Weekly Digests
‹ Back to November

The good (T cells), the bad (T cells), and the ugly (tumor)

November 28, 2018

A wide variety of factors are known to contribute to the success or failure of cancer immunotherapy. In an effort to identify specific factors, including cell states, Sade-Feldman and Yizhak et al. profiled the transcriptomes of immune cells isolated from 48 tumor samples from 32 patients with melanoma who had been treated with checkpoint blockade therapy (anti-PD-1, anti-CTLA-4, or combination) with known clinical outcomes. The results of these analyses and related functional studies were recently published in Cell.

To begin their analysis, Sade-Feldman and Yizhak et al. categorized patients and individual lesions as responders (complete response, partial response) or non-responders (progressive disease, stable disease). They then performed single-cell RNA sequencing (scRNAseq) on CD45+ immune cells, identifying 16,291 cells suitable for analysis. Unsupervised clustering of the cells identified 11 distinct clusters. In responding lesions, one cluster of B cells and one cluster of T cells were significantly enriched, while in non-responders a cluster of monocytes/macrophages, a cluster of dendritic cells, a cluster of T cells, and a mixed cluster of T and NK cells were enriched. Signatures of lymphocyte activation and exhaustion were identified in both responding and non-responding lesions both before and after therapy. All individual patients showed changes in their immune profiles between baseline and post-treatment samples, though interestingly, none of the observed changes were shared between patients.

Based on their high frequency in tumors and their known role in mediating checkpoint blockade, Sade-Feldman and Yizhak et al. focused their analysis on CD8+ T cells. Clustering of CD8+ T cells identified two distinct CD8+ T cell states: CD8_G had increased expression of genes linked to memory, activation, and cell survival and reduced expression of co-inhibitory molecules, while CD8_B was enriched for genes linked to exhaustion. While CD8_G and CD8_B cells coexisted in all lesions, CD8_G cells were enriched in responders and CD8_B cells were enriched in non-responders, and the CD8_G/CD8_B ratio could predict the regression or progression of tumors.

In an even finer analysis of clustering, CD8_G and CD8_B could be further subcategorized. Within CD8_G cells, two clusters were marked by memory and effector functions, while a third was marked by memory and early activation. Within CD8_B cells, one cluster was marked by exhaustion and cell cycle genes, another was marked by exhaustion and heat shock genes, and a third was marked by exhaustion alone.

Looking into individual markers, the researchers noted that the transcription factor TCF7 was a top marker associated with responding lesions and that it was often expressed in CD8_G cells. TCF7 has previously been identified for its key role in differentiation, self-renewal, persistence of memory CD8+ T cells, and reinvigoration of exhausted CD8+ T cells in response to checkpoint blockade. In the current dataset, more TCF7+CD8+ T cells were found in responding samples and more TCF7-CD8+ T cells were found in non-responding samples, and a higher TCF7+CD8+/TCF7-CD8+ ratio corresponded with longer survival, indicating that TCF7 expression in CD8+ T cells was associated with response to checkpoint blockade and survival.

Another marker noted by researchers in this study was CD39, which was identified as an exhaustion marker that was co-expressed with TIM-3. CD39+TIM3+CD8+ T cells showed reduced expression of TNFα and IFNγ and reduced antitumor efficacy ex vivo compared to CD39-TIM3-CD8+ T cells (which also expressed TCF7). In a mouse model of melanoma, dual blockade of CD39 and TIM3 reduced tumor size and enhanced survival compared to either monotherapy. The addition of a CD39 inhibitor to either anti-PD-1 or anti-PD-1/anti-CTLA-4 treatments also showed a synergistic effect evidenced by further reduction of tumor growth and enhanced survival. The effects of CD39 inhibition were dependent on CD8+ T cells and led to increased IFNγ production and proliferation in response to TCR stimulation.

To better understand the underlying transcriptional regulation behind the observed CD8+ T cell states, Sade-Feldman and Yizhak et al. turned to analysis of the epigenetic landscape. Patterns of open chromatin (identified by ATAC-seq) aligned with previously identified upregulated transcripts and showed that key regulatory elements and transcription factors including TCF7 and BATF regulate memory-like and exhaustion-like programs, respectively, in CD8+ T cells in human melanoma.

Finally, looking at the TCR clonal landscape, the researchers found that non-responders were enriched for persistent TCRs (observed both before and after therapy), but that those T cells did not fall into memory or effector clusters. They also observed a rise in TCR clones (typically singlets) in effector clusters that did not exist prior to therapy, indicating that lymphoid-derived T cells may play a role in effective immunotherapy. Enriched TCRs (defined as being detected in multiple T cells) were more likely to be associated with exhausted T cell phenotypes, which may suggest persistent exposure to tumor antigen. Further, identical TCRs could be found in exhausted and memory states, suggesting that T cells can transition between states.

Overall, Sade-Feldman and Yizhak et al. identified markers, transcription factors, and immune cell states – particularly CD8+ T cell states – associated with response or lack of response to immunotherapy in melanoma and identified TCF7 expression as potentially predictive of outcome. With more information about the immune contexture within melanoma and other types of tumors, researchers may be able to more effectively predict response to therapies and treat cancer.

by Lauren Hitchings


Sade-Feldman M., Yizhak K., Bjorgaard S.L., Ray J.P., de Boer C.G., Jenkins R.W., Lieb D.J., Chen J.H., Frederick D.T., Barzily-Rokni M., Freeman S.S., Reuben A., Hoover P.J., Villani A.C., Ivanova E., Portell A., Lizotte P.H., Aref A.R., Eliane J.P., Hammond M.R., Vitzthum H., Blackmon S.M., Li B., Gopalakrishnan V., Reddy S.M., Cooper Z.A., Paweletz C.P., Barbie D.A., Stemmer-Rachamimov A., Flaherty K.T., Wargo J.A., Boland G.M., Sullivan R.J., Getz G., Hacohen N. Defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma. Cell. 2018 Nov 1.

In the Spotlight...

Combination cancer immunotherapy targeting PD-1 and GITR can rescue CD8+ T cell dysfunction and maintain memory phenotype

Using bioinformatic and clustering analysis of single cell RNAseq and flow cytometry data, Wang and Zhang et al. deeply analyzed the functional/dysfunctional state of antigen-specific CD8+ T cells in two mouse tumor models following anti-PD-1 and/or agonist-GITR therapy. Combination therapy resulted in durable tumor control, enhanced T cell functionality, and uniquely induced a memory precursor T cell population. Anti-PD-1 prevented dephosphorylation of CD226 and agonist-GITR reduced surface TIGIT presence, tipping the balance of these positive and negative T cell regulators toward stimulation and memory.

Modulation of NKG2D, NKp46, and Ly49C/I facilitates natural killer cell-mediated control of lung cancer

In a murine model system in which NK cells are the dominant cytotoxic effector cells, Shi and Li et al. show that host MHC-I expression reversibly affects NK cell functionality and is required for NK cells to control MHC-1+ lung cancer. NK cell licensing by environmental MHC-I through Ly49C/I led to upregulation of NKG2D and NKp46. Stimulation of NKG2D and NKp46 through stress ligands on tumor cells resulted in shedding of Ly49C/I, eliminating interference with tumor MHC-I. Bone marrow transplant from MHC-1+/+ mice to MHC-I-/- mice enhanced NK cell activation and control of MHC-I+ tumor growth, suggesting clinical applications for re-licensing NK cells.

IFNγ-activated dermal lymphatic vessels inhibit cytotoxic T cells in melanoma and inflamed skin

Using PD-L1-/- bone marrow chimeras, Lane et al. found that both hematopoietic and non-hematopoietic PD-L1 expression contributes to immunosuppression in mouse melanoma. Notably, lymphatic endothelial cells (LECs) locally upregulate PD-L1 in cancer and viral infection in response to CD8+ T cells. Mice with LEC-specific IFNγR knockout showed reduced LEC PD-L1 expression and increased T cell infiltrate, resulting in elevated pathology in infection and improved tumor control. In human melanoma, lymphatic cell and CTL gene expression correlated, and CD8+ T cells spatially organized with lymphatic vessels in the tumor periphery.

IL-15 enhanced antibody-dependent cellular cytotoxicity mediated by NK cells and macrophages

To enhance the clinical efficacy of ADCC-inducing antibodies, Zhang and Wen et al. evaluated the common γ-chain cytokine IL-15, which is functionally related, but not identical, to the previously studied IL-2. In two mouse models, combination of IL-15 administration and ADCC-inducing antibodies dramatically enhanced tumor control, which depended on NK cells, macrophages, and FcγR. IL-15 upregulated NK cell NKG2D, granzyme B, FcγRIV, and antibody-independent and -dependent cytotoxicity. Interaction with macrophages was required to optimally induce NK cells. A clinical trial of IL-15 plus alemtuzumab has been initiated.

Integrin αvβ8-expressing tumor cells evade host immunity by regulating TGF-β activation in immune cells

Takasaka et al. demonstrated that integrin αvβ8 is expressed on tumor cells, while the latent form of TGF-β, which is activated by αvβ8, is expressed on T cells and macrophages within the tumor. Antibody blockade of αvβ8 reduced tumor growth and improved survival in mice by increasing tumor apoptosis and infiltration of proinflammatory TAMs as well as IFNγ-producing CD8+ T cells and NK cells, while decreasing Tregs and angiogenesis. These effects were independent of the PD-1/PD-L1 pathway. In human epithelial cancers, high β8 expression rarely coincided with high PD-L1 expression and was associated with decreased survival.

Evolution of Metastases in Space and Time under Immune Selection

Using high-resolution sequencing and immunohistochemistry, Angelova et al. performed a longitudinal analysis on 31 metastases derived from two patients with stage IV colorectal carcinoma with long-term survival. The researchers delineated the evolution of metastases, observed significant heterogeneity between and within metastases, and determined that progression arose from immune-escaping (non-immunogenic or unedited) clones, shaped under immune pressure at different sites. For each individual metastasis, high immunoscore and immunoediting, and smaller tumor size predicted the lowest risk of recurrence.

Everything New this Week In...

Close Modal

Small change for you. Big change for us!

This Thanksgiving season, show your support for cancer research by donating your change.

In less than a minute, link your credit card with our partner RoundUp App.

Every purchase you make with that card will be rounded up and the change will be donated to ACIR.

All transactions are securely made through Stripe.