Application of prostaglandin PGE1 or PGE2 in preparation of product for promoting proliferation of depleted precursor T cell

Abstract

The invention discloses application of prostaglandin PGE1 or PGE2 in preparation of a product for promoting proliferation of depleted precursor T cells. Specifically, prostaglandin PGE1 or PGE2 is added into a culture system of the T cells for culture, so that the efficiently amplified depleted precursor T cells can be obtained, meanwhile, the depletion degree of the CD8 + T cells can be inhibited, and when the prostaglandin PGE1 or PGE2 is stopped, the depleted precursor T cells can be rapidly and directionally differentiated into the CD8 + T cells. Therefore, when the prostaglandin PGE1 or PGE2 is applied to the in-vitro preparation process of adoptive T cell therapy, the total amount of CD8 + T cells can be remarkably increased, the proportion of Tpex is increased, the expression of a depletion marker is reduced, and a technical support is provided for clinical popularization of solid tumor infiltration lymphocyte therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immune cell therapy, in particular, the application of prostaglandin PGE1 or PGE2 in the preparation of a product for promoting the proliferation of exhausted precursor T cells. BACKGROUND

[0002] Adoptive cell therapy (ACT) is a general term for a class of tumor immunotherapy, the core principle of which is: immune active cells are isolated from the body of a patient or a donor, activated, expanded or genetically modified in vitro, and then returned to the patient's body, so as to kill tumor cells by using the anti-tumor activity of these cells. The tumor-infiltrating lymphocyte (TIL) therapy is an important subtype of adoptive cell therapy, which specifically refers to a treatment method in which the infiltrating lymphocytes (mainly CD8 + T cells, and also containing CD4 + T cells, etc.) isolated from the tumor tissue of a patient are expanded in vitro in a large amount, and then returned to the patient's body. It is one of the most promising immunotherapy methods for solid tumors at present. However, it is difficult to obtain sufficient number of CD8 + T cells with good function (sufficient number and maintaining low exhaustion and high survival rate) during the in-vitro culture of TIL, which limits its clinical application.

[0003] Recent studies have shown that CD8 + T cells with “stem cell” characteristics, i.e. exhausted precursor T cells (Tpex), can self-renew and differentiate into effector T cells. Maintaining and expanding Tpex in vitro is crucial to improve the effect of TIL therapy.

[0004] Prostaglandin PGE1 or PGE2 is an unsaturated fatty acid with multiple physiological activities, which can be synthesized through the COX pathway. It is known that it can inhibit the differentiation of myeloid progenitor cells, promote the proliferation of erythroid progenitor cells, and has an important regulatory effect on the hematopoietic system. Recent studies have shown that PGE2 can increase the number and function of hematopoietic stem cells (HSCs) and reduce the apoptosis rate, thereby improving the long-term transplantation and homing ability. The article “PGE2 limits effector expansion of tumour infiltrating stem-like CD8+ T cells” also mentions that prostaglandin E2 (PGE2) limits the proliferation and effector function of these cells in tumors through the EP2 and EP4 receptor signaling pathways on CD8 + T cells, thereby promoting the immune escape of cancer. That is, the prior art discloses that PGE2 can inhibit CD8 +T cell proliferation. However, there are currently no reports on the effects of prostaglandins PGE1 or PGE2 on the proliferation of exhausted precursor T cells. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide an application of prostaglandin PGE1 or PGE2 in the preparation of products that promote the proliferation of exhausted precursor T cells.

[0006] A second objective of this invention is to provide prostaglandin PGE1 or PGE2 in the preparation of CD8-promoting prostaglandin B1 or PGE2. + Application in products that promote T-cell proliferation.

[0007] A third objective of this invention is to provide a method for promoting the proliferation of exhausted precursor T cells.

[0008] The fourth object of the present invention is to provide a method for promoting CD8 + Methods for T cell proliferation.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution:

[0010] This invention uses T cells from OT1 and TCF1-GFP double-positive mice (a type of CD8 cell that combines antigen specificity and TCF1 expression visualization markers). + T cells, ideal tool cells for studying exhausted precursor T cells in immunological research, were cultured in activation medium for a period of time. Then, a certain concentration of prostaglandin PGE1 or PGE2 (prostaglandin PGE1 / 2) was added to the culture system. After 8 days of culture, flow cytometry results showed that TCF1 was present in the T cells. + TIM-3 - The significantly increased cell clustering (high TCF1 expression is a marker of Tpex) indicates that adding prostaglandins PGE1 or PGE2 to the culture system can significantly increase the number and proportion of exhausted precursor T cells (Tpex) (Tpex proportion increased 3-fold). Exhausted precursor T cells are CD8+ cells with "stem cell" characteristics. + T cells are capable of self-renewal and differentiation into effector T cells. Simultaneously, PD-1 in T cells... + TIM-3 + Clustering was significantly reduced (TIM3 is CD8) + T cell surface exhaustion markers indicate that the addition of prostaglandins PGE1 or PGE2 to the culture system can also significantly inhibit CD8. +T cell exhaustion level (proportion of exhausted T cells decreased by ≥30%). After discontinuing administration and continuing culture for 6 days, flow cytometry results showed that the number of T cells in the prostaglandin PGE1 or PGE2 treatment group was significantly higher than that in the control group, indicating that adding prostaglandin PGE1 or PGE2 to the culture system for a period of time and then stopping administration can significantly promote T cell proliferation (total cell number increased by ≥30%, cell viability increased by 3-fold). Simultaneously, the CD8+ in the prostaglandin PGE1 or PGE2 treatment group... + Clear T cell clustering with a percentage ≥90% indicates that CD8 cells can be directionally expanded after adding prostaglandin PGE1 or PGE2 to the culture system for a period of time and then stopping the drug administration. + T cells. Therefore, this invention provides a simple and scalable method for TIL expansion, which, by adding prostaglandins PGE1 or PGE2, can maintain / increase the proportion of exhausted precursor T cells and reduce exhaustion levels, significantly enhancing CD8+. + The number of T cells provides technical support for the clinical promotion of tumor-infiltrating lymphocyte therapy for solid tumors.

[0011] Therefore, this invention provides the use of prostaglandin PGE1 or PGE2 in the preparation of products that promote the proliferation of exhausted precursor T cells.

[0012] This invention provides the use of prostaglandin PGE1 or PGE2 in the preparation of CD8-promoting prostaglandin B1 or PGE2. + Application in products that promote T-cell proliferation.

[0013] Furthermore, the method of promoting the proliferation of exhausted precursor T cells involves upregulating TCF1-positive CD8+. + T cell ratio.

[0014] Furthermore, the promotion of CD8 + T cell proliferation is characterized by downregulated TIM3-positive CD8+. + T cell ratio.

[0015] Furthermore, the promotion of CD8 + T cell proliferation inhibits CD8 + T cell exhaustion promotes T cell proliferation and cell viability, and increases CD8+. + The proportion of T cells.

[0016] The present invention also provides a method for promoting the proliferation of exhausted precursor T cells, wherein the method comprises adding prostaglandin PGE1 or PGE2 to an in vitro T cell activation culture system.

[0017] The present invention also provides a method for promoting CD8 +The method for T cell proliferation involves adding prostaglandin PGE1 or PGE2 to an in vitro T cell activation culture system, culturing for 6-10 days, stopping the addition of prostaglandin PGE1 or PGE2, and then culturing for another 4-8 days.

[0018] Furthermore, the final concentration of the prostaglandin PGE1 or PGE2 is 1–10 μM.

[0019] Furthermore, the final concentration of the prostaglandin PGE1 or PGE2 is 5–10 μM.

[0020] Furthermore, the activation culture system is a basic 1640 medium containing 10% v / v FBS, 1‰ w / v IL-2, 1‰ v / v β-mercaptoethanol and 1‰ w / v OVA short peptide.

[0021] Furthermore, the density of the T cells is 1×10⁻⁶. 5 ~5×10 6 cells / mL.

[0022] Furthermore, when adding prostaglandin PGE1 or PGE2, the culture medium is changed and supplemented with the same concentration of prostaglandin PGE1 or PGE2 every 48–72 hours.

[0023] Furthermore, the culture time after adding prostaglandin PGE1 or PGE2 is 8 days.

[0024] Furthermore, the time for stopping the addition of prostaglandin PGE1 or PGE2 during culture is 6 days.

[0025] Furthermore, the product is a culture medium or a drug.

[0026] This invention also provides exhausted precursor T cells and CD8 cells cultured by the above method. + Application of T cells in the preparation of anti-tumor drugs.

[0027] Furthermore, the tumor is a solid tumor, including but not limited to melanoma, non-small cell lung cancer, and colorectal cancer.

[0028] This invention also provides a method for enhancing the anti-tumor function of TILs cells, the method comprising contacting the TILs cells with an effective amount of prostaglandin before reinfusing them into a patient. This method can be applied to the in vitro preparation process of adoptive T-cell therapy to pre-improve the functional state of T cells.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of prostaglandin PGE1 or PGE2 in the preparation of products that promote the proliferation of exhausted precursor T cells. Specifically, by adding prostaglandin PGE1 or PGE2 to a T cell culture system and culturing, highly efficient expansion of exhausted precursor T cells can be obtained, while simultaneously inhibiting CD8. + The degree of T cell exhaustion is such that when prostaglandin PGE1 or PGE2 is discontinued, exhausted precursor T cells can rapidly differentiate into CD8 cells. + T cells. Therefore, using prostaglandin PGE1 or PGE2 in the in vitro preparation process of adoptive T cell therapy can significantly increase CD8+. + Increasing the total number of T cells, raising the proportion of exhausted precursor T cells (Tpex), and reducing the expression of exhaustion markers provide technical support for the clinical promotion of tumor-infiltrating lymphocyte therapy for solid tumors. Attached Figure Description

[0030] Figure 1 The results showed an increase in the number and proportion of exhausted precursor T cells (Tpex) for PGE1 or PGE2. Figure 1 In the table, A represents the results of the cell loss assay; B and C represent the statistical results of the effects of PGE1 or PGE2 treatment on exhausted precursor T cells, respectively.

[0031] Figure 2 To suppress CD8 by PGE1 or PGE2 + The result of T cell depletion.

[0032] Figure 3 The results show that PGE1 or PGE2 promotes T cell proliferation and cell viability.

[0033] Figure 4 CD8 was directionally amplified for PGE1 or PGE2. + The results of T cell analysis. Among them, Figure 4 In the diagram, A represents the result of a cytometer test; B represents the effect of PGE1 or PGE2 treatment on CD8. + Statistical results of the influence of T cells. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Prostaglandin E1 (PGE1): also known as alprostadil, is an organic compound, CAS Registry Number 745-65-3, with the chemical formula C. 20H 34 O5. The structural formula is shown below: .

[0037] Prostaglandin E2 (PGE2): also known as dinoprostone, is a natural prostaglandin, CAS Registry Number 363-24-6, chemical formula C 20 H 32 O5. The structural formula is shown below: .

[0038] Example 1: Prostaglandins PGE1 or PGE2 increase the number and proportion of exhausted precursor T cells (Tpex). I. Experimental Methods 1. Acquisition and activation of mouse spleen T cells This embodiment uses T cells from OT1 and TCF1-GFP double-positive mice (mouses that are double-positive by hybridization of OT1 and TCF1 mice). To obtain mouse spleen T cells, the spleen was first placed in a culture dish containing 2% FBS solution in a sterile operating table, then transferred to a 70 μm filter screen, 2 mL of 2% FBS solution was added, and the cells were gently ground with the bottom of a syringe plunger. The cell suspension was collected into a 15 mL centrifuge tube, and another 3 mL of 2% FBS was added to repeat the grinding process. The suspensions were combined and the volume was adjusted to 5 mL. After centrifugation at 4°C and 1500 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in 1 mL of erythrocyte lysis buffer and lysed at room temperature for 5 min. 2 mL of 2% FBS was added to stop the lysis, and the cells were centrifuged again and the supernatant was discarded. Cells were resuspended in 1 mL of 2% FBS, centrifuged, and the supernatant was discarded. The supernatant was replaced with 1 mL of activation medium (basal 1640 medium containing 10% v / v FBS, 1‰ w / v IL-2, 1‰ v / v β-mercaptoethanol, and 1‰ w / v OVA short peptide). The suspension was filtered through a 40 μm filter into 10 cm culture dishes, and 9 mL of activation medium was added before incubation. The medium was changed every other day. Cells were collected on day 4, centrifuged, resuspended in 1 mL of activation medium, and counted. The cell concentration was adjusted to 1×10⁶ cells / mL. 6 / mL.

[0039] 2. Effects of prostaglandins PGE1 or PGE2 on exhausted precursor T cells Press 5×10 5 Cells were seeded into culture plates and treated with drugs according to their respective groups (control group, 5 μM prostaglandin PGE1 or PGE2, 10 μM prostaglandin PGE1 or PGE2 (final concentration)); the medium was changed and the drugs were administered again every other day. On day 8, the number and proportion of exhausted precursor T cells (Tpex) were detected by flow cytometry.

[0040] II. Experimental Results After adding prostaglandin PGE1 or PGE2 to the culture medium, the flow cytometry results are as follows: Figure 1 As shown, TCF1 in T cells + TIM-3 - The significantly increased cell clustering (high TCF1 expression is a marker of Tpex) indicates that adding prostaglandins PGE1 or PGE2 to the culture system can reduce the expression of exhaustion markers, improve cell function and survival, and significantly increase the number and proportion of exhausted precursor T cells (Tpex), with the proportion of exhausted precursor T cells increasing more than threefold. Exhausted precursor T cells are CD8+ cells with "stem cell" characteristics. + T cells are capable of self-renewal and differentiation into effector T cells, namely, prostaglandin PGE1 or PGE2 maintains CD8. + Stem cells in T cells.

[0041] Example 2: Prostaglandin PGE1 or PGE2 inhibits CD8 + T cell exhaustion I. Experimental Methods 1. The acquisition and activation of mouse spleen T cells are the same as the steps described above.

[0042] 2. Effects of prostaglandins PGE1 or PGE2 on T cells Press 5×10 5 Cells were seeded into culture plates and treated with drugs according to their respective groups (control group, 5 μM prostaglandin PGE1 or PGE2, 10 μM prostaglandin PGE1 or PGE2 (final concentration)); the medium was changed and the drugs were administered again every other day. On day 8 of culture, CD8+ was detected by flow cytometry. + The number and proportion of T cells.

[0043] II. Experimental Results After adding prostaglandin PGE1 or PGE2 to the culture medium, the flow cytometry results are as follows: Figure 2 As shown, PD-1 in T cells + TIM-3 + Clustering was significantly reduced (TIM3 is CD8) + T cell surface exhaustion markers indicate that the addition of prostaglandins PGE1 or PGE2 to the culture system can significantly inhibit CD8. + The degree of T cell exhaustion is defined as a decrease in the proportion of exhausted T cells ≥30%.

[0044] Example 3: Increased CD8 after culture with prostaglandin PGE1 or PGE2 + Number and proportion of T cells I. Experimental Methods 1. Acquisition and activation of mouse spleen T cells This invention uses T cells from OT1 and TCF1-GFP double-positive mice for experiments. To obtain mouse spleen T cells, the spleen was first placed in a culture dish containing 2% FBS solution in a sterile operating table, then transferred to a 70 μm filter screen, and 2 mL of 2% FBS solution was added and gently ground with the bottom of a syringe plunger. The cell suspension was collected into a 15 mL centrifuge tube, and another 3 mL of 2% FBS was added to repeat the grinding process. The suspensions were combined and the volume was adjusted to 5 mL. After centrifugation at 4°C and 1500 rpm for 5 min, the supernatant was discarded, and the pellet was resuspended in 1 mL of erythrocyte lysis buffer and lysed at room temperature for 5 min. Lysis was terminated by adding 2 mL of 2% FBS, centrifuged again, and the supernatant was discarded. Cells were resuspended in 1 mL of 2% FBS, centrifuged, and the supernatant was discarded. The supernatant was replaced with 1 mL of activation medium (basal 1640 medium containing 10% v / v FBS, 1‰ w / v IL-2, 1‰ v / v β-mercaptoethanol, and 1‰ w / v OVA short peptide). The suspension was filtered through a 40 μm filter into 10 cm culture dishes, and 9 mL of activation medium was added before incubation. The medium was changed every other day. Cells were collected on day 4, centrifuged, resuspended in 1 mL of activation medium, and counted. The cell concentration was adjusted to 1×10⁶ cells / mL. 6 / mL.

[0045] 2. Effects of prostaglandins PGE1 or PGE2 on T cells Press 5×10 5 Cells were seeded into culture plates and treated according to their respective groups (control group, 10 μM prostaglandin PGE1 or PGE2 (final concentration)). The medium was changed, cells were counted, and the treatment repeated every other day. On day 8, cells were collected, centrifuged, and treated again without further administration. The cells were resuspended in 1 mL of activation medium and counted using trypan blue. The cells were then seeded into culture plates. The medium was changed and cells were counted every other day for a total of 14 days. CD8+ was detected by flow cytometry. + The number and proportion of T cells.

[0046] II. Experimental Results Trypan blue cell count results as follows Figure 3 As shown, the number of T cells in the drug-treated group was significantly higher than that in the control group, indicating that adding prostaglandin PGE1 or PGE2 to the culture system for a period of time and then stopping the drug treatment can significantly increase T cell viability and promote T cell proliferation (total cell count increased by ≥30%, cell viability increased by 3 times). Further, the flow cytometry results are as follows... Figure 4 As shown, CD8 in the drug-treated group + T cell clusters were clearly defined and accounted for ≥90% (proliferation rate >10-fold), while the control group had CD8. +The low T-cell content indicates that the addition of prostaglandin PGE1 or PGE2 to the culture system for a period of time followed by discontinuation of drug administration can lead to the targeted expansion of CD8 cells. + T cells. Specifically, to relieve the effects of prostaglandins PGE1 or PGE2 on CD8. + Maintaining the "stem nature" of T cells allows depleted precursor T cells to exert stronger functions and exhibit a stronger tumor immune effect through rapid proliferation.

[0047] In summary, this invention provides a simple and scalable method for TIL amplification. By adding prostaglandins PGE1 or PGE2, the proportion of exhausted precursor T cells can be maintained / increased while reducing exhaustion levels, and CD8 levels can be significantly increased. + The number of T cells provides technical support for the clinical promotion of tumor-infiltrating lymphocyte therapy for solid tumors.

Claims

1. Use of prostaglandin PGE1 or PGE2 for the preparation of a product for promoting the proliferation of exhausted T cells.

2. Use of prostaglandin PGE1 or PGE2 in the manufacture of a product for promoting CD8 + T cell proliferation.

3. Use according to claim 1, characterized in that, Said promoting proliferation of pre-exhaustion T cells is upregulating TCF1 positive CD8 + T cell ratio.

4. Use according to claim 2, characterized in that, The promoting CD8 + T cell proliferation is down-regulating TIM3 positive CD8 + T cell proportion.

5. A method of promoting proliferation of exhausted precursor T cells, characterized in that, The method is to add prostaglandin PGE1 or PGE2 in an in vitro T cell activation culture system.

6. A method of promoting CD8 + T cell proliferation, characterized in that, The method is to add prostaglandin PGE1 or PGE2 in an in vitro T cell activation culture system, stop adding prostaglandin PGE1 or PGE2 after 6-10 days of culture, and then culture for another 4-8 days.

7. The method of any of claims 5 or 6, wherein, The final concentration of the prostaglandin PGE1 or PGE2 is 1-10 μM.

8. The method of any of claims 5 or 6, wherein, The activation culture system is a basic 1640 culture medium containing 10% v / v FBS, 1‰ w / v IL-2, 1‰ v / v β-mercaptoethanol and 1‰ w / v OVA short peptide.

9. The method of any of claims 5 or 6, wherein, The density of the T cells is 1 x 10 5 ~5 x 10 6 cells / mL.

10. Use according to claim 1 or 2, characterized in that, The product is a culture medium or a pharmaceutical preparation.

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