JPH0436656A - Separating material for activated t cell and/or helper t cell - Google Patents

Abstract

PURPOSE:To allow simple sepn. without depending on monoclonal antibodies by forming the above separating material of a water-insoluble solid having a basic functional group on the surface. CONSTITUTION:The basic functional group is nitrogenous arom. ring groups, such as primary to tertiary amino groups, quaternary ammonium group, pyridyl group and imidazoryl group, and the amt. thereof on the surface of the water- insoluble solid is generally 0.2 to 10% basic nitrogen atom content per weight of the dry state. Activated T cells and/or helper T cells are captured in the separating material when a lymphocyte suspension is passed in a separator packed with this separating material. The liquid contg. the remaining components flows out of the outlet of the separator. The simple sepn. is executed in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention is directed to helper T, which is one of the components constituting lymphocytes.
The present invention relates to a separation material for separating cells or activated T cells. The separation material described above can be used for diagnosis of diseases involving immune abnormalities, extracorporeal blood circulation treatment, and the like.

(Prior art and its problems) It is well known that lymphocytes play a central role in the immune system of living bodies. Lymphocytes are B cells, T cells,
Non-B non-T cells are broadly divided into three subpopulations, and functionally important T cells are further divided into helper TwU cells, helper TwU cells,
They are classified into a subset of suppressor T cells. Furthermore, T@ cells are differentiated into activated T cells and non-activated T cells by binding with anti-HLA-DR antibodies, and each type includes helper T cells and suppressor T@ cells classified into the aforementioned subsets. .

Therefore, the activated T cells and/or helper TMJ cells to be separated in the present invention refer to activated suppressor T cells, activated helper T cells, and/or non-activated helper T cells.

In recent years, activated T@cysts and rheumatoid arthritis (Arthri
tis Rheum 31:834-843.19
88), systemic lupus erythematosus (J, Rheumat.
ol 15:946-951.1988) Scleroderma (A
nn Rheum Dis 49:40-45.19
90), Multiple Sclerosis (Immunology To
day 10:104-107.1989)HTL
V-1-associated Myelopath
y (RAM) (Ann Neurol 24:28
0-282, 1988), type 1 diabetes mellitus (N.

Engl, J. Med, 306:785-78
7.982) has been reported to be deeply involved in the pathology of diseases such as. It is also known that the ratio of the number of helper T cells to the number of suppressor T cells is involved in the pathology of various autoimmune diseases.

If it becomes possible to easily separate activated T cells and/or helper TwJ cells in diseases involving immune abnormalities such as those described above, it will provide powerful clues for diagnosis and extracorporeal blood circulation therapy. However, so far,
There have been few reports of materials and methods for easily separating activated T cells and/or helper T cells.

Regarding the conventional technology, separation, recovery, and removal of leukocytes using fibers (JP-A-54-119013, JP-A-60-1
93468, WO37-05812, etc.) and separation, collection, and removal of lymphocytes (Japanese Patent Application Laid-open No. 55-6452, etc.), separation, collection, and removal of B1B cells and T cells by materials (Japanese Patent Application Laid-Open No. 55-1999)
8-170717, JP-A-59-216584, etc.), but none of them disclose anything regarding the separation of lymphocyte subsets. In addition, theoretically, by immobilizing the corresponding monoclonal antibodies such as Leu HLA-DR for activated T cells and 0KT4 and Leu3a for helper T cells on an insoluble carrier, activated T cells and/or Alternatively, a separation material for separating helper T cells can be obtained. However, with this method, monoclonal antibodies are expensive and difficult to stably obtain in large quantities; denaturation and inactivation of antibodies during immobilization on insoluble carriers and sterilization; and trace amounts of antibodies eluted during extracorporeal blood circulation treatment. There are many problems such as the risk of

(Objective of the Invention) An object of the present invention is to provide a separation material for easily separating activated T cells and/or helper T cells from lymphocytes without using monoclonal antibodies.

(Structure and Effect of the Invention) The present inventors have developed a method to selectively activate activated T cells and/or helper T cells and B cells among lymphocytes, which are thought to be deeply involved in the pathology of various diseases. As a result of developing a separation material for adhesion and separation, we have finally completed the present invention.

That is, the present invention is a material for separating activated T cells and/or helper T cells, which is made of a water-insoluble solid having a basic functional group on its surface.

In the present invention, a water-insoluble solid refers to a solid that is substantially insoluble in water as a whole, in which a water-soluble low-molecular substance or a high-molecular substance is bonded to a water-insoluble solid. Including those that are designed not to dissolve in water. A measure of being substantially insoluble in water is passing an eluate test using water as an extraction solvent, which is prescribed for various medical devices.

The separation material of the present invention needs to have a basic functional group on the surface of the water-insoluble solid. The reason is not necessarily clear, but in normal lymphocyte suspensions, from phosphate buffers to blood, basic functional groups have a positive charge and generally act to statically hold various negatively charged cells, especially lymphocytes. It is thought that it is electrically attracted. Among various lymphocytes, activated T cells and/or helper T cells selectively adhere to each other because activated T cells and/or helper T cells have a negative charge compared to other lymphocyte components. It is presumed that the amount is large and that the adhesiveness itself to the solid surface is high.

The basic functional group referred to in the present invention includes a primary amino group,
Examples include secondary amino groups, tertiary amino groups, quaternary ammonium groups, nitrogen-containing aromatic ring groups such as pyridyl groups and imidazolyl groups.

In the separation material of the present invention, it is preferable that the surface of the water-insoluble solid has a hydrophilic group as well as a basic functional group. By having a hydrophilic group, the surface of the water-insoluble solid becomes more philic, suppressing the adhesion of non-specific lymphocyte subsets, and selecting for selective adhesion of activated TMJ cells and/or helper T cells. Improves sex.

Examples of the hydrophilic group in the present invention include a hydroxyl group, an amide group, and a polyethylene oxide chain.

In the separation material of the present invention, it is also preferable that the surface of the water-insoluble solid has an acidic functional group as well as a basic functional group.

Since the acidic functional group has a negative charge in normal lymphocyte suspension, adhesion of non-specific lymphocyte components is suppressed,
Selectivity in selective adhesion of activated T cells and/or helper T cells is improved.

Examples of acidic functional groups in the present invention include carboxyl groups, sulfonic acid groups, and phosphoric acid groups.

In the lymphocyte subset separation material of the present invention, the preferred range of the amount of basic functional groups on the surface of the water-insoluble solid is influenced by the types and amounts of coexisting hydrophilic groups and acidic groups; is 0.2 to 10% by weight as the content of basic nitrogen atoms in the basic functional groups per dry weight in the surface layer of the separation material. If the total amount of the above basic nitrogen atoms is less than 0.2% by weight, the adhesiveness of activated T cells and/or helper T cells to the separation material will be small, and separation of these cells by adhesion will not be performed efficiently. It ends up. In addition, if the content of the basic nitrogen atoms is greater than 10% by weight, non-specific lymphocyte components will adhere to the target, so that selection in the isolation of the desired activated T@cells and/or helper T cells may occur. Sexuality becomes low. The content of the above basic nitrogen atoms is more preferably in the range of 0.5 to 5% by weight, and even more preferably in the range of 1 to 3% by weight. In addition, when a large amount of hydrophilic groups or acidic groups are present on the surface of the water-insoluble solid, the preferable range of the above basic nitrogen atom content is as compared to when a large amount of hydrophilic groups or acidic groups are not present. , the basic nitrogen atom content increases.

The surface layer of the separation material in the present invention refers to the surface area of the separation material that can interact with lymphocytes.In the separation material of the present invention, the distribution state of basic functional groups on the water-insoluble solid surface is as follows: It may be uniformly distributed or non-uniformly distributed. Micro-heterogeneity distribution may be preferable because it suppresses non-specific adhesion of lymphocyte components.

As for the shape of the separation material of the present invention, any known shape such as particulate, fibrous, hollow fiber, membrane, porous membrane, etc. can be used.

In the case of particulate separation material, it is easier to fill the separation material evenly when filling a specific container to create a separator.
Therefore, it is preferable in that precise separation with little variation becomes easy. Regarding particle size, 30 to 600 μm
is preferred.

In the case of fibrous separation materials, they are easy to handle, and when filled into a specific container to create a separator, they generally have a large porosity and a small pressure loss, so separation can be performed quickly. preferable. As for the aggregate state of the fibers, a woven fabric or a non-woven fabric is preferable to a cotton-like fiber mass because it is easier to uniformly fill the fibers and the pressure loss can be further reduced. In particular, when the diameter of the fibers is small, a nonwoven fabric shape is preferable. Furthermore, the fiber diameter is preferably 1 to 60 μm. If the diameter of the m group is less than 1 μm, non-specific lymphocyte subsets will adhere to a large number, resulting in low selectivity in separating the target activated Ti1 cells and/or helper T cells. On the other hand, the fiber diameter is 60 μm
If it exceeds , the surface area of the separation material per unit volume will be too small and the separation efficiency will become low. A more preferable range of fiber diameter is 1.5 to 15 μm, and even more preferably 3 to 8 μm.

In the case of a porous membrane-like separation material, in addition to the adhesion effect of lymphocytes to the material surface, it is possible to utilize the sieving effect due to the pore size of the porous material, which is preferable. The pore diameter of the porous portion is preferably 30 to 60 μm.

The separation material of the present invention can be obtained by introducing basic functional groups, hydrophilic groups, acidic groups, etc. as necessary, into the surface of various known water-insoluble solids through chemical reactions, or by separately manufacturing materials having these groups. The water-insoluble solid is then manufactured by a method such as physically covering the surface of the water-insoluble solid by coating or the like.

As a specific method for the above chemical reaction, active groups for chemical reactions are introduced as necessary on the surface of the water-insoluble solid, and basic functional groups, woody groups and acidic groups are introduced as necessary. Examples include a method of coupling a compound having a group, etc., and a method of surface graft polymerization of a compound having a basic functional group.

The separation material of the present invention is generally used as a separator by filling it into a suitable container having a population and an outlet for the lymphocyte suspension.

When the lymphocyte suspension is passed through the separator containing the separation material of the present invention thus obtained, activated T cells and/or helper T cells are captured by the separation material, and the liquid containing the remaining components is separated into It flows out from the outlet of the container. As a method for bringing the lymphocyte suspension into contact with the separation material of the present invention, the above-mentioned chromatographic operation is preferred, but a method of immersing the separation material of the present invention in the lymphocyte suspension may also be considered. The operating temperature when performing lymphocyte suspension treatment using the separation material of the present invention may be any temperature that does not damage cell components, and is preferably from room temperature (15 to 25°C) to about 37°C.

When separating activated T cells and/or helper T cells from a lymphocyte suspension using the separation material of the present invention, after bringing the lymphocyte suspension into contact with the separation material of the present invention, the surface is coated with an acidic functional It may be preferable to contact a water-insoluble solid with groups.

When analyzing lymphocyte components by binding a fluorescent substance-conjugated antibody to a human lymphocyte suspension that has passed through the separation material of the present invention, the lymphocytes that have passed through the separation material of the present invention are Antibodies are non-specifically adsorbed,
Analysis of lymphocyte components may become difficult.

In such cases, nonspecific adsorption of antibodies to lymphocytes can be eliminated by subsequently bringing the lymphocytes that have passed through the separation material of the present invention into contact with a water-insoluble solid having an acidic functional group on its surface. When a water-insoluble solid having an acidic functional group on its surface is used in combination with the separation material of the present invention, the water-insoluble solid having an acidic functional group is placed together with the separation material of the present invention in one separation downstream of the separation material. It may be filled in a container, or it may be filled in a separate container and connected in series downstream of the separator made of the separation material of the present invention.

In the present invention, the lymphocyte suspension refers to lymphocytes suspended in a physiological fluid such as physiological saline, phosphate buffer, or plasma, and human or animal peripheral blood or blood is separated by centrifugation or the like. It also includes fractions containing lymphocytes and lymph fluid obtained by

For peripheral blood, etc., it is common to add anticoagulants such as citric acid and heparin.

When a lymphocyte suspension is separated using the separation material of the present invention, activated TllllI cells and/or helper T cells selectively adhere to and are captured by the separation material of the present invention. Adhering cell components can also be detached from the separation material and recovered by an appropriate method.

Activated T cells and/or helper T cells that have adhered to the separation material can be detached and recovered by flowing physiological fluid vigorously into the separator, by flowing it while applying vibration to the separator, or by removing it from the separator. This is carried out in the form of obtaining a suspension of detached cell components by removing the separation material and shaking it vigorously in a physiological fluid.

The present invention will be explained in more detail below using Examples.

(Example 1 and Comparative Example 1.2) 2-Hydroxyethyl methacrylate (HEMA)
Homopolymer HE-0, copolymer HE-0 of HEMA and diethylaminoethyl methacrylate (DEAEMA)
20 (20 represents the content mol% of DEAEMA units in the copolymer), and HEMA and methacrylic acid (MAA
) copolymer HA-20 (20 is the content of MAA units in mol % in the copolymer) was synthesized by ordinary solution radical polymerization.

The polymerization conditions included azoisobutyronitrile (AI
A polymerization reaction was carried out at 60° C. for 8 hours in the presence of 1/200 mol/β (BN). The content of basic nitrogen atoms in the obtained polymer was determined by elemental analysis. A nonwoven fabric (10
0 g/m2 fabric weight) was cut into a circle with a diameter of 13 mm, and after soaking it in a 0.1% ethanol solution of the above polymer, the excess polymer solution absorbed by the nonwoven fabric was squeezed out, and finally dried. Dry with air.

The surface-coated nonwoven fabric obtained in this way was used as a separation material, and it was used as a filter holder (Shibata Kagaku Kikai Kogyo Co., Ltd.).
A separator was created by setting it in a

In Example 1, two sheets of HE-20 nonwoven fabric (population side) and two sheets of HA-20 nonwoven fabric (outlet side) were stacked and set, and in Comparative Example 1, one HE-0 nonwoven fabric was set. In Comparative Example 2, four sheets of HA-20 nonwoven fabric were set.

Peripheral blood collected from patients (patients with rheumatoid arthritis, patients with scleroderma, HTLV
A total of 10 blood samples from patients with -I-associated Myelopathy, carriers of HTLV-I, and patients with polyarteritis nodosa) (10%
Citric acid addition) was flowed at room temperature using a syringe pump at a rate of 0.6 mj 27 min.

Analysis of lymphocyte components in blood before and after flowing it into a separator
Fluorescein 1sothiocyana
CD4 antibody conjugated with te (FITC), FITC
CD8 antibody conjugated with phycoerythr
HLA-DR antibody (PE)-conjugated antibody (
The above antibodies are manufactured by Coulter Immunology.
After adding hemolytic agent (manual from Coulter) to the blood sample and incubating at 4°C for 30 minutes,
After washing thoroughly, the obtained cells were suspended in a phosphate buffer, and then transferred to a flow cytometer (FACStar).

(manufactured by Becton Dickinson).

Table 1 shows changes in the contents of various cell components before and after the separators when the blood collected from the patient was passed through the separators of Example 1 and Comparative Examples 1 and 2.

ft O, CD4+, CD8+ and DR+ in Table 1
have the following meanings.

DR”: Anti-HLA-DR antibody positive cells (representing activated T cells and other cell components) CD4”:
CD4 antibody positive cells (representing helper T cells and other cell components) CD8”:
CD8 antibody-positive cells (representing suppressor T cells and other cellular components) In Example 1, the ratio of CD4 antibody-positive cells to CD8 antibody-positive cells was significantly decreased compared to helper T cells or suppressor T cells. It can be seen that it selectively adheres to the separation material. Furthermore, the ratio of anti-HLA-DR antibody-positive cells was also significantly reduced, indicating that activated T cells were selectively adherent. Furthermore, the ratio of CD4 antibody-positive cells to CD8 antibody-positive cells among anti-HLA-DR antibody-positive cells was also significantly reduced, indicating that activated suppressor T! B
IW! It can be seen that activated helper T cells are selectively adherent compared to .

On the other hand, in Comparative Example 1.2, there was no significant change as seen in Example 1, indicating that selective adhesion did not occur. In addition, when comparing the components of lymphocytes in 10 blood samples from patients and 10 blood samples from healthy individuals, anti-HLA-DR antibody-positive lymphocytes (activated Tl1l
fI cells, etc.) and C in anti-HLA-DR antibody-positive cells
It can be seen that the ratio of D4 antibody-positive lymphocytes (helper T cells, etc.) to CD8 antibody-positive lymphocytes (suppressor T cells, etc.) is significantly higher in patients.

(Effects of the Invention) By using the separation material of the present invention, activated T cells and/or helper T cells can be selectively separated from a lymphocyte suspension with a simple operation.

The separation material of the present invention provides a powerful means for diagnosis of various diseases involving immune abnormalities, extracorporeal blood circulation therapy, and basic research on immunity.

Hirijin Patent Attorney Shuntetsu Sasaki

Claims (1)

[Claims] A material for separating activated T cells and/or helper T cells, comprising a water-insoluble solid having a basic functional group on its surface.