A type of CD8 + T cell function-related Tox gene transcript variants and their applications

Abstract

This invention provides a method with CD8 + T cell function-related Tox Gene transcript variants and their applications. Specifically, this invention discovers a variant... Tox Gene transcripts ( Tox iso-2), this transcript is located in Tox This transcript variant originates from a novel transcription start site approximately 3 kb before exon 2 of the gene. Experimental studies have shown that this transcript variant does not affect T cell development; however, its reduced levels can delay CD8 cell growth. + T cell exhaustion. Therefore, this Tox Gene transcript variants around CD8 + T-cell function has promising applications in fields such as auxiliary diagnosis and treatment, and drug development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method related to CD8. + T cell function-related Tox Gene transcript variants and their applications. Background Technology

[0002] TOX (thymocyte selection-associated high mobility group box protein) is a key molecule determining T cell fate and function regulation. Early studies have established TOX's role in CD4+. + The irreplaceable role of T cells in normal development. Simultaneously, they also play a crucial regulatory role in the development and maturation of lymphoid tissue inducers (LTi) and NK cells. In recent years, TOX has been used in CD8... + The function of T cells in the process of T cell exhaustion has attracted much attention and has become a research hotspot in the field of tumor immunology.

[0003] Exhausted T cells are a dysfunctional state of T cells in chronic infection and tumor microenvironments, characterized by the progressive loss of effector functions (such as cytokine secretion and proliferative capacity) accompanied by persistently high expression of multiple inhibitory receptors (such as PD-1, TIM-3, and LAG-3), ultimately leading to the body's inability to effectively clear pathogens or tumor cells. Multiple studies have confirmed that TOX is a key transcription factor driving the T cell exhaustion program. In chronic infection and tumor models, TOX is involved in the depletion of CD8+ cells. + It is specifically highly expressed in T cells and promotes and stabilizes the establishment of the exhaustion phenotype by regulating the exhaustion-related gene network. While TOX deficiency can reverse CD8 to some extent... + T cell exhaustion and the recovery of their effector function, but also lead to CD8 deficiency in the tumor microenvironment. + T cells, due to overactivation, rapidly undergo activation-induced cell death (AICD), thereby losing their long-term residence and sustained anti-tumor capabilities. This indicates that TOX, while driving the exhaustion program, also plays a role in maintaining T cell persistence.

[0004] Furthermore, aberrant TOX expression is closely associated with the progression of various malignant tumors. In T-cell acute lymphoblastic leukemia (T-ALL), TOX is highly expressed as an oncogene, promoting genomic instability and accelerating leukemia progression by inhibiting non-homologous end junction repair pathways. In solid tumors, high TOX expression in tumor-infiltrating lymphocytes is also positively correlated with T cell exhaustion. Based on this functional duality, TOX has been recognized as a highly promising target for immunotherapy. However, while current strategies for complete inhibition or knockout of TOX can restore T cell killing activity in the short term, they also lead to a rapid reduction in the T cell pool due to the weakening of TOX-mediated survival signals, severely impacting the long-term sustainability of immunotherapy. This suggests that current molecular understanding of TOX functional regulation is insufficient to support the design of precise intervention strategies.

[0005] Recent studies have shown that Tox Genes possess sophisticated post-transcriptional regulatory mechanisms. At least two TOX protein isoforms are currently known: full-length TOX (TOX-FL) and N-terminal truncated TOX (TOX-S). However, whether an independent transcript directly encodes this truncated isoform, and whether these two isoforms play differentiated functions in the T cell exhaustion process, remains unclear. Currently, there is a lack of systematic research on the specific functional divisions of full-length and truncated TOX in promoting exhaustion phenotypes and maintaining cell survival, and how they synergistically or competitively regulate downstream signaling networks.

[0006] In summary, there is an urgent need in this field to study transcriptomics at the transcript level. Tox The gene expression patterns were analyzed in depth, particularly clarifying the actual existence and independent regulatory function of short TOX transcripts. The differential regulatory mechanisms of full-length and truncated TOX transcripts in T cell exhaustion and persistence maintenance were systematically elucidated. This not only helps to understand the intrinsic basis of the functional duality of TOX from a molecular perspective, but also provides key theoretical basis and potential targets for developing precision immunotherapy strategies based on selective intervention of TOX isoforms. Summary of the Invention

[0007] The purpose of this invention is to provide a CD8 + T cell function-related Tox Gene transcript variants and their applications.

[0008] In a first aspect, the present invention provides a method for using T cells. Tox Variant transcripts ( Tox The use of iso-2 and its detection reagents for the preparation of a diagnostic reagent or kit for evaluating CD8 in a given object. +T cell depletion status; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

[0009] In another preferred embodiment, the Tox The nucleotide sequence of the variant transcript is shown in SEQ ID NO:31.

[0010] In another preferred embodiment, the T cells include tumor-infiltrating T cells.

[0011] In another preferred embodiment, the T cells include tumor-infiltrating CD8 cells. + T cells.

[0012] In another preferred embodiment, the object includes a human or a non-human mammal.

[0013] In another preferred embodiment, the subject is a patient with a chronic infection and / or a tumor.

[0014] In another preferred embodiment, the detection is performed on an ex vivo sample.

[0015] In another preferred embodiment, the ex vivo sample contains CD8. + Ex vivo samples of T cells.

[0016] In another preferred embodiment, the ex vivo sample includes a tumor sample.

[0017] In another preferred embodiment, the ex vivo sample is a transcriptome sample.

[0018] In another preferred embodiment, the detection reagent is selected from the group consisting of sequencing libraries, primers, antibodies, probes, nucleic acid chips, or combinations thereof.

[0019] In another preferred embodiment, the detection reagent is the... Tox Specific primers for variant transcripts.

[0020] In another preferred embodiment, the diagnostic reagent includes: antibodies, primers, probes, or nucleic acid chips.

[0021] In another preferred embodiment, the diagnostic reagent further includes a pharmaceutically acceptable carrier, diluent, or excipient.

[0022] In another preferred embodiment, the kit also includes a label or instructions.

[0023] In another preferred embodiment, the evaluation of a certain object's CD8 + T cell exhaustion includes the following steps: (a1) Detect the CD8 of the object+ T cells Tox Levels / contents of variant transcripts; (a2) The above Tox The level / content of variant transcripts was compared with reference / standard values ​​to assess the CD8 content of the subject. + T cell depletion status; Wherein, if the Tox If the level / content of the variant transcript is higher than the reference / standard value, it indicates that the target CD8 + Increased T cell exhaustion, or CD8 + The risk of T cell exhaustion is increased; conversely, it indicates that the CD8+ of the target cell is depleted. + The T cell exhaustion phenomenon is weakened, or CD8 + The risk of T cell depletion is reduced.

[0024] In another preferred embodiment, the evaluation of a certain object's CD8 + T cell exhaustion also includes: detecting the CD8+ of the subject. + T cells Tox The level / content of full-length transcripts was used to assess the CD8 levels of the subjects. + T cell depletion.

[0025] A second aspect of the present invention provides a method for preparing cells that do not express TOX protein at all, the method comprising the steps of: (1) Provide a cell to be modified; (2) In the cells described Tox The genes were treated as follows to obtain cells that did not express TOX protein at all: (2.1) Knockout of exon 1; and (2.2) Knock out the 3 kb sequence upstream of exon 2, and / or mutate the start codon on exon 2 to a non-start codon.

[0026] In another preferred embodiment, the cells are derived from human or non-human mammals.

[0027] In another preferred embodiment, the non-human mammals include rodents, monkeys, rabbits, dogs, and pigs.

[0028] In another preferred embodiment, the rodents include mice and rats.

[0029] In another preferred embodiment, the knockout includes methods selected from the group consisting of: CRISPR gene editing systems, TALEN, ZFN, conditional knockout, or combinations thereof.

[0030] In another preferred embodiment, the mutation is to mutate the three start codons into non-start codons.

[0031] In another preferred embodiment, the mutation is to mutate the methionine start codon to the alanine codon.

[0032] In another preferred embodiment, the three start codons are M40, M42, and M44 relative to the nucleotide sequence shown in SEQ ID NO:33.

[0033] A third aspect of the present invention provides a method for slowing down / inhibiting T cell exhaustion, the method comprising the steps of: (A) Provide a T cell to be modified; (B) Downregulate / reduce the levels of these T cells Tox The level / content of transcripts, thereby slowing / inhibiting T cell exhaustion; Tox Transcripts were selected from the following group: (b1) Tox Variant transcripts; (b2) Tox Full-length transcript; or combinations thereof; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

[0034] In another preferred embodiment, the Tox The nucleotide sequence of the variant transcript is shown in SEQ ID NO:31.

[0035] In another preferred embodiment, the Tox The nucleotide sequence of the full-length transcript is shown in SEQ ID NO:34.

[0036] In another preferred embodiment, the T cells are CD8. + T cells.

[0037] In another preferred embodiment, the T cells are tumor-infiltrating T cells.

[0038] In another preferred embodiment, the down-adjustment / reduction includes knockout or knockdown.

[0039] In another preferred embodiment, the CD8 + T cells and Tox Exposure to variant transcript inhibitors downregulates / reduces CD8 + T cells Tox Levels of variant transcripts.

[0040] In another preferred embodiment, theTox Inhibitors of variant transcripts include: antisense oligonucleotides, siRNA, shRNA, and CRISPR interference combinations.

[0041] In another preferred embodiment, the depletion-mitigating / suppressing CD8 + T cells also have the following characteristics: reduced persistence.

[0042] A fourth aspect of the invention provides a modified T cell that expresses... Tox Full-length transcript and not expressed Tox Variant transcripts, wherein, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

[0043] In another preferred embodiment, the Tox The nucleotide sequence of the variant transcript is shown in SEQ ID NO:31.

[0044] In another preferred embodiment, the Tox The nucleotide sequence of the full-length transcript is shown in SEQ ID NO:34.

[0045] In another preferred embodiment, the T cells are tumor-infiltrating T cells.

[0046] In another preferred embodiment, the T cells are CD8. + T cells.

[0047] In another preferred embodiment, the T cell has one or more of the following characteristics: (c1) The exhaustion phenomenon is reduced / delayed; (c2) Reduced durability.

[0048] A fifth aspect of the present invention provides a method for constructing an animal model of reduced / inhibited T cell exhaustion, the method comprising the steps of: (S1) Provide an individual animal to be modified; (S2) Knock out or knock down the levels in the said animal individuals Tox Variant transcripts were used to construct animal models of reduced / inhibited T cell exhaustion; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

[0049] In another preferred embodiment, the Tox The nucleotide sequence of the variant transcript is shown in SEQ ID NO:31.

[0050] In another preferred embodiment, the model animal expresses Tox Full-length transcript.

[0051] In another preferred embodiment, the Tox The nucleotide sequence of the full-length transcript is shown in SEQ ID NO:34.

[0052] In another preferred embodiment, the model animal is a non-human mammal.

[0053] In another preferred embodiment, the non-human mammals include rodents, monkeys, rabbits, dogs, and pigs.

[0054] In another preferred embodiment, the rodents include mice and rats.

[0055] In another preferred embodiment, the knockout includes methods selected from the group consisting of: CRISPR gene editing systems, TALEN, ZFN, conditional knockout, or combinations thereof.

[0056] In another preferred embodiment, the knockdown includes methods selected from the group consisting of RNA interference, CRISPR interference, antisense oligonucleotides, or combinations thereof.

[0057] A sixth aspect of the present invention provides Tox Use of variant transcript inhibitors in the preparation of drugs or drug compositions for slowing / inhibiting CD8. + T cell depletion; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

[0058] In another preferred embodiment, the Tox The nucleotide sequence of the variant transcript is shown in SEQ ID NO:26.

[0059] In another preferred embodiment, the drug or drug composition is also used to reduce CD8. + T cell persistence.

[0060] In another preferred embodiment, the CD8 + T cells are CD8 cells that infiltrate tumors. + T cells.

[0061] In another preferred embodiment, the Tox Inhibitors of variant transcripts include: antisense oligonucleotides, siRNA, shRNA, and CRISPR interference combinations.

[0062] In another preferred embodiment, the drug or drug composition does not affect the development of T cells.

[0063] The seventh aspect of the present invention provides Tox The use of variant transcripts and their detection or regulatory reagents in screening immunomodulatory drugs, the aforementioned Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

[0064] In another preferred embodiment, the Tox The nucleotide sequence of the variant transcript is shown in SEQ ID NO:31.

[0065] In another preferred embodiment, the features of the immunomodulatory drug are selected from the group consisting of: (a1) Specific inhibition of the above Tox Levels / contents of variant transcripts; or (a2) Specifically promotes the Tox Levels / content of variant transcripts.

[0066] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0067] Figure 1 The image shows (A) wild-type mice and mice with exon 1 deletion. Tox ΔE1 mice Tox (B) Schematic diagram of gene structure; Successfully constructed Tox Gel electrophoresis patterns of ΔE1 mice; (C) Wild-type mice and Tox Western blot results of TOX protein in the thymus and spleen of ΔE1 mice.

[0068] Figure 2 (A) is shown. Tox Schematic diagram of dKO mouse construction; (B) Compared to wild-type mice, Tox Sequencing profile of the 4 bp deletion in dKO mice.

[0069] Figure 3 Showing (A) wild-type mice, Tox dKO mice and Tox Western blot results of TOX protein in the thymus and spleen of ΔE1 mice; (B) Tox A schematic diagram of the upstream and downstream primer design for the new transcript; (CD) Tox Gel electrophoresis patterns of new transcripts in ΔE1 mouse and human Jurkat cells.

[0070] Figure 4 5' RACE detection was shown Tox A schematic diagram and results of primer design for the transcription start site of the new transcript.

[0071] Figure 5 (A) is shown. Tox (A) Schematic diagram of the construction of ΔE1 & iso2-KO mice; (B) Wild-type mice, Tox dKO mice, Tox ΔE1 mice and Tox Western blot results of TOX protein in the thymus and spleen of ΔE1 & iso2-KO mice.

[0072] Figure 6 (A) is shown. Tox (A) Schematic diagram of the construction of ΔE1 & M40 / 42 / 44A mice; (B) Wild-type mice, Tox dKO mice, Tox ΔE1 mice and Tox Western blotting results of TOX protein in the thymus and spleen of ΔE1 & M40 / 42 / 44A mice.

[0073] Figure 7 Showing Tox A schematic diagram of two transcriptional regulation modes of genes.

[0074] Figure 8 Wild-type mice and Tox (A) Comparison of thymus morphology in iso2-KO mice; (B) Comparison of spleen morphology; (CD) Flow cytometry analysis results of SP4, SP8, DP, and DN developmental populations in the thymus; (EF) Flow cytometry analysis results of dividing thymus development into four stages using CD3 and CD69; (GH) CD4 in the spleen + T and CD8 + Flow cytometry analysis results of T; (IJ) CD4 in lymph nodes + T and CD8 + Flow cytometry analysis results of T.

[0075] Figure 9 Wild-type mice and Tox Flow cytometry analysis results of SP4, SP8, DP, and DN developmental populations in the thymus of ΔE1 mice (AB); (CD) Flow cytometry analysis results of thymus development divided into four stages using CD3 and CD69; (EF) CD4 in the spleen. + T and CD8 +Flow cytometry analysis results of T.

[0076] Figure 10 Displayed CD8 in the GEO database + Comparison of accessibility of the exon 2 front region in T cells in chronic infection and tumors.

[0077] Figure 11 Wild-type mice and Tox (A) Comparison of tumor morphology in iso2-KO mice; (BC) Tumor volume and weight; (D) CD8 + T cell percentage; (EF) PD-1 and TIM-3 expression levels; (G) ProEx and TermEx cell percentage. Detailed Implementation

[0078] Through extensive and in-depth research, the inventors made their first discovery. Tox A new transcript of a gene ( Tox (iso-2), further revealing a novel transcriptional regulation mechanism in this gene: a new transcription start site exists between its first intron and second exon, located 2-3 kb before exon 2. Functional experiments demonstrated that this... Tox The new transcript does not affect T cell development, but it can effectively affect CD8. + T cell exhaustion; through knockout Tox The new transcript can effectively suppress T cell exhaustion; further knockout... Tox New transcripts and Tox The full-length transcript can achieve complete knockout of TOX protein in cells. Based on this, the present invention was completed.

[0079] Specifically, the present invention discovered Tox The gene exhibits a previously unreported mechanism of transcriptional regulation: a novel transcription start site exists between its first intron and second exon. This reveals... Tox This represents a completely new level of gene expression regulation, encompassing not only known translational regulation but also complex transcriptional regulation. This novel transcription initiation site can drive the generation of a new... Tox Transcripts ( Tox iso-2).

[0080] Sequence analysis revealed that this transcript encodes an N-terminally truncated TOX protein, which is similar in sequence to previously reported truncated proteins generated through translational regulation, but its generation mechanism is distinctly different, suggesting that it may be subject to different regulation and perform unique functions under different physiological and pathological conditions. More importantly, the functional studies in this study revealed this novel…Tox The transcript has unique biological significance. The results showed that the presence of this transcript is important for CD4. + Normal T cell development is not essential. However, in the specific context of the tumor microenvironment, Tox iso-2 transcripts exhibit a crucial function: they play a vital role in maintaining tumor-invasive CD8. + The exhaustion state and persistence of T cells are crucial. This discovery directly links a novel transcriptional regulatory event to a key cellular dysfunction phenotype, providing a completely new perspective for understanding the molecular basis of T cell exhaustion.

[0081] The discovery of this transcript not only enriches our understanding of the complex regulatory network of the key transcription factor TOX, but more importantly, it specifically maintains the tumor-invasive CD8... + The function of T cell exhaustion state and persistence provides a promising new target for precision immunotherapy, enabling detection and drug development. Traditional strategies for broadly inhibiting T cell function may be limited due to their impact on CD4+. + The impact on fundamental functions such as T cell development can lead to unpredictable side effects. Intervention strategies targeting this specific transcript or its regulatory elements hold promise for addressing depleted CD8+ cells. + The "precision strike" of T cell status reverses their functional exhaustion to enhance anti-tumor immunity while minimizing interference with the normal function of other T cell subsets. This lays a solid theoretical foundation for developing a new generation of more targeted and safer tumor immunotherapy methods and opens up a promising new path.

[0082] It should be understood that the specific methods and experimental conditions of the invention described below in varying degrees of detail are intended to provide a substantive understanding of the invention. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0083] the term As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed-ended. In other words, the terms also include “consistently made of” or “made of”.

[0084] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0085] As used in this article, the term "significant" means that, in a hypothesis test, the observed effect (such as the difference between the experimental and control groups) is unlikely to be caused solely by random error. A hypothesis test includes: the null hypothesis (H0), which assumes that the observed effect does not exist (such as no difference between the experimental and control groups); the p-value, which is the probability of observing the current or more extreme effect when H0 is true; and the significance threshold (α). The significance threshold is typically used to determine whether a hypothesis test is significant. Generally, the significance threshold is 0.05. If the p-value ≤ α, then H0 is rejected, meaning the observed effect exists, and the result is called "significant."

[0086] As used in this article, the term "persistence" refers to the ability of immune cells to survive in the host body for an extended period of time and maintain a functional response after antigen stimulation or adoptive transfer.

[0087] As used in this article, the term "exhaustion" refers to the depletion of immune cells (especially T cells, particularly CD8 cells). + Cytotoxic T cells (TCCs) have evolved a protective mechanism to avoid excessive immune activation and immunopathological damage when exposed to persistent antigen stimulation (such as in the tumor microenvironment). This mechanism allows T cells to maintain long-term cell survival under strong stimulation. Exhaustion is not the same as cell death.

[0088] TOX TOX is an important nuclear DNA binding factor, belonging to the high-mobility cassette protein superfamily. This gene was first discovered and identified in 2002 by Kaye's laboratory and is highly conserved evolutionarily. Human origin. TOX The gene is located on chromosome 8q12.1, is approximately 313 kb in length, contains 9 exons, and encodes 526 amino acids. (Mouse-derived) Tox The gene is located on chromosome 4, is approximately 304 kb in length, contains 9 exons, and encodes 526 amino acids. Currently, regarding... Tox Regarding gene expression regulation, only the translational regulation mechanism of the classic transcript has been discovered. Specifically, Tox Classic transcripts ( Tox iso-1 can generate full-length TOX protein (TOX-FL) and N-terminal truncated TOX protein variant (TOX-S) through variable translation. The NCBI accession number for human TOX-FL is NP_055544.1; the NCBI accession number for mouse TOX-FL is NP_001364007.1.

[0089] As used in this article, the terms "TOX classic transcript", " Tox “Classic transcript”, “TOX full-length transcript” and “ Tox "Full-length transcript" can be used interchangeably, both referring to... ToxThe transcript ENSMUST00000039987.5 contains a 9-exon transcribed sequence. In a preferred embodiment, the 5' UTR of the classical TOX transcript is shown in SEQ ID NO:32, the coding sequence is shown in SEQ ID NO:33, and the 3' UTR is shown in SEQ ID NO:35. In a preferred embodiment, the complete sequence of the classical TOX transcript is shown in SEQ ID NO:34.

[0090] Tox Variant transcripts As used in this article, the terms "TOX variant transcript", " Tox "variant transcripts", Tox New transcripts Tox "New transcripts", "new" Tox Transcripts Tox "gene transcript variants" and " Tox "iso-2" can be used interchangeably, both referring to a new [organism / method] discovered in this invention. Tox Transcript. This new Tox Transcripts are produced via a transcription start site located 2-3 kb before exon 2, and after translation, they express an N-terminal truncated TOX protein (TOX-S). Compared to Tox The classic transcript, this new Tox The transcript lacks partial sequences of exons 1 and 2 and has a different 5' UTR; in other words, the 5' UTR sequence is located before exon 2 of the classical transcript, and translation begins with the start codon in exon 2 to produce TOX-S. The sequence following exon 2 (including the 3' UTR) is identical to that of the classical transcript. In a preferred embodiment, Tox The 5' UTR of the variant transcript is shown in SEQ ID NO:29, the coding sequence is shown in SEQ ID NO:30, and the 3' UTR is shown in SEQ ID NO:35. In a preferred embodiment, Tox The complete sequence of the variant transcript is shown in SEQ ID NO:31.

[0091] The new Tox Transcripts ( Tox The discovery of iso-2 will help in understanding Tox Gene expression regulation and function, and based on the fact that it only affects CD8. + T cell exhaustion and persistence without affecting CD4 + This characteristic of T cell development holds promise for enabling the treatment of depleted CD8 cells. +The "precision strike" of T cell status reverses their functional exhaustion to enhance anti-tumor immunity while minimizing interference with the normal function of other T cell subsets.

[0092] Cells that do not express TOX protein at all and their preparation method This invention provides a cell that does not express TOX protein at all and a method for preparing the same. The method includes the steps of: (1) providing a cell to be modified; (2) adding the cell to the modification process. Tox Cells that do not express TOX protein are obtained by knocking out or mutating the following sequences of the gene: (2.1) knocking out exon 1; and (2.2) knocking out the 3 kb sequence upstream of exon 2, and / or mutating the start codon on exon 2 to a non-start codon. Preferably, the mutation is to mutate all three start codons to non-start codons. Preferably, the mutation is to mutate the methionine start codon to the alanine codon. Preferably, the three start codons are M40, M42, and M44 relative to the nucleotide sequence shown in SEQ ID NO:33.

[0093] In this field, Tox The conventional method for constructing gene deletion mice is to knock out... Tox Exon 1 of the gene can simultaneously knock out both TOX protein isoforms. However, this invention unexpectedly discovered that the gene constructed according to this method... Tox In gene-deleted mice, a variant of the TOX protein with an N-terminus truncated was still present in the spleen; this protein variant is related to... Tox The TOX protein variant obtained from the full-length transcript through variable translation processing is consistent. Experimental verification revealed that this TOX protein variant is derived from a... Tox It is translated from a variant transcript. Tox The variant transcript is transcribed from a transcription start site located 3 kb before exon 2, and its translation initiation depends on the three methionine start codons on exon 2 in the classical transcript.

[0094] Therefore, based on this, a method for preparing cells that do not express TOX protein at all is provided, which involves simultaneously knocking out exon 1 (which cannot be transcribed). Tox (Full-length transcript) and the 3 kb sequence upstream of exon 2 (cannot be transcribed) Tox (mutated transcript) or by mutating three methionines in exon 2 to alanine ( Tox The variant transcript cannot be translated, thus achieving complete non-expression of TOX protein, thereby preparing cells that do not express TOX protein at all.

[0095] Methods to slow down / inhibit T cell exhaustion and modified T cells This invention provides a method for slowing down / inhibiting T cell exhaustion, and a modified T cell prepared using this method. The method includes the steps of: (A) providing a T cell to be modified; (B) downregulating / reducing the levels of certain substances in the T cell. Tox The level / content of transcripts, thereby slowing / inhibiting T cell exhaustion; Tox Transcripts were selected from the following group: (b1) Tox Variant transcripts; (b2) Tox Full-length transcript; or combinations thereof; wherein, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

[0096] This invention has discovered that, Tox Full-length transcripts and Tox Variant transcripts all play an irreplaceable role in T cell exhaustion, among which, Tox Full-length transcripts can initiate the T cell exhaustion process, causing T cells to enter a progenitor cell exhaustion state, while Tox Variant transcripts can sustain the exhaustion process, causing T cells to differentiate into terminally exhausted cells. Therefore, [the following is a possible interpretation:] Tox Full-length transcripts and / or Tox Both knockout and knockdown of variant transcripts can slow down or inhibit the T cell exhaustion process.

[0097] Based on this method, a modified T cell is provided. This is achieved by knocking out [certain components] in the T cell. Tox Variant transcripts, without affecting Tox The level / content of full-length transcripts is used to prepare the modified T cells. The exhaustion phenomenon of the modified T cells is reduced / delayed. Preferably, the T cells are CD8. + T cells. Preferably, the T cells are tumor-infiltrating T cells.

[0098] Due to the highly conservative nature of TOX, those skilled in the art can reasonably expect that the discoveries of this invention... Tox New transcripts can also be found in other mammalian species besides rodents (including but not limited to humans, pigs, dogs, and non-human primates). Although specific embodiments of the present invention are described in detail using mouse models (such as mouse gene knockout and related functional verification experiments) as representative examples, based on the fundamental principle of functional conservation of homologous genes in biology, the technical solutions, regulatory mechanisms, and their resulting technical effects disclosed in this embodiment are entirely reasonable to expect for other animal individuals (especially other mammalian individuals).

[0099] Inspired by the disclosure of this invention, those skilled in the art can, without inventive effort, utilize well-known techniques to explore the transcriptional initiation range of the TOX variant transcript in other species, and to regulate its expression or validate its function in cells / tissues, achieving the same or similar technical effects as the embodiments of this invention. Therefore, the scope of protection of this invention should not be limited to the specific species (e.g., mice) listed in the embodiments, but should cover all equivalent embodiments reasonably conceived by those skilled in the art based on the teachings of this invention.

[0100] The main advantages of this invention include: (1) This invention is the first to discover Tox A novel transcript of a gene. This novel transcript has a transcriptional pattern different from existing transcripts, with its transcription start site located 3 kb before exon 2.

[0101] (2) The new transcripts of the present invention do not affect the development of T cells in the thymus, spleen and lymph nodes.

[0102] (3) The novel transcripts of the present invention are significantly enhanced in chronic infections and cancers, and can promote CD8 in tumors. + T cells are depleted and their persistence is maintained.

[0103] (4) The novel transcripts of the present invention can be used around CD8 without affecting T cell development. + Diagnosis, detection, and drug development of T cell function.

[0104] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0105] Example 1: Tox Novel transcripts ( Tox The discovery of iso-2 Known Tox The classic transcript can be translated into full-length TOX protein (TOX-FL) and N-terminal truncated TOX protein (TOX-S) through variable translation. Using CRISPR / Cas9 technology, a transcript with exon 1 deletion was constructed. Tox ΔE1 mice ( Figure 1 (A and B) can theoretically blockTox The expression of the classic transcript was inhibited, thereby knocking out these two TOX protein isoforms. Western blotting revealed the absence of TOX protein, unexpectedly showing that although... Tox TOX is not expressed in the thymus of ΔE1 mice, but a truncated TOX protein is still present in the spleen. Figure 1 C). This result indicates that Tox In addition to the reported translational regulation mechanisms that generate TOX-S, there are also classical transcript-independent transcriptional regulation mechanisms that enable TOX-S expression.

[0106] Further utilizing CRISPR / Cas9 technology, in Tox A frameshift mutation was generated on exon 3 to construct Tox dKO mice ( Figure 2 (A and B). WB results show that, compared with... Tox The spleen of ΔE1 mice contains truncated TOX protein, which differs from other mice. Tox In dKO mice, no TOX protein subtype was expressed in either the thymus or spleen. Figure 3 A). The above results suggest that this truncated TOX generated through transcriptional regulation depends on the presence of exon 3, and its transcription start site is located upstream of exon 3.

[0107] To verify the existence of this transcriptional regulation, in Tox An upstream primer was designed in intron 1, and a downstream primer was designed in exon 3. The presence of a new transcript was determined by RT-PCR. Figure 3 B). The results showed that the transcript was present but poorly expressed in the spleen of wild-type mice; in Tox In the spleen of ΔE1 mice, the expression of this transcript is enhanced. Figure 3 C) suggests that in the event of disruption of the canonical transcript, the expression of this non-canonical transcript is upregulated, potentially compensating for some functions of TOX. Therefore, Tox The classic transcript is defined as Tox iso-1, a newly discovered non-classical transcript defined as Tox iso-2. Meanwhile, a specific target was designed in human Jurkat cells. TOX Primers for iso-2 were used to confirm the presence of the new transcript via RT-PCR. Figure 3 (D), which is consistent with what has been observed in mice, indicating that this novel transcript is conserved in humans and mice.

[0108] In order to analyze this new type Tox The specific transcription start site of iso-2 transcripts, using ToxThe transcription start site of RNA from the spleen of ΔE1 mice was identified using 5' RACE. A specific 5' RACE downstream primer was designed in intron 1, and PCR was used to determine that the transcription start site of this transcript was located 2-3 kb before exon 2. Figure 4 AB).

[0109] To verify that this transcription initiation site is the starting point... Tox The key to iso-2 transcript expression, in Tox Based on the ΔE1 mouse model, CRISPR / Cas9 technology was used to knock out 3 kb upstream of exon 2 to construct... Tox ΔE1 &iso2-KO mice ( Figure 5 A) Disruption of the potential transcription start site of this transcript. Western blotting results showed that, regardless of whether in the thymus or spleen, Tox ΔE1 & iso2-KO mice and Tox Consistent with dKO mice, no TOX protein subtype was expressed. Figure 5 B) indicates that the absence of this region successfully achieved the desired result. Tox ISO-1 and Tox Double knockout of iso-2.

[0110] Known Tox The classical transcript, through alternative translation, produces an N-terminal truncated TOX-S dependent on three methionine residues in exon 2. To verify... Tox Whether the TOX truncated protein produced by the iso-2 transcript depends on three methionine residues in exon 2 of the classical transcript, i.e., whether the TOX truncated protein translated from this transcript is the same protein as TOX-S, remains to be determined. Tox Based on the ΔE1 mouse, CRISPR / Cas9 technology was used to mutate three methionines in exon 2 to alanine, successfully constructing a new cytokine-based mouse model. Tox ΔE1 & M40 / 42 / 44A mice ( Figure 6 A). Western blotting results showed that, regardless of whether it was in the thymus or spleen, Tox ΔE1 & M40 / 42 / 44A mice and Tox Consistent with dKO mice, no TOX protein subtype was expressed. Figure 6 B), indicating Tox TOX-S is a truncated protein produced by the iso-2 transcript, and its production depends on three methionine residues in exon 2.

[0111] Therefore, this study found ToxA novel transcriptional regulation mechanism has been discovered, with the transcription start site located 2-3 kb before exon 2. Combining existing reports with the results of this study, it can be demonstrated that… Tox There are two regulatory mechanisms in gene expression ( Figure 7 ), its well-known classic transcript ( Tox iso-1 can generate a full-length TOX protein (TOX-FL) and an N-terminal truncated TOX protein variant (TOX-S) through variable translation. Furthermore, Tox It can also generate novel transcription start sites located 2-3 kb before exon 2. Tox The iso-2 transcript also expresses an N-terminally truncated TOX protein (TOX-S).

[0112] Example 2: Tox The novel transcript does not affect T cell development. Given that TOX is related to CD4 + Normal T cell development is essential; therefore, this embodiment involves exploring this new… Tox Does the transcript play a role in T cell development? The specific steps are as follows: In Tox Based on ΔE1 mice Tox While using ΔE1 & iso2-KO mice, a PCR model was also constructed based on WT mice. Tox iso2-KO mice. Wild-type mice and... Tox Immunodevelopmental phenotypes were examined in the thymus, spleen, and lymph nodes of iso2-KO mice. Morphologically, wild-type mice and... Tox The morphology of the thymus and spleen was not different in iso2-KO mice. Figure 8 A and B). Changes in the proportions of various cell populations in the thymus were detected by flow cytometry, and the results showed that... Tox The proportions of CD4SP, CD8SP, DP, and DN in iso2-KO mice were not significantly different from those in wild-type mice. Figure 8 (C and D).

[0113] Furthermore, based on the expression levels of CD3 and CD69 proteins on the cell membrane, the entire thymocyte population was divided into four developmental stages: pre-positive selection, positive selection stage, post-positive selection, and mature single-positive thymocytes. Flow cytometry analysis revealed that... Tox The proportions of cells in the four stages of positive selection in iso2-KO mice were not different from those in wild-type mice. Figure 8 E and F).

[0114] In addition, by comparing wild-type mice and ToxThe composition of immune cells in the spleen and lymph nodes of iso2-KO mice also showed that CD4+ was present. + T cells and CD8 + There was no difference in the proportion of T cells. Figure 8 GJ).

[0115] and Tox Different iso-2 transcripts Tox Iso-1 transcript loss ( Tox ΔE1) will cause CD4 + T cell development is impaired. Compared to wild-type mice, Tox The proportion of CD4 SP cells in the thymus of ΔE1 mice is extremely low. Figure 9 AB), and positive selection is impaired during thymus development, resulting in a negative CD3 concentration in the positively selected cell population. + CD69 + and CD3 - CD69 + The proportions all decreased significantly ( Figure 9 CD). Correspondingly, Tox CD4 in the spleen of ΔE1 mice + The proportion of T cells also decreased significantly. Figure 9 EF).

[0116] In summary, this new type of non-classical Tox Transcripts ( Tox iso-2 is different from the classic transcript ( Tox iso-1), Tox The loss of iso-2 transcripts has no significant effect on T cell thymus development.

[0117] Example 3: Tox Novel transcript promotes CD8 + T cell depletion and maintenance of its persistence This embodiment involves further exploring the physiological significance of this novel transcript.

[0118] TOX is known to promote CD8 in chronic infections and cancer. + T cell exhaustion. In order to investigate Tox To investigate the role of novel transcripts in chronic infections and cancer, ATAC-seq data (GSE87646, GSE126970, and GSE93013) from the GEO database were analyzed. Tox The front end of exon 2, i.e. ToxAccessibility of novel transcripts near transcription start sites. Results showed that the region near the 2 kb anterior end of exon 2 is inaccessible in Naïve T cells, but its accessibility is significantly enhanced during chronic infection. Furthermore, in hepatocellular carcinoma and melanoma, non-antigen-specific CD8+... + T cells have weak accessibility to the proximal region of exon 2, but antigen-specific CD8+ + The accessibility of this region to T cells is significantly enhanced. Figure 10 The above analysis suggests that chronic infections and tumors significantly increase the risk of infection. Tox Generation of iso-2 transcripts.

[0119] Based on the above analysis results, it is inferred that... Tox The presence of iso-2 transcripts is essential for CD8. + T cell exhaustion has a regulatory function. To verify this conclusion, in wild-type mice and Tox Subcutaneous injection of B16F10-OVA melanoma cells into iso2-KO mice and monitoring of tumor progression. Results showed that... Tox The tumors in iso2-KO mice were larger and heavier than those in wild-type mice. Figure 11 AC). Flow cytometry analysis showed that Tox CD8 in iso2-KO mice + The proportion of T cells decreased significantly ( Figure 11 D), suggesting that the deletion of this transcript may affect CD8. + T cells accumulate or survive locally in the tumor. Simultaneously, further investigation of CD8... + The detection of T cell exhaustion revealed... Tox iso2-KO mice with tumor-infiltrating CD8 + T-cell inhibitory receptor PD-1 + and TIM-3 + CD8 + The positive rate of T cells was significantly downregulated. Figure 11 EF). It is worth noting that CD8 + T cells with exhausted progenitor cells (ProEx, SLAMF6) + TIM-3 - The proportion of ) increased significantly, while the proportion of terminally exhausted cells (TermEx, SLAMF6) increased significantly. - TIM-3 + The proportion decreased () Figure 11 G), the aforementioned PD-1 + / TIM-3 + The decrease in the positive rate and the reduction in the proportion of the terminal exhaustion subgroup are logically consistent, suggesting... ToxThe loss of the iso-2 transcript did not completely block the initiation of the exhaustion program, but rather hindered the differentiation process of exhausted T cells from the progenitor stage to the terminal stage. Simultaneously, it bound CD8... + The overall decrease in the proportion of T cells suggests, as indicated by the above results, that... Tox iso-2 may also participate in CD8 activation while regulating terminal differentiation due to exhaustion. + The persistent maintenance of T cells in the tumor microenvironment.

[0120] The above results indicate that Tox Different transcripts play a non-redundant role in the regulation of T cell exhaustion. Based on the above results, it can be inferred that... Tox still present in iso2-KO mice Tox ISO-1 is sufficient to initiate the exhaustion process, driving T cells into a progenitor exhaustion state and upregulating the expression of inhibitory receptors. However, simply relying on... Tox iso-1 is insufficient to support the complete process of an exhaustive program; Tox The absence of iso-2 causes the exhaustion process to be arrested at the progenitor stage, preventing effective differentiation into terminal exhaustion cells. This finding is consistent with observations in tumor and chronic infection microenvironments. Tox This is consistent with the phenomenon of increased accessibility to iso-2 chromatin, i.e. Tox iso-2 is induced to be expressed under continuous antigen stimulation, and its function is to drive exhausted T cells to advance along the differentiation trajectory toward the terminal stage.

[0121] In summary, it is known that TOX drives CD8. + This invention further discloses that: T cell exhaustion programs are key factors in their initiation and maintenance, and TOX-induced exhaustion programs are considered an adaptive protective mechanism against T cell overactivation leading to AICD. Tox The absence of iso-2, although preserved Tox While iso1-mediated depletion initiation capacity exists, it leads to partial arrest of terminal differentiation, with a significant proportion of cells remaining in the progenitor depletion stage. These retained progenitors did not exhibit the expected antitumor effects, presumably due to a lack of... Tox iso-2-mediated differentiation protection predisposes to AICD, leading to intratumoral CD8... + The number of T cells decreased significantly, resulting in a loss of control over tumor growth. Therefore, Tox iso-2 transcripts are essential for maintaining CD8. + T cells are essential for survival and functional integrity in the tumor microenvironment.

[0122] DNA sequence Note: The bolded part in the sequence is the start codon.

[0123] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A type of T cell Tox Variant transcripts ( Tox The uses of iso-2 and its detection reagents are characterized by, This is used to prepare a diagnostic reagent or kit for evaluating CD8 in a given subject. + T cell depletion status; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

2. The use as described in claim 1, characterized in that, The Tox The nucleotide sequence of the variant transcript is shown in SEQ ID NO:

31.

3. The use as described in claim 1, characterized in that, The CD8 evaluation of a certain object + T cell exhaustion includes the following steps: (a1) Detect the CD8 of the object + T cells Tox Levels / contents of variant transcripts; (a2) The above Tox The level / content of variant transcripts was compared with reference / standard values ​​to assess the CD8 content of the subject. + T cell depletion status; Wherein, if the Tox If the level / content of the variant transcript is higher than the reference / standard value, it indicates that the target CD8 + Increased T cell exhaustion, or CD8 + The risk of T cell exhaustion is increased; conversely, it indicates that the CD8+ of the target cell is depleted. + The T cell exhaustion phenomenon is weakened, or CD8 + The risk of T cell depletion is reduced.

4. A method for preparing cells that do not express TOX protein at all, characterized in that, The method includes the following steps: (1) Provide a cell to be modified; (2) In the cells described Tox The genes were treated as follows to obtain cells that did not express TOX protein at all: (2.1) Knockout of exon 1; and (2.2) Knock out the 3 kb sequence upstream of exon 2, and / or mutate the start codon on exon 2 to a non-start codon.

5. The use as described in claim 3, characterized in that, The mutation involves changing the methionine start codon to the alanine codon.

6. A method for slowing down / inhibiting T cell exhaustion, characterized in that, The method includes the following steps: (A) Provide a T cell to be modified; (B) Downregulate / reduce the levels of these T cells Tox The level / content of transcripts, thereby slowing / inhibiting T cell exhaustion; Tox Transcripts were selected from the following group: (b1) Tox Variant transcripts; (b2) Tox Full-length transcript; or combinations thereof; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

7. A modified T cell, characterized in that, The T cells express Tox Full-length transcript and not expressed Tox Variant transcripts, wherein, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

8. A method for constructing an animal model of reduced / inhibited T cell exhaustion, characterized in that, The method includes the following steps: (S1) Provide an individual animal to be modified; (S2) Knock out or knock down the levels in the said animal individuals Tox Variant transcripts were used to construct animal models of reduced / inhibited T cell exhaustion; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

9. A kind Tox The use of variant transcript inhibitors is characterized by, Used to prepare a drug or drug composition for slowing / inhibiting CD8. + T cell depletion; Among them, the Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

10. Tox The use of variant transcripts and their detection or regulatory reagents in screening immunomodulatory drugs, characterized in that, The Tox The transcription start site of the variant transcript is located at Tox Within 3 kb upstream of exon 2 of the gene.

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