CA1333887C

CA1333887C - Immunity memory cell suspension and method of preparing same

Info

Publication number: CA1333887C
Application number: CA000596787A
Authority: CA
Inventors: Mitsuru Doi
Original assignee: GEO-RESEARCH COMPANY, LIMITED
Current assignee: GEO-RESEARCH COMPANY Ltd
Priority date: 1988-11-30
Filing date: 1989-04-14
Publication date: 1995-01-10
Anticipated expiration: 2012-01-10
Also published as: CH678337A5; JP2639834B2; FR2646778B1; FR2646778A1; GB2230790B; GB8908241D0; GB2230790A; JPH02150274A; DE3913438A1; AU603950B1

Abstract

A method of preparing an immunity memory cell suspension, wherein cells consisting of T-cells are separated from cells collected from an immunity sensitized individual immunized against a particular pathogen and are mixed with an antibody reactive to a histocompatibility antigen specific to a pre-scribed species of cells for producing antigen-antibody complexes by the antibody and those of the separated cells which have the histocompatibility antigen, whereupon a blood serum is added to the cells containing the antigen-antibody complexes for causing the complement in the blood serum to bind to the antigen-antibody complexes and thereby destroying the cells having the histocompatibility antigen and allowing the remaining cells to persist. To these persisting cells is then added an antibody reactive to a histocompatibility antigen specific to immunity memory cells and from the resul-tant cells are separated the immunity memory cells which have the histocompatibility antigen specific to immunity memory cells. Immunity memory cells may be cultured and subcultured successively through a plurality of generations to establish a continuous line of cells which originates in the immunity memory cells thus separated from the initial immunity sensi-tized individual.

Description

-"IMMUNITY MEMORY CELL SUSPENSION AND METHOD OF PREPARING SAME"

FIELD OF THE INVENTION
The present invention relates to a method of preparing an immunity memory cell suspension useful for the prevention and/or treatment of an infectious disease or a malignant tumor such as typically cancer. The present invention also relates to an immunity memory cell suspension prepared by such a method.
BACKGROUND OF THE INVENTION
A human being or other animal that has once experienced a viral or bacterial disease and has reacted against a particu-lar species of antigen exhibits a prominent defensive response to the antigen when the individual is for a second time subjected to stimulation with the same type of antigen. This defensive reaction is caused as a result of the immunity conferred to the individual against the particular disease and is commonly called secondary immune response or anamnestic response of an organism to the antigen. As well known in the art, such an immune response of an organism to an antigen results from excessive multiplication of the lymphocytes involved in the reaction of the organism to the stimulation with the antigen that has contributed to the outbreak of the disease. The lymphocytes thus developed in response to the stimulation by an antigen are generally called immunological . . ~ . . ., ... ... . ... . . . ... . " , . . ~ ~ .. ; .

~ - 2 -1 memory cells.
Immunological memory cells produced in the body of an organism are a species of lymphocytes inhabiting the lymph and blood circulating through the vessels of the circulatory system of the organism. The lymphocytes in the circulatory system immigrate into the thymus gland of the organism where they develop into T-lymphocytes or T-cells which confer an immune ability to the organism. As well known in the art, such immunologically competent T-cells, sometimes also called thymus-dependent or thymus-derived cells, are distributed extensively in almost all the peripheral tissues of the circulatory system of an organism, as in the tissue of the spleen, around the post-capillary venules in the paracortical areas of the lymph nodes, and in the central artery of the spleen white pulp of the organism.
As for the im.munity memory cells contained in the T-cells thus developed from lymphocytes in the body of an organism, evidence in the art seems to indicate that such cells are recognized merely conceptually as ~immunological" memory cells as above mentioned. There have indeed been no practical attempts to isolate or extract such cells or at least any fraction containing such cells from a tissue of an organism and collecting the resultant immunity memory cells or cell-cont~;n;ng fraction in a practically usable state or in vitro.
Neither a positive prospect for utilizing immllnnological memory cells for the prevention and/or treatment of an _ - 3 -1 infectious disease or a malignant tumor has ever been held out or suggested on a realistic basis, nor any clinical or even experimental approach toward such a practical use of immuno-logical memory cells has been ambitiously undertaken.
The present invention contemplates realization of a method of separating immunity memory cells directly from a tissue of an organism at a significantly high efficiency.
Such a method according to the present invention depends for its effect on various specific natures and attributes of immunity memory cells which confer cell-mediated immunity to an organism. The present invention further contemplates provision of a method of cultivating immunity memory cells through a plurality of generations to provide a continuous, established cell line originating in the immunity memory cells initially separated from a tissue of an organism.
SUMMARY OF THE INVENTION
It is accordingly an important object of the present invention to provide a method of preparing a cell suspension cont~;ning immunity memory cells separated from a tissue of an organism that has been immunologically sensitized against a bacterial or viral infectious disease or a malignant tumor such as typically cancer.
It is another important object of the present invention to provide a method of cultivating the immunity memory cells separated from a tissue of an organism to establish a conti-nuous line of immunity memory cells in vitro.

1 It is still another important object of the present invention to provide a method of preparing an immunity memory cell suspension from a tissue of an organism and thereafter cultivating the immunity memory cells in the cell suspension to provide an established line of immunity memory cells in vitro.
It is still another important object of the present invention to provide a cell line originating in the immunity memory cells separated from a tissue of an immunity sensitized individual in accordance with the present invention or in any immllnity memory cells otherwise separated from a tissue of an individual.
In accordance with one outstanding aspect of the present invention, there is provided a method of preparing an immunity memory cell suspension, comprising a) separating cells sub-stantially consisting of T-cells from cells collected from an immtlnity sensitized individual immunized against a particular pathogen, b) adding to the separated cells an antibody reac-tive to a histocompatibility antigen specific to a prescribed20 species of cells for producing antigen-antibody complexes by . the antibody and those of the separated cells which have the histocompatibility antigen, c) adding a blood serum to the cells cont~i n; ng the antigen-antibody complexes for causing the complement in the blood serum to bind to the antigen-antibody complexes and thereby destroying the cells having the histocompatibility antigen and allowing the remaining cells to I persist, d) adding to the persisting cells an antibody reac-tive to a histocompatibility antigen specific to immunity memory cells, and e) separating from the resultant cells those which have the histocompatibility antigen specific to immunity memory cells.
In this method according to the first outstanding aspect of the present invention, the cells which have the histocom-patibility antigen specific to immunity memory cells may be separated in the form of a first-stage immunity memory cell suspension from the cells which have resulted from the addi-tion of the antibody to the persisting cells. In this in-stance, the method according to the first outstanding aspect of the present invention further comprises f) preparing normal cells syngeneic with the cells collected from the immunity sensitized individual, g) exposing the normal cells to a radioactive radiation for inhibiting the divisional ability of the cells, h) preparing pathogenic cells of a pathogen of the type identical with the particular pathogen, i) inactivating the cells separated from the pathogen identical with the particular pathogen, j) preparing a mixture of the first-stage immunity memory cell suspension, the normal cells having their divisional ability inhibited and the inactivated cells, and k) incubating the mixture to allow the cells in the first-stage immunity memory cell suspension to multiply and produce a second-stage i~.unity memory cell suspension.
In accordance with another outstanding aspect of the ., . ~ . . , . .. , . , ~ , , 1 resent invention, there is provided a method of preparing an immunity memory cell suspension, comprising a) separating cells substantially consisting of T-cells from cells collected from an immunity sensitized individual immunized against a particular pathogen, b) adding to the separated cells an antibody reactive to a histocompatibility antigen specific to a prescribed species of cells for producing antigen-antibody complexes by the antibody and those of the separated cells which have the histocompatibility antigen, c) adding a blood serum to the cells containing the antigen-antibody complexes for causing the complement in the blood serum to bind to the antigen-antibody complexes and thereby destroying the cells having the histocompatibility antigen and allowing the remain-ing cells to persist, d) adding to the persisting cells antibody molecules reactive to a histocompatibility antigen specific to immunity memory cells, and e) preparing a first-stage immunity memory cell suspension from the resultant cells, the first-stage immunity memory cell suspension con-taining cells having the histocompatibility antigen specific to immunity memory cells, f) preparing normal cells syngeneic with the cells collected from the immunity sensitized indi-vidual, g) exposing the normal cells to a radioactive radia-tion for inhibiting the divisional ability of the cells, h) preparing pathogenic cells of a pathogen of the type identical with the particular pathogen, i) inactivating the cells separated from the pathogen identical with the particular ~ 1~33887 1 pathogen, j) preparing a mixture of the first-stage immunity memory cell suspension, the normal cells having their divi-sional ability inhibited and the inactivated cells, and k) incubating the mixture to allow the cells in the first-stage immunity memory cell suspension to multiply and produce a second-stage immunity memory cell suspension In a method according to the present invention as herein-before described, the cells having the histocompatibility antigens specific to immunity memory cells are separated preferably by e/l) plating an anti-IgG antibody preparation onto a surface to form a coating of the anti-IgG antibody preparation on the surface, e/2) applying to the coating the cells which have resulted from the addition of the antibody molecules to the persisting cells so that the cells having the histocompatibility antigens specific to immunity memory cells adhere to the surface, and e/3) removing from the surface the cells which have adhered thereto.
Furthermore, the cells substantially consisting of T-cells as above mentioned are separated from the cells collected from the immunity sensitized individual preferably by a/l) subjecting the cells substantially consisting of T-cells to a first stage of affinity column chromatography using a solid adsorbent of glass wool, and a/2) thereafter subjecting the resultant cells to a second stage of affinity column chromatography using a solid adsorbent of nylon wool.
In accordance with still another outstanding aspect of .

_ - 8 --1 the present invention, there is provided an immunity memory cell suspension containing cells which are collected from an ~ nity sensitized individual immunized against a particular pathogen and which substantially consist of T-cells, wherein cells accounting about 98 per cent by number of the T-cells contain at least lx103 ;mmtln;ty memory cells.
It may be noted that the term "pathogen" herein referred to may be a cancerous or other malignant tumor or any patho-genic microbe such as a bacterium or a virus that is known to be causative of a disease or a toxic response in a human being or other animal.
An immunity memory cell suspension prepared in a method according to the present invention as hereinbefore described can be separated, purified and cultured from any tissue of an l~ organism that has been immunologically sensitized against an infectious disease or a malignant tumor. This means that immunological prevention and/or treatment of a disease can be effected against any bacterial or viral infectious disease or a malignant tumor insofar as an immunity sensitized org~ni cm that has been cured of such a disease or tumor is available.

Typical of the diseases that can be coped with by such immN-nological prevention and/or treatment are those caused by pathogens including Cancer cells, Salmonella, Corynebacterum, Pseudmbnas, Pasteurella, Streptococcus, ectromelia virus, and 2~ Sendai virus (mouse parainfluenza virus type 1).

It may also be noted that the immune mechanism evoked by -1 an immunity memory cell suspension prepared in a method according to the present invention basically differs from that of the humoral immunity that depends on the antigen-antibody reaction represented by vaccination. No such adverse effects that are to inevitably result from vaccination could be involved in the prevention and/or treatment of a disease by the use of an immunity memory cell suspension proposed by the present invention.
Where immunity memory cells provided by the present n invention are cultivated from the cells obtained in the form of the first-stage immunity memory cell suspension and further subcultured successively through a plurality of generations from the second-stage immunity memory cell suspension, a continuous line of immunity memory cells can be established to make it possible to manufacture such cells stably and econo-mically on a quantity basis.
DETAILED DESCRIPTION OF THE lNv~NlION
For preparing an immunity memory cell suspension in accordance with a first outstanding aspect of the present, cells containing lymphocytes are first collected from an individual (hereinafter referred to as immunity sensitized individual) that is known to have been cured of and accord-ingly immunized against any bacterial or viral infectious disease or a cancerous or other malignant tumor. These lymphocyte-containing cells may for example be extracted from the peripheral blood collected from the heart or the vein of . . . . . . . . . .. , . . ~ . . . , -- ... . . .. .

133~887 1 the immunity sensitized individual. Alternatively, the lymphocyte-containing cells may be collected from a slice of the immunity sensitized individual's spleen tissue.
T-cells are then separated from the lymphocyte-cont~ining cells which have thus been collected from the peripheral blood or the spleen tissue of the i~ nity sensitized individual used. For this purpose, the lymphocyte-cont~ining cells are processed by column affinity chromatography using a suitable solid adsorbent allowing passage of T-cells selectively. Also contained in the lymphocyte-containing cells herein in use are B-cells which confer cell-mediated immunity to an organism.
These B-cells are in a major proportion caused to adhere to the solid adsorbent and, as a consequence, a cell suspension containing cells largely consisting of T-cells is fractionally lS eluted from the column. Preferred as the solid adsorbent for use in this column affinity chromatography process is nylon wool packed in a column tube.
A solid adsorbent of nylon wool adsorbs not only B-cells but also other unwanted cells such as typically macrophages and leucocytes as well known in the art and, for this reason, the affinity chromatography using a solid adsorbent of nylon wool alone could not provide a satisfactorily high adsorption efficiency for B-cells. In a method according to the present invention, it is for this reason preferable that the affinity chromatography process using the solid adsorbent of nylon wool be performed subsequently to a column affinity chromatography 1 process using another solid adsorbent such as typically glass wool. As well known in the art, both T-cells and B-cells hardly adhere to a solid adsorbent of glass wool, which is however capable of arresting macrophages and leucocytes with significantly high degrees of affinity. Through use of the two-stage affinity chromatography process using glass wool prior to nylon wool, the B-cells in the initial lymphocyte-containing cells can be removed at a satisfactorily high efficiency from the cells which have been preliminarily cleared of macrophages and leucocytes.
In substitution for such a two-stage affinity chromato-graphy process may be used a T-cell separation process which depends for its effect on the difference between the surface antigens (histocompatibility antigens) specific to T-cells and B-cells, respectively. Where a mouse is employed as the immunity sensitized individual as previously noted, Thyl surface antigen may be used to identify T-cells in such a T-cell separation process.
The cells contained in the cell suspension resulting from the two-stage column affinity chromatography process consist largely of T-cells as above noted but still contain a small quantity of unwanted species of cells such as typically premature lymphocytes lacking in an immune ability. Accord-ingly, the process to be performed subsequently is to remove such unwanted premature cells by causing the cells to experi-ence an antigen-antibody reaction using the cells in the cell . . ~ .

1 suspension as the antigen.
For this purpose, an antibody which specifically reacts with a particular histocompatibility antigen is added to the cell suspension which has resulted from the two-stage column affinity chromatography process. The antibody preparation to be added to the cell suspension is selected to be capable of reacting with the cells which have histocompatibility antigens specific to the unwanted lymphocytes contained in the cell suspension.
As a result of the antigen-antibody reaction thus caused between the antibody preparation and the cells having such histocompatibility antigens, antigen-antibody complexes are formed by the antibody molecules united with the cells having the histocompatibility antigens. To the antigen-antibody complexes produced in this fashion is added a suitable blood serum such as typically rabbit serum so that the complement in the rabbit serum binds to the antigen-antibody complexes and destroys the cells having the histocompatibility antigens specific to the unwanted lymphocytes. The result is that only the cells exhibiting a negative surface antigenic activity against such histocompatibility antigens are allowed to survive in the resultant cell suspension. It will be apparent that these cells largely consist of immunity memory cells.
Where a mouse is used as the immunity sensitized individ-ual as herein assumed to be the case, Ia (I-region-associatedJ
antigen may typically be used as the histocompatibility .. ... .
... . . . .

1 antigen in the reaction to produce the antigen-antibody complexes. In this instance, anti-Ia antigen monoclonal antibody is added to the cell suspension to form the antigen-antibody complexes by the Ia antigen molecules united with the cells exhibiting a positive Ia antigenic activity. To these antigen-antibody complexes is added a suitable blood serum so that the complement in the serum binds to the antigen-antibody complexes. The cells exhibiting the positive Ia antigenic activity are thus destroyed and as a consequence only the cells having a negative Ia antigenic activity are allowed to persist in the resultant cell suspension. It may be noted that the Ia antigen in mouse corresponds to the HLA-D
(or HLA-DR) histocompatibility antigen in man.

The cell suspension obtained at this stage consist of cells which exhibit the negative surface antigenic activity against the histocompatibility antigens specific to the unwanted lymphocytes and which largely consist of immunity memory cells. To such a cell suspension is further added an antibody preparation typically containing rat antibody reac-tive to the surface antigens specific to immunity memory cells. Coatings of the rat antibody are formed on the surface antigen molecules to produce antigen-antibody complexes by the rat antibody formed on the molecules of the antigen specific to the immunity memory cells.
To the immunity memory cells collected as a result of the antigen-antibody and complement fixation reactions is then . .

1 added an antibody, such as for example rat antibody, which is reactive particularly to the surface antigens specific to immunity memory cells. A coating of, for example, the rat antibody is thus formed on the surface antigen of each immu-nity memory cell.
Separately of the immunity memory cell suspension thusobtained is prepared an anti-rat IgG (gamma immunogloblin) antibody preparation with use of a suitable anti-rat IgG serum as a starting material. The anti-rat IgG antibody preparation is applied to the inner surface of a glass vessel, into which the cell suspension containing the immunity memory cells is poured for reaction with the anti-rat IgG antibody plated in the glass vessel. The anti-rat IgG antibody reacts with the rat antibody wrapping the surface antigen on each immunity memory cell and, accordingly, plays the role of an antigen in the antigen-antibody reaction which is caused between the anti-rat IgG antibody and the rat antibody. The result is that the cells each having the rat antibody binding to the surface antigen of the cell adhere to the inner surface of the 2~ glass vessel coated with the anti-rat IgG antibody suspension.
The cells which have failed to adhere to the inner surface of the glass vessel are apparently lacking in the ability to bind to the anti-rat IgG antibody on the surface of the glass vessel and are thus considered to have no surface antigens to form the antigen-antibody complexes that would otherwise be for~ed by the rat antibody combined with the .... .. . ... . .. .. . .

-l surface histocompatibility antigens specific to ;mm1ln;ty memory cells. This means that the cells not adherent to the inner surface of the vessel substantially consists of cells other than immunity memory cells and may be discarded as useless from the glass vessel. In a method according to the first outstanding aspect of the present invention, only the cells which are deposited on the inner surface of the glass vessel and which are accordingly considered to consist of ~ nity memory cells are therefore collected as useful cells.
The cell suspension contAining such useful cells is herein re-ferred to as "first-stage" im~llnity memory cell suspension.
Known examples of the histocompatibility antigens specific to immunity memory cells of a mouse include Thyl, Lytl, Lyt2, Lyt3 and Lyt3 antigens.
1~ Tests conducted with the first-stage ir~nnity memory cell suspension have revealed that the cells contained in the first-stage ;mmllnity memory cell suspension account for approximately 0.1 per cent of the cells in the cell suspension resulting from the two-stage column affinity chromatography.
The results of the tests further indicate that the immunity memory possessed by the cells in the first-stage immunity memory cell suspension prepared in a method according to the first outstanding aspect of the present invention is almost perfectly transferred to a non-immunized individual when the ;mm~lnity memory cell suspension contains at least lx103 ;mmllnity memory cells, as will be discussed in more detail.

, . . . . .. . .. . . . . .. . . .. . ... . .. . . . .

1333~7 Now, in accordance with a second outstanding aspect of the present invention, immunity memory cells are cultivated efficiently from the cells obtained in the form of the first-stage immunity memory cell suspension prepared in accordance with the first outstanding aspect of the present invention as hereinbefore described. Immunity memory cells may thus be cultured and subcultured successively through a plurality of generations to establish a continuous line of cells which originates in the "first-generation" immunity memory cells separated from an immunity sensitized individual in accordance with the first outstanding aspect of the present invention.
For cultivating cells or "second-generation" immunity memory cells from the first-generation immunity ~emory cells, normal diploid cells are first separated from a ~ouse synge-neic with the mouse herein used as the immunity sensitizedindividual. These normal diploid cells are, after multipli-cation, exposed to a suitable radioactive radiation such as, for example, cobalt-60 gamma rays inhibiting the divisional and multiplicative activity of the cells but allowing the cells to live on. The normal diploid cells having their divisional and multiplicative ability thus inhibited are herein referred to as mitosis-inhibited normal diploid cells.
On the other hand, pathogenic cells of the type that has caused the disease contracted by the immunity sensitized individual herein in use are inactivated to produce an inacti-vated pathogenic antigen suspension as herein so referred to.

.
.. , . . . ~ . . , .. . . . . , . ~

`- 1333887 1 These pathogenic cells are used as the antigen of the type memorized by the immunity memory cells now provided in the form of the first-stage immunity memory cell suspension. For this purpose, such pathogenic cells are collected from an 5 individual presently contracted by a disease caused by the pathogen under consideration is suspended in a culture medium and the resultant cell suspension is subjected to electro-magnetic or radioactive radiation with, for example, ultra-violet or cobalt-60 gamma rays.
The mitosis-inhibited normal diploid cells are then added to the first-stage immunity memory cell suspension which has been prepared as hereinbefore described. To the mixture of these is further added the inactivated pathogenic antigen suspension. The resultant mixture is incubated to promote multiplication of the im~unity memory cells in the suspension to thereby yield a n second-stage" immunity memory cell suspen-sion in a method according to the second outstanding aspect of the present invention. It may be herein noted that this second-stage immunity memory cell suspension is the product of the primary culture of the first-generation immunity memory cells separated from the immunity sensitized individual that is herein assumed to be in use. It may be further noted that immunity memory cells of a subsequent generation can be easily subcultivated when the second-stage immunity memory cell suspension prepared as above described is processed in a manner similar to that used for the primary culture of the .

133388~
l cells from the first-stage immunity memory cell suspension.
Thus, immunity memory cells of the third generation can be obtained with the resultant mixture incubated under conditions similar to those used for the cultivation of the second-generation immunity memory cells from the first-generation immunity memory cells. Immunity memory cells according to the present invention can be in these manners easily cultivated from one generation to another to establish a strain of immunity memory cells at a satisfactorily high efficiency when a procedure similar to that used for the cultivation of the immunity memory cells of the second generation from the those of the first generation is followed repeatedly.
According to the results of the experiments conducted by the inventor, the immunity memory cells according to the 1~ present invention multiply at a rate which, in terms of the number of cells, increases with a factor of approximately three for five days of incubation during cultivation of the cells (the second-generation immunity memory cells) starting with the cells obtained by the primary culture. Such a high rate of multiplication of cells will provide an assurance of creating an established line of immunity memory cells and manufacturing such cells stably and economically on a quantity basis.
It may be further noted that the immunity memory cells of 2~ the seccnd generation cultivated from those of the first generation appear generally globular in shape and are . _. .. . .

1 approximately three times larger than the first-generation immunity memory cells when observed microscopically. It may be added that the second-generation immunity memory cells have nuclei almost all of which are larger in size than those of the first-generation immunity memory cells and capsular antigen molecules which are largely identical with those of the first-generation immunity memory cells.
Tests were further conducted with the second-stage immunity memory cell suspension, the results of the tests indicating that the immunity memory possessed by the cells in the second-stage immunity memory cell suspension prepared in a method according to the second outstanding aspect of the present invention is also almost perfectly transferred to a non-immunized individual when the immunity memory cell suspen-t5 sion contains at least lx103 immunity memory cells, as will bediscussed in more detail.
A method of preparing an immunity memory cell suspension in accordance with the present invention as hereinbefore described may be put into practice in any of various manners which fall within the purview of the present invention.
Hereinafter described is a preferred example of such various manners of carrying out a method according to the first and second outstanding aspects of the present invention.
EXAMPLE
(l) Separating Immunity Memory Cells from Organism A mouse recovered from and therefore immunologically .. . . . ... .. . .

1 sensitized against salmonellosis was used as the immunity sensitized individual in this Example. It will be however apparent that, if desired, a mouse immunized against any other bacterial infectious disease or cured of any viral infectious disease or a cancerous or other malignant tumor may be used in substitution for the immunity sensitized individual herein selected for use.
Preparing Initial Cell Suspension (1-1) Peripheral blood was collected from the heart of the mouse for use as the starting material in this Example.
The blood collected was diluted with a five-fold volume of Hanks' solution to dissociate the cell aggregates into a suspension of single cells, followed by centrifugation at 2000 rpm for 15 minutes at 4C. A buffy coat formed by the super-natant in the resultant preparation was removed from theremainder of the preparation by gently sucking in the super-natant.
(1-2) The buffy coat fraction, containing leucocytes in a major proportion, was further diluted with a five-fold volume of Hanks' solution. The resultant cell suspension was centri-fuged at 2000 rpm for 15 minutes at 4C, followed by removal of the supernatant. The cell suspension thus obtained was further centrifuged in two steps each under conditions similar to those used for the immediately earlier step of centrifuga-tion. The supernatant formed on the surface of the cellsuspension produced during each of these two steps of , . . . ... _ .. .. ... . . .

1 centrifugation was removed at the end of each of the centrifugation steps. To the cells which had been washed through the total of three steps of centrifugation each followed by removal of the supernatant was added a Hanks' solution in a proportion selected to yield a cell density of 4x107 cells/ml and containing 5% by volume of fetal cow serum (FCS). The cell suspension obtained at this stage of processing will be hereinafter referred to as "initial" cell suspension.
n (1-3) In substitution for the peripheral blood herein used as the starting material may be used the spleen tissue of an immunity sensitized individual if desired. Where the spleen tissue of a mouse is used as the starting material, a slice of spleen tissue is excised from the mouse's spleen and is forced through a steel strainer having perforations con-forming to, for example, Tyler standard screen scale No. 100.
The cells passed through the perforations in such a steel strainer are dispersed into an appropriate volume of Hanks' solution and may be thereafter processed similarly to the cell suspension prepared from the peripheral blood of the mouse herein assumed to be used. Thus, the cell suspension con-taining the spleen-derived lymphocyte-containing cells may also be centrifuged at 2000 rpm for 15 minutes at 4C, fol-lowed by two further steps of centrifugation each under conditions similar to those used for the first step of centri-fugation of the cell suspension prepared from the peripheral 1 blood of the mouse as previously described.
Separating T-Cells (1-4) The initial cell suspension prepared from the peripheral blood of the mouse as hereinbefore described was applied to a glass wool column having 10 grams of glass wool packed in a vertical glass tube (measuring 20mm in inside diameter and lOOmm in length), followed by addition of a Hanks' solution containing 5% by volume of fetal cow serum.

The cell suspension was then heated in the glass wool column at 37C for 45 minutes for incubation of the cells in the suspension. After the incubation, the cell suspension was allowed to slowly drip from the column tube with a similarly proportioned Hanks' solution being continuously added to the column which was maintained at 37C. The cell suspension thus fractionally eluted from the glass wool column was collected in a centrifuge tube.
(l-S) As previously noted, T-cells and B-cells tend to adhere to fibers of glass wool less firmly than other types of cells do and, for this reason, cells other than T-cells and, generally, B-cells contained in a cell suspension added to a glass wool column are selectively retained to the fibers of the glass wool. This means that the cells contained in the cell suspension resulting from the glass wool column affinity chromatography used in this Example largely consisted of T-cells and B-cells. Typical of the cells thus isolated from the T-cells and B-cells and left in the column are macrophages .. = . . . .

and leucocytes as previously noted.
(1-6) Tests were conducted with the cell suspension collected by the glass wool column affinity chromatography process as hereinbefore described. The results of the tests indicated that the T-cells and B-cells separated through the glass wool column affinity chromatography accounted for approximately 30 per cent of the total quantity of the cells which had been contained in the initial cell suspension applied to the glass wool column.
1 (1-7) Subsequently to the glass wool col D affinity chromatography, the cell suspension collected in the centri-fuge tube was subjected to centrifugation at 2000 rpm for 15 minutes at 4C, followed by removal of the supernatant from the resultant suspension. To the cell suspension thus ob-tained was added a Hanks' solution in a proportion selected to yield a cell density of 4x107 cells/ml and containing 5% by volume of fetal cow serum. The resultant preparation was applied to a nylon wool column having 10 grams of nylon wool (manufactured by Fenwal Laboratories) packed in a vertical glass tube (measuring 20mm in inside diameter and lOOmm in length), followed by addition of a Hanks' solution containing 5% by volume of fetal cow serum. The cell suspension was then heated in the nylon wool column at 37C for 45 minutes for incubation of the cells in the suspension. After the incuba-tion, the cell suspension was allowed to drip from the column tube with a similarly proportioned Hanks' solution being .. . . .. . ..

1 continuously added to the column which was maintained at 37C.
The cell suspension thus fractionally eluted from the nylon wool column was also collected in a centrifuge tube.
(1-8) Tests were conducted with the cell suspension collected as a result of this nylon wool column affinity chromatography, the results of which indicated that the cells separated through the nylon wool column affinity chromato-graphy accounted for approximately 10 per cent of the total quantity of cells which had been contained in the initial cell suspension applied to the glass wool column.
(1-9) The cells contained in the cell suspension result-ing from the two-stage column affinity chromatography process contain T-cells in a major proportion which, according to the results of the tests conducted, amounted to approximately 95 per cent of the total cell population in the cell suspension.
Most of those cells other than the T-cells that had been contained in the initial cell suspension prior to the two stages of column affinity chromatography are considered to have been adsorbed partially to the fibers forming the glass wool and partially to the fibers forming the nylon wool. As noted previously, however, the cell suspension obtained through the two-stage column affinity chromatography process still contains lymphocytes and other cells unwanted in a method according to the present invention.
Purifying T-Cells (1-10) For the purpose of removing these unwanted l species of cells, the cell suspension collected in the centri-fuge tube as a results of the nylon wool column affinity chromatography was subjected to centrifugation at 2000 rpm for 15 minutes at 4C, followed by removal of the supernatant therefrom. The resultant cell suspension was added to a culture medium in a proportion selected to yield a cell density of 8x107 cells/ml. The culture medium herein used consisted of a mixture of Medium 199 containing 2~ by volume of fetal cow serum. A culture medium having such a composi-tion will be hereinafter referred to as medium "A".

(l-11) To one part by volume of the cell suspension thus obtained were then added two parts by volume of an anti-Ia (anti-I-region-associated) antigen monoclonal antibody prepa-ration (manufactured by Cedarlane Laboratories Ltd.) diluted with a thirty-fold volume of medium "A" and one part by volume of rabbit serum diluted with a two-fold volume of medium "An.
The mixture of these three component preparations was heated for reaction at 37C for 30 minutes.
(1-12) As a result of the reaction thus caused in the mixture, antigen-antibody complexes were formed by the anti-Ia monoclonal antibody united with the cells having a positive Ia antigenic activity. To the resultant mixture was added rabbit serum so that the complement in the rabbit serum bound to the antigen-antibody complexes. The cells having the positive Ia antigenic activity were thus destroyed by the rabbit serum and, accordingly, only the cells having a negative Ia .. , , . . . , ~, .. ..

-1 antigenic activity were allowed to survive. The cell suspension, now containing a concentrated population of immunity memory cells, was centrifuged at 2000 rpm for 15 minutes at 4C to collect the immunity memory cells.
(1-13) Centrifugation was further performed for the resultant cell suspension in three successive steps each at 2000 rpm for 15 minutes at 4C in medium "A". The cell suspension obtained at the end of the three steps of centri-fugation was diluted with medium "A" in a proportion selected to yield a cell density of 8x107 cells/ml.

(1-14) Besides this cell suspension, an anti-immunity memory cell rat antibody suspension was prepared in a manner to be described (chapter (4)) and was diluted with a five-fold volume of medium "An. The rat antibody suspension and the cell suspension each prepared as described above were mixed together in substantially equal volumes and the resultant mixture was allowed to react at 4C for 60 minutes. As a result of this reaction, the rat antibody bound to the antigen molecules specific to immunity memory cells. Antigen-antibody complexes were produced by the coatings of the rat antibody formed on the antigen molecules specific to immunity memory cells. The cell suspension containing the immunity memory cells carrying such antigen-antibody complexes was centrifuged at 2000 rpm for 15 minutes at 4C in medium "A". Centrifuga-tion was further performed on the resultant cell suspension inthree successive steps each under conditions similar to those 1 used for the preceding step of centrifugation. To the cells obtained through these steps of centrifugation was added medium "A" in a proportion selected to yield a cell density of 8X106 cells/ml.
(1-15) In addition to this immunity memory cell suspen-sion was prepared an anti-rat IgG goat antibody preparation.
For this purpose, 10 ml of anti-rat IgG goat serum diluted with a thirty-five fold volume of medium "A" was prepared in a sterile plastic petri dish (manufactured by Becton Dickinson Co., Ltd.) measuring lOOmm in inside diameter and was allowed to stand overnight at 4C. An excess of antiserum was then washed away from the petri dish so that an anti-rat IgG goat antibody was allowed to remain uniformly in the dish in the form of a thin liquid coating deposited on the inner surface 15 of the dish.
(1-16) The immunity memory cell suspension prepared as described above was poured into the petri dish having the liquid coating of the anti-rat IgG goat antibody deposited therein. The petri dish was then maintained horizontally and 20 was allowed to stand undisturbed for 60 minutes. Thereupon, the cells which had failed to adhere to the coating on the inner surface of the dish were removed and discarded from the dish by trembling the dish lightly. As has been noted, the cells that had failed to bind to the coating of the anti-rat 25 IgG goat antibody have no antigen-antibody complexes which would have otherwise been formed by the rat antibody combined ., . , .. . . . , , . . . .. . , j . . . ., ~ .. ~ .

_.

1 with the antigens specific to ;mmlln;ty memory cells. Most of the cells floating on the coating of the anti-rat IgG goat antibody on the surface of the dish are thus considered to consist of non-;mmlln;ty memory cells.
(1-17) The inner surface of the dish was then rinsed in three steps each with a mixture of Medium RPMI 1640 containing 10% by volume of fetal cow serum, followed by spraying of the same medium over the inner surface of the petri dish. The cells which adhered to the inner surface of the petri dish were in this manner stripped from the surface of the dish and were collected as the first-stage immunity memory cell suspen-sion in a method according to the present invention.
(1-18) The cells collected at this stage of the process accounted for approximately 0.1 per cent of the cells that had 1~ been contained in the cell suspension resulting from the two successive stages of column affinity chromatography. Tests were conducted with this first-stage immunity memory cell suspension, the results of which showed that the cells ac-counting for about 98 per cent by number of the cells con-2~ tained in the first-stage immllnity memory cell suspension (1) belong to a family of small lymphocytes;
(2) exhibit a positive antigenic activity in respect of the antigen molecules specific to immunity memory cells; and (3) exhibit a positive antigenic activity in respect of 25 the Thyl, Lytl, Lyt2 and Lyt3 antigens and a negative antigenic activity in respect of the Ia antigen.

.

_ - 29 -l (1-19) Tests were further conducted wherein the immunity memory cells in the form of the first-stage immunity memory cell suspension were introduced into a non-immunized individ-ual syngeneic with the mouse herein used as the immunity sensitized individual. The results of the tests showed that the immunity memory possessed by the immunity memory cells was transferred to the recipient perfectly, viz., with a probabil-ity of 100 per cent when the immunity memory cell suspension contains at least lx103 immunity memory cells, as will be described in more detail. Tests were further conducted with the first-stage immunity memory cell suspension, wherein individuals into which the immunity memory cells had been introduced were irradiated with gamma rays from cobalt-60.
The results of these tests indicated that the immunity memory which each of the individuals had acquired remained unimpaired when the individual was exposed to cobalt-60 gamma rays of 600 roentgens or less but was destroyed when the individual was subjected to irradiation with cobalt-60 gamma rays of more than 1100 roentgens. This evidences that the immunity memory cells contained in the first-stage memory cell suspension prepared in this Example belong to a family of cells which are significantly resistant to radioactive radiations.
(2) Cultivating Cells for Establishing Cell Line Cells were then cultivated from the immunity memory cells obtained in the form of the first-stage immunity memory cell suspension prepared as hereinbefore described. It may be - 13338~7 1 noted that the steps of cultivating cells as will be herein-after described are useful not only for the establishment in vitro of a cell line originating in the immunity memory cells contained in the first-stage immunity memory cell suspension prepared in accordance with the first outstanding aspect of the present invention but also for the culture and subculture of immunity memory cells in general which may be otherwise separated or extracted from an immunity sensitized individual.
Cultivating Syngeneic Normal Cells (2-1) Normal diploid cells were separated in the form of a cell suspension from a mouse syngeneic with the mouse used as the immunity sensitized individual in this Example and were grown in a sterile petri dish. Approximately when it was observed that a single layer of cells was formed in the petri 1~ dish, the cells were exposed to cobalt-60 gamma rays of 2000 roentgens to inhibit the divisional and multiplicative ability of the cells.
Preparing Inactivated Pathogenic Antigen (2-2) On the other hand, pathogenic cells of the type that caused the disease (salmonellosis) contracted by the immunity sensitized individual herein used was inactivated to produce an inactivated pathogenic antigen suspension. For this purpose, bacterial cells of Salmonella were added to a culture medium in a proportion selected to yield a cell density of lx107 cells/ml. The culture medium used for this prccess consisted of a mixture of Medium RPMI 1640 containing 1 10% by volume of fetal cow serum, 2 grams/liter of sodium hydrogencarbonate (NaHCO3), 0.11 gram/liter of sodium pyruvate (NaOOCCOCH3), and 0.1% by volume of yeast oleate. A culture medium having this composition will be hereinafter referred to as medium "Bn.
(2-3) The suspension containing the bacterial cells of Salmonella dispersed in the medium "B" was irradiated with ultraviolet rays of an intensity of 3.6x103 erg/cm2.sec for five minutes to inactivate the bacteria. The use of medium "B" in this inactivation process is merely by way of example and, as such, any other medium having a desired composition may be used in substitution for medium "Bn.
(2-4) Where an ;mmunity sensitized individual that has contracted a viral infectious disease is used for the separa-tion of immunity memory cells therefrom, virus crystals may bedispersed in medium "B" in a proportion selected to yield a density of lx10 crystals/ml.
(2-5) On the other hand, if it is desired to use malig-nant tumor cells such as cancer cells, a slice of a tissue involving tumor is excised from the body of an individual and is cut into fragments with use of a knife or scissors. The fragments of the tumor cell tissue may then be immersed in a phosphate buffered physiological salt solution containing 0.02% by volume of ethylenediaminetetraacetic acid (EDTA) and agitated for 60 minutes at 4C. The resultant pieces of tissue may be pressed against a steel strainer having l perforations conforming to, for example, Tyler standard screen scale No. 100. The cell suspension containing the cells forced through the perforations in the strainer is centrifuged at 700 rpm to 800 rpm for five minutes at 4C. The tumor cells collected in this manner is dispersed into medium "B" in a proportion selected to yield a cell density of 2x103 cells/ml and the resultant cell suspension is exposed to cobalt-60 gamma rays of 2000 roentgens to produce an inacti-vated pathogenic antigen suspension equivalent to that used in this Example. It will be apparent that, where an inactivated pathogenic antigen suspension is to be prepared from a tumor cell tissue, any desired culture medium other than medium "B~
may be used for the processing of the cells extracted from the tissue.
Cultivating Tmmlln;ty Memory Cells (2-6) To the first-stage immunity memory cell suspension which had been prepared as hereinbefore described was then added medium "B" in a proportion selected to yield a cell density of 8x105 cells/ml. The resultant cell suspension was gently poured into the petri dish cont~;n;ng the mitosis-inhibited cells separated from the syngeneic mouse. The volume of the cell suspension thus added to the mitosis-inhibited cells may be selected depending on the size of the petri idish used and was, in this Example, selected at S
2~ milliliters in consideration of the size of the petri dish which measured lOOmm in inside diameter. If a petri dish , . . . . .. . . . . . . . . .. .

- 13338g7 1 having another inside diameter of, for example, 60mm is to be used, it is advisable to use 2.5 milliliter of the first-stage immunity memory cell suspension in the dish.
(2-7) To the mixture of the immunity memory cell suspen-sion and the mitosis-inhibited cells plated in the petri dish was added an equal volume of the inactivated pathogenic antigen suspension which had been prepared as described above.
The resultant mixture was incubated at 37C in an atmosphere containing 5% of carbon dioxide to promote multiplication of the immunity memory cells in the mixture. The preparation resulting from this centrifugation is the second-stage immu-nity memory cell suspension prepared in a method according to the second outstanding aspect of the present invention.
(2-8) When the second-stage immunity memory cell suspen-sion prepared as hereinbefore described is processed in amanner similar to that used for the primary culture of the cells from the first-stage immunity memory cell suspension, immunity memory cells of a subsequent generation can be easily subcultivated, as previously noted. For this purpose, the second-stage immunity memory cell suspension may be diluted with medium "B" in a proportion selected to yield a cell density of 8x105 cells/ml. The resultant cell suspension is mixed with the mitosis-inhibited cells of the syngeneic individual and the inactivated pathogenic antigen suspension prepared as previously described. Immunity memory cells of the subsequent, viz., third generation can thus be obtained .. . . . . ..

- 133388~

l when the resultant mixture is incubated and centrifuged under the specified conditions. As has been noted, the experiments conducted by the inventor have revealed that, during cultiva-tion of the ; mm~n; ty memory cells starting with the cells 5 obtained by the primary culture, the immunity memory cells multiply at a rate which, in terms of the number of cells, increases with a factor of approximately three for five days of incubation.
(3) Preventive Effect of Tm~t~n;ty Memory Cells Tests were conducted with ;mmll~; ty memory cell suspen-sions each prepared in a method according to the present invention for assaying the disease preventive effects of the cells against various pathogens. The cell suspensions used for the assay include those represented by the first-stage 5 imm~ln;ty memory cell suspension cont~;n;ng ;mm~ln;ty memory cells separated from an ;mmlln;ty sensitized individual and those represented by the second-stage i-m-~unity memory cell suspension containing ;mmlln;ty memory cells grown from the cells in the first-stage immunity memory cell suspension. The 2~-~ cell suspensions used for the tests contained different quantities of ; mmlln; ty memory cells ranging in nu-mber from lx102 to lx104. These cell suspensions were introduced into non-;mmllni7ed mice as recipient individuals and, thereafter, the re`cipient individuals were attacked by different pathogens to evaluate the disease preventive effects in percentage. The pathogens which were used in the tests include cancer cells, 1 Salmonella, Corynebacterum, Pseudmonas, Pasteurella, Streptococcus, ectromelia virus and Sendai virus. These bacterial, viral and cancer cell pathogens were introduced into the individuals in quantities selected to provide the incidence rate of 100% for each of the pathogens. The results of the tests are demonstrated in the following table.

Pathogens No. of CellsPreventive Effect Cancer cells 1 x 104 100%
1 x 103 100%
6 x 102 70%
3 x 102 42%
1 x 102 34%

Salmonella 1 x 10 100%
1 x 103 100%

6 x 10 68%
3 x 1o2 34%
1 x 102 20%

Corynebacterum 1 x 10 100%
1 x 103 100%
6 x 10 48%
3 x 102 26%

1 x 102 14%

Pseudmonas 1 x 104 100%
1 x 103 100%
6 x 102 41%
3 x 102 23%
1 X 102 11%

_ - 36 -1 Pasteurella 1 x 104 100%
1 x 103 100%
6 x 102 78%
3 x 1o2 53%
1 x 102 35%

Streptococcus 1 x 10 100%
1 x 103 100%
6 x 102 44%
3 x 102 31%
1 x 102 17%

ectromelia virus 1 x 104 100%
1 x 103 100%
6 x 102 51%
3 x 102 27%
1 x 102 13%

Sendai virus 1 x 104 100%
1 x 103 100%
6 x 102 88%
3 x 102 63%
1 x 102 29%

As will be understood from these results of the tests conducted, the immunity memory possessed by the cells in a immunity memory cell suspension prepared in a method according to the present invention is successfully transferred to a non-immunized individual with the probability of 100% when the immunity memory cell suspension contains lx103 immunity memory cells or more. The results of the tests further indicate that .. .. .

_ ~ 37 ~ 1333 88~

1 considerably high degrees of disease preventive effect can be achieved when an immunity memory cell suspension containing less than lx103 immunity memory cells is introduced into an individual. For example, when an immunity memory cell suspen-sion containing less than 6X102 immunity memory cells isintroduced into a non-immunized individual, the imml7n;ty memory is transferred to the recipient individual with the probability of about 50% and, if an immunity memory cell suspension containing less than 3X102 immunity memory cells is used, the immunity memory is transferred to the recipient individual with the probability of about 30%, as will be seen from the results of the tests indicated in the table.

(4) Preparing Anti-Memory Cell Rat Antibody Description will be hereinafter made regarding the process of preparing the anti-immunity memory cell rat anti-body suspension used in the steps hereinbefore described in paragraph 1-14.
For this purpose, the immunity memory cell suspension prepared as described in paragraph 1-13 was diluted with phosphate buffered physiological salt solution and the resul-tant preparation was centrifuged under appropriate conditions.
The cell suspension obtained by three steps of centrifugation was, after washing, further diluted with similar salt solution in a proportion selected to yield a cell density of lx108 cells/ml. A 0.25 ml fraction of the resultant immunity memory cell suspension was injected into the femoral subcutaneous .. . . . , . . , . , , . ~ . . .. . . . .. ~., . ~ . ..

_ - 38 -1 tissue of each of a plurality of female rats (of Wister species). In ten days and twenty days, respectively, after the cell suspension was thus administered to each rat, similar fractions of the cell suspension were injected into the femoral subcutaneous tissue of the rat.
The ;mm-lnity memory cell suspension diluted with phos-phate buffered physiological salt solution and centrifuged in three successive steps as described above was also diluted with phosphate buffered physiological salt solution this time in a proportion selected to yield a cell density of lx109 cells/ml. A 0.25 ml fraction of the resultant immunity memory cell suspension was injected into the abdominal cavity of each of the female rats in forty days after the cell suspension had been administered to the rat for the first time. A total volume of blood was then collected from each of the rats in fifty to sixty days after the cell suspension had been for the first time administered to the rat. Five to six milliliters of blood sera were obtained from each of the rats.
On the other hand, a slice of spleen tissue was excised from a newborn mouse syngeneic with the mouse from which the immunity memory cells had been collected. The slice of the spleen tissue was forced through a steel strainer and the spleen cells passed through the steel strainer were dispersed into phosphate buffered physiological salt solution. The cell suspension thus prepared was centrifuged in three successive steps and, thereafter, an approximately 1.5 part by volume of . .

_ - 39 -1 the rat serum prepared as described above was added to the resultant population of cells, followed by thorough agitation.
The preparation thus obtained was then allowed to stay in iced water for 60 minutes with agitation at time intervals of 15 minutes and was thereafter centrifuged under appropriate conditions.
From the resultant preparation was then collected the supernatant, which was diluted with phosphate buffered physio-logical salt solution, followed by steps similar to those10 taken for the cell suspension initially prepared from the spleen tissue. Supernatant was further collected from the preparation thus obtained finally and was used as the anti-immunity memory cell rat antibody preparation in the steps hereinbefore described in paragraph 1-14.
From the foregoing description it will have been under-stood that an immunity memory cell suspension to be prepared in a method according to the present invention can be sepa-rated, purified and cultured from any tissue of an organism that has been immunologically sensitized against an infectious disease or a malignant tumor. Immunological prevention and/or treatment of any bacterial or viral infectious disease or a malignant tumor or tumor can be effected if an ~ n;ty sensitized organism that has cured of the disease or tumor is practically available.
It is also important that the im~une mechanism evoked by an immunity memory cell suspension prepared in a method . , ~ . . , . ,, ~ ~ . . . . ....

1 according to the present invention basically differs from that of the humoral immunity that depends on the antigen-antibody reaction represented by vaccination. No such adverse effects that are to inevitably result from vaccination could be involved in the prevention and/or treatment of a disease by the use of an immunity memory cell suspension proposed by the present invention.
It may be further added that where immunity memory cells provided by the present invention are cultivated from the cells obtained in the form of the first-stage immunity memory cell suspension and further subcultured successively through generations from the second-stage immunity memory cell suspen-sion, a continuous line of immunity memory cells can be established to make it possible to manufacture such cells stably and economically on a quantity basis.

. .... . ... , ~ .. . ... . .... . .

Claims

1. A method of preparing an immunity memory cell suspension, comprising a) separating cells substantially consisting of T-cells from cells collected from an immunity sensitized individual immunized against a particular pathogen, b) adding to the separated cells an antibody reactive to a histocompatibility antigen specific to a prescribed species of cells for producing antigen-antibody complexes by said antibody and those of said separated cells which have said histocompatibility antigen, c) adding a blood serum to the cells containing said antigen-antibody complexes for causing the complement in the blood serum to bind to said antigen-antibody complexes and thereby destroying the cells having said histocompatibility antigen and allowing the remaining cells to persist, d) adding to the persisting cells an antibody reactive to a histocompatibility antigen specific to immunity memory cells, and e) separating from the resultant cells those which have said histocompatibility antigen specific to immunity memory cells.

2. A method as set forth in claim 1, in which the cells which have said histocompatibility antigen specific to immu-nity memory cells are separated in the form of a first-stage immunity memory cell suspension from the cells which have resulted from the addition of said antibody to said persisting cells, the method further comprising f) preparing normal cells syngeneic with the cells collected from said immunity sensitized individual, g) exposing said normal cells to a radioactive radiation for inhibiting the divisional ability of the cells, h) preparing pathogenic cells of a pathogen of the type identical with said particular pathogen, i) inactivating the cells separated from the pathogen identical with said particular pathogen, j) preparing a mixture of said first-stage immunity memory cell suspension, the normal cells having their divi-sional ability inhibited and the inactivated cells, and k) incubating said mixture to allow the cells in said first-stage immunity memory cell suspension to multiply and produce a second-stage immunity memory cell suspension.

3. A method of preparing an immunity memory cell suspension, comprising a) separating cells substantially consisting of T-cells from cells collected from an immunity sensitized individual immunized against a particular pathogen, b) adding to the separated cells an antibody reactive to a histocompatibility antigen specific to a prescribed species of cells for producing antigen-antibody complexes by said antibody and those of said separated cells which have said histocompatibility antigen, c) adding a blood serum to the cells containing said antigen-antibody complexes for causing the complement in the blood serum to bind to said antigen-antibody complexes and thereby destroying the cells having said histocompatibility antigen and allowing the remaining cells to persist, d) adding to the persisting cells antibody molecules reactive to a histocompatibility antigen specific to immunity memory cells, e) preparing a first-stage immunity memory cell suspen-sion from the resultant cells, said first-stage immunity memory cell suspension containing cells having said histocom-patibility antigen specific to immunity memory cells, f) preparing normal cells syngeneic with the cells collected from said immunity sensitized individual, g) exposing said normal cells to a radioactive radiation for inhibiting the divisional ability of the cells, h) preparing pathogenic cells of a pathogen of the type identical with said particular pathogen, i) inactivating the cells separated from the pathogen identical with said particular pathogen, j) preparing a mixture of said first-stage immunity memory cell suspension, the normal cells having their divi-sional ability inhibited and the inactivated cells, and k) incubating said mixture to allow the cells in said first-stage immunity memory cell suspension to multiply and produce a second-stage immunity memory cell suspension

4. A method as set forth in claim 1, in which the cells having said histocompatibility antigens specific to immunity memory cells are separated by e/1) plating an anti-IgG antibody preparation onto a surface to form a coating of the anti-IgG antibody preparation on said surface, e/2) applying to said coating the cells which have resulted from the addition of said antibody molecules to said persisting cells so that the cells having said histocompati-bility antigens specific to immunity memory cells adhere to said surface, and e/3) removing from said surface the cells which have adhered thereto.

5. A method as set forth in claim 1, in which said cells substantially consisting of T-cells are separated from the cells collected from said immunity sensitized individual by a/1) subjecting the cells substantially consisting of T-cells to a first stage of affinity column chromatography using a solid adsorbent of glass wool, and a/2) thereafter subjecting the resultant cells to a second stage of affinity column chromatography using a solid adsorbent of nylon wool.