Hijacking Sexual Immuno-Privilege in GBM—An Immuno-Evasion Strategy

Regulatory T-cells (Tregs) are immunosuppressive T-cells, which arrest immune responses to ‘Self’ tissues. Some immunosuppressive Tregs that recognize seminal epitopes suppress immune responses to the proteins in semen, in both men and women. We postulated that GBMs express reproductive-associated proteins to manipulate reproductive Tregs and to gain immune privilege. We analyzed four GBM transcriptome databases representing ≈900 tumors for hypoxia-responsive Tregs, steroidogenic pathways, and sperm/testicular and placenta-specific genes, stratifying tumors by expression. In silico analysis suggested that the presence of reproductive-associated Tregs in GBM tumors was associated with worse patient outcomes. These tumors have an androgenic signature, express male-specific antigens, and attract reproductive-associated Related Orphan Receptor C (RORC)-Treg immunosuppressive cells. GBM patient sera were interrogated for the presence of anti-sperm/testicular antibodies, along with age-matched controls, utilizing monkey testicle sections. GBM patient serum contained anti-sperm/testicular antibodies at levels > six-fold that of controls. Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are associated with estrogenic tumors which appear to mimic placental tissue. We demonstrate that RORC-Tregs drive poor patient outcome, and Treg infiltration correlates strongly with androgen levels. Androgens support GBM expression of sperm/testicular proteins allowing Tregs from the patient’s reproductive system to infiltrate the tumor. In contrast, estrogen appears responsible for MDSC/TAM immunosuppression.

The response of the patient immune system to the GBM also affects patient outcome, with poor outcome being associated with tumor infiltration of myeloid-derived suppressor cells (MDSC) [16,17], tumor-associated macrophages (TAM) [18,19] and regulatory Tcells (Tregs) [20,21]. There may be a link between GBM tumor hypoxia and MDSCs and immunosuppressive macrophages. These are typically localized uterocervically, an area characterized by low glucose, low oxygen, low pH, and high lactate levels, much like a hypoxic tumor. Many of the components that induce uterocervical colonization by MDSCs/macrophages and their immunosuppressive phenotypes are also typically found in tumors, including GBM [22].
MUC16 is a membrane-bound protein which generates a soluble mucin following proteolytic action, with the solubilized fragment sometimes called carcinoma antigen-125 (CA-125). Uterocervical CA-125 is generated in situ from MUC16 expressed in the cervix/uterus/endometrium [35] and high levels are an indicator of endometrial receptivity and a predictor of pregnancy [36]. Uterocervical CA-125 levels can be directly augmented from a sexual partner as seminal plasma has high levels of CA-125 [37]. This mucin fragment drives epitope-specific Treg infiltration and activation in normal reproductive biology and cancer, with CA-125 also expressed by ovarian tumors, generating an immunosuppressive niche [38]. The physiological function of the uterocervical RORC-Treg population is to aid the granting of immuno-privilege to 'non-Self' male-specific, partner-specific and offspring-specific seminal and gestational antigens [39][40][41]. In males, mucin expression within the reproductive tract begins at puberty, corresponding with their period of RORC-Treg colonization [42][43][44].
The generation of sex steroids by GBM and other cancers initiates expressional changes in both cancer and infiltrating immune cells. Estrogen drives the epithelial to mesenchymal transition in glioma cultures and could act in the same manner in vivo [60]. Androgen receptor is elevated in both men and women's GBM tumors and blockage of androgen signaling with enzalutamide in vitro leads to a loss of 'stemness', causing a reduction in cancer stem cell markers CD133, Nanog and Oct4 [61].
Reportage of tumor infiltration by macrophages was calculated from the averaged, normalized levels of ITGAM and CD68. Polarization into M1 and M2 states was calculated by dividing total macrophage levels with ITGAX [M1] and with CD163 (M2) transcript levels.

HiF and RORC-Treg Gene Cross Correlation, Tumor Microenvironment and Impacts GBM Patient Outcome Validation of HiF and RORC-Treg Gene Markers
There is a high degree of cross correlation between each of the five genes chosen to report HiF status across the four GBM transcriptome databases utilized.
In Supplemental Figure S1A, we show the cross-correlation plots, correlation coefficients and p values (from t-tests) of the five genes used for HiF reportage, with data taken from the Agilent (left) and Gravendeel (right) databases, respectively.
In Supplemental Figure S1B, we show the cross-correlation plots, correlation coefficients and p values (from t-tests) of the five genes we selected for RORC-Treg reportage, with data taken from the Aglient (left) and Gravendeel (right) databases, respectively.
These data clearly indicate that in these two databases, the genes chosen for the HiF and for the RORC-Treg are clearly correlated. Similar statistically significant correlations are found in the CGGA and U133 databases.
As RORC-Tregs of reproductive origin are normally associated with sperm/testicularspecific proteins and found in mucin rich tissues, we examined the cross-correlation plots, correlation coefficients and p values (from t-tests) of representative RORC-Treg-markers CTLA4 and RORC with sperm/testicular-specific and mucin transcripts, Supplemental Figure S2.
In Supplemental Figure S2, we show that in GBM tumors, the expression of sperm/testicular-specific proteins, ACRBP, SPATA12, TSSK6, HSPB9, and CATSPER1 are highly cross-correlated with each other and with the representative RORC-Treg markers CTLA4 and RORC. A similar pattern emerges when examining the cross-correlations of CTLA4 and RORC with the mucin genes, MUC5B, MUC6, MUC13, MUC16 and MUC17, Supplemental Figure S2B.
Supplemental Figures S1 and S2, drawing on representative datasets Agilent and Gravendeel, show that RORC-Tregs can be present in GBM tumors, and that levels of RORC-Tregs are strongly and significantly correlated with sperm/testicular-specific proteins and mucins.

HiF and Tregs Impacts on Survival and Tumor Microenvironment
We examined the effect of RORC-Treg infiltration in hypoxic and normoxic tumors, by gating transcriptomes for previously described quintets of genes into high/low HiF and RORC-Treg. High/low cuts in each of the gene transcripts were made so as to obtain four roughly sized groups; Treg, Treg&HiF, HiF and neither. This was achieved by utilizing four databases: The Firehose Legacy Agilent microarray (201 patients), the CGGA database (180 patients) [99], the Gravendeel database (122 patients) [100] and the Firehose Legacy U133 platform (389 patients). This stratification methodology allows for assessment of both infiltrating Tregs (a property of the patient's immune response) and tumor HiF status (derived from tumor metabolism and vascularization). Figure 1A shows four Kaplan-Meier survival curves stratified into Treg, Treg&HiF, HiF and neither categories. With each survival curve, the calculated p values and median survival (months) are indicated, along with dataset ID. We have omitted markers to identify censored patient endpoints (alive at time of archiving), but these data were used in the statistical analysis. Color-coded Kaplan-Meier survival curves of GBM patients stratified for high Treg (red), high HiF (blue), high Treg&HiF (violet), and low Treg&HiF, known as 'neither' (black), Agilent, CGGA and Gravendeel and U133 databases. All p values are statistically significant, p < 0.05, when compared to the 'neither' group, and median overall survival is indicated in months. (B) The distribution of GBM subtypes in the four stratified groups, compared with the global distribution (37% classical, 32% mesenchymal, and 31% proneural), n = 892, as pie charts. Pie charts are color-coded with classical (light blue), mesenchymal (light green) and proneural (burnt orange). p values calculated from utilizing the χ 2 -test are shown. (C) Pie charts indicating seven markers of interest, n = 892, from normalized transcripts reporting medians. From left to right; HiF and Treg gene baskets validate stratification methodology. Total microglia (MG) are highest in normoxic tumors without Tregs, whereas both inflammation and NF-kB activation markers are elevated in HiF tumors. Myeloid cell marker CD33 is elevated in hypoxic tumors. ACRBP, which cross-correlates with other sperm/testicular-specific proteins, correlates with RORC-Treg infiltration. p < 0.05 in all cases utilizing t-tests. (D) Data from U133 database, examining sex steroid and macrophage phenotypes in stratified groups in the form of pie charts. Androgen reportage (KRT37 transcript levels) associates with RORC-Tregs, whereas hypoxia correlates with estrogen reportage. Macrophage infiltration (ITGAM and CD68) tracks hypoxia, and M2/M1 polarization markers CD163 and ITGAX indicate RORC-Tregs appear to drive M1 > M2. IL10, EBI3 and CCL18 levels are biased toward hypoxic tumors. p < 0.02 in all cases utilizing t-tests. Individual t-test results in Supplemental Table S1.

Survival and HiF/RORC-Treg Status
Patients whose tumors have infiltrating RORC-Treg or the HiF phenotype have a significantly worse outcome than normoxic and non-Treg-infiltrated tumors. Patients with Warburg phenotype tumors highly infiltrated by Tregs generally tend to have the worst outcome, with median survival <50% of the neither group.
We analyzed the reportage levels of HiF and RORC-treg gene baskets from all patients in the four databases, n = 892. Stratification by HiF/Treg results in the median HiF gene-basket reportage at levels 2.4 ± 0.7 times greater in the selected HiF subgroups than normoxic subgroups. The median RORC-Treg gene basket is expressed at levels 2.3 ± 0.9 times greater in the selected Treg subgroups than the negative pair; calculated p-values are p < 10 −10 between pairs in both cases.

CMP Subtypes
The distribution of CMP-subtypes in the stratified tumors are shown in Figure 1B, along with χ 2 calculated p-values. In the hypoxic-Treg infiltrated tumors, the mesenchymal subtype dominates, in contrast to the normoxic-Treg infiltrated tumors with elevated classical and proneural sub-types.

Stratification Reportage and Microglial Phenotypes
Levels of HiF and RORC-Treg markers of the four stratified groups are displayed in Figure 1C, using pie charts derived from 892 patients (U133, Aglient, Gravendeel and CGGA datasets). Total microglia levels were elevated in the neither subgroup, the only immunological cell type correlated with improved patient outcome, p < 0.01, Figure 1C. Poor patient outcome is correlated with microglia activation state with both proinflammatory and elevated NF-κB signaling over-represented in the at-risk subgroups.

MDSCs
CD33 expression is a proxy for MDSC levels [101], and levels are overrepresented in hypoxic tumors, potentiated by RORC-Treg infiltration.

Sperm/Testicular Proteins
Li and coworkers found antibodies toward sperm-specific protein ACRBP in the serum of their glioma patients [76]. We chose to examine ACRBP as a representative of sperm-specific gene expression in GBM tumors (see Supplemental Figure S2A). Expression of ACRBP is concordant with degree of Treg infiltration, but independent of HiF status, as shown in Figure 1C.

Androgen and Estrogen Reporters and Macrophage Status
The expression of KRT37 is controlled by three androgen response elements, and transcript levels can serve as an androgen/androgen receptor signaling proxy [102][103][104][105]. GBM tumor levels of KRT37 are highly correlated with Treg infiltration. Supplemental Figure S3 shows the correlations between expression of KRT37 (i.e., androgen expression) and the RORC-Treg gene basket, using the U133 dataset. Individual RORC-Treg markers are highly correlated with KRT37, all correlations p < 0.001, suggesting androgen-driven expansion and activation of Treg populations in GBM tumors [66]. In Figure 1D, we show pie charts representing the sex-steroid reporter levels and macrophage status using the U133 dataset. Using KRT37 androgen reportage, it is clear that there is a strong correlation between androgen reportage and RORC-Tregs.
We calculated total macrophages levels using normalized levels of classical markers ITGAM and CD68. Hypoxia has been shown to aid macrophage recruitment and polarization toward the M2-phenotype [106], which is what we observe. The M2 > M1 hypoxic tumors are also noteworthy in their expression of anti-inflammatory, macrophageassociated, cytokine/chemokines IL10, EBI3 (co-protein in IL33) and CCL18.
In pursuit of a mechanistic explanation for immune responses in the subgroups, we examined the correlation of stratified subtypes with > 2200 genes. Figure 2 shows selected genes found highly expressed in the Treg, Treg&HiF and HiF subtypes (using the U133 dataset), p < 0.05. . Genes clustered and highlighted are sperm/testicular proteins, including ACRBP and gestational genes such as PSGs. The androgen-responsive gene KRT37 is associated with Tregs, as are many reproduction-associated genes, including sperm-specific, placental-associated and mucins genes. In contrast, aromatase (CYP19A1), which converts testosterone to estrogen, is associated with hypoxia. Noteworthy too is the HiF association with immunosuppressive M2 macrophage markers, including CD68, CD69, CD163, MS4A4A, and MDSC inducer MUC1. We also observe M2 macrophage cytokines CCL2 and CCL13 and IL10 which can give rise to this polarization in hypoxic GBM.
The genes are clustered into groups based on cell type and function. The Treg subtype is to the left, HiF is on the right and Treg&HiF is centered, Figure 2. Bolded for emphasis are: (a) Keratin 37, our androgen reporter, correlates with Tregs. (b) Aromatase (CYP19A1), which converts testosterone to estrogen is elevated with HiF. (c) Sperm/testicular-specific gene transcripts ACRBP, CATSPER1, LY6K, SPATA12 and THEG all correlate with Tregs.

Steroidogenesis in GMB
An examination of steroidogenic enzyme gene levels in the stratified GBM tumors, Figures 1 and 2, indicated that there were two pathways for steroidogenesis in GBM tumors: the canonical and 'backdoor' pathways. Based on this emergent property, we stratified the tumors from the four databases based on the expression of enzymes required for steroido-genesis. GBMs were first stratified by ≈50% based on the high expression of canonical steroidogenic pathway genes. Those with the canonical pathway were then subdivided, based on aromatase expression, into androgen-(with progesterone) and estrogen (with progesterone)-generating tumors. The remaining GBMs with low levels of the enzymes of the canonical pathway were then stratified into two equally sized fractions of those expressing the non-canonical 'backdoor' pathway genes, which generated dihydrotestosterone and estrogen, and the tumors which appear to be asteroidogenic (See Methods). Figure 3A shows the Kaplan-Meier survival curves of GBM patients stratified into steroidogenic phenotypes; androgen and progesterone, with low estrogen (A&P), estrogen and progesterone, with low androgen (E&P), estrogen and androgen, low progesterone (E&A), and asteroidogenic groups. Also shown is the median survival in months and the calculated p values with respect to the asteroidogenic group. Markers identifying censored patient endpoints were again omitted but used in statistical analysis. GBM steroidogenesis is highly detrimental to patient outcome, with median survival time of the E&A group only 75% that of the asteroidogenic group. Patients with A&P/E&P tumors fare worse, with only ≈60% of the longevity of the low steroid group.

CMP Subtypes
In Figure 3B, we show the distribution of CMP subgroups, stratified by steroidogenic output, capturing the role of sex steroids in GBM etiology. Levels of mesenchymal cells in the three steroidal groups are consistent with reported effects of sex steroids on epithelialmesenchymal transition. Reports state progesterone fetters the epithelial-mesenchymal transition [107], whereas androgen's effects are much weaker [108]. In contrast, estrogen was shown to drive the epithelial-mesenchymal transition in many tissues, including GBM cells [60]. We find estrogen causes over-representation of mesenchymal phenotype GBMs, whereas progesterone favors the classical CMP subgroup.

HiF and Tregs
The first two pie charts of Figure 3C show there is a small, statistically significant elevation in HiF reportage in tumors with the ability to generate androgen and estrogen, p < 0.05. Androgenic GBMs clearly show elevated levels of infiltrating RORC-Tregs, p < 0.001.

Microglial Phenotypes
The two estrogenic subtypes induce microglial proliferation and have elevated NF-κB activation markers, which matches studies performed in vitro and in vivo [109,110]. In contrast, both progesterone and androgen suppress NF-κB pathway activation with progesterone acting directly through its receptor, but with androgen indirectly acting via cytokine signaling [111,112].

MDSCs
GBM tumor infiltration by MDSCs is estrogen sensitive, with marker CD33 overrepresented, p < 0.001, Figure 3C [113,114]. Elevated expression of the estrogen signaling reporter basket of genes correlates with MDSC and TAM marker genes [115], Supplemental Figure S5, all correlations p < 0.001. This is consistent with our postulate that GBMs are steroidogenic, allowing the tumors to mimic reproductive tissues that attract either RORC-Tregs or MDSCs/TAMs. with tumors stratified by high androgen/progesterone; low estrogen (red), high estrogen/progesterone; low androgen (violet), high estrogen/androgen production; low progesterone (blue), and overall low steroid production, asteroidogenic (black). All p-values are statistically significant compared to the low group. (B) CMP subtypes of GBMs correlate with androgen, estrogen and progesterone expression. (C) Data from all four databases in the form of pie charts indicating the amount of steroidal influence on seven markers of interest, all statistically significant at p < 0.05 except microglial activation. Estrogenic GBMs have elevated HiF and androgenic GBMs have infiltrating RORC-Treg reporter levels. Estrogenic GBMs are associated with microglial infiltration and NF-κB signaling. Myeloid cell infiltration reporter CD33 tracks estrogen, with a 2:1 ratio of CD33 levels in estrogenic tumors compared with tumors with low estrogen. Sperm-specific ACRBP clearly correlates with androgenic GBM subtypes. (D) U133 database pie charts indicating that androgen and estrogen reporter genes track the steroidogenic stratification. Total macrophage levels are low in androgen and progesterone. Sex steroids clearly affect the M2/M1 polarization, with androgen driving M1 > M2, but estrogen driving M2 > M1. Levels of immunosuppressive IL10 and CCL18 correlate with estrogen, but EIB3 generation is inhibited by androgen. p < 0.02 in all cases utilizing t-tests. Individual t-test results are in Supplemental Table S2. 2.3.7. Sperm/Testicular Proteins Expression of ACRBP is concordant with the degree of steroidogenesis, Figure 3C. Expression of ACRBP is strong in all three sex-steroid groups, all statistically significantly different from the asteroidogenic group, p < 0.001, but the two androgenic types are indistinguishable from each other. This observation supports the postulate that sperm-specific proteins are expressed by GBM to lure testicular/uterocervical Treg pools. From an analysis of the relative levels of different steroidogenic enzyme transcript levels, we present the routes and relative fluxes of steroidogenesis for the A&P, E&P and E&A groups in Supplemental Figure S6. Additionally, the ability to block steroidogenesis with off-the-shelf clinical inhibitors is shown.

GBM Steroidogenesis Validation, Macrophage Infiltration and Polarization
Estrogen acts as a potent anti-inflammatory regulator in healing and causes macrophage polarization M2 > M1 [116]. Progesterone acts in a similar manner to estrogen, driving M2 > M1 polarization [117]. The E&P group has the highest fraction of both total macrophages and M2-polarized macrophages. Androgens can initially aid M2 > M1 polarization; however, prolonged androgen exposure has been shown to cause M1 > M2 macrophage polarization [118], consistent with our stratification of GBM by steroidogenesis. Macrophage levels and polarization in the four types of tumors is consistent with the reported estrogen anti-inflammatory M2-phenotype, driving also the production of Il10 and IL33, but androgen driving M2 > M1 polarization. CCL18 generation by M2 macrophages is elevated by estrogen [119], and our data suggest that it is inhibited by androgen.

Elevation of Sperm/Testicular Specific Antibodies in GBM Patients
Emboldened by this in silico data, we reasoned that GBMs shed sperm/testicularspecific proteins into the peripheral lymphatic system. In adults, the levels of antisperm/testicular antibodies in circulation are very low, as reactive B-cell lineages are either deleted or converted into Bregs, either in testicles or by the seminized cervix (Appendix A). Shed sperm/testicular-specific antigens from a GBM tumor should generate anti-sperm/testicular-specific antibodies in patient serum. It has been demonstrated that following vasectomy or vaccination, sperm introduced in the periphery induce an immune response, and so high titers of anti-sperm antibodies develop [120][121][122][123][124][125]. Men, following vasectomy, have elevated circulating anti-sperm antibodies, but do not suffer from autoimmune orchitis because testicular immune privilege is maintained by Tregs [120][121][122]126].
We devised a methodology to interrogate GBM patient serum for multiple antisperm/testicular antibodies, whereby we labeled human antibodies with specificity toward proteins present in primate testicle slices. We used primate testicle as sperm-specific proteins generally have very high rates of evolution due to sperm competition, especially in small mammals [127]. This testicular tissue was obtained from control animals from a different study that had come to an end, and we were able to obtain and fix the tissue within minutes of euthanasia.
We incubated monkey testicular slices with serum from GBM patients and agematched controls. Then, we labeled the tissue for the presence of human IgG. As a privileged organ, anti-sperm/testicular antibody binding to the monkey testicle should be low in both women and in unvasectomized men. Figure 4 shows that the levels of anti-testicular antibodies were elevated in GBM patients compared with gallbladder surgery patients. In Figure 4A, we show a plot comparing the intensity of DAB-staining intensity in anti-sperm/testicular antibodies binding to monkey testicular tissue. We find nearly sevenfold greater levels of anti-sperm/testicular antibodies in GBM patient serum compared to control serum, p < 10 −10 . Figure 4B,C allows the comparison of DAB (brown) labeling of monkey testicle with anti-sperm/testicular antibodies from the serum from two male and two female gallbladder surgery patients (B) and that from the serum of four male and four female GBM patients (C). The nuclei counter-stained blue. The images in Figure 4B,C clearly show a heterogenous pattern of patient-derived antibody testicular labeling, which can be seen in the high magnification images, suggesting that different testicular epitopes are targeted/labeled in each patient's serum. These antibodies arise from differential sperm/testicular-specific proteins expressed by an individual patients' GBM, recruiting a separate pool of reproductive Tregs. The labeling of testicular slices with serum samples taken from other GBM and control patients are shown in Supplemental Figure S7. We conclude two things: first, sperm/testicular-specific IgGs are present in GBM patient serum, and second, these antibodies arise due to sperm/testicular-specific proteins being expressed by the tumors.

Discussion
We have used a gene-basket approach to stratify GBM tumor transcriptomes, which has been used to examine the levels of specific cell types and signaling/synthetic path-ways in tumors. A drawback of this type of analysis is that although we can identify correlations between gene transcripts, we cannot be sure that the transcripts are present in the same cells. The tumor-infiltrating RORC-Tregs we have shown to be linked to poor patient outcomes and associated with tumor androgenesis were selected based on elevated levels of FOXP3, CTLA4, GITR, RORC and GATA3 transcripts in the whole tumor, not from individual T-cells. A reasonable criticism of the in silico analysis performed is that these transcripts could be expressed by different cells within the tumor, and the correlations between the transcripts are spurious, in the same manner that rates of forest fires and ice-cream sales are spuriously correlated. With respect to the identification of tumor-infiltrating RORC-Tregs, additional evidence for this supposition is that the relationship between levels of the identified infiltrating RORC-Tregs and patient outcome is consistent utilizing data drawn from four different GBM transcriptome databases. Additionally, these RORC-Tregs transcripts are not only highly cross-correlated with each other, but also highly correlated with transcripts known to be recruiters of this Treg subclass, 'male-specific' ACRBP, SPATA12, TSSK6, HSPB9 and CATSPER1, and with mucins MUC5B, MUC6, MUC13, MUC16 and MUC17.
Our in silico analysis identified tumor steroidogenesis as a means for GBM to acquire an immunosuppressive phenotype, with the generation of androgens appearing to drive RORC-Treg infiltration and the generation of estrogen driving infiltration/activation of MDSCs/TAMs. Steroidogenesis can either be supported by the GBM cells, from non-cancer cells within the tumor, or an interplay between both. It has recently been reported that tumors can cause the expression of CYP11A1 in T-cells, causing them to metabolize cholesterol to pregnenolone. This hormone is then released, resulting in immunosuppression [128]. The stratification of GBM tumors based on the synthesis of androgen and estrogen can be, and has been, validated by monitoring the transcripts of androgen-dependent (KRT37) and estrogen-dependent (THBD, THEMIS2, SERPINA1, PIK3CG and VAV1) reporter transcripts. We have presented a multiplicity of evidence for the generation of androgens by GBM tumors to be correlated with levels of RORC-Tregs, and for estrogen synthesis by these tumors to correlate with infiltration by immunosuppressive MDSCs and macrophages.
In GBM patients, we observe compartmentalization between an immune-privileged tumor microenvironment and a peripheral immune response, evident from the >6-fold increase in circulating anti-sperm/testicular antibodies. A similar discordant immune response is observed post-vasectomy. Vasectomy does not cause autoimmune orchitis, but the release of 'male-specific' proteins into the periphery causes elevated anti-sperm antibodies. Testicular Tregs maintain immune privilege following vasectomy, despite a peripheral immune response. This same immune compartmentalization appears to occur in GBM patients.
The heterogenous pattern of patient-derived antibody testicular labeling, which can be seen in the high magnification images, suggests different testicular epitopes are targeted/labeled in each patient's serum. These antibodies arise from differential sperm/testicular-specific proteins expressed by an individual patients' GBM tumors, recruiting a separate pool of reproductive Tregs. The reproductive Treg populations must be lured into the tumor microenvironment to become activated toward malespecific epitopes, and one of the most promising candidates is the mucins expressed by GBM. MUC16 has previously been shown to generate a Treg-attracting fragment after proteolysis, which has previously been shown to result in tumor aggressiveness in ovarian cancers [38].

Materials and Methods
The in silico transcriptome analysis we report is derived from four publicly available datasets using only IDH wild-type GBM tumors: the Firehose Legacy U133 (389 patients), the Firehose Legacy Agilent microarray (201 patients), the CGGA [99] database (180 patients) and the Gravendeel [100] database (122 patients). The classification of GBM sub-types refined by Wang et al. was taken from the analysis furnished by the Gliovis platform [86].

Stratification of GBM Tumors by HiF
We used a basket approach to stratify tumors into positive/negative HiF based on expression of the hypoxia-response genes: Glut1 (SLC2A1) [9], Glut3 (SLC2A3) [10,11], ADM [14], VLDLR [13] and VEGFA [12]. Normalized mRNA expression levels of the five genes were rescaled between 0 (min) and 1 (max) and then averaged. The 5-10% fraction of tumors expressing low levels of each marker were relegated into HiF negative status, and the ≈50% tumors displaying the highest average expression were deemed as HiF positive. GBM tumor expression of HIF signature gene cross-correlations is shown in Supplemental Figure S1 using data obtained from the Firehose Legacy Agilent microarray [129] and the Gravendeel databases [100] with plots generated utilizing Gliovis [86]. SLC2A1, SLC2A3, ADM, VLDLR and VEGFA expression levels in GBM populations are highly cross-correlated with the other HiF markers, with p < 0.001 in all cases. To identify HiF status, we normalized and rescaled the expression levels of the quintet; the 5-10% fraction of tumors expressing low levels of each marker were deemed HiF negative; the ≈50% tumors displaying the highest average expression were deemed as HiF positive.

Stratification of GBM Tumors by Tregs
After the transcriptiome data had been stratified into two similar groups representing hypoxic and normoxic, it was once more restratified. In the second round of stratification, the samples were divided on the basis of high/low levels of RORC-Treg markers. The GBM-infiltrating RORC-Tregs were selected on the basis of elevated expression of a 5-gene signature; FOXP3 [23], CTLA4 [24,87], GITR [20,26,88], RORC [29,45,89] and GATA3 [27,90,91]. The cross-correlations of GBM tumor expression of these Treg signature genes is shown in Supplemental Figure S2, with data obtained from the Firehose Legacy Agilent microarray and Gravendeel GBM tumor databases [100,129] and plots generated using the Gliovis platform [86]. The quintet of genes used to identify tumor infiltrating Tregs are highly cross-correlated with the other, with p < 0.001 in all cases.
To identify RORC-Treg high/low status, we normalized and rescaled the expression levels of the quintet; the 5-10% fraction of tumors expressing low levels of each marker were deemed RORC-Treg low, and the ≈50% tumors displaying the highest average expression were deemed as Treg high.

Selection of Treg&HiF and Neither Groups
After assignment of HiF and Treg status, the data fell into four distinct groups: high Treg, high HiF, high Treg&HiF, and 'neither' (low Treg and low HiF). The mRNA levels of highlighted gene expression levels with respect to the neither group is found in Supplemental Table S1A (Excel Sheet, Treg&HiF Stratification), along with the fractional size of the four groups, for analysis from the four presented databases. The normalized average HiFs are 2.4 ± 0.7 times greater in the HiF-positive groups than in the HiF-negative groups and the Treg groups are 2.3 ± 0.9 times greater in the Treg high groups than in the Treg low groups across the four databases.

Assaying Microglia Levels and Status
Given that microglia exhibit sex-specific phenotypes (Yanguas-Casás [130]), these immune cells were a major target of investigation. Tumor-infiltrating microglia and their inflammatory status were interrogated using the gene expression of 4 separate baskets, averaging the products of normalized/rescaled mRNA expression levels of each gene in a basket, with baskets drawn from transcriptome changes induced by LPS treatment or stimulation of NF-κB signaling in human microglial cultures (total MG: ADORA3, IGSF6, TBXAS1, SASH3 and P2RY13; downregulated during inflammation, P2RY12, TMEM119 and GPR34; upregulated by NF-κB: GCLC, NQO1, GCLM, NFKBIA and SLC39A8; and downregulated by NF-κB: ZDHHC22, BCL7A and GNG4) [92].

Choice of Genes for Stratification of Tumors by Steroidogenesis
We initially analyzed GBMs based on the genes of the canonical steroidogenesis pathway and planned to divide GBM tumors into androgenic, estrogenic and asteroidogenic groups. However, upon examining the outputs of androgen and estrogen reporter genes, it was evident that some GBMs had low transcript levels of critical canonical steroidogenic pathway genes, and yet appeared to be generating androgen and estrogen. This emergent property of the tumors led us to examine the non-canonic 'backdoor' steroidogenic pathway, which is utilized during gestation and in castration-resistant prostate cancers. We discovered that in some GBMs, this non-canonical pathway appears to be present, and so we used a stratification procedure that gave rise to four steroidogenic phenotypes: androgen (testosterone) with progesterone (A&P), estrogen with progesterone (E&P), estrogen with androgen (DHT) (E&A), and asteroidogenic.

Canonical Steroidogenesis Groups A&P and E&P
The A&P and E&P subgroups use the canonical steroidogenesis pathways, expressing high levels of canonical pathway genes CYP11A1, CYP17A1, HSD17B3 and HSD3B1 alongside low levels of the progesterone catabolic enzyme AKR1C1. We selected the ≈50% of tumors with high levels of the first four genes and low AKR1C1. These were then stratified based on expression of aromatase (CYP19A1), which converts testosterone into estrogen, delineating the A&P and the E&P subtypes.

Selection of Androgen/Estrogen in the Four Groups
An emergent property was apparent during an initial analysis of the steroidogenic enzyme/transporter gene levels, indicating that GBM can be stratified into 4 groups: A&P (testosterone and progesterone), E&P (estrogen and progesterone), E&A (Estrogen and dihydrotestosterone), and 'neither' (asteroidogenic). The mRNA levels with respect to the neither group are found in Supplemental Table S1B (Excel Sheet, Sex-Steroid Stratification), along with the fractional size of the four groups for each of the four databases analyzed. Normalized average androgen reportage is 2.4 ± 0.7 times greater for two androgenpositive groups than in the asteroidogenic group, and estrogen reportage in the two estrogen groups is 2.0 ± 0.6 times greater than the asteroidogenic group.

Visualization of Anti-Sperm Antibodies from Patient Sera
Unless otherwise stated, reagents were obtained from Sigma (Sigma-Aldrich, Inc., St. Louis, MO, USA). We probed primate testicle with patient serum, then visualized human antibodies with DAB staining. At necropsy, Cynomolgus monkey testicle was removed and fixed in 4% paraformaldehyde buffer for 7 days at 4 • C. The tissue was then washed and dehydrated using graded alcohol and xylene and then wax embedded. The block was sliced into 5 µm sections that were affixed to slides and dried. Slides were dewaxed four times in xylene, then twice in isopropanol, and rehydrated using graded ethanol. The slides were washed and permeabilized using Phosphate Buffered Saline (PBS, Fisher Scientific, Waltham, MA, USA) containing 0.1% Triton X-100. The slides were placed in Na-citrate buffer (100 mM, pH 6.0) and heated to <100 • C in a steamer for 30 min, followed by cooling slowly to bench temperature for epitope retrieval. After washing in PBS, endogenous peroxidase activity was eliminated using mild conditions: 1.8% H 2 O 2 for 5 min, then in 1% sodium periodate for 5 min, followed by 0.02% NaBH 4 for 2 min. Serum-Free Protein Block (Dako North America, Inc., Carpinteria, CA, USA) was applied for 1 h, and after washing, 50 µL of GBM or gallbladder surgery patient serum was applied and incubated at 4 • C overnight. After washing in PBS, 1:500 mouse anti-human IgG antibody (HP-6017 was applied and incubated for four hours. After washing, the HiDefTM HRP-polymer system (Cell Marque, Rocklin, CA, USA) was used to functionalize with peroxidase, and a DAB chromogen kit (Dako North America, Inc., Carpinteria, CA, USA) was used for visualization. Slices were then counter-stained using hematoxylin and sealed. Images were taken with a Carl Zeiss microscope (Oberkochen, Germany), and DAB labeling levels were quantified using ImageJ, as previously described [141,142]. We used an age-matched control group, as anti-sperm antibody titers have a slight age dependency [120,121]. Gallbladder surgery patient serum was used for control studies as these are demographically akin to GBM patients, and these samples were obtained from the Houston Methodist Biorepository. Anti-testicular antibodies were assayed using serum from GBM patients (10♀, 12♂) and gallbladder surgery controls (5♀, 6♂). All samples were treated simultaneously, in parallel, at each step of the immunohistochemistry process, incubated for the same time and using the same solutions.

Conclusions
Tumor hypoxia and infiltration by Tregs and also by CD33 + MDSCs are independent risk factors in GBM. Steroidogenesis is central to tumor infiltration by immunosuppressive cells. Estrogenicity creates a niche filled by MDSCs/TAMs. Androgenicity is correlated with expression of male-specific antigens and RORC-Treg infiltration and activation. These processes can potentially be halted with steroid inhibitors, improving patient out-come. Sperm/testicular-specific protein shedding by GBM can be assessed by examining levels of anti-sperm/testicular antibodies in the patient serum. These discoveries unlock novel methods for detection and aiding the treatment of GBM. The data herein strongly support the view that the main Treg subtype supporting GBM ferocity is the RORC-Treg class, a class of Tregs normally associated with mucin-rich cancers such as colorectal and pancreatic.

Acknowledgments:
We are grateful to the many patients and families who have helped us with our research. Special thanks to Jeanne Manalo and Aaron Thorne for editorial input.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Appendix A
Supplemental Text: Granting of sexual privilege. Epitope-Specific Tregs by Gender in Health and Disease: Background Immunology: Epitopes are uniquely recognized by T-cell receptors (TCRs) and B-cell receptors (BCRs), which are generated in T-and B-cells via the recombination of somatic genes [143]. Vast numbers of different TCRs and BCRs expressed by T and B cells allow these immune cells to interrogate the whole body for presence of 'non-Self'. When these receptors bind to an epitope, they either behave in a pro-or anti-inflammatory manner, dependent on the context. Naive CD4 + T cells can become anti-inflammatory regulatory T cells (Tregs) by following three steps known as signals 1, 2, and 3 [144]. Signal 1 involves presenting epitopes to the TCR on the major histocompatibility complex (MHC) class II molecule of an antigen presenting cell, typically a dendritic cell (DC) or B cell. Signal 2 must follow, for proper activation and proliferation, from costimulatory molecules on the antigenpresenting cell, for example, CD80/CD86 with CD28 expressed on the T-cell surface. Signal 3, the cytokine environment, determines classification as either pro-or anti-inflammatory. IL-10 and TGFβ drive activated CD4 + T-cells into Tregs, whereas IL-6 and IL-23 drive pro-inflammatory class-switching.
Determining 'Self' and 'non-Self': Estimates of potential numbers of TCRs produced by the recombination process range from >10 20 to 10 61 [145,146]. CD4 + T cells, generated in the bone marrow, travel to the thymus and mature, undergoing thymopoiesis. Here, medullary thymic epithelial cells (mTECs) screen candidate CD4 + T-cells for 'Self' recognition. The mTECs synthesize most 'Self' proteins produced in the body, chop them into epitopic peptides and present these epitopes on MHCII to candidate CD4 + T-cells as 'Self'. Candidate CD4 + T-cells whose TCR recognizes a proffered 'Self' epitope are either subject to clonal deletion (apoptosis) or become a Treg (provided presence of IL-2/IL-15/NFκβ signaling). The interrogation of CD4 + T-cells by the mTECs with 'Self' epitopes is the major means of suppression of autoimmune T-cell activity [147]. Negative selection failure gives rise to autoreactive CD4 + T-cells and autoimmune diseases [65]. When a B cell binds an antigen on its BCR, the antigen is internalized, directed toward lysosomes, proteolyzed into peptidyl fragments, bound to MHCII, exposed to the extracellular environment, and presented to immune cells. The proffered MHCII bound epitopes can be interrogated by the TCRs of CD4 + T-cells. If the TCR of a T cell binds to an epitope presented on MHCII, then signal one is activated. Mutualistic signaling results in proliferation of these epitopespecific BCR/TCR interactions between the B cell and CD4 + T cell, and the two cell types drive the proliferation and pro-inflammatory activation of the other. Activated B cells drive activated CD4 + T-cell class switching into pro-inflammatory T-helper cells and B-cell proliferation, and differentiation is driven by the interaction with CD4 + T-helper cells. B cells that bind and present 'Self' epitopes can give rise to autoimmune diseases. B-cell linages that recognize 'Self' can be terminated or undergo class-switching into anti-inflammatory, regulatory B cells (Bregs). This lineage-editing or class-switching generally occurs when B cells present epitopes that are recognized by the TCR of a Treg. Normally, when a Treg recognizes a presented epitope via its TCR, it responds by sending a variety of death signals toward the B cell, causing apoptosis and the termination of the B-cell lineage [148]. However, sometimes the signaling directs the B cells to undergo a class-switch, and the B cell is directed to become an anti-inflammatory Breg.
Sexual privilege in males: Prepubescent boys have B cells whose BCRs recognize 'non-Self' sperm/testicular-specific epitopes. This results in circulating anti-sperm antibodies with CD4 + T-cells that can bind sperm/testicular-specific epitopes [124]. At puberty, immune responses to 'non-Self' sperm/testicular-specific epitopes must be arrested. This is partially achieved by compartmentalization, with a blood-testis barrier created by tightjunctions between Sertoli cells. Additionally, Sertoli cells present male-specific epitopes on their MHCII [149], and drive TCR-activated CD4 + T-cell class-switching into immunosuppressive Tregs. These Tregs maintain the anti-inflammatory environment of the testicle and edit the B-cell lineages which give rise to anti-sperm antibodies. This is achieved either by lineage deletion or by class-switching into Bregs, resulting in anti-sperm antibodies disappearing after puberty [124]. The immunological 'self' is established long before puberty. At puberty, the maturation of testicles and the onset of sperm production presents boy's immune systems with a myriad of testicular and sperm specific autoantigens. If testicles are not afforded 'immune privilege', the immune system attacks the tissue, giving rise to autoimmune orchitis. This can occur spontaneously in patients with AIRE gene mutations, due to dysfunctional Tregs [150]. Tregs control tolerogenic versus autoimmune responses, with respect to sperm/testicular-specific antigens, as was demonstrated by Wheeler [122,151]. Vasectomy is a well-established form of birth control that does not lead to autoimmune orchitis, due to the presence of tolerogenic Tregs that are responsive to sperm/testicular-specific antigens [122,151,152]. Following vasectomy, the maintenance of tolerance toward sperm/testicular antigens leaking from the severed vas deferens requires a rapid response by Tregs [122,151,152]. The Tregs arrest the elevation of auto-antibodies and pathogenic CD4 + T cells. This Treg tolerance to sperm/testicular antigens can be overcome by vaccination with sperm/testicular antigens. When volunteers with androgensensitive prostate cancer were injected with human testicular homogenate prior to their scheduled orchidectomy, they quickly developed autoimmune orchitis [125]. Experimental autoimmune orchitis (EAO) in animal models are well explored and give insights into the role of Tregs in immunotolerance toward sperm/testicular-specific antigens. Immune system attacks on testicles are seen when male mice are vaccinated with testicular homogenate or with sperm/testicular-specific proteins such as Zonadhesin (ZAN). EAO is caused by the expansion of sperm/testicular-specific CD4 + T cells, sensitized toward sperm/testicular-specific epitopes, as was demonstrated by Feng and co-workers in elegant transplantation studies [153]. BALB/c donor mice were vaccinated with epididymal spermatozoa, giving rise to EAO. When sperm-specific CD4 + T cells were extracted from these animals and transferred into naïve recipient mice, these unvaccinated males then developed EAO. Vasectomy exposes sperm/testicular antigens to the general immune system, away from a site of immune privilege. This provokes a B-cell response which elevates the levels of anti-sperm/testicular-specific antibodies, but does not lead to autoimmune orchitis. In contrast, when men were vaccinated with testicular homogenate, the immune response to the large bolus of sperm/testicular antigens overwhelmed the normal Treg maintenance of testicular privilege, resulting in autoimmune orchitis.
Sexual privilege in females. Following sexual activity, a woman's reproductive tract hosts a variety of male-specific antigens present in semen. Mounting an immune response toward male-specific antigens, leading to anti-sperm antibody production, renders women infertile. The presence of sperm/testicular-specific antibodies in women is a leading cause of infertility, generally treatable with steroids [124,154,155]. Semen's effect on the cervix allows women to host 'non-Self' male-specific antigens without mounting a proinflammatory response, through bestowing privilege. Upon encounter with healthy semen, it provides an exploitable immunological-niche geared toward the generation of epitopespecific Tregs and Bregs. Seminal fluid has high levels of prostaglandins and transforming growth factor-β (TGFβ 1 , TGFβ 2 and TGFβ 3 ) [156,157], Figure A1A. The cervix can also produce immunotolerant dendritic cells (tDCs) and myeloid-derived suppressor cells (MDSCs) [158][159][160][161]. The high levels of prostaglandin E2 (PGE2) in semen have been demonstrated to cause cervical DCs to take up a tolerogenic role [162]. PGE2 signaling alters cytokine production during antigen presentation, driving the production of IL-10 and TGFβ, promoting T-cell class-switching. Elevated levels of PGE2/TGFβ elicit the release of chemokines CXCL3 and CXCL8, which signal recruitment of monocytes from the periphery. These recruited monocytes are then presented with PGE2/TGFβ and undergo class-switching, becoming tDCs [158], which further release PGE2/TGFβ and IL-10 [159]. In the semenized cervix, large amounts of IL-11 are produced by the ectocervical cells. The combination of IL-11, via GP130 activation, with PGE2/TGFβ, has been shown to drive monocytes to become MDSCs [163]. Moreover, tDCs and MDSCs interact with each other and the ectocervix to generate positive feedback cytokine signaling, with tDC and MDSC generation of PGE2, TGFβ and IL-10, to further enhance epitope-specific Treg generation. Women's dendritic cell response to sperm/testicular antigens in the presence of PGE2 gives rise to tolerogenic Treg linages, sensitized to sperm/testicular-specific epitopes [158,164]. PGE2/TGFβ in semen also cause the ectocervix's endothelial and fibroblast cells to release immunomodulators and growth factors [165]. The apoptosis of activated B cells is driven by Tregs partially through CTLA4 and partially due to death signals granzyme, adenosine and FasL. CTLA4 can pluck CD80 and CD86 ligands out of the plasma membrane of B-cells by trans-endocytosis [166], triggering B-cell apoptosis, depicted in Supplemental Review Figure 1B. This CTLA4 mechanism drives the conversion of DCs from pro-inflammatory CD80 Hi /CD86 Hi into tolerogenic DCs CD80 Low /CD86 Low cells [167].
These Tregs allow women to tolerate sperm/testicular-specific antigens which are presented to them during coitus. In addition to mediating women's tolerogenicity toward sperm/testicular-specific antigens, these Tregs also form the basis of maternal fetal tolerance [156,168]. Fetal maternal tolerance is essential for a successful pregnancy. Tregs play an essential role in pregnancy and a women's tolerance to her unborn baby, primed by seminal fluid, as described above. Natural killer [NK] cells, which are CD16-CD56 bright , CD4 + T cells, and CD8 + T cells, are all found to recognize trophoblasts that start to invade the endometrial tissue and uterine spiral artery after implantation, inducing a tolerogenic environment allowing for fetal development [169].
As is the case in men, the privilege women afford to sperm/testicular-specific antigens is conditional and women who are vaccinated with sperm/testicular-specific antigens exhibit an anti-immune response. Some 90 years ago, Baskin et al. injected women intramuscularly with their husbands' semen to cause production of anti-sperm antibodies; he proposed that this technique could be a means to allow women to control their fertility [170]. Such vaccination strategies to control fertility are being pursued currently [171].