Ten years ago, scientists have announced the development of a cancer immunotherapy called CAR (chimeric antigen receptor)-T, which involves genetic modification of T cells from patients, enabling them to have powerful anti-tumor effects, and then sent these genetically modified T cells back into the patient's body. Ever since its emergence, CAR-T (one of several strategies collectively referred to as "adoptive T cell metastasis") has made headlines as a new approach to cellular immunotherapy. To date, they have been most successfully used against the so-called "liquid cancers" such as leukemias and lymphomas.

Sarcomas and cancer are more resistant to these methods, in part because genetically modified T cells gradually lose their ability to fight tumors once they infiltrate into the tumor. Immunologists refer to this cellular fatigue as a T cell "exhaustion" or "dysfunction."

To understand why, Dr. Anjana Rao and Dr. Patrick Hogan of the La Jolla Institute of Immunology in the United States have published a series of papers over the past few years, reporting a transcription factor called NFAT that regulates gene expression turns on the cell's "downstream" gene expression against the tumor response, thus leading to T cell failure. One group of downstream genes encodes the transcription factor NR4A, and a former graduate student Joyce Chen has found that genetic elimination of tumor-infiltrating NR4A protein in CAR-T cells improves tumor rejection. However, the identity of other participants who play a synergistic role with NFAT and NR4A in this pathway remains unknown.

Today, in a new study, Rao Labs and Hogan Labs provide a more complete list of participants in a broad gene expression network that establishes and maintains T cell failure. The study used a mouse model to find that the removal of two new transcription factors, TOX and TOX2, by genetic means would also improve the eradication of "solid" melanoma in the CAR-T approach. It suggests that similar interventions targeting NR4A and TOX factors in patients may extend the application of CAR-T-based immunotherapy to solid tumors. The relevant research results were published online in the PNAS journal on May 31, 2019.

They first compared the gene expression profiles in normal and "depleted" T cell samples, looking for factors that were up-regulated with NR4A as co-ordinators of T cell dysfunction. Dr. Hyungseok Seo, the first author of the paper and a postdoctoral researcher at Rao Labs, said they found that two DNA-binding proteins called TOX and TOX2 are highly expressed together with the NR4A transcription factor. This finding suggests that factors such as NFAT or NR4A may control the expression of TOX.

They then reproduce the CAR-T protocol in mice: first inoculate melanoma cells into mice to establish tumors, and then infuse these mice with one of two T cell populations one week later. There are two samples: "Control" T cell samples from normal mice; T cell samples from mice that did not express TOX and TOX2 in genetically engineered T cells.

Notably, mice receiving CAR-T cell perfusion lacking TOX showed a more pronounced melanoma regression compared to mice receiving normal T cell perfusion. Furthermore, mice treated with CAR-T cells lacking TOX showed significantly increased survival, indicating that the lack of TOX factor is resistant to T cell failure and allows T cells to destroy tumor cells more efficiently.

They found that the TOX factor in combination with NFAT and NR4A promotes expression of an inhibitory receptor called PD-1. PD-1 is present on the surface of depleted T cells and sends an immunosuppressive signal. PD-1 can be blocked by a number of monoclonal antibodies called immune checkpoint inhibitors that resist immunosuppression and activate innate anti-cancer immune responses.

The convergence of TOX, NFAT and NR4A on PD-1 is both molecularly and immunologically meaningful, which will enable the integration of cellular immunotherapy and antibody immunotherapy.

Currently, CAR-T cell therapy has shown amazing effects for patients with 'liquid tumors' such as leukemia and lymphoma. However, due to T cell failure, they still cannot be used to treat patients with solid tumor. If we use small molecules to treat CAR-T cells so as to inhibit TOX or NR4A, this strategy may have a strong therapeutic effect on solid tumors such as melanoma.

Scientists at Creative Peptides are always keeping close watch on the latest development of cancer therapy.

Hyungseok Seo el al. TOX and TOX2 cooperate with NR4A transcription factors to impose CD8+ T cell exhaustion. PNAS, 2019, doi:10.1073/pnas.1905675116.

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