An Inconvenient Variable: Sex Hormones and Their Impact on Responses Melissa A. Brown and Maureen A. Su This information is current as J Immunol 2019; 202:1927-1933; ; of September 26, 2021. doi: 10.4049/jimmunol.1801403 http://www.jimmunol.org/content/202/7/1927 Downloaded from References This article cites 71 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/202/7/1927.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. An Inconvenient Variable: Sex Hormones and Their Impact on T Cell Responses Melissa A. Brown* and Maureen A. Su†,‡ Epidemiologic data demonstrate sex differences in au- by female congressional representatives and scientists, that the toimmune diseases, immune responses against infec- National Institutes of Health announced a policy to ensure that tion, and antitumor immunity, and accumulating sex is considered as a biological variable and that all National evidence suggests a major role for sex hormones in me- Institutes of Health–funded preclinical studies would include diating these differences. In this study, we review recent both males and females. This policy has resulted in a wealth advances in understanding how sex hormones regulate of new data, and we are beginning to uncover the underlying T cell responses to alter susceptibility to . immune mechanisms that dictate these differences. In this Although sex hormones can directly alter tran- study, we provide a brief overview of recent advances in our Downloaded from scriptional programs of T cells, we focus in this study understanding of sex-dependent immune responses with a fo- on how sex hormones alter T cell development and cus on how sex hormones differentially regulate T lymphocytes function through their effects on thymic stromal cells to alter susceptibility to disease. and innate cell types. In addition to contributing to Sex hormones and their receptors our understanding of sex differences, these findings also http://www.jimmunol.org/ have implications for the therapeutic use of sex hor- , , and are the major gonadal mones and modulators, which are now being sex hormones (reviewed in Ref. 6). Estrogens include , prescribed to increasing numbersofpatientsforawide 17b-, and and are derived from aromatization variety of indications. The Journal of Immunology, of androgens by a single aromatase (P450aro) enzyme. P450aro is expressed in steroidogenic tissue (ovarian gran- 2019, 202: 1927–1933. ulosa cells in premenopausal women as well as the placenta during pregnancy) and in nonglandular tissue (fat and bone). ex differences, defined by distinct con- Progesterone is also produced by ovarian granulosa cells, the

tent, unique reproductive organs, and sex-determined adrenal glands, the corpus luteum during the menstrual cycle, by guest on September 26, 2021 S steroid hormone levels, dictate many aspects of our and the placenta. The major sources of androgens are the testes being. It is now well-documented that our immune responses and adrenal glands. Leydig cells of the testes are the major are critically determined by sex, as illustrated by the pre- source of testosterone in males, and zona reticularis of the ad- dominance of females with multiple autoimmune diseases renal gland produces dehydroepiandrosterone sulfate in males in which female to male ratios can approach 11:1 (1). Sex and females. Testosterone is converted to , a dimorphism in antitumor immunity and responses to more biologically active form of testosterone, by 5a-reductase in infection/vaccination are also apparent (reviewed in Ref. 2). testosterone target tissues (scalp and other peripheral tissues and For instance, in a recent study of men and women receiving male reproductive tissues). trivalent-inactivated seasonal influenza vaccine, increased The classical sex hormone receptors, the receptors proinflammatory cytokines and Ab responses were seen in (ER) ERa and ERb, the progesterone (PR) isoforms females (3). Yet, despite the preponderance of evidence, PRA and PRB, and the receptor (AR), function as disease-related studies have historically ignored the contri- hormone-activated factors that bind to hor- bution of sex (4). Males or male-derived cells have routinely mone response elements in target to elicit gene ex- been used to study many aspects of human health, and until the pression (reviewed in Ref. 7). As such, sex hormone/receptor 1990s, females of childbearing age were excluded from drug complexes can regulate transcription through direct inter- trials (5). It was not until 2015, after much lobbying, primarily actions with specific DNA sequences. Known as hormone

*Department of Microbiology and Immunology, Feinberg School of Medicine, NS079683 and R01 NS107851, the APS1 Foundation, and the Parker Institute for Northwestern University, Chicago, IL 60611; †Department of Microbiology, Im- Cancer Immunotherapy (all to M.A.S.). munology and Molecular Genetics, David Geffen School of Medicine, College of Address correspondence and reprint requests to Dr. Maureen A. Su, University of Life Sciences, University of California at Los Angeles, Los Angeles, CA 90095; and California at Los Angeles, Biomedical Sciences Research Building, 615 Charles E. Young ‡Department of Pediatrics, David Geffen School of Medicine, University of California Drive East, Los Angeles, CA 90095. E-mail address: [email protected] at Los Angeles, Los Angeles, CA 90095 Abbreviations used in this article: AIRE, ; AR, ; ORCID: 0000-0002-3680-9982 (M.A.S.). cTEC, cortical ; Dll, d-like; EAE, experimental autoimmune en- Received for publication October 17, 2018. Accepted for publication December 5, 2018. cephalomyelitis; ER, ; ILC, ; MS, multiple sclero- sis; mTEC, medullary thymic epithelial cell; PR, ; SLE, systemic This work was supported by National Institutes of Health (NIH), National Multiple lupus erythematosus; Treg, regulatory T cell; TSA, tissue self-. Sclerosis Society Grants RG 4684A5/1 and RG 5281-A-3, and NIH Grants R21 NS081598 and RO1AI12829 (all to M.A.B.) as well as NIH Grants R01 Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801403 1928 BRIEF REVIEWS: SEX HORMONES AND T CELL RESPONSES response elements, these sequences have been identified at promoters of several genes with critical roles in immune responses. For instance, the IFN-g promoter possesses four putative estrogen response elements, and 17b-estradiol drives the expression of promoter–reporter constructs in transiently transfected lymphoid cells (8). This finding suggests the possibility that higher estrogen levels in females drive in- creased T cell IFN-g production and, in this way, predispose females to IFN-g2mediated autoimmune conditions. At the same time, androgen/AR action in CD4+ T cells may also prevent autoimmunity in males by directly increasing ex- pression of Ptpn1, a,phosphatase that inhibits Th1 differen- tiation (9). Androgen/AR complexes can also directly induce anti-inflammatory IL-10 expression by CD4+ T cells, which has been proposed to underlie male protection from CNS autoimmunity (10). These findings suggest that sex differ- ences in autoimmunity may be attributed to direct alteration of T cell transcriptional profiles by sex hormones. Downloaded from It is now clear, however, that this paradigm is overly sim- FIGURE 1. Sex hormone effects on cell types. Sex hormones plistic. First, sex hormone–receptor interactions can exert their induce of double-positive (DP) progenitors. Additionally, sex effects through DNA-independent mechanisms, such as the hormones directly modulate in thymic epithelial cells. activation of cytoplasmic signal transduction pathways (11). Androgens downregulate Dll expression in cTECs to inhibit thymopoiesis GPER1, for example, is a G –coupled ER localized while upregulating Aire in mTECs to promote negative selection of self- reactive T cells. Estrogen downregulates Aire in mTECs to inhibit thy- to the cell membrane that elicits the activation of a variety mocyte negative selection. http://www.jimmunol.org/ of cytoplasmic signaling molecules, including ERK/MAPK, PKC, PI3K and cAMP (12). Moreover, ERa can also exert its effects through cytoplasmic signaling (13), and activation of In the thymus, androgens can directly mediate apoptosis of nontranscriptional signaling mechanisms have also been de- developing T cells (especially double-positive ) scribed for PR and AR (14, 15). In addition to activating signal through the release of TNF-a (26). However, sex steroid transduction cascades, sex hormones can also alter gene ex- receptor expression is significantly higher on thymic epithelial pression through their effects on epigenetic modifications (16). cells than thymocytes, and thymic atrophy has consequently

In regulatory T cells (Tregs), for example, androgen exposure been attributed to sex steroid interactions with thymic epi- by guest on September 26, 2021 alters acetylation of histone H4 at the FOXP3 locus (17). thelial cells (18). These cells can be further categorized into Second, sex hormones shape T cell responses not only through cortical thymic epithelial cells (cTECs) and medullary thymic direct effects on T cells but also indirectly through modulation of epithelial cells (mTECs), based on their anatomic localization other immune cell types. Whereas there are some notable differ- within the thymus and their distinct functions in shaping the ences between human and mouse cells in sex hormone–receptor T cell repertoire. Notably, recent studies have demonstrated expression patterns, ERs, PRs, and ARs have been identified at the that sex hormones control both cTEC and mTEC function. RNA and/or protein level not only in T cells, but also in thymic An important function of cTECs is to support thymopoiesis epithelial cells (18–20), B cells (21), dendritic cells, macro- through the expression of d-like (Dll) 4 (27). Dll4 plays a phages and monocytes, NK cells, Type 2 innate lymphoid cells nonredundant role in thymic T cell development because (ILCs), and granulocytes, including neutrophils, eosinophils, thymic epithelial cell–specific deletion of Dll4 resulted in a 2 and mast cells (reviewed in Ref. 22). In the following sections, complete block of T cell development at the CD44+ CD25 we highlight recent studies showing how sex hormones control double-negative one stage. Recently, it was reported that T cell phenotype/function during T cell development in the testosterone administration decreased Dll4 expression by thymus by modulating thymic epithelial cells. We also discuss cTECs in 3.5-wk-old mice (28). The mechanism for this the hormone-driven interactions of mast cells and ILC2s that downregulation appears to be direct because androgen re- + can alter the differentiation of CD4 T cells in the periphery. sponse elements are found in the promoter of the Dll4 gene and androgen/AR complexes were localized to the Dll4 pro- Sex hormones, thymic epithelial cells, and T cell development moter by chromatin immunoprecipitation (28). Consistent The thymus is a critical site for generating a diverse T cell with a role for androgens in downregulating Dll4 expression, repertoire while maintaining self-tolerance, and sex hor- lower Dll4 gene expression has been noted in male cTECs mones play a critical role in shaping thymic function (Fig. 1). compared with female (29). Mice heterozygous for thymic It has been recognized for decades that sex hormones influ- epithelial cell–specific Dll4 deletion showed decreased total ence the thymus: thymic enlargement occurs with castration thymic cellularity and a lack of progression of thymocytes, of male mice, whereas shrinkage occurs with androgen ad- suggesting that quantitative decreases in Dll4 expression nega- ministration (23). In parallel, increased thymic output is seen tively affect thymopoiesis. Conversely, sex steroid blockade was in hypogonadal men, and this is reversed with androgen re- associated with increased Dll4 expression, increased thymic placement therapy (24). Indeed, multiple studies have linked size, and increased numbers of all subsets. sex steroids with thymic involution that occurs with puberty These findings suggest that sex steroid ablation may be ben- and normal aging (reviewed in Ref. 25). eficial in clinical conditions characterized by loss of thymic The Journal of Immunology 1929 function. Loss of thymic function not only occurs with aging negative selection of ubiquitous self-antigen–reactive T cells but is also consequence of radiation, chemotherapy, gluco- also occurs in the thymic cortex (35). Interestingly, cTECs are corticoid administration, infection, and other states of stress. not the main APC mediating of T cells in the Decreased thymic function, particularly in the setting of bone cortex. Instead, dendritic cells appear to play a major role marrow transplantation, can be associated with increased because ablation of dendritic cells reduced the number of susceptibility to opportunistic infection and malignancy. Thus, thymocytes expressing activated caspase 3. Whether sex there is a need for therapeutic strategies that enhance thymic steroids also play a role in T cell negative selection within function in these states. Indeed, sex steroid ablation accelerated the cortex and whether this mechanism contributes to sex recovery of thymic cellularity in mice subjected to suble- differences in autoimmune predisposition is currently not thal total body irradiation and resulted in improved T cell– known and will require further study. mediated viral clearance in these mice (28). These findings add to a previous report that sex steroid blockade enhances Sex hormones, mast cells, ILC2s, and regulation of T cells in thymopoiesis and thymic output in mice after conditioning the periphery regimen–induced thymic epithelial cell damage (30, 31). In Sex hormones exert profound and direct effects on most innate this study, sex steroid blockade and keratinocyte growth factor immune cell populations. Cell development, cytokine and administration in combination had additive effects, suggesting chemokine production, expression of TLRs and PGs, migra- that this regimen may be an effective means to improve thymic tory abilities, and signaling intermediates have all been iden- function. tified as targets of sex hormone modulation in these cells Downloaded from In addition to regulating thymocyte development through (reviewed in Ref. 36). In this study, we focus on a hormone- their effects on cTECs, sex steroids also regulate mTEC mediated interaction between mast cells and ILC2s that reg- function. mTECs play a major role in the maintenance of self- ulates Th cell differentiation and contributes to protection in tolerance toward tissue-specific Ags through their expression a sex-dimorphic . of the autoimmune regulator (AIRE) gene. AIRE promotes Mast cells are tissue-resident cells that are best studied in the the promiscuous expression of tissue self- (TSAs) by context of allergic inflammation (reviewed in Ref. 37). Upon http://www.jimmunol.org/ mTECs (32), and presentation of these TSAs to developing FceRI cross-linking by IgE/Ag complexes, mast cells release T cells results in negative selection of developing T cells preformed mediators, such as histamine, lysozymes, and pro- that recognize these TSAs with high affinity. In addition to teases. Expression of newly synthesized immune modulators eliminating autoreactive T cell clones, AIRE also maintains can also be elicited by cytokine receptor and TLR activation. self-tolerance by diverting autoreactive T cells into the Treg Importantly, mast cell activation is also under the control of sex lineage. The importance of AIRE in self-tolerance is clearly hormones. Estrogen, in particular, has been implicated in the illustrated by the development of multiorgan autoimmunity increased severity of allergic disease in females (38). Estrogen in Aire-deficient patients and mice. can directly elicit mast cell degranulation corresponding to by guest on September 26, 2021 Notably, recent work has demonstrated a prime role for sex worsening of allergic asthma during high estrogen phases of the hormones in modulating Aire expression. Androgens enforce menstrual cycle in females. thymic self-tolerance mechanisms by upregulating Aire expres- ILC2s also play a seminal role in allergic inflammation and are sion in mTECs (33, 34). mTECs express AR, and androgen/AR members of a heterogeneous group of tissue-resident innate complexes bind directly to the Aire promoter to upregulate immune cells that include the noncytotoxic subsets (ILC1 and its expression (33). Increased Aire expression drives increased ILC3), as well as the cytotoxic NK cell subset (39). ILC2s are 2 TSA expression, which results in more efficient negative selec- distinguished from other ILC subsets (CD45+,lineage ,IL7R+) tion of self-reactive T cells. As a consequence, mice administered by the expression of GATA3, ST2, IL-33R, and production of dihydrotestosterone express higher levels of Aire and are pro- Th2-associated cytokines, including IL-5, IL-13, and IL-9. They tected from CNS autoimmunity in an Aire-dependent manner. are required for the development of strong Th2 responses that At the same time, estrogens inhibit thymic self-tolerance drive pathologic inflammation in allergic disease. Somewhat mechanisms by decreasing Aire expression (34). Interest- surprisingly, there is still relatively limited information regard- ingly, this inhibition does not occur through a direct effect of ing the direct effects of hormones on ILC subsets. Uterine ILC1 2 2 2 estrogen/ER on the AIRE promoter. Instead, estrogen appears subpopulations (CD45+CD3 CD19 CD11blow/ NK1.1+ to downregulate Aire expression through epigenetic effects NKp46+), distinguished by their expression of Eomes, on DNA methylation, although the mechanism by which CD49a, and CXCR6, exhibit differing abundance and tran- estrogen alters DNA methylation remains to be determined. scriptomes during prepuberty, sexual maturity, pregnancy, Estrogen administration increased the production of anti- and postpartum, suggesting hormonal exposure affects num- thyroid Abs in experimental autoimmune thyroiditis, and bers and functional potential (40). ERs and ARs are expressed thymectomy abolished this effect. Importantly, Aire ex- by ILC2s and regulate population size in the uterus and lungs pression in mice and human thymus is higher in males (41, 42). Consistent with increased allergic asthma prevalence compared with females (33, 34). Thus, sex hormone mod- in females, numbers of circulating ILC2s are higher in females ulation of Aire in the thymus is an important mechanism by compared with male asthmatics (43). In mice, postpubescent which sex bias occurs in autoimmunity: increased levels of female mice have significantly more ILC2 precursors and lung androgen and decreased levels of estrogen promote Aire ILC2s than age-matched males (42, 44, 45). expression in males to protect against autoimmunity. A sex-specific interaction between mast cells and ILC2s was Although negative selection of self-reactive T cells is clas- recently observed in studies of experimental autoimmune sically considered an event that occurs in the thymic medulla encephalomyelitis (EAE), the mouse model of the CNS au- under the control of Aire+ mTECs, it is worth noting that toimmune demyelinating disease, multiple sclerosis (MS) (46) 1930 BRIEF REVIEWS: SEX HORMONES AND T CELL RESPONSES

(Fig. 2). EAE is induced by immunization with myelin pep- for robust Th2 cell differentiation (57, 58). It is hypothesized tides in CFA or adoptive transfer of encephalitogenic T cells. that the lower testosterone levels in females are not sufficient As in MS, myelin-specific Th1 and Th17 cells enter the brain to induce a threshold amount of IL-33 production by mast and/or spinal cord and orchestrate an immune attack on the cells to trigger this IL-33/ILC2 Th2 differentiation pathway. myelin sheath, a structure that assists in nerve impulse con- Indeed, an immunization-induced increase in ILC2s in the duction, as well as on the myelin-producing oligodendrocytes. CNS is observed in males but is much reduced in females. Although most EAE studies have used C57BL/6 mice, SJL The protective consequences of this mast cell–IL-33/ILC2 mice uniquely recapitulate two important features of MS pathway was validated in experiments in which susceptible (47). First, they develop a relapsing–remitting course of dis- mice were treated with IL-33. Administration of IL-33 to ease, the most common form of MS. In addition, they exhibit female mice induces significant increases in IL-13–producing sex dimorphism: females are more susceptible to severe disease ILC2s in the lymph nodes and CNS. If given prior to dis- than males. Immunized SJL females generate a strong Th17 ease, a primary Th2 anti-myelin response is generated and anti-myelin response, but surprisingly, resistant males do EAE is diminished. Strikingly, IL-33 treatment of mice with not fail to respond but rather produce a Th2 response that established disease converts the ongoing Th17 response to is nonpathogenic in this setting (46). This difference is one dominated by Th2 cells, demonstrating the plasticity of clearly influenced by testosterone: males are more suscep- these encephalitogenic T cells, and prevents relapses (46). tible to disease after castration or treatment with AR an- The testosterone effects are direct, as shown by the ability of + tagonists (48, 49). Male mice also show increased EAE this hormone to elicit IL-33 in AR male–derived mast cell Downloaded from prevalence with advancing age corresponding to decreasing lines. However, it is likely that testosterone has other effects testosterone levels (50). Administration of testosterone to SJL during mast cell development that poise mast cells for IL-33 females prior to EAE induction attenuates the pathologic T cell expression potential, perhaps at the epigenetic level. This idea response to one that is IL-4 and IL-10 dominated (48, 51–54). is based on the observation that, although both male- and The Th2 anti-myelin response in SJL males is influenced by female-derived mast cells express the AR, testosterone elicits testosterone-dependent actions on mast cells. Within the CNS, inducible IL-33 only in male-derived cells (46). This male- http://www.jimmunol.org/ mast cells are normal residents of the meninges, brain, and and female-specific cytokine pattern, IL-33 induction in males spinal cord and are activated early postdisease induction. In and TNF/IL-1b production in females, also occurs with other females, mast cells produce proinflammatory cytokines, such as modes of mast cell activation, including FceR1 cross-linking. TNF and IL-1b, which collectively alter the normally restrictive These data indicate that the ability to express a particular blood–brain barrier (55). Mast cell–derived IL-1b also pro- array of cytokines may be programmed by prolonged sex motes T cell encephalitogenicity (56). In contrast, under the hormone exposure starting early in life and is not solely be- influence of relatively high testosterone levels, mast cell acti- cause of acute sex hormone–receptor interactions.

vation in males results in the production of IL-33, an IL-1 Other T cells may be targets of testosterone-induced IL-33 by guest on September 26, 2021 family cytokine, but not TNF or IL-1b (46). IL-33 activates production. A subset of IL-33R+ (ST2+)Tregswasrecently ILC2s to proliferate and to produce IL-13, a cytokine essential described that limits inflammation in a model of inflammatory

FIGURE 2. Proposed regulation of periph- eral T cell responses by androgens. Under conditions of relatively high testosterone in males, this hormone acts directly to induce IL-33 expression by mast cells. IL-33 activates the preferential expansion of IL-13–producing ILC2s. IL-13, in turn, drives Th2 cell differ- entiation. Normal adult females express tes- tosterone but at levels 7–8 times lower than adult males. This reduced level is below the threshold to trigger a strong IL-33 response. Exposure to high testosterone during cell de- velopment may alter the Il33 locus, increasing chromatin accessibility and expression potential in males. Testosterone promotes Treg expan- sion and function indirectly; through induction of IL-33, an ST2+ subset of Tregs undergoes differentiation and local accumulation in tis- sues. Testosterone may also influence acquisi- tion of chromatin modifications in the Foxp3 gene that promote increased Foxp3 expression. The Journal of Immunology 1931 boweldisease,anactivitydependentonIL-33(59).De- Clinical implications of sex hormones and the immune response + creases in Foxp3 Tregs also correspond with declining tes- Understanding the role of sex hormones has recently taken on tosterone levels and increased EAE severity in aging C57BL/ new urgency, given the rise in the number of patients un- 6mice(50). dergoing treatment with sex hormones and sex hormone Finally, there is evidence that the effects of sex hormones on modulators for a variety of conditions. Androgen preparations, mast cells and ILC2s are tissue specific. In contrast to the for example, are currently FDA approved for a number of increased numbers and activity of ILC2s in the CNS of males androgen deficiency syndromes (e.g., Klinefelter syndrome, after EAE induction, testosterone limits ILC2 expansion and bilateral torsion, and testicular failure because chemotherapy) activation in the lung in allergic asthma models (44). This in males. Additionally, testosterone is also being used exten- inhibitory effect is thought tocontributetothereduced sively by men with low testosterone levels because of aging, prevalence of allergic asthma in postpubescent males relative despite the lack of evidence for its safety and efficacy for this to females. condition. From 2009 to 2013, the number of patients re- ceiving a prescription for testosterone increased from 1.3 to Chromosomal and environmental contributions to sex differences 2.3 million (65). Another example is the increasing use of Although our discussion in this review has focused on aromatase inhibitors for disorders of growth and development hormonal differences, evidence now exists that other sex in children, estrogen-responsive cancers, and other indications differences also contribute to sex dimorphism in the immune (66). Given their widespread use, it is important from a response. X chromosome contributions to autoimmunity are public health perspective to understand the effects of sex Downloaded from suggested by the observation that 47XXY males with Kli- hormone modulation on multiple physiologic systems. nefelter syndrome are equally as susceptible to systemic lupus With the accumulating evidence for their importance in erythematosus (SLE) as XX females (60). In contrast, 45X regulating immune function, it is not surprising that admin- females with Turner syndrome rarely develop SLE (61). istration of sex hormone–modulating therapies have had un- Studies using uniquely informative mouse models strongly intended immune consequences. An excellent example of this is support the hypothesis that the X chromosome promotes with prostate cancer, in which androgen-ablative therapy is a http://www.jimmunol.org/ autoimmune disease susceptibility independent of hor- standard palliative treatment for advanced prostate cancer for mones (62). “Four core genotype” mice allow the effects of the purpose of depriving hormone-dependent prostate cancer the sex chromosome complement to be determined without cells of testosterone. Concomitantly, androgen ablation has been the confounding effects of gonadal type and are derived reported to increase T cell infiltration of prostate tumor tissue by crossing XX females and XY-Sry males. In XY-Sry mice, 7–28 d after administration (67), suggesting that its efficacy Sry, the testes-determining factor gene, is deleted from the Y as a treatment may also be immune dependent. In support of chromosome and inserted as a transgene onto an autosomal this, androgen ablation in a mouse model of prostate cancer chromosome. Because Sry is autosomal, the chromosomal promoted effector function of prostate-specific T cells (68). by guest on September 26, 2021 sex of these mice is independent of gonadal (hormone- Interestingly, androgen ablation has also been associated with related) sex: both XX-Sry and XY-Sry progeny are gonadal upregulation of PD-L1 on dendritic cells and PD-1 on T cell males, and mice lacking Sry, XX and XY progeny, are go- immune cells (69). Whether combination of anti–PD-1 IgG4 nadal females. Voskuhl and colleagues (63) showed that XX (pembrolizumab) and an anti-androgen (enzalutamide) will females are more susceptible to EAE and SLE than XY fe- have additive effects in prostate cancer is currently under in- male mice. Furthermore, gonadectomized XX females and vestigation (NCT02861573). Thus, understanding how sex XX-Sry males exhibit more severe disease than gonadecto- hormones affect immune responses is important for making mized XY females or XY males, suggesting X chromosome– informed decisions about the use of sex hormone modulators encoded genes do not depend on a female hormonal and for optimizing treatment strategies for immune-mediated environment to exert their influence. The mechanism by diseases. which the XX chromosome complement predisposes to autoimmunity is not completely clear, but more severe Conclusions disease in XX and XX-Sry mice was associated with in- Sex differences in immune responses are well-documented, and creasedmyeloidcellexpressionofIL-13Ralpha2,anX what underlies these mechanisms is the subject of active in- chromosome–linked gene that limits Th2 responses (63). vestigation. It is now clear that sex hormones play a major role In addition to distinct sex chromosome complements, sex in shaping T cell responses. In addition to their direct effects differences in microbiota may also underlie differences in on T cell transcriptional profiles, sex hormones also influence autoimmune susceptibility. Thisisnicelyillustratedbya T cell responses through controlling gene expression in thymic seminal study in the NOD mouse model of type 1 diabetes, epithelial cells and regulating innate immune cells. Despite which exhibits a female bias in susceptibility (64). Danska recent progress, however, multiple outstanding questions et al. (64) showed that males and females have distinct remain regarding sex hormones and their influence on im- populations of commensal bacteria in their gut. Treatment mune cells and their mediators. Estrogen effects in auto- with broad-spectrum antibiotics or housing in a germ-free immunity are highly complex, for instance, and can be either facility eliminates this sex difference, and transfer of male pro- or anti-inflammatory in different contexts. In EAE gut extracts to females confers protection. It is worth not- mouse models, estrogens have paradoxical anti-inflammatory ing that the male-associated microbes that confer protec- roles by inhibiting Th1/Th17 differentiation and decreasing tion in NOD mice appear to be androgen-dependent, chemokine expression (70, 71). In SLE models, in contrast, suggesting that hormones play a pervasive role in mediat- estrogens have apparent proinflammatory roles and are suffi- ing sex differences. cient to induce disease phenotype (72). These distinct effects 1932 BRIEF REVIEWS: SEX HORMONES AND T CELL RESPONSES may reflect differential activation of ERa versus ERb versus 24. Olsen, N. J., and W. J. Kovacs. 2011. Evidence that androgens modulate human thymic T cell output. J. Investig. Med. 59: 32–35. GPER1, although how this leads to reciprocal outcomes re- 25. Hince, M., S. Sakkal, K. Vlahos, J. Dudakov, R. Boyd, and A. Chidgey. 2008. The quires further clarification (73). Another major area of interest role of sex steroids and gonadectomy in the control of thymic involution. Cell. Immunol. 252: 122–138. is the mechanisms by which sex hormones act to influence 26. Guevara Patin˜o, J. A., M. W. Marino, V. N. Ivanov, and J. Nikolich-Zugich. 2000. immune cell development and function. For instance, one Sex steroids induce apoptosis of CD8+CD4+ double-positive thymocytes via TNF- way in which sex hormones function in immune cells is by alpha. Eur. J. Immunol. 30: 2586–2592. 27. Koch, U., E. Fiorini, R. Benedito, V. Besseyrias, K. Schuster-Gossler, M. Pierres, altering epigenetic modifications, but how sex hormones N. R. Manley, A. Duarte, H. R. Macdonald, and F. Radtke. 2008. Delta-like 4 is bring about this change remains unclear. Finally, given the the essential, nonredundant ligand for Notch1 during thymic T cell lineage com- mitment. J. Exp. Med. 205: 2515–2523. effects of sex hormones on multiple tissue types, devising 28. Velardi, E., J. J. Tsai, A. 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