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Make the most of what you’ve got: Vδ1+ γδ T cells

October 30, 2019

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

Meet the researcher

This week, first co-authors Fernanda Kyle-Cezar and Yin Wu answered our questions.

First co-authors Fernanda Kyle-Cezar and Yin Wu and lead author Adrian Hayday on the screen

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.


Wu Y., Kyle-Cezar F., Woolf R.T., Naceur-Lombardelli C., Owen J., Biswas D., Lorenc A., Vantourout P., Gazinska P., Grigoriadis A., Tutt A., Hayday A. An innate-like Vδ1+ γδ T cell compartment in the human breast is associated with remission in triple-negative breast cancer. Sci Transl Med. 2019 Oct 9.

In the Spotlight...

REVIEW: Developing neoantigen-targeted T cell-based treatments for solid tumors

Yamamoto et al. review the status of T cell-based adoptive cell transfer (ACT) for the treatment of solid tumors. Current, mostly unsatisfactory, approaches include CARs, engineered TCRs, and TIL therapies. Challenges of ACT include: identifying tumor-specific neoantigens; identifying and expanding neoantigen-reactive T cells and determining their in vivo antitumor reactivity; and maintaining the appropriate T cell phenotype during extensive expansion. The efficacy of ACT could be improved by recruiting patients soon after initial diagnosis, creating individualized TCRs, using iPSCs, reducing T cell apoptosis, and combining ACT with ICB and/or vaccines.

Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer

Joshi, de Massy, and Ismail et al. examined the heterogeneous TCR repertoire within early-stage, untreated non-small cell lung tumors, focusing on the top 1% most frequent TCR sequences. The number of expanded TCRs found ubiquitously in the tumor and the number of expanded TCRs found in only some regions correlated with the number of ubiquitous and regional mutations, respectively, suggesting a neoantigen-driven immune response. Patients with a high number of total or ubiquitous mutations had longer disease-free survival. Ubiquitous TCRs could be non-invasively detected in peripheral blood at the time of tumor resection.

Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity

To boost antitumor immunity by enhancing antigen expression, Wang and Chow et al. utilized targeted gene activating CRISPRa methodology and built multiplexed genomic and precision mutant (targeting 1116 mutated genes) CRISPRa libraries from TNBC E0771 tumors. When delivered as cell-based vaccines (prophylactic and therapeutic) or i.t. using AAV, potent antitumor efficacy was seen with all modes of delivery. Therapeutic i.t. AAV delivery was effective in three tumor types and in distant uninjected sites. Antitumor responses were T cell dependent and associated with increases in TCR diversity, T cell infiltration, and effector function.

Contributed by Katherine Turner

CLINICAL TRIAL: Pembrolizumab in Relapsed or Refractory Primary Mediastinal Large B-Cell Lymphoma

In two clinical trials (phase Ib and phase II), 74 adult patients with relapsed or refractory primary mediastinal large B cell lymphoma were treated with pembrolizumab for up to 2 years. In the phase Ib study, the ORR was 48% (33% CR), and median PFS was 10.4 months. In the phase II study, the ORR was 45% (13% CR), and median PFS was 5.5 months. Responses were durable – none of the patients with CR had progressed by data cutoff. Grade 3 or 4 treatment-related adverse events occurred in 23-24% of patients. Higher PD-L1 expression by IHC was related to the magnitude of 9p24 gene abnormality and was significantly associated with longer PFS.

Take a look at this ReFigure on this topic.

Neoantigen Dissimilarity to the Self-Proteome Predicts Immunogenicity and Response to Immune Checkpoint Blockade

Richman and Vonderheide et al. evaluated the relationship of metrics of neoantigen quality to immunogenicity, and identified dissimilarity to the non-mutated self-proteome as a key predictive determinate of neoantigen immunogenicity and of PFS to PD-1 blockade in NSCLC. A rare, unique subset of predicted, distinctly hydrophobic, immunogenic neoantigens with high dissimilarity to self-proteome was observed to correlate with clinical response independently of predicted MHC affinity. These data identify dissimilarity and high dissimilarity to self-proteome as metrics to improve selection of immunogenic neoantigens with predictive clinical activity.

Contributed by Samuel Goldman

Migratory DCs activate TGF-β to precondition naive CD8+ T cells for tissue-resident memory fate

Utilizing skin vaccination in mice that lack DCs expressing TGFβ-activating αV integrins (αV-ΔDC mice), Mani et al. show that formation of epidermal CD8+ resident memory T (eTRM) cells requires prior sustained exposure to αV+ DCs. Genome-wide chromatin accessibility analysis of resting CD44lo naive CD8+ T cells from wildtype vs αV-ΔDC mice revealed a pattern of TGFβ-induced epigenetic conditioning conducive to forming eTRm cells. Using LN-deficient mice or blocking lymphocyte tissue egress mapped eTRM differentiation capability to MHC class I-dependent interactions with migratory tissue-derived αV+ DCs that traffic via CCR7 to LNs, not spleen.

Contributed by Paula Hochman

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