DE3913438A1 - METHOD FOR PRODUCING AN IMMUNITY MEMORY CELL SUSPENSION - Google Patents

Abstract

The suspension is prepared by (a) separating T cells from cells collected from an immunity sensitized individual immunized against a particular pathogen, (b) mixing the separated cells with an antibody to a histocompatibility antigen specific to a prescribed species of cells to produce Ag-Ab complexes with such cells, (c) adding a blood serum so that complement binder to the complexes and thereby destroys the cells having said antigen, (d) adding to the persisting cells an antibody to a histocompatibility antigen specific to immunity memory cells and (e) separating from the resultant cells the immunity memory cells which have the histocompatibility antigen specific thereto. The cells may be cultured and subcultured successively to establish a continuous line of cells originating in the separated immunity memory cells. They may be useful in the prevention and treatment of infectious diseases or malignant tumours.

Description

The present invention relates to a Process for producing and cultivating a Immunity memory cell suspension, which for the Prophylaxis and / or treatment of an infectious Disease or malignant tumor, typically Cancer, is useful. The invention relates continue to an immunity memory cell suspension, which is produced by such a method.

A human or an animal that one day has gone through viral or bacterial disease and has responded to a particular type of antigen exhibits a striking defensive response against that Antigen on if the individual is a second time Stimulation with the same type of antigen is subjected. This defensive response is that Result of immunity conferred on the individual against this particular disease and is commonly "secondary immune response" or "anamnestic response" of the organism called the antigen. It is well known that such an immune response is the result of excessive proliferation of lymphocytes that is in the Response of the organism to the stimulation with the Antigen that contributed to the onset of the disease has been involved. The lymphocytes, so called Response to antigen stimulation will be developed in general called "immunological memory cells".  

The generated in the body of an organism immunological memory cells are one in the Lymph and occurring in the blood and through the vessels of the Circulatory system of the organism circulating Lymphocyte species. The lymphocytes of the circulatory system immigrate to the organism's thymus where it develop into T lymphocytes or T cells that Give the organism immunity. These become immunologically competent T cells known as sometimes "thymus dependent" or "thymus-derived" cells and are in large scale in almost all peripheral tissues of the Distribute circulatory tissue of an organism.

As for the immunity memory cells that look in the way Lymphocyte-containing T cell population included are concerned, there are references in the state of the Technique that the cells are only conceptual (conceptually) as "immunological" memory cells be recognized. So far it has no practical Attempts given such cells or at least any fraction containing such cells from which To isolate or isolate tissues of an organism extract and the resulting Immunity memory cells or those cells containing fraction in a practical use State or collect in vitro. Neither is one yet positive outlook for using immunological Memory cells for prophylaxis and / or treatment an infectious disease or malignant tumor given or is such on a realistic Basis has been proposed, nor are any clinical and even experimental trials regarding the practical use of such  immunological memory cells seriously undertaken been.

The present invention relates to a method for Detach immunity memory cells directly from a tissue of an organism with a significant high efficiency. Such an inventive The method depends on its effectiveness specific characteristics and attributes of Immunity memory cells that affect the organism confer cell-mediated immunity. The present The invention also relates to the provision a method of cultivating Immunity memory cells through a variety of Generations to create a continuous, established To provide cell line by the Immunity memory cells that come from the tissue of a Organism have been separated.

The present invention is further concerned with Providing a Immunity memory cell suspension coming from the tissue of an immunosensitive individual in accordance with of the present invention or from any Immunity memory cells that are otherwise from the Tissues of an individual are made becomes.

According to a first embodiment of the present invention is a method for Prepare an immunity memory cell suspension for Provided with cells that are essentially consist of T cells, one of the cells immunosensitized individual with a certain pathogen has been immunized  an antibody is added to the separated cells with one for a specific cell type specific histocompatibility antigen is reactive, the more antigen-antibody complexes with the antibody and those of the separated cells that the Have histocompatibility antigen to generate where being further to the cells that the Antigen-antibody complexes contain a blood serum is added so that complement in the blood serum too cause to get to the antigen-antibody complexes bind, and so destroy the cells that do that Histocompatibility antigen, while it has the remaining cells is possible to persist and an antibody was added to the persistent cells that is specific with a histocompatibility antigen responds to immunity memory cells, and from the cells those are separated which are suitable for Immunity memory cells specific histocompati have balance-sheet antigen.

In a second embodiment of the present Invention becomes an immunity memory cell suspension provided that the from a immunosensitive individual who is against a certain pathogen has been immunized Contains cells consisting essentially of T cells consist of cells that are approximately in number Make up 98% of the T cells, at least 1 × 10³ Immunity memory cells included.

It should be noted that the term "Pathogen" as used here carcinomatous or other malignant tumor or any can be pathogenic microorganism, for example a bacterium or virus that is known to  it is a disease or toxic response in one Humans or an animal.

A manufactured according to the invention Immunity memory cell suspension can be from anyone Tissue of an organism that is immunological against a infectious disease or a malignant tumor has been sensitized, separated, cleaned and be cultivated. That means the immunological Prophylaxis and / or treatment of a disease against any bacterial or viral infectious disease or any malignant tumor can be caused insofar as an immunity-sensitized immunized Organism from this disease or the tumor has been cured is available. Typical examples for diseases with such an immunological Prophylaxis and / or treatment can be combated, are those caused by pathogens, including cancer cells, Salmonella, Corynebacteria, Pseudomonas, Pasteurellias, streptococci, ectromeliavirus and Sendai virus (mouse parainfluenza virus type 1) diseases caused.

It should also be noted that the Immune mechanisms caused by a Immunity memory cell suspension elicited be aware of humoral immunity distinguish that from the antigen-antibody response, as represented by vaccination, depend. Such adverse effects as they do could be inevitable result of vaccination the prophylaxis and / or treatment of a disease by using a Immunity memory cell suspension not involved be.  

Where the immunity memory cells of Cells are cultivated in the form of a First stage - Immunity memory cell suspension can be obtained and continue successively over a multitude of generations from the second-stage immunity memory cell supers sion can be subcultivated, a continuous Line of immunity memory cells to be established to enable such cells to be stable and economically in large quantities.

For the production of a Immunity memory cell suspensions are first Lymphocyte-containing cells from an individual which is known to be from any bacterial or viral infectious diseases or a cancerous or other malignant tumor is cured and therefore immunized against it. These Lymphocyte-containing cells can, for example from peripheral blood that comes from the heart or a vessel the immune-sensitized individual is obtained, be extracted. Alternatively, the lymphocyte-containing cells from a tissue section of the Spleen obtained from the immunosensitized individual will.

Then T cells from the cells separated from lymphocytes. To this end the lymphocyte-containing cells Affinity column chromatography using a processed suitable solid adsorbent, the selective allows the passage of T cells. In the Lymphocyte-containing cells are also found B cells, the cell-mediated to the organism Give immunity. The main part of this  B cells will attach to the solid adsorbent and consequently, a cell suspension, the cells in the essentially consist of T cells, fractionated by the column eluted. The preferred solid adsorbent for is the use in this chromatography process nylon wool packed in a tube.

A solid nylon wool adsorbent does not adsorb only B cells, but also other undesirable ones Cells, for example, as well as in the prior art is known, typically macrophages and leukocytes; because of this, affinity chromatography when using a solid nylon wool adsorbent alone not a satisfactorily high level Adsorption efficiency for B cells available put. In the method according to the invention, it is over for this reason preferred that the Affinity Chromatography Using Nylon wool following one Affinity column chromatography procedures performed that is another solid adsorbent, for example typically glass wool. It is in the state well known in the art that both T cells and B cells hardly adhere to glass wool, but they do Macrophages and leukocytes with a significantly high Level of affinity. By using this two-step affinity chromatography process, under Using glass wool in front of nylon wool, the B cells of the initial lymphocyte-containing cells with a satisfactorily high efficiency from the Cells cleaned by macrophages and leukocytes are removed.

As a replacement for such a two-stage Affinity chromatography procedures can be a  T cell separation methods are used, the Effect on the difference between those for T cells or B-cell specific surface antigens (Histocompatibility antigens). In that case, in which a mouse is an immune-sensitized individual can be used, the Thy1 surface antigen used to be in such T cells Identify T cell separation methods.

The in as a result of the two-tier Affinity column chromatography method obtained Cells containing cells suspension exist in essentially from T cells, but still contain one small amount of unwanted cell types, for example typically premature lymphocytes, each of which Immunity is lacking. So then there is procedures to be performed in such undesirable to remove premature cells by removing the cells from a Antigen-antibody reaction are subjected to the cells in the cell suspension as the antigen be used.

Because of this, an antibody that is specific with a special histocompatibility antigen reacts, added to the cell suspension, which the Result of the two-stage Affinity column chromatography procedure. The the Antibody preparation to be added to cell suspension selected so that it is able to deal with cells react that to the unwanted in the Cell suspension obtained cells specific Have histocompatibility antigen.

As a result of the so evoked Antigen-antibody reaction between the  Antibody preparation and the cells that make up such Have histocompatibility antigen Antigen-antibody complexes through the Antibody molecules that work with the cells that do that Have histocompatibility antigen, are connected, educated. To the antigen-antibody complexes is a suitable blood serum, for example typically Rabbit serum, added so that the complement in the Rabbit serum binds to the antigen-antibody complexes and destroyed the cells, one for the unwanted Lymphocyte specific histocompatibility antigen to have. As a result, only one cell survives negative surface antigenicity activity against such Exhibit histocompatibility antigens in which resulting cell suspension. It is obvious, that the cells are essentially made up Immunity memory cells exist.

If a mouse is an immunosensitive individual Ia (I-range-associated) Antigen as a histocompatibility antigen in the reaction used to make the antigen-antibody complex produce. In this case, an anti-Ia antigen monoclonal antibody to the cell suspension added to the antigen-antibody complexes by Ia antigen molecules that interact with cells that have a have positive Ia antigenic response, are united, to build. This antigen-antibody complex is a appropriate blood serum is added so that the complement binds to the antigen-antibody complexes in the serum. The cells that have the positive Ia antigen activity will be destroyed and as a result only the cells that have negative Ia antigen activity have in the resulting cell suspension persist. It should be noted that the  Ia antigen in mouse the HLA-D (or HLA-DR) - Corresponds to histocompatibility antigen in humans.

The cell suspension obtained in this step exists from cells that have a negative Surface antigen activity against the Histocompatibility antigens responsible for the unwanted Lymphocytes are specific, have and large Part consist of immunity memory cells. One such a cell suspension will continue to be Antibody preparation added, typically one for the surface antigens of Immunity memory cells specific Contains rat antibody. Coatings of the Rat antibodies are found on the surfaces of the Antigen molecules formed to form antigen-antibody complexes by generating the rat antibody that is on the for Immunity memory cells specific antigens Molecules are formed.

As a result of the antigen-antibody reaction and Complement fixation reaction obtained Immunity memory cells become an antibody added, for example a rat antibody, the especially with those for immunity memory cells specific surface antigens is reactive. A Coating of the rat antigen body is therefore on the Surface antigens of every immunity memory cell educated.

Separated from the one so obtained Immunity memory cell suspension becomes one Anti-rat IgG (Gamma immunoglobin) antibody preparation under Use a suitable anti-rat IgG serum as  Starting material manufactured. The Anti-rat IgG antibody preparation is used on the inner surface of a glass vessel applied, in which the cell suspension which the Contains immunity memory cells for the response with the anti-rat IgG antibodies in the vessel are plated, is poured. The Anti-rat IgG antibody reacts with the Rat antibody, the surface antigen of everyone Immunity memory cell covered, and, accordingly the role of an antigen in the Antigen-antibody reaction between the Anti-rat IgG antibody and the rat antibody is caused, plays. The result is that the Cells, each of which has a rat antibody, attached to the cell's surface antigen binds to the inner one Stick surface of the vessel.

The cells that are not on the inner surface of the vessel are obviously missing Ability to target the anti-rat IgG antibody on the Surface of the vessel to bind and from them therefore assumed that they have no surface antigens for have the formation of an antigen-antibody complex. This means that they have cells that are not attached to the Pin the surface of the jar, essentially other than immunity memory cells and as can be uselessly discarded from the vessel. in the the method according to the invention is used only by the cells, which deposited on the inner surface of the vessel are supposed to be from Immunity memory cells exist and become obtained as useful cells. A cell suspension that contains such useful cells is referred to below as "First stage" immunity memory cell suspension  designated. Known examples of Histocompatibility antigens specific for Immunity memory cells in the mouse are included Thy1, Lyt1, Lyt2, Lyt3 and Lyt antigens.

Studies have shown that they are in the First stage immunity memory cell suspension contained approximately 0.1% of the cells in the cells Cell suspension consisting of the two-stage Affinity column chromatography method results, represent. The results of these studies show further that the immune memory of the cells in the First-stage immunity memory cell suspension almost perfect for a non-immunized individual is passed on if the cell suspension at least Contains 1 × 10³ immunity memory cells.

According to a second embodiment of the present Invention is the immunity memory cells efficiently cultivated from the cells in the form of the First stage immunity memory cell suspension be preserved. Immunity memory cells can therefore cultivated and through a variety of Generations are successively sub-cultivated to one to establish continuous cell line by the Immunity memory cells of the "first generation", the separated from the immunosensitive individual are descended.

For the cultivation of cells or "Second generation" immunity memory cells from the First generation immunity memory cells first normal diploid cells from a mouse separated, which is syngeneic with the mouse, which as immunosensitive individual was used. These  normal diploid cells become reproductive radioactive radiation exposed, for example cobalt 60 gamma rays, which is the division and multiplication activity of Inhibit cells, but cell survival allow. The normal diploid cells with such inhibited division and multiplication activity are hereinafter referred to as "mitosis-inhibited" normal called diploid cells.

On the other hand, pathogenic cells of the Tpyes, the has caused the disease that immune-sensitized individual has contracted inactivated to an inactivated pathogenic Generate antigen suspension. These pathogen cells are used as the antigen of the type to which the immunity memory cells remember that in the form a first-stage immunity memory cell suspension to be provided. For this purpose, such pathogenic cells from an individual that is currently one caused by the pathogen Disease has contracted, gained and in one Culture medium resuspended; the resulting Cell suspension becomes an electromagnetic or radioactive radiation, for example ultraviolet or cobalt 60 gamma radiation.

The mitosis-inhibited normal diploid cells will be then the first-stage immunity memory cell sion added. This mixture is further one added inactivated antigen suspension of the pathogen. The resulting mixture is used to promote the Proliferation of immunity memory cells in the Suspension suspended so as to "Two-stage" immunity memory cell suspension too  produce. It should be noted that this Two-stage immunity memory cell suspension that Product of the primary culture of the First generation immunity memory cells is which are separated from the immunosensitive individual will. Immunity memory cells of a following Generation can easily be subcultivated if the Two-stage immunity memory cell suspension in one of the similar ones used for the primary culture Way out of the cells of the First stage immunity memory cell suspension be processed. So they can Third generation immunity memory cells be obtained with the resulting mixture under Condition that are similar to those that are incubated for the cultivation of the immunity memory cells of the second generation were applied. According to the invention Immunity memory cells can do this can be easily cultivated over generations, so one Line of high immunity memory cells Establish efficiency when a process is similar for cultivating Second generation immunity memory cells has been used, is applied repeatedly.

Experiments related to the present Invention have shown that the invention Immunity memory cells grow at a rate multiply, which, expressed in cell numbers, with a Factor of approximately 3 during 5 days of incubation as cell cultivation increases, starting with the cells obtained from the primary culture. Such a high rate of cell growth is one Security for creating an established line of immunity memory cells for the stable and  economical production of such cells in one larger scale.

It is further noted that the Immunity memory cells of the second generation in generally spherical in shape approximately three times larger than the immunity memory cells of the are first generation when using the microscope to be watched. It can be added that the Second generation immunity memory cells Have cores that are almost all larger than that of the First generation immunity memory cells, and Capsule antigen molecules that essentially match those of first generation immunity memory cells are identical.

The following example illustrates the invention.

example 1. Separation of immunity memory cells from the organism

A mouse was identified as an immune-sensitized individual used who had recovered from salmonellosis and was immunologically sensitized to Salmonella. However, it is apparent that, if desired, one against any other bacterial infectious disease immunized mouse or a mouse from a viral infectious disease or a carcinomatous or other malignant tumor is cured instead of immunosensitive individual who chose here was used.  

1-1. Prepare an initial cell suspension

Peripheral blood from the heart became the starting material won the mouse. The blood obtained was with a 5 times the volume of Hank 's solution diluted to the Cell aggregates in a suspension of individual cells to dissociate, followed by centrifugation of 15 minutes at 4 ° C and 2000 rpm. One of the supernatant the resulting preparation of formed "butty coat" was carefully removed from the rest of the preparation Aspirate removed from the supernatant.

1-2.

The "buffy coat" fraction, for the most part Leukocytes contained was further increased 5-fold Volume of Hank's solution diluted. The resulting one Cell suspension was 15 minutes at 4 ° C and 2000 rpm centrifuged, followed by removal of the supernatant. The cell suspension thus obtained was used in two more Centrifuged steps, each under conditions that those for the previous two Centrifugation steps used were similar. The on the surface of the cell suspensions during this supernatant formed in both centrifugation steps was removed at the end of each centrifugation step. To the through a total of three centrifugation steps washed cells, each of which removes the Supernatant followed, Hank's solution with one Content of 5 vol% fetal calf serum in one proportion added to a cell density of 4 × 10⁷ cells / ml leads. Which is at this point in the process cell suspension obtained is in the following as "Initial" cell suspension can be referred to.

1-3.

As a replacement for peripheral blood, if that is desired, the splenic tissue one  immunosensitive individual can be used. If the spleen tissue of a mouse as a starting material a slice of splenic tissue is used cut out the mouse spleen and through a steel sieve with perforations, for example the Tyler standard sieve, size no. 100, pressed. The perforations in such a steel screen passed cells are in a reasonable volume Hank's solution dispersed and can in the following similar to that from mouse peripheral blood prepared cell suspension can be processed. The means that the cell suspension that comes from the spleen derived cells containing lymphocytes, similar to that from mouse peripheral blood prepared cell suspension can be processed.

1-4. Detach the T cells

The initial cell suspension made up of peripheral blood Mouse had been manufactured on a Glass wool column with 10 g glass wool in a vertical Glass tube (20 mm inside diameter and 100 mm length) applied, followed by the addition of Hank's solution with 5 vol% fetal calf serum. The cell suspension was then in the column at 37 ° C for 45 minutes Incubation of the cells of the suspension warmed. To the cell suspension was allowed to incubate, slowly dripping from the column tube, being the same Shares of Hank's solution continuously in the column were fed, and the column was kept at 37 ° C has been. The cell suspension eluted from the column was collected in a centrifuge tube.

1-5.

As can be seen from the explanations above has been confirmed that the in the from the  Glass wool column chromatography resulting Cell suspension essentially contains cells T cells and B cells passed. Typical for that of the T cells and B cells isolated cells in the The remaining pillars are macrophages and leukocytes.

1-6.

With the cell suspensions created by the Column chromatography method obtained with glass wool investigations were carried out. The Results of these investigations indicated that the T cells and B cells by the Chromatography had been separated, approximately 30% the total amount of cells in the on the column applied initial cell suspension were contained, shown.

1-7.

Following affinity column chromatography with glass wool that was in the centrifuge tube collected cell suspension 15 minutes one Subjected to centrifugation at 4 ° C and 2000 rpm, followed by the removal of the supernatant from the resulting suspension. The so obtained Cell suspension became Hank's solution containing 5% by volume fetal calf serum contained in one portion added to a cell density of 4 × 10⁷ cells / ml leads. The resulting preparation was made on a Nylon wool pillar with 10 g nylon wool (manufactured by Fenwal Laboratories), packed in a vertical Glass tube (20 mm inner diameter and 100 mm length), applied, followed by the addition of Hank's solution with 5 vol% fetal calf serum. The cell suspension was then heated in the column to 37 ° C. for 45 minutes, to incubate the cells in the suspension perform. After the incubation, the Cell suspension dropped from the column tube,  with equal proportions of Hank's solution continuously the column was fed, which was kept at 37 ° C. has been. The cell suspension eluted from the column was also collected in a centrifuge tube.

1-8.

With the as a result of Nylon wool affinity column chromatography Cell suspensions were examined the results of which indicated that the Nylon wool affinity chromatography separated Cells approximately 10% of the total in the Initial cell suspension on the glass wool column cells were included.

1-9.

The in the cell suspension, which by the two-stage chromatography procedures have been obtained was contained within cells main proportion of T cells that are in line with the results of the investigations carried out approximately 95% of the total cell population of the suspension turn off. Most of the T cells different cells in the initial cell suspension before the two-step chromatography, is believed, in part, to the fibers that make up the Form glass wool, and partly to the fibers that make up the Form nylon wool to have adsorbed. As before previously noted, contains through the two-stage Chromatography method obtained cell suspension however, lymphocytes and other undesirable ones Cells.

1-10. T cell cleaning

To remove these unwanted cell types, the in the centrifuge tube as a result of  Nylon wool affinity column chromatography collected Cell suspension with a centrifugation of 15 minutes Subjected to 4 ° C and 2000 rpm, followed by the Removal of the supernatant. The resulting one Cell suspension became a part of a culture medium added to a cell density of 8 × 10⁷ cells / ml led. The culture medium consisted of a mixture of Medium 199, containing 2% by volume fetal calf serum. Culture medium with such a composition is in hereinafter referred to as medium "A".

1-11.

To a part by volume of the so obtained Cell suspension then became two volumes of one Anti-Ia antigen monoclonal antibody preparation (manufactured by Cedarlane Laboratorien, Ltd.), diluted with 30 times the volume of medium "A" added, as well as a volume of rabbit serum, diluted with twice the volume of medium "A". The mixture of this three-component preparation was heated to 37 ° for 30 minutes for the reaction.

1-12.

As a result of that in the mix evoked reaction were the Antigen-antibody complexes through the anti-Ia monoclonal antibody combined with the Cells that have positive Ia antigen activity educated. The resulting mixture was Rabbit serum added so that the complement in the Rabbit serum bound to the antigen-antibody complexes. Cells with positive Ia antigen activity were screened the rabbit serum was destroyed and accordingly could only the cells with negative Ia activity survive. a cell suspension, which is now a concentrated one Population of immunity memory cells contained  was centrifuged at 4 ° C and 2000 rpm for 15 minutes to win the immunity memory cells.

1-13.

A centrifugation for the resulting cell suspension in three successive Steps, 15 minutes each at 4 ° C and 2000 rpm in Medium "A" performed. The cell suspension, the obtained at the end of the three centrifugation steps was diluted in a proportion with medium "A", which resulted in a cell density of 8 × 10⁷ cells / ml.

1-14.

In addition to this cell suspension, a Anti-immunity memory cell rat antibody suspensions sion in a manner that will be described later (Chapter 4) manufactured and with 5 times the volume of Diluted medium "A". The rat antibody suspension and the cell suspension, each made as above were described in equal parts by volume mixed together and the resulting mixture was allowed to react at 4 ° C for 60 minutes. As The result of this reaction bound the rat antibody those specific for immunity memory cells Antigen molecules. By the on of the rat antibody the specific for the immunity memory cells Antigen molecules were formed Antigen-antibody complexes generated. The Cell suspension that contains the immunity memory cells such antigen-antibody complexes contained, was 15 minutes at 4 ° C and 200 rpm in medium "A" centrifuged. Centrifugation was continued with the resulting cell suspension in three successive Steps, each under conditions similar to those were for the previous one Centrifugation step were used. To those obtained through these centrifugation steps  Medium "A" was added to cells in a proportion that led to a cell density of 8 × 10⁶ cells / ml.

1-15.

In addition to this Immunity memory cell suspension became one Anti-rat IgG goat antibody preparation. For this purpose, 10 ml of anti-rat IgG Goat serum diluted with 35 times the volume of Medium "A", in a sterile plastic petri dish (manufactured by Becton Dickinson Co., Ltd.) with a Inside diameter of 100 mm manufactured and overnight left at 4 ° C. The antiserum excess was then washed out of the bowl so that the Goat anti-rat IgG antibody evenly in the Shell could remain and a thin liquid, on the inner surface of the bowl dejected coating could form.

1-16.

The one made as described above Cell suspension was in the dish with the liquid Coating of the anti-rat IgG goat antibodies that poured on it. The shell was then held horizontally and undisturbed for 60 minutes ditched. Then the cells that not the coating on the surface of the bowl bound, removed and discarded from the shell, by gently shaking the bowl. As before has been found to have cells that are not attached the coating of the goat anti-rat IgG antibody have bound no antigen-antibody complex that otherwise combined with the rat antibody the antigens specific for Immunity memory cells are formed. Most of the cells on the coating of the Goat anti-rat IgG antibody on the surface of the  Float shell, it is believed to be out Nonimmunity memory cells exist.

1-17.

The inner surface of the bowl was then in three steps each with a mixture of the Medium RPMI 1640, containing 10 vol% fetal Calf serum, rinsed, followed by spraying the same medium over the inner surface of the Bowl. The cells that adhered to the dish became removed from the shell and as inventive First stage immunity memory cell suspension collected.

1-18.

The ones obtained in this stage of the process Cells represent approximately 0.1% of the cells in the from two-step affinity column chromatography obtained cell suspension were included. With this first-stage immunity memory cell suspension Investigations carried out showed that these Cells that number approximately 98% of those in the First stage immunity memory cell suspension represent contained cells

• 1) belong to a family of small lymphocytes;
• 2) a positive antigenic activity in terms of specific for immunity memory cells Have antigen molecules; and
• 3) a positive antigenic activity in terms of Thy1, Lyt1, Lyt2 and Lyt3 antigens and a negative Antigenic activity of the Ia antigen exhibit.

1-19.

Further investigations have been carried out in which the immunity memory cells in the form of First stage immunity memory cell suspension in one  nonimmunized individual was introduced, the with the one here as an immune-sensitized individual mouse used was syngeneic. The results of this Studies have shown that immunity memory, that these immunity memory cells had had been completely transferred to the recipient, d. H. with a 100% probability if the Immunity memory cell suspension at least 1 × 10³ Contains immunity memory cells. Further Investigations were carried out with First stage immunity memory cell suspensions carried out, the individuals into which the Immunity memory cells had been introduced were irradiated with cobalt 60 gamma radiation. The Results of these studies showed that the Immunity memory cells that each of the individuals had been preserved unchanged if that Individual of a cobalt 60 gamma radiation of 600 X-ray or less has been exposed but destroyed was when the individual underwent radiation with cobalt 60 gamma rays exposed to more than 1100 X-rays has been. This proves that those in the First stage immunity memory cell suspensions contained immunity memory cells to a family belong to cells that are significantly resistant to is radioactive radiation.

2. Culturing cells for the establishment of Cell lines

From the in the form of First stage immunity memory cell suspensions Immunity memory cells obtained became cells cultured.  

2-1. Cultivate syngeneic normal cells

Normal diploid cells were in the form of a Cell suspension from one with the as immunosensitive individual used syngeneic mouse separated and in a sterile Bred Petri dish. Approximately when observed was that a unicellular cell layer in the Had formed a petri dish, the cells became one Exposed to cobalt 60 gamma radiation from 2000 X-rays, in order for the cells to divide and multiply inhibit.

2-2. Production of inactivated pathogenic antigens

Pathogenic cells of the type that causes the disease from which the immunosensitive individual were inactivated to an inactivated to generate pathogenic antigen suspension. To this Purpose were bacterial Salmonella cells Culture medium added in a proportion that to a Cell density of 1 × 10⁷ cells / ml resulted. The Culture medium consisted of a mixture of RPMI 1640 Medium containing 10 vol% fetal calf serum, 2 g / l Sodium bicarbonate (NaHCO₃), 0.11 g / l Sodium pyruvate (NaOOCCOCH₃) and 0.1 vol% yeast oleate contained. The culture medium with this composition will be referred to below as medium "B".

2-3.

The suspension, which is the bacterial Contained Salmonella cells dispersed in medium "B", was made with ultraviolet rays of an intensity of 3.6 × 10³ erg / cm² · s irradiated for 5 minutes to obtain the Inactivate bacteria. The use of medium "B" in this inactivation procedure is only one  Example and can by using each other Medium, which has a desired composition, to be replaced instead of the medium "B".

2-4.

In the case where an immunity-sensitive Individual who has a viral infectious disease has moved for the separation of Immunity memory cells are used on it can virus crystals in the medium "B" in one Ratio resulting in a density of 1 × 10⁹ crystals / ml leads, to be dispatched.

2-5.

If desired, tumor cells, for example Using carcinoma cells will be a slice of the tumor-infested tissue from the body of the individual cut out and cut into fragments. The Fragments of the tumor cell tissue can then be placed in a Phosphate-buffered saline, the Contains 0.02% by volume of ethylenediaminetetraacetic acid (EDTA), be immersed and shaken at 4 ° C. for 60 min will. The tissue pieces obtained can be against one Steel sieve with perforations, for example the Tyler standard sieve size No. 100 correspond, pressed will. The cell suspension that contains the cells that were pressed through the perforation in the sieve at 700 rpm to 800 rpm for 5 minutes at 4 ° C centrifuged. The so obtained Tumor cells are in the medium "B" in a ratio dispersed that to a cell density of 2 × 10³ Cells / ml leads, and the resulting cell suspension becomes a cobalt-60 gamma radiation of 2000 X-rays exposed to an inactivated pathogenic antigen Suspension equivalent to that in this example used to generate. It is obvious that if an inactivated pathogenic antigen suspension  a tumor cell tissue is to be produced, each other desired culture medium than medium "B" for the Processing of those extracted from this tissue Cells can be used.

2-6. Culturing Immunity Memory Cells

The first-stage immunity memory cell suspension, made as previously described Medium "B" added in a ratio that corresponds to one Cell density of 8 × 10⁵ cells / ml leads. The resulting one Suspension was carefully poured into the bowl the mitosis-inhibited, from the syngeneic mouse contained separated cells. The volume of the so Mitose-inhibited cells added suspension can in Depends on the size of the shell used be chosen and was given the size of the Bowl of 100 mm inner diameter with 5 ml selected. If one petri dish with another Inner diameter of, for example, 60 mm is used it is advisable to add 2.5 ml of the First stage immunity memory cell suspensions to use in the bowl.

2-7.

The mixture of the Immunity memory cell suspensions and the Mitosis-inhibited cells that are plated in the dish the same volume became inactivated pathogenic antigen suspension, as described above had been added. The resulting one Mixture was at 37 ° C in an atmosphere with a Carbon dioxide content of 5% incubated to increase to promote immunity memory cells. From this incubation (centrifugation) resulting Preparation is that prepared according to the invention  Second stage immunity memory cell suspensions.

2-8.

If the Second stage immunity memory cell suspension in a manner similar to that for the primary culture of cells from the First stage immunity memory cell suspension was used, can be further processed Immunity memory cells of the following generations easily subcultivated. For this purpose, the Second stage immunity memory cell suspensions with medium "B" in a ratio that to a Cell density of 8 × 10⁵ cells / ml leads, are diluted. The resulting cell suspension is with Mitosis-inhibited cells of the syngeneic individual mixed and in inactivated pathogenic Antigen suspension prepared as described above. Immunity memory cells of the following, i.e. H. the third generation, so can be obtained if the resulting mixture among the specified Conditions are incubated and centrifuged. How has already been established by the inventor experiments showed that during the Cultivation of Immunity Memory Cells, Starting with the cells obtained from the primary culture had been using the immunity memory cells multiply at a rate that is a factor of approximately 3 rises during 5 days of incubation.

3. Preventive effect of immunity memory cells

With the manufactured according to the invention Immunity memory cell suspensions were made Investigations carried out to the disease-preventing effects of the cells against  to test different pathogens. The one for this test Cell suspensions used include those by the First stage immunity memory cell suspension represented by one immunity-sensitized individual Immunity memory cells contain, and those that through the Second stage immunity memory cell suspension represent the immunity memory cells contains that from cells of the First stage immunity memory cell suspension were bred. The ones used for these tests Cell suspensions contained different amounts of Immunity memory cells in the range of 1 × 10² to 1 × 10⁴. These cell suspensions were in non-immunized mice as recipient individuals introduced and then these recipients Attacks of various pathogens exposed to the disease-preventing effect in percent evaluate. The antigens used for these tests include carcinoma cells, salmonella, corynebacteria, Pseudomonas, pasteurellias, streptococci, Ectromeliavirus and Sendai virus. These bacterial, viral and carcinoma cell pathogens were identified in the Individuals introduced in such amounts that so were selected to have a disease rate of 100% for to provide each of the pathogens. The results these tests are shown in the following table.  

From the results of the tests performed is obvious that the immunity memory successfully on a non-immunized individual 100% probability is transmitted if the cell suspension is 1 × 10³ or more Contains immunity memory cells. The results of these tests further indicate that a whole considerable extent of disease preventers Effects can be achieved if a Cell suspension containing less than 1 × 10³ Immunity memory cells, introduced into an individual becomes. For example, if a Immunity memory cell suspension less than Contains 6 × 10² immunity memory cells in one non-immunized individual is introduced to this Recipient-individual with an immunity memory a probability of approximately 50% and then when an immunity memory cell suspension,  the less than 3 × 10² immunity memory cells contains, is used, the immunity memory on the recipient with a probability of about 30% transmitted.

4. Manufacture of Anti-memory cell rat antibodies

The following is the process for making the Anti-immunity memory cell rat antibody suspension described in the in paragraphs 1-14 described steps is used.

The immunity memory cell suspension, as described in Paragraph 1-13 had been established with Phosphate-buffered physiological saline diluted and the resulting preparation under centrifuged in suitable conditions. The through three Cell suspension obtained was obtained by centrifugation after washing continue with a similar saline solution diluted in a ratio that corresponds to a cell density of 1 × 10⁸ cells / ml. A fraction of 0.25 ml the resulting immunity memory cell suspension has been in the thigh subcutaneous tissue of a variety of female rats [Wistar rats (Wisterspecies)] injected. 10 to 20 days after the cell suspension so each rat had been given similar ones Fractions of the cell suspension in the Thigh subcutaneous tissue injected into each rat.

The one with phosphate-buffered physiological Saline diluted in three, as above described successive steps Centrifuged cell suspension was also included Phosphate buffered physiological saline in  diluted a ratio chosen this time was that there was a cell density of 1 × 10⁹ cells / ml led. A fraction of 0.25 ml of the resulting Immunity memory cell suspension was in the Abdominal cavity of each of the female rats after 40 days the first administration of the cell suspension to the Rats injected. Each of the rats was 50 to 60 Days after the first administration of the cell suspension the total blood volume obtained. From each of the rats 5 to 6 ml of blood serum were obtained.

On the other hand, a slice of splenic tissue became one with the mouse, from which the immunity memory cell had been obtained from a syngeneic newborn mouse cut out. The tissue section was cut through a Pressed steel screen and those pressed through the steel screen Spleen cells were buffered in phosphate dispersed physiological saline. The so prepared cell suspension was in three successive steps and centrifuged then about 1.5 parts by volume of the same as above Rat serum produced to the resulting cell population, followed by violent movement. The preparation thus obtained was 60 minutes with movement at intervals of 15 Let stand in ice water for minutes and then centrifuged under suitable conditions.

The resulting preparation was then the Supernatant obtained with phosphate-buffered physiological saline was diluted, followed by Steps that were similar to those for originally made from the spleen tissue Cell suspension had been carried out. Of the the preparation finally obtained was again the  Obtained supernatant and as an anti-immunity memory cell-rat antibody preparation in those previously described in paragraphs 1-14 Steps used.

Claims (5)

1. A method for producing an immunity memory cell suspension, characterized by
the separation of cells consisting essentially of T cells from cells which have been obtained from an immunity-sensitized individual who has been immunized against a particular pathogen,
the addition of an antibody reactive with a histocompatibility antigen specific for a particular cell type to the separated cells, antigen-antibody complexes being formed from the antibody and those of the separated cells which have the histocompatibility antigen,
adding a blood serum to the cells containing the antigen-antibody complex so as to cause the complement in the blood serum to bind to the antigen-antibody complexes, thereby destroying the cells with the histocompatibility antigen and the remaining cells allow to persist
adding an antibody reactive with the histocompatibility antigen specific for immunity memory cells to the persistent cells and
separating the cells having the histocompatibility antigen specific for immunity memory cells from the resulting cells. 2. The method of claim 1, wherein the cells having the histocompatibility antigen specific for immunity memory cells are separated in the form of a first-stage immunity memory cell suspension from the cells resulting from the addition of the antibody to the persistent cells, the method further characterized is through
the production of normal cells that are syngeneic with the cells obtained from the immune-sensitized individual,
exposing these normal cells to radioactive radiation in order to inhibit the ability of the cells to divide,
the production of pathogenic cells of a pathogen of the type that is identical to the specific pathogen,
inactivating the cells which have been separated from the pathogen identical to the particular pathogen,
making a mixture of the first stage immunity memory cell suspension, the normal cells with inhibited dividing ability and the inactivated cells and
incubating this mixture to allow the cells of that first-stage immunity memory cell suspension to multiply and produce a second-stage immunity memory cell suspension. 3. The method according to claim 1 or 2, characterized in that the cells are separated with the histocompatibility antigens specific for immune memory cells by applying an anti-IgG antibody preparation to a surface in order to coat the anti-IgG antibody preparation on the surface form,
onto this coating are applied the cells resulting from the addition of the antibody molecules to the persistent cells so that the cells with histocompatibility antigens specific for immunity memory cells adhere to the surface, and
the cells attached to the surface are removed from the surface. 4. The method according to at least one of claims 1 to 3, characterized in that the cells consisting essentially of T cells are separated from the cells obtained from the immune-sensitized individual by
the cells consisting essentially of T cells are subjected to a first stage of affinity column chromatography, glass wool being used as the solid adsorbent, and
the resulting cells are then subjected to a second stage of affinity column chromatography using nylon wool as the solid adsorbent. 5. Immunity memory cell suspension, thereby characterized in that the cell suspension contains cells, by an immunity-sensitive individual who is immunized against a specific pathogen and that they are essentially made up of T cells consist of cells that number approximately 98% of the total Represent T cells, at least 1 × 10³ Immunity memory cells included.