JPS6260106B2 - - Google Patents

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for removing cells with immunosuppressive functions from body fluids. Prior art and its problems Suppressor T cells, a type of immunosuppressive cell, exist in the blood at a low rate even in healthy people, and prevent reactions with self-antigens caused by autoantibody production to maintain the body's life. contributes to However, in patients with diseases where the immune system is abnormal, such as cancer patients or immunodeficiency patients, these suppressor T cells are abnormally enhanced and suppress the functions of cells that promote antibody production, such as helper T cells and B cells. . For this reason, these patients have a decline in not only humoral immunity but also cellular immunity, and as a result, the body's defenses against non-self or modified self-antigens do not work, accelerating the progression of the disease. This will worsen the symptoms. Conventionally, chemotherapy, radiation therapy, surgical therapy, and the like have been used to prevent the enhancement of suppressor T cells, but these treatments also damage other cells, so they cannot fully achieve their purpose. Although it is possible to remove immunosuppressive cells by separating them from the blood, conventional techniques also remove other useful cells. OBJECTS OF THE INVENTION An object of the present invention is to provide a method and device for selectively removing suppressor T cells, which are immunosuppressive cells. A method for removing suppressor T cells that achieves the above objective involves bringing a body fluid containing suppressor T cells into contact with a carrier on which an anti-suppressor T cell antibody is immobilized, and allowing the carrier to capture and remove the suppressor T cells in the body fluid. It is. In addition, the anti-suppressor T cell antibody is obtained by absorbing immobilized serum that is immunoreactive against antigens with immunosuppressive ability with red blood cells, and then separating it from this serum by a 1/3 ammonium sulfate saturation method. - Preferably of the globulin fraction. Furthermore, what achieves the above object is a suppressor T cell removal device comprising a container through which a body fluid containing suppressor T cells can flow, and a carrier on which an anti-suppressor T cell antibody contained in the container is immobilized. Further, in the present invention, the carrier is a fibrous material, a tubular material, a granular material, a spherical material, or a film-like material, and the material thereof is activated carbon, a polysaccharide, a protein, or a synthetic resin. Furthermore, antibodies can be obtained by absorbing inactivated serum, which is immunoreactive against antigens with immunosuppressive ability, with red blood cells, and then extracting the γ-globulin fraction from this serum using the 1/3 ammonium sulfate saturation method. It is something. DETAILED DESCRIPTION OF THE INVENTION FIG. 1 shows an apparatus for removing immunosuppressive cells from body fluids, which contains a carrier 2 in a container 1. This container 1 has a blood inlet 3 and a blood outlet 4 formed at both ends, and filters 5 and 5 are provided at the inlets and outlets, respectively. These filters 5,
5 prevents the carrier 2 from flowing out of the container 1. The carrier 2 is a solidified product of a cell membrane antigen of immunosuppressive cells, that is, an antibody that specifically reacts with the cell membrane as an antigen, and is formed of a fibrous material, a tubular material, a granular material, or a spherical material. This carrier 2 is
It is preferable to fill the container to such an extent that body fluids such as blood passing through the container can efficiently contact the antibody. The material of the carrier 2 having these shapes is as follows:
Activated carbon, polysaccharides, proteins, synthetic resins, etc. are suitable, and examples of polysaccharides include cellulose, agarose, fibrin, and collagen. Examples of synthetic resins include polystyrene, polyvinyl chloride, polyvinyl alcohol, polyvinylpyrrolidone, polycarbonate, nylon, silicone, and polyoxymethylene. Note that these materials are not limited to the entire carrier 2, and may be formed only on the surface to which the antibody is attached. The antibody immobilized on carrier 2 is an anti-suppressor T cell antibody when the immunosuppressive cells to be removed are suppressor T cells. This anti-suppressor T cell antibody is obtained by immunizing a given animal with cells that have been confirmed to have immunosuppressive ability as an antigen, separating and inactivating the serum from the post-immunized blood, and then absorbing the serum with red blood cells. This is a γ-globulin fraction extracted by the 1/3 ammonium sulfate saturation method. This antibody can be immobilized on a carrier using a known method using, for example, glutaraldehyde. Therefore, the container 1 containing the carrier 2 is
As shown in the figure, the blood inflow port 3 is connected to the human body 7 or the blood storage device through the drip chamber 6, and the blood outflow port 4 is connected to the human body 7 or the blood storage device through the drip chamber 8. Connected. Although not shown, this device is provided with a mechanism for supplying an anticoagulant such as heparin into the blood to prevent blood from coagulating. Specific Functions and Effects of the Invention In the apparatus for removing immunosuppressive cells in body fluids configured as described above, blood is returned to the body through the container 1 by the operation of the blood pump 9. Inside container 1,
Since a predetermined antibody is immobilized on the carrier 2, it specifically reacts with the cell membrane antigen of immunosuppressive cells and selectively captures these cells. In this case, cells other than immunosuppressive cells are not captured by this antibody and are returned to the body. Furthermore, since immunosuppressive cells are removed by immobilizing antibodies on the carrier 2, no operational problems such as blockage of the channel system or reduction in throughput occur. The effects of the present invention as described above were confirmed by the experiments shown below. First, suppressor T cells used as antigens in this experiment were obtained as follows. 200 ml of blood was collected from a human elbow vein, layered with Fico-Conley specific gravity solution, and heated at 2000γpm for 20 minutes.
After centrifugation for a minute, the intermediate lymphocyte layer is separated and suspended in physiological saline at 2×10 ⁶ cells/ml. This suspension was mixed with 2% neuraminidase-treated sheep red blood cells and centrifuged at 400 x g for 5 minutes.
This is left to stand at 4°C for 1 hour. Here, the lymphocytes are gently suspended, applied again to the Fico-Conrey density solution, and centrifuged at 2000 γpm for 20 minutes.
After hemolyzing sheep red blood cells in the separated rosette-forming cells with a 0.83% ammonium chloride solution, the cells are washed with physiological saline (1000 γpm, 10 minutes, 3 times). The T cells obtained by this procedure are prepared by adding physiological saline to a concentration of 2×10 ⁶ cells/ml. Next, an equal amount of 1% rabbit anti-human IgG-antibody-sensitized chicken red blood cells is mixed therein, and the mixture is allowed to stand at 4°C for 24 hours. This was layered on Ficoll-Conrey density solution, centrifuged at 2000 γpm for 10 minutes to remove the precipitated rosette-forming cells, then chicken red blood cells were hemolyzed with 0.83% ammonium chloride solution, and washed with physiological saline (1000 γpm, 3 times for 10 minutes). In order to examine the immunosuppressive ability of the suppressor T cells obtained by such operations, the following measurements were performed. Lymphocytes obtained from another healthy person were separated into B cells and T cells by Fuico-Conrey gravity centrifugation, and the number of cells was determined to be 2 x 10 ⁵ cells/ml and 8 x 10 ⁵ cells/ml.
Add 20% FCS (fetal bovine serum) to ml.
Prepared with RPMI1640-HEPES and used as a control cell group. In addition, when adding isolated T cells to healthy human lymphocytes, B cells are 2×10 ⁵ cells/ml, T cells are 2×
10 ⁵ cells/ml, and the isolated T cells were prepared in the above medium to a concentration of 8×10 ⁵ cells/ml to form a test cell group. Add 10 μg of PWM (porkweed mitogen) to 1 ml each of control cell group and test cell group,
Culture for 7 days at 37°C in a 5% _CO2 incubator. These control and test cell groups were fluorescently stained with rabbit anti-human Ig (IgG + IgA + IgM) serum and washed with MEM medium (1000 γpm, 5 minutes, 3
After that, more than 500 cells were observed under a fluorescence microscope, and cells with specific fluorescence in the cytoplasm were counted as positive, and the immunosuppressive ability was determined from the appearance rate of Ig-bearing cells. As a result, the number of antibody-producing cells (Ig-bearing cells) was 6% in the control cell group, while it was as low as 1.5% in the test cell group, indicating that the isolated T cells had immunosuppressive ability. On the other hand, suppressor T cell antibodies were obtained as follows. Rabbits were immunized twice at 10-day intervals using 1×10 ⁷ lymphocytes with confirmed immunosuppressive ability as an antigen.
Seven days after the final immunization, whole blood was collected, and the separated serum was collected at 56
It was inactivated by treatment at ℃ for 30 minutes. This was absorbed using an equal amount of human AB type red blood cells at 37°C for 1 hour, and then similarly absorbed using human B cells. This rabbit anti-human suppressor T cell serum was subjected to a 1/3 saturation method using ammonium sulfate to obtain a γ-globulin fraction, which was used as a rabbit anti-human suppressor T cell antibody (hereinafter abbreviated as antibody). Next, in order to examine this antibody, the following cytotoxicity test was performed. The aforementioned suppressor T cells and non-suppressor T cells were each prepared at 2500 cells/mm ³ and added to a microtest plate (#3034, Falcon, trade name) in an amount of 1 μ using a microsyringe. Next, add 1μ of the above antibody and incubate at 22°C for 45 minutes. Then add 5μ of rabbit complement and heat at 22°C.
Incubate for 90 minutes. Add 2μ of eosin to this and further add formalin (PH7.0) for staining and fixation. Then, as a result of counting live cells and dead cells under a microscope and calculating the dead cell rate, suppressor T
The cell group had 95% dead cells, while the non-suppressor T cells had 5% dead cells. From the above results, it was confirmed that this antibody specifically reacts with and kills suppressor T cells. In addition, in order to examine the cytotoxicity of this antibody, we tested rabbit anti-human suppressor T using a microtest plate.
A human lymphocyte cytotoxicity test using cell antibodies was conducted. As a result, as shown in Table 1, this antibody
It showed cytotoxicity up to a 2500-fold dilution and was found to have a high titer.

【table】

[Table] Next, examples of the present invention will be described. The antibody used in the above-mentioned experimental example was immobilized on a carrier made of nylon particles with an average particle diameter of 250 μm by a known method using glutaraldehyde. This carrier was placed in a container at a filling rate of 40%, and a removal device (capacity 10 ml) as shown in FIG. 2 was assembled. Next, a suppressor T cell removal test was conducted using 30 ml of blood collected from a gastric cancer patient. Here, the container used was one that had been sufficiently washed with physiological saline and had no residual harmful substances such as pyrogens and bacteria. Blood was circulated through the container at a flow rate of 120 ml per hour for 2 hours, and suppressor T cells in the blood before and after the test were measured by the double rosette method. As a result, the proportion of suppressor T cells in lymphocytes was 20% before the experiment, but it was found to have decreased to the normal range of 4% after the experiment. In addition, the appearance rate of antibody-producing cells due to PWM stimulation was measured by cytoplasmic immunoglobulin fluorescence staining before and after the experiment. As a result, it was confirmed that before the implementation, the rate was low at 2.5%, but after the implementation, it returned to 7%, which is within the normal range. As is clear from the above results, according to the present invention, since an antibody that specifically reacts with the cell membrane antigen of immunosuppressive cells is used, the cells can be selectively and significantly removed, and the immune system is not involved. It is of great significance in treating certain diseases.

[Brief explanation of the drawing]

FIG. 1 is a sectional view of a removing device showing an embodiment of the present invention, and FIG. 2 is an explanatory diagram of a system incorporating the removing device. 1... Container, 2... Carrier.

Claims (1)

[Scope of Claims] 1. A suppressor characterized in that a body fluid containing suppressor T cells is brought into contact with a carrier on which an anti-suppressor T cell antibody is immobilized, and the suppressor T cells in the body fluid are captured by the carrier and removed. T cell removal method. 2. The anti-suppressor T cell antibody is obtained from immobilized serum that is immunoreactive with immunosuppressive antigens after absorption with red blood cells.
The suppressor T cell removal method according to claim 1, which is a γ-globulin fraction separated by a 1/3 ammonium sulfate saturation method. 3 A container through which body fluid containing suppressor T cells can flow, and an anti-suppressor T cell housed in the container.
A suppressor T cell removal device comprising a carrier on which a cell antibody is immobilized. 4. The suppressor T cell removal device according to claim 3, wherein the carrier is a fibrous material, a tubular material, a granular material, a spherical material, or a membranous material. 5. The suppressor T cell removal device according to claim 3, wherein the carrier is activated carbon, polysaccharide, protein, or synthetic resin. 6. The anti-suppressor T cell antibody is obtained from immobilized serum that is immunoreactive with immunosuppressive antigens after absorption with red blood cells.
The suppressor T cell removal device according to any one of claims 3 to 5, which is a γ-globulin fraction separated by a 1/3 ammonium sulfate saturation method.