Aged murine hematopoietic stem cells drive aging

Regular Article

HEMATOPOIESIS AND STEM CELLS

Aged murine hematopoietic stem cells driveaging-associated immune remodelingHanna Leins,1,2 Medhanie Mulaw,3 Karina Eiwen,2 Vadim Sakk,2 Ying Liang,4 Michael Denkinger,5,6 Hartmut Geiger,2,7,*

and Reinhold Schirmbeck1,*

1Department of Internal Medicine I, University Hospital of Ulm, Ulm, Germany; 2Institute of Molecular Medicine, Stem Cell and Aging, Ulm University, Ulm,Germany; 3Institute of Experimental Cancer Research, University Hospital of Ulm, Ulm, Germany; 4Department of Toxicology and Cancer Biology, University ofKentucky, Lexington, KY; 5AGAPLESION Bethesda Hospital, Geriatric Research Unit and 6Geriatric Center Ulm/Alb-Donau, Ulm University, Ulm, Germany; and7Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

KEY PO INT S

l Phenotypic andfunctional changes inT and B cells of oldmice are primarilydriven by aging ofHSCs.

l CASIN-treated agedHSCs reconstitute animmune system with afunction similar to thatin young animals.

Aging-associated remodeling of the immune system impairs its functional integrity andcontributes to increased morbidity and mortality in the elderly. Aging of hematopoieticstem cells (HSCs), from which all cells of the adaptive immune system ultimately originate,might play a crucial role in the remodeling of the aged immune system. We recently re-ported that aging of HSCs is, in part, driven by elevated activity of the small RhoGTPaseCdc42 and that aged HSCs can be rejuvenated in vitro by inhibition of the elevated Cdc42activity in aged HSCs with the pharmacological compound CASIN. To study the quality ofimmune systems stemming selectively from young or aged HSCs, we established a HSCtransplantation model in T- and B-cell-deficient young RAG12/2 hosts. We report that bothphenotypic and functional changes in the immune system on aging are primarily a con-sequence of changes in the function of HSCs on aging and, to a large extent, independentof the thymus, as young and aged HSCs reconstituted distinct T- and B-cell subsets in

RAG12/2 hosts that mirrored young and aged immune systems. Importantly, aged HSCs treated with CASIN rees-tablished an immune system similar to that of young animals, and thus capable of mounting a strong immune responseto vaccination. Our studies further imply that epigenetic signatures already imprinted in aged HSCs determine thetranscriptional profile and function of HSC-derived T and B cells. (Blood. 2018;132(6):565-576)

IntroductionAging is associated with a variety of changes in the immunesystem, summarized as aging-associated immune remodelingand dysfunction (AAIR). Although the immune system in theelderly seems to be at least partially competent to respond tonewly encountered antigens, age-related alterations impair itsfunctional integrity, resulting in an increased susceptibility toinfections and decreased responsiveness to vaccines.1,2 AAIR isthus thought to be a major cause of the increased morbidity andmortality in the elderly.3

Almost all constituents of the human andmurine immune systemare affected in the elderly. T cells present with prominentchanges, primarily including a decrease in the number of naiveT cells that are competent to respond to new antigens.4 ForT cells, the involution of the thymus has been seen as a criticalfactor contributing to reduced lymphopoiesis on aging, as theinvolution precedes T-cell-related immune incompetence.5

However, novel emerging data suggest that the reduced pro-duction of lymphocytes in both aged mice and humans mightalso depend on cell intrinsic changes in hematopoietic stem cells(HSCs) from which lymphocytes are ultimately derived.6-8 Aged

HSCs show an altered differentiation capacity: they are skewedtoward the myeloid lineage, resulting in an overall reducedproduction of lymphoid precursors.9 Although the role of thymicinvolution for AAIR has been described in detail,5,10,11 there isonly limited information available on the extent to which agedHSCs contribute to AAIR.

We recently demonstrated that an increase in the activity of thesmall RhoGTPase cell division control protein 42 (Cdc42) in agedHSCs is causative for their aging.12,13 Cdc42 cycles betweenan inactive, GDP-bound and an active, GTP-bound form. A shiftfrom canonical to noncanonical Wnt signaling resulting fromelevated expression of Wnt5a in old HSCs results in elevatedCdc42 activity in these HSCs.13 The elevated activity of Cdc42 iscausative for a loss in HSCs polarity for several proteins (eg,tubulin and Cdc42 itself). Pharmacological inhibition of the el-evated Cdc42 activity in aged HSCs to the level found in youngones with the Cdc42 activity-specific inhibitor CASIN revertsthese age-associated phenotypes, and in serial transplantationexperiments, CASIN-treated aged HSCs function similar toyoung HSCs.12 CASIN reduces Cdc42 activity by interfering withthe GTP loading exchange reaction in a dose-dependent andreversible manner.14 We thus hypothesized that CASIN-treated

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Figure 1. Transplantation of DY andDOHSCs in RAG12/2 recipients. (A) Schematic representation of the experimental setup: 600 HSCs were isolated from young (Y) and old(O) B6.SJL (CD45.1) mice and subsequently transplanted into irradiated young RAG12/2 (CD45.2) recipients. (B) Contribution of DY and DO HSCs (CD45.1) to white blood cellswas analyzed in peripheral blood every 4 weeks up to 12 weeks (DY, DO, n5 15). (C) 12 weeks posttransplantation, recipient mice were scarified and donor contribution to totalwhite blood cells in spleen was analyzed (DY, n5 16; DO, n5 19). (D) Absolute number of donor-derived splenic CD191 B cells, CD31CD41 and CD31CD81 T cells in recipientanimals and nontransplanted young and aged B6.SJL mice (Y, n5 8; O, n5 7; DY, n5 16; DO, n5 19). (E) Representative gross anatomy of thymi taken from a nontransplantedRAG12/2 mouse and RAG12/2 recipients transplanted with DY or DO HSCs. (F) Weight of thymus glands isolated from nontransplanted young and aged B6.SJL mice, RAG12/2

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aged HSCs might reconstitute an immune system that is similarto that found in young mice.

In this study, we demonstrate that HSCs from young or agedmice regenerate distinct adaptive immune systems on trans-plantation into RAG12/2 mice that resemble the altered T- andB-cell systems of young and aged mice. CASIN-treated agedHSCs reconstitute a functional immune system in RAG12/2 hoststhat was similar to the one found in recipients of young HSCs,as indicated by reestablishing a CD81 T-cell response to DNAvaccination, as well as gene expression profiles in HSC-derivedT and B cells.

Materials and methodsMiceFemale young (2-3months) B6.129S7-Rag1tm1Mom/J (RAG12/2), young(2-3 months) and aged (.20 months) B6.SJL-PtprcaPepcb/BoyJ(B6.SJL), and young (2-3 months) C57BL/6Jmice were used. Micewere kept under standard pathogen-free conditions in the animalfacility of Ulm University.

Cell sortingHSCs were sorted as lin2Sca-11c-kit1CD342Flk-22 cells from thebonemarrow of young and agedmice, using a BD FACS Aria II ora BD FACS Aria III (BD Bioscience).12

Noncompetitive HSC transplantationSorted HSCs were cultured for 16 hours, where indicated, with5 mM CASIN (Xcessbio).12 Six hundred HSCs were transplantedinto sublethally (6.5 Gy) irradiated RAG12/2 mice. Characterizationof the recipients’ immune system was performed 12 weeksposttransplantation except where otherwise specified.

Flow cytometryFor characterization of the immune systems, peripheral bloodand spleen immunostaining was performed according to stan-dard protocols, using the antibodies listed in supplementalTable 1, available on the Blood Web site.

DNA immunizationMice were immunized with 100 mg pCI/C vector DNA intra-muscularly (tibialis anterior muscles).15 At 12 to 13 dayspostvaccination, Kb/C93-100-specific CD81 T-cell frequenciesin spleen and liver were determined using Kb/C93-100-loadedH2Kb dimers.15

More detailed and additional materials and methods can befound in the supplemental Material.

The RNA Seq raw data of the study are available in the GeneExpression Omnibus repository (accession no. GSE107007).

ResultsReconstitution of RAG12/2 mice with HSCsrecapitulates reduced lymphoid contribution ofaged HSCsWe initially set out to investigate the role of aging of HSCsfor AAIR. The outcome of standard HSC transplantation intoimmune-competent recipients is difficult to interpret because ofinterference of radiation-resistant cells from the recipient thatmight confound results16 (supplemental Figure 1A-B). To over-come this limitation, 600 HSCs (Lin2Sca-11c-kit1CD342Flk-2-;supplemental Figure 1C) from the bone marrow of young andold CD45.11 B6.SJL mice were transplanted into sublethallyirradiated T- and B-cell-deficient young CD45.21 RAG12/2

mice (Figure 1A). In this setting, young and aged donor HSCsare exposed to a young microenvironment, and the devel-opment of HSC-derived T cells proceeds in the presence of ayoung thymus.

Twelve weeks posttransplantation, recipients of aged (DO) HSCsshowed fewer donor-derived B cells but more myeloid cells inperipheral blood compared with recipients of young (DY) HSCs(Figure 1B). Overall reconstitution with DO HSCs resulted infewer donor-derived cells in the spleen than with DY HSCs(Figure 1C). Furthermore, absolute numbers of B, CD41, andCD81 T cells in recipients of DO HSCs were decreased com-pared with recipients of DY HSCs (Figure 1D).

Transplantation of aged HSCs resulted in fewernaive T cellsThe development of mature single positive naive T cells de-pends on a functional thymus.5,10,11 In recipients of DY HSCs,thymus weight was significantly increased compared with innontransplanted RAG12/2 mice, and these thymi containedhigh numbers of distinct T-cell progenitors including double-negative, double-positive, and single-positive CD41 and CD81

T cells (Figure 1E-G). In contrast, in recipients of DO HSCs,there was only a slight increase in thymus weight, and thenumbers of double-positive T-cell progenitors were reduced(Figure 1E-G). These findings demonstrated that the “dor-mant” thymus in RAG12/2 mice at least partially resumed itsappropriate function after adoptive transfer of DY and DOHSCs.

One hallmark of AAIR in mice (and man) is a reduced frequencyof naive (CD442CD62L1) T cells.17 The CD81 T-cell pool in thespleen of young B6.SJL mice contained 50% to 70% naive CD81

T cells while their frequency decreased to 3% to 10% in old mice(Figure 2A-C). Concurrently, the frequency of CD441 memoryCD81 T cells increased on aging, filling about 90% of the CD81

T-cell pool in aged mice but only 30% to 50% in young mice(Figure 2A-B). The frequencies of naive andmemory CD81 T-cellsubsets in recipients of DY or DO HSCs resembled the CD81

T-cell pools of young and old mice, respectively (Figure 2A-C).A similar distribution of CD81 T-cell subsets evolved in RAG12/2

Figure 1 (continued)mice, and RAG12/2 recipients transplanted with DY and DO HSCs (Y, n5 4; O, RAG12/2, n5 3; DY, n5 5; DO, n5 8). (G) Representative thymic profile ofnontransplanted RAG12/2 mice and RAG12/2 hosts of DY and DO HSCs. Total thymic cells from transplanted and nontransplanted animals were analyzed for CD4 and CD8expression, using flow cytometry. CD41, CD81, and CD41CD81 T cells were gated as mononucleated cells within the lymphocyte population. *P , .05; **P , .01; ***P , .001;and ****P, .0001. (B) Two-tailed unpaired Student’s t-test, mean6 SEM. (C-D) Two-tailed unpaired Student’s t-test, mean1 SEM. (D) Only means of nontransplantedyoung vs aged mice and recipients of DY vs DO HSCs were compared statistically. (F) Two-tailed unpaired Students’s t-test, one-way ANOVA, mean 1 SEM.

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Figure 2. Phenotypic characterization of splenic CD81andCD41T cells in RAG12/2 recipients. (A-B) To identify naive andmemory CD81 T cells, splenocytes were stained forCD3, CD8, CD62L, and CD44 expression. Shown are representative flow cytometry profiles of naive (CD442CD62L1), central memory (CM; CD441CD62L1), and effector memory(EM; CD441CD62L2) T cells within the CD31CD81 T cells from young and aged nontransplanted mice and RAG12/2 recipients of DY and DO HSCs. (C) Quantification of naiveCD31CD81 T cells within the total CD31CD81 population (Y, n5 8; O, n5 7; DY, n5 16; DO, n5 19). (D) Proliferation of CD31CD81 T cells isolated from young and old animals


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mice transplanted with C57BL/6-derived (CD45.21) DY HSCs(supplemental Figure 2), validating that this phenotype is in-dependent of the congenic CD45.1 interval in B6.SJL ani-mals.18 Aged B6.SJL mice and recipients of DO HSCs showeda higher frequency of CD441CD62L2 effector memory CD81

T cells compared with young mice and recipients of DY HSCs(Figure 2A-B). The higher frequencies of memory T cells in agedmice and DO HSC recipients might imply a higher proliferativeactivity of these T cells.19 However, in comparison with naiveCD81 T cells, the CD441memory CD81 T-cell pool in young andold mice and in recipients of DY and DO HSCs showed elevatedfrequencies of cells that express the proliferation marker Ki-67(Figure 2D). The higher number of memory CD81 T cells in agedmice might thus not be a direct consequence of a different cellcycle activity on aging. Finally, the frequency of CD81 T cells thatexpress inhibitory receptors usually associated with exhaustion,such as PD-1, CD244.2, LAG-3, or TIM-3,20,21 was higher in oldmice and in recipients of DO HSCs when compared with youngmice and recipients of DY HSCs (Figure 2E-F).

The CD41 T-cell pool in young mice contained about 70%naive CD442CD41 T cells, which declined to less than 10% inaged mice, whereas the frequency of memory CD441CD41

T cells was elevated in aged animals (Figure 2G-I). Similar tothe reconstitution of CD81 T cells by HSCs, the frequencies ofnaive and memory CD41 T-cell subsets in recipients of DY andDO HSCs resembled the CD41 T-cell pools of young andaged mice, respectively (Figure 2A-B,G-H). The frequenciesof naive CD41 T cells were significantly higher in recipients ofDY HSCs compared with recipients of DO HSCs (Figure 2G-I).Young and aged HSCs thus directly contribute to the gen-eration of distinct naive and memory CD81 and CD41 T-cellsubsets in transplanted hosts that are also found in young andold mice.

Aged HSCs confer an increase in the frequency ofregulatory T cellsRegulatory Foxp31CD41 T cells (Tregs) are critical for the reg-ulation of adaptive immune responses.22 In line with previousreports,23 aged mice showed a higher frequency of Tregs. TheCD41 T-cell pool in the spleen of young mice contained about10% to 15% Tregs, whereas their frequency increased up to 35%in old mice (Figure 3A-B). Higher frequencies of Tregs were alsofound in recipients of DO HSCs compared with recipients of DYHSCs, whereas the difference was less pronounced but stillsignificant (Figure 3A-B). Moreover, the frequency of Tregs thatcoexpress the transcription factor Helios24 was higher in agedmice and recipients of DO HSCs (Figure 3C-D). Helios expres-sion is prominent but not exclusive in thymus-derived Tregs, andHelios1Foxp31 Tregs are known to exhibit superior suppressiveactivities compared with Helios2Foxp31 Tregs.25 Aged HSCsalso contribute to the increase in the frequency of distinct Tregsin old animals.

Increased frequency of age-associated B cells inrecipients of DO HSCsAge-associated B cells (ABCs)26 are characterized by a lackof both CD21/35 and CD23 expression and represent anexhausted B-cell subset.27 The percentage of ABCs in the CD191

B-cell pool was higher in the spleen of old compared with youngmice (Figure 3E-F).26 The frequency of CD21/351CD231 follic-ular B cells was lower in old animals, whereas the frequency ofCD21/35hiCD232 marginal zone B cells did not differ betweenyoung and old animals (Figure 3E-F). Similarly, compared withDY HSC recipients, transplants of DO HSCs presented with ahigher frequency of ABCs, a lower frequency of follicular B cells,and no difference in marginal zone B cells (Figure 3E-F). Micetransplanted with HSCs thus showed a reconstitution of B-cellsubsets resembling those found in young and old mice(Figure 3E-F). Therefore, aged HSCs also contribute to the age-associated changes in CD191 B-cell subsets.

The immune systems in aged mice andrecipients of DO HSCs show a reduced DNAvaccination responseTo examine the function of the immune systems, vaccinationexperiments were performed. DNA-based vaccination is anattractive technique to elicit antigen-specific CD81 T-cell re-sponses in mice because they allow direct antigen expressionand MHC class I-restricted epitope presentation by in vivotransfected APCs.28-30 A hepatitis B virus Core antigen-expressingpCI/C vector induced core-specific CD81 T cells in young andaged mice, but the frequency of de novo primed Kb/C93-100

dimer1 CD81 T cells15 was significantly lower in aged mice(supplemental Figure 3). Similarly, compared with recipients ofDY HSCs, a significantly lower frequency of Kb/C93-100 dimer1

CD81 T cells was found in vaccinated recipients of DO HSCs(Figure 4A-C). These data support a novel concept in which pri-marily the age of HSCs determines the magnitude of the CD81

T-cell response to vaccination.

CASIN-treated aged HSCs restore an efficientCD81 T-cell response to DNA vaccinationWe previously reported that the elevated activity of the smallRhoGTPase Cdc42 is causative for aging of HSCs. Ex vivotreatment of aged HSCs with the Cdc42 inhibitor CASIN revertsthe aging-associated phenotypes of HSCs.12 CASIN is thusthought to rejuvenate aged HSCs.12 As a consequence, wehypothesized that the functional decline of the old immunesystem in recipients of DO HSCs (Figure 4B-C) might be at-tenuated by CASIN-treatment of aged HSCs.

We transplanted untreated aged HSCs (DO) or CASIN-treatedaged HSCs (DO1C) into RAG12/2 mice (Figure 4D), followedby a single injection of the pCI/C vector at 12 weeks post-transplantation. Compared with recipients of DO HSCs, thefrequencies of Kb/C93-100-specific dimer1 CD81 T cells were

Figure 2 (continued) and RAG12/2 recipients of DY and DO HSCs was analyzed by expression of the nuclear protein Ki-67. Percentage of naive (CD442) and memory (CD441)CD31CD81 T cells, which are Ki-671, are depicted (Y, O, n5 4; DY, n5 5; DO, n5 9). (E) CD31CD81 T cells isolated from nontransplanted young and old B6.SJL mice andRAG12/2 hosts transplanted with DY or DO HSCs were stained for the exhaustion marker PD-1, CD244.2, TIM-3, and LAG-3. Representative flow cytometric profiles ofindividual stains are depicted. (F) Percentages of the different inhibitory receptors within CD31CD81 T cells are shown (Y, n5 8; O, n5 7; DY, n5 16; DO, n5 19). (G-H)Splenic naive CD41 T cells from recipients of DY and DOHSCs and nontransplanted young and agedmice were identified as CD442CD62L1 cells within the CD31CD41

T-cell population. Representative dot plot profiles of the flow cytometric analysis are depicted. (I) Quantification of naive CD31CD41 T cells within the total CD31CD41

population (Y, n 5 8; O, n 5 7; DY, n 5 16; DO, n 5 19). *P , .05; **P , .01; ***P , .001; ****P , .0001 2-tailed unpaired Student’s t-test, mean 1 SEM.


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significantly higher in spleen and liver of recipients of DO1CHSCs (Figure 4E-F). The frequencies of Kb/C93-100-specific CD81

T cells in vaccinated recipients of DO1C and DY HSCs were verysimilar (Figure 4C,F). DO1C HSCs thus generate an immunesystem capable of mounting a strong CD81 T-cell response toDNA vaccination. Recipients of DO and DO1C HSCs showedsimilar frequencies of donor-derived white blood cells in the

spleen and a slight increase in the frequencies of B cells (sup-plemental Figure 4A-B). However, we observed a significantincrease in the frequency of naive CD81 T cells and a strongtrend toward a higher frequency of CD41 T cells in recipientsof DO1C HSCs when compared with recipients of DO HSCs(supplemental Figure 4C-D). These elevated frequencies ofT-cell subsets in DO1C recipients might explain, at least

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Figure 3. Regulatory T- and B-cell subsets in HSC trans-planted RAG12/2 mice. To identify regulatory T cells,splenocytes were stained for surface expression of CD3 andCD4 as well as intracellular FoxP3 expression. Representativegraphs of individual stains for FoxP3 as well as quantificationof FoxP31 cells within the CD31CD41 population are shownin (A) and (B), respectively (Y, n5 8; O, n5 7; DY, n5 15; DO,n5 17). Regulatory T cells coexpressing the Ikaros zinc fingertranscription factor Helios were identified within the FoxP31

population. (C) Representative dot plot profiles of the un-derlying flow cytometric analysis and (D) quantification ofHelios1 regulatory T cells within the FoxP31 population(Y, n 5 5; O, n 5 5; DY, n 5 4; DO, n 5 7). For characteriza-tion of splenic B cells, CD191 cells from young and old non-transplanted B6.SJL mice and RAG12/2 recipients of DY andDO HSCs were stained for follicular B cells (FOB; CD21/351

CD231), marginal zone B cells (MZ; CD21/35highCD232), andage-associated B cells (ABC; CD21/352CD232). Shown arerepresentative graphs of individual stains (E) and quantifi-cation of these B-cell populations within the total CD191

population (Y, n 5 8; O, n 5 7; DY, n 5 13; DO, n 5 16)(F). *P , .05; **P , .01; ***P , .001; ****P , .0001, 2-tailedunpaired Student’s t-test, mean 1 SEM.


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partially, the ability to mount the elevated frequency of Kb/C93-

100-specific CD81 T cells in these mice.

DY and DO1C HSCs generate comparable geneexpression profiles in T and B cellsTo obtain information on the extent to which CASIN treatment ofaged HSCs affects the gene expression profile of daughter B andT cells, RNAseq analyses were performed on naive CD41 T cellsand CD191 B cells isolated from DO, DO1C, or DY HSC re-cipients (Figure 5). In RNAseq analysis, we identified 137 sig-nificantly differentially expressed protein-coding genes bycomparing CD41 T cells isolated from recipients of DY to DO

HSCs. Similarly, comparison of CD41 T cells isolated from re-cipients of DO1C and DO HSCs revealed 67 significantly dif-ferentially expressed genes. Thirty genes were in commonbetween the 2 analyses, where 26 genes were up- and 4 weredownregulated (Figure 5B; supplemental Table 2). Unsupervisedclustering-based heat map of these differentially expressedgenes showed that DO1C HSC recipients primarily clusteredwith DY HSC recipients, with only 1 of the 3 samples falling intothe DOHSCs cluster (Figure 5B). In the analysis of CD191 B cells,we identified 169 and 103 protein-coding genes significantlydifferentially expressed by comparing cells isolated fromrecipients of DY to DO HSCs and DO1C to DO HSCs(Figure 5C). Forty-seven of these genes were in common

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Figure 4. Priming CD81 T-cell responses in HSC-transplantedRAG12/2 hosts by DNA vaccination. (A) Schematic repre-sentation of the experimental set-up: 12 weeks after trans-plantation, recipient mice were immunized with pCI/C DNAencoding the hepatitis B virus Core antigen. Thirteen days afterimmunization, splenic Kb/C93-100 dimer1CD81 T-cell frequencieswere determined by flow cytometry. Representative graphs ofindividual stains as well as quantification of the KbC93-100 dimerstaining are shown in (B) and (C), respectively (DY, n 5 9; DO,n5 8). (D) Schematic representation of the experimental set-up.HSCs were isolated from old B6.SJL mice and cultured for16 hours 6 5 mM CASIN. Subsequently, 600 HSCs were trans-planted in subleathally irradiated young RAG12/2 mice. Twelveweeks after transplantation, recipient mice were immunizedwith themammalian expression vector pCI/C. Thirteen days afterimmunization, transplanted mice were sacrificed and the per-centage of Kb/C93-100 dimer1 cells was determined within theCD31CD81 population by flow cytometry. Quantification as wellas representative graphs of the Kb/C93-100 dimer staining forspleen (DO, n5 11; DO1C, n5 9) and liver (DO, n5 8; DO1C,n 5 6) are shown in (E) and (F), respectively. *P , .05; **P , .01,2-tailed unpaired Student’s t-test, mean 1 SEM.


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DYvs. DO

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Rplp2–ps1

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Figure 5. Gene expression profiles of naive CD41 T cells and CD191 B cells isolated from HSC-transplanted RAG12/2 mice. (A) Schematic representation of the ex-perimental setup: HSCs were isolated from young and old B6.SJL (CD45.1) mice and cultured for 16 hours6 5 mMCASIN. 600 HSCs were transplanted into sublethally irradiatedyoung RAG12/2 (CD45.2) recipients. Twelve weeks after transplantation, naive CD41 T and CD191 B cells were isolated and RNA sequencing was performed (B) Venn diagram ofsignificantly differentially expressed genes in naive CD41 T cells between DY vs DO (blue circle) and DO1C vs DO (green circle). The heat map shows unsupervised clusteringof the genes expressed differentially in the same direction between the 2 groups. (C) Venn diagram of significantly differentially expressed genes in CD191 B cells between DY vsDO (orange circle) and DO1C vs DO (violet circle). Genes, differentially expressed in the same direction between the 2 groups, are depicted as heat map (unsupervisedclustering). (D) Venn diagram shows GSEA of significantly enriched GO gene sets differentially expressed in naive CD41 T cells between DY vs DO (blue circle) and DO1C vs


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between the 2 comparisons and showed the same directionof change in expression (33 genes upregulated, 14 genesdownregulated; Figure 5C; supplemental Table 3). Un-supervised hierarchical clustering of these genes revealedthat B cells isolated from recipients of DY and DO1C HSCsclustered together, whereas those from DO HSCs formed aseparate cluster (Figure 5C).

Most prominent in the upregulated gene expression profile ofnaive CD41 T cells from DO HSC recipients were genes im-plicated in Treg functions (itgae, ikzf2, folr4, lag3) and in-flammation (penk, adam19, coro2a)31 (Figure 5B). In CD191

B cells from DO HSC recipients, we found altered expression ofgenes associated with cellular senescence (abl1, irs2, rrm2,s100a8), the cytokine/chemokine network (ccl5, scimp), cellproliferation (mki67, racgap1, top2a), cell migration (ccr7,cmtm6, cnr2), and B-cell receptor signaling (fosb). Furthermore,the expression of the transcription factor t-bet, a key attribute ofABCs,32 was upregulated in CD191 B cells from DO HSC re-cipients (Figure 5C).

To identify pathways and biological processes altered inrecipients of DY, DO1C, and DO HSCs, we performed geneset enrichment analysis (GSEA). Gene ontology (GO) analysisrevealed significant enrichment (false discovery rate [FDR]q value , 0.05) of 29 and 20 gene sets in naive CD41 T cellsisolated from recipients of DY and DO1C HSCs whencompared with recipients of DO HSCs (Figure 5D). Weidentified 6 overlapping gene sets downregulated in recip-ients of DY and DO1C HSCs compared with recipients ofDO HSCs (Figure 5D). Chromatin organization (GO_DNAPackaging Complex and GO_DNA Packaging) and mitosis(GO_Mitotic Sister Chromatid Segregation) were among thesignificantly deregulated gene sets, implying that naiveCD41 T cells isolated from recipients of DO HSCs might showan altered chromatin or mitotic transition structure as com-pared with T cells isolated from recipients of DY and DO1CHSCs. Importantly, CASIN-treatment of aged HSCs (DO1C)resulted in a gene expression profile for chromatin structurein naive CD41 T cells similar to the profile found in CD41

T cells of DY HSC recipients.

Figure 5 (continued) DO (green circle). Normalized enrichment scores (NES) of GO categories differentially expressed in the same direction between the 2 groups are depicted.(E-F) Venn diagrams showGSEAof significantly enrichedGOand REACTOMEgene sets, respectively, differentially expressed in CD191B cells betweenDY vsDO (orange circle) andDO1C vs DO (violet circle). NES of GO and REACTOM categories differentially expressed in the same direction between the 2 groups are depicted. (G) GSEA enrichment plots ofcorrelation between gene sets previously identified byMirza et al.33 to be differentially expressed in naive CD41 T cells from nontransplanted young and agedmice and differentiallyexpressed genes between DY and DO HSC recipients as well as DO1C and DO recipients. DY, DO and DO1C, n 5 3. (D-F) FDR q-value , 0.05; (G) FDR q-value , 0.25.

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GSEA of CD191 B cells revealed 47 (DY vs DO HSC recipients)and 15 (DO1C vs DO HSC recipients) significantly deregu-lated GO gene sets (Figure 5E). In a similar analysis, 21 and17 REACTOME gene sets were significantly enriched in DY vsDO HSC recipients and DO1C vs DO HSC recipient com-parisons (Figure 5F). Ten of the GO terms and 11 of theREACTOME gene sets were enriched (FDR q value , 0.05) inboth DY vs DO HSC recipients and DO1C vs DO HSC re-cipients comparisons (Figure 5E-F). Interestingly, all theoverlapping gene sets were upregulated in CD191 B cellsisolated from recipients of DO HSCs. Of significance, 9 of the10 GO gene sets and all of the 11 REACTOME gene sets wererelated to cell cycle, proliferation, and mitosis (Figure 5E-F),indicating that B cells developing from DO HSCs mightcontain an enhanced proliferation activity. In contrast, DO1CHSCs generated B cells characterized by a proliferationprofile similar to that seen in B cells repopulated from DYHSCs, indicating that CASIN-treatment of aged HSCs re-stored a young B-cell compartment.

We also performed GSEA on genes previously identified byMirza et al.33 to be differentially expressed between naive CD41

T cells from young and aged mice. Our analysis showed anenrichment of those genes to differentially expressed genes(P value , .05 and FDR , 0.25) between DY and DO HSC re-cipients (Figure 5G; supplemental Figure 5). This demonstratesthat naive CD41 T cells isolated from young mice and recipientsof DY HSCs are very similar. When comparing the differentiallyexpressed genes between young and aged naive CD41 T cells ofMirza et al and differentially expressed genes between naiveCD41 T cells from DO1C and DO HSC recipients, we couldobserve the same enrichment. This supports that the tran-scriptional profile of naive CD41 T cells in DO1C HSC recipientsmimics that of CD41 T cells in DY HSC recipients, and even moreimportant, that naive CD41 T cells in DO1C HSC recipientspresent with a transcription profile very similar to CD41 T cellsfrom young mice.

DiscussionOur data support that AAIR (both phenotypic and functional) isdriven to a large extent by aging of HSCs. As a consequence,rejuvenated DO1C HSCs restore an immune system capable ofmounting a strong vaccination response, which is similar to that ofrecipients of DY HSCs. Changes in the number of specific typesof immune cells (eg, CD81 T cells) developing in recipients ofDO1C HSCs, most likely in combination with an improvedfunction of these cells, might be a crucial driving force forattenuating AAIR and mounting a strong vaccination response.In this study, we primed CD81 T-cell responses by DNA-basedimmunization. After a single injection of antigen-expressingvector DNA, CD81 T-cell responses peaked at 11 to 13 dayspostinjection.15 Thus, this vaccine allows the determination ofde novo primed CD81 T cells in nontransplanted mice andrecipients of HSCs under standardized conditions. DNA-basedvaccination is also of clinical interest and is tested in variousclinical trials.30

Using young RAG12/2 mice as hosts, we are able avoid con-founding effects from cells that resist irradiation in trans-plantation models that use immunocompetent recipients.16 InRAG12/2 recipients, all distinct types of T and B cells are

offspring of transplanted donor HSCs in an environment initiallydevoid of lymphoid cells. Transplantation into RAG12/2 miceis thus an attractive tool to directly compare DY, DO, andDO1C HSCs with respect to lymphoid contribution and function.RAG12/2 mice reconstituted with DO or DY HSCs showed animmunization response that was almost identical to that in youngand aged mice. However, the immune systems established fromtransplanted HSCs in the complete absence of host T and B cellsmight not be fully identical to immune systems established bythe normal developmental route and might limit the ultimateextent of the generalization of our findings. Furthermore, miceare housed under specific pathogen-free conditions, and themurine immune system, although regarded as a valued modelalso for AAIR in humans, is still distinct from the human immunesystem.34 Therefore, some of our findings might be, with limi-tations, also relevant for humans.

We report here that aged HSCs provide lower numbers of naiveT cells to the periphery. These findings show that aged HSCspresent with intrinsic defects that, at least in part, cause AAIR andsuggest either that aged HSCs are impaired in the production ofT-cell progenitors or that T-cell progenitors derived from agedHSCs are impaired in thymus homing and/or interactions withthe thymic stroma,5,35 even in an environment conducive forproper maturation of T cells in a young thymus. Aging-associatedchanges in T-lymphopoiesis have been linked to thymic in-volution.5 Our data demonstrate that aging of HSCs is able tocause changes in T-lymphopoiesis, and in transplantationexperiments, aged HSCs mimic at least in part aged T-lym-phopoiesis. In combination with published reports on the roleof the thymus for AAIR, the data therefore support a role forboth HSC intrinsic as well as extrinsic processes that contributeto AAIR.

To the same extent as the frequency of naive T cells declines inaged mice, memory T-cell subsets accumulate.17,36 The memoryT-cell pool in aged mice is thought to arise from naive CD81

T cells in response to self-peptide/MHC ligands and signalingthrough various cytokines (eg, interleukin 7, interleukin 15).17

Memory-phenotype CD81 T cells are found before birth inhumans and in unimmunized mice housed under germ-free oreven antigen-free conditions, indicating that the bulk of memoryT cells in mice, and possibly to some extent also in humans, isdistinct from other types of antigen-specific memory T cells.17,37

We showed that unimmunized aged mice and recipients of DOHSCs, but not young mice and recipients of DY HSCs, con-tained a high number of effector memory CD81 T cells. Thethymus-independent generation of this T-cell subset may bedirectly linked to the reduced numbers of naive T cells in re-cipients of DO HSCs, and thus indirectly conferred by agedHSCs.

Distinct interventional approaches similar to, in our example, theinhibition of the elevated Cdc42 activity in old HSCs withCASIN12 have been reported to attenuate aging of HSCs.38-40 AsDO1C HSCs restore an immune system capable of mounting astrong vaccination response similar to that primed in recipientsof DY HSCs, our data further provide rationale for testing CASINin vivo in aged animals. Clinical relevance for a role of inhibitionof Cdc42 activity upon aging is implied by the fact that also inhumans Cdc42 activity in blood cells positively correlates withaging, whereas gene expression association studies revealed


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a correlation between elevated Cdc42 expression in hemato-poietic cells and morbidity.41,42

Most interestingly, the transcriptional profile of T and B cellsreconstituted from DY and DO HSCs mimicked that of T andB cells in young and aged animals. The profile of T and B cellsderived from DO1C HSCs was more like the profile of youngT and B cells. The differentially expressed genes and pathwaysidentified in our analyses are thus likely to be causatively linkedto AAIR in T and B cells. The data also imply that expressionprofiles down to terminally differentiated T and B cells are al-ready decided in HSCs themselves and are, at least partially,regulated by changes in the activity Cdc42. As HSCs do notexpress T- and B-cell genes, the mechanisms therefore will needto be based on yet-to-be-identified epigenetic mechanisms thatdo not directly regulate transcription. Previous publications al-ready implied a role of epigenetic dysfunction as a contributor toHSC aging.43 We previously showed that CASIN treatment ofaged HSCs restored the apolar distribution of the epigeneticmarker histone 4 acetylated on lysine 16 (H4K16) in the nucleusof aged HSCs back to the polar distribution found in youngHSCs, and also restored the level of H4K16 to the higher levelseen in young HSCs. Such changes of the epigenetic make-upin HSCs might thus direct phenotypic and functional changesin immune cells on HSC differentiation that include a multitudeof other types and levels of epigenetic mechanisms awaitingfurther investigation. In conclusion, we present an attractivestrategy to attenuate or even revert AAIR by CASIN-mediatedrejuvenation of aged HSCs.

AcknowledgmentsThe authors thank the Core Facility fluorescence-activated cell sorting atUlm University for cell sorting support, the DNA Sequencing and Gen-otyping Core at Cincinnati Children’s Hospital Medical Center (CCHMC)for assistance with RNA sequencing, and the Tierforschungszentrum ofthe University of Ulm, as well as the Comprehensive Mouse and CancerCore at CCHMC, for support with animal experiments. Furthermore, theauthors thank Nadine Schweizer and Kalpana Nattamai for excellenttechnical assistance.

This work was supported by grants from the Bundesministerium furBildung und Forschung (systems biology analysis of impaired stem cell

function and regeneration during aging [SyStaR]) (H.G. and M.D.) andfrom theDeutsche ForschungsgemeinschaftGraduiertenkolleg (GRK) 1789Cellular and Molecular Mechanisms in Aging (H.G. and R.S.). The labo-ratory of H.G. was further supported by National Institutes of Health grantsHL076604, DK077762, AG040118, and DK104814; the DeutscheForschungsgemeinschaft (GRK 2254 HEIST and SFBs 1074, 1149, and1275); and the Excellence-Initiative of the Baden-Wurttemberg Foun-dation. The laboratory of R.S. was further supported by grants from theDeutsche Forschungsgemeinschaft (GRK 2254 HEIST; SCHI505/6-1) andthe Bohringer Ingelheim Ulm University Bio Center. H.L. is a PhD can-didate at Ulm University. This work is submitted in partial fulfilment of therequirement for the PhD.

AuthorshipContribution: H.L., M.D., H.G., and R.S. designed and interpreted ex-periments and wrote the manuscript; H.L. analyzed and performed ex-periments; K.E. and V.S. performed experiments; M.M. performedbiocomputational analyses; and Y.L. provided reagents.

Conflict-of-interest disclosure: The authors declare no competing fi-nancial interests.

Correspondence: Hartmut Geiger, Institute of Molecular MedicineStem Cell and Aging, Ulm University, Meyerhofstrasse, 89081 Ulm,Germany; e-mail: [email protected]; and Reinhold Schirmbeck,Department of Internal Medicine I, University Hospital Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany; e-mail: [email protected].

FootnotesSubmitted 1 February 2018; accepted 30 May 2018. Prepublished onlineas Blood First Edition paper, 11 June 2018; DOI 10.1182/blood-2018-02-831065.

*H.G. and R.S. share senior authorship.

The online version of this article contains a data supplement.

There is a Blood Commentary on this article in this issue.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked “advertisement” in accordance with 18 USC section 1734.

REFERENCES1. Black S, De Gregorio E, Rappuoli R.

Developing vaccines for an aging population.Sci Transl Med. 2015;7(281):281ps8.

2. Boraschi D, Aguado MT, Dutel C, et al. Thegracefully aging immune system. Sci TranslMed. 2013;5(185):185ps8.

3. Nikolich-Zugich J. The twilight of immunity:emerging concepts in aging of the immunesystem. Nat Immunol. 2018;19(1):10-19.

4. Decman V, Laidlaw BJ, Doering TA, et al.Defective CD8 T cell responses in agedmice are due to quantitative and qualitativechanges in virus-specific precursors.J Immunol. 2012;188(4):1933-1941.

5. Aw D, Silva AB, Maddick M, von Zglinicki T,Palmer DB. Architectural changes in the thy-mus of aging mice. Aging Cell. 2008;7(2):158-167.

6. Young K, Borikar S, Bell R, Kuffler L, Philip V,Trowbridge JJ. Progressive alterations in

multipotent hematopoietic progenitors un-derlie lymphoid cell loss in aging. J Exp Med.2016;213(11):2259-2267.

7. Ergen AV, Goodell MA. Mechanisms of he-matopoietic stem cell aging. Exp Gerontol.2010;45(4):286-290.

8. Geiger H, de Haan G, Florian MC. The ageinghaematopoietic stem cell compartment. NatRev Immunol. 2013;13(5):376-389.

9. Wahlestedt M, Pronk CJ, Bryder D. Concisereview: hematopoietic stem cell aging and theprospects for rejuvenation. Stem Cells TranslMed. 2015;4(2):186-194.

10. Scollay RG, Butcher EC, Weissman IL. Thymuscell migration. Quantitative aspects of cellulartraffic from the thymus to the periphery inmice. Eur J Immunol. 1980;10(3):210-218.

11. Bredenkamp N, Nowell CS, Blackburn CC.Regeneration of the aged thymus by a singletranscription factor. Development. 2014;141(8):1627-1637.

12. Florian MC, Dorr K, Niebel A, et al. Cdc42activity regulates hematopoietic stem cellaging and rejuvenation. Cell Stem Cell. 2012;10(5):520-530.

13. Florian MC, Nattamai KJ, Dorr K, et al. A ca-nonical to non-canonical Wnt signalling switchin haematopoietic stem-cell ageing. Nature.2013;503(7476):392-396.

14. Lin Y, Zheng Y. Approaches of targeting RhoGTPases in cancer drug discovery. ExpertOpin Drug Discov. 2015;10(9):991-1010.

15. Riedl P, Reiser M, Stifter K, Krieger J,Schirmbeck R. Differential presentation ofendogenous and exogenous hepatitis B sur-face antigens influences priming of CD8(1)T cells in an epitope-specific manner. Eur JImmunol. 2014;44(7):1981-1991.

16. Rajasekaran N, Wang N, Hang Y, et al. B6.g7mice reconstituted with BDC2×5 non-obesediabetic (BDC2×5NOD) stem cells do notdevelop autoimmune diabetes. Clin ExpImmunol. 2013;174(1):27-37.


Dow


ATUR

VERW

ALTUN

G user on 12 D

ecember 2019

mailto:[email protected]



https://doi.org/10.1182/blood-2018-02-831065

https://doi.org/10.1182/blood-2018-02-831065

http://www.bloodjournal.org/content/132/6/553

17. Nikolich-Zugich J. Aging of the T cell com-partment in mice and humans: from no naiveexpectations to foggy memories. J Immunol.2014;193(6):2622-2629.

18. Waterstrat A, Liang Y, Swiderski CF, SheltonBJ, Van Zant G. Congenic interval of CD45/Ly-5 congenic mice contains multiple genesthat may influence hematopoietic stem cellengraftment. Blood. 2010;115(2):408-417.

19. Neujahr DC, Chen C, Huang X, et al.Accelerated memory cell homeostasis duringT cell depletion and approaches to overcomeit. J Immunol. 2006;176(8):4632-4639.

20. Lee K-A, Shin K-S, Kim G-Y, et al.Characterization of age-associated exhaustedCD81 T cells defined by increased expression ofTim-3 and PD-1.Aging Cell. 2016;15(2):291-300.

21. Lages CS, Lewkowich I, Sproles A, Wills-KarpM, Chougnet C. Partial restoration of T-cellfunction in aged mice by in vitro blockade ofthe PD-1/PD-L1 pathway. Aging Cell. 2010;9(5):785-798.

22. Shevach EM, Thornton AM. tTregs, pTregs,and iTregs: similarities and differences.Immunol Rev. 2014;259(1):88-102.

23. Jagger A, Shimojima Y, Goronzy JJ, WeyandCM. Regulatory T cells and the immune agingprocess: a mini-review. Gerontology. 2014;60(2):130-137.

24. Sugimoto N, Oida T, Hirota K, et al. Foxp3-dependent and -independent moleculesspecific for CD251CD41 natural regulatoryT cells revealed by DNA microarray analysis.Int Immunol. 2006;18(8):1197-1209.

25. Sebastian M, Lopez-Ocasio M, Metidji A,Rieder SA, Shevach EM, Thornton AM. Helioscontrols a limited subset of regulatory T cellfunctions. J Immunol. 2016;196(1):144-155.

26. Ratliff M, Alter S, Frasca D, Blomberg BB, RileyRL. In senescence, age-associated B cellssecrete TNFa and inhibit survival of B-cellprecursors. Aging Cell. 2013;12(2):303-311.

27. Hao Y, O’Neill P, Naradikian MS, Scholz JL,Cancro MP. A B-cell subset uniquely re-sponsive to innate stimuli accumulates in agedmice. Blood. 2011;118(5):1294-1304.

28. Gurunathan S, Klinman DM, Seder RA. DNAvaccines: immunology, application, and opti-mization. Annu Rev Immunol. 2000;18(1):927-974.

29. Saade F, Petrovsky N. Technologies for en-hanced efficacy of DNA vaccines. Expert RevVaccines. 2012;11(2):189-209.

30. Ferraro B, Morrow MP, Hutnick NA, Shin TH,Lucke CE, Weiner DB. Clinical applications ofDNA vaccines: current progress. Clin InfectDis. 2011;53(3):296-302.

31. Franceschi C, Bonafe M, Valensin S, et al.Inflamm-aging. An evolutionary perspectiveon immunosenescence. Ann N Y Acad Sci.2000;908(1):244-254.

32. Rubtsova K, Rubtsov AV, Cancro MP, MarrackP. Age-associated B cells: a T-bet-dependenteffector with roles in protective and patho-genic immunity. J Immunol. 2015;195(5):1933-1937.

33. Mirza N, Pollock K, Hoelzinger DB,Dominguez AL, Lustgarten J. Comparativekinetic analyses of gene profiles of naıveCD41 and CD81 T cells from young and oldanimals reveal novel age-related alterations.Aging Cell. 2011;10(5):853-867.

34. den Braber I, Mugwagwa T, Vrisekoop N, et al.Maintenance of peripheral naive T cells issustained by thymus output in mice but nothumans. Immunity. 2012;36(2):288-297.

35. Famili F, Wiekmeijer A-S, Staal FJ. The de-velopment of T cells from stem cells in miceand humans. Future Sci OA. 2017;3(3):FSO186.

36. Fortner KA, Bond JP, Austin JW, Boss JM,Budd RC. The molecular signature of murineT cell homeostatic proliferation reveals bothinflammatory and immune inhibition patterns.J Autoimmun. 2017;82:47-61.

37. Sprent J, SurhCD.Normal T cell homeostasis: theconversion of naive cells intomemory-phenotypecells. Nat Immunol. 2011;12(6):478-484.

38. Chen C, Liu Y, Liu Y, Zheng P. mTOR regulationand therapeutic rejuvenation of aging hema-topoietic stem cells. Sci Signal. 2009;2(98):ra75.

39. Chang J, Wang Y, Shao L, et al. Clearance ofsenescent cells by ABT263 rejuvenates agedhematopoietic stem cells in mice. Nat Med.2016;22(1):78-83.

40. Cheng C-W, Adams GB, Perin L, et al.Prolonged fasting reduces IGF-1/PKA topromote hematopoietic-stem-cell-based re-generation and reverse immunosuppression.Cell Stem Cell. 2014;14(6):810-823.

41. FlorianMC, Klenk J, Marka G, et al. Expressionand activity of the small RhoGTPase Cdc42 inblood cells of older adults are associated withage and cardiovascular disease. J Gerontol ABiol Sci Med Sci. 2017;72(9):1196-1200.

42. Kerber RA, O’Brien E, Cawthon RM. Geneexpression profiles associated with aging andmortality in humans. Aging Cell. 2009;8(3):239-250.

43. Wahlestedt M, Norddahl GL, Sten G, et al. Anepigenetic component of hematopoietic stemcell aging amenable to reprogramming into ayoung state. Blood. 2013;121(21):4257-4264.


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Aged murine hematopoietic stem cells drive aging