WO1996001891A1

WO1996001891A1 - Method for generating a population of cells having a high surface density of a mhc molecule-associated specific exogenous peptide, and cell population

Info

Publication number: WO1996001891A1
Application number: PCT/FR1995/000907
Authority: WO
Inventors: Pierre Langlade Demoyen; Philippe Kourilsky; Jean-Pierre Abastado
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm); Institut Pasteur
Priority date: 1994-07-07
Filing date: 1995-07-06
Publication date: 1996-01-25
Also published as: FR2722207B1; FR2722207A1; AU2930395A

Abstract

A method for generating a population of cells having a high surface density of an MHC molecule-associated specific exogenous peptide. A population of living non-tumour mammalian cells, particularly human peripheral blood lymphocyte splenic cells, ganglion cells, cord blood cells or placental cells, wherein said cells have a surface density of one MHC molecule-associated specific exogenous peptide with the same allele restriction as MHC molecules, whic h restriction is substantially higher than that of the corresponding cells that express said native MHC molecule-associated exogenous peptide.

Description

Method for generating a population of cells having on their surface a high density of a specific exogenous peptide associated with MHC molecules; cell population. The subject of the invention is a method for producing a population of living cells of the immune system having on their surface a high density of a specific exogenous peptide, useful in therapy in particular, for carrying out anti-tumor or anti-infectious immunotherapy in a patient or to treat an autoimmune disease.

The treatment of cancers by immunotherapy has been the primary objective of many studies for several years.

Cytotoxic lymphocytes (CTL) destroy cells infected with an infectious host, such as a virus with high efficiency, and can be useful in killing tumor cells. While the identification of specific tumor antigens and the prediction of peptides capable of binding to molecules of the Major Histocompatibility Complex (MHC) has progressed during the recent period, the induction of CTL has been the main limiting factor tumor immunotherapy.

Many practical and theoretical difficulties have been encountered in the induction of anti-tumor CTLs. Restriction of MHC and immunological tolerance to the self limit the number of possible target peptides. However, the major obstacle seems to be the quantitatively different afference requirements for the activation of T lymphocytes. While T lymphocytes activated by the antigen require only a few hundred MHC Class I complexes / peptides to exert their action, quiescent T cells require higher amounts of antigens for their activation. Since tumor antigens, with regard to the presentation of the antigen, can be considered as normal cellular constituents, their peptides are likely to compete with other peptides derived from cellular proteins for binding to class I molecules. Their number may be less than that of peptides originating from of viral particles which usually actively synthesize their constituents and often stop cell synthesis. This could explain why tumor antigens have a relatively low capacity to elicit T cell responses in vivo.

Dendritic cells loaded with peptide are capable of initiating a primary CTL response in vitro (Nair et al. (1)), that is, lymphocyte responses to CTL in unimmunized mice. The expression of B7, B7-2, HSA or other unidentified co-stimulant molecules is probably an important factor, but the density of peptide / MHC complexes is also critical, since defective human or murine mutant cells which can be efficiently loaded with exogenous peptides can also initiate primary CTL responses. Likewise, it has been shown that Drosophila melanogaster cells transfected with H-2 and HLA genes expressing "empty" MHC Class I molecules can be loaded with an exogenous peptide and can induce a response in vitro. CTL specific in primary cultures.

The inventors of the present invention have now shown that it is possible to detach endogenous peptides from MHC molecules expressed on the cell surface of living cells having MHC molecules associated with endogenous peptides and possibly peptides on their surface. exogenous (for a small part of them) without affect cell viability or the ability of MHC molecules to bind peptides again. Such populations of cells "re-loaded" with an exogenous peptide are effective in inducing a response to CTL CD8 ⁺ , directed against this peptide in the case of MHC Class I, or to CTL CD4 ⁺ directed against this peptide in the case MHC Class II both in vitro and in vivo. This approach makes it possible to avoid the prior techniques consisting in cloning and expressing the numerous antigens of the HLA system, insofar as it allows the use of autologous cells to obtain a high proliferation in CTL, or to activate the B lymphocytes in the case of CMH Class II.

The subject of the invention is a method for generating a population of living cells having on their surface a high density of a specific exogenous peptide associated with MHC molecules, characterized in that a population of native cells having molecules is treated MHC loaded with peptides of endogenous and possibly exogenous origin, to detach said peptides, and in that the MHC molecules are loaded with said specific exogenous peptide with the same allelic restriction as the MHC molecules.

By high density of exogenous peptide is meant that at least a part of the cells of the treated population has at least 10% and preferably at least 90% of allelic MHC molecules loaded with the specific peptide. Thus, for a cell carrying 10 ⁶ molecules of MHC, the cell will present after treatment at least 10 ⁵ molecules of the same peptide associated with the molecules of MHC.

According to the invention, the MHC molecules include the conventional MHC molecules of Class I, Class I non-class and Class II. Non-classical class I molecules (or class Ib molecules by as opposed to classical class 1 molecules) are proteins whose gene structure is similar to that of classical class I genes, but they are much less polymorphic and have a more limited tissue distribution. This family, to which the molecules of the M and Q regions of the MHC belong for example, has been described in particular by Ojcius et al (2). The peptides of endogenous and possibly exogenous origin associated with the MHC molecules are advantageously detached from these by treatment at pH <5 or at pH> 9 for a duration varying between 10 seconds and 1 minute, preferably approximately 30 seconds. .

MHC Class I binds a wide variety of short peptides (8-10 amino acids) through mostly conserved interactions between the peptide backbone and the non-polymorphic terminal residues of the αl-α2 domain of the MHC molecule. Much of the binding energy appears to be provided by hydrogen bridges between the ends of the peptide and the polar or charged residues of the MHC molecules.

Furthermore, Ojcius et al. (3) have shown that the recombinant MHC Class I-peptide complexes dissociate at a pH below 5 or above 9.

Advantageously, the "stripping" of the peptides of the MHC molecules of Class I Class I, I unconventional or II takes place in citric acid medium buffered to pH about 3, in the presence of a concentration of about 20 μM per 10 ⁶ cells / ml of the exogenous peptide of interest. Thanks to the method according to the invention, it is possible to charge the MHC molecules with a peptide of low affinity for these molecules (that is to say having a Kd greater than 0.5 μM ^"1 ).

Advantageously, the exogenous peptide is a viral or tumor antigen peptide having from 8 to 10 amino acids for peptides presented by MHC Class I and 12 to 15 amino acids for peptides presented by MHC Class II.

The cells obtained by the method according to the invention loaded in a homogeneous or quasi-homogeneous manner (more than 90% of the endogenous peptides are detached by treatment in acid medium) with a given peptide can become cells presenting the antigen with a high efficiency. . The method according to the invention is implemented on any source of lymphoid cells preferably on lymphocytes of peripheral human blood, human spleen cells or cells of any other origin (lymph nodes, cord blood, placenta, etc. .), due to the fact that some of them are already "professional" antigen presenting cells, for example dendritic cells and consequently will become after the step of "stripping" and loading with a specific antigen peptide cells presenting the antigen with great efficiency.

The subject of the invention is also a population of non-tumor living cells of mammals, in particular of human origin, in particular of spleen cells, of peripheral blood lymphocytes, of lymph node cells, of cord or placenta blood cells. , characterized in that the cells have on their surface a density of the same specific exogenous peptide associated with MHC molecules and of the same allelic restriction as the latter substantially greater than that of corresponding cells expressing said exogenous peptide associated with molecules of the MHC in the native state, the MHC molecules being able to be of Class I classic, Class I nonclassical or Class II. Advantageously, the cells according to the invention tion include an amount of MHC molecules greater by a factor of about 10, preferably about 100 and up to more than 1000 for peptides of low affinity compared to cells presenting these peptides in the native state.

Thus, the cells comprising 10 ⁶ molecules of the MHC can comprise at least 10 ⁵ molecules of the said exogenous peptide associated with the molecules of the MHC and advantageously from 10 ⁵ to 10 ⁶ molecules of the said peptide, preferably 9.10 ⁵ molecules of the said peptide.

The invention also relates to a mixture of cells comprising a population as defined above.

This population can be used as a therapeutic composition in order to stimulate the production of cytotoxic lymphocytes against a bacterial, viral, parasitic or tumor antigen or of the immunological self, in a patient suffering from an infection, a tumor affection or autoimmune, for which there are identified antigenic peptides capable of binding to the MHC.

Examples of viral conditions are those caused by HIV or influenza. An example of a bacterial condition is tuberculosis. An example of a parasitic condition is malaria. An example of a tumor condition is melanomas.

It can also be used for the treatment of autoimmune diseases for which the idiom has been characterized, in particular systemic lupus erythematosus or rheumatoid arthritis.

In this case, the specific antigen will be a B cell idiocyte antigen.

The invention also relates to a method of therapeutic treatment of a host suffering from a condition for the treatment of which intervenes. stimulation of cytotoxic lymphocytes, in particular an infectious, tumor or autoimmune disease, characterized in that a sample of host cells expressing MHC molecules is taken, for example a sample of ganglion cells, of cells peripheral blood or spleen cells of the host, which these cells are subjected to a treatment to detach the peptides of endogenous and possibly exogenous origin associated with MHC molecules, in the presence of an antigenic peptide specific for the condition to be treated in order to charge the MHC molecules with said specific antigenic peptide, and in that the sample composed of a population of cells having a high density of said specific antigenic exogenous peptide associated with the molecules of the MHC to said host, in order to obtain> the proliferation and activation of cytotoxic lymphocytes specific for said antigenic peptide.

Advantageously, the host is injected with an amount of approximately 10 ⁴ to 10 ⁹ cells highly charged with exogenous peptide of interest.

Insofar as the cells injected into the host are autologous cells, this method makes it possible to overcome the allelic contingencies linked to the MHC (HLA system in humans), provided that at least one peptide presentable by at least one of the MHC alleles is known. If such a peptide is not known, possible alternative methods consist in bringing the population of host cells in the presence of a mixture of antigenic peptides, at least one of the peptides of the mixture being capable of being presented by MHC molecules, or to put the host cells in the presence of the tryptic digestion products of a polypeptide or protein, and to load the "empty" MHC molecules with these peptides. These peptides can possibly be non-optimal. In this case, after their binding to the MHC molecules, the unbound part can be cleaved, by digestion, using suitable proteases well known to those skilled in the art. Another advantage of this method is that it is not necessary to carry out an additional step of purification of the rare cells presenting the "professional" antigen.

There will be described in detail below embodiments of the invention using peptides presentable by MHC molecules of Class I with reference to the appended figures in which:

- Figure 1 shows the results of the binding of a peptide bank to mouse P815 cells before (column 1) and after treatment (column 2) according to the invention;

- Figure 2 shows the results of the binding of a peptide bank to RMA-S cells treated according to the invention; - Figure 3 shows the results of the activation of a T cell hybridoma by P815 cells treated according to the invention;

- Figure 4 shows the results of primary induction of CTL from naive mouse splenocytes;

- Figure 5 shows the in vivo results of inhibition of tumor growth obtained with cells treated according to the invention;

- Figure 6 shows the results of primary induction of CTL from human peripheral blood.

EXAMPLES A table will be given below describing the characteristics of the exogenous peptides used in the examples. PEPTIDE SEQUENCE RESTRICTION

Cw3 RYLKNGKETL K "

NP TYQRTRALV κ ^>

P198 KYQAVTTTL K *

Ova8 SIINFEKL κ ^b

P1A LPYLGWLVF L ^d

VSV RGYVYQGL K ^b

Gag QMKDCTERQ HLA-A2

Env LWVTVΎYGV HLA-A2

Pol ILKEPVHGV HLA-A2

NP2 ASNENMETM D ^b

P53A (25-34) LLPENNVLS HLA-A2.1

P53B (65-73) RMPEAAPPV HLA-A2.1

P53C (187-197) GLAPPQHLIRV HLA-A2.1

P53D (339-347) EMFRELNEA HLA-A2.1

MAGE-3 (101) ALSRKVAEL HLA-A2.1

MAGE-1 (150) SLQLVFGIEL HLA-A2.1

HER2 / neu A HLYQGCQW HLA-A2.1

HER2 / neu B PLTSIISAV HLA-A2.1

HER2 / neu C DLAARNVLV HLA-A2.1

FLU GILGFVFTL HLA-A2.1 These synthetic peptides were synthesized by NEOSYSTEM (Strasbourg) and purified by the manufacturer by HPLC.

The peptides were radiolabelled by iodization catalyzed by chloramine T.

EXAMPLE 1

Preparation of H-2 K ^d MHC mastocytoma cells loaded with exogenous peptides

The cell line used was the mastocytoma cell line P815 (H-2 ^d ) (ATCC TIB64). The test used was a quantitative binding assay of radiolabelled ^d- bound peptides K originating from a peptide bank described by Abastado (9).

The P815 cells (10 ⁷ ) were treated for 30 seconds with a stripping buffer (0.13 M citric acid, 66 mM Na ₂ H P0 ₄ , 150 mM NaCl, 17 μg / ml of phenol red) containing 4 μM from the radiolabelled peptide bank, in the absence or presence of Cw3 or VSV peptide (40 μM each).

After adjusting the pH to neutral by adding dropwise a saturated solution of Na ₂ HP0 ₄ (300 μl), the cells were washed three times with a phosphate buffered saline in order to remove the unbound, lysed peptides on ice for 5 minutes in 10 mM Tris-HCl, pH 7.6, 1% Triton X-100, 140 mM NaCl, 1 mM PMSF. The nuclei were separated by centrifugation at 15000 g for 10 minutes and the K ^d molecules immunoprecipitated with the monoclonal antibody SF1-1.1.1 (ATCC HB159).

As shown in Figure 1, the treated cells (Column 2) bound twice as much peptide as the untreated cells (Column 1). The peptide CW3 (K ^d restricted) present in an excess of 10 times during the treatment was in competition for the binding of the peptide bank to P815 cells (col. 3) unlike the peptide VSV (K ^b -restreint) (col. 4).

These results show that the stripping of the peptides of the MHC Class I molecules does not make their ability to bind peptides disappear and that

Increasing the binding of the peptide retains allelic specificity.

EXAMPLE 2

Preparation of MHC H- ^2b lymphoma cells loaded with exogenous peptide

The RMA-S cell line (Lijunggren et al. (10) is a B cell lymphoma with a mutation in the Tap2 gene.

In this cell line, the peptides derived from the peptides synthesized by the endogenous route are not transported in the lumen of the endoplasmic reticulum and the MHC class I molecules are not loaded with peptides of high affinity.

As a result, MHC Class I molecules that reach the cell surface are thermolabile. At 37 ° C, RMA-S cells express very few Class I molecules on their surface, but expression can be induced by culturing RMA-S cells at 26 ° C or by incubating them with peptides of strong affinity.

RMA-S cells were cultured for 16 hours at 26 ^β C in the presence of 2 μM of the VSV peptide. This peptide, derived from protein G of the Vesicular Stomatitis Virus, binds with a strong affinity to the K ^b molecules, but not to the D ^b molecules. As shown in FIG. 2, these culture conditions induce a high level of expression of K ^b , but not of D ^d (not shown tee).

The cells were treated as above (Example 1) in the presence or absence of different peptides (20 μM), then washed and incubated in a culture medium (complete RPMI) for 1 hour at 37 ° C., and Class I expression was tested by flow cytometry after labeling of cells by indirect immunofluorescence using the monoclonal antibody 5F1 (Sherman et al. (11)) specific for K ^b and a polyclonal antibody IgG anti-mouse goat labeled with fluorescein (Immunotech, Marseille). Figure 2 shows that the treatment in an acid medium completely abolishes the expression of K ^b at a level comparable to that of RMA-S cultured at 37 ° C. When the VSV peptide was present during the treatment in an acid medium, a high level of expression of K ^b was observed, this level being in fact higher than that obtained with untreated cells, which suggests that some of the complexes K ^b - peptide expressed on untreated cells dissociate during the two hours of incubation at 37 ^β C. On the other hand, when the peptide CW3 (binding to K ^d ) was present during the treatment, the expression profile of K ^b was superimposable on that obtained in the absence of peptide. These results show that the treatment in acid medium detaches the peptides from MHC Class I molecules and confirm that such MHC Class I molecules treated in acid medium can bind peptides and that this bond is allele specific.

EXAMPLE 3

Ability to present the antigen of cells treated according to the invention

Although cells with molecules K ^d and K ^b treated in an acid medium were capable of binding peptides in a specific way and were efficiently recognized by antibodies sensitive to the conformation (Example 1 and Example 2), it remained possible that the MHC-peptide complexes obtained from this way either had a slightly different structure or was not fully functional with respect to the presentation of the peptide.

The inventors therefore studied the recognition of cells treated in an acid medium in the presence of exogenous peptide by a specific T lymphocyte.

The P815 cells were treated in an acid medium in the presence of an excess of C 3 peptide as described above and tested for their capacity to activate the T lymphocyte hybridoma 9.4 specific for Cw3 obtained by fusing the CTL clone CW3 / 1.1 ( Maryanski et al. (12)) and the T58 ^α " ^p lymphocyte hybridoma (Letourneur et al. (13)).

For this purpose, 10 ⁵ T lymphocytes of the hybridoma were cultured in 96-well plates with the quantity of APC (cells presenting the antigen) required for 48 hours. The culture supernatants were combined and tested to assess the IL-2 content as a function of their ability to maintain in culture an IL-2 dependent CTL-L line. The proliferation of CTL-L cells was measured by the incorporation of tritiated thymidine.

As a control, untreated P815 cells were incubated in the presence of the same concentration of peptide C 3 and tested for their ability to activate the hybridoma 9.4.

As shown in FIG. 3, 300 P815 cells treated in an acid medium / well were sufficient to stimulate the secretion of IL-2 by hybridoma 94 (col. 2) while the same number of cells stimulated untreated women only provided a background signal (compare col. 2 and col. 2). Three to four times the amount of untreated P815 cells was required to achieve a similar level of stimulation of hybridoma 9.4 (compare cols 2 and 4).

EXAMPLE 4

Primary induction of CTL by autologous cells obtained according to the invention

The following peptides were used: a K-restricted peptide: Ova8 of ovalbumin and three K ^d -restricted peptides: NP, C 3 and 0198 derived from the nucleoprotein of the influenza virus, from HLA-CW3 (Maryansky et al. ( 12)) and the P815 variant antigen, respectively. Con-A blasts of naive DBA / 2 and C57 bI / 6 mice obtained by incubation for two days of 5 x 10 ⁶ splenocytes in 15 ml RPMI 1640 supplemented with 5% fetal calf serum and 5 μg / ml of Concanavalin A were treated as described above in the presence of the appropriate peptides (20 μM) and co-cultured in the presence of syngeneic splenocytes.

After 5 days, the proliferation of cytotoxic lymphocytes was tested on target cells labeled with Cr ⁵¹ . As shown in FIG. 4 (top left), the splenocytes of naive C57 / B16 mice cultured in the presence of blasts treated according to the invention loaded with 0va8, lyzed the RMA-S (H-2 ^b ) cells in the presence of 'Ovaδ but not in his absence. P815 cells, even in the presence of Ovaδ, were not lysed. Similarly, naive DBA12 mouse splenocytes cultured in the presence of blasts treated according to the invention loaded with C 3, NP or P198 lyzed the P815 cells transfected with HLA-CW3 (P815-C 3) loaded with the NP peptide or the variant P198 Tuirf of P815, respectively- is lying.

Untransfected, uncharged or wild-type P815 cells were not lysed (Figure 4, top right, and bottom, right and left). These results show that the blasts treated according to the invention loaded with peptides effectively generate primary CTLs in vitro which are specific for MHC and the peptide of interest.

EXAMPLE 5

In vivo primary immunization test in mice

ConA blasts (2 x 10 ⁷ ) of naive C57BL / 6 mice, which were treated as described above in the presence of 20 μM of Ovaδ or only loaded with 0va8 without having been treated in an acid medium were injected by the peritoneal to the same mice. After 7 days, the mice were injected with 10 ⁷ cells of the EG7 transfectant (Moore et al. (14)). These cells are derived from murine EL4 (H- ^2b ) lymphoma (ATCC TIB 39) by transfection of chicken ovalbumin cDNA. Control mice were immunized with 2 × 10 ⁷ irradiated EG7 cells, known to generate CTL specific for Ovaβ in vivo (Moore et al. (14)). As shown in FIG. 5A, mice to which the blasts treated according to the invention had been injected rapidly rejected the EG7 tumor. In fact, rejection was faster than for mice immunized with the irradiated tumor itself. Mice which had received no injection or which had been injected with conA blasts loaded with the peptide in the absence of treatment in an acid medium, did not reject the tumor during the duration of the experiment (30 days).

Additional studies were carried out in order to determine whether the method according to the invention could be effective in immunizing an animal against a syngenic tumor, in this case P815 (a more physiological system). To this end, 10 ⁷ conA blasts treated as described above and loaded with the peptide P1A (tumor system A, B, Lethe et al .; (7)) were injected 4 times over 3 days. .

These mice were then injected with 10 ⁶ P815 tumor cells. As shown in Figure 5B, these mice quickly rejected the tumor. Mice receiving injections of untreated blasts were unprotected, while mice receiving irradiated P815 cells rejected the tumor.

EXAMPLE 6 Results of primary induction of CTL in

Man

PHA-blasts derived from peripheral human blood from healthy HLA A2-1 donors were used.

Peripheral blood lymphocytes (PBL) were isolated on a Ficoll, Hypaque gradient. The pHA blasts were obtained by incubation for 2 days of 3 x 10 ⁶ PBL in 15 ml of RPMI 1640 supplemented with 20% fetal calf serum, 1 mM sodium pyruvate, phytohemagglutin (PHA) (dilution 1 / 1000, Difco) and 10 " ⁸ of phorbol acetate myristate (PMA). Primary human CTLs were obtained in a mixed culture of lymphocytes. PBL (10 ⁶ ) from HIV-negative donors were mixed with 10 ⁶ autologous blasts treated as described above, loaded with 20 μM of peptide Gag or Env of HIV in 2 ml of RPMI 1640 supplemented with 1 mM of sodium pyruvate in the presence of fetal calf serum.

After 5 days of co-culture in the presence of naive autologous PBLs, the primary CTL activity was detected. As shown in FIG. 6A, the PBLs co-cultivated with the PHA-blasts treated according to the invention in the presence of the Gag peptide lyzed the T2 lymphocytes loaded with Gag, but not the T2 lymphocytes loaded with an indifferent peptide (Pol). Similarly, the PBLs co-cultivated with the PHA blasts treated in the presence of Env, specifically lyzed the T2 lymphocytes loaded with Env (FIG. 6B).

The same type of experiment was carried out with four peptides of the suppressed oncogene suppressor P53 (P53 A, B, C, D), two peptides of melanoma (MAGE-1 and MAGE-3), three peptides of the HER2 / neu oncogene (HER2 / neu A, B, C) and the influenza peptide. In each of the experiments carried out, the peptide used, after loading onto PBLs, also allowed induction of autologous CD8 ⁺ T lymphocytes.

BIBLIOGRAPHICAL REFERENCES

1. NAIR S. et al., 1992, J. Exp. Med. 175: 609

2. OJCIUS D. et al., 1994, Crit. Reviews in Immunol. 14 (324) 193: 220

3. OJCIUS D. et al., 1993, J. Immunol.

4. ROMERO P. et al., 1991, J. Exp. Med. 174: 603

5. ROETZSCHKE O., 1990, Nature (London) 348: 252

6. ENGELHARD V., 1994, Curr. Opin. in Immunol. 6:13

7. LETHE B. et al., 1992, Eur. J. Immunol. 22: 2283

8. FALK K. et al., 1991, Nature 351: 290

9. ABASTADO J.-P. et al., 1993, Eur. J. Immunol. 23: 1776

10. LJUNGGREN H.-G., 1985, J. Exp. Med. 162: 1745

11. SHERMAN L. et al., 1981, Immunogenetics 12: 183

12. MARYANSKI J. et al., 1986, J. Immunol. 136: 4340

13. LETOURNEUR F. et al., 1989, Eur. J. Immunol. 19: 2269

14. MOORE M. et al., 1990, Cell 60: 397

Claims

1. Method for generating a population of living cells having on their surface a high density of a specific exogenous peptide associated with MHC molecules, characterized in that a population of native cells having MHC molecules loaded with peptides is treated of endogenous and possibly exogenous origin, in order to detach said peptides therefrom, and in that the MHC molecules are loaded with said specific exogenous peptide with the same allelic restriction as the MHC molecules.

2. Method according to claim 1, characterized in that the peptides associated with the molecules of

MHCs are detached from said molecules by treatment in an acid medium at pH <5 or in a basic medium at pH> 9.

3. Method according to claim 1 or claim 2, characterized in that the MHC molecules are class I classics and non-classics.

4. Method according to claim 2 or 3, characterized in that the treatment is carried out in citric acid medium buffered to pH about 3 for a period of between 10 seconds and 1 minute, preferably for about 30 seconds, in the presence of 'an excess of the specific antigenic peptide.

5. Method according to any one of the preceding claims, characterized in that the exogenous antigenic peptide is a peptide of low affinity for MHC molecules with the same allelic restriction.

6. Method according to claim 5, caracté¬ ized in that the specific exogenous peptide is an antigenic, bacterial, parasitic, viral or tumor peptide.

7. Method according to any one of the preceding claims, characterized in that the cells are lymphocytes from human peripheral blood, human spleen cells, lymph node cells, cord blood cells or placenta cells.

8. Method according to claim 7, caracté¬ ized in that the cells are cells presenting the antigen in the native state.

9. Population of non-tumor living mammalian cells, in particular spleen cells of peripheral blood lymphocytes, lymph node cells, cord blood cells or placenta cells, of human origin, characterized in that the cells have on their surface a density of the same specific exogenous peptide associated with MHC molecules and with the same allelic restriction as these substantially greater than those of corresponding cells expressing said exogenous peptide associated with MHC molecules in their native state.

10. Population according to claim 9, characterized in that the cells comprise at least 10% and preferably at least 90% of allelic MHC molecules loaded with the specific peptide.

11. Population according to claim 9 or

10, characterized in that the exogenous peptide is a peptide of low affinity for MHC molecules, in particular a viral or tumor antigen.

12. Population according to any one of claims 9 to 11, characterized in that the cells are cells presenting the antigen in the native state.

13. Population according to any one of claims 9 to 12, characterized in that it is obtained by the method defined in any one of claims 1 to 8.

14. Therapeutic composition characterized in that it comprises a population of cells according to any one of claims 9 to 13.

15. The therapeutic composition according to claim 14, intended to be administered to a determined human being, characterized in that the cell population consists of cells autologous of said human being.

16. Method for the therapeutic treatment of a host suffering from a disease for the treatment of which a stimulation of cytotoxic lymphocytes, in particular an infectious, tumor or autoimmune disease, takes place, characterized in that a sample of cells is taken of the host expressing MHC molecules, for example a sample of lymph node cells, peripheral blood cells or host spleen cells, which are subjected to treatment to detach the original peptides endogenous and possibly exogenous associated with MHC molecules, in the presence of an antigenic peptide specific for the condition to be treated to charge the MHC molecules with said specific antigenic peptide, and in that the compound sample is reinjected a population of cells having a high density of said specific antigenic exogenous peptide associated with MHC molecules to said host, in order to obtain to stop the proliferation and activation of specific cytotoxic lymphocytes of said antigenic peptide.