Nature: The new T cell signaling checkpoint RASA2 is available! Three top research institutions found that knocking out RASA2 can inhibit T cell depletion, making T cell anti-tumor more effective and durable

*For medical professionals only

Adoptive T cell therapy based on T cells has greatly improved the treatment status of many tumors

For example, chimeric antigen receptor T cell (CAR-T) therapy, which has played a prominent role in a variety of malignant hematologic tumors, and transgenic T cell (TCR-T) therapy, which has emerged in a variety of solid tumors

However, due to the presence of various immunosuppressive factors, there are still many types of tumors, especially solid tumors, that do not respond to

Therefore, enhancing T cell function and persistence by editing specific genes in T cells is a hot topic

Recently, a research team led by Julia Carnevale, Eric Shifrut, Justin Eyquem and Alexander Marson of the UCSF Institute of Genetic Immunology, Alan Ashworth of the University of California, San Francisco, and Giedre Krenciute of St.

They found that knocking out the RASA2 gene in T cells promoted T cell activation, enhanced their antigen sensitivity, and proliferative capacity and effector function in multiple immunosuppressive environments

In addition, knockout RASA2 can inhibit the T cell depletion process induced by repeated antigen stimulation, allowing T cells to maintain tumor killing function

Overall, the study identified RASA2, a new signaling checkpoint in T cells that regulates T cell function, providing new ideas

Screenshot of the first page of the paper

Let's take a look at how Alexander Marson and Alan Ashworth's team conducted
this research.

The researchers first used their previously constructed whole-genome CRISPR knockout screening framework SLICE[4] to screen for genes that may affect human primary T cell proliferation under conditions that include five different immunosuppressive environments
.

The methods used to mimic these five immunosuppressive environments are based on two calcineurin inhibitors (tacrolimus and cyclosporine) that inhibit T cell activation,[5] the immunosuppressive cytokines TGFβ[6] and the immunosuppressive metabolite adenosine[4], and regulatory T cells (Treg) in the tumor microenvironment [7].

By comparing and analyzing sgRNAs enriched in proliferating active T cells in different screening models, the researchers found that knockout of some genes confers on T cells a greater ability to
proliferate in specific immunosuppressive environments.

For example, PDE4C found in the adenosine model, TGFBR1 in the TGFβ model, tacrolimus and NFKB2
in the cyclosporine model.

Knockout of some genes promotes T cell proliferation
in a variety of models.

TMEM222 and RASA2, on the other hand, were found
under all 5 immunosuppressive filters.

CRISPR screening models in immunosuppressive settings and their main findings

Given that the team also found in previously published articles that RASA2 may affect the proliferative capacity of T cells and tumor killing function[4], the researchers decided to further investigate the effects
of knockout RASA2 on adoptive T cell therapies.

The researchers found that RASA2-deficient (RASA2^KO) cells also had a stronger tumor killing ability
in the context of immunosuppression than normal TCR-T cells.

Since RASA2 is a Ras GTPase-activating protein, and the RAS signaling pathway plays an important role in both T cell activation, proliferation, and differentiation[8], the researchers analyzed
the changes in RAS-GTP levels and T cell function that may result from knockout RASA2.

They found that under TCR-stimulated conditions, RASA2-deficient human primary T cells had higher levels of RAS-GTP, and their downstream proteins pMEK, pERK, and pS6 levels were also elevated
.

In addition, RASA2^KO T cells are also able to secrete higher levels of effector cytokines IFNγ, IL-2, and TNF
.

These changes do not exist in the absence of TCR stimulation, suggesting that RASA2's regulation of the RAS signaling pathway relies on TCR
.

We all know that how to reduce side effects such as uncontrolled T cell proliferation and cytokine release during CAR-T treatment has always been a difficult problem [9].


Thus, this dependence of RASA2 on TCR stimulation in regulating T cell proliferation and effector function means that there is greater potential
to enhance the efficacy of adoptive T cells by knocking out RASA2 in the clinic.

In addition, the researchers also found that these phenotypes in RASA2^KO T cells are widely present in different TCR stimulation signal intensities, which may mean that RASA2^KO T cells are more sensitive to antigenes
.

The killing experiments of tumor cells with different CD19 expression levels also proved that RASA2^KO T cells can better recognize and kill tumor cells
.

Knockout RASA2 can upregulate the RAS signaling pathway in T cells, increasing the sensitivity of T cells to antigens

Next, the researchers analyzed the consequences
of knocking out RASA2 at the transcriptional level.

By using a series of Jurkat T cell transcriptional reporting systems, the researchers found that knockout RASA2 significantly upregulated transcriptional levels
of AP-1 and NFкB induced by TCR stimulation.

Genome-wide RNA sequencing analysis showed that genes associated with cell proliferation, oxidative phosphorylation, and glycolysis were upregulated
in RASA2^KO T cells.

Combined with reduced levels of SELL and TCF7 expression, the researchers suggest that knockout RASA2 may facilitate the transformation
of T cells into effector memory T cells.

The researchers also analyzed
the transcription level of RASA2 itself.

They found that after T cells were activated, RASA2 levels were first rapidly downregulated
.

In the setting of chronic infection or persistent antigenic stimulation, RASA2 levels are gradually raised
.

In addition, the RASA2 expression level of infiltrated T cells in tumors was also higher
than that of T cells in peripheral blood.

These results suggest that RASA2 may be a new intrinsic signaling checkpoint that inhibits T cell function
.

To test this hypothesis, the researchers analyzed
the tumor killing function of TCR-T cells under conditions of T cell depletion induced by repeated antigen stimulation.

They found that as the number of antigen stimuli increased, T cells would gradually enter a depleted state, manifested by a gradual decline in vitality, effector function, and tumor killing ability; Knockout RASA2 inhibits this process
of T cell depletion induced by repetitive antigen stimulation.

Compared with normal T cells, RASA2^KO T cells still have higher levels of cytoresin expression and secretion (including granulase A, granulase B, IFNγ and IL-10), higher levels of oxidative phosphorylation, and higher tumor killing capacity
after multiple antigen stimulation.

These results were also replicated in CAR-T cells, illustrating that knockout RASA2 can enhance T cells' resistance to depletion signals
.

RASA2^KO T cells are resistant to T cell depletion induced by repetitive antigen stimulation

Finally, the researchers analyzed the clinical relevance and translatability of knockout RASA2 in a variety of mouse models, including a model of melanoma (A375) treated with TCR-T cells, a model of B-cell precursor leukemia (Nalm6) treated with TCR-T cells, a Nalm6 model that received CAR-T cell therapy, and a model
of intraperitoneal local osteosarcoma (LM7) that received CAR-T cell therapy.

The researchers found that in the above model, compared with normal TCR-T cells and CAR-T cells, RASA2^KO T cells were able to significantly inhibit tumor development and prolong mouse survival
.

By comparing healthy mice treated with CAR-T cells, Nalm6 tumor mice treated with normal TCR-T cells, and Nalm6 tumor mice treated with RASA2^KO CAR-T cells, the researchers demonstrated that knockout of RASA2 did not increase safety risks
.

This also echoes the previously mentioned finding that RASA2 is dependent on TCR stimulation in regulating T cell proliferation and effector function
.

RASA2^KO's TCR-T and CAR-T cells are able to control the development of a variety of tumors

Overall, the study identified RASA2, an important gene that improves the efficacy and persistence of adoptive T-cell therapy, and provides new ideas
for the development of more effective TCR-T and CAR-T therapies, as well as their application in some tumors with immunosuppressive microenvironments.

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