroid plexus, isolated tissues were fixed with 2.5% Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pepti- paraformaldehyde for several hours and subsequently transferred dylprolyl isomerase A (PPIA), and hypoxanthine guanine phos- to PBS containing 0.05% sodium azide. Before staining, the dis- phoribosyltransferase (HPRT) were chosen as reference genes sected tissues were washed and blocked (20% horse serum, 0.3% according to their stability in the target tissue. The amplification Triton X-100, and PBS) for 1 h at room temperature with shaking. cycles were 95 °C for 5 s, 60 °C for 20 s, and 72 °C for 15 s. At the Whole-mount staining with primary (in PBS containing 2% horse end of the assay, a melting curve was constructed to evaluate the serum and 0.3% Triton X-100) and secondary antibodies (in PBS) specificity of the reaction. For some genes, the cDNA was pre- was performed for 1 h at room temperature with shaking. Each step amplified in 14 PCR cycles with nonrandom PCR primers, thus was followed by three washes in PBS. The tissues were mounted increasing the sensitivity of the subsequent real-time PCR analysis onto slides, using Immu-Mount (9990402; Thermo Scientific), and (PreAmp Master Mix Kit; Applied Biosystems). For these genes, sealed with coverslips. For staining of sectioned brains, two dif- expression was determined using TaqMan Real-Time PCR, ac- ferent tissue preparation protocols (paraffin-embedded and cording to the manufacturer’s instructions (Applied Biosystems). microtomed frozen sections) were applied, as previously described All quantitative real-time PCR reactions were performed and (4). For immunocytochemistry, CP cells were isolated as described analyzed using a 7500 Real-Time PCR System (Applied Biosystems). above and grown on PLL-coated coverslips for 7 d, replacing the The following TaqMan probes were used: Mm02342430_g1 (ppia), medium every 3 d. Cytokines were added for the last 24 h of culture. Mm00446968_m1 (hprt), Mm00445260_m1 (il-4), Mm01168134_m1 Following 7 d of culture, the wells were washed with PBS and the (ifn-γ), Mm00441238_m1 (ccl11), and Mm00475988_m1 (arg-1). In cells were fixed either with 2.5% paraformaldehyde for 20 min or addition, the following primers were used: gapdh forward 5′-A- methanol:acetone (1:1) for 10 min at −20 °C, following by two ATGTGTCCGTCGTGGATCTGA-3′ and reverse 5′-GATGCCT- washing steps with PBS. For CCL11 staining, cells were treated with GCTTCACCACCTTCT-3′; ppia forward 5′-AGCATACAGGTC- Brefeldin A 2 h before fixation to arrest protein secretion. The CTGGCATCTTGT-3′ and reverse 5′-CAAAGACCACATGCT- coverslips of the cultured CP cells were incubated with the indicated TGCCATCCA-3′; ccl11 forward 5′-CATGACCAGTAAGAAG- antibodies and mounted with GVA mounting solution (Invitrogen). ATCCC-3′ and reverse 5′-CTTGAAGACTATGGCTTTCAGG-3′; The following primary antibodies were used: rat anti-CD3 (Abcam); arg-1 forward 5′-AAGACAGGGCTCCTTTCAG-3′ and reverse 5′- mouse anti-MHCII (Abcam); mouse anti-arginase 1 (BD Bio- TGTTCACAGTACTCTTCACCT-3′; bdnf1 forward 5′-CCTGC- sciences); mouse anti-cytokeratin (Covance); rat anti-CCL11 ATCTGTTGGGGAGAC-3′ and reverse 5′-GCCTTGTCCGTG- (R&D Systems); rabbit anti-BDNF (Alomone Labs); rabbit or GACGTTTA-3′; ho1 forward 5′-AGATGACACCTGAGGTCAA- mouse anti–ZO-1 (Invitrogen); and rabbit anti-Claudin 1 (In- GCACA-3′ and reverse 5′-GCAGCTCCTCAAACAGCTCAATG- vitrogen). Secondary antibodies included Cy2/Cy3-conjugated T-3′;andpgc1α forward 5′-GCCAAACCAACAACTTTATCTC-3′ donkey anti-rat, -rabbit, or -mouse antibody and Cy3-conjugated and reverse 5′-GTTCGCTCAATAGTCTTGTTCTC-3′. donkey anti-goat (1:200; all from Jackson ImmunoResearch). The slides were exposed to Hoechst stain (1:2,000; Invitrogen) for 1 min. Irradiation and Bone Marrow Transplantation. Aged C57BL/6 wild- Two negative controls were routinely used in the immunostaining type mice were subjected to total-body γ-irradiation from a cobalt procedures: staining with an isotype control antibody followed by source (a single dose of 950 rad), while their heads were pro- a secondary antibody, and staining with a secondary antibody alone. tected to avoid radiation-induced brain damage. Transplanted For morphological quantification, the average cross-sectional area bone marrow (BM) was composed of either whole BM or BM of epithelial cells was measured following Nissl staining. Cell bor- depleted of T cells, which was prepared by magnetic depletion of + ders (20–25 cells per picture; 20 pictures per group) were marked, CD3 cells on a MACS column (Miltenyi Biotec) according to and the area was quantified using Image-Pro Plus software (Media the manufacturer’s protocol. BM transplantation was performed Cybernetics, Inc.) in a blinded fashion. on the following day by i.v. injection of 5 × 106 bone marrow cells suspended in PBS (total volume 0.15 mL). For detection of BM Multiplex Cytokine Analysis System. CPs were isolated from lateral, cells following transplantation, cells were prelabeled with car- third, and fourth ventricles and pooled in groups of four, due to boxyfluorescein succinimidyl ester (CFSE; Molecular Probes) the limited amount of protein extracted from a single CP. The before transplantation. For labeling, 5 × 106 BM cells (20 × 106 + + excised tissues were homogenized in PBS containing protease cells/mL) were incubated in PBS (without Ca2 /Mg2 ) supple- inhibitors (1:100; P8340; Sigma). Four freeze–thaw cycles (3 min mented with 5 μM CFSE (Molecular Probes) for 8 min at 25 °C. each) were performed to break the cell membranes. Homoge- Following incubation, the cells were washed with RPMI con- nates were then centrifuged for 10 min at 500 × g, and the total taining 8% FBS. protein quantities in supernatants were determined by Bradford reagent. Frozen supernatants were assayed in duplicate using Morris Water Maze. Acquisition and probe trials were performed as a multiplex bead-based Luminex assay (MILLIPLEX Mouse previously described (5). Following the probe trial, mice were Cytokine/Chemokine Panel; Millipore), performed by out- given three additional trials without the platform to extinguish sourcing (American Medical Laboratories) according to the their initial memory of the platform’s position. In the reversal manufacturer’s instructions. Results are expressed as picograms phase, the platform was placed in a new location in the pool of protein per milligram of total tissue protein. (opposite to where it was located in the acquisition phase) and the mice were given three trials/d on 2 consecutive days, con- RNA Purification, cDNA Synthesis, and Real-Time Quantitative PCR. ducted in a similar manner to the initial acquisition. Position and Total RNA of the hippocampus was extracted with TRI Re- movement of the mice were recorded using an EthoVision au- agent (Molecular Research Center) and purified from the lysates tomated tracking system (Noldus). using an RNeasy Kit (Qiagen). Total RNA of the choroid plexus was extracted using an RNA MicroPrep Kit (Zymo Research). Statistical Analysis. The specific tests used to analyze each set of mRNA (1 μg) was converted to cDNA using a High Capacity experiments are indicated in the figure legends. Data are expressed cDNA Reverse Transcription Kit (Applied Biosystems). The ex- as mean ± SEM. In the graphs, y axis error bars represent the pression of specific mRNAs was assayed using fluorescence-based SEM. Statistical analysis was performed using Prism 5.0 software real-time quantitative PCR (qPCR). Quantitative PCR reactions (GraphPad Software). Means between two groups were compared were performed using Power SYBR Green PCR Master Mix by two-tailed, unpaired Student’s t test. One-way ANOVA was (Applied Biosystems). Quantification reactions were performed used to compare several groups, and the Newman–Keuls test for

Baruch et al. www.pnas.org/cgi/content/short/1211270110 2of7 pairwise comparisons was used for follow-up post hoc compari- age and number of days as between-subject factors. All histology son of groups. Data from behavioral tests were analyzed using and behavioral experiments were conducted in a randomized two-way repeated-measures ANOVA with group treatment or and blinded fashion.

1. Smith TF, Waterman MS (1981) Identiﬁcation of common molecular subsequences. J 4. Ziv Y, Avidan H, Pluchino S, Martino G, Schwartz M (2006) Synergy between immune Mol Biol 147(1):195–197. cells and adult neural stem/progenitor cells promotes functional recovery from spinal 2. Lefranc MP, et al. (2009) IMGT, the international ImMunoGeneTics information system. cord injury. Proc Natl Acad Sci USA 103(35):13174–13179. Nucleic Acids Res 37(Database issue):D1006–D1012. 5. Ron-Harel N, et al. (2008) Age-dependent spatial memory loss can be partially restored 3. Heyer LJ, Kruglyak S, Yooseph S (1999) Exploring expression data: Identiﬁcation and by immune activation. Rejuvenation Res 11(5):903–913. analysis of coexpressed genes. Genome Res 9(11):1106–1115.

OVA- SCH- Naive Vββ distributions Naive A B immunized immunized choroid spleen spleen plexus 30 spleen 1 * OVA-immunized spleen zed A- *** Naive spleen ni 1.0000 0.8422 0.5938 0.5011 OV spleen 0.9

SCH-immunized spleen mmu i 20 Naive choroid plexus ***

** n e 0.8422 1.0000 0.5861 0.4224 0.8 ple Naive s

* 10 * *** 0.7

* n *** H- 0.5938 0.5861 1.0000 0.9332 C * * unized

*** S m splee 0.6 im Distribution frequency (%) *** 0 roid s o

7 2 9 0 1 2 3 4 5 6 8 9 xu .1 . .3 1 1 1 1 20 0.5011 0.4224 0.9332 1.0000 0.5 V1 V2 V4 V6 V 8 8 V e V3.1 /5.2 V V V8 V V1 V1 V1 V V1 V1 V V V 1 pl ive ch

V5. a N C Jβ distributions D 1 30 A-

OVA-immunized spleen V 1.0000 0.9028 0.8341 0.7557 O ) * spleen **

Naive spleen immunized 0.95 SCH-immunized spleen 20 Naive choroid plexus 0.9028 1.0000 0.9308 0.7978

Naive 0.90 spleen **

10 0.8341 0.9308 1.0000 0.9537 0.85 CH- unized ibution frequency (% S m

* spleen r * im * Dist 0.80 us

0 x 0.7557 0.7978 0.9537 1.0000

.1 .2 3 4 6 1 2 .3 .4 .5 .7 ple 1 1.5 1.7 2. 2. 2 2 J1 J J1. J1. J J1. J J J J J2 J J2 Naive choroid

Fig. S1. The choroid plexus CD4+ TCR repertoire is enriched with CNS-specific clones. (A and B)Vβ use (A) and correlation coefficients (B) for spleens of OVA- immunized mice, SCH-immunized mice, naïve spleen, and naïve choroid plexus (n = 3 per group; *P < 0.05; **P < 0.01; ***P < 0.001; one-way ANOVA, Newman–Keuls post hoc). (C and D)Jβ use (C) and correlation coefficients (D) for the same groups as in A and B.

Baruch et al. www.pnas.org/cgi/content/short/1211270110 3of7 Fig. S2. Comparison of the clonal expansion induced by OVA and SCH immunization. A Venn diagram showing the intersection between libraries of clones that were signiﬁcantly up-regulated in OVA- or SCH-immunized spleens (n = 3 per group) compared with spleens of naïve nonimmunized mice (n = 3). A small overlap is observed between these two libraries.

A B

1500 ) 2 area m

( *** onal Young i

s 1000 l ct l ial ce l e 500 th ecross-se g ra of epi ve Old A 0

ld O Young

Fig. S3. Th2-mediated inﬂammation of the CP epithelium during the aging process. (A) Representative micrographs showing morphological Nissl staining in young (3-mo-old) and old (20-mo-old) mouse CP. (Scale bars, 50 μm.) (B) Nissl staining was used to measure the average cross-sectional area of epithelial cells in μm2 (***P < 0.001; Student’s t test) and to assess epithelial hypertrophy.

Baruch et al. www.pnas.org/cgi/content/short/1211270110 4of7 2.0 *

1.5

1.0

mRNA fold change 0.5 rg1 a 0.0

O R-K - Young WT ng IFN u o Y

Fig. S4. Changes in arg1 mRNA levels, measured by real-time qPCR, in the CP of young wild-type (3-mo-old) and young IFN-γR-KO mice (3-mo-old) (n = 5–6 per group; *P < 0.05; Student’s t test).

50 sue)

s * 40

(pg/mg ti Young Old 20 vels e 10 *

Protein l 0 6 1 - IL IL- TNF-

Fig. S5. IL-1β, IL-6, and TNF-α protein levels (pg/mg tissue) in the CP of young (3-mo-old) and old (20- to 22-mo-old) wild-type mice (n = 16 per group; *P < 0.05; Student’s t test).

Baruch et al. www.pnas.org/cgi/content/short/1211270110 5of7 1,000,000 Young *** Old 800,000

600,000 cells

+ 400,000 4 D 200,000 C

g + n ld u O CD4 o TCR Y CD45 200,000 ls

l *

ce 150,000 +

D4 100,000 C y 50,000

+ mor CD4 / 0 high Me ld CD44 ung O Yo

+ + Fig. S6. Representative FACS histograms and quantitative analysis for CD4 T cells and CD4 /CD44high memory T cells in the bone marrow of young (3-mo-old) + and old (22-mo-old) mice. Cells were pregated by CD45 and TCRβ (n = 4–5 per group; *P < 0.05; ***P < 0.001; Student’s t test).

Young BM Young recipient Young recipient donor (d0) spleen (d3) spleen (d5) Young

Old BM Old recipient Old recipient donor (d0) spleen (d5) lymph nodes (d5) ld O SE CF TCRβ

+ Fig. S7. Representative ﬂow cytometry plots demonstrating resting (CFSEhigh) and dividing (CFSElow) T cells (TCRβ ) in donor BM on the day of transplantation (d0), in the spleen of young recipients on days 3 and 5 posttransplantation (Upper), and in the spleen and cervical lymph nodes of aged recipients on day 5 posttransplantation (Lower).

Baruch et al. www.pnas.org/cgi/content/short/1211270110 6of7 Fig. S8. Cognitive ability induced by homeostatis-driven proliferation of pseudoautologous BM is T cell-dependent. Aged mice with memory impairments were transplanted with either complete BM, derived from donor mice of identical age to the recipients, or with T cell-depleted BM from the same donors. The mice were tested in a Morris water maze 8 wk following transplantation, together with nontreated young mice of the same genetic background. In both the acquisition and the reversal phases of the test, the performance of aged mice transplanted with aged BM depleted of T cells (aged-BMT-T) was signiﬁcantly impaired compared with young mice, whereas the performance of aged mice transplanted with whole BM (aged-BMT) was similar to that of the young mice [acquisition: repeated-measures ANOVA: groups: P = 0.005, days: P = 0.0005, groups x days: P = 0.02; reversal: analysis of covariance, comparing groups re- garding differences between the performance of each mouse in the 2 d of the test (day 2 − day 1), with the day 1 measure as the covariant: P = 0.0002, *P < 0.05 (young vs. aged-BMT-T)]. Reversal: performance in the reversal phase according to the swimming path length to the platform [P < 0.0001, #P < 0.05 (aged- BMT-T vs. aged-BMT), *P < 0.05 (aged-BMT-T vs. young)].

Baruch et al. www.pnas.org/cgi/content/short/1211270110 7of7