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Cell: Continuous antigen stimulation can lead to CAR-T exhaustion
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Cell: Continuous antigen stimulation can lead to CAR-T exhaustion.
The progress of chimeric antigen receptor T cell therapy (CAR-T) in the treatment of hematological tumors is obvious to all, but the treatment of solid tumors has been difficult and the effect is not satisfactory.
The reason for this also starts from the tumor microenvironment .
Unlike flowing blood, solid tumors are in a relatively closed and stuffy environment . In order for CAR-T cells to take effect, they must first have enough contact with tumor cells: this requires it to break through numerous obstacles, penetrate into solid tumors, and become tumor infiltrating lymphocytes (TIL).
Unfortunately, not all CAR-Ts can become TILs. These warriors CAR-Ts who can enter the tumor go deep into the hinterland and are attacked on all sides.
In addition to working hard to resist the immune-suppressing cells and factors in the microenvironment, they also suffer To continuous stimulation from tumor antigens.
CAR-T is exhausted for a long time, and eventually leads to abnormal functions, which can only end in exhaustion (flatness) .
The specific manifestations of exhaustion are the decrease of T cell proliferation, the abnormal expression of the apparent and transcriptome, the continuous expression of multiple inhibitory receptors, the decrease of effector function, the decrease of cytokines, and the increase of chemokine expression. However, the specific mechanism of CAR-T failure due to continuous antigen stimulation is still unknown .
So can you build a model to study the causes of CAR-T function abnormalities?
If the mechanism of CAR-T cell failure can be found, then CRISPR can be used to edit genes to eliminate this inducement, thereby solving the problem of CAR-T function abnormalities in solid tumors, thereby improving the efficacy.
Recently, a team led by Regina Young, Shelley Berger and Carl June of the University of Pennsylvania published a major research results in Cell .
They found that continuous antigen stimulation can lead to CAR-T exhaustion, and CD8+ T cells gradually show the characteristics of NK cells .
In dysfunctional CAR-T, the transcription factors ID3 and SOX4 are up-regulated , and they regulate exhaustion-related genes including NK receptors.
By knocking out ID3 or SOX4, the previously affected cytotoxicity is partially restored! This provides infinite possibilities for making better CAR-T.
There may be hope for the future treatment of solid tumors!
▲ Screenshot of the paper’s homepage
This study first constructed an in vitro model, using tumors expressing specific antigens to repeatedly stimulate CAR-T cells to simulate the pressure of CAR-T in the tumor microenvironment, and then observe the corresponding gene expression and expression of CAR-T after being affected. Functional changes.
The continuous antigen stimulation in this abnormal model is kindly provided by the pancreatic cancer cell line AsPC-1, which expresses low mesothelin antigen. CAR-T uses CAR-T that specifically recognizes mesothelin.
Every 3-4 days, CAR-T cells will be co-cultured with brand new tumor cells and continue to be stimulated. After the whole process lasts for 20-35 days, CAR-T cells basically no longer proliferate .
Although 70 to 80% of CAR-T cells are still alive, after 18 days of stimulation, CAR-T cells have been characterized by late apoptosis.
In terms of tumor-killing cytotoxicity, compared with unstimulated (Day 0) CAR-T, CD8+ CAR-T cells on the 28th day after stimulation can neither control tumor growth nor produce cytokines, indicating a specific antigen The CAR-T cell after continuous stimulation is already a waste CAR-T .
In this way, the model of CAR-T dysfunction has been successfully constructed. But there is one more verification: Is the abnormal function displayed by CAR-T a unique response to the CAR signal pathway?
The study found that the depletion phenomenon is actually specific to the CAR signal !
After 24 days of stimulation, CD8+ CAR-T cells are stimulated with phorbol ester (PMA) + ionomycin (ionomycin).
The purpose is to bypass TCR and directly activate the downstream signaling pathways of T cells, and still produce a large amount of IL2 and IFNg.
However, there is no response when the pancreatic cancer AsPC1 cells with specific antigen are used to stimulate it. This shows that although CAR is lying flat, the downstream signal pathway is still intact .
It’s like we are overworked and lose the highlight in our eyes. Although we are already a salted fish, we are not sleepy when it comes to playing!
Let’s look at the expression of CAR on the surface of CD8+ T cells: After repeated antigen stimulation, the expression of CAR is also decreasing.
Can the CAR lost due to continuous antigen stimulation be recovered?
From the cells that were continuously stimulated by the antigen for 23 days, the cells that did not express CAR were isolated and cultured with IL-15 alone for one day, and immediately 38% of the cells could express CAR on the cell surface again, and they are also very effective in anti-tumor. Can fight .
Just take one more day off, it is a good CAR again!
▲ After CD8+ CAR-T is stimulated by specific antigen for 26 days (before rest), rest in the culture medium with IL-15 for one day (After rest+IL-15), the ability to lyse the tumor is restored
The model representing the abnormal function of CAR-T has been constructed. Compared with the previous exhaustion model, what are the similarities and differences?
First, let’s make a bulk RNAseq, and compare the unstimulated (Day 0) cells with the CD8+ CAR-T cells on the 28th day after stimulation .
After obtaining the transcriptional change characteristics of this abnormal model, we compared it with the two exhaustion models plus the failed CD8+ TIL RNAseq data in the four groups of patients, and found that there was a significant overlap of some up-regulated genes, including T cell exhaustion. Related genes [3, 4].
After confirming the eyes with the old model, let’s talk about the new findings in this research. Researchers use pathway analysis software IPA to further analyze the RNAseq data and explore the biological significance of the signaling pathways where the genes whose expression is dysregulated.
In addition to the well-known T cell depletion signals PD-1/PD-L1 and CTLA4, some pathways related to NK cells also appear in the list of up-regulated genes for continuous antigen stimulation of CD8+ CAR-T, including a number of NK receptors: KLRC1, KLRC2, KLRC3, KLRB1, KLRD1 and KIR2DL4.
Single-cell RNAseq also provides important evidence support. And did not received stimulation (Day 0) as compared to 20 days after chronic stimulation of the CAR-T showed two groups (Cluster1 / 4), and the bulk RNAseq upregulated genes were focused together, comprise NK related genes .
Among the two groups representing abnormal CAR-T, the 30 genes with the largest expression changes were selected and tentatively designated as the characteristic genes of abnormal CAR-T.
In this way, a set of common characteristics of CAR-T abnormalities was found from the overall and single-cell transcription level.
▲ In CAR-T cells stimulated for 20 days, the expression of abnormal characteristic genes increased.
Each column in the dot plot represents a group found in single-cell sequencing
The next step is to verify the characteristic expression of this group of abnormal CAR-T from multiple angles. In mice, tumors caused by pancreatic cancer cells AsPC-1 can be successfully eliminated within two weeks of CAR-T injection. However, after 2-4 months, the tumor recurred in some mice.
The recurring tumor still expresses mesothelin, an antigen specifically recognized by CAR-T, indicating that CAR-T is indeed dysfunctional and can no longer control tumor growth .
CAR-T cells extracted from recurrent tumors have high expression of NK receptors, which are also in line with the previously summarized characteristics of disorders .
This is the case in the mouse model, but what about the patient?
In clinical trials of diffuse large B-cell lymphoma (DLBCL), CAR-T can be expanded normally in most patients, except for 3/17 patients who have a large number of these within 10 days of reinfusion of CAR-T.
A kind of CAR-T cell like NK. Among them, the patients with the most NK-like CAR-T cells did not even respond to CAR-T treatment .
In short, the above in vivo experiments and patient data have confirmed the correlation between NK-shaped CAR-T and dysfunction.
▲ In the clinical trial for the treatment of DLBCL (NCT02030834), when the proliferation of CAR-T with CD19 as the target reaches its peak, the expression level of CD56 on the surface of CD8+CAR-T is shown in the figure
At the protein level, in CD8+CAR-T cells, NK receptors do increase with the time of continuous antigen stimulation, and they are not NKT cells .
Mass flow cytometry data showed that a small group of unstimulated (Day 0) T cells gradually evolved to image NK T cells with chronic stimulation .
So the question arises: Does this NK-like T cell originally existed and grew by an overwhelming advantage in continuous antigen stimulation, or was it transformed from other cells?
To answer this question, they first determined the characteristic markers of NK-like T cells: there are two sets of markers that co-express the characteristics of T cells and NK cells, CD3+CD56+ or CD3+KLRB1+ . Knockout of CD56+ is basically equivalent to the removal of NK cells and NK-like T cells.
Next, in order to verify the source of the cells, CD56+ was knocked out from CAR-T cells that had not been stimulated (Day 0), and then the continuous antigen stimulation experiment was repeated.
The results were no different from the experiment where CD56+ was not deleted . All NK-related cells were deleted from the source, and they can be reproduced after stimulation, which can only be converted from T cells.
▲The left picture shows the frequency of NK-like T cells (CD3+CD56+) in the initial (Day0) CAR-T before and after CD56 is knocked out.
The picture on the right shows the same number of NK-like T cells under chronic antigen stimulation regardless of whether CD56 is present in the initial state.
In order to confirm this T to NK conversion, scRNA-seq once again appeared on the scene, sequencing the TCR that was not stimulated (Day 0) and 28 days after the stimulation to track the lineage of T cells.
It is confirmed that the cells are still the same cell after long-term stimulation, but NK receptors have grown on them .
It is important to understand the mechanism, it is better to find the target.
So far, the reasons for CAR-T dysfunction under continuous antigen stimulation have been observed, and the relevant abnormal expression characteristics have been summarized.
So can you find transcription factors that are specifically responsible for regulating these abnormal genes and responsible for dysfunction?
The researchers again used IPA to analyze the differentially expressed genes of scRNA-seq, and ID3 and SOX4 stood out from the related transcription factors .
Finally, the most exciting moment has arrived. Can the targets obtained through massive data analysis actually improve CAR-T and repair functions?
After the researchers knocked out ID3 or SOX4, they used the upgraded CAR-T to perform the continuous antigen stimulation experiment again.
In order to facilitate the evaluation and comparison of the degree of CAR-T dysfunction, the researchers packaged the 30 characteristic genes of abnormal CAR-T obtained before and made an operation, named it the dysfunction score (dysfunction score).
The CAR-T dysregulation index that knocked out ID3 or SOX4 was lower, which effectively improved the cytotoxicity and tumor lethality of abnormal CAR-T cells after continuous antigen stimulation .
▲ Under continuous antigen stimulation, CAR-T with ID3 or SOX4 knocked out is more effective against tumor cells than the original abnormal CAR-T
Compared with the mouse model, the advantage of the in vitro model is that it is more flexible and easy to operate, and it is convenient to sample and observe at multiple time points.
This study discovered a set of characteristic genes of CAR-T cells that are dysfunctional due to chronic stimulation: continuous antigen stimulation can cause CAR-T cell depletion and promote the conversion of CD8 T cells to NK-like T cells.
During this transition, the transcription factors ID3 and SOX4 were up-regulated, regulating genes related to cell failure, including the NK receptor. Knockout of ID3 or SOX4 partially repaired the problem of CAR-T cell dysfunction and improved the anti-tumor efficacy.
The up-regulation of NK receptors on the surface of CAR-T has become a new sign of cell exhaustion, which will have a profound impact on the field of immunotherapy .
Of course, there are still many questions to be further studied, such as: What is the biological significance of NK receptors for CAR-T cells? Does the strategy of knocking out ID3 and SOX4 from CAR-T in turn affect the tumor microenvironment? Can this strategy also be applied to treat other tumors?
In addition, this disorder index used to evaluate CAR-T function is also worthy of further extensive verification.
If it is universal, it can be promoted as a reference standard for judging or predicting CAR-T function in the future.
1.Marofi F, Motavalli R, Safonov VA, Thangavelu L, Yumashev AV, Alexander M, et al. CAR T cells in solid tumors: challenges and opportunities. Stem Cell Research & Therapy. 2021;12(1). doi: 10.1186 /s13287-020-02128-1.
2.Good CR, Aznar MA, Kuramitsu S, Samareh P, Agarwal S, Donahue G, et al. An NK-like CAR T cell transition in CAR T cell dysfunction. Cell. 2021. Epub 20211126. doi: 10.1016/j. cell.2021.11.016. PubMed PMID: 34861191.
3.Pauken KE, Sammons MA, Odorizzi PM, Manne S, Godec J, Khan O, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science. 2016;354(6316):1160- 5. Epub 20161027. doi: 10.1126/science.aaf2807. PubMed PMID: 27789795; PubMed Central PMCID: PMC5484795.
4.Beltra JC, Manne S, Abdel-Hakeem MS, Kurachi M, Giles JR, Chen Z, et al. Developmental Relationships of Four Exhausted CD8. Immunity. 2020;52(5):825-41.e8. Epub 20200511. doi: 10.1016/j.immuni.2020.04.014. PubMed PMID: 32396847; PubMed Central PMCID: PMC8360766.
Cell: Continuous antigen stimulation can lead to CAR-T exhaustion
(source:internet, reference only)