I believe that everyone is no stranger to CAR-T cell therapy, it has an extremely significant effect on some blood tumors, but CAR-T cell therapy has little effect in the treatment of solid tumors, and even in blood tumors, in addition to acute B-cell leukemia, most other types of blood tumor patients have not had sustained remission due to inactivation of CAR-T cells in vivo [1].
So many scientists have worked to find the genes that inactivate T cells and the biological mechanisms involved. But some researchers have done the opposite.
Recently, a research team led by Peter Smibert and Neville E. Sanjana of New York University published important research in the journal Nature[2].
Using OverCITE-seq, a single-cell level, high-throughput technique, they tested the overexpression of approximately 12,000 human genome open reading frames (ORFs), which had an enhancing effect on the proliferation and function of T cells. They found ORFs that increaseD cell activity and proliferation, and demonstrated that using them to engineer chimeric antigen receptors in existing CAR-T cells can enhance the anti-cancer ability of CAR-T cells.
Screenshot of the paper
As a "live" drug for the treatment of cancer, six different CAR-T therapies have been approved for marketing by the FDA [3] and have achieved very significant results in the treatment of hematological tumors, with patients who have not relapsed after ten years of treatment, and even CAR-T cells are still present in the body [4].
Even so, there are still many patients with hematological tumors who do not achieve sustained remission after treatment, the main reason is that the CAR-T cells infused back into the body are inactivated and enter a depleted state [1].
To solve this problem, many scientists have turned to the biological mechanisms that cause T cells to inactivate. For example, CRISPR technology knocks out all genes on the genome to determine which genes play a dominant role [5].
This approach is challenging due to the sheer number of genes, and because of the CRISPR system's own properties, it is possible that the edited T cells are rejected by the patient's own immune system [6].
So Peter Smibert and Neville E. Sanjana's team came up with a completely different solution — since CAR-T cells are easily inactivated in vivo, add them genes that enhance proliferation and anti-cancer activity, making CAR-T cells more "aggressive" to cancer cells.
To avoid the added genes not being expressed properly in T cells, the researchers used lentiviruses as vectors to transfer full-length sequences of about 12,000 genes into pre-stimulated CD4+ and CD8+ T cells from three healthy donors, each with an average of 6 "barcodes" for subsequent recognition.
After 14 days of in vitro culture of these cells, they are fluorescently labeled, and then re-stimulated to allow T cells to proliferate, and then the effect of these genes on T cell proliferation can be obtained by comparing the changes in the number of genes Barcode before and after stimulation.
Experimental design principles
Among the top-ranked genes that enhance T cell expansion, many are genes known to be involved in immune processes, including MAPK3, BATF, IL12B, and IL23A[7]. The gene that promotes the most is LTBR (receptor encoding lymphotoxin β), which is widely expressed in the matrix and bone marrow cells, but is rarely expressed in lymphocytes.
The researchers then further explored the effects of the 33 Top-ranked genes on other functions of T cells, this time choosing a nerve growth factor receptor (tNGFR) that lacks intracellular domains as a control, and transducing tNGFR and 33 genes into CD4+ and CD8+ T cells using vectors, respectively.
After going through the same experimental process as before, the researchers not only compared the differences in T cell expansion between the experimental and control groups, but also compared the ability of T cells to secrete cytokines and the differences in surfactive marker expression.
They found that in CD4+ and CD8+ T cells, the Top-ranked gene was highly correlated with proliferative capacity (r=0.61, p=0.002), and that most of these genes contributed to increased expression of CD25 and CD154 in T cells after activation.
There are also genes that enhance the ability of T cells to secrete cytokines, the most significant gene is still LTBR, which can increase the cytokines secreted by T cells by as much as 5 times.
AMONG THE GENES THAT PROMOTE T CELL SECRETION OF CYTOKINES, LTBR IS THE STRONGEST
So far, the researchers have shown that the Top-ranked gene can promote T cell proliferation, surfactant marker expression, and cytokine secretion, so they want to know the specific mechanism by which these genes work.
Peter Smibert and Neville E. Sanjana's team developed the OverCITF-seq sequencing platform to quantify the amount of gene ORF expression transduced into T cells at the single-cell level. The principle of this technique is to design an RNA primer for the gene ORF Barcode to guide the orf's mRNA reverse transcription, so that during RNA sequencing, orf can form a separate cDNA library, and other genes in the cell form another cDNA library.
Technical principles of OverCITE
Using OverCITE-seq technology, the researchers sequenced CD8+ T cells from the same healthy donor who had each transduced 30 genes orf, and some of these cells were operated according to the previous experimental procedure - after a re-stimulation process, and some of them were not re-stimulated after transduction and were in a "dormant state".
After unsupervised clustering of these CD8+ T cells according to gene expression profiles, they found that activated and inactive T cells could be clearly divided into two categories, and the two categories could be divided into 10 sub-categories. Some of these species are related to a specific T cell state or function, indicating that the same class of T cells has a similar state or function, such as class 1 and cell cycle related, class 10 related to T cell activation and proliferation.
Cluster T cells according to gene expression profiles
The researchers then analyzed the orf enrichment of transduction in 10 T cells to determine the effect of ORF on the cell transcriptome and found significant enrichment of CDK1 and CLIC1 in category 1 and LTBR in category 10.
Combining the relationship between the different types of T cells described above with specific states and functions, this further suggests that LTBR achieves the ability to both promote T cell proliferation and enhance T cell activity and cytokine secretion by remodeling the cell transcriptome.
Since LTBR can single-handedly reshape the transcriptome of T cells, it's worth looking at how it does it.
The researchers used burk-seq to compare gene expression profiles between T cells that transduced LTBR and tNGFR (hereinafter referred to as LTBR cells and tNGFR cells), respectively, and found that LTBR cells not only upregulated MHC-I. and MHC-II. related genes, but also expressed MHC-II. constant chains encoded by CD74, which has been shown to activate the anti-apoptotic NF-kB pathway in B cells, while LTBR cells also upregulate BATF3. This is a gene that promotes the survival of CD8+ T cells.
In subsequent experiments, the researchers also found that LTBR cells have a stronger ability to resist activation induced apoptosis compared with tNGFR cells, as well as retain greater functionality under continuous stimulation and are not easily depleted.
Activated LTBR cells have a stronger ability to resist apoptosis compared to tNGFR cells (upper);
LTBR remains low in PD-1 levels after multiple stimuli (Lower)
Having said so much about the characteristics of LTBR cells, back to LTBR itself, it is expressed in bone marrow cells, and the signaling pathway it involves is activated by the trimer or LIGHT (encoded by TNFSF14) composed of LTA and LTB, while LTA, LTB, and LIGHT are expressed by activated T cells.
So the researchers explored whether activated LTBR cells would be affected by the above three molecules from the outside, and found that LTBR cells were not affected, suggesting that the high expression of LTBR, although it has an enhancing function to promote proliferation of T cells, does not drive its own activation. This also eliminates a safety issue in clinical applications - LTBR cells do not lose the function of specifically recognizing tumor antigens due to the external environment.
Subsequently, to find out the key domains of LTBR, the researchers introduced mutations in the LTBR sequence. Overall, the N-terminus of LTBR is more tolerant of deletion mutations than the C-terminus, with residues missing 393-435 having little effect on LTBR function, while missing residues of 377-435 renders LTBR completely defunctional.
The exploration of the regulatory mechanism of LTBR continues, and the researchers analyzed the epiomic differences between LTBR cells and tNGFR cells using ATAC-seq and found that NF-kB p65 (RELA) is the most concentrated transcription factor in LTBR cells. At the same time, they observed that the p65 (RELA) of the NF-kB signaling pathway in LTBR cells after stimulation would be rapidly phosphorylated, and the corresponding inhibitor phosphorylation level would also increase, both of which enhanced the activity of the NF-kB pathway and the transcriptional activity of the genes involved in the pathway.
In addition to the classical NF-kB pathway, the researchers also found that p52 (RELB) of the non-classical NF-kB pathway was also enriched in LTBR cells, but they found by knocking out two signaling pathway-related genes bound to burk-seq analysis that only the deletion of RELA would cause the LTBR to involve the core gene of the pathway to be downregulated, while the deletion of RELB did not have a significant effect, indicating that LTBR most likely functioned through the classical NF-kB pathway.
The deletion of RELA causes the core gene of LTBR cells to be downregulated, while the deletion of RELB does not
Since LTBR cells and tNGFR cells in the previous experiment were activated by CD3/CD28 stimulation, which is a non-specific activation process, the researchers wanted to know whether LTBR cells would have the same effect as before under the stimulation of specific antigens.
They engineered T cells to build co-expression vectors with CARs from two FDA-approved CAR-T therapies, both of which specifically target CD19, but one CAR uses CD28 as the co-stimulation domain and the other 4-1BB as the co-stimulation domain.
After co-culturing the engineered CAR-T cells with Nalm6 (CD19+ lymphoma cells), the researchers found that almost all of the Top-ranked genes promoted the expression of CD25 in T cells, as well as antigen-specific cytokine secretion.
Although secreted cytokines are an important indicator of T cell anti-tumor activity, there is also a key part of the direct killing effect on tumor cells, that is, cytotoxicity. The Top-ranked gene has a greater enhancing effect on CD28 CAR-T cytotoxicity than 4-1BB CAR-T cells. And CAR-T cells expressing LTBR are less likely to enter a depleted state.
The Top-ranked gene has a greater enhancing effect on CD28 CAR-T cells (left); CAR-T cells expressing LTBR are more functional
Considering that the T cells in all of the above experiments are from healthy people, these T cells are easy to engineer and will not be inactivated during the culture process, but in fact CAR-T cell therapy is modified with T cells in tumor patients.
So the researchers collected peripheral blood from patients with diffuse large B-cell lymphoma, transduced LTBR and CARs into monocytes, and after co-culture with CD19+ target cells, they found that LTBR CAR-T cytokine secretion increased, consistent with the phenomenon observed in healthy human T cells.
It is worth mentioning that the researchers also tried to perform the above modification experiment in a "non-mainstream" T cell, γδT cells, and the final result is still an increase in the ability of the modified T cells to secrete cytokines.
From the perspective of "gain of function", the study found genes in 12,000 genes that can enhance the function of existing CAR-T cells without losing specificity, the most significant gene LTBR can even enhance the proliferation ability of T cells, but also increase the expression of T cell surface activity markers and cytokine secretion.
Dr Andrew Sewell, an expert in the field of cellular immunotherapy at Cardiff University in the UK, commented [8]: "Finding genes that allow T cells to function at the genome-wide scale has great potential to make immunotherapy more successful, especially in the field of solid tumor treatment, where CAR-T therapy is currently ineffective."
bibliography:
[1] Fraietta JA, Lacey SF, Orlando EJ, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia [published correction appears in Nat Med. 2021 Mar;27(3):561]. Nat Med. 2018;24(5):563-571. doi:10.1038/s41591-018-0010-1
[3] https://www.cancer.gov/about-cancer/treatment/research/car-t-cells
[5] Shifrut E, Carnevale J, Tobin V, et al. Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Cell. 2018;175(7):1958-1971.e15. doi:10.1016/j.cell.2018.10.024
[8] https://www.nygenome.org/programming-the-immune-system-to-supercharge-cancer-cell-therapies/
Responsible editor 丨IoTalker