CN110221068B

CN110221068B - Application of reagent for detecting Kyn content

Info

Publication number: CN110221068B
Application number: CN201810175576.XA
Authority: CN
Inventors: 黄波; 刘玉英
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2018-03-02
Filing date: 2018-03-02
Publication date: 2020-09-18
Anticipated expiration: 2038-03-02
Also published as: CN110221068A

Abstract

The disclosure provides an application of a reagent for detecting Kyn content. The disclosure specifically provides an application of a reagent for detecting Kyn content in preparation of a reagent or a kit for diagnosing or assisting in diagnosing whether a person to be detected has a disease accompanied by increased expression of PD-1 or has a high risk of having the disease. The technical scheme provided by the disclosure can be used for accurately diagnosing whether a person to be detected has a tumor, preferably whether the person to be detected has the tumor with increased PD-1 expression level in vitro.

Description

Application of reagent for detecting Kyn content

Technical Field

The present disclosure belongs to the field of biological detection, and relates to the application of detection reagent for biological marker in body fluid. In particular, the disclosure relates to the use of detection reagents that can detect Kyn in bodily fluids.

Background

Tumors remain one of the most fatal threats to human health. Solid tumors are responsible for most of those deaths. Although there has been significant progress in the medical treatment of certain tumors, the overall 5-year survival rate of all tumors has improved only about 10% over the last 20 years. Tumors or malignancies grow and metastasize rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult.

The tumor immunotherapy is another tumor therapy method which has definite effect on treating tumors after operations, radiotherapy and chemotherapy, resists and kills tumor cells by activating the human body autoimmune system, and is one of the development directions of future tumor therapy.

Different patients will show different information with different immunotherapies, which requires specific immune system evaluation of tumor immunotherapy patients to monitor tumor immunotherapy effectiveness.

Programmed death receptor 1(PD-1) is a protein that has been linked to chronic infection, pregnancy, tissue allografts, autoimmune disease, and suppression of immune system responses during tumors. PD-1 modulates immune responses by binding to an inhibitory receptor called programmed death receptor 1(PD-L1) expressed on the surface of T cells, B cells, and monocytes. The action mechanism of PD-1 immunotherapy is to design specific protein antibodies against PD-1 or PD-L1, prevent the recognition process of PD-1 and PD-L1, and partially recover tumor-specific cytotoxicity CD8 with antigen specificity⁺Lymphocytes (CTL) function, thereby making it possible to kill tumor cells.

However, despite the unprecedented success of clinical application of programmed cell death receptor 1(PD-1) antibodies against many types of tumors, PD-1 is tumor-specific cytotoxic CD8 in the tumor microenvironment⁺The underlying mechanisms how to regulate in lymphocytes (CTLs) are still not well characterized. Meanwhile, considering the relationship between tumor recurrence and residual tumorigenic cells, even after alleviation from the inhibitory effect of PD-1 signal, CTL does not destroy stem cell-like cancer cells [ Tumor Regenerative Cells (TRCs) of regenerative tumors]. These TRCs are a self-renewing, highly tumorigenic subpopulation of cancer cells that play a key role in the initiation, promotion, and progression of tumorigenesis. In addition, part of the treated patient population that initially responded and exhibited excellent therapeutic outcome still recurs, and therefore medical personnel were required to predict, evaluate, and monitor the therapeutic effect after treatment with immunotherapy to determine the subsequent treatment regimen。

Therefore, it is desirable to provide a convenient and rapid tumor diagnosis, and a scheme for predicting, evaluating and monitoring the tumor immunotherapy effect, which provides a non-invasive method for disease diagnosis and immunotherapy effect evaluation for patients, and helps medical workers and tumor patients to predict the tumor immunotherapy effect at an early stage, so that patients can select a therapy more effectively in the fight against tumors, and the time and money for tumor patients to treat are not wasted.

Disclosure of Invention

Problems to be solved by the invention

In order to solve the above problems, the present disclosure provides a method for rapidly diagnosing whether a subject has a tumor in vitro, and provides a technical solution that can be used for predicting, evaluating, and monitoring the immunotherapy effect of a tumor.

Means for solving the problems

The present disclosure provides an application of a reagent for detecting Kyn content in the preparation of a reagent or a kit for diagnosing or assisting in diagnosing whether a subject has a disease accompanied by an increase in PD-1 expression or has a high risk of having the disease.

In the present disclosure, the disease is selected from diseases caused by tumors accompanying an increase in PD-1 expression; preferably, the disease caused by the tumor generation accompanied by the PD-1 expression increase is one or more selected from melanoma accompanied by the PD-1 expression increase, liver cancer accompanied by the PD-1 expression increase, breast cancer accompanied by the PD-1 expression increase, lung cancer accompanied by the PD-1 expression increase, colorectal cancer accompanied by the PD-1 expression increase, ovarian cancer accompanied by the PD-1 expression increase, and leukemia accompanied by the PD-1 expression increase.

The present disclosure also provides an application of a reagent for detecting Kyn content in preparing a reagent or a kit for predicting, evaluating, or monitoring the effect of disease immunotherapy of a patient having a disease accompanied by an increase in PD-1 expression.

The present disclosure also provides a method for diagnosing or assisting in diagnosing whether a subject has a disease accompanied by an increase in PD-1 expression or has a high risk of having the disease, characterized in that the method comprises a detection step of detecting the content of Kyn in a body fluid of the subject using a detection reagent.

In the disclosure, the method further comprises a comparison step of comparing the content of Kyn in the body fluid of the person to be tested with the content of Kyn in the body fluid of a normal person;

optionally, the method further includes a determining step, where the determining step is:

(1) when the content of Kyn in the body fluid of the subject is significantly higher than the content of Kyn in the body fluid of the normal person, the subject suffers from the disease or has a high risk of suffering from the disease; optionally, the content of Kyn in the body fluid of the person to be tested is more than 2 times higher than that of the body fluid of the normal person;

(2) when the content of Kyn in the body fluid of the subject is lower than 2 times the content of Kyn in the body fluid of the normal human, the subject does not suffer from the disease or has a low risk of suffering from the disease.

The present disclosure also provides a method for predicting, evaluating or monitoring the effect of disease immunotherapy in a patient having a disease accompanied by an increase in PD-1 expression, characterized in that the method comprises a detection step of detecting the content of Kyn in a body fluid of a subject using a detection reagent.

In the present disclosure, the method further comprises a comparison step of comparing the content of Kyn in the body fluid of the patient with the content of Kyn in the body fluid of the patient having a good disease immunotherapy effect;

(1) when the content of Kyn in the body fluid of the patient is significantly higher than the content of Kyn in the body fluid of the patient with a good disease immunotherapy effect, the patient has a good disease therapy immunotherapy effect; optionally, the content of Kyn in the body fluid of the patient is more than 2 times higher than that in the body fluid of the patient with good disease immunotherapy effect;

(2) when the content of Kyn in the body fluid of the patient is less than 2 times of the content of Kyn in the body fluid of the patient with a good disease immunotherapy effect, the subject has a poor disease therapy immunotherapy effect.

The present disclosure also provides a kit for preparing a reagent for detecting Kyn content in diagnosing or assisting in diagnosing whether a subject has a disease accompanied by an increase in PD-1 expression, or predicting, evaluating, or monitoring an effect of immunotherapy on a disease of a subject having a disease accompanied by an increase in PD-1 expression, wherein the kit contains a reagent for detecting Kyn content.

ADVANTAGEOUS EFFECTS OF INVENTION

The technical scheme provided by the disclosure can be used for accurately diagnosing whether a person to be detected has a tumor, preferably whether the person to be detected has the tumor with increased PD-1 expression level in vitro.

Meanwhile, the method for predicting, evaluating and monitoring the immunotherapy effect can accurately judge the tumor immunotherapy effect.

Drawings

FIG. 1 shows that IFN-. gamma.stimulation of TRC from CTL up-regulates PD-1 expression in CTL.

FIG. 2 shows Kyn in CD8⁺Up-regulated PD-1 expression in T cells.

FIG. 3 shows Kyn activated aromatic hydrocarbon receptor (AhR) for PD-1 upregulation.

FIG. 4 shows that AhR up-regulates the expression of SLC7A8 and PAT4 to facilitate Kyn transport.

FIG. 5 shows that TCR signaling facilitates up-regulation of PD-1 by Kyn.

FIG. 6 shows that Kyn upregulates PD-1 expression in vivo by activating AhR.

FIG. 7 shows that PD-1 expression is regulated by the Kyn-AhR pathway in patients.

FIG. 8 shows peripheral blood CD8 of tumor patients⁺And (3) carrying out correlation analysis on the expression quantity of the T cell PD-1 and the level of the plasma Kyn. Wherein, FIG. 8A shows the peripheral blood CD8 of three tumor patients of breast cancer, colon cancer and lung cancer and healthy volunteers⁺(ii) a T cell PD-1 expression level; FIG. 8B shows the peripheral blood Kyn levels in three tumor patients with breast, colon, and lung cancer and in healthy volunteers; FIG. 8C shows the peripheral blood CD8 of breast cancer patients⁺Correlation of the expression level of PD-1 in T cells and the Kyn level in plasma; FIG. 8D shows peripheral blood CD8 of healthy volunteers⁺Correlation of expression levels of PD-1 in T cells and Kyn levels in plasma.

FIG. 9 shows CD8 in tumor tissue of a patient⁺And (3) analyzing the correlation between the expression quantity of PD-1 of the T cells and the Kyn level. Among them, FIG. 9A shows CD8 infiltrated in breast and colon cancer tissues⁺T cell PD-1 expressionAn amount; FIG. 9B shows Kyn levels in breast and colon cancer and their corresponding paracarcinoma tissues; FIG. 9C shows CD8 in breast cancer tissue⁺Correlation of expression levels of PD-1 and Kyn levels in T cells.

FIG. 10 shows the peripheral blood plasma Kyn levels before and after treatment of acute B-lymphoblastic leukemia patients showing efficacy and ineffectiveness of CAR-T cell therapy.

Detailed Description

Definition of

The terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification can mean "one," but can also mean "one or more," at least one, "and" one or more than one.

As used in the claims and specification, the terms "comprising," "having," "including," or "containing" are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Throughout this specification, the term "about" means: a value includes the standard deviation of error for the device or method used to determine the value.

Although the disclosure supports the definition of the term "or" as merely an alternative as well as "and/or," the term "or" in the claims means "and/or" unless expressly indicated to be merely an alternative or a mutual exclusion between alternatives.

The term "kynurenine (Kyn)" is a metabolic intermediate of tryptophan in the human body.

The term "good disease immunotherapy effect" means: the patient is cured or less ill when receiving immunotherapy for the disease.

The term "poor disease immunotherapy effect" means: when patients receive disease immunotherapy, the treatment is ineffective or poorly responsive to the treatment, or the disease recurs during the course of the treatment.

Examples

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

The experimental animals, reagents and consumables adopted by the present disclosure are as follows:

female C57BL/6 mice (6-8 weeks old), OT-I transgenic mice, Pmel-1 transgenic mice, AhR-/-mice, and MMTV-PyMT mice were purchased from commercial establishments. These animals were kept under pathogen-free conditions in the corresponding facilities of the applicant. All studies involving mice were approved by the animal care and use committee.

Mouse tumor cell lines OVA-B16 (melanoma), H22 (hepatocellular carcinoma) and ID8 (ovarian cancer) and human tumor cell lines a375 (melanoma) and HepG2 (hepatocellular carcinoma) were purchased from the chinese type culture collection (beijing, china) and cultured in DMEM (Thermo Scientific) with 10% Fetal Bovine Serum (FBS) (Gibco, USA) except that H22 cells were grown in rp1640 mi medium (Gibco, USA) containing 10% FBS.

pGFP-C-shLenti-shIDO1, pGFP-C-shLenti-shSLC1A5, pGFP-V-RS-shPAT4, pGFP-V-RS-shSLC7A8 and pCMV6-Entry-DDK-SLC1A5 were purchased from origin (MD, USA). Kynurenine, kynurenic acid, indoxyl sulfate, melatonin, 1-L-MT, 3',4' -Dimethoxyflavone (DMF), TCDD, inosinic acid (SAR), 2-amino-2-norbornanecarboxylic acid (BCH), L-gamma-glutamyl-3-carboxy-4-nitroaniline (GPNA) and anti-PAT 4 antibodies were from Sigma-Aldrich (ST, USA). Cyclosporin a is from selockchemicals. Neutralizing antibodies to anti-IFN-. gamma., TNF-. alpha., PD-1 and PD-L1 and anti-PD-1, CD3, CD8, IFN-. gamma., TNF-. alpha.and CD107a antibodies were purchased from BioLegend. IFN-. gamma.was purchased from R & D Systems. Salmon fibrinogen, thrombin and dispase were from Reagent Proteins (CA, USA). Puromycin was from Invitrogen (SD, USA).

Human peripheral blood, resected human breast or colon cancer tissue was from a hospital patient. Ethical approval was granted by the ethical committee of the clinical trials in the respective hospitals. All patients provided written informed consent to participate in the study.

Experimental methods

(1) Method for detecting biomarkers

In any of the foregoing methods, the presence and/or expression level/amount of the biomarker is measured by determining the protein expression level of the biomarker. In certain embodiments, the methods comprise contacting the biological sample with an antibody that specifically binds to a biomarker described herein (e.g., an anti-PD-1 antibody) under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods may be in vitro or in vivo. In some cases, the antibody is used to select subjects eligible for treatment with a PD-1 axis binding antagonist, e.g., biomarkers for selecting individuals. Any method of measuring protein expression levels known in the art or provided herein can be used. For example, in some embodiments, the protein expression level of a biomarker (e.g., PD-1) is determined using a method selected from the group consisting of: flow cytometry (e.g., fluorescence activated cell sorting (FACTM)), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Immunohistochemistry (IHC), immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, surface plasmon resonance, spectroscopy, mass spectrometry, and HPLC.

(2) Tumor cells cultured in 2D hard culture dishes or 3D fibrin gel

For conventional 2D cell culture, tumor cells were maintained in rigid culture dishes with intact medium. Specifically, fibrinogen (Searun Holdings company, Freeport, ME) was diluted to 2mg/ml with T7 buffer (pH 7.4,50mM Tris, 150mM NaCl). Then, 1: 1 (volume) mixture of fibrinogen and cell solution. Mu.l of the cell/fibrinogen mixture was seeded in a 24-well plate and mixed well with 5. mu.l of thrombin (0.1U/. mu.l, Searn Severn) previously added. After incubation at 37 ℃ for 30 minutes, the cells were supplemented with 1ml of complete medium. After 5 days of incubation, dispase II was added and the incubated gel was digested for 10 minutes at 37 ℃. Spheroids were harvested and further digested with 0.25% trypsin for 3 minutes to obtain single cell suspensions for the following experiments.

(3) Statistical analysis method

All experiments were performed at least three times. Results are expressed as mean ± SEM. And analyzed by Student's t test. P values <0.05 were considered statistically significant. Analysis was performed using Graphpad 6.0 software. Sample exclusion was never performed.

⁺Example 1: PD-1 expression in tumor-specific CD8T cells induced by IFN-gamma stimulated TRC

The steps and corresponding results of the technical scheme adopted by the embodiment are as follows:

(1) anti-CD 3/CD28 bead-activated OVA-CTL was cultured for 24 hours either alone or with OVA-B16 cells (differentiated OVA-B16) from conventional flasks or OVA-B16TRC grown in 3D fibrin matrix. PD-1 expression was determined by flow cytometry. The measurement results are shown in FIG. 1A.

(2) The co-culture supernatant or complete medium of CTL with differentiated OVA-B16 cells or OVA-B16TRC (negative control) was concentrated by freeze-drying and incubated with anti-CD 3/CD28 bead-activated OVA-CTL for 24 hours. PD-1 expression was determined by flow cytometry. The measurement results are shown in FIG. 1B.

(3) CD3/CD28 bead activated OVA-CTL was cultured for 24 hours and the supernatant was analyzed by ELISA. The measurement results are shown in FIG. 1C.

(4) anti-CD 3/CD28 bead-activated OVA-CTL was co-cultured with OVA-B16TRC for 24 h. IFN- γ production in T cells and tumor cells was analyzed by flow cytometry. The measurement results are shown in FIG. 1D.

(5) anti-CD 3/CD28 bead-activated OVA-CTLs were treated with PBS or conditioned media from either CTLs stimulated with IFN- γ or OVA-B16TRC for 24 h. PD-1 expression of OVA-CTL was subsequently determined by flow cytometry. The measurement results are shown in FIG. 1E.

(6) OVA-B16 cells from conventional flasks or OVA-B16TRC from 3D fibrin gel were co-cultured with anti-CD 3/CD28 bead activated OVA-CTL at a ratio of 1:30 for 4 h. CD 45-tumor cell apoptosis was determined by flow cytometry. The measurement results are shown in FIG. 1F.

(7) OVA-B16TRC was co-cultured with anti-CD 3/CD28 bead-activated OVA-CTL in the presence of neutralizing anti-PD-1 antibody or IgG isotype control (isotype control) at a ratio of 1: 30. After 4h, cell viability was analyzed by flow cytometry. The measurement results are shown in FIG. 1G.

From the results of example 1, using B16 melanoma TRC expressing model tumor antigen albumin (OVA) and OVA-specific CTLs derived from OT-1T-cell receptor transgenic mice, we found that TRC strongly induced anti-CD 3/CD28 bead-activated (bead-activated) OT-1T cells to up-regulate PD-1 expression during co-culture (co-culture) (fig. 1A). Such PD-1 upregulation is also at gp 100-specific CD8⁺T cells and OVA peptide-activated OT-1 cells. Notably, PD-1⁺The above increase in cells is attributed to PD-1^-To PD-1⁺Transformation of T cells, but not by PD-1^-Depletion of cells (depletion) or PD-1⁺Expansion of cells based on PD-1^-Co-culture of OT-1 cells with OVA-B16TRC, wherein PD-1⁺T cell appearance, and PD-1⁺Co-culturing the cells with or without TRC, wherein PD-1⁺Proliferation of T cells was not altered, whereas PD-1 expression was upregulated. Furthermore, the above co-culture supernatant can be in irrelevant CD8⁺in line with this, we found that IFN- γ neutralization blocks the above-described PD-1 upregulation, while TNF- α neutralization does not, we found that IFN- γ is expressed only by OT-1 cells but not by TRCs (fig. 1D), has been identified as a key molecule, we treated T cells and TRCs separately with recombinant IFN- γ, and found that supernatant from TRCs treated only can up-regulate PD-1 on T cells (fig. 1E).Thus, these data indicate that during interaction of TRCs with tumor-infiltrating T cells, IFN- γ produced by T cells directly stimulates TRCs to release factors that promote PD-1 expression. Here we also verified that the up-regulated PD-1 is functional. We found that OVA-specific CTLs were difficult to kill against OVA-TRCs in the above co-culture (fig. 1F), whereas addition of neutralizing anti-PD-1 antibody significantly improved the killing of TRCs by CTLs (fig. 1G). The anti-PD-1 antibody effect was not due to an up-regulation of PD-L1. Indeed IFN- γ upregulates PD-L1 expression; however, the additional supplementation of the co-culture with anti-PD-L1 antibody had no effect on killing of TRCs.

Example 2: recognition of kynurenine-mediated upregulation of PD-1 by TRC and upregulation of IFN- γ in TRC The amino acid transporter SLC1A5 thereby facilitating Trp uptake and Kyn production

(1) anti-CD 3/CD28 bead-activated OVA-CTL was co-cultured with OVA-B16 cells from conventional flasks or OVA-B16TRC grown in 3D fibrin matrix for 24 hours. Kyn in the supernatant was determined by ELISA. The measurement results are shown in FIG. 2A.

(2) CD3/CD28 bead-activated OVA-CTL was co-cultured with OVA-B16TRC for 24 hours in the presence of neutralizing anti-IFN- γ antibodies or isotype controls. Kyn levels in the supernatants were determined by ELISA. The measurement results are shown in FIG. 2B.

(3) OVA-B16 cells or OVA-B16TRC grown in flasks were treated with IFN-. gamma. (10ng/ml) for 24 h. The level of Kyn in the supernatant was determined by ELISA. The measurement results are shown in FIG. 2C.

(4) Separating spleen CD8 in the presence of different concentrations of Kyn⁺T cells were incubated with anti-CD 3/CD28 beads. PD-1 expression was determined by flow cytometry. The measurement results are shown in FIG. 2D.

(5) Same as (4) but with CD8⁺T cells were treated with 200 μ M Kyn for various times as indicated. The measurement results are shown in FIG. 2E.

(6) anti-CD 3/CD28 bead-activated CTLs were treated with PBS, 200 μ M Kyn, or other metabolites from the tryptophan pathway (500 μ M) (melatonin, kynurenine (kynurinic acid), and indole sulfate). After 24h, PD-1 expression was determined by flow cytometry. The measurement results are shown in FIG. 2F.

(7) Control (scrambles) or shIDO1- (Sh1 or Sh2) -OVA B16TRC were co-cultured with anti-CD 3/CD28 bead-activated OT-1T cells. After 4H or 24H, TRC apoptosis (as in fig. 2G) or PD-1 expression on T cells (as in fig. 2H) were analyzed by flow cytometry. The analysis results are shown in fig. 2G and fig. 2H, respectively.

(8) Tryptophan levels in cell lysates were measured by ELISA in the presence or absence of IFN- γ for 24h in control and shSLC1a5- (Sh1 or Sh2) -B16 TRC. The measurement results are shown in fig. 2I.

(9) Control and shSLC1A5- (Sh1 or Sh2) -OVA B16TRC were co-cultured with anti-CD 3/CD28 bead activated OT-1T cells. After 4h or 24h, TRC apoptosis (as in fig. 2J) or PD-1 expression (as in fig. 2K) was analyzed by flow cytometry. The analysis results are shown in fig. 2J and fig. 2K, respectively.

(10) B16TRC was transiently transfected with vector or Flag-SLC1A5 plasmid. Overexpression of SLC1A5 was confirmed by immunoblotting (western blot) (left). Tryptophan levels in the supernatant were determined by ELISA and Trp consumption was calculated (right). The confirmation result is shown in FIG. 2L.

(11) The SLC1A5-B16 cells cultured in vector (Vec-) or in rigid plates (rigid plates) were treated with PBS or IFN-. gamma. (10ng/ml) for 24 h. Kyn levels in the supernatants were determined by ELISA. The results of the measurement are shown in FIG. 2M. P <0.05, p < 0.01. Data represent mean ± SEM.

From the results of example 2, higher levels of Kyn were found in the supernatant of B16TRC-T cell co-culture (fig. 2A) compared to the supernatant of differentiated B16 cell-T cell co-culture, and IFN- γ neutralization inhibited Kyn production (fig. 2B). Consistent with this result, we found that IFN- γ can directly stimulate B16TRC, rather than differentiated B16 cells, releasing high levels of Kyn (fig. 2C). In addition, TRCs produced from other tumor types (murine H22 liver cancer and human a375 melanoma) also released significant amounts of Kyn under IFN- γ stimulation. Kyn in CD8 to confirm OVA-B16TRC⁺Up-regulated PD-1 expression in T cells, weDirected to CD8 in the presence of anti-CD 3/CD28 microbeads⁺The addition of Kyn by T cells showed that the expression of PD-1 was significantly upregulated by T cells in a dose and time dependent manner (FIGS. 2D and 2E). We also found increased Kyn PD-1⁺The cells are due to PD-1^-To PD-1⁺Transformation of T cells, but not by PD-1^-Depletion or PD-1 of⁺in addition, supernatants from the tryptophan coculture were not able to upregulate PD-1 expression in OT-1T cells resistant to activation by CD3/CD 28. in conclusion, these data indicate that Kyn released from TRC stimulated by IFN- γ is able to upregulate PD-1 expression in T cells.

in addition, blocking IDO1 activity or knocking down (knock down) IDO1 by inhibitor 1-MT in OVA-B16 TRCs resulted in more TRC apoptosis in the co-culture system (fig. 2G), and down-regulation of PD expression-1 (fig. 2H), but up-regulation of IFN- γ, TNF- α, and CD107a, indicating that IDO1-Kyn specifically regulates CD8 25, relative to the differentiated counterpart, IDO1 expression was much higher in all types of TRCs, and it could be further up-regulated by IFN- γ, and it was not detected by tdo- γ⁺PD-1 expression in T cells. Although induction of IDO1 expression is clearly critical for the initial phase of rapid Kyn production, maintaining high Kyn production in TRC requires the input of sufficient extracellular tryptophan as a substrate for IDO1, particularly given that Kyn can be further metabolized and released into the extracellular space. Although tryptophan consumption in TRCs is high due to high IDO1 activity, cytoplasmic tryptophan levels are not reduced relative to differentiated tumor cells,we continued to study the expression of three common neutral amino acid transporters (SLC7A5, SLC7A8, and SLC1A5) that transport essential amino acids into cells by active transport, we found that TRCs had higher SLC1A5 expression but not SLC7A5 or SLC7A8, and that expression of SLC1A5 was further enhanced by IFN- γ treatment at both the mRNA and protein levels, furthermore, blocking SLC1A5 activity by shRNA knockdown SLC1A5 or by the inhibitor L-glutamate γ - (p-nitroaniline) hydrochloride (GPNA) reduced TRC tryptophan intracellular levels (fig. 2I), with more TRC cell apoptosis and PD-1 down-regulation of expression of IFN- γ, TNF- α and CD107a (fig. 2J, 2K) conversely, overexpression of SLC1 A4 increased tryptophan consumption (fig. 2L) and promoted up-regulation of expression of IFN- γ, TNF- α and CD107a in cells differentiation (fig. 2J, thus increasing the uptake of SLC1a tryptophan by active amino acid in the SLC1A5, maintaining the SLC- γ uptake map 5 and the activity map 5.

Example 3: kyn activates PD-1 upregulated aryl hydrocarbon receptors (AhR) in T cells

(1) CD8 isolated from spleen⁺T cells were stimulated with 200. mu.M Kyn or Kyn + anti-CD 3/CD28 beads for 48 h. Will CD8⁺T cells were fixed, stained with anti-AhR antibody and imaged by confocal microscopy. The imaging results are shown in fig. 3A. Error Bar (Bar), 10 μ M.

(2) The same as (1) except that the cytoplasmic protein and the nuclear protein were partially separated before immunoblot analysis of AhR. The intensity was calculated relative to the resting/Kyn group. The calculation results are shown in fig. 3B.

(3) Treatment of 48h resting CD8 with PBS or Kyn (200. mu.M)⁺T-cell and anti-CD 3/CD28 bead activated CD8⁺Immunoblot analysis of AhR expression in T cells. The AhR ratio for Nuc/Cyt was calculated relative to the Kyn (-) group. The analysis results are shown in fig. 3C.

(4) CD8 activated by anti-CD 3/CD28 beads⁺T cells were treated with PBS, Kyn or Kyn/DMF (10 or 20. mu.M) for 72 h. Expression of PD-1 was determined by flow cytometry. The measurement results are shown in FIG. 3D.

(5) Wild type or AhR activated anti-CD 3/CD28 beads^-/-OT-1 mice were co-cultured with OVA-B16 cells or OVA-B16 TRC. Tumor cell apoptosis (as in fig. 3E) or PD-1 expression in T cells (as in fig. 3F) were analyzed by flow cytometry. The analysis results are shown in fig. 3E and 3F, respectively.

(6) HEK293T cells were transiently co-transfected with luciferase reporter PGL3 coupled to PD-1 promoter together with AhR or empty plasmid for 24h and then treated with Kyn for an additional 48 h. The detection results are shown in fig. 3G.

(7) Activated CD8⁺T cells were treated with TCDD for 48h and PD-1 expression was analyzed by qPCR (left) and flow cytometry (right). The analysis results are shown in FIG. 3H. P<0.01. Data represent mean ± SEM.

From the results of example 3, we found that addition of Kyn resulted in transport of cytoplasmic AhR to the nucleus of activated T cells as demonstrated by fluorescence microscopy and western blot (fig. 3A and 3B). Interestingly, in addition to AhR activation, we also found that AhR, although low expressed in naive and activated T cells, was upregulated in resting T cells under Kyn stimulation and further increased in activated T cells (fig. 3C). On the other hand, inhibition of AhR activity using DMF or CH-223191 prevented Kyn-dependent elevation of PD-1 (fig. 3D). To further specifically confirm the role of AhR in PD-1 upregulation, we used AhR^-/-OT-1T cells were co-cultured with OVA-B16 TRC. Indeed, AhR deficiency resulted in more TRC apoptosis (fig. 3E), with PD-1 down-regulation (fig. 3F). Furthermore, when we co-transfect HEK293T cells with PD-1 luciferase reporter and a plasmid driving overexpression of AhR, we found that addition of Kyn resulted in a 4-fold increase in luciferase activity in HEK293T cells with intrinsic AhR expression (transfected with empty control plasmid) and a 12-fold increase in cells overexpressing AhR (fig. 3G). Since AhR functions to recognize various exogenous toxins, we also tested TCDD, the exogenous ligand for AhR. Consistent with our hypothesis, TCDD also upregulates PD-1 expression in T cells (fig. 3H). Taken together, these data indicate that Kyn can up-regulate CD8 by inducing and activating AhR⁺PD-1 expression in T cells.

⁺Practice ofExample 4: AhR regulates expression of CD8T cell transporter at transcription level so as to take up exogenous Kyn

(1) CD8 activated by resting or anti-CD 3/CD28 beads⁺T cell (1 × 10)⁴Individual cells) were lysed and Kyn levels were analyzed by ELISA. The analysis results are shown in FIG. 4A.

(2) Activated CD8⁺T cells were treated with PBS or Kyn (200. mu.M) for 24 h. Expression of PAT4 and SLC7a8 was determined by real-time PCR (left) and immunostaining (right). The measurement results are shown in FIG. 4B. Error Bar (Bar), 10 μ M.

(3) Delivery of control shRNAs or shRNAs against SLC7A8 and PAT4 to activated CD8 via retrovirus⁺T cells were treated for 24h, then transduced T cells were treated with Kyn (200. mu.M) for 48 h. Kyn levels in the supernatants were determined by ELISA and Kyn consumption was calculated. The measurement results are shown in FIG. 4C.

(4) Same as (3), but the expression of PD-1 was analyzed by flow cytometry. The analysis results are shown in fig. 4D.

(5) Activated OT-1T cells were transfected with control shRNA, PAT4shRNA (Sh1 and Sh2) or SLC7A8 shRNA (Sh1 and Sh2) for 72h, and then co-cultured with OVA-B16 TRC. TRC apoptosis was analyzed by flow cytometry. The analysis results are shown in fig. 4E.

(6) Activated CD8 at 48h treatment with PBS, Kyn/CH-223191(10 μ M) or Kyn/DMF (20 μ M)⁺mRNA expression of PAT4 and SLC7a8 was determined in T cells. The measurement results are shown in FIG. 4F.

(7) Resting or activated CD8⁺T cells were treated with PBS or Kyn for 48 h. Chip-qPCR analysis was performed using anti-AhR antibody and qPCR primers specific for SLC7A8 or PAT4 promoter. The analysis results are shown in FIG. 4G.

(8) HEK293T cells were transiently co-transfected with AhR vector and luciferase reporter PGL3 coupled to SLC7A8 or PAT4 promoter, followed by treatment with Kyn for an additional 48 h. The measurement results are shown in FIG. 4H. P < 0.01. Data represent mean ± SEM.

From the results of example 4, we followedHow exogenous Kyn entered T cells was investigated. Little endogenous Kyn was detected in CD8+ T cells (fig. 4A), and these T cells were found to express very low levels of IDO1, indicating CD8⁺The T cells activate AhR by virtue of exogenous Kyn so as to up-regulate PD-1 expression. Several transporters including SLC1a5, SLC7a5, SLC7A8 and PAT4 that transport Kyn into cells. Although these four transporters were found in CD8⁺Expression in T cells, but addition of Kyn led surprisingly to upregulation of expression of SLC7A8 and PAT4 (fig. 4B). Notably, with the resting CD8⁺Expression of SLC7A8 and PAT4 in anti-CD 3/CD28 bead activated CD8 in comparison to T cells⁺T cells are upregulated and further enhanced by Kyn treatment. Furthermore, we found that IFN- γ treatment did not affect CD8⁺Expression of PAT4 and SLC7a8 in T cells. These results prompted us to speculate that these two transporters are CD8⁺Major Kyn import in T cells. Thus, we used retroviruses to deliver SLC7A8 and PAT4shRNA to primary T cells or T cells treated with the inhibitor 2-amino-2-norbornanecarboxylic acid (BCH) against SLC7A8 or sarcosine against PAT 4. The results show that inhibition or knock-down of SLC7A8 and PAT4 blocked Kyn entry into T cells (fig. 4C), inhibited AhR activity, abolished PD-1 upregulation (fig. 4D), and increased the cytotoxicity of OT-1T cells against OVA-B16TRC (fig. 4E). At the same time, we found that AhR inhibitors DMF or CH-223191 were effective in inhibiting Kyn-induced up-regulation of SLC7a8 and PAT4 (fig. 4F). chip-PCR analysis further confirmed that AhR did bind to the SLC7A8 and the promoter of the PAT4 gene (FIG. 4G). Luciferase assays also demonstrated enhanced expression of SLC7A8 and PAT4 upon AhR binding (fig. 4H). With respect to the reverse trafficking function in many trafficking systems, we also analyzed the effects of SLC7A8 and PAT4 on the trafficking of other essential amino acids. CD8 activated by anti-CD 3/CD28 beads 48 hours after treatment with or without Kyn⁺T cells were used to analyze essential amino acids in the cytosol by LC-QE-MS. The results show that Kyn treatment has no effect on most essential amino acids except for some reduction in threonine strength. Taken together, these data indicate that AhR transcriptionally activates the Kyn transporter and promotes uptake of exogenous Kyn by T cells.

Example 5: Kyn-AhR regulated PD-1 pathway is independent ofTCR signaling but can be facilitated by TCR signaling

(1) will CD8⁺T cells were treated with Kyn (200. mu.M) for the indicated times. Expression of PD-1 was determined by flow cytometry. The measurement results are shown in FIG. 5A.

(2) In the presence of a wild-type or AhR^-/-Mouse isolated resting or anti-CD 3/CD28 bead activated CD8⁺PD-1 expression was determined by flow cytometry in T cells. The measurement results are shown in FIG. 5B.

(3) Resting and CD3/CD28 bead activated CD8⁺PD-1 expression in T cells was determined by flow cytometry at the time points indicated. The results of the measurement are shown in FIG. 5C.

(4) Will CD8⁺T cells were pre-treated with anti-CD 3/CD28 beads for 4h, followed by PBS or Kyn for 24 h. PD-1 expression was determined by flow cytometry. The results of the measurement are shown in FIG. 5D.

(5) Resting or anti-CD 3/CD28 bead activated CD8⁺mRNA levels of SLC7A8 or PAT4 in T cells. The results of the measurement are shown in FIG. 5E.

(6) CD8 activated by anti-CD 3/CD28 beads⁺T cells were treated with CsA (1. mu.M) for 48 h. mRNA expression of PAT4 and SLC7a8 was determined by real-time PCR. The measurement results are shown in FIG. 5F.

(7) Activated CD8 in the Presence or absence of CsA (1. mu.M)⁺T cells were treated with Kyn (200. mu.M) for 48 h. Cells were lysed and Kyn levels determined by ELISA assay. The measurement results are shown in FIG. 5G.

(8) Resting or activated CD8⁺T cells were treated with PBS, Kyn (200. mu.M), CsA (1. mu.M), Kyn/CsA for 48 h. PD-1 levels were determined by flow cytometry. The measurement results are shown in FIG. 5H.

(9) CD8 in resting or activated Using an anti-NFATc 1 antibody and qPCR primers specific for the PAT4 or SLC7A8 promoter⁺Chip-qPCR analysis was performed in T cells. The analysis results are shown in FIG. 5I.

(10) The pulses were released (pulsed with) in the presence of IgG or anti-PD-1 neutralizing antibody (10. mu.g/ml))OVA_257-264(2. mu.g/ml) splenic APC with OT-1CD8⁺T cells were co-cultured for 72 h. IFN- γ expression was measured by flow cytometry (left). Or CD8 from OT-1 mice⁺T cells were treated with Kyn (200 μ M) for 72h, then co-cultured with OVA-peptide loaded APCs and treated with or without anti-PD-1 neutralizing antibody for 72h (right). The measurement results are shown in fig. J. P<0.01. Data represent mean ± SEM.

From the results of example 5, we found that Kyn-AhR pathway and TCR signaling can induce PD-1 expression independently of each other. Regardless of the aforementioned PD-1 upregulation by Kyn in the presence of T cell activation signals (fig. 2D and 2E), Kyn alone is actually sufficient to upregulate resting CD8⁺PD-1 in T cells (FIG. 5A). However, such induction is a slow process, starting to increase at the 48 hour time point. On the other hand, CD8 with AhR defect⁺T cells also upregulated PD-1 expression when T cells activated signaling (fig. 5B). Notably, we found that TCR signaling-induced PD-1 expression was a rapid process, beginning to increase at the initial hour time point (fig. 5C). We will read CD8⁺T cells were incubated with anti-CD 3/CD8 microbeads for 4 hours. After bead removal, the cells were treated with Kyn. We found that this transient TCR-activation resulted in higher PD-1 expression (fig. 5D), suggesting that TCR signaling promotes PD-1 induction by Kyn-AhR. Since Kyn import is a limiting step in the Kyn-AhR dependent pathway, we investigated whether and how TCR signaling affects Kyn-transporters. We found that primary (naive) T cells expressed low levels of SLC7A8 and PAT4, whereas activated T cells had significantly higher expression of both transporters (fig. 5E). In CD8⁺During T cell activation, a number of transcription factors triggered by TCR signaling are activated and may be involved in the enhanced expression of PD-1. Among them, nuclear factor (NFATc1) activating T cell c1 seems to be crucial. NFATc1 is normally localized in the cytosol in a phosphorylated inactive form but translocates into the nucleus upon dephosphorylation. Inhibitor cyclosporin A (CsA) blocks nuclear translocation of NFATc1 and inhibits PD-1 in CD8⁺Expression in T cells. When we used CsA to block NFATc1, we found SLC7A8And PAT4 in activated CD8⁺Upregulation in T cells was also blocked (fig. 5F) and in agreement therewith, we also observed a reduction in Kyn uptake (fig. 5G). The promotion of Kyn/AhR-induced PD-1 expression by TCR signaling was also inhibited by CsA (FIG. 5H). Furthermore, analysis using Jaspar and Genomatrix software revealed the presence of multiple shared cis-elements for binding of NFATc1 to the promoters of SLC7a8 and PAT4, confirmed by ChIP assay, indicating that NFATc1 did bind to the promoter (fig. 5I). By co-culturing CD8⁺T cells and Antigen Presenting Cells (APC), we found that PD-1 blockade inhibited IFN-. gamma.production, whereas Kyn treatment upregulated IFN-. gamma.production upon PD-1 blockade (FIG. 5J). Taken together, these data indicate that the Kyn-AhR regulated PD-1 pathway is independent of TCR signaling but can be facilitated by TCR signaling.

⁺Example 6: Kyn/AhR regulates PD-1 expression of CD8T cells in tumor-bearing mice

(1) WT C57BL/6 mice were injected intraperitoneally with PBS or Kyn (50 μ g, 1 time per day) for 5 days, and CD8 was sorted by FACS from spleen and mesenteric lymph nodes⁺T cells. PD-1 expression was determined by flow cytometry (fig. 6A). Some CD8 from spleen⁺T cells were immunostained with AhR and DAPI (fig. 6B), and mRNA expression of PAT4 and SLC7a8 was determined by real-time PCR (fig. 6C). The above experimental results are shown in fig. 6A, 6B, and 6C, respectively. Error Bar (Bar), 10 μ M.

(2) Except for using AhR^-/-The same as (1) except for the mouse. The results of the experiment are shown in his 6D.

(3) AhR from 5 days of PBS or Kyn injection^-/-CD8 isolated from C57BL/6 mice from resting or anti-CD 3/CD28 bead-activated spleen⁺PD-1 expression in T cells. The results of the expression measurement are shown in FIG. 6E.

(4) WT or AhR^-/-C57BL/6 mice were injected with or without anti-CD 3 antibody (5 μ g, i.p.) at various times and PD-1 expression was determined by flow cytometry. The measurement results are shown in FIG. 6F.

(5) C57BL/6 mouse injectionanti-CD 3 antibody was administered for 72h, and then Kyn (50 μ g) was administered to these mice. Spleen CD8⁺PD-1 expression in T cells was determined by flow cytometry after 48 h. The measurement results are shown in FIG. 6G.

(6) C57BL/6 mice with 5 × 5mm OVA-B16 melanoma were CFSE-labeled OT-1CD8⁺T cell (4 × 10)⁶One per mouse, 1 time every 3 days) 3 adoptive transfers. Meanwhile, some mice were injected intraperitoneally with Kyn (50. mu.g/mouse, 1 time every 2 days) 3 times. On day 7 after transfer, CFSE was administered⁺CD8⁺T cells were isolated from tumors for flow cytometric analysis of PD-1 expression. The analysis results are shown in fig. 6H.

(7) will be 4X 10⁶adoptive transfer of CD45.2OT-1T cells transfected with scrambled, shPAT 4-or shSLC7a 8-retrovirus to CD45.1 mice with 5 × 5mm OVA-B16 melanoma 3 times (1 time every 3 days) tumor growth was measured (left) and long-term survival analyzed (right) the results of the measurements are shown in fig. 6I.

(8) Same as (7) except C57BL/6 mice were treated with Kyn, DMF (5 μ g/mouse, o.t., 2 times daily) or Kyn + DMF for 6 days. CFSE from tumor⁺CD8⁺T cell isolation was used for flow cytometric analysis of PD-1 expression. The analysis results are shown in fig. 6J.

(9) C57BL/6 mice with 5 × 5mm B16 melanoma were treated with PBS, Kyn (50. mu.g/mouse, i.p., 1 time every 2 days), DMF (5. mu.g/mouse, o.t., 1 time every day), or Kyn + DMF for 6 days, CD8 isolated from the tumor⁺PD-1 expression in T cells was determined by flow cytometry (n ═ 5). The measurement results are shown in FIG. 6K.

(10) C57BL/6 mouse intraperitoneal injection of 1X 10⁶Individual ID8 ovarian cancer cells. After 10 days, mice were dosed intraperitoneally with PBS, Kyn, DMF, or Kyn + DMF for 6 days. Mixing CD8 from ascites⁺T cells were isolated and PD-1 expression was determined by flow cytometry (n-5). The measurement results are shown in FIG. 6L.

(11) Sorting CD45 from spontaneously developing breast tumors in MMTV-PyMT mice^-ALDH⁺And CD45^-ALDH^-A tumor cell. Cells were seeded separately into 90Pa soft 3D fibrin gel. After 4 daysAnd the number of colonies was counted. The counting results are shown in fig. 6M.

(12) Will CD45⁺、CD45^-ALDH⁺And CD45^-ALDH^-Cells were lysed and Kyn levels were determined by ELISA. The measurement results are shown in fig. 6N.

(13) C57BL/6 mice with 5 × 5mm OVA-B16 melanoma were used 1 time per 3 days or not OT-1CD8⁺T cell (4 × 10)⁶Individual cells/mouse) were adoptively transferred 3 times. Meanwhile, these mice were treated with PBS, DMF (5 μ g/mouse, o.t., 2 times per day) or anti-PD-1 neutralizing antibody (250 μ g/day, 1 time 2 times per 2 days) for 20 days. Tumor growth was measured (left) and long-term survival analyzed (right). The measurement results are shown in fig. 6O.

(14) Same as (9), but the tumors were weighed. The weighing results are shown in fig. 6P. P <0.05, p < 0.01; relative to CTL group # p <0.05, # # p < 0.01. Data represent mean ± SEM.

From the results of example 6, analysis of spleen and lymph nodes showed CD8⁺T cells did not undergo proliferation but upregulated PD-1 expression on day 5 (fig. 6A). At the same time, we observed these CD8⁺AhR in T cells translocated to the nucleus (fig. 6B) and Kyn transporters were upregulated with SLC7A8 and PAT4 (fig. 6C). Consistent with the in vitro data above, this exogenous Kyn was unable to induce AhR^-/-PD-1 expression in mice (FIGS. 6D and 6E). In another aspect, in WT or AhR^-/-Injection of the stimulatory anti-CD 3 antibody in mice rapidly upregulated splenic CD8 observed at the 6 hour time point⁺PD-1 of T cells, but began to decline after 72 hours (fig. 6F). -at this 72 hour time point we injected mice with Kyn we found that Kyn-induced PD-1 expression was enhanced by first TCR signaling (fig. 6G), further suggesting that TCR signaling promotes PD-1 induction by Kyn-AhR hi order to study the up-regulation of PD-1 in an in vivo tumor model, we CD45.2OT-1T cells were adoptively transferred to subcutaneous injections of OVA-B16TRC (5 × 10 TRC)⁵/mouse) three days later, at different time points, we analyzed PD-1 expression and measured Kyn concentration at the tumor site. We found that Kyn levels gradually increased and reached a plateau on day 9. Coincidently, CD45.2OT-1T cells also gradually increased PD-1 expression and reached a plateau at day 9, suggesting an intrinsic relationship between Kyn and PD-1 expression then, after adoptive transfer of CFSE-labeled OT-1T cells, we treated mice bearing OVA-B16 melanoma (5 × 5mm) with Kyn consistent with the above data, we had tumor-infiltrated CFSE⁺Higher levels of PD-1 expression were observed in T cells (FIG. 6H).

Next, we adoptively transferred control shPAT4 or shSLC7A8 OT-1T cells into CD45.1 mice bearing 3 × 3mm OVA-B16 melanoma We found that PAT4 or SLC7A8 knockdown significantly reduced PD-1 expression, but increased IFN-. gamma.and TNF-. alpha.expression in OT-1T cells, with delayed tumor growth and prolonged survival of mice (FIG. 6I), indicating that Kyn triggers PD-1 expression in the tumor microenvironment⁺This Kyn triggered tumor specific CD8 in the presence of the AhR-inhibitor DMF, observed in T cells⁺Up-regulation of PD- α expression in T cells was abolished (FIG. 6J). consistent, up-regulation of IFN-. gamma.and TNF-. alpha.expression was observed in T cells in addition, B16 melanoma tumor-bearing mice also received treatment with Kyn, Kyn + DMF or DMF⁺PD-1 expression in T cells, however it was blocked by DMF (fig. 6K). Peritoneal tumors, including ovarian cancers, are normally upregulated by CD8 for PD-1 expression⁺T cell infiltration. Here, we additionally used two mouse peritoneal tumor models of ID8 ovarian cancer and H22 liver cancer ascites to verify the above PD-1 expression results. Likewise, Kyn promotes peritoneal, spleen or lymph node CD8⁺PD-1 expression in T cells suppressed the expression of IFN-. gamma.and TNF- α (FIG. 6L), while the PD-1, IFN-. gamma.and TNF-. alpha.expression patterns were reversed by DMF.

In spontaneously developing MMTV-PyMT mice with breast cancer, CD45-ALDH has been isolated⁺The tumor cells are identified as tumorigenic cells. We further identified them as TRCs, since only CD45 is present in 90Pa 3D flexible fibrin gel^-ALDH⁺But not CD 45-ALDH-tumor cells grew into colonies (fig. 6M). As expected, CD45^-Swelling of ALDHTumor cells and CD45⁺IDO1 expression and Kyn levels in immune cells were very low, but in CD45-ALDH⁺Significantly high in TRC (fig. 6N). Consistently, CD45 from IFN- γ treatment^-ALDH⁺But not CD 45-ALDH-cell supernatant to up-regulate CD8⁺PD-1 expression in T cells. On the other hand, we injected the same number of OVA-B16TRC or OVA-B16 cells intratumorally into CD45.1C57BL/6 melanoma-bearing mice while adoptively transferring CD45.2OT-1 cells. We found that OT-1T cells in the OVA-B16TRC group expressed higher PD-1 after 3 days than in the OVA-B16 group. At the same time, IDO1 knockdown in melanoma formed by B16TRC resulted in adoptively transferred gp-100 specific CD8⁺a significant reduction in PD-1 expression in T cells, and an increase in IFN- γ and TNF- α expression in these T cells.

We reacted OVA-B16TRC, OVA-B16 or B16 cells with OVA-specific CD45.2OT-1T cells and OVA-non-specific CD45.1CD8⁺T cells were co-cultured for 24 hours. We found that CD45.1CD8 is non-specific⁺PD-1 upregulation in T cells is not induced by the tumor cell types described above; OVA-B16 cells only mildly induced PD-1 upregulation in antigen-specific OT-1T cells; however, PD-1 up-regulation in OT-1T cells was clearly induced by OVA-B16TRC, indicating that PD-1 expression is not only restricted by antigen but also controlled by local Kyn concentrations. OVA-specific CD8⁺Adoptive transfer of T cells produced a therapeutic effect against mouse OVA-B16 melanoma, which was enhanced by administration of PD-1 antibody (fig. 6O). The use of DMF also resulted in the same treatment effect compared to PD-1 antibody (fig. 6O). In addition, the accelerated tumor growth seen with Kyn treatment was also reversed by DMF treatment (fig. 6P). Taken together, these data indicate CD8 in tumor-bearing mice⁺PD-1 expression by T cells is regulated by the Kyn-AhR pathway.

⁺Example 7: PD-1 expression in CD8T cells of cancer patients is upregulated by the Kyn-AhR pathway

(1) from healthy subjects (n-50) or patients with breast (n-84), colon (n-28) or lung (n-15) cancerSeparation of peripheral blood CD8⁺T cells. Alternatively, CD8 was isolated from breast (n ═ 9) or colon (n ═ 17) tumor tissue⁺T cells. PD-1 expression was analyzed by flow cytometry. The analysis results are shown in FIG. 7.

(2) Kyn levels in the serum of healthy subjects (n-31) and patients with breast (n-73), lung (n-10) or colon (n-26) cancer were measured. Alternatively, lysates from fresh human breast (n-11) or colon (n-8) tumor tissue and adjacent normal tissue were prepared and Kyn levels were measured. The measurement results are shown in fig. 7B.

(3) Correlation between PD-1 expression and Kyn levels in healthy humans (left, n-37) and patients with breast cancer (right, n-62). The analysis results are shown in fig. 7C.

(4) CD8 isolated from PBMCs of patients with breast or lung cancer⁺T cells were activated by anti-CD 3/CD28 beads and treated with PBS, Kyn (200. mu.M) or Kyn/DMF (20. mu.M) for 48 h. Expression of PD-1 was determined by flow cytometry. The measurement results are shown in FIG. 7D.

(5) CD8 isolated from breast or lung cancer patients⁺T cells were treated with DMF (20. mu.M) for 48 h. PD-1 expression was determined by flow cytometry (n ═ 6). The measurement results are shown in FIG. 7E.

(6) Sorting PD-1 from PBMCs of healthy donors or patients with breast or lung cancer^{Height of}CD8⁺Or PD-1^{Is low in}CD8⁺T cells. Expression of AhR mRNA was analyzed by real-time PCR (n-8, fig. 7F). These cell populations were also immunostained with anti-AhR antibodies and DAPI and imaged by confocal microscopy (fig. 7G). The experimental results are shown in fig. 7F and 7G, respectively. Error Bar (Bar), 10 μ M.

(7) Chip-qPCR analysis of AhR binding to PD-1 promoter. The analysis results are shown in fig. 7H.

(8) CD8 from breast cancer patients⁺T cells were treated with PBS, Kyn (200. mu.M), Kyn/DMF (20. mu.M) or Kyn/CH (50. mu.M) for 48 h. Expression of PAT4 and SLC7a8 was determined by real-time PCR. The measurement results are shown in FIG. 7I.

(9) PD-1 from breast and lung cancer patients^{Height of}CD8⁺T cells or PD-1^{Is low in}CD8⁺mRNA expression of PAT4 or SLC7a8 in T cells. The expression results are shown in FIG. 7J.

(10) PD-1 isolated from PBMC of breast cancer patients^{Height of}CD8⁺T cells or PD-1^{Is low in}CD8⁺Chip-qPCR analysis was performed in T cells using anti-AhR antibodies and qPCR primers specific for the SLC7A8 or the promoter of PAT 4. The analysis results are shown in fig. 7K. Data represent mean ± SEM.

(11) PD-1 is shown at CD8⁺Schematic of how it is upregulated in T cells. The results are shown in FIG. 7L. P<0.01，***p<0.001. Data represent mean ± SEM.

From the results of example 7, flow cytometry analysis showed peripheral blood CD8 from breast, colon, and lung cancer patients compared to healthy donor controls⁺T cells had higher PD-1 expression (FIG. 7A). In addition, tumor-infiltrated CD8 in breast and colon cancer patients⁺Very high PD-1 expression was found in T cells (fig. 7A). Consistent with these results, Kyn levels were significantly elevated in plasma and tumor tissues of these cancer patients (fig. 7B). Notably, Kyn levels were strongly correlated with PD-1 expression in breast and colon cancer patients, but not in healthy populations (fig. 7C). To elucidate the source of Kyn-producing cells, CD45 isolated from breast (n-10) and colon (n-14) tumor tissues of patients was analyzed⁺Immune cells and CD45^-A tumor cell. IDO1 expression and Kyn levels at CD45^-Cells other than CD45⁺Predominate in the cell. We then treated CD45 with IFN-gamma in vitro in the presence or absence of 1-MT^-Tumor cells were used for 24 hours. The supernatant was used to treat peripheral blood T cells of the patient for 24 hours. Flow cytometry analysis showed that PD-1 is expressed in CD8⁺T cells are upregulated and this process is blocked by the addition of 1-MT, indicating that Kyn is produced predominantly by tumor cells in human tumor tissue, which promotes PD-1 at CD8⁺Expression in T cells. Given that Kyn levels are regulated by Trp transporters, we have additionally found that SLC1a5 expresses high levels in breast and colon tumor tissues of patients.

We used Kyn in vivoTreatment of the above human samples with or without stimulation with anti-CD 3/CD28 microbeads of CD8⁺T cells. Kyn upregulated resting and activated CD8 from healthy donors and patients with breast and lung cancer (FIG. 7D)⁺PD-1 expression in T cells. In addition, Kyn treatment promotes resting and activated peripheral CD8 in patients from breast and lung cancer⁺AhR translocates from the cytosol to the nucleus in T cells. Consistently, AhR nuclear translocation was also found in tumor-infiltrating T cells in breast and colon cancer patients. We used DMF or CH-223191 to block AhR activity. We found that the addition of DMF or CH-223191 inhibited human CD8⁺Kyn-induced PD-1 upregulation in T cells (fig. 7D). More importantly, DMF alone can directly down-regulate the CD8 of the patient⁺PD-1 expression in T cells (FIG. 7E), suggesting that AhR may be CD8 driving cancer patients⁺A key signal molecule for PD-1 expression in T cells.

We compared PD-1 in cancer patients^{Height of}And PD-1^{Is low in}CD8⁺AhR activity between T cells. We found PD-1 selected from lung and breast cancer patients but not from healthy controls^{Height of}CD8⁺T cells expressing PD-1 in a corresponding setting^{Is low in}CD8⁺Much higher AhR mRNA levels by T cells (fig. 7F). Further immunostaining analysis confirmed PD-1 from patients as evidenced by nuclear translocation^{Height of}T cells did have more AhR activity (fig. 7G), and such nuclear AhR bound strongly to CD8 from breast cancer patients compared to healthy controls⁺The promoter of PD-1 in T cells (FIG. 7H). SLC7A8 and PAT4 are involved in PD-1 expression by transporting exogenous Kyn and are present in murine CD8⁺T cells were regulated by AhR (fig. 4D and 4E).

SLC7A8 and PAT4 in human CD8 when Kyn is added⁺T cells were upregulated (fig. 7I). Immunohistochemical staining showed that tumor-infiltrating T cells highly expressed PAT4 in breast and colon cancer patients. However, this up-regulation could be blocked by the AhR inhibitors DMF or CH-223191 (fig. 7I). Consistently, only PD-1 was sorted from cancer patients^{Height of}But not PD-1^{Is low in}CD8⁺T cells with SLC7A5 and PAT4 highExpression (fig. 7J). In addition, in human CD8⁺Binding of AhR to the promoters of SLC7A8 and PAT4 and regulation of their expression was demonstrated in T cells (fig. 7K). Taken together, these data indicate CD8 in cancer patients⁺Elevated PD-1 expression in T cells may be driven primarily by the Kyn-AhR pathway.

⁺Example 8: PD-1 is highly expressed by peripheral blood CD8T cells of tumor patients and is positively correlated with the Kyn level in plasma

1. Detection of peripheral blood CD8 in breast, colon, lung cancer patients and healthy volunteers⁺T cell PD-1 expression

1) Experimental procedure

Collecting peripheral blood of breast cancer (30 cases), colon cancer (25 cases), lung cancer (25 cases) and healthy volunteers (20 cases) about 2mL, separating with lymphocyte separation solution to obtain lymphocytes, labeling with flow-type antibodies CD3, CD8, PD-1, and analyzing CD3⁺CD8⁺PD-1 expression in T cells.

2) Results of the experiment

FIG. 8A shows the CD8 of peripheral blood of patients with breast, colon, and lung cancer⁺T cells expressed higher levels of PD-1 molecules than healthy volunteers, indicating CD8 in tumor patients⁺T cell function is mostly in a state of inhibition.

2. Detection of peripheral blood plasma Kyn levels in breast, colon, lung cancer patients and healthy volunteers

1) Experimental procedure

Peripheral blood of breast cancer (30 cases), colon cancer (25 cases), lung cancer (25 cases) and healthy volunteers (20 cases) is collected at about 2mL, 400g is centrifuged for 5min to obtain upper plasma, and Kyn level is detected by ELISA kit.

2) Results of the experiment

FIG. 8B shows that the plasma of breast, colon, and lung cancer patients contains higher levels of Kyn than healthy volunteers, indicating that the tumor cells produce large amounts of Kyn.

3. Peripheral blood CD8 of breast cancer patients and healthy volunteers⁺Correlation analysis of expression level of T cell PD-1 and plasma Kyn level

1) Experimental procedure

Peripheral blood CD8 was analyzed in 30 breast cancer patients and 20 healthy volunteers using GraphPad Prism 6.0 software, respectively⁺Correlation of expression levels of PD-1 in T cells and Kyn levels in plasma.

2) Results of the experiment

As can be seen in FIGS. 8C and 8D, the peripheral blood CD8 of breast cancer patients⁺The expression level of PD-1 of T cells is positively correlated with the Kyn level of plasma, while the expression level of PD-1 of healthy volunteers is not correlated with the Kyn level.

From the results of example 8, CD8 in peripheral blood and tumor tissue of tumor patients⁺The T cells highly express PD-1 and simultaneously generate high level of Kyn, and the two are in positive correlation. Therefore, whether the tumor disease exists can be detected by detecting the Kyn content; that is, Kyn can be used as a diagnostic index for tumors.

⁺Example 9: CD8T cells in tumor tissues of patients highly express PD-1 and are positively correlated with tissue Kyn level

1. Detection of infiltration of CD8 in breast and colon cancer tissues in patients⁺PD-1 expression in T cells

1) Experimental procedure

Fresh breast (20) and colon (15) cancer tissues were harvested, minced, digested with collagenase II and IV for 2 hours, lysed for erythrocytes to give a single cell suspension, flow labeled CD3, CD8, PD-1, analyzed for CD3⁺CD8⁺PD-1 expression in T cells.

2) Results of the experiment

FIG. 9A shows CD8 infiltrating in breast and colon cancer tissues⁺High expression of PD-1 by T cells, which indicates that CD8 is present in tumor patients⁺T cell function is mostly in a state of inhibition.

2. Detection of Kyn levels in Breast and Colon cancer and its corresponding paracarcinoma tissues

1) Experimental procedure

Fresh breast cancer (20 cases) and colon cancer (10 cases) and corresponding paracarcinoma tissues are collected, the mass is weighed, an electric homogenizer is used for preparing tissue homogenate, and an ELISA kit is used for detecting the Kyn level.

2) Results of the experiment

As can be seen in fig. 9B, breast and colon cancer tissues contain higher levels of Kyn than the corresponding paraneoplastic tissues, indicating that tumor cells can produce large amounts of Kyn.

3. CD8 in breast cancer tissue⁺Correlation analysis of PD-1 expression amount and Kyn level of T cells

1) Experimental procedure

Analysis of 20 breast cancer tissues for CD8 using GraphPad Prism 6.0 software⁺Correlation of PD-1 expression and Kyn levels in T cells.

2) Results of the experiment

FIG. 9C shows the CD8 infiltrated in breast cancer tissue⁺The expression level of PD-1 of the T cells is positively correlated with the Kyn level of the tissues.

From the results of example 9, in tumor patients, CD8⁺There is a correlation between PD-1 expression by T cells and Kyn production by tumor cells.

Example 10: significant reduction in plasma Kyn levels following treatment of therapeutically effective neoplastic patients

1. Experimental procedure

Collecting about 2mL of peripheral blood of the patients with the acute B lymphocyte leukemia and the acute B lymphocyte leukemia with effective and ineffective CAR-T treatment, centrifuging for 5min at 400g to obtain upper plasma, and detecting the Kyn level by using an ELISA kit.

2. Results of the experiment

As can be seen in FIG. 10, plasma Kyn levels are significantly lower after treatment in leukemia patients who are CAR-T therapeutically effective than before treatment, while plasma Kyn levels remain substantially unchanged in patients who are not therapeutically effective.

From the results of example 10, it is shown that the expression level of Kyn reflects the therapeutic effect of tumor.

The above examples of the present disclosure are merely examples provided for clearly illustrating the present disclosure and are not intended to limit the embodiments of the present disclosure. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present disclosure should be included in the protection scope of the claims of the present disclosure.

Claims

1. The application of the reagent for detecting the Kyn content in preparing a reagent or a kit for diagnosing or assisting to diagnose whether a person to be detected has a disease accompanied with the increase of PD-1 expression, wherein the disease is selected from one or more of breast cancer accompanied with the increase of PD-1 expression, lung cancer accompanied with the increase of PD-1 expression and colon cancer accompanied with the increase of PD-1 expression.

2. Use of an agent for detecting Kyn content for the preparation of a reagent or kit for predicting, assessing or monitoring the effect of disease immunotherapy in a patient having a disease accompanied by an increase in PD-1 expression, wherein the disease is selected from one or more of breast cancer accompanied by an increase in PD-1 expression, lung cancer accompanied by an increase in PD-1 expression, and colon cancer accompanied by an increase in PD-1 expression.

3. Use of a reagent for detecting the Kyn content in a body fluid of a subject for the preparation of a kit for diagnosing or aiding in the diagnosis of whether the subject suffers from a disease accompanied by an increase in PD-1 expression by a method comprising a detection step of detecting the Kyn content in the body fluid of the subject using the detection reagent; wherein the disease is selected from one or more of breast cancer accompanied by increased PD-1 expression, lung cancer accompanied by increased PD-1 expression, and colon cancer accompanied by increased PD-1 expression.

4. The use according to claim 3, wherein the method further comprises a comparison step of comparing the Kyn content in the body fluid of the subject with the Kyn content in the body fluid of a normal human.

5. Use according to claim 4, characterized in that the method further comprises a step of determining:

6. Use of an agent for detecting the Kyn content in a body fluid of a subject for the preparation of a kit for predicting, evaluating or monitoring the effect of disease immunotherapy on a patient having a disease accompanied by an increase in PD-1 expression by a method comprising a detection step of detecting the Kyn content in the body fluid of the subject using a detection agent; wherein the disease is selected from one or more of breast cancer accompanied by increased PD-1 expression, lung cancer accompanied by increased PD-1 expression, and colon cancer accompanied by increased PD-1 expression.

7. The use according to claim 6, wherein the method further comprises a comparison step of comparing the amount of Kyn in the patient's body fluid with the amount of Kyn in the patient's body fluid having a good disease immunotherapy effect.

8. Use according to claim 7, characterized in that the method further comprises a step of determining: