CN111773380A - Application of PLPP1 in preparation of T cell immune tumor related medicament - Google Patents

Abstract

The application belongs to the technical field of cellular immunology biology, and particularly relates to an application patent application of PLPP1 in preparation of a T cell immune tumor related medicament. The PLPP1 is a key enzyme for regulating and controlling the phospholipid metabolism of T cells; in a tumor microenvironment, the reduction of the expression of a metabolism key enzyme PLPP1 causes abnormal phospholipid metabolism of CD8+ T cells, further causes abnormal metabolism of CD8+ T cells and finally causes abnormal T cell function; that is, the expression of PLPP1 has a positive correlation with the activation, infiltration and function of CD8+ T cells in the tumor microenvironment. The intensive study shows that: after the PDL1/PD-1 signal channel is activated, an AKT/mTOR signal channel is further activated, the expression of a key enzyme PLPP1 for the phospholipid metabolism of T cells is regulated and controlled through a transcription factor GATA1, and finally the function regulation of the T cells is realized through the change of the metabolic function of the T cells.

Description

Application of PLPP1 in preparation of T cell immune tumor related medicament

Technical Field

The application belongs to the technical field of cellular immunology biology, and particularly relates to an application patent application of PLPP1 in preparation of a T cell immune tumor related medicament.

Background

In recent years, in order to inhibit the threat of malignant tumor to human health, chimeric antigen receptor T (CAR-T) cell therapy technology and immune checkpoint blockade antibody (ICB) technology (ICB therapy is mainly applied to various solid tumors such as lung cancer, esophageal cancer, gastric cancer and melanoma) are rapidly developed, certain success is achieved in the treatment of partial types of blood and solid tumor patients, and the 5-year survival rate of the patients is well improved. Practical applications have shown that these T cell-based immunotherapy techniques are effective in only a few patients, especially in solid tumor patients.

It has been considered that: the interaction between the solid tumor microenvironment and T cells is a key factor affecting the efficacy of immunotherapy, whereas there are numerous immunosuppressive molecules in the solid tumor microenvironment, such as PD-L1, CTLA-4, IDO, etc., where PD-L1 is considered to be a key molecule inhibiting T cell activity and PD-L1 inhibits tumor killing activity of T cells by activating PD-1 on T cells. Based on the application, a part of monoclonal antibody medicaments targeting PDL1/PD-1 and CTLA4 are applied to a certain extent, and show a certain application effect. For example: iplilimumab (anti-CTLA 4), Pembrolizumab (anti-PD-1), Nivolumab (anti-PD-1), and the like.

With respect to T cell development, the main physiological processes are: after T cells in thymus are mature, the T cells can enter the peripheral circulation system of human and play a main role in adaptive immune response, wherein the T cells in the resting stage are further differentiated into effector T cells and memory T cells after being stimulated by external antigens or self-mutation antigens so as to play an immune function.

With respect to T cell physiological metabolic processes, when T cells are in the resting phase, catabolism of glucose and amino acids provides a source of energy for the cells' vital activities; when T cells enter an activated state from a resting stage, the metabolic characteristics of the T cells are changed, and the cells can obtain and consume a large amount of nutrients from the outside for synthesis of substances in the cells and energy demand, and the metabolic pathways mainly comprise aerobic glycolysis, glutamine catabolism, oxidative phosphorylation and the like.

Theoretically, the blocking antibody therapy targeting PD-L1 or PD-1 can improve tumor therapy, but actually, clinical studies show that environmental factors can cause the metabolism of T cells to be changed, and further influence the responsiveness of the T cells to the blocking therapy and the tumor therapy effect. Thus, a prerequisite for improving T cell immunotherapy is a deep understanding of the interactions between immunosuppressive factors and T cell metabolism and their impact on blocking therapy, finding key metabolic molecules to develop more effective combination therapy strategies.

Disclosure of Invention

The application aims to lay a certain technical foundation for the prevention or treatment of related tumors through the research on the effect of PLPP1 in the preparation of T cell metabolism and the exertion of immune functions.

The technical solution adopted in the present application is detailed as follows.

Use of PLPP1 in the manufacture of a medicament for treating a T cell immune tumor, said PLPP1 (phospholiphatase 1 ) being a key enzyme for regulating T cell (CD 8 +) phospholipid metabolism, said reduction of PLPP1 expression being a key enzyme for the metabolism of T cell phospholipid in the tumor microenvironment, said reduction of PLPP 6324 expression being positively correlated with CD8+ T cell phospholipid metabolism, which in turn leads to CD8+ T cell global metabolism and ultimately to T cell dysfunction, or said reduction of PLPP1 expression being positively correlated with the activation, infiltration and function of CD8+ T cells in the tumor microenvironment, i.e. said reduction of PLPP1 expression in CD8+ T cells results in T cell proliferation, metabolism and function reduction, whereas said high PLPP1 expression promoting T cell infiltration and function in tumor tissues, said phospholipid metabolism including but not limited to phospholipid metabolism (PA), diglyceride (diacylglycerol, phosphatidylcholine (phosphatidylcholine, Phosphatidylcholine (PDG), and cholesterol metabolism (PDL-PC) including but not limited to reduction of TNF-cholesterol metabolism and said reduction of intracellular metabolism including but not limited to TNF-PDL, cholesterol metabolism, and said reduction of intracellular metabolism including but not limited to TNF-PDL-cholesterol metabolism, and cholesterol metabolism, said reduction of IFN, and cholesterol metabolism including but also to decrease of TNF-cholesterol metabolism including TNF-cholesterol metabolism, and cholesterol metabolism including the metabolism in the metabolism of TNF-cholesterol metabolism including but also including the metabolism of TNF-mediated by IFN cells²⁺The flux concentration is used as a regulating path, so that the proliferation function, the immune function and the metabolic function of T cells are obviously reduced, or when the expression of PLPP1 is reduced, the proliferation, the killing function, the metabolism and the intracellular Ca of the T cells are reduced²⁺The stream concentration is significantly reduced.

In a specific signal pathway, PDL1/PD-1 is combined, activation of an AKT/mTOR signal pathway is inhibited, nuclear translocation of GATA1 is increased, and expression of PLPP1 in cells is reduced finally; in specific application, after the PDL1 blocking monoclonal antibody is applied, the expression of PLPP1 is increased by restoring AKT/mTOR signal pathways in CD8+ T cells, so that the infiltration and the function of the T cells are promoted, and the growth of tumor cells is finally delayed.

The use of PLPP1 in the preparation of a T cell immune tumor-associated medicament, wherein the tumor includes but is not limited to: solid tumors such as esophageal cancer, lung cancer, colon cancer or melanoma.

In addition to the high expression of co-stimulatory molecules on the surface of T cells during the activation process, the high expression of co-inhibitory molecules on the cell surface, such as PD-1, etc., these co-stimulatory molecules and co-inhibitory molecules are involved in the activation of the cells in a synergistic manner. After the T cell is activated, a corresponding signal channel in the cell is activated, so that various activation-related transcription factors in the T cell are subjected to nuclear translocation, and the proliferation, survival and effect function of the T cell are promoted finally.

It has been suggested that the signaling pathways associated with T cell activation are mainly PI3K/AKT, PKC, RAS/ERK, and calmodulin/NFAT, among others. The main reason for the inhibition of T cell function and proliferation ability in the tumor microenvironment is due to the presence of a number of immunosuppressive factors, such as immunosuppressive cells (tumor-associated macrophages or myeloid-derived suppressor cells, etc.), cytokines (IL 6, IL10, etc.) in the tumor microenvironment. These immunosuppressive factors will eventually act on T cells, resulting in abnormal signaling pathways within the T cells. Although the PDL1/PD-1 signal pathway in the tumor microenvironment plays a crucial role in the tumor killing process of T cells, clinical trials find that even if the PDL1/PD-1 signal pathway is blocked, a part of patients still have no clinical response. Therefore, the inventor conducts further intensive research on the action mechanism of the PDL1/PD-1 signal path through a series of experiments, and the result shows that: after the PDL1/PD-1 signal channel is activated, the AKT/mTOR signal channel is further inhibited, the expression of a key enzyme PLPP1 for the phospholipid metabolism of T cells is regulated and controlled through a transcription factor GATA1, and finally the function regulation of the T cells is realized through the change of the metabolic function of the T cells.

Drawings

FIG. 1 shows the T cell function and proliferation (PBMC obtained by density gradient centrifugation and TIL (tumor infiltrating lymphocytes) obtained by tumor tissue digestion) in peripheral blood and tumor tissue sites of patients with lung cancer, wherein A: expression of CD69+ CD8+ T cell surface co-suppressor molecules in PBMCs and TILs of lung cancer patients; b: secretion of CD69+ CD8+ T cell killer cytokines in PBMCs and TILs of lung cancer patients; c: expression of cell proliferation-related molecules in CD69+ CD8+ T cells in PBMCs and TILs of lung cancer patients;

FIG. 2 shows the tumor tissue site PLPP1 of a patient with lung cancer^highAnd PLPP1^lowCD69+ CD8+ T cell function of (a); wherein, A: PLPP1 in tumor tissue of patients with lung cancer^highAnd PLPP1^lowFlow-through display of CD69+ CD8+ T cell function of (a);

b: PLPP1 in tumor tissue of patients with lung cancer^highAnd PLPP1^lowA statistical map of CD69+ CD8+ T cell function of (a);

FIG. 3 shows PLPP1 in mouse melanoma tumor tissue^highAnd PLPP1^lowAntigen-specific T cell function and proliferation; wherein, A: establishing a mouse malignant melanoma model; b: mouse tumor growth curve; c: PLPP1 in malignant melanoma tumor tissue^highAnd PLPP1^lowSecretion of IFN- γ from antigen-specific T cells of (1); d: PLPP1 in malignant melanoma tumor tissue^highAnd PLPP1^lowExpression of Ki67 in antigen-specific T cells;

FIG. 4 is a graph showing the change in CD8+ T cell metabolism-associated enzymes following PLPP1 knockdown; wherein, A: changes in glycolytic related enzymes in PLPP1-sh1 and PLPP1-sh2CD8+ T cells; b: alterations in lipid metabolism-associated enzymes in PLPP1-sh1 and PLPP1-sh2CD8+ T cells; the ordinate value is relative expression quantity of PLPP1-sh group metabolic enzyme/relative expression quantity of NC group metabolic enzyme, the left side is PLPP1-sh1, the right side is PLPP1-sh2, the upper dotted line represents the enzyme with increased expression of PLPP-sh group, and the lower dotted line represents the enzyme with decreased expression of PLPP-sh group;

FIG. 5 is the change in ECAR and OCR in CD8+ T cells following PLPP1 knockdown; wherein, A: changes in OCR of PLPP1-sh1 CD8+ T cells; b: changes in ECAR in PLPP1-sh1 CD8+ T cells; c: mitochondrial changes in PLPP1-sh1 and PLPP1-sh2CD8+ T cells;

FIG. 6 shows the proliferation of CD8+ T cells following PLPP1 knockdown; wherein, A: apoptosis of PLPP1-sh1 and PLPP1-sh2CD8+ T cells; b: the number of PLPP1-sh1 and PLPP1-sh2CD8+ T cells; c: the proportion of PLPP1-sh1 and PLPP1-sh2CD8+ T cell proliferating cells; d: expression of Ki67 from PLPP1-sh1 and PLPP1-sh2CD8+ T cells;

FIG. 7 is a functional change in CD8+ T cells following PLPP1 knockdown; wherein, A: secretion of IFN-gamma and TNF-alpha from PLPP1-sh1 and PLPP1-sh2CD8+ T cells following PMA/ionomycin stimulation; b: changes in Ca2+ flux in PLPP1-sh1 and PLPP1-sh2CD8+ T cell cytoplasm following OKT3 stimulation; c: secretion of IFN-gamma and TNF-alpha from PLPP1-sh1 and PLPP1-sh2CD8+ T cells after OKT3 stimulation;

FIG. 8 is a change in PLPP1 after co-incubation of CD8+ T cells with tumor cells; wherein, A: expression of PLPP1 after co-incubation of healthy human activated CD8+ T cells with lung cancer cell lines H460 and a 549; b: expression of PLPP1 following co-incubation of activated OT-1 cells in mouse spleen with mouse malignant melanoma B16-OVA; c: activation of PLPP1+ in tumor tissue of lung cancer patients expression of co-inhibitory molecules PD-1, CTLA4 and Tim3 in CD8+ T cells;

FIG. 9 is the change in CD8+ T cell proliferation and PLPP1 expression following PDL1/PD-1 binding; wherein, A: expression of PD-1 on the surface of healthy human activated CD8+ T cells and after co-incubation with lung cancer cell line a 549; b: expression of PDL1 on the surface of tumor cells after healthy human activated CD8+ T cells and co-incubation with lung cancer cell line a 549; c: expression of PLPP1 in activated CD8+ T cells and activated CD8+ T cells cultured with PDL1Fc fragment; d: expression of MKi67 in activated CD8+ T cells and activated CD8+ T cells cultured with PDL1Fc fragment;

FIG. 10 is a graph of the change in PLPP1 expression and function in CD8+ T cells following PDL1/PD-1 binding; wherein, A: expression of PLPP1 and IFN- γ in healthy human activated CD8+ T cells and activated CD8+ T cells cultured with PDL1Fc fragment; b: expression of PLPP1 in healthy human activated CD8+ T cells and activated CD8+ T cells cultured with PDL1Fc fragment; c: knockdown efficiency of CRISPR/Cas9 technology on activated CD8+ T cell surface PD-1; d: effect of PDL1Fc fragment on PLPP1 expression in activated CD8+ T cells as well as CRISPR-PD-1 CD8+ T cells; e: effect of PDL1Fc fragment and PD1 blocking antibody on PLPP1 expression in activated CD8+ T cells;

FIG. 11 is the change in CD8+ T cell metabolism following PDL1/PD-1 binding and CD8+ T cell metabolism following PLPP1 knockdown; wherein, A: changes in OCR in healthy human activated CD8+ T cells and activated CD8+ T cells cultured with PDL1Fc fragment; b: changes in OCR in healthy human activated PLPP1-sh1 CD8+ T cells and activated PLPP1-sh1 CD8+ T cells cultured with PDL1Fc fragment; c: flow schematic of intracellular mitochondrial changes in CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells with and without treatment of PDL1Fc fragment; d: statistical plots of intracellular mitochondrial changes in CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells with and without treatment of the PDL1Fc fragment;

FIG. 12 is a change in CD8+ T cell function following PDL1/PD-1 binding and CD8+ T cell function following PLPP1 knockdown; wherein, A: intracellular Ca in PDL1Fc fragment treated and untreated CD8+ T cells, PLPP1-sh1 CD8+ T cells²⁺The change of concentration, B is a statistical graph of the change of intracellular TNF- α of CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells treated and untreated with PDL1Fc fragment, C is a statistical graph of the change of intracellular IFN-gamma of CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells treated and untreated with PDL1Fc fragment;

FIG. 13 shows the expression and functional changes of PLPP1 in antigen-specific T cells in malignant melanoma of mice after PDL1 monoclonal antibody treatment; wherein, A: a flow chart of a mode of mouse malignant melanoma inoculation, OT-1 adoptive reinfusion and monoclonal antibody treatment; b: PDL1 mab treated and untreated mouse tumor volume change curves; c: expression of PLPP1 in PD-1highTCRV beta 5+ CD45.2+ CD8+ T cells and PD-1-TCRV beta 5+ CD45.2+ CD8+ T cells in PDL1 monoclonal antibody treated and untreated mouse tumor tissues; d: expression of IFN- γ, granzyme b and CD107a in TCRV β 5+ CD45.2+ CD8+ T cells in PDL1 mab-treated and untreated mouse tumor tissues;

FIG. 14 is a graph of the effect of PDL1/PD-1 binding on the AKT/mTOR signaling pathway in CD8+ T cells; wherein, A: the relationship between the expression of PLPP1 in CD8+ T cells in the tumor microenvironment of lung cancer patients in the GSE90728 database and the PI3K/AKT/mTOR signaling pathway; b: changes in the AKT/mTOR signaling pathway following treatment of Jurkat and Jutkat-PD1 cell lines with the PDL1Fc fragment; c: change in the proportion of p-AKT following treatment of activated CD8+ T cells of healthy human origin with PDL1Fc fragment; d: changes in the AKT/mTOR signaling pathway and PLPP1 following treatment of activated CD8+ T cells of healthy human origin with PDL1Fc fragment; NC is a control group, i.e., PDL1Fc untreated group; PDL1Fc is PDL1Fc treatment group;

FIG. 15 is the effect of PDL1/PD-1 binding on transcription factors in CD8+ T cells; wherein, A: transcription factor change heatmap after PDL1Fc fragment treatment of activated CD8+ T cells; b: fold change in transcription factors following treatment of activated CD8+ T cells with PDL1Fc fragment (PDL 1Fc group/NC group); c: wein analysis of the transcription factor changed 2 times after the PDL1Fc fragment is processed and the transcription factor predicted by the PROMO website; d: changes in PLPP1 following transcription factor silencing in activated CD8+ T cells; FIG. 16 is a binding assay of GATA1 with the PLPP1 promoter region in CD8+ T cells; wherein, A: figure shows the binding sites of GATA1 to the PLPP1 promoter region; b: analysis of the binding ability of GATA1 to the binding site of PLPP1 promoter region; c: analysis of the binding ability of GATA1 to the binding site of PLPP1 promoter region after AKT inhibitor treatment, wherein Control represents the AKT inhibitor untreated group;

FIG. 17 is a binding assay of GATA1 at different sites in the PLPP1 promoter region in CD8+ T cells; wherein, A: schematic target sequence for GATA1 recognition and binding to DNA; b: wild type and mutant forms of GATA1 recognition sequence; c: binding assay of GATA1 to the Site1 of the PLPP1 promoter region in CD8+ T cells; d: binding assay of GATA1 to the Site2 of the PLPP1 promoter region in CD8+ T cells;

FIG. 18 is a nuclear translocation analysis of GATA1 in CD8+ T cells; wherein, A: imaging flow diagrams of GATA1 nuclear translocation in CD8+ T cells following PDL1Fc fragment or PD-1 blocking antibody treatment; b: flow-scale map of GATA1 nuclear translocation in CD8+ T cells following PDL1Fc fragment or PD-1 blocking antibody treatment; c: nuclear translocation of GATA1 and expression of PLPP1 in CD8+ T cells following AKT inhibitor treatment; in each figure, P < 0.05, P < 0.01.

Detailed Description

The present application is further explained below with reference to the drawings and examples. Before describing the embodiments, the background of some of the experiments involving some of the biological materials, reagents, devices, etc. described in the following examples is briefly described as follows.

Biological material:

peripheral blood of patients with esophageal cancer, lung cancer or colon cancer, and paracancerous normal tissues and tumor tissues are approved by an ethical committee and come from a department related to the first subsidiary hospital of Zhengzhou university; peripheral blood of healthy donors from the red cross blood center of zheng zhou city, han province; lung cancer tumor cell lines H460 and a549 from the department of chinese academy of sciences; SPF grade CD 45.1C 57BL/6N wild-type female mice from Beijing university Hospital's basic medical college; SPF grade CD45.2 OT-1 mice, as well as B16-OVA cell line, 293T cell line, Jurkat cell line, from Beijing cooperative medical college of Chinese medical sciences; plasmid pSIH 1-H1-concGFP-shRNA, plasmid pSpCas9(BB) -2A-GFP (PX458), purchased from Biovector plasmid vector bacterial cell Gene Collection; plasmid pcDNA3.1(+) -3 Flag from Nanjing PPL plasmid and protein shared library; plasmid pGL3-basic and plasmid pRL-TK from Promega, USA; the related PCR primers and short hairpin RNA (shRNA) sequences, small interfering RNA (siRNA) sequences and guide RNA (gRNA) sequences are as follows (all provided by Shanghai's synthetic synthesis, and the primers are not listed in the experimental process and refer to the conventional design or the conventional primers in the prior art):

beta-actin upstream: 5'-CTCCATCCTGGCCTCGCTGT-3', downstream of β -actin: 5'-GCTGTCACCTTCACCGTTCC-3', respectively; upstream of PLPP 1: 5'-ACGCCCCACACTGCAATTT-3', PLPP1 downstream: 5'-TGAGTCCAGTCAACACATCGC-3', respectively; upstream of PLPP1-shRNA 1: 5'-GCTGTTTGTGGCACTTTATCTTCCTGTCA-3', PLPP1-shRNA1 downstream: 5'-GAATAAAGTGCCACAAACAGCTTTTT-3', respectively; upstream of PLPP1-shRNA 2: 5'-GCTGTATATGTATCGGATTCTTCCTGTCA-3', PLPP1-shRNA2 downstream: 5'-GAAATCCGATACATATACAGCTTTTT-3', respectively; upstream of PD-1-gRNA: 5'-GTCTGGGCGGTGCTACAACT-3', PD-1-gRNA downstream: 5'-AGTTGTAGCACCGCCCAGAC-3', respectively; upstream of GATA 1: 5'-ATGGAGTTCCCTGGCCTGGG-3', GATA1 downstream: 5'-TCATGAGCTGAGCGGAGCCA-3', respectively; GATA1-siRNA1 (si 1): 5 '-CAGGYGYACCCAUGCUCAACUGUA-3', GATA1-siRNA1 (si 1) downstream: 5'-UACAGUUGAGCAAUGGGUACACCUG-3', respectively; GATA1-siRNA2 (si 2): 5'-GGAAGGAUGGUAUUCAGACUCGAAA-3', GATA1-siRNA2 (si 2): 5'-UUUCGAGUCUGAAUACCAUCCUUCC-3', respectively;

the main reagents are as follows:

NucleoSpin RNA XS (microtissue RNA extraction kit), germany MN; CD8 MicroBeads (human), MACS buffer, Tumor Dissociation Kit (human), T cell TransAct^TNMiltenyi biotec, germany; RT Profile PCR Array and RT²First Strand Kit, Germany QIAGEN, FastStartEssential DNA Green Master, Switzerland Roche, PPAP2A antisense, British Biorbyt, JETPRIME (transfection reagent), France Ployplus, reverse transcription Kit, Japan TAKARA, Anti-CD8 alpha antisense, Anti-PPAP2A antisense, GATA1 antisense, Abcam company, Alexa Fluor 555 Donkey antisense-rat IgG, Alexa Fluno TCR 488 Golgi-Mouse IgG, Alexa Fluno 488 Donkenti-rat IgG, APC/Cyaniine 7 antisense-APC CD8, Ser 5-CD 7 antisense-Mouse CD 5, CD-14-APC-rabbit IgG, CD 14-14 Goat IgG, APC/Cyani-CD 7 antisense-APC 8, CD 7 antisense-Mouse CD 5, CD 19-Mouse IgG, CD 14-Goat 14-rat IgG, CD 14 Goat IgG, CD 5-Goat 14 Goat-Goat 14-rat IgG, CD 14-Goat 14, CD 14-Goat antisense DNA 5, CD 14, CD 5-Goat antisense DNA 5-5, CD 5-5, CD 5-Goat IgG, CD 5-5, CD III, CD 5-III, 5-III, 5-III, 5-III, III-III, III-III, III-III, III-III, III-III, III-III, III-IV, III-III, III-IV, III-IV, IIIeBioscience, usa; recombinant Human PD-L1 Fc Chimera Protein, U.S. R&D; CellProlification Dye eFluor 670, MitoTracker Deep Red FM, Rhod-2 (AM), ECL emitting fluid, Thermo Fisher Scientific, USA; restriction enzymes BamH1, EcoR1, Age1, Xho1, Nhe1 and Quick Ligation Kit, U.S.A.NEB; XF Cell Mito Stress Test Kit, XF Glycosys Stress Test Kit, Agilent, USA;

HRP-coupled goat anti-rabbit IgG, HRP-coupled goat anti-mouse IgG, and Beijing Zhonghuai Jinqiao; extracting kit of nuclear protein and plasma protein, Shanghai Beyotime;

part of the experimental equipment:

ultra-high performance liquid phase, NanoDrop 2000, seahorse XFe96 cell energy metabolism analyzer, Agilent, usa; high resolution mass spectrometry, usa AB Sciex; six color flow cytometric instrument, U.S. BD; a fluorescent quantitative PCR amplification instrument, a PCR amplification instrument, American Bio-Rad; imaging analysis flow cytometer, Merck, usa; mouse in vivo fluorescence imager, U.S. PerkinElmer; chemiluminescence imaging system, Bio-Rad, USA;

part of the experimental detection methods (the operation is not detailed and the prior art can be referred to):

CD8+ T cell function assay: after the FACS buffer solution is used for resuspending the cells, adding APC/Cyanine7 anti-human CD8 antibody and PE/Cyanine7 anti-human CD69 (containing APC/Cyanine7 anti-human CD8, PerCP/Cy5.5 anti-human TCRV beta 5.1/5.2 and PE/Cyanine7 anti-human CD 45.2), and incubating for 15 minutes at 4 ℃ in a dark place; then, fixing and breaking membranes, adding PPAP2A antibody (1: 1000 dilution), APC-human TNF-alpha (or APC anti-human IFN-gamma (containing APC anti-mouse IFN-gamma)), and incubating for 30 minutes at 4 ℃ in the absence of light; adding PE Donkeyanti-rabbitIgG, and incubating for 15 minutes at 4 ℃ in the dark; after centrifugal cleaning, resuspending FACS buffer solution, and detecting on a flow type computer;

t cell proliferation assay: detecting the proliferation of CD8+ T cells by adopting a dye eFluor 670 labeling method or a Ki67 staining method; and (3) detecting cell apoptosis: processing by using APC-annexin V, and detecting the apoptosis ratio of CD8+ T cells on a computer; t cell Ca²⁺Flow detection: detection of CD8+ T cells by calcium ion probe Rhod-2 AMIntracellular Ca²⁺A stream; ECAR and OCR detection: using a special ECAR (sea horse XF Base Medium) for detecting energy metabolism of hippocampus to cooperate with 2-DG or antimycin A to respectively detect the extracellular acidification rate (ECAR) of CD8+ T cells and the oxygen consumption rate (O2 neutralization rate, OCR) of CD8+ T cells; t cell mitochondrial detection: the CD8+ T cell mitochondria are detected and judged by using MitoTracker Deep Red FM treatment and detecting the change condition of fluorescence intensity on a computer.

Example 1

The inventor thinks that the microenvironment of the solid tumor has a close correlation function with the function of the T cells, and the formation of the microenvironment is the result of the combined action of the T cells and the tumor tissues, and the inventor carries out a series of experiments for researching and determining the key influencing factors of the T cell metabolism related to the tumor in the tumor microenvironment, and the detailed description is as follows.

(ii) related expression molecule differences

In order to determine the actual molecular expression difference, the inventor respectively takes a lung cancer patient peripheral blood and a tumor tissue sample, treats the lung cancer patient peripheral blood and the tumor tissue sample into 2-4mm small fragments, utilizes three enzymes (enzyme H, enzyme R and enzyme A) to treat and digest the tissue, extracts human Peripheral Blood Mononuclear Cells (PBMC) by a density gradient centrifugation method, further utilizes CD8 Microbeads to separate and obtain CD8+ T cells by a magnetic sorting method, and finally utilizes anti-human CD69 antibody to perform flow sorting to obtain CD69+ T cells, namely activated CD8+ T cells (namely CD69+ CD8+ T cells). Subsequently, the differences of the CD8+ T cell expression co-suppression molecules (PD-1, CTLA4 and Tim 3), the functional molecules (Granzyme B and IFN-gamma) and the proliferation related molecules Ki67 are detected and analyzed (the specific operation refers to the prior art or the instruction of a kit). The statistical results for 5 samples are shown in FIG. 1. Analysis can see that the expression of CD69+ CD8+ T cell surface PD-1, CTLA4 and Tim3 in Tumor Infiltrating Lymphocytes (TIL) is significantly up-regulated compared to PBMCs (fig. 1A); reduction of secretion of granzyme B and IFN- γ (fig. 1B); in addition, Ki67 expression was down-regulated in CD69+ CD8+ T cells in TIL (fig. 1C). These results all show that: PD-1, CTLA4 and Tim3 were expressed at higher levels in tumor tissue, while granzyme b, IFN- γ and Ki67 were expressed at lower levels, indicating that CD8+ T cells are inhibited in the tumor microenvironment; namely: the TIL has the function disorder of CD8+ T cells, and the proliferation capacity of the cells is obviously reduced, thereby finally reducing the killing capacity of the T cells to tumor cells.

(II) differences in specific physiological indices

Based on the preliminary flow cytometry analysis, the functional exhaustion state of T cells in a tumor microenvironment can be found, and based on the existing research, the inventor deduces that the distribution of lipid molecules in the T cells in the tumor microenvironment is probably in an abnormal state. Therefore, after quenching the metabolic substances in the CD69+ CD8+ T cells, the lipid molecule type and abundance in the cells are detected by using the ultra-high performance liquid mass spectrometry (specifically referring to the prior art). As a result, it was found that: the distribution of lipid molecules in CD69+ CD8+ T cells in TIL was significantly different from that in PBMC, and further analysis of the differential lipid molecules revealed: in tumor tissue, CD8+ T cells have a higher abundance of Phosphatidic Acid (PA) and lower abundance of Diglycerides (DG), Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE). The above results indicate that the PA catabolic pathway is inhibited and cannot be effectively utilized in T cells, and PA is the main substrate for the synthesis of both PC and PE phospholipid molecules, where the expression of DG as an intermediate product is also reduced during the synthesis of the phospholipid molecules. This result indicates that: decreased ability to synthesize phospholipid molecules in CD8+ T cells; in other words: the TIL has low DG content in CD69+ CD8+ T cells, and the content of the main components of the cell membrane, namely PC and PE, is also at a low level, so that the TIL can be preliminarily determined as follows: abnormal lipid metabolism of CD8+ T cells in TIL is involved in the regulation of T cell function and proliferation.

Because the metabolic process of synthesizing two phospholipid molecules, namely PC and PE by PA is mainly regulated and controlled by PLPP1, namely PLPP1 is a key enzyme for changing the phospholipid metabolism of T cells, the inventor further utilizes a NucleoSpin RNA XS kit and refers to the prior art to extract RNA of CD69+ CD8+ T cells and then carries out reverse transcription on the RNA to obtain cDNA, and the inventor utilizes an RT-PCR (internal reference gene beta-actin) technology and combines with flow cytometry and a cell immunofluorescence technology to detect the expression condition of an activated CD8+ T cell phospholipid metabolism related enzyme PLPP1 in peripheral blood and tumor tissues of tumor patients. The expression detection result of PLPP1 in activated CD8+ T cells in paracancerous normal tissues and tumor tissues shows that: expression of activated CD8+ T cells PLPP1 was down-regulated in tumor tissue compared to peripheral blood and paracancerous normal tissue. The result of the expression of PLPP1 protein level in activated CD8+ T cells in TIL by immunofluorescence and flow cytometry shows that: PLPP1 expression was lower in activated CD8+ T cells in TIL.

Correlation of PLPP1 expression with signaling pathways and T cell functional molecules

Based on the above analysis, it can be seen that a decrease in PLPP1 in T cells of the tumor microenvironment results in an impairment of cellular phospholipid anabolic processes. To fully understand the effect of PLPP1 expression on T cells, the inventors re-analyzed CD8+ T cell profiles in the existing GEO database GSE90728 data (which was primarily RNA sequencing analysis of CD8+ T cells from non-small cell lung cancer patients in tumor tissues and paracancerous normal tissues) to analyze the correlation of PLPP1 expression with signaling pathways and T cell functional molecules. In the analysis process, after grouping according to the levels of PLPP1 expression, GO enrichment analysis and GSEA analysis are carried out on differential genes, and the results show that PLPP1 is related to fatty acid metabolism, oxidative phosphorylation and IFN-gamma secretion in cells, and further prove that PLPP1 plays a main role in regulating and controlling the proliferation and activation of T cells. Further, the tissue is divided into TIL high infiltration (TIL) according to the infiltration of CD8+ T cells at the tumor tissue part^hi) Moderate infiltration (TIL)^int) And low infiltration group (TIL)^low) Later, it was found that PLPP1 was in TIL^hiThe relative expression level of the group was high. In combination with previous studies (T cells in tumor tissue of tumor patients with more TIL infiltration mainly present tissue-resident memory cell subtypes, of which CD103 is one of the main marker molecules), the TIL was characterized by CD8+ T cellsExpression of CD103 in cells, which is classified as: CD103 high expression group (CD 103)^hi) CD103 intermediate expression group (CD 103)^int) And CD103 Low expression group (CD 103)^low). The results show that PLPP1 is on CD103^hiThe relative expression level of the group was high.

The association between PLPP1 and activation and function-related molecules in CD8+ T cells was further analyzed in conjunction with the results of the pre-binding GSEA assay (finding that PLPP1 is associated with IFN- γ secretion), showing that: PLPP1 has positive correlation with genes related to cell proliferation and cell cycle such as MKI67, TOP2A and CDK4, etc., and has positive correlation with genes related to cell activation and function, such as ITGAE, ID3, LCK, CD27, GAMB and CXCL13, etc.

It has been found that IFN- γ secreted by T cell activation is capable of causing apoptosis of T cells by binding to its own receptor. By analyzing the correlation between PLPP1 and IFNGR1, we found that the two molecules have a negative correlation, which indicates that: PLPP1 prevents IFN-gamma induced apoptosis while promoting T cell function.

(IV) other solid tumor impact and prognostic relevance

Similar to the above study method, the inventors further examined and analyzed the expression of PLPP1 in CD69+ CD8+ T cells in peripheral blood and tumor tissues of patients with esophageal cancer and colorectal cancer. The results show that: the decreased expression of PLPP1 in CD69+ CD8+ T cells at the tumor tissue site compared to peripheral blood in patients with either esophageal or colorectal cancer suggests: the effect of PLPP1 on phospholipid metabolism and its function of T cells is extensive and not tumor specific. Further, in conjunction with the existing GEO database with patient prognosis information, the inventors analyzed the effect of TIL infiltration and PLPP1 expression on their prognosis in tumor tissues of different tumor patients. The results were found by analyzing the effect of PLPP1 on TIL infiltration and prognosis in patients with colorectal cancer, malignant melanoma, renal cancer and lung cancer: patients with tumors with high expression of PLPP1 with high TIL infiltration had better prognosis, whereas patients with tumors with low expression of PLPP1 with high TIL infiltration had poor prognosis. This indicates that: the high expression of PLPP1 can promote the infiltration and function of T cells in tumor tissues, thereby controlling the progress of tumors, while the low expression of PLPP1 can not improve the tumor killing effect of T cells. In other words: the expression level of PLPP1 is positively correlated with the number of tumor infiltrating CD8+ T cells, and the high expression of PLPP1 can increase infiltration and tumor killing capacity of T cells in a tumor microenvironment.

In general, the functional deletion of T cells in the tumor microenvironment has been reported, and the depletion of T cells in the tumor microenvironment is the result of the combined action of various factors in the tumor microenvironment. In the present example, the inventors considered through a series of experiments and analyses that: abnormal phospholipid metabolism of T cells in the tumor microenvironment is closely linked to the exertion of its function, further: in a solid tumor patient, the lipid metabolism level of tumor infiltrating CD8+ T cells is inhibited, while the phospholipid metabolism related enzyme PLPP1 is closely related to the infiltration level and activation degree of the T cells at a tumor site, and the PLPP1 expression is down-regulated to be a key enzyme for the change of the lipid metabolism of the T cells.

Example 2

Based on example 1, the inventors further performed research analysis on the effect of PLPP1 on the metabolism and function of activated CD8+ T cells by knocking down PLPP1 by using genetic engineering technology, and the specific research process is summarized as follows.

(one) flow analysis of functional molecule differences in low-and high-expression of PLPP1 in CD8+ T cells

In example 1, the use of existing database statistics has preliminarily shown that: the expression of PLPP1 in CD8+ T cells is associated with its function and metabolism. To further validate this association, the inventors examined PLPP1 in the tumor microenvironment of lung cancer patients using flow cytometry^highAnd PLPP1^lowThe relevant factor in CD8+ T cells (see the prior art for details). The results are shown in FIG. 2. Analysis can see that: and PLPP1^lowPLPP1 in comparison to CD8+ T cells^highCD8+ T cells were able to secrete higher levels of the killer cytokines IFN-. gamma.and TNF- α (FIGS. 2A and 2B). that is, by comparative analysis of function in CD8+ T cells with low and high expression of PLPP1 at the lung cancer tumor tissue siteThe difference of the molecules (IFN-gamma and TNF- α) further proves that the CD8+ T cell with high expression of PLPP1 has better immune function compared with the CD8+ T cell with low expression of PLPP 1.

(II) constructing a malignant melanoma mouse model and further verifying and analyzing

On the basis of the analysis, the inventor further constructs a malignant melanoma mouse model, and contrasts and analyzes the expression difference of a functional molecule (IFN-gamma) and a proliferation related molecule (Ki 67) in the CD8+ T cells with low expression and high expression of PLPP1 at the tumor tissue site of the mouse by flow cytometry.

It should be explained that the mouse T cell-depleted tumor model (i.e. malignant melanoma model, which is mainly used for the research of T cell depletion and the tumor volume is not resolved due to the return transfusion of OT-1 cells) is constructed by (1) digesting the B16-OVA cells of mouse melanoma cell line, resuspending PBS, and adjusting the cell concentration to 3-4 × 10⁶About/ml, (2) taking CD45.1-C57BL/6N wild type female mice, and inoculating 100 mul of B16-OVA cells, namely 3-4 × 10, to the subcutaneous part of each mouse⁵The cells are used for subcutaneous tumor formation of mice, (3) OT-1T cells are sorted on the 8 th day after subcutaneous injection of tumor cells and are stimulated to activate the T cells for 12 hours by using Ultra-purified anti mouse CD3, (4) the activated OT-1T cells are reinfused by tail veins (reinfusion 2 × 10) when the tumor growth diameter of the mice reaches 0.25-0.40cm (about the 8 th-9 th day after subcutaneous injection of tumor cells), and the activated OT-1T cells are reinfused by tail veins (reinfusion 2 ×)⁶One); (5) at day 9 of cell reinfusion, the mice were sacrificed under anesthesia, the tumor tissues of the mice were ground, and the cell suspension was filtered using a 70 μm filter and centrifuged; PBS after cell precipitation, 4 ℃ storage for use.

The CD8+ OT-1T cells of the mice are obtained by utilizing streptavidin labeled magnetic bead negative selection separation (OT-1T cells are mainly used for caudal vein back transfusion, and the negative selection separation method is mainly adopted to reduce experimental interference).

Observations at day 9 of cell reinfusion revealed that the tumor volume in the mice did not regress, but rather showed a steady increasing trend (fig. 3A and 3B). Further treatment of tumor groups by flow cytometryWoven PLPP1^highAnd PLPP1^lowThe functional difference analysis of the antigen-specific T cells (CD 45.2+ TCRV β 5+ CD8+ T) can find that the PLPP1 is in malignant melanoma tissues of mice^highThe IFN-gamma secretion capacity of the CD45.2+ TCRV β 5+ CD8+ T cells is obviously higher than that of PLPP1^lowCD45.2+ TCRV β 5+ CD8+ T cells, while PLPP1^highThe proliferation capacity of CD45.2+ TCRV β 5+ CD8+ T cells is also obviously increased (FIG. 3C and FIG. 3D). briefly, the function and proliferation capacity of the CD8+ T cells with high expression of PLPP1 at the tissue site of malignant melanoma of a mouse are higher than those of the cells with low expression of PLPP 1.

Effect of (tri) PLPP1 knockdown on CD8+ T cells

On the basis of the previous research, in order to further determine the effect of PLPP1 on the functions of CD8+ T cells, the inventor constructs a PLPP1-sh plasmid (namely: pSIH 1-H1-consGFP-shPLPP 1 plasmid) by using a molecular cloning technology, transfects activated CD8+ T cells derived from peripheral blood of a healthy person, and detects and analyzes the types and the abundances of lipid molecules in the activated CD8+ T cells, the PLPP1-sh1 and the PLPP1-sh2CD8+ T cells by using an ultra-high performance liquid chromatography technology. The specific experimental procedures and results are summarized below.

The pSIH 1-H1-consGFP-shPLPP 1 plasmid (comprising two plasmids, PLPP1-sh1 and PLPP1-sh 2) is constructed by the following steps (the specific operation refers to relevant kit specifications and the prior art): firstly, carrying out double enzyme digestion on pSIH 1-H1-consGFP-shRNA plasmid by BamH1 and EcoR1, and recovering and purifying enzyme digestion products; secondly, adding homologous arms at two cut ends of pSIH 1-H1-consGFP-shRNA (provided by Shanghai's synthesis) at the upstream and downstream ends of PLPP1-sh1 and PLPP1-sh2, and then annealing; thirdly, the enzyme-cut pSIH 1-H1-consGFP-shRNA plasmid is respectively connected with the annealed PLPP1-sh1 and PLPP1-sh2 sequences by using quick-acting ligase; and finally, transforming the connected pSIH 1-H1-concGFP-shPLPP 1 plasmid into competent cells, screening and identifying by using an LB culture medium to ensure correct recombination, and extracting the plasmid with correct recombination for later use.

Specifically infected CD8+ T cells: first, transfection reagent, packaging plasmid PMD2G and package were transfected with JETPIMEPacking plasmid PSPAX2, transfecting plasmid pSIH 1-H1-concGFP-shPLPP 1 to 293T cells for packaging and infection, culturing for 72 hours after infection, collecting supernatant for later use or directly infecting cells (the supernatant is packaged virus particles), and secondly, packing the virus particles into the cells according to the proportion of 1 × 10 to 10⁷ Add 100. mu.l of T cell TransAct to individual CD8+ T cells^TNIn proportion, activated T cells were used for cell infection (cell state: first enlarged and rounded, and later deformed) for 48h, and finally, in 24-well plates, at a rate of 5 × 10 per 5 h⁶Adding 100 mul of virus supernatant (namely packaged virus particles) and 5 mul/ml Polybrene proportion into each activated CD8+ T cell, infecting to finally obtain PLPP1-shCD8+ T cells, culturing (by using RPMI-1640 complete culture medium) for 72 hours, detecting fluorescence intensity of a FITC channel and expression of PLPP1 by flow cytometry, and simultaneously detecting the transfection efficiency of the T cells and the knocking efficiency of target proteins.

PLPP1 is a catalytic enzyme in the phospholipid metabolism process and is capable of regulating the synthesis of DG molecules as well as the phospholipid molecules PE and PC. The test results show that after in vitro activation of CD8+ T cells in healthy humans, PLPP1 has increased protein level expression after T cell activation, which indicates that: activated T cells are possibly involved in the regulation of cell proliferation, function and metabolism through PLPP1 molecules. The detection result of the infection efficiency of the T cells in the virus infection process shows that the infection efficiency of PLPP1-sh1 and PLPP1-sh2 reaches more than 80 percent; further flow cytometry examination of protein expression of PLPP1 in GPF + CD8+ T cells showed that the expression level of PLPP1 was down-regulated in PLPP1-sh1 and PLPP1-sh2CD8+ T cells compared to Negative Control (NC).

After GPF + CD8+ T cells are purified by a flow sorting method, the knocking efficiency is detected by using an RT-PCR method, and the distribution condition of intracellular phospholipid is detected by combining an ultra-high performance liquid chromatography mass spectrometry technology. The results show that: compared with a Negative Control (NC), the level of the PLPP1 knocked down in PLPP1-sh1 and PLPP1-sh2CD8+ T cells reaches about 50 percent; mass spectrometry shows that the content of most of PC and PE in PLPP1-sh1 and PLPP1-sh2CD8+ T cells is reduced compared with that in an NC group; after further path enrichment analysis, the glycerol phospholipid metabolism path in the PLPP1-sh1 and PLPP1-sh2CD8+ T cells is obviously reduced, and the sphingomyelin metabolism and the biosynthesis process of glycosylated phosphatidylinositol are also reduced, and the results further prove that PLPP1 can really regulate the phospholipid metabolism of the T cells, and particularly has the most obvious regulation on PC and PE molecules.

(IV) specific metabolic differential analysis

Aiming at PLPP1-sh CD8+ T cells subjected to PLPP1 knocking down in the step (III), the inventor further detects the expression condition of metabolic pathway related enzymes, ECAR (extracellular acidification rate) and OCR (optical character recognition) metabolic rate in the activated CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells respectively by using an RT-PCR method and a hippocampal energy detection technology; meanwhile, the proliferation, apoptosis and functions of activated CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells and intracellular Ca are detected by using flow cytometry²⁺And (4) concentration. The specific case is briefly described as follows.

(1) Expression of enzymes associated with cellular metabolism

As previously mentioned, based on existing database analysis it can be found that: in addition to affecting cellular lipid metabolism, PLPP1 also affects cellular oxidative phosphorylation. To further determine specific influence conditions, the inventors used RT-PCR technology to detect key enzymes in sugar metabolism, cholesterol metabolism and fatty acid metabolism in CD8+ T cells after PLPP1 knock-down. The results show that: compared with the NC group, the expression of the glycolytic associated enzymes HK2, GPI, PFKM, PKM2, TPI, ENO1 and LDHA of PLPP1-sh1 and PLPP1-sh2CD8+ T cells was reduced (FIG. 4A). In addition, the expression of cholesterol metabolism related enzymes HMGCR, HMGCS1, SQLE and IDI1 is also reduced; fatty acid metabolism was also in a decreased state, as expression of CPT-1, ACACA and FASN was decreased in PLPP 1-knocked down CD8+ T cells (fig. 4B).

Although the metabolic level of the whole cell is influenced after the knockdown of PLPP1, in general, PLPP1 mainly influences the utilization of glucose in the glycolysis process of T cells, such as the expression of HK2 is 20-30% lower; while the influence on the anabolism of fatty acid in the process of lipid metabolism is far greater than the influence on the metabolism of cholesterol and the oxidation of fatty acid, such as the reduction of 20-30 percent of the expressions of ACACACA and FASN. Thus, overall it can be assumed that: the effects of PLPP1 knockdown on cell metabolism are mainly manifested in phospholipid anabolism, fatty acid anabolism, and glycolysis. In other words, the overall metabolic level of CD8+ T cells also showed a significant decrease following the knockdown of PLPP 1.

(2) ECAR and OCR profiles of cellular metabolism

Further, to better reflect changes in the metabolic levels of CD8+ T cells, we examined the ECAR and OCR rates of T cells. In general, ECAR reflects primarily changes in cellular glycolytic metabolism, and if the glycolytic capacity of a cell is enhanced, the cellular ECAR increases; OCR mainly reflects the oxygen consumption rate of cells, and the oxygen of the cells is consumed by the progress of oxidative phosphorylation metabolism of the cells, so that the OCR represents the strength of the oxidative phosphorylation process of the cells.

The foregoing analysis of the existing tumor patient database showed that: the higher the expression of PLPP1 in T cells, the higher the level of oxidative phosphorylation. The results of the assays of this example show that the glycolytic activity of the cells is reduced and the fatty acid oxidation is reduced after in vitro knockdown of PLPP 1. The oxidative phosphorylation requires the participation of acetyl-CoA, and the decrease of glycolysis and fatty acid oxidative metabolism reduces the content of acetyl-CoA in cells, thereby reducing the oxidative phosphorylation of the cells. Specifically, ECAR and OCR analyses performed on one of the SH knockdown CD8+ T cells showed: rates of ECAR and OCR were significantly reduced in PLPP1-sh1 CD8+ T cells, with both basal and maximal respiration rates reduced in OCR (FIGS. 5A and 5B). In addition, since the increase of the whole cellular metabolism, especially the oxidative phosphorylation, can cause the replication, deformation, fusion and enlargement of the intracellular mitochondria, we further examined the activity of the intracellular mitochondria. The results show a significant reduction in mitochondrial activity in PLPP1-sh1 and PLPP1-sh2CD8+ T cells compared to the NC group (FIG. 5C), which also reflects a reduction in the oxidative phosphorylation metabolic processes of the cells. Based on these results it can be assumed that: the metabolic rates of ECAR and OCR were also significantly reduced in CD8+ T cells after PLPP1 knockdown.

(3) Cell proliferation status

The analysis result of the existing database preliminarily identifies that: the CD8+ T cells with high PLPP1 expression in a tumor microenvironment have stronger cell division and DNA replication capacity. And the detection of the apoptosis condition of the CD8+ T cells after the PLPP1 is knocked down shows that: reduction of PLPP1 molecules did not affect apoptosis of T cells (fig. 6A). However, the method does not affect apoptosis and does not show that the method also has no influence on the proliferation condition, and therefore, the proliferation condition of the T cells after the PLPP1 is knocked down needs to be further analyzed.

When the specific proliferation condition is analyzed, 1 × 10 is respectively added⁶NC, PLPP1-sh1 and PLPP1-sh2CD8+ T cells were plated on cell culture plates, added with T cell activating reagent and cultured for 3 days, and cells were counted again. As a result, it was found that: PLPP1-sh1 and PLPP1-sh2CD8+ T cells hardly proliferated (FIG. 6B). In addition, NC, PLPP1-sh1 and PLPP1-sh2CD8+ T cells were labeled with fluorescent dyes, respectively, and after in vitro culture for 3 days, the proliferation ratio of the cells was detected by flow measurement, and as a result, it was found that: the proliferation capacity of PLPP1-sh1 and PLPP1-sh2CD8+ T cells is only about 50% (FIG. 6C); after further examination of the expression of Ki67 in CD8+ T cells by flow cytometry, it was found that the expression ratio of Ki67 was reduced in both PLPP1-sh1 and PLPP1-sh2CD8+ T cells compared to NC (fig. 6D). In conclusion, the results can be identified as follows: after the knockdown of PLPP1, the proliferation capacity of CD8+ T cells is obviously weakened.

(4) Specific cellular functional Effect

In example 1, the assay of the DG content in T cells indicated that the DG content in T cells in tumor tissues of lung cancer patients was low, whereas the assay of the present application after in vitro PLPP1 knockdown indicated that the DG content in PLPP 1-knocked down CD8+ T cells was still reduced, which indicated that: PLPP1 may influence T cell function through DG molecules.

Binding of PLPP1 molecule to Ca already present²⁺Ca in the control of flow and activation of T cell function²⁺Flow effect studies, the inventors have shown whether PLPP1 affects Ca in T cells²⁺In particular, after activation of T cells with PMA and ionomycin, no significant decrease in IFN-. gamma.and TNF- α secretion by CD8+ T cells was observed following PLPP1 knockdown, with only a slight down-regulation of TNF- α secretion by PLPP1-sh2CD8+ T cells (FIG. 7A).

In general, ionomycin is capable of upregulating intracellular Ca²⁺In order to activate calmodulin downstream of the T cell, enhancing the function of the cell, and therefore it can be assumed that: ionomycin neutralized Ca caused by PLPP1 knockdown²⁺Flow changes, resulting in no difference in T cell function. To this end, we used the T cell activation reagent CD3 mab (OKT 3) in subsequent T cell function assays to better mimic the overall process of T cell activation. Further labeling Ca in T cells with cell dye²⁺The detection result of the change of the flow shows that T cells have intracytoplasmic Ca after being stimulated by OKT3 by PLPP2-sh1 and PLPP1-sh2CD8+ T cells²⁺The concentration did not increase significantly (fig. 7B), whereas the amount of IFN- γ and TNF- α secreted by PLPP 1-knockdown CD8+ T cells stimulated with OKT3 (fig. 7C).

CD8+ T cells, which are a killer cell in the adaptive immune system of the body, recognize foreign substances and protect the body from damage, such as viruses, mutant antigens, etc. The tumor cells are generated by the malignant change of normal cells of the body, and can escape the immune surveillance function of the body and escape the killing of CD8+ T cells, thereby causing the local amplification and metastasis of the tumor. With the continuous expansion of tumor cells, the formation of tumor microenvironment further inhibits the function of CD8+ T cells, resulting in the exhaustion of T cells. In this example, by constructing a mouse depleted tumor model and knocking down PLPP1 expression, it was further demonstrated that: the expression of PLPP1 was significantly elevated after CD8+ T cell activation, whereas PLPP1 in the tumor microenvironment^highThe function and the proliferation capacity of the CD8+ T cells are higher; when PLPP1 is knocked down, the overall metabolic level of CD8+ T cells is down-regulated, such as phospholipid metabolism, glycolysis, fatty acid anabolism and the like; however, the knocking-down of PLPP1 does not affect the apoptosis of CD8+ T cells, but the proliferation capacity of the T cells is obviously reduced; further analysis showed that: activated CD8+ T cell intracytoplasmic Ca following PLPP1 knockdown²⁺In short, as a multi-transmembrane protein, PLPP1 is used for catalyzing the decomposition of fatty acid and has direct regulation and control effects on the functions and metabolism of CD8+ T cells,proliferation, killing function, metabolism of T cells and intracellular Ca2 when PLPP1 expression is reduced⁺The stream concentration is significantly reduced.

Example 3

Combining the results of examples 1 and 2, it has been clarified that PLPP1 has an important effect on the regulation of T cell proliferation and function, and therefore, further, the inventors have conducted further studies on key molecules that regulate PLPP1 expression in CD8+ T cells in tumor microenvironment. The specific study is briefly described as follows.

Preliminary detection of related molecules

First, differences in expression of activated CD8+ T cell surface co-suppression molecules (PD-1, CTLA4, and Tim 3) that contrasts high and low expression of PLPP1 in tumor tissues of lung cancer patients were examined by flow cytometry. Subsequently, with reference to the prior art and the procedures described previously, healthy human peripheral blood-derived CD8+ T cells activated for 48 hours were co-incubated with lung cancer tumor cell lines H460 and a549 for 24 hours in vitro; expression of PLPP1 in the T cells after co-incubation and expression of PD-1 on the surface of the T cells and PDL1 on the surface of the tumor cells are detected by RT-PCR and flow cytometry, and specific experimental procedures can be referred to as follows (specific operations refer to relevant kit instructions or the prior art): after the FACS buffer solution is used for resuspending the cells, APC/Cyanine7 anti-humanCD8 antibody, PE/Cyanine7 anti-humanCD 69 antibody, PE anti-humanPD-1/PE anti-humanCTLA 4/PE anti-humanTim 3 (containing PE anti-humanPD-1 and PE anti-humanPDL 1) are added, and the cells are incubated for 15 minutes at 4 ℃ in a dark place; then fixing and breaking membranes, adding PPAP2A antibodyy (diluted 1: 1000) and Alexa Fluor 488Donkey anti-rabbitIgG, and incubating for 15 minutes in a dark place at 4 ℃; after centrifugation, the FACS buffer was resuspended and tested on a flow machine. In the experiment of incubating the activated T cells with the lung cancer tumor cell lines H460 and a549, it was found that: the expression of PLPP1 was significantly reduced in CD8+ T cells co-incubated directly with H460 and a549 compared to CD8+ T cells cultured alone or in tumor supernatant (fig. 8A). Further, after incubating mice OT-1T cells with B16-OVA at different ratios (5: 1, 10: 1, 20:1, and 50: 1) for 4 hours and 12 hours, respectively, statistics showed that: with increasing co-incubation timeAnd increased ratio, the expression of PLPP1 was decreased in OT-1T cells (fig. 8B). When analyzed by expression detection of PLPP1+ CD69+ CD8+ T cell surface associated co-suppression molecules in the tumor microenvironment of lung cancer patients, the results show: PLPP1^lowThe expression level of PD-1 on the surface of T cells is higher, however, PLPP1^highThe expression level of PD-1 on the surface of the T cell is lower, and the difference has statistical significance; at the same time, PLPP1^lowExpression of CTLA4 was higher and expression of Tim3 was lower, but the difference was not statistically significant (fig. 8C).

The results show that: after co-incubation of tumor cells and T cells, binding of cell surface associated molecules can reduce the expression of PLPP1 in T cells.

To determine the specific molecular type, the inventors further examined the expression of PD-1 in CD8+ T cells and the expression of PDL1 on a549 cells in a system in which activated CD8+ T cells were incubated with a549 cells. As a result, it was found that: the expression of PD-1 on the surface of CD8+ T cells in the co-incubation system is obviously higher than that of CD8+ T cells cultured alone (FIG. 9A), and the expression of PDL1 on the surface of A549 in the co-incubation system is also obviously higher than that of A549 tumor cell line cultured alone (FIG. 9B). This result indicates that: the binding of PD-1 on the surface of T cells to its ligand PDL1 may be involved in the regulation of PLPP 1.

Based on the sequencing data in the existing GSE122149 database (in the database, T cells are mainly divided into two groups, one group activates CD8+ T cells through CD3/CD28 monoclonal antibody, and the other group activates CD8+ T cells and adds PDL1Fc fragment), the statistical analysis finds that: the expression of PLPP1 was increased after CD8+ T cell activation, but the expression of PLPP1 was suppressed after culture of PDL1Fc fragment (fig. 9C); at the same time, expression of MKi67 in activated CD8+ T cells was also reduced at different activation time points after culture of PDL1Fc fragment (fig. 9D).

The inventor thinks that: the expression of PD-1 on the surface of a CD8+ T cell with high expression of PLPP1 is low, and the expression of PD-1 in a CD8+ T cell with low expression of PLPP1 is high; and the experimental results of the combined clinical sample analysis and in vitro co-incubation can be preliminarily determined as follows: binding of PD-1 to its ligand PDL1 reduced the expression of PLPP1 in T cells.

(II) PDL1Fc fragment addition

The foregoing statistical results indicate that the addition of PDL1Fc fragment has a direct effect on PLPP1 expression, for which the inventors added PDL1Fc fragment for binding to PD-1 on the T cell surface during the in vitro activation of CD8+ T cells. The expression of PLPP1 and the expression of a functionally related molecule IFN-gamma in activated CD8+ T cells after PDL1Fc fragment culture were further analyzed by an imaging flow cytometer. The results show that: the expression of PLPP1 and IFN-. gamma.in CD8+ T cells was significantly reduced by culturing with the PDL1Fc fragment (FIG. 10A), and flow cytometry analysis also demonstrated the same results, with the differences being statistically significant (FIG. 10B).

To further verify the results, the inventors knocked out the expression of PD-1 on the surface of activated CD8+ T cells by using CRISPR/Cas9 technology, and the procedures are as follows (the specific procedures are as follows: firstly, carrying out double digestion on pSpCas9(BB) -2A-GFP (PX458) plasmid by using Age1 and EcoR1 restriction enzymes, and recovering and purifying; secondly, homologous arms (provided by Shanghai's synthesis) at two ends of the cut pSpCas9(BB) -2A-GFP (PX458) are respectively added at two ends of the upstream and downstream of the PD-1 gRNA, and annealing treatment is carried out (namely, the upstream of the PD-1 gRNA containing the homologous arms and the downstream of the PD-1 gRNA containing the homologous arms are placed in boiling water and then naturally cooled to room temperature); thirdly, the recovered plasmid fragment of pSpCas9(BB) -2A-GFP (PX458) after enzyme digestion is connected with the annealed PD-1 gRNA sequence by using a quick-acting ligase (namely, pSpCas9(BB) -2A-GFP (PX458) -PD-1 gRNA plasmid is constructed and obtained); thirdly, transforming the ligation product into competent cells, screening and identifying to ensure correct construction, and extracting plasmids with correct construction for later use; finally, the magnetically sorted CD8+ T cells were used with T cell TransAct^TNAfter activation, the pSpCas9(BB) -2A-GFP (PX458) -PD-1 gRNA plasmid was transfected by using JETPIME transfection plasmid, and after culturing for 48-72 hours, the knockout efficiency of the T cells (namely the expression condition of PD-1 on the cell surface) was detected.

The detection result of the expression condition of the activated CD8+ T cell surface PD-1 knocked out by the CRISPR/Cas9 technology shows that: expression of PD-1 on the T cell surface was reduced from 37.4% to 12.3% (fig. 10C). Further Western blot detection finds that: protein expression of PLPP1 was not reduced after culturing CD8+ T cells with PD1 knocked out by CRISPR/Cas9 on PDL1Fc fragment (fig. 10D). Further, when a PD-1 blocking antibody was added while culturing CD8+ T cells using PDL1Fc fragment, it was found that: expression of PLPP1 was restored in CD8+ T cells following PD-1 blocking antibody treatment (fig. 10E).

Further analysis of the Effect of PDL1Fc fragment on CD8+ T cell metabolism and function

The above experimental results have preliminarily shown that: the PDL1/PD-1 binding can regulate the expression and function of PLPP1 in T cells, and the inventor carries out further analysis and verification on whether the PDL1/PD-1 binding regulates the metabolism and function of the T cells and depends on the expression of PLPP1 molecules.

First, we knocked down the expression of PLPP1 in CD8+ T cells, followed by culture of CD8+ T cells and PLPP1-sh1 CD8+ T cells using PDL1Fc fragment (see the foregoing and prior art for specific operations). Through a hippocampal energy detection technology and flow cytometry, the metabolic rate of OCR, the change of mitochondrial function in cells and the change of cell function and intracellular Ca2+ concentration in CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells after PDL1Fc treatment are detected. The research result shows that: PDL1Fc fragment was able to reduce the OCR rate of T cells after CD8+ T cells were cultured (fig. 11A), but there was no significant change in OCR rate in PLPP 1-knocked down CD8+ T cells (fig. 11B). Further detection of mitochondrial changes in CD8+ T cells indicated that: after the culture of the PDL1Fc fragment, the fluorescence intensity of mitochondria was not changed significantly in the PLPP1-sh1 and PLPP1-sh2CD8+ T cells, while the fluorescence intensity of mitochondria was significantly reduced after the treatment of the CD8+ T cells of the control group (FIG. 11C and FIG. 11D). These results show that: the regulation of CD8+ T cell metabolism and mitochondria after PDL1/PD-1 binding is mainly exerted through the expression of PLPP 1.

On the other hand, the results of the detection of functional changes in CD8+ T cells after PDL1Fc culture and CD8+ T cells after PLPP1 knockdown indicate that: intracellular Ca of CD8+ T cells cultured by PDL1Fc²⁺The concentration is obviously reduced, but when the CD8+ T is thinIntracellular Ca after cell PLPP1 knockdown and PDL1Fc culture²⁺There was no significant change in concentration (FIG. 12A). after culturing activated CD8+ T cells, PLPP1-sh1 and PLPP1-sh2CD8+ T cells for 24 hours using PDL1Fc fragment, experimental results showed that the PDL1Fc fragment reduces the secretion of the T cell killer cytokines IFN-. gamma.and TNF- α after culturing CD8+ T cells, whereas the expression of IFN-. gamma.and TNF- α did not change significantly after culturing PLPP1-sh1 and PLPP1-sh2CD8+ T cells in PDL1Fc fragment (FIGS. 12B and 12C).

Combining the results of these experiments, it is also demonstrated that: PDL1/PD-1 also functions to reduce the function of CD8+ T cells by expression of PLPP 1.

(IV) animal model experiment combining PDL1 blocking monoclonal antibody

The in vitro experiments all show that: PDL1/PD-1 binding can reduce the expression of PLPP1 in CD8+ T cells, thereby reducing the metabolism and function of the T cells. To further verify, the inventors constructed a malignant melanoma mouse model, after tumor growth, performed OT-1 cell reinfusion treatment and PDL1 blocking monoclonal antibody treatment at different time points (after mice OT-1T cells were reinfused into mice via tail vein, 200 μ g of InVivoMAb anti mPDL1 (10 f.9g2) or InVivoMAb rat IgG2b isotype control was intraperitoneally injected on day 3 and day 7, respectively), and finally detected the expression and functional status of PLPP1 in OT-1T cells in the tumor microenvironment (the flow is shown in fig. 13A). The results show that: the volume growth rate of melanoma was slower in mice in the PDL1 mab-treated group compared to the control group (fig. 13B).

And for T cells in tumor tissues (PD-1 is obtained by flow sorting)^highFunctional detection analysis of TCRV β 5+ CD45.2+ CD8+ T cells and PD-1-TCRV β 5+ CD45.2+ CD8+ T cells, RT-PCR method for detecting PLPP1 expression in sorted T cells) shows that PD-1^highExpression of PLPP1 in TCRV β 5+ CD45.2+ CD8+ T cells was up-regulated after PDL1 monoclonal antibody treatment, while expression of PLPP1 in T cells was not significantly changed in PD-1-TCRV β 5+ CD45.2+ CD8+ T cells (FIG. 13C), which indicates that the expression of PLPP1 in T cells is directly regulated by PDL1/PD-1, and the increase of T PP1 expression is simultaneously achieved by PDL1 monoclonal antibody treatmentExpression of functional molecules of CRV β 5+ CD45.2+ CD8+ T cells, such as IFN- γ, granzyme b and CD107a (fig. 13D), in other words, PDL1 blocking monoclonal antibody treatment restored the expression of PLPP1 in antigen-specific T cells in the tumor microenvironment and restored its killing function and eventually reduced the growth rate of tumor cells.

With respect to the above-described related experimental results, the inventors summarized as follows: after the CD8+ T cells are activated, besides the high expression of co-stimulatory molecules on the cell surface, the expression of co-inhibitory molecules is increased, such as PD-1 and CTLA4, and the depletion of CD8+ T cells in the tumor microenvironment is one of the causes of the further development of the tumor, that is: t cell depletion in the tumor microenvironment is accompanied by high expression of various immune co-inhibitory molecules, such as PD-1, CTLA4 and Tim 3. Based on the results of the studies of the foregoing examples 1 and 2, it has been clarified that: the reduction of the expression of PLPP1 in CD8+ T cells in a tumor microenvironment can reduce intracellular phospholipid anabolism, cholesterol metabolism, fatty acid metabolism and the like; simultaneously by regulating Ca of T cells²⁺Changes, in turn, modulate T cell function. In this embodiment, it is further demonstrated through related experiments that: PD-1 can affect the mitochondrial state of T cells after being combined with a ligand PDL1 thereof, simultaneously reduces the OCR rate of the T cells and finally reduces the functions of the T cells, and PDL1/PD-1 regulates the functions of the T cells by regulating the expression of a key enzyme PLPP1 in an intracellular phospholipid metabolic pathway. That is, PLPP1 is a key molecule in the PDL1/PD-1 signaling pathway in the tumor microenvironment that regulates T cell function and metabolism.

Example 4

Based on example 3, it is clear that the PDL1/PD-1 signal pathway is dependent on PLPP1, a key molecule for regulating T cell function and metabolism, but the specific signal pathway in the regulation pathway is still not clear enough, and therefore, the inventors have made further intensive research and study. The specific experiments and results are summarized below.

(one) Effect of AKT/mTOR Signal pathway after PDL1Fc processing

When GSEA analysis was performed on RNA sequencing data from CD8+ T cells in the tumor microenvironment of lung cancer patients in the GEO database GSE90728 as described above, it was found that the degree of PI3K/AKT/mTOR signaling pathway activation was higher in T cells in patients with higher PLPP1 expression (fig. 14A). To further validate this pathway, the inventors treated activated CD8+ T cells derived from healthy human peripheral blood with PDL1Fc, as well as Jurkat and Jurkat-PD1 cell lines, and finally detected changes in the AKT/mTOR signaling pathway in these cells after PDL1Fc treatment by flow cytometry and Western blot techniques.

After culturing Jurkat and Jurkat-PD1 cell lines with PDL1Fc fragment and detecting the degree of activation of the intracellular AKT/mTOR signaling pathway at different time points, experimental results showed: the inhibition of AKT/mTOR signaling pathway was weak in cells at 15 min after PD-L1 Fc culture, but the inhibition of AKT/mTOR signaling pathway was significant after 1 hour of culture, with the most significant difference at 1 or 2 hours (FIG. 14B), and the phosphorylation of late-stage detection signaling pathway protein was detected at about 1 hour. In contrast, when CD8+ T cells derived from peripheral blood of healthy humans were cultured and activated in vitro and the T cells were cultured using PDL1Fc fragment, it was found that: the expression ratio of p-AKT in PD-1+ CD8+ T cells was significantly decreased in the PDLI Fc treated group (PDL 1Fc group) compared with the untreated group (NC group) (FIG. 14C). Meanwhile, the result of detecting the phosphorylation of signal protein in cells by using the western blot technology shows that: phosphorylation of AKT and S6 in cells of PDL1Fc group decreased, while expression of cellular PLPP1 decreased (fig. 14D).

Combining the above results, it can be preliminarily determined that: after PDL1/PD-1 is combined, an AKT/mTOR signal path in a CD8+ T cell is inhibited, and the expression of PLPP1 is reduced.

(II) analysis of related transcription factors

In order to determine the change condition of related transcription factors in a signal path, the inventor utilizes PCR array and the prior art to predict and analyze the change of the transcription factors in CD8+ T cells derived from peripheral blood of healthy people after PDL1Fc treatment, and simultaneously detects the expression change of PLPP1 in the cells through an experiment of knocking down the related transcription factors in CD8+ T cells in vitro. The results show that: significant changes in transcription factors common in cells were detected by the method of pcraray (fig. 15A), specifically: after PDL1Fc treatment, there were 14 significant changes (fold change over 2-fold) in T cells, of which 5 were upregulated and 9 were downregulated (fig. 15B). Further, the promoter region of PLPP1 gene was analyzed in the PROMO website for prediction of transcription factor binding sites, and found that there were 76 predicted transcription factors capable of binding to it, and we performed intersection analysis of these 76 and predicted transcription factors with the transcription factors with changes after culture of PDL1Fc, and finally found that only 5 transcription factors (GATA 1, E2F1, HNF1A, HNF4A and ARNT) might be involved in regulating the expression of PLPP1 (fig. 15C). Further, we designed, synthesized and transfected these transcription factors into activated CD8+ T cells to determine if these transcription factors could affect the expression of PLPP1 (of which 2 transcription factors were expressed at very low levels in T cells and were therefore directly excluded). The results show that: HNF1A and HNF4A were hardly expressed in T cells, and thus these two altered transcription factors were considered not major regulatory factors; while the expression of PLPP1 was significantly up-regulated after inhibition of GATA1 expression in CD8+ T cells (fig. 15D). Based on this, it can be preliminarily assumed that: PDL1/PD-1 can up-regulate the expression of GATA1 after binding, thereby finally reducing the expression of PLPP 1.

(III) Chip analysis (Chromatin Immunoprecipitation, Chromatin Co-Immunoprecipitation technique, also known as binding site analysis)

GATA1 acts as a transcription factor, and plays a regulatory role mainly by binding to the DNA sequence of the gene. For this reason, we tested the binding of the transcription factor GATA1 to the promoter region upstream of the target gene PLPP1 using Chip technology, and further verified whether GATA1 could bind to the promoter region of PLPP1 by a dual luciferase assay. Since GATA1 generally needs to enter nucleus to play a role in regulating the expression of a target gene, the inventor also detects the nuclear translocation condition of GATA1 in CD8+ T cells after PDL1Fc treatment by an imaging flow method and a Western blot technology.

Through the binding analysis of PROMO and JASPAR websites, two sites with highest binding scores of GATA1 and PLPP1 promoter, namely Site1 (-1453 bp to-1443 bp) and Site2 (-1145 bp to-1135 bp) can be found (FIG. 16A). The Chip experiment found that GATA1 was able to bind to both sites of the PLPP1 promoter region (FIG. 16B). After treatment of activated CD8+ T cells with AKT inhibitors in vitro, it was again found by Chip experiments that: following inhibition of the AKT signaling pathway, GATA1 bound to two sites of the PLPP1 promoter with a significant increase (fig. 16C).

To further verify whether GATA1 was able to regulate expression of this gene by binding to the PLPP1 promoter region, we performed a dual luciferase experiment. Specifically, we mutated the promoter region binding site of PLPP1 and transfected the wild-type and mutant promoter regions of PLPP1 into 293T cells overexpressing GATA1 or control by molecular cloning and cell transfection techniques (fig. 17A and 17B). Unlike the Chip experimental results, we found that: the fluorescence intensity in 293T cells overexpressing GATA1 was increased following mutation in the PLPP1 Site2 promoter region (fig. 17D), whereas the Site1 Site was not significantly changed (fig. 17C), indicating that: GATA1 was able to bind to Site2 of the promoter region of PLPP1, thereby reducing the expression of PLPP 1.

In summary, on the basis of preliminary identification that GATA1 is able to regulate the expression of PLPP1 by binding to the PLPP1 promoter region, we cultured in vitro activated CD8+ T cells again using PDL1Fc fragment or PD-1 blocking antibody, and found by imaging flow cytometry analysis: the nuclear translocation ratio of GATA1 increased from 23.8% to 40.1% after T cells were cultured with PDL1Fc fragment, but at the same time, the nuclear translocation ratio of GATA1 decreased after treatment with PD-1 blocking antibody (fig. 18A and 18B). Further treatment of in vitro activated CD8+ T cells with AKT inhibitors and western blot detection revealed: expression of PLPP1 was reduced in T cells following AKT inhibitor treatment, while nuclear translocation of GATA1 (N-GATA 1/C-GATA 1) was increased (FIG. 18C). Combining the above results, all show that: PDL1/PD-1 reduces PLPP1 expression in CD8+ T cells primarily by inhibiting the AKT/mTOR signaling pathway leading to increased GATA1 nuclear translocation; or the following steps: PDL1/PD-1 enhances the nuclear translocation of GATA1 in CD8+ T cells by inhibiting the activation of AKT/mTOR, thereby reducing the expression of PLPP 1.

(IV) mouse malignant melanoma model experiment

On the basis that PDL1/PD-1 can reduce the expression of PLPP1 by inhibiting the activation of an AKT/mTOR signal pathway through the in vitro experiment, a malignant melanoma mouse model is further constructed, after lung tumors grow out, OT-1 cell reinfusion treatment is carried out through reinfusion of B16-OVA-luc cells through tail veins (reinfusion of mouse OT-1T cells in a tail vein injection mode), and PDL1 blocking type monoclonal antibody treatment is carried out at different time points (intraperitoneal injection of 200 mu g of InVivoMAb anti m PDL1 (10 F.9G2) or InVivoMAb rat IgG2B isotype control on days 3 and 7 respectively).

The results show that: the tumor growth rate of the tumor-bearing mice is obviously reduced after the tumor-bearing mice are treated by the PDL1 blocking monoclonal antibody.

On day 19 after tumor cell inoculation, mice were sacrificed under anesthesia and lung tumors were paraffin embedded, sectioned, and H & E stained to determine mouse lung tumor formation. Analysis shows that the flow result of the previous animal experiment is consistent, and tissue immunofluorescence shows that the infiltration number of PLPP1+ CD8+ T cells at the lung tumor tissue part of the mouse is obviously increased after the PDL1 blocking monoclonal antibody is treated. We have also found that: the expression ratio of p-AKT and p-mTOR in these antigen-specific T cells is obviously increased at the tumor tissue site.

The combination of the above results also indicates that: after the PDL1 blocking monoclonal antibody is treated, AKT/mTOR signal pathways in CD8+ T cells at a tumor tissue part can be activated again, so that infiltration of PLPP1+ CD8+ T cells in a tumor microenvironment is improved, and the growth speed of tumor cells is reduced, namely: the reduction of the expression of PLPP1 in CD8+ T cells by PDL1/PD-1 binding is caused by a reduction of the AKT/mTOR signaling pathway.

Based on this embodiment, the following can be summarized: there are a variety of transcription factors that regulate T cell activation or depletion, and in this example, it is basically demonstrated through a series of experiments that: PDL1/PD-1 promotes nuclear translocation of a transcription factor GATA1 mainly by inhibiting activation of AKT/mTOR signaling pathway in CD8+ T cells, finally results in reduction of PLPP1 expression, and finally inhibits proliferation, metabolism and function of the T cells.

Claims (6)

1. The application of PLPP1 in preparing a T cell immune tumor related medicament is characterized in that the PLPP1 is a key enzyme for regulating and controlling T cell phospholipid metabolism; in a tumor microenvironment, the reduction of the expression of a metabolism key enzyme PLPP1 causes abnormal phospholipid metabolism of CD8+ T cells, further causes abnormal metabolism of CD8+ T cells and finally causes abnormal T cell function;

   that is, the expression of PLPP1 has a positive correlation with the activation, infiltration and function of CD8+ T cells in the tumor microenvironment, or, the reduction of the expression of PLPP1 in CD8+ T cells leads to the reduction of T cell proliferation, metabolism and function, while the high expression of PLPP1 can promote the infiltration and function of T cells in tumor tissues.
2. 2. Use of PLPP1 in the manufacture of a T cell immunotumor-associated medicament according to claim 1 wherein phospholipid metabolism includes but is not limited to: the contents of phosphatidic acid PA, diglyceride DG, phosphatidylcholine PC and phosphatidylethanolamine PE;

   such T cell functions include, but are not limited to, secretion of IFN- γ and TNF- α;

   the T cell metabolism includes, but is not limited to: sugar metabolism, cholesterol metabolism, fatty acid metabolism, and oxidative phosphorylation.
3. 3. Use of PLPP1 for the manufacture of a T cell immunotumor related medicament according to claim 1 wherein the reduction of CD8+ T cell metabolism and function after PDL1/PD-1 binding is dependent on the reduction of PLPP1 expression; and when the expression of PLPP1 is reduced, intracellular Ca is reduced through regulation²⁺The flow concentration is taken as a regulating path, so that the proliferation function of T cells, the immune function of T cells and the metabolic function of T cells are obviously reduced.
4. 4. The use of PLPP1 in the manufacture of a T cell immunotumor-associated medicament according to claim 1, wherein PDL1/PD-1, upon binding, increases nuclear translocation of GATA1 by inhibiting activation of the AKT/mTOR signaling pathway, and ultimately decreases PLPP1 expression in the cell.
5. 5. The use of PLPP1 in the manufacture of a T cell immunotumor-associated medicament according to claim 4, wherein the PDL1 blocking mab when applied increases the expression of PLPP1 by restoring the AKT/mTOR signaling pathway in CD8+ T cells, thereby promoting infiltration and function of T cells and ultimately delaying the growth of tumor cells.
6. 6. Use of PLPP1 in the manufacture of a medicament for use in association with a T cell immune tumor according to claim 1, wherein the tumor includes but is not limited to 4 solid tumors selected from the group consisting of esophageal cancer, lung cancer, colon cancer and melanoma.

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