Sex bias in the development, aggressiveness, and mortality of cancer is a well documented phenomenon that cannot be fully explained by behavioral, physical, or genetic differences. Looking into potential immunological mechanisms as to how biological sex, as defined by differential sex chromosomes and gonadal hormones, influences cancer development, Kwon et al. uncovered a mechanism through which androgens, which are differentially produced in males and females, contribute to alterations in CD8⁺ T cell-mediated antitumor immunity. Their results were recently published in Science Immunology.

For their investigation, Kwon et al. focused on bladder cancer, which is both more common and more deadly in males. This phenomenon was recapitulated in mice exposed to the carcinogen N-butyl-N, despite similar rates of mutation in mice of both sexes, and in mice transplanted with syngeneic MB49 bladder cancer. Depletion of various immune cell types in mice revealed that sex bias was dependent on CD8⁺ T cells.

After showing that CD8⁺ T cells did not mediate differential outcomes due to recognition of H-Y minor histocompatibility antigens, or chromosomal differences between males and females, the researchers began investigating immune phenotypes. In male and female mice ~10 days following implantation with MC39 bladder tumors (which is about the time when sex bias becomes apparent) males had a two-fold lower frequency of polyfunctional T cells producing IFNγ, TNFα, and granzyme B. Similarly, adoptively transferred T cells underperformed in male mice, particularly when the donor T cells were also derived from males.

Single-cell RNAseq data from CD8⁺ T cells from day 10 MB49 tumors from male and female mice identified 11 T cell clusters, 4 of which were more frequent in males. Two of these male-biased clusters showed striking sex difference in gene expression patterns, including Tcf7, Sell, Bcl2, and Jun compared to female mice, where the same clusters were instead enriched for costimulatory receptors, inhibitory surface receptors, effector molecules, transcription factors, chemokines, cytokines, and migratory receptors. The other two male-biased clusters were the only two clusters that did not show signs of activation, and were instead enriched for stem-like properties, which again included Tcf7, Sell, and Bcl2. They were also enriched for a progenitor exhausted (PE) signature.

Tcf7 is a transcription factor known to antagonize effector programming early in T cell differentiation, so the researchers hypothesized that expression of Tcf7 could influence male T cells to adopt a PE phenotype, while female T cells adopt an effector-like phenotype. In pseudotime analysis, Tcf7 was expressed earlier and was more likely to be expressed and retained in male CD8⁺ T cells. Flow cytometry analysis of T cells from MB49 tumors identified one cluster of T cells that expressed TCF1, BCL2, and SLAMF6, resembling the CD8⁺ PE T cells from the scRNAseq data. This cluster showed male bias frequency that was most prominent at day 7 and continued through day 13. Further, among CD44⁺CD62L^- “antigen-experienced” cells, there was a male bias in the frequency of TCF1⁺ cells, which later manifested as TOX⁺ cells. Male tumors also showed high expression of PE-associated proteins and a protein-level PE signature.

Adoptive transfer of CD8⁺ PE T cells into Rag2^-/- mice showed that these cells had progenitor capacity, but gave rise to bona fide exhausted cells that failed to respond to restimulation. These cells showed less antitumor efficacy in males and resulted in a higher frequency of terminally exhausted T cells in male tumors. A male bias for increased terminally exhausted CD8⁺ TILs was also identified in data from both human BCC and treatment-naive NSCLC.

Using the Four Core Genotype model, which provides all 4 combinations of sex chromosome distribution and gonadal sex, the researchers showed that MB49 tumors grew more aggressively in mice with high testosterone, regardless of their sex chromosomes, suggesting that the differences in T cells may be mediated by androgens. Consistent with this, either castration, CD8⁺ T cell-specific androgen receptor (AR) knockout, or use of an AR inhibitor limited tumor aggressiveness to what is typically observed in females. In the knockout model, CD8⁺ PE and exhausted T cells were also shown to be reduced. Additionally, the efficacy of anti-PD-1 was found to be increased with concurrent castration, even in mice with well established tumors, suggesting that reducing androgen-driven T cell exhaustion sensitizes mice to checkpoint blockade.

To determine how androgen mediates changes in T cell phenotypes, the researchers analyzed CD8⁺ TILs from MB49 tumors and virus-specific CD8⁺ T cells and found that genetic, epigenetic, and molecular signatures of androgen response were uniquely enriched in Tcf7⁺ PE T cells. Investigating this further, the researchers found that AR expression decreased upon T cell activation. Androgen response signatures correlated negatively with signatures of type I IFN, a cytokine known to repress TCF1 and inhibit the formation of CD8⁺ PE T cells in MB49 tumors and human bladder cancer, BCC, and NSCLC.

Given that lack of AR expression reduced CD8⁺ PE T cells, the researchers hypothesized that AR signaling increases Tcf7 expression, inducing the associated exhaustion program. Identifying 10 sex-specific transcriptional regulators of the Tcf7 gene module in CD8⁺ PE TILs, 5 of which were unique to males, 2 of which were unique to females, and 3 of which were shared, the researchers noted that all of the male and shared transcription factors had confirmed androgen-response elements (AREs).

AR motif scanning identified 4 and 5 putative AREs within one kilobase of promoters immediately upstream of human and mouse Tcf7 transcriptional start sites, respectively. Exposure to testosterone and DHT stimulated Tcf7 promoter activity in an manner dependent on AR and one particular ARE, hARE3; induced a 7-fold increase in AR binding to the Tcf7 promoter at the hARE3 site, increased Tcf7 in male CD8⁺ T cells (which could be  blunted by type I IFN); and decreased proliferation in an AR-overexpressing Jurkat cell line, consistent with driving exhaustion.

Overall, these results uncover an important role for androgens and androgen receptor signaling in CD8⁺ T cells, promoting expression of Tcf7 and driving an exhaustion program that favors differentiation towards a PE phenotype that gives rise to terminally exhausted vs. effector cells. This effect limits antitumor immunity and response to checkpoint blockade. This research helps to explain the sex bias towards the development, aggressiveness, and mortality of cancer in males, and provides insights into novel targets that could be utilized to improve immunotherapy.

By Lauren Hitchings

Meet the researcher

This week, both first author Tony Kwon and lead senior author Zihai Li took the time to answer our questions.

What prompted you to tackle this research question?
TK: In 2016, I joined Dr. Zihai Li’s laboratory as a graduate student to pursue my passion for cancer immunology. During my studies, I was intrigued to learn that nearly all non-reproductive malignancies show a male bias in their incidence and mortality, yet the underlying immunological mechanisms were unclear. Consistent with an increasing appreciation for the role of immune system in modulating tumor development, we hypothesized that investigation of sex differences, especially at the level of tumor microenvironment, would provide fundamental insights on cancer immunity. In addition to superb mentorship from Dr. Li, I was also fortunate to have support from the co-authors (with a special shout-out to Dr. Johanna Schafer) to efficiently navigate through this new research direction in our laboratory.
ZL: My initial interest was based on a conversation I had with Dr. Xue (Sean) Li during a family trip to Boston in December 2016. He was a faculty member and developmental biologist at Boston Children’s Hospital, working on understanding the roles of chromosome-mediated epigenetic modifier, such as KDM6A, in driving sex bias in bladder cancer. I told him we must look at the immunological basis. Subsequently, he introduced to me the sex altering Four Core Genotype mice, including XX male (due to transgenic expression of Sry), and XY female mice (due to Sry knockout). At the time, my student, Hyunwoo (Tony) Kwon, had just joined my lab to work on his PhD thesis around cancer immunology. It did not take much convincing for Tony to earnestly initiate the project of deciphering the immunological basis of sex bias in cancer. We got a lot of help from Sean and our data science collaborator Dr. Dongjun Chung, both of whom are co-senior authors of this paper. It has been a fun ride ever since.

What was the most surprising finding of this study for you?
TK: In this work, we characterized sex differences in CD8⁺ T cell fate within the tumor microenvironment and an underlying androgen–AR-driven mechanism of T cell exhaustion. One of the most important findings is that ablation of the androgen–AR axis favors effector CD8⁺ T cell differentiation and potentiates the efficacy of anti-PD-1 therapy. I was quite surprised to find that genetic deletion of AR in male CD8⁺ T cells conferred tumor protection similar to that observed in wild-type females. Many outstanding questions remain with regards to AR and CD8⁺ T cells, such as how its expression is regulated and what its other downstream targets are. It has been an exciting journey to see our project based on a fundamental basic science question grow and establish androgen–AR as a possible new target for cancer immunotherapy.
ZL: Our approach was unbiased. I did not predict initially that we would zoom onto CD8⁺ T cells. I thought more about the innate contribution, such as monocyte activation in females that led to enhanced immunity. This was probably due to my earlier interests in lupus, an autoimmune condition with a profound female bias. I was thus surprised that tumor-infiltrating CD8⁺ T cells from female mice were more functional than male mice. The real surprise came after we did single-cell RNA sequencing analysis of CD8⁺ T cells from the tumor. We found the progenitor exhausted T cells were not only more enriched in males, but also has had evidence of more androgen signaling. This led us to pursue the T cell-intrinsic roles of androgen receptor in orchestrating T cell exhaustion – which turned out to be fruitful.

What was the coolest thing you’ve learned (about) recently outside of work?
TK: I like to travel, explore new cultures, and find a new hobby in the area. In Charleston, SC, I have learned to go crabbing with a hand line. It’s nice to simply sit back, enjoy the breeze and quality time with friends and family, and (sometimes) get a free dinner.
ZL: The COVID-19 pandemic, the geopolitical regionalization, and recent conflicts reminded me that it is actually the little things that matter more dearly to us. It gave me a sense of peace and fulfillment by simply strolling through the neighborhood, having a haircut in a local barber shop, and shopping in a few remaining little bookstores, etc.. Recently I developed interest in visiting some old towns in Ohio, such as Cambridge and Athens, which were both settled around the 1790s. These seemingly aimless trips somehow reminded me that our purpose in life can often be traced back to our roots.

Follow these authors on Twitter at @t_kwon92 and @Zihai.