Inhibition of anticancer lymphocytes
A protein that shortens the antitumor service life of T-lymphocytes has been identified
Vyacheslav Kalinin, "Elements"
One of the immunotherapeutic methods of cancer treatment, adaptive cell therapy, consists in taking a small part of the anti-cancer T-lymphocytes from the patient, multiplying and activating them in a test tube, and then injecting them back. The disadvantage of this method is that the injected lymphocytes soon die. In experiments on mice, it was shown that switching off the gene controlling the REGNASE-1 protein significantly increases the survival of lymphocytes, increases their accumulation in tumors and enhances antitumor activity.
Fig. 1. Scheme of antitumor immune response. The T-cell response begins with the activation of naive T-lymphocytes recognizing the target protein. Activated cells generate T cells similar to memory cells, which can self-renew or generate cytotoxic CD8+T-cells that are already able to directly fight the tumor. In the work under discussion, it was shown that switching off the Regnase-1 gene prolongs the life of lymphocytes inside the tumor and increases the positive effect of them. A drawing from the popular synopsis to the article under discussion in Nature.
Inactivation of the Ptpn2 and Socs1 genes further enhanced the effect: the development of tumors was sharply suppressed, and the mice lived much longer. The development of drugs that suppress the activity of these proteins will be a significant breakthrough in immunotherapy.
Currently, oncologists have at their disposal an extensive arsenal of means to fight cancer. These are, for example, the latest technologies that allow performing the most precise surgical operations with minimizing damage to the body, and various conditionally non–invasive methods - radiotherapy, chemotherapy, immunotherapy. Radiotherapy is based on the fact that exposure to ionizing radiation damages the DNA of cells and causes them to lose their ability to divide or simply die. At the same time, the more actively the cell divides, the more harmful radiation is for it. Since cancer cells usually divide much more actively than healthy cells of the body, it is possible to cause great damage to the tumor without damaging healthy tissues too much. In chemotherapy, strong cellular poisons are used to fight tumors, which are either delivered directly to the target, or designed to act primarily on cancer cells. Despite the obvious progress in these methods, they have many side effects and even if they help to defeat the disease (which, alas, does not always happen), they are accompanied by severe damage to healthy cells and cause severe harm to the patient's body. In contrast, immunotherapy is characterized by a much higher selectivity of action directed at cancer cells and tumors.
In immunotherapy, two main approaches can be distinguished: immunotherapy with antibodies directed against cancer-specific antigens, and cellular immunotherapy with immune lymphocytes. Antibodies (immunoglobulins) recognize antigens specific to cancer cells and lead to the destruction of malignant cells – either independently causing their apoptosis, or attracting other components of immunity.
Cell therapy is carried out with the help of immune lymphocytes, which can be divided into three main types: B-lymphocytes associated with the production of antibodies, NK-lymphocytes related to innate immunity, as well as T-lymphocytes involved in the acquired immune response. T-lymphocytes, in turn, are divided into T-helpers that stimulate the production of antibodies and participate in the presentation of antigens, T-suppressors that regulate these processes, as well as T-killers or cytotoxic T-cells whose task is to destroy damaged and malignant cells of the body, as well as foreign cells. T-killers are the main component of cellular immunity, which also works against cancer cells. T-lymphocytes are formed in the thymus, hence the letter "T" in the name.
The development of methods for artificially enhancing the anti-cancer immune response with the help of T-lymphocytes has made a real revolution in the treatment of cancer. The basis was laid by the pioneering works of James Ellison and Tasuku Honjo, completed 30 years ago, for which they received The Nobel Prize in 2018.
One of the approaches in cancer immunotherapy is the so-called adaptive cell therapy (adaptive cell therapy, ACT). In response to the development of a cancerous tumor, the body turns on the cellular immunity system. One of the components of this immunity, T-lymphocytes labeled with transmembrane glycoprotein CD8, directly bind to cancer cells and kill them.
But often the forces of their own CD8+The body's T-cells – their number and activity – are not enough to fight the tumor. The simplest AST therapy involves the isolation of activated lymphocytes from the blood or tumor, multiplying them in vitro and injecting them back to the patient. This procedure gives at least a partial effect. On the one hand, the body receives a large number of ready-to-fight immune cells, many of which have time to benefit. On the other hand, artificially diluted CD8+T-cells are counteracted by the tumor's own self-defense mechanisms and die quickly enough.
Exactly how the protective mechanisms of the tumor work is not really known. It has been suggested that the viability of CD8+T cells depend on some features of their metabolism (R. J. Kishton et al., 2017. Metabolic regulation of T cell longevity and function in tumor immunotherapy). But from what specific features? How do these features affect? All this was absolutely unknown. Adaptive cell therapy methods are constantly being improved and are increasingly widely used in clinical practice. But the most important problem of instability of newly injected antitumor lymphocytes remains unresolved.
Some time ago, American scientists conducted a study to determine how various metabolic products and factors (enzymes, activators and transcription inhibitors, as well as transport proteins) affect the anti-cancer activity of CD8+T-cells. The main idea of the work was the inactivation of genes controlling proteins and enzymes of interest to scientists, followed by tracking how this inactivation will affect the viability of T-lymphocytes. Modern methods of genomic engineering have made it possible to selectively inactivate 3017 genes. To do this, the authors have developed an elegant system based on the principles of CRISPR-Cas9 mutagenesis. CRISPR-Cas9 systems are currently widely used in genetic engineering research for gene modification and genome editing both in eukaryotic cells and in lower organisms.
CD8+ sourceA specially designed line of mice served as T-cells, in whose genome the Cas9 gene was embedded and expressed, intended for subsequent mutagenesis. Such mice were inoculated with human melanoma cells, and CD8+T lymphocytes were isolated from the formed tumors. These cells (carrying the Cas9 gene) were multiplied in vitro and their transduction was carried out by a library of lentiviruses. Specially designed lentiviruses allow the most effective introduction of foreign genes into cells. In this case, lentiviruses were injected into CD8+T cells with individual guide RNAs, each of which was designed to inactivate one of the genes of metabolic enzymes, small molecule transport or transcription regulators. A total of 9551 guide RNAs were targeted at 3017 genes.
After CD8+ transductionT-cells were injected into mice with melanoma, lymphocytes infiltrating tumors were isolated after 7 days and compared with the original ones using high-coverage genome sequencing (see Coverage). With the help of such sequencing, it is possible not only to determine the presence of certain DNA sites, but also to estimate their number immediately in a large set of CD8+T-cells. This made it possible to track the enrichment of the CD8+ populationT cells with certain mutant genes, which occurred due to the fact that lymphocytes with damaged vital genes did not survive until the end of the seven-day period. Accordingly, among the remaining cells, you can look for genes whose work after mutation improved survival.
The analysis showed that 218 genes were severely damaged in lymphocytes infiltrating tumors, including a number of well-known regulators of survival and CD8+ divisionT-cells. Among CD8+The proportion of T cells isolated from melanoma or from the spleen of diseased mice was especially significantly increased by the proportion of cells with mutations in the Regnase-1 gene. Previously, this gene was found in mammals, including humans, and was identified as a transcription activation factor associated with cell death. It was also known that the REGNASE-1 protein encoded by this gene participates in the regulation of immune cell activation, but its function in antitumor immunity was not clear.
The authors suggested that the REGNASE-1 protein is the main negative regulator of the T-cell antitumor immune response. To understand in detail the effect of REGNASE-1 protein function loss, the researchers transduced CD8+ labeled with various fluorescent proteinsT cells either neutral guide RNA or guide RNA exclusively against the Regnase-1 gene. The cells were mixed, grown, and injected into mice with melanoma. After 7, 14 and 21 days, the number of differently labeled cells in the spleen and in animal tumors was determined. It turned out that the number of CD8+T-cells with the Regnase-1 gene turned off exceeded the number of cells with the active gene by thousands of times (Fig. 2, left). This means that T-lymphocytes without the REGNASE-1 protein are more resilient and therefore much better able to accumulate in the tumor and destroy it.
2. On the left, T cells transduced by a guide RNA lentivirus against the Regnase–1 gene accumulate in the tumor (TIL, tumour-infiltrating lymphocyte) and in the spleen (spleen) in significantly larger quantities than control cells (they are shown by black and blue labels). In each case, it is indicated how many times more cells there were with the Regnase-1 gene disabled. On the right – the introduction of T-lymphocytes without the REGNASE-1 protein increases the effectiveness of adaptive cell therapy: tumor growth slows down (a) and the survival rate of mice increases (b). Graphs from the discussed article in Nature.
Experiments confirmed this conclusion: after adaptive cell therapy of mice, it turned out that the introduction of cells with the Regnase-1 gene turned off greatly slowed the growth of solid melanoma tumors and increased the survival of animals (Fig. 2, right). Similar effects were observed in mice "infected" with leukemia.
To track the processes affected by the Regnase-1 gene, scientists conducted comparative RNA sequencing in cells in which this gene was turned off, and in wild-type cells. Cells of both types were isolated from the microenvironment of melanomas (that is, these cells were in direct contact with the tumor) and from the blood. In cells with the Regnase-1 gene switched off from the microenvironment, the set of RNA turned out to be shifted in the direction characteristic of memory cells (forming a rapid secondary immune response). Thus, the absence of the REGNASE-1 protein allows lymphocytes used for ATS to be reprogrammed, accumulate in the tumor microenvironment and remain active for a long time, performing their antitumor function.
Inspired by fundamentally new and important results from both theoretical and practical points of view, scientists have tried to better understand the mechanisms of CD8+ reprogrammingT cells with REGNASE-1 protein deficiency. To do this, they conducted a CD8+ part transductionT cells are lentiviruses carrying guide RNAs against REGNASE-1, and the other part is lentiviruses carrying guide RNAs against almost all mouse genes. Further, after growing and activation, the cells were mixed and injected into the bloodstream of mice with melanoma tumors. Then the sequenced genomes of cells obtained from the tumor environment were compared again with the genomes of the original cells.
Cells with the Regnase-1 gene turned off, passed through the mouse body, contained more active Batf genes and fewer active Ptpn2 and Socs1 genes than the original ones. Additional experiments have shown that double inactivation of Regnase-1 and Batf shortens the life span of cells, and inactivation of Regnase-1 in combination with inactivated Ptpn2 or Socs1, on the contrary, increases the accumulation of CD8+ in the tumorT-cells and their antitumor activity, and also prolongs their life. The BATF protein is a transcription regulation factor, activates mitochondrial functions and plays the role of a key regulator of CD8+ differentiationT-cells. It was also already known that the SOCS1 protein inhibits the proliferation of T cells in cultures, and the deletion of the Ptpn2 gene enhances the effect of immunotherapy. Now it has been found that the combination of inactivation of the Regnase-1 gene and inactivation of at least one of the Ptpn2 or Socs1 genes further enhances the antitumor activity of CD8+T-cells.
Thus, scientists have established that the REGNASE-1 protein involved in the regulation of CD8+ activationT-cells, is a very powerful and almost the main negative regulator of the anti-cancer cellular immune response. As a result of switching off the synthesis of this protein, CD8+ activityT-cells used for adaptive anti-cancer immunotherapy can be enhanced many times. This effect is observed for both solid and hematological cancers. Additional shutdown of SOCS1 or PTPN2 protein synthesis enhances the effect even more. In addition, it turned out that REGNASE-1 defective cells produce much more cytotoxic proteins (interferon gamma, interleukin 2, tumor necrosis factor) compared to conventional T-lymphocytes. Moreover, this is characteristic not only of mature CD8+T cells, but also memory cells, although normally this is not typical for them. Whether the modified memory cells are involved in the attack on the tumor is still unknown. And, most importantly, it remains to be seen how switching off the Regnase-1 gene will affect the activity of lymphocytes against many other tumors other than melanoma. But it is unlikely that the effect will be different, since the mechanisms of the cellular antitumor response in cases of various cancers are very similar.
These results look very promising, although for the time being, of course, the introduction of treatment methods based on them into practice is still far away. If it is possible to develop means that suppress the activity of the corresponding proteins, then this will be a real breakthrough in cancer immunotherapy.
Sources:
1) Jun Wei et al., Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy // Nature. 2019.
2) Reina-Campos et al., Antitumour T cells stand the test of time // Nature. 2019. (Popular synopsis to the article under discussion.)
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