In order to effectively utilize immunotherapy against solid tumors, it is important to understand the underlying immune landscape of both tumors and the healthy tissues they grow in. To expand the understanding of the immune landscape in breast cancer, Wu and Kyle-Cezar et al. analyzed healthy and malignant breast samples from prophylactic and therapeutic mastectomies and identified an influential Vδ1+ γδ T cell subset, which may play a role in protection from breast cancer growth and relapse. Their results were recently published in Science Translational Medicine.
To explore the immune landscape of breast cancer, Wu and Kyle-Cezar et al. used a “grid” explant culture system to recover and characterize large numbers of lymphocytes, first from disaggregated breast tissue samples from 29 healthy subjects. Investigating the γδ T cell compartment, the researchers noticed that while most γδ T cells in peripheral blood made use of the Vδ2 TCR, γδ T cells from breast tissue tended to incorporate Vδ1 into their TCRs, as has been observed in resident γδ T cells in the skin and gut.
Looking more closely at breast Vδ1+ T cells, the researchers found that they expressed a consistent phenotype that included expression of NKG2D and CD69 and lacked CD28. Analysis of functional potential showed that upon non-specific PMA/ionomycin stimulation, these cells produced CD107a, TNF, and IFNγ, indicating a function profile skewed towards cytolysis and resembling that of CD8+ αβ T cells. Notably, breast Vδ1+ T cells cells did not produce IL-17A, which is commonly produced by γδ T cells in mice and may support tumor growth.
To determine how Vδ1+ γδ T cell are activated, Wu and Kyle-Cezar et al. tried stimulating the cells with MICA (an NKG2D ligand) in vitro. Vδ1+ γδ T cells exhibited a cytolytic response to MICA stimulation that was dependent on NKG2D, while αβ CD8+ T cells did not respond to MICA. Vδ1+ γδ T cell also responded to a combination of STAT-signaling cytokine and an IL-1 family member (IL-12 and IL-18, respectively). Overall, these results indicate that human breast Vδ1+ T cells respond in a TCR-independent, innate-like manner.
Having observed Vδ1+ γδ T cells in healthy tissue, Wu and Kyle-Cezar et al. wondered whether these cells might play a role in breast cancer. Much like in healthy breast tissue, γδ T cells from breast cancer samples preferably incorporated Vδ1 over Vδ2, and these Vδ1+ cells showed a similar phenotype and functional profile – especially when looking at paired cancer and healthy tissue samples – and similarly responded to innate signals in the absence of overt γδ TCR signaling.
Investigating whether breast γδ T cells were responsive to tumors, the researchers tested healthy tissue-derived and breast cancer-derived γδ T cells against two lines of human breast cancer, MCF7 and HCC1854. γδ T cells derived from either source were able to target and kill cancer cell targets in vitro, and this effect was at least partially mediated by NKG2D, as NKG2D blockade reduced, but did not abrogate killing by γδ T cells. Interestingly, NKG2D blockade had a stronger effect on γδ T cells derived from healthy breast tissue versus those derived from breast cancer. In the one sample for which primary breast tumor cells could be cultured, autologous tumor-derived γδ were also effective at killing cancer cells.
Given that breast γδ T cells showed antitumor efficacy, the researchers investigated whether this subset could play a role in clinical outcomes by analyzing paired healthy and malignant samples from 11 patients with triple-negative breast cancer who underwent surgery that was intended to be curative and for whom long-term follow-up data was available. Using quantitative genomic sequencing of rearranged TCRα and TCRδ chain genes, the researchers were able to infer absolute counts of αβ and γδ T cells from samples. Both T cell types were abundant in tumor tissue, and were more abundant in tumors from patients who remained in remission following surgery. αβ T cells and Vδ1+ T cells (but not total γδ T cells nor Vδ2+ T cells) both correlated with progression-free survival, but Vδ1+ T cells alone correlated with overall survival.
Further exploring the antitumor immune response, the researchers assessed TCR repertoires of tumor-infiltrating T cells. They found that while αβ T cells became more clonal in cancer, Vδ1+ T cells in tumors showed little evidence of an adaptive-driven response, supporting their innate-like role in this tissue. The researchers did not identify any shared Vδ1 sequence between donors, though there was some overlap in Vδ1 sequences between tumors and tissues within individuals.
Overall, the researchers identified an important subset of Vδ1+ γδ T cells that are resident in breast tissue, show a consistent phenotype and a cytolytic functional profile, and respond in an innate cell-like manner independent of TCR signaling. Based on their correlation with PFS, OS, and the presence of TCRα, it is possible that these cells may play a role in the control of breast cancer.
by Lauren Hitchings
This week, first co-authors Fernanda Kyle-Cezar and Yin Wu answered our questions.
What prompted you to tackle this research question?
Having previously been a rather unfashionable area of research, the field of tumour immunology has seen an exponential growth in research activity over recent years. A lot of research activity thus far has centered around the biology of tumour-reactive αβ T cells. This has led to iterative advances, but given that the immune system is multicomponent, and no cell acts in a vacuum, it is likely that other cells may also hold keys to whether or not anti-tumour responses succeed or fail. Adopting this logic, we focused on γδ T cells, whose role in human cancer is mostly unresolved, as previous clinical studies have been limited by the unavailability of appropriate technologies to rigorously identify, isolate and examine these rare cells.
Through the use of an ex vivo “grid” culture system, we knew we could extract enough of these cells in a functional state from primary tissues and tumours to allow extensive ex vivo characterisation of their phenotype and functional potential. With next generation T cell receptor (TCR) sequencing, we were confident in identifying these rare cells, with heretofore unparalleled sensitivity and specificity, from precious clinical samples. Armed with the appropriate toolbox, we wanted to see if these cells were indeed protective in human cancers because the majority of patients with triple negative breast cancers do not benefit from contemporary immunotherapies that target αβ T cells. By contrast, γδ T cell therapy remains an untapped and potentially fruitful avenue for further clinical development.
What was the most surprising finding of this study for you?
At the time we were quite surprised that Vδ1 cells expressed substantial amounts of PD-1 and yet, were very much functional in terms of effector cytokine production and cytolysis of transformed cells. Of course, with a bit more reflection, some hindsight, and the benefit of other papers in the literature describing the same phenomenon in CD8+ PD1+ tumour-infiltrating αβ T cells, this now seems quite logical. Indeed, we ourselves show that these cells are most likely responding to cancer via conserved innate signals (such as via the NKG2D receptor) as opposed to their TCRs, which are far more subject on PD-1 inhibition. On reflection, the seeming collaboration of an innate γδresponse and an adaptive αβ response was both surprising and exciting and an area we are actively following up on.
What was the coolest thing you’ve learned (about) recently outside of work?
Besides being lab colleagues, Yin and I (Fernanda) are good friends outside the lab. This summer we went to Fringe Festival in Edinburgh whilst Adrian was busy reviewing the paper. Although I have lived in the UK since 2012, it was the first time I had been to Fringe (Yin is a regular). I was really impressed with how Edinburgh was completely transformed for the festival and also with the variety and quality of the shows. We went to see an incredible number and variety of shows in just two days including comedy, theatre, acrobatics, magic and improv. It was really inspiring to see passionate artists giving it their all, day in and day out. I was particularly astonished by “Whose line is it anyway?”. I didn’t realise that they did all of their sketches completely on the fly. They must think so fast to be able to come up with such witty and entertaining acts at the drop of a hat.