Effects of Sex and Aging on the Immune Cell Landscape As Assessed by Single-Cell Transcriptomic Analysis

Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis

Zhaohao Huanga,1, Binyao Chena,1, Xiuxing Liua,1,HeLia,1, Lihui Xiea,1, Yuehan Gaoa,1, Runping Duana, Zhaohuai Lia, Jian Zhangb, Yingfeng Zhenga,2, and Wenru Sua,2

aState Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China; and bDepartment of Clinical Research Center, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, China

Edited by Lawrence Steinman, Stanford University School of Medicine, Stanford, CA, and approved June 22, 2021 (received for review November 19, 2020) Sex and aging influence the human immune system, resulting in the immune system integrates numerous interconnected compo- disparate responses to infection, autoimmunity, and cancer. How- nents, pathways, and cell types in sex and aging. ever, the impact of sex and aging on the immune system is not yet To this end, we profiled the transcriptomes of peripheral im- fully elucidated. Using small conditional RNA sequencing, we mune cells sampled from men and women of two distinct age found that females had a lower percentage of natural killer (NK) groups. Then, we investigated sex- and age-related differences in cells and a higher percentage of plasma cells in peripheral blood PBMC immune cell composition, molecular programs, and com- compared with males. Bioinformatics revealed that young females munication network. Extensive differences in all of these aspects exhibited an overrepresentation of pathways that relate to T and were observed between sexes, particularly in aging. Compared – B cell activation. Moreover, cell cell communication analysis revealed with males, females exhibited a higher expression of T cell (TC)– evidence of increased activity of the BAFF/APRIL systems in females. and B cell (BC)–activated signaling at the transcriptional level. Notably, aging increased the percentage of monocytes and reduced Overall, this work expands our knowledge of sex differences in the percentage of naïve T cells in the blood and the number of immune cells. differentially expressed genes between the sexes. Aged males expressed higher levels of inflammatory genes. Collectively, the re- Results sults suggest that females have more plasma cells in the circulation Study Design and Analysis of Single Immune Cell Profiling by Sex and and a stronger BAFF/APRIL system, which is consistent with a stron- Aging. To study the effects of sex and aging on the immune mi- IMMUNOLOGY AND INFLAMMATION ger adaptive immune response. In contrast, males have a higher per- croenvironment, we conducted single-cell RNA sequencing centage of NK cells in blood and a higher expression of certain (scRNA-seq) analysis on the peripheral blood mononuclear cells proinflammatory genes. Overall, this work expands our knowledge of sex differences in the immune system in humans. (PBMCs) of five young females (YF), five old females (OF), five young males (YM), and five old males (OM) (Fig. 1A and SI Appendix A B sex | aging | single-cell sequencing | immune responses | , Table S1 and ). The age range of the young group cell–cell communication was 20 to 30 y, while that of the old group was 60 to 80 y. The single-cell suspensions were converted to barcoded scRNA-seq ex and aging are biological variables that influence immune Ssystem composition, development, and the repertoire of re- Significance sponses to pathogenic insults. The type and severity of immune disorders differ greatly between males and females (1). For in- Differences in immune functioning stem from multiple factors, stance, in the United States, ∼80% of the autoimmune diseases, including sex and aging. However, the specific roles of these including Sjögren syndrome and systemic lupus erythematosus variables in immunity remain elusive. We profiled immuno- (SLE), affect females (2). At the same time, males are twice as cytes from young and old males and females at single-cell vulnerable as females to fatal malignancies (3). Additionally, sex- resolution and constructed a precise atlas of blood-circulating based differences in the responses to many vaccines have been immunocytes. T cell– and B cell–activated signals were higher reported for children and adults (4). Females are generally more in young females than males, while aging increased the sex- susceptible to experiencing adverse side effects from vaccina- related differences in immunocytes, cellular composition, and tions and generate more antibodies than men, which has led to inflammatory signaling. Additionally, males showed a higher the recommendation of different dose protocols for men and accumulation of inflammatory factors during aging, whereas – women (5). Moreover, males are more affected by severe viral, cell cell communication analysis revealed different trends in bacterial, and fungal infections than females (6). Studies have gene expression between females and males with aging. These demonstrated a higher COVID-19–related fatality rate in males findings might aid in the understanding of the mechanisms than in females, particularly in aged males (7). Sex-related dif- underlying sex-based differences in immunity and disease ferences in susceptibility to immunity disorders originate from susceptibility across the lifespan. the differential composition and signaling pathways between male Author contributions: Y.Z. and W.S. designed research; Z.H., B.C., X.L., H.L., L.X., Y.G., Y.Z., and female immune compartments (8, 9). Sex and aging affect the and W.S. performed research; Z.H., B.C., X.L., H.L., L.X., Y.G., R.D., Z.L., J.Z., Y.Z., and W.S. composition of the immune microenvironment, thereby contrib- analyzed data; and Z.H., B.C., X.L., H.L., L.X., Y.G., Y.Z., and W.S. wrote the paper. uting to sex-based differences in the response to infection and The authors declare no competing interest. development of autoimmune disease and cancer. This article is a PNAS Direct Submission. Seminal studies have yielded important insights into the sex- This open access article is distributed under Creative Commons Attribution-NonCommercial- and age-related differences in immune responses. However, NoDerivatives License 4.0 (CC BY-NC-ND). these studies have focused on detecting developmental and cell 1Z.H., B.C., X.L., H.L., L.X., and Y.G. contributed equally to this work. lineage markers of limited cell types, obscuring more complex 2To whom correspondence may be addressed. Email: [email protected] or hierarchies (1, 8, 10–12). Furthermore, the interactive and over- [email protected]. lapping effects of sex and aging require further elucidation. Thus, a This article contains supporting information online at https://www.pnas.org/lookup/suppl/ comprehensive immune cell atlas that encompasses the influences doi:10.1073/pnas.2023216118/-/DCSupplemental. of sex and aging at a single-cell resolution is needed to reveal how Published August 12, 2021.

PNAS 2021 Vol. 118 No. 33 e2023216118 https://doi.org/10.1073/pnas.2023216118 | 1of11 Downloaded by guest on September 24, 2021 A

clustering

young female old female subset proportions

10x Genomics PBMCs acquisition scRNA-seq differential expression analysis

I L

2 R2 3

3 R

CC C

C IL 1 6 2 R R C B C 1 XC R C 3 C C R 2 C X C L 16

6 CL1 CX CC R2 R2 CC C XCL 16 3 CCR

B1 IL12R

IL12RB1

CCR3 CXCL16

CCR2 young male old male CCR2 L16 CXC C CR3

IL 12R B1

C CR 2 CC R healthy subjects 3 C X C 4 L 2 C 1 C 6 CL C R C C 2 R 3 cell-cell communication

B TC MC BC NK DC MegakaryocytesRedCD34 blood cells Z score TC CD4 Naive CCR7 2 IL7R CD4 Tcm CD69 TCF7 CD4 Tem CCR6 1 LEF1 FOXP3 0 LTB CD4 Treg CCR7 −1 CD8 Naive CD8 Tem GZMK −2 CD8 Cytotoxic GZMB T cells CD4-8- TRDV2 CD4+8+ TYROBP T-mito MKI67 MC CD14 CD14 S100A8 Inter CSF1R S100A9 LYZ CD16 FCGR3A BC NBC IL4R CD79A MBC CD27 CD79B MZB1 MS4A1 PC Age-associated ITGAX BC NK NK1 KLRC1 GNLY NK2 SPON2 GZMB KLRC2 NKG7 NK3 CLEC9A DC cDC1 IRF8 cDC2 CD1C CD1C Plasmacytoid CD123 CLEC10A DC AXL pre−DC Megakaryocytes PPBP Red blood cells HBB CD34 CD34

Fig. 1. Overview of the approach and clustering strategy used for analyzing the circulating immune cell profiling of aging and sex. (A) Schematic of the experimental design for scRNA-seq. PBMCs were collected from YF, YM, OF, and OM then processed by scRNA-seq using the 10× Genomics platform. (B) Heatmaps showing scaled expression of discriminative genes for each cell type and subset.

libraries using 10× Genomics. The sequencing data were pro- expression database and subclustered immune cells into tran- cessed by CellRanger software and passed stringent, high-quality scriptionally classical subtypes (SI Appendix, Figs. S9 and S10). filtering. In total, 20 samples were sequenced, and 174,684 cells (YM, 39,828 cells; YF, 43,552 cells; OM, 47,101 cells; and OF, The Cellular Composition Changes in the Cellular Sex and Aging 44,203 cells) were collected for subsequent analyses. Ecosystem. To determine the differences between male and fe- Using t-distributed stochastic neighbor embedding (t-SNE), male PBMC immune cell composition, we matched the proportion we identified red blood cells, megakaryocytes (MEGA), CD34+ of immune cells in each group subset (Dataset S2). Generally, the cells, and five major immune cell lineages (TCs, natural killer composition of immune cell subsets in PBMCs differed by sex [NK] cells, BCs, monocytes [MCs], and dendritic cells [DCs]) via (Fig. 2A)andaging(SI Appendix,Fig.S11A). By comparing the expression of canonical lineage markers gene expression (Fig. 1B general proportions of total PBMCs, males exhibited a higher B C and SI Appendix, Figs. S1 A–C and S2–S7). For the t-SNE of general NK count (Fig. 2 ), verified by flow cytometry (Fig. 2 and SI Appendix B each individual subject, the number of cells and the mean reads ,Fig.S11 ). Consistent with a previous study (12), female TC count was higher than in males (Fig. 2A), though the per cell could be seen in SI Appendix, Fig. S8. Differentially difference was not significant. Also, MCs were more abundant in expressed genes (DEGs) of each individual subject are shown in aged PBMCs than in young groups (SI Appendix,Fig.S11C and D). Dataset S1. The detailed marker genes used can be found in our Importantly, the scRNA data affirmed that older male PBMCs also SI Appendix previous studies ( , Table S2) (13, 14). The gene ex- contained elevated MC counts compared with younger males pressions of canonical lineage markers in individual cell clusters (Fig. 2D). Flow cytometry revealed that MC count increases with on t-SNE plots are shown in SI Appendix, Fig. S1C. The four age in both males and females (Fig. 2E and SI Appendix,Fig.S11E). groups exhibited characteristic immune cell types based on The CD4 and CD8 TC populations in males and females t-SNE (SI Appendix, Fig. S1B), suggesting consistency in cell type varied greatly. Hence, aging-induced lymphopenia might be at- definition across sex and aging. Next, we analyzed the gene tributed to a decrease in CD8+ (Fig. 2 F and G) and CD4+ naïve

2of11 | PNAS Huang et al. https://doi.org/10.1073/pnas.2023216118 Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis Downloaded by guest on September 24, 2021 A % of total PBMC BCNK of total PBMCs (%) NK of total PBMCs (%) 100 cDC1 20 cDC2 40 M plasmacytoid DC pre-DC 0.045 10.8 0.022 NBC MBC PC 30 15 Age-associated BC CD14 CD16 Inter· NK1 50 NK2 20 10 NK3 F CD4 Naive CD4 Tcm 6.24 CD4 Tem CD4 Treg CD8 Naive 10 5 CD8 Tem CD8 Cytotoxic T cells CD4-CD8- CD4+CD8+ T mito

CD56 0 0 0 MF MF CD16 MF

D MC of total PBMCs (%) E MC of total PBMCs (%) YM OM 0.175 20 0.029 30 0.028 0.001 0.044 6.83 14.2 15 20 0.423

YF OF 10

10 6.41 12.8 5 IMMUNOLOGY AND INFLAMMATION

0 SSA 0 YM YF OM OF CD14 YM YF OM OF

F CD8 naive of total CD8 TC (%) G CD8 naive of total CD8 TC (%) YM OM 0.028 80 0.023 100 0.026 73.4 25.8 0.002 60 80

60 40 YF OF 74.1 44.4 40 20 20

0 0 CCR7 YM YF OM OF CD45RA YM YF OM OF

HIJTreg of total CD4 TC (%) PC of total PBMCs (%) PC of total BC (%)

15 1.5 0.024 M 8

0.023 0.023 1.28 0.023 6 10 1.0

F 4

5 0.5 4.36 2

0 0.0 0 CD20 YM YF OM OF M F CD38 MF

Fig. 2. Changes in cell proportions associated with aging and sex. (A) Relative cluster abundance in males and females. Male group includes YM (n = 5) and OM (n = 5); female group includes YF (n = 5) and OF (n = 5). (B) Percentage of NK in PBMCs between males (n = 10) and females (n = 10). (C) Flow cytometry results for the proportion of NK in PBMCs between males (n = 20) and females (n = 20). The scatter plot is a statistical representation of the results. (D) Percentage of MC in PBMCs among the four groups (n = 5, per group). (E) Flow cytometry results for the proportion of MC in PBMCs among the four groups (n = 10, per group). The scatter plot is a statistical representation of the results. (F) Percentage of naïve CD8 TC in CD8 TC among the four groups (n = 5, per group). (G) Flow cytometry results for the proportion of CD8 naïve in CD8 TC among the four groups (n = 10, per group). The scatter plot is a statistical representation of the results. (H) Percentage of Treg in CD4 TC among the four groups (n = 5, per group). (I) Percentage of PC in PBMCs between males (n = 10) and females (n = 10). (J) Flow cytometry results for the proportion of PC in BC between males (n = 20) and females (n = 20). The scatter plot is a statistical representation of the results. Two-way ANOVA was used for the differences between sexes and age groups; F statistic and P value of sex, aging, and interaction could be seen in SI Appendix, Table S3; and false discovery rate (<5%) was corrected using the Benjamini–Hochberg method.

Huang et al. PNAS | 3of11 Effects of sex and aging on the immune cell landscape as assessed by single-cell https://doi.org/10.1073/pnas.2023216118 transcriptomic analysis Downloaded by guest on September 24, 2021 TCs (SI Appendix, Fig. S12 A–C). Additionally, in contrast to the messenger RNA (mRNA) translation initiation, and cytokine reduced naïve TC count, we observed an increase of Treg pro- production (SI Appendix,Fig.S13C), suggesting enriched protein portions with aging (Fig. 2H), consistent with previous research synthesis processes in females. (15). Moreover, OM exhibited a higher proportion of prolifer- By comparing the up-regulated NK DEGs in aging individuals, ating mitotic TC (T-mito) among the TC population. Further- in the male group, aging was associated with the up-regulation of more, the sex-related differences in T-mito increased with aging ISG20, CCL4L2, DUSP2, and S100A10 (Fig. 3G). Subsequently, (SI Appendix, Fig. S12D). the biological implications of age-related DEGs were investigated Although BC populations in PBMCs were similar between the using GO and KEGG pathway analyses for each NK subset. We sexes, females had more plasma cells (PCs) than males (Fig. 2 I found that females and males underwent different immune and J). However, this trend weakened with aging, indicating changes during aging, as evidenced by the increased IFN- differential antigen–antibody responses to aging between males γ–mediated signaling pathway in males (Fig. 3H). As shown and females (SI Appendix, Fig. S12 E and F). No significant in the heatmap, inflammatory genes were primarily expressed differences in DC counts were observed between the different in males, particularly in OM (Fig. 3I). In summary, males could groups (Dataset S2). Cumulatively, these results demonstrate show an increased proinflammatory background and NK-mediated that there are differences in the composition and ratio of pe- immunity compared with females, which increased during aging. ripheral blood immune cells between males and females. Females Exhibit Up-Regulated TC- and BC-Activated Signaling. It has Aging Increases Sex Differences in Circulating Immune Cells, been reported that sex and aging play vital roles in TC devel- Particularly NK Cells. To explore the effect of sex- on age-related opment and function (17). We observed that CD4+8+ and CD8+ changes, we compared the aging DEGs between different age TC were among the subsets with the most sex-specific DEGs (Fig. groups (OM: YM and OF: YF; P adjusted < 5%) in each cell 4A). Moreover, YF CD8+ TC were enriched in several processes type (Dataset S3). Age-related transcriptional expression changes related to TC activation. Consistent with this, flow cytometry were observed in both females and males. However, the number of revealed that a higher percentage of CD4+ and CD8+ TC exhibited DEGs was amplified in males (Fig. 3A). Moreover, in males, aging expression of the early activation marker, CD69, compared with increased the expressions of aging-related genes in all immune cells male counterparts (Fig. 4 B and C and SI Appendix,Fig.S14A). In (Fig. 3B). To separate the direct effects of sex on each cell type, we contrast, older males exhibited an increased expression of genes compared the DEGs between sexes (YM: YF and OM: OF) in associated with TC receptor and MAPK signaling (Fig. 4D). YF each subset. Remarkably, the sex differences were widespread exhibited increased expression of GNLY, CCL5, NKG7,and across immune cells and generally enhanced with aging (Fig. 3C). IFITM3 in CD8+ TC (Fig. 4E). Meanwhile, older males exhibited Specifically, based on DEG analysis, sex impacted BC and MC at a increased expression of DUSP2, CXCR4, DDIT4, NFKBIA,and young age. Moreover, the number of sex-specific DEGs in NK cells JUNB in CD4+8+ and CD8+ TC (SI Appendix,Fig.S14B). was dramatically increased upon aging (Fig. 3C). Among CD4+ TC, Treg gene expression was most influenced We generated UpSet plots (16) of up-regulated (males) or by sex, with these DEGs being maintained during aging (Fig. 4F down-regulated (females), sex-related DEGs for the elderly and SI Appendix, Fig. S14C). Moreover, females showed an in- group (Fig. 3D and SI Appendix, Fig. S13A). Based on the creased enrichment of pathways that included T helper 17 cell number of DEGs, NK and MC genes showed the highest, sex- differentiation, TC activation, and IL-6 production (Fig. 4G), related differences in the elderly group (Fig. 3C). Compared with which were driven by the up-regulation of CYBA, CCR4, and OF, OM had up-regulated DDIT4, HLA-DRB5, ZFP36, JUNB, CD74 (Fig. 4H), compared with males. Compared with OM, OF and CXCR4 expression in all immune cells (Fig. 3D), indicating had an increased expression of IL-2RG (Fig. 4H), an X an increased oxidative stress and inflammatory state in males. chromosome–encoded gene that is important for TC activation Additionally, compared with OM, OF showed increased ex- and Treg homeostasis. This indicated that Treg in females might pression of CSNK2B, XIST, and EEF1G in immune cells (SI lose their immune regulatory capacity and transition into an Appendix, Fig. S13A). Generally, the results highlight the im- activated state. Additionally, we found that changes in CD4+ TC portance of sex and aging in determining the immune system. caused by aging differed between females and males, which was We determined the sex- and aging-related transcription sig- observable as an aging-induced increase in GO pathways related natures of NKs. Compared with females, we found that the ex- to mRNA stability and protein transport and increased IFN- pression of DDX3Y, HLA-B, and DUSP1 was up-regulated in γ–mediated signaling in males (SI Appendix, Fig. S14D). More- YM and OM (Fig. 3E). Meanwhile, these sex differences were over, CD4+ TC of OM were enriched in pathways related to increased in the elderly groups, as evidenced by the increased inflammation, compared with OF (SI Appendix, Fig. S14D). expression of GZMH, PRF1, JUNB, IFITM3, and DUSP2 in OM With respect to the BC compartment, the effects of sex and compared with OF (Fig. 3E). In addition, we found ARL4C, aging on gene expression were analyzed using GO and KEGG CRIP1, CYBA, RHOC, and XCL2 expression increased in OF analysis. YF showed an overrepresentation of pathways associ- compared with OM (Fig. 3E). Moreover, the NK2 (CD16high ated with BC-activated signaling compared with YM (Fig. 4I). CD56dim CD57low cytotoxic NK) was most influenced by aging, Interestingly, the pathways associated with BC activity decreased as the expression of sex DEGs in NK2 was dramatically in- with age (SI Appendix, Fig. S15A). By comparing the DEGs of creased with aging (SI Appendix, Fig. S13B). OM and OF, several inflammatory and cellular senescence genes Next, we explored the biological implications of sex DEGs were expressed at higher levels in males (Fig. 4J). BC marker (YM: YF and OM: OF) using the Gene Ontology (GO) and genes were primarily expressed in females, particularly in the YF Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway group. Conversely, genes related to inflammation were increased analyses for each NK subset. The generally up-regulated (male in OM compared with the other groups (Fig. 4K). biased) genes across subsets were enriched in relation to NK- Next, we compared the sex-related DEGs (YM: YF and OM: mediated cytotoxicity (Fig. 3F). Moreover, several pathways, such OF) and explored the cell subset–specific gene programs in BC as the IFN-γ–mediated signaling pathway, and the positive regu- subtypes. Naïve BCs (NBCs) were among the subsets with the lation of immune effector processes were up-regulated in males; most sex-specific DEGs (SI Appendix, Fig. S15B). Among the however, this difference was not observed in the young group YF-biased DEGs, we identified a range of subtype-specific gene (Fig. 3F). Meanwhile, the commonly down-regulated genes across programs, such as IFI44L and TXNIP in NBC, LTB and ZFP36 subsetswereenrichedingenesrelatedtocell–substrate junctions, in memory BC (MBC), and IFITM1 and BST2 in PC (SI

4of11 | PNAS Huang et al. https://doi.org/10.1073/pnas.2023216118 Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis Downloaded by guest on September 24, 2021 ABUp-regulated DEGs in aging Intersection of upregulated DEGs 40 39 180

OM 30 150 110 71

73 YM YM OM vs. 20 19 120 OF 18 52 DDIT4, ZFP36, HLA-DQA2 YF 90 10 8 79 7 66 5555444 24 22 59 36 1111111 60 53 17 25 8 0 40 NK 39 36 14 44 TC 30 26 24 25 BC MC DC 0 4080 0 NK TC BC DC MC Set Size CDUp-regulated DEGs in sex Intersection of upregulated DEGs 20 18 17 38 120 OM OM vs. OF 15 DDIT4, HLA-DRB5, ZFP36, JUNB, CXCR4

39 YM 11 83 OF 10 80 24 7 23 YF 55 21 62 5 4 3 46 26 2 42 111111 40 15 39 14 0 15 25 10 20 22 NK 20 TC 11 BC MC 0 DC NK TC BC DC MC 10203040 0 Set Size

EF G IMMUNOLOGY AND INFLAMMATION Sex related DEGs in NK GO analysis of upregulated Upregulated age sex related DEGs in NK subsets related DEGs in NK

DDX3Y, HLAB, DUSP1 TNF signaling pathway GZMH, PRF1, positive regulation of -LogP 39 JUNB, IFITM3, immune effector process Young Old DUSP2 peptide antigen binding 6 Female 8 7 Natural killer cell 4 31 1 mediated cytotoxicity 2 MAP kinase 0 phosphatase activity leukocyte chemotaxis Count Male male-biased 52 interferon−gamma−mediated 0 ARL4C, CRIP1, signaling pathway 2 CYBA, RHOC, cellular defense response 4 Young Old XCL2 6 Apoptosis 8 18 2 81 Antigen processing and presentation ISG20, CCL4L2, Old Young Old Young Old Young DUSP2, FGFBP2, S100A10 female-biased NK1 NK2 NK3

HIGO analysis of upregulated Gene expression in NK age related DEGs in NK subsets Z score NK3_Y 1.5 response to interferon−beta protein targeting to 1.0 -LogP YF endoplasmic reticulum 0.5 20 0.0 mRNA catabolic process 15 -0.5 mitogen−activated 10 YM protein kinase binding 5 -1.0 leukocyte cell−cell adhesion 0 -1.5 intrinsic apoptotic Count signaling pathway OF IFNγ−mediated signaling pathway 0 5 I−kappaB kinase/NF−kappaB signaling 10 cellular response to 15 20 tumor necrosis factor OM antigen receptor−mediated signaling pathway MFMFMF PRF1 JUNB NKG7 ISG20 NK1 NK2 NK3 GZMH IFITM3 DUSP2 CXCR4 CCL4L2 S100A10

Fig. 3. Changes in blood immune cell and NK cell transcriptional profiles. (A) Numbers of age-related DEGs (OM: YM and OF: YF) in the five immune cell subsets (see Dataset S4 for the gene lists). (B) Integrated comparative analysis of up-regulated DEGs in immune cell subsets between OM and YM. UpSet plots is an alternative to the Venn Diagram used to deal with more than three sets. (C) Numbers of sex-related DEGs (YM: YF and OM: OF) in the five immune cell subsets (see Dataset S5 for the gene lists). (D) Integrated comparative analysis of up-regulated DEGs in immune cell subsets between OM and OF. (E) Inte- grated comparative analysis of up-regulated (Top, male biased) and down-regulated (Bottom, female biased), sex-related DEGs in NK between young and old groups. (F) Representative GO terms and pathways enriched in up-regulated, sex-related DEGs, based on functional enrichment analysis in NK subsets of young and old. (G) Integrated comparative analysis of up-regulated, age-related DEGs in NK between females and males. (H) Representative GO terms and pathways enriched in up-regulated, age-related DEGs, based on functional enrichment analysis in NK subsets of males and females. (I) A heatmap representing scaled expression values of the 10 inflammatory genes in NK among groups.

Huang et al. PNAS | 5of11 Effects of sex and aging on the immune cell landscape as assessed by single-cell https://doi.org/10.1073/pnas.2023216118 transcriptomic analysis Downloaded by guest on September 24, 2021 A Up-regulated DEGs in TC B Activated CD4 TC of total CD4 TC (%)

100 0.034 160 OM 65.8

YM 80

120 OF 53 60 YF M 50 80 44 40 40 36 78.7 45 27 16 40 29 14 27 20 22 12 23 19 14 12 12 3 6 F 0 + + - - CD69 0 CD4 CD8 CD4 8 CD4 8 T-mito CD8 M F

C Activated CD8 TC DEGO analysis of sex related Relative expression in CD8 TC of total CD8 TC (%) DEGs in CD8 TC type I interferon CCL5 GNLY signaling pathway -LogP p<0.001 p<0.001 translational initiation 100 0.021 4 47.1 9 4 T cell receptor signaling pathway 6 2 2 80 T cell receptor complex 3 0 0 0 T cell proliferation YF YM YF YM M 60 T cell activation Count NKG7 IFITM3 0 p<0.001 40 response to interferon−beta p<0.001 64.1 2 3 MAP kinase tyrosine/serine/ 4 4 2 20 threonine phosphatase activity 6 2 C−C chemokine binding 8 1 F 0 Antigen processing 0 0 CD69 M F and presentation YF YM YF YM CD8 YF OF YM OM

FGNumbers of sex related GO analysis of sex related H Relative expression in Treg DEGs in CD4 TC DEGs in Treg CCR4 translational initiation CYBA -LogP p<0.001 p<0.001 CD4 Naive 14 24 13 36 Th17 cell differentiation 10.0 3 3 7.5 T cell activation 2 5.0 2 CD4 Tcm 16 27 11 49 RNA splicing 2.5 1 1 0.0 regulation of T 0 0 CD4 Tem cell proliferation Count YF YM YF YM 17 23 10 45 multi−organism 0 cellular process 3 CD74 IL-2RG mRNA catabolic process 6 p<0.001 p<0.001 CD4 Treg 13 25 29 45 9 interleukin−6 production 12 4 cellular response 3 2 YoungOld Young Old to interleukin−4 2 1 Antigen processing 1 Upregulated Downregulated and presentation 0 0 YF OF YM OM YF YM OF OM IJKGO analysis of sex related GO analysis of sex related Gene expression in BC DEGs in BC DEGs in BC -LogP -LogP Z score lymphocyte mediated immunity stress−activated protein 1.5 kinase signaling cascade YF 20 6 1.0 immunoglobulin production RNA splicing 0.5 15 4 0.0 humoral immune response response to 10 interferon−gamma -0.5 Fc−gamma receptor 2 YM 5 response to hypoxia -1.0 signaling pathway -1.5 defense response to bacterium 0 protein targeting to ER 0 complement activation Count classical pathway p38MAPK cascade 0 OF Fc−gamma receptor way 0 1 4 signaling pathway B cell mediated immunity Cellular senescence 2 8 3 OM B cell activation Cell adhesion molecules 12 4 MYC CD69 antigen binding XBP1 16 JUNB

C−C chemokine binding CALR MZB1 DDIT4 HLA-B 5 ZFP36 IGHG2 IFITM3 DUSP1 BACH2 CXCR4 JCHAIN

YF YM OF OM 6 NFRSF17 HLA-DQA2 T

Fig. 4. Changes in transcriptional profiles of TC and BC. (A) Numbers of sex-related DEGs (YM: YF and OM: OF) in the TC subsets (see Dataset S6 for the gene lists). (B) Flow cytometry results for the proportion of activated CD4 TC in CD4 TC between males (n = 20) and females (n = 20). The scatter plot is a statistical representation of the results. (C) Flow cytometry results for the proportion of activated CD8 TC in CD8 TC between males (n = 20) and females (n = 20). The scatter plot is a statistical representation of the results. (D) Representative GO terms and pathways enriched in sex-related DEGs, based on functional enrichment analysis in CD8 TC of the young and old groups. (E) Distribution of normalized expression levels of selected genes in the CD8+ TC cluster between YF and YM. (F) Numbers of sex-related DEGs in CD4+ TC subsets in the young and old groups. (G) Representative GO terms and pathways enriched in sex-related DEGs, based on functional enrichment analysis in Treg of the young and old groups. (H) Distribution of normalized expression levels of selected genes in Treg clusters between YF and YM, and IL2RG between OF and OM. (I) Representative GO terms and pathways enriched in sex-related DEGs, based on functional enrichment analysis in BC of the young group. (J) Representative GO terms and pathways enriched in sex-related DEGs, based on functional enrichment analysis in BC of the old group. (K) A heatmap representing scaled expression values of the seven BC markers (on the Left) and 10 inflammatory genes (on the Right) in BC among the four groups. Two-way ANOVA was used for the differences between sexes and age groups; F statistic and P value of sex, aging, and interaction could be seen in SI Appendix, Table S3; and false discovery rate (<5%) was corrected using the Benjamini–Hochberg method.

6of11 | PNAS Huang et al. https://doi.org/10.1073/pnas.2023216118 Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis Downloaded by guest on September 24, 2021 Appendix, Fig. S15C). By comparing the NBC transcriptional expression of select genes (CCL4, TNF, JUNB, and HLA-B) in the differences between males and females, we found that the IFN- OM versus OF (Fig. 5G) and observed a higher expression of these γ–mediated signaling pathway and the MAPK pathway were genes in OM, further reinforcing that certain inflammatory pro- overactive in OM than those in OF, and OF showed greater up- grams may be enhanced more in males with aging. regulation of pathways related to BCR signaling, proliferation, SI Appendix D and activation compared with OM ( , Fig. S15 ). Enhanced BAFF/APRIL System in the Cell–Cell Cross-talk of Young Furthermore, our in vitro results demonstrate that females Females. Complex cellular responses are initiated by ligand– produce more IgG following stimulation, consistent with previ- receptor binding and the subsequent activation of specific sig- SI Appendix E ous studies ( , Fig. S15 ) (12). These gene expression naling pathways. To better understand the effect of sex and age patters suggest that NBCs of males are exposed to more proin- on cellular communication, using our dataset, we conducted a flammatory cytokines in their in vivo niches, whereas female BC, bioinformatics analysis of cell–cell communication using iTALK in general, have a higher potential for activation and antibody and CellChat (18, 19), which could quantitatively characterize secretion. and compare the inferred cell–cell communication, based on the In summary, these results demonstrated the presence of sex average expression of the ligands and receptors in cell pop- differences in lymphocyte populations at single-cell resolution ulations. We compared the number of interactions between cells (i.e., females tend to have higher TC counts and up-regulated, in all groups (SI Appendix, Fig. S16A) and found that YF had BC-activated signaling, while males tend to exhibit a heightened increased cell–cell interactions between DC, TC, BC, and MC, inflammatory state). compared with the other three groups. Moreover, in the old group, the number of interactions between MC, TC, DC, and BC Sex Differences in Myeloid Cells Are Affected by Aging. Human- were higher in males compared with females. circulating myeloid cells (such as DC and MC) are the primary To compare the differences in immune cell communication cells that mediate antigen presentation and inflammatory activity. between sexes, we combined ligand–receptors and cell pairs to As reported, the capacity of DC to present antigens gradually de- form a unique identification (ID) for YF and YM groups. In YF, teriorates with age, and this is accompanied by the up-regulation of DC exhibited a higher expression of CCL3–CCR1 (Fig. 6A). In proinflammatory gene programs (14). To explore the sex-specific, addition, the females exhibited an increased expression of aging-induced changes, we studied the biological implication of ICOSLG, OSM, and their corresponding receptors among cells aging DEGs in DC (OM: YM and OF: YF), by conducting GO (Fig. 6A). In YM, IL18 and its receptors were highly expressed in the DC–BC and MC–BC cell interactions (SI Appendix, Fig. pathway analysis, and found that aging decreased pathways involved IMMUNOLOGY AND INFLAMMATION in MHC II antigen processing and presentation in females, while S16B). In addition, males showed a unique pattern of gene ex- decreasing pathways involved in the positive regulation of leukocyte pression of C3–IFITM1 in MC–TC, C3–ITGAM in MC–MC, differentiation in males (Fig. 5A). Upon comparing the down- IGF1–IGF1R in BC–BC, and IL7–IL7R in NK–TC interactions regulated, aging DEGs, we found that TXNIP and MALAT1 were (SI Appendix, Fig. S16B). down-regulated with aging in both males and females (Fig. 5B). In We then explored potential, sex-based differences in cellular addition, several antigen presentation-related genes were down- communication using CellChat, which could analyze intercellular regulated during female aging (Fig. 5C). Interestingly, the main communication networks and predict main signaling inputs and effect of aging on males was represented by increased inflammatory outputs (19). These studies suggested a potential increase in BAFF pathways, including TNF, IFN-γ, and IL-1 pathways (Fig. 5A). and APRIL signaling in females (Fig. 6 B–E and SI Appendix,Fig. These results indicate that the aging process might differentially S16 C and D), which has been shown to play a key role in the influence DC in males and females. development of autoimmunity (20). Furthermore, the network To explore the sex differences in DC-related genes or in- centrality analysis of interactions identified cDC2 as the major flammatory genes at the transcriptional level, we compared their contributor to the BAFF gene signal acting on MBC and PC (SI expression among the four groups. We found that inflammatory Appendix,Fig.S16E and F). Moreover, the autocrine BAFF sig- genes were primarily expressed in OM, while DC-related genes naling pathway was up-regulated in MBC and PC of females versus were expressed in YF (Fig. 5D). Moreover, OM showed an up- males. Flow cytometry further demonstrated that BAFFR and regulation in pathways related to p38/MAPK and cellular se- BCMA (BAFF and APRIL receptors) expression was higher in YF nescence in classical DC2 (cDC2) cells compared with OF (SI BC compared with YM BC, supporting the notion that the BAFF/ Appendix, Fig. S15F), with higher GADD45B and JUNB expres- APRIL system is enhanced in YF (Fig. 6F and SI Appendix,Fig. sion in the OM versus the other three groups (Fig. 5D). Hence, S17A). In addition, the patterns of gene expression suggested that + + sex-specific differences in DC function occur that are further CD4 and CD8 TChadincreasedinteractionwithplasmacytoid influenced by aging. MC are primarily involved in innate im- DC and PC in females and with cDC2 and pre-DC in males, in munity and mediate inflammation (14). Although the proportion relation to the IL-10 signaling pathway (SI Appendix,Fig.S17B–D). of MC was similar between sexes, differential transcriptional states This cell–cell communication analysis highlighted potential sex were observed for MC. Specifically, in relation to females, males differences in cell signaling pathways and immune cell interac- exhibited increased pathways related to innate immunity and in- tions, pointing to increased BAFF/APRIL signaling between DC flammation; this was observed in both YM and OM. Meanwhile, and BC in females, which may provide insights into the higher the aging process further enhanced these patterns, as evidenced by incidence of BC-mediated autoimmunity in females (21). up-regulated NF-κB pathway, cellular response to IL-1 pathway, and chronic inflammatory response pathway in OM compared Sex Heterogeneity in Cell–Cell Communication During Aging. Next, with OF (Fig. 5E). Additionally, aging-related changes in MC we explored the impact of age on cell–cell interactions. We showed the differences between females and males with the in- found that age was associated with the up-regulation of certain flammatory responses and pathways enhanced by aging (Fig. 5E). CCL–CCR receptor pairings in the immune cells, which differed Next, we analyzed the sex DEGs in the old group and observed between females and males. While in OF, CCL3L1 expression that all MC subtypes exhibited elevated expression of TNF, was significantly increased in CD4+ TC compared with YF, there was JUNB, and DDIT4 in OM (Fig. 5F). A range of subtype-specific no significant difference in the expression of the corresponding re- patterns were identified, including IFITM3 and S100A10 in CD14+ ceptors (SI Appendix,Fig.S18A). In contrast, CCL–CCR pairing was MC, FOS and IFITM1 in CD16+ MC, and HLA-F and NCF1 in enhanced in OM (SI Appendix,Fig.S18B). By comparing the unique intermediate MC (Fig. 5F). Subsequently, we calculated the average ID in aging, we found that females and males shared aging-specific

Huang et al. PNAS | 7of11 Effects of sex and aging on the immune cell landscape as assessed by single-cell https://doi.org/10.1073/pnas.2023216118 transcriptomic analysis Downloaded by guest on September 24, 2021 A GO analysis of age related BC DEGs in DC Downregulated DEGs in aging Relative expression in DC HLA−DRB5 HSPA5 tumor necrosis factor−mediated -LogP HLA-DRB5, HSPA5, signaling pathway 6 CLEC12A, UBC p<0.001 p<0.001 translational initiation 4 4 toll−like receptor signaling pathway 2 2 Female regulation of T cell mediated cytotoxicity 9 2 1 NIK/NF−kappaB signaling 0 0 0 Count MALAT1， MHC class II protein complex YF OF YF OF 0 5 TXNIP， PTPRC CLEC12A UBC interleukin−1−mediated signaling pathway 1 p<0.001 p<0.001 interferon−γ−mediated signaling pathway 2 3 positive regulation of 3 31 Male 2 leukocyte differentiation 4 2 Antigen processing and presentation 1 5 1 6 FMFM HLA-DQA1 0 0 Downregulated Upregulated YF OF YF OF

D Gene expression in DCE GO analysis of upregulated sex related DEGs Z score and age related DEGs in MC 1.5 YF 1.0 Toll−like receptor signaling pathway -LogP 8 0.5 TNF signaling pathway 0.0 6 NOD−like receptor signaling pathway YM -0.5 4 -1.0 NF−kappa B signaling pathway 2 -1.5 intrinsic apoptotic signaling pathway 0 OF interferon−gamma−mediated signaling pathway Count ERK1 and ERK2 cascade 0.0 OM chronic inflammatory response 2.5 5.0 Chemokine signaling pathway 7.5 IFI6 UBC IRF7 cellular response to interleukin−1 10.0 JUNB CYBA ISG15 DDIT4 HLA-B ZFP36 IFITM3 HSPA5 DUSP1 CXCR4 NFKBIA CLEC12A GADD45B HLA-DRB5 HLA-DQA2 YM:YF OM:OF OF:YF OM:YM

FGupregulated DEGs in MC subsets Relative expression in MC NFKBIA, TNF, JUNB, HLA-DRB5, DDIT4, ZFP36, LSP1, HLA-B, HLA-DQA2 CCL4 TNF JUNB HLA-B

CD14 0 CD16

14 10 15

17 1 p<0.001 p<0.001 p<0.001 p<0.001 TNFAIP3, FOS, DUSP1, 4 CCL3L1, 13 4 IFITM3, KLF6, 4 Inter IFITM1, 3 4 PSMB9, 3 3 S100A10 FCN1 2 2 2 2 1 1 1 HLA-F, CLIC3, FGFBP2, SPON2, NCF1, CTSW, HLA-DMA, LAIR2, HLA-DPA1 0 0 0 0 OF OM OF OM OF OM OF OM

Fig. 5. Changes in DC and MC transcriptional profiles. (A) Representative GO terms and pathways enriched in age-related DEGs, based on functional enrichment analysis in DC of females and males. (B) Integrated comparative analysis of down-regulated, age-related DEGs in BC between females and males. (C) Distribution of normalized expression levels of selected genes in the DC cluster between YF and OF. (D) Heatmap representing scaled expression values of the 10 inflammatory genes (on the Left) and eight DC markers (on the Right) in DC among all the groups. (E) Representative GO terms and pathways enriched in up-regulated, sex-related DEGs and age-related DEGs, based on functional enrichment analysis in MC. (F) Integrated comparative analysis of up-regulated (male biased), sex-related DEGs in MC subsets. (G) Expression of selected genes was projected onto the t-SNE plot and are shown as violin plots. Violin plots are showing the distribution of normalized expression levels of selected genes in the MC cluster between OF and OM.

8of11 | PNAS Huang et al. https://doi.org/10.1073/pnas.2023216118 Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis Downloaded by guest on September 24, 2021 cell-cell communication in YF group A CCL3_CCR1 Normalization 1.0 CCL5_CCR1 0.8 CCL5_CCR5 0.6 CCL5_CXCR3 0.4 0.2 CD24_SIGLEC10 0.0 CD40LG_CD40 ICOSLG_ICOS LTA_TNFRSF1A LTA_TNFRSF1B LTB_CD40 OSM_IL6ST TNFSF4_TNFRSF4 TC_TC BC_TC NK_TC TC_BC TC_NK DC_TC TC_DC BC_BC NK_BC DC_BC DC_NK BC_DC TC_MC MC_TC DC_DC MC_BC BC_MC NK_MC DC_MC MC_DC MC_MC BAFFR 9000 0.021 B BAFF D APRIL F - - - - CD4 8 CD4 8 + + + + CD4 8 CD4 8000 CD4 8 CD4 500 CD8 CD8 400 PC PC 300 7000 CDC1 CDC1 200

Age-associated Age-associated Count CDC2 BC BC 100 6000 CDC2 T-mito T-mito 0 -100 10 10 10 5000 MBC BAFFR YM YF MBC pre-DC pre-DC NBC NBC plasmacytoid YF groupplasmacytoid YF group DC DC YF BAFF APRIL 3.18 BCMA CE 5 0.041 CD4-8- CD4-8- CD44+8+ CD4 CD4+8+ CD4 CD8 4 CD8 PC PC 3 CDC1 IMMUNOLOGY AND INFLAMMATION CDC1 YM Age-associated Age-associated 1.96 BC 2 BC CDC2 T-mito CDC2 T-mito 1 MBC pre-DC MBC pre-DC BCMA 0 NBC plasmacytoid YM group YM group NBC plasmacytoid CD19 YM YF DC DC GIBTLA LIGHT K BMP CD16 CD16 CD16 CD4 CD14 CD4 CD14 CD4 CD14

CD4-8- PC CD4D4-8- PC CD44-8- PPC

Age-associated + + Age-associated + + CD44 8 Age-associated + + CD44 8 CD4 8 BC BC BC T-mito T-mito CD8 T-mT-mito CD8 CD8 NNBC Inter OF group InterInt OF group OF group MBC NBC MBMBC Internte NBC MBC H BTLA JLIGHT L BMP CD16 CD16 CD16 CD4 CD14 CD4 CD14 CD4 CD14

CD44-8- PC CD4-8- CD44-8- PC PC

CD44+8+ Age-associated CD44+8+ Age-associated CD44+8+ Age-associated BC BC BC T-mito T-mito T-mito CD8 CD8 CD8 InterInt NBNBC OM group OM groupIntertee OM group MBMBC InterInt NNBC NBC MBC MBC

Fig. 6. Heterogeneous, sex-based differences in cell–cell communication. (A) A heatmap depicting selected cell–cell interactions enriched in YF but absent in YM. (B) Inferred BAFF signaling networks in YF (circle plot). (C) Inferred BAFF signaling networks in YM. (D) Inferred APRIL signaling networks in YF. (E) Inferred APRIL signaling networks in YM. (F) Flow cytometry results for the proportion of BAFFR and BCMA of BC between YM and YF. The scatter plot is a statistical representation of the results. (G) Inferred BTLA signaling networks in OF. (H) Inferred BTLA signaling networks in OM. (I) Inferred LIGHT signaling networks in OF. (J) Inferred LIGHT signaling networks in OM. (K) Inferred BMP signaling networks in OF. (L) Inferred BMP signaling networks in OM.

patterns (SI Appendix,Fig.S18C). However, several interactions (SI Appendix,Fig.S18D). Additionally, the ephrin EFNB1 and its were higher in OM than OF, including IL1B–IL1R2 in MC–MC receptor (EPH family) were dominant interactions between NK and CXCL10–CXCR3 in DC–TC interactions (SI Appendix,Fig. and other cells. As the largest family of receptor tyrosine kinases, S18C). Moreover, aging resulted in interactions in males that were EPHs play a role in the development of inflammation in optic absent in females. Specifically, immune cells of OM showed in- nerve injury and atherosclerosis (22–24). creased ligand–receptor pairings that suggested the potential cel- To determine if any sex-based influences were exacerbated by lular communication pathways in OM via the chemokine family aging, we explored sex differences in signaling between OM and

Huang et al. PNAS | 9of11 Effects of sex and aging on the immune cell landscape as assessed by single-cell https://doi.org/10.1073/pnas.2023216118 transcriptomic analysis Downloaded by guest on September 24, 2021 OF using CellChat. The gene expression of BTLA and LIGHT TC-mediated immunity between males and females is mediated by signaling pathways were up-regulated in OM compared with OF the down-regulation of transcriptional processes of genes associ- (Fig. 6 G–J and SI Appendix, Fig. S19 A and B). Known to inhibit ated with TC development and activity in males. ZFP36 and BC and TC functions, BTLA and HVEM interactions inhibit TC DUSP2 were the most noticeable male-biased DEGs in various activation and cytotoxic activity and promote TC survival, pro- immune cell subsets and showed the highest expression in OM. As moting their differentiation to memory TC. The LIGHT signal- an RNA-binding protein, ZFP36 suppresses TC activation in mice ing pathway transduces proinflammatory signals to immune cells by attenuating marker gene activation, limiting TC expansion, and (25, 26). Our network centrality analysis of inferred BTLA and promoting apoptosis (31). Accordingly, the high level of ZFP36 in LIGHT interactions revealed that in OF CD4+ and CD8+ TC, all OM cell subsets partly accounts for the restrained TC immunity the gene expression patterns suggested that autocrine signaling and reduced proportions. Meanwhile, elevated expression of was increased compared with that in males. In OM, CD16+ MC DUSP2 in male TC might explain the sex differences in tumor were an important intercellular communication regulator (SI susceptibility and prognosis. Data from the Cancer Genome Atlas Appendix, Fig. S19 C–F). revealed that DUSP2 was selectively up-regulated in exhausted, Several pathways showed vast signaling interactions among tumor-infiltrating lymphocytes and associated with the poor cells, particularly MC in OM, including BMP, CD46, CD40, IL- prognosis of cancer patients. Furthermore, DUSP2high effector TC 4, IGF, and MHC II pathways (Fig. 6 K and L and SI Appendix, reportedly lose their proliferative and effector capacities, devel- Figs. S19G, S20, and S21 A and B), which regulate inflammatory oping an exhausted phenotype with impaired anti-tumor functions processes. Moreover, CellChat detected evidence of active TNF (32). Therefore, the male-biased expression of ZFP36 and DUSP2 and WNT signaling pathways in immune cells of OM (SI Appen- might account for the weaker adaptive immunity in males. dix,Fig.S21C and D) but not OF, suggesting that these classic The female bias in autoimmune disease susceptibility is well inflammatory signaling pathways were activated in aging males. documented (12). We mapped the ligand–receptor interactions In summary, cell–cell communication patterns presented dif- that mediate cell–cell communication to depict the sex-specific ferent trends in aging males and females. These communication immune pathways and found that the BAFF and APRIL path- patterns suggested increased proinflammatory signaling between ways were more up-regulated in females than males. BAFF, a MC, TC, and BC in aged males. BC-activating factor, and APRIL, a proliferation-inducing ligand, are essential for sustaining the BC pool and humoral immunity Discussion (33). The binding of BAFF and APRIL to their respective recep- Sex-based differences in the immune system widely impact the tors, BAFFR and BCMA, contributes to the pathogenesis of vari- pathology, susceptibility, and prognosis of diseases, including auto- ous BC-mediated autoimmune diseases, including SLE (34, 35), immune diseases and cancer. For example, females are less vul- IgA nephropathy (36), and Sjögren’s syndrome (37). In some pa- nerable to the severe acute respiratory syndrome coronavirus 2 virus tients, higher serum BAFF and APRIL levels are associated with and experience lower mortality compared with males, particularly in autoimmune disease severity, including elevated levels of patho- aged populations (7). However, the current understanding of the genic autoantibodies (38, 39). A genome-wide association study (40) differences between the immune systems of males and females is found that the TNFSF13B variant (encoding BAFF) causes an in- based primarily on flow cytometry and bulk sequencing data, which crease in soluble BAFF, increased BC count, and up-regulated can mask the slight yet key differences observable in small subsets. humoral immunity, which is associated with SLE. Belimumab, a The current study depicts a sex-specific immune system atlas—at BAFF inhibitor, has been approved as an add-on therapy for SLE a single-cell resolution—that analyzes the immunocyte composi- patients (41). Atacicept is a BAFF and APRIL coinhibitor that has tions and the dominating pathways in both sexes. Furthermore, we undergone phase IIb clinical trials in SLE patients (42). Thus, we explored the impact of aging on sex-specific immune responses. We propose that the up-regulated BAFF and APRIL pathways might observed that male PBMCs contained more NK cells than female be primary factors responsible for the increased incidence of certain PBMCs, whereas females had more PCs than males. Furthermore, autoimmune diseases in females than males. our pathway analysis of DEGs expression suggested that females As an inhibitory receptor widely expressed on lymphocytes and exhibit more TC and BC activity, whereas males exhibit more ac- antigen-presenting cells, BTLA (B and T lymphocyte attenuator) tivation of the IFN-γ receptor and MAPK pathways compared with provides an inhibitory signal to BC and TC upon activation by its females. Furthermore, we revealed that age exerts a more signifi- unique ligand HVEM (43, 44). BTLA activation inhibits TC cant impact on the immune system than sex, with aging further proliferation and effector function during immune responses in amplifying the observed sex-related differences. human and animal models (45, 46). Moreover, reduced BTLA Generally, the composition and proportion of immune cell levels hyperactivated TC in HIV patients (47). BTLA inhibited subsets in PBMCs are roughly the same for males and females. innate immune responses in patients with sepsis, thus promoting However, we observed a number of sex-based differences be- disease development and progression (48). Elevated levels of tween male and female PBMCs; NK cells were present at a BTLA have also been observed in various carcinomas. Hepato- higher percentage in males, and PCs were present at a higher cellular carcinoma patients’ CD4+ TCs overexpress BTLA, and percentage in females. Differences in sex hormones are the main the BTLA/HVEM blockade increases IFN-γ production (46). In determinants of the sex-biased NK proportion and function (27). gastric cancer patients, increased BTLA and HVEM levels cor- Specifically, estrogen and progesterone repress NK cells activity; relate with increased disease development and poor prognosis no effects of androgen treatment on NK cells are seen in mice (49). Additionally, in diffuse large BC lymphoma, increased (28). Moreover, higher estrogen levels and compromised NK BTLA levels impair TC cytotoxic functions and are associated cytotoxicity were observed in ovarian tumors and endometriosis with poor prognosis (45). Previous reports have found that (29). In addition, females taking oral contraceptives have a re- BTLA blockers enhance human TC responses when used alone duced proportion of NK in PBMCs (30). In the current study, we or in combination with antibodies against PD-1 (50). Consider- observed that aging was associated with a decreased TC count, ing the high correlation between BTLA expression and tumor increased MC counts, and markers of inflammation in PBMCs, development, BTLA may be a key immunotherapeutic target for which might account for the higher susceptibility of the elderly to treating cancer. We found that the aging process of the immune COVID-19 (14). We found that the aging-induced proinflammatory system, termed immunosenescence (51), was enhanced in older genes were more apparent in older males. males, characterized by low-level, systemic, and chronic inflam- The superior female adaptive immune response has been rec- mation. DEG analysis further demonstrated that the proin- ognized in previous studies (12). We propose that the disparity in flammatory genes DDIT4, HLA-DRB5, ZFP36, JUNB,andCXCR4

10 of 11 | PNAS Huang et al. https://doi.org/10.1073/pnas.2023216118 Effects of sex and aging on the immune cell landscape as assessed by single-cell transcriptomic analysis Downloaded by guest on September 24, 2021 were the main male-biased DEGs in the older group across all and written informed consent was obtained from all subjects. Detailed methods immune cell subsets. Up-regulated BTLA pathways in males, ac- are provided in SI Appendix and include the following: human subjects; single- companied by high-ZFP36 and -DUSP2 expression, might inhibit cell collection and scRNA-seq; scRNA-seq data alignment and sample aggre- TC proliferation and activation, making males potentially more gating; dimensionality reduction and clustering; differential analysis for clus- susceptible to tumorigenesis. ters and groups; gene functional annotation; cell–cell communication; flow Taken together, the results of this study have described the cytometry data generation and analyses; enzyme-linked immunosorbent assay sex-based differences in multiple facets of the immune system, data generation; statistical analysis; and data availability statement. including the cellular composition, DEGs, enriched pathways, and cell–cell communication, while also providing potential mecha- Data Availability. The fastq data have been deposited in the National Ge- nisms for sex-based differences in diseases. nomic Data Center (HRA000624). All other study data are included in the article and/or supporting information. Materials and Methods We recruited 20 healthy subjects at the Zhongshan Ophthalmic Center. Our ACKNOWLEDGMENTS. This study was supported by the National Key R&D study was approved by the Ethics Committee of Zhongshan Ophthalmic Center Program of China (Grant 2017YFA0105804).

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