METHOD FOR PRODUCING CYTOLYTIC T CELLS SPECIFIC FOR A COMPLEX OF PEPTIDE AND MHC-MOLECULE

Abstract: This invention relates to methods of obtaining cytolytic T cell subpopulations specific for a peptide/MHC complex.

Description: METHOD FOR PRODUCING CYTOLYTIC T CELLS SPECIFIC FOR A COMPLEX OF PEPTTDE AND MHC-MOLECULE Field of the Invention This invention relates to methods of obtaining cytolytic T cell subpopulations specific for a peptide/MHC complex. Background of the Invention The basis of immunity is the immune system's ability to recognize foreign molecules (antigens) and react with them, while at the same time tolerating molecules of the body's own tissues. Lymphocytes are cells responsible for recognizing and differentiating the various antigens which the immune system encounters. Each lymphocyte carries a surface receptor which is capable of recognizing a particular antigen. Although each lymphocyte carries only one type of receptor and therefore can only recognize one type of antigen, different lymphocytes have different receptors, so that the lymphocyte population as a whole can recognize a wide range of antigens. Lymphocytes are of two main types; namely B cells which develop in the bone marrow or fetal liver and can differentiate into antibody-producing plasma cells, and T cells which differentiate in the thymus and serve a number of different functions. The antigen receptors used by B and T cells are different. B cells can recognize an unmodified antigen molecule, either free in solution or on the surface of other cells, whereas T cells can recognize antigen only when it is presented to them in association with molecules encoded by the major histocompatibility complex (MHC). Cells which present the antigen are called antigen presenting cells ("APCs"). In humans, MHCs are called "HLAs" or "human leukocyte antigens". Antigen presenting cells take up, sequester in intracellular compartments, and process the antigen, and then present the processed antigen at the cell surface membrane in an energy-requiring, dynamic process that requires cell viability. There is considerable circumstantial evidence that for most soluble protein antigens, the re- expressed surface antigen that is recognized by T cells is not in native form. Kinetic studies have shown an approximately 1 hour delay between initial antigen binding to presenting cells and effective antigen presentation to T cells. Agents that raise lysosomal pH such as ammonia and chloroquine inhibit the development of effective antigen presentation when given immediately after antigen binding to presenting cells but not at later times. All of these findings have led to the concept of "antigen processing", which, based on the information referred to above, has been interpreted to involve the obligatory partial degradation of internalized antigen by presenting cells followed by the re-expression on the surface membrane of antigenic fragments in a form that can be recognized in association with la molecules by T cells. In fact, the mechanism by which a protein is processed and presented on a cell surface has now been fairly well documented. A cursory review of the development of the field may be found in Barinaga, "Getting Some 'Backbone': How MHC Binds Peptides", Science 257: 880 (1992); also, see Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992). These papers generally point to a requirement that the peptide which binds to an MHC/HLA molecule be about nine amino acids long (a "nonapeptide") and that certain MHC molecules are prefer certain amino acids at certain positions, e.g., the importance of the first and ninth residues of the nonapeptide. Recent work suggests that other amino acids may also act as so-called "anchor" positions. Prior methods of generating cytolytic T cells (CTLs) to specific antigen/MHC complexes have exposed a mixed lymphocyte culture to an antigen presenting cell that has already processed the antigen of interest intracellularly. The antigen presenting cell initiates a response from cytolytic T cell precursors having T cell receptors that recognize the antigen/MHC complex, whereby those T cells are activated and begin cell division and proliferation. This method of generating cytolytic T cell clones specific to a certain antigen/MHC complex suffers from a number of drawbacks. In particular, it relies upon the ability of the processing cell to generate the peptide of interest, as well as a source (e.g., a larger antigen) of the peptide presented. Thus, there exists a need for improved methods of generating cytolytic T cells to specific antigen/MHC complexes. The inventors have surprisingly discovered that exposing blood mononuclear cells to peptides will bind the peptide directly to MHC molecules present on the surface of the blood mononuclear cells and that the exposed cells are capable of stimulating cytolytic T-cells specific to the complex of the peptide and MHC molecule. The blood mononuclear cells do not process the peptide intracellularly. As a result, the method of the invention provides for a method to obtain cytolytic T- cells specific to a particular peptide/MHC complex as well as a method to identify antigenic peptides. SUMMARY OF THE INVENTION It is an object of the invention to provide an improved method for obtaining cytolytic T-cells specific for a peptide/MHC complex. It is a further object of the invention to provide a method for identifying antigenic peptides capable of generating a cytolytic T cell response. In accomplishing these and other objects, there has been provided a method of obtaining a cytolytic T cell subpopulation specific for a peptide/MHC complex, comprising contacting a sample of blood mononuclear cells with said peptide to bind said peptide directly to MHC molecules present on the surface of said blood mononuclear cells, and contacting said sample to a population of CD8β + cells under conditions favoring stimulation of any CD8β + cells specific to complexes of said peptide and said MHC molecule to which it binds. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Detailed Description of the Invention The present invention relates to an improved method of obtaining a cytolytic T cell subpopulation specific for a peptide/MHC complex by contacting a sample of blood mononuclear cells with said peptide to bind said peptide directly to MHC molecules present on the surface of said blood mononuclear cells, and contacting said sample to a population of CD8β + cells, under conditions favoring stimulation of any CD8β + cells specific to complexes of said peptide and said MHC molecule to which it binds. The peptide bound to the MHC molecule can be any peptide capable of eliciting a cytolytic T-cell response. Certain MHC molecules are known to prefer certain amino acids at certain positions in peptides. See Barinaga, Matsumura et al., and Latron, supra. PCT application PCT/US92/04354, filed on May 22, 1992, assigned to the same assignee as the subject application, and incorporated herein by reference, teaches a family of human tumor rejection antigen precursor coding genes, referred to as the MAGE family. Several of these genes are also discussed in van der Bruggen et al., Science 254: 1643 (1991). It is now clear that the various genes of the MAGE family are expressed in tumor cells, and can serve as markers for the diagnosis of such tumors, as well as for other purposes discussed therein. See also Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254:1643(1991). Studies on the MAGE family of genes have now revealed that certain peptides are presented on the surface of tumor cells, and that the presentation of the nonapeptide requires that the presenting molecule be HLA-Al. Complexes of the MAGE-1 tumor rejection antigen (the "TRA") lead to lysis of the cell presenting it by cytolytic T cells. In a preferred embodiment, the peptide is a MAGE derived peptide. Especially preferred is the MAGE-1 peptide MZ2-E, or a peptide derived from MAGE-3 and presented by HLA-A2 molecules, depicted herein as SEQ ID NOS: 2, 3 & 4. The cytolytic T cells raised in vitro according to the present invention can be administered to a patient utilizing known techniques to treat tumor-cell related conditions. The cytolytic T-cells lyse tumor cells, thereby achieving the desired therapeutic goal. The cytolytic T cells are also useful in a diagnostic context. For example, once one has obtained CTLs specific to a peptide/MHC complex, assays can be developed for the presence of that particular peptide/MHC complex, using well known techniques such as chromium release assays, and TNF release assays. The present invention also can be used to identify antigenic peptides. In this embodiment, the peptide of interest is contacted to blood mononuclear cells to bind the peptide to the MHC molecules on the surface of the cell. The cells are then contacted to a population of CD8β + cells under conditions favoring stimulation of any CD8β + cells specific to complexes of the peptide/MHC complex. The peptides are preferably 9-20 amino acids in length and, more preferably, 9-12 amino acids in length. Most preferably, they are nonamers, i.e., 9 amino acids in length. EXAMPLES EXAMPLE I Generation of CTLs Specific to Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr/HLA-Al Complex A. Reagents The medium used in the examples is "Iscove's medium" (GIBCO, Grand Island, NY), supplemented with 10% pooled ABO decomplemented human serum, L-arginine (0.55 mM), L-asparagine (0.24 mM), L-glutamine (1.5 mM) and 2- mercaptoethanol (5 x 10 "5 M). The MZ2-E peptide consists of Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr (SEQ ID NO. 1) that was lyophilized, resuspended at 2mM in water and stored at -80°C. This peptide is known to be presented by HLA-Al molecules (Traversari, et al., supra The anti-CD8β antibody is monoclonal antibody 1 A3.3 coupled to fluorescein. However, any anti-CD8β antibody may be used. Interleukin-2(IL-2) was used as an agent to stimulate the proliferation of CD8β + cells. One unit/ml is the concentration that allows half-maximal proliferation of the IL-2 dependent cell line CTLL-2. Other agents that stimulate the proliferation of CD8β + can also be used, such as other interleukins or cytokines, T cell growth factors, mitogens, and other substances well known to the art. Clone C1R-Al.cl3 was generated by cotransfecting plasmid pcDSRα having the HLA-Al gene cloned therein and plasmid pSVtkneoβ conferring resistance to geneticin into EBV-transformed B cell line C1R (HLA-A " , HLA-B ,HLA-Cw4 + ) using well known techniques not reiterated here. Clones were isolated from the genticin- resistant transfectants. Clone C1R-Al.cl3 expressed HLA-Al, as established by labelling with an anti-HLA-Al monoclonal antibody. R Isolation of CD8β + Cells Blood mononuclear cells from patient MZ2 were isolated by density gradient centrifugation and stored at -80°C in medium containing 10% DMSO. These were thawed and labelled with mAb 1A3.3 coupled to fluorescein. The CD8β + lymphocytes (20% of cells in this case) were sorted on an ATC 3000 flow cytometer, and seeded at 800 cells/well in 96 N-bottom microwells in 25 ml of Iscove's medium. C Stimulation of CD8β + lymphocytes As stimulator cells, blood mononuclear cells were thawed and resuspended at 5xl0 6 cells/ml in serum-free medium X-NIVO 10 containing 5 mg/ml of human β2- microglobulin and lOOmM of the MZ2-E peptide referred to supra. After incubating for 2 hours at 37°C, blood mononuclear cells were irradiated with 3,000 rads from a cesium source, and adjusted to 4x10 s cells/ml with medium. IL-2 was added (20 U/ml) and 25 ml of the suspension (corresponding to 10,000 stimulator cells) were added to the microwells containing the CD8β + lymphocytes obtained from step (B). The microcultures were incubated at 37°C in 8% CO 2 . On day 3, the microcultures were restimulated by the addition of 25 ml of medium containing 3 mM of the MZ2-E peptide and 10 U/ml of IL-2. On day 7, autologous blood mononuclear cells were contacted with the MZ2-E peptide exactly as above. These stimulator cells (10,000/microculture) were added in 50 ml of medium, supplemented with 10 U/ml of IL-2. On day 11, the stimulator cells were transferred into flat-bottom microwells (Νunc), and 160 ml of medium containing 10 U/ml of IL-2 was added. D. Lytic Activity of CD8β + lymphocytes On days 14, 18 and 23, aliquots of 50 ml of responder cells were transferred to V-bottom microwells to assay their lytic activity. Their volume was replaced by medium containing 5 U/ml of IL-2. On day 15 microcultures were restimulated by E peptide as follows: cells were resuspended at 1-5X10 5 cells/ml in medium containing 600 nM of peptide, and incubated at 37°C for 30 minutes. Control cells were incubated in medium without peptide. Cells were centnfuged, resuspended in medium, counted, and added (1000 target cells/microwell) to the effector cells. Microcultures with a peptide-specific lytic activity were found. For most of them this activity was found on day 14, on day 18, and on day 23. Lytic activities of microcultures of MZ2 CD8B + lymphocytes stimulated with autologous irradiated blood mononuclear cells contacted with the MAGE-1 MZ2-E nonapeptide. Percentages of specific lysis are shown for two sets of 36 microcultures assayed on days 14, 18 and 23 of the stimulation. Target cells are the EBN- transformed B cell line C1R-Al.cl3, that expresses HLA-Al but not the MZ2-E antigen, and the same cells contacted with the MZ2-E peptide. Lytic activities of microcultures that show specificity for the peptide are boxed. Twenty-three microcultures that showed specificity for the MZ2-E peptide were amplified in vitro using well known techniques. Cytolytic T cell clones were transferred into 2 ml wells and restimulated on days 28, 37 and 44 by the addition of medium supplemented with MZ2-E peptide (200 nM), IL-2 (30 U/ml) and irradiated (10,000 rads) allogeneic EBN-B cells (lOVwell) as feeder cells. On day 48 the lytic activities of the CTL clones were measured against C1R- Al.cl3 cells, contacted with the MZ2-E peptide, against the MZ2-MEL tumor cells and against antigen-loss variant MZ2-MEL E- tumor cells that had lost the expression of the MZ2-E antigen. Thirteen microcultures still lysed ( > 20% lysis at an E/T ratio of 30) the C1R-Al.cl3 cells contacted with peptide significantly. They also lysed the autologous melanoma cells, but did not lyse variant that did not present the MZ2-E antigen (See Table 2). Table 2 Lytic activities of anti-MZ2 nonapeptide microcultures and of the stable anti-MZ2-MEL CTL clones derived from these microcultures. Only microcultures that were specific for the MZ2-E peptide at day 23 are shown. Values of lysis observed at day 23 are indicated. These microcultures were then amplified and their lytic activities were assayed on days 48 and 68, with defined Effector/target ratios and with additional targets: the autologous MZ2-MEL melanoma cell line and its E- antigen-loss variant. As a control, lytic activity of anti-MZ2-E CTL clone 82/30 is shown. This CTL clone is described in SUBSTITUTE SHEET (RULE 26} PCT/US92/04354, commonly assigned, and, the contents of which are incorporated herein by reference. On days 49 and 58, the clones from the 13 microcultures were restimulated by the addition of medium supplemented with IL-2 (50 U/ml); irradiated (10,000 rads) EBN-B cells (lOVwell), and irradiated MZ2-MEL tumor cells (10 5 /well). Their lytic activities were tested again on day 68. All still lysed the MZ2-MEL tumor cells (Table 2). EXAMPLE π Generation of CTLs Specific to MAGE-3 HLA-A2 Complexes Aj. Reagents The medium used in the examples is "Iscove's medium" supplemented with 10% pooled ABO decomplemented human serum, L-arginine (0.55 mM), L- asparagine (0.24 mM), L-glutamine (1.5 mM) and 2-mercaptoethanol (5 x 10 "5 M). MAGE-3 peptides Phe-Leu-Trp-Gly-Pro-Arg-Ala-Leu-Nal (SEQ ID NO: 2) (codons 271-279; hereinafter "LB-373-1"), Ala-Leu-Ser-Arg-Lys-Val-Ala-Glu-Leu- Val (SEQ ID NO: 3) (codons 108-117; hereinafter "LB-373-2"), and Ala-Leu-Val- Glu-Thr-Ser-Tyr-Nal-Lys-Val (SEQ ID NO: 4) (codons 277-286; hereinafter "LB- 373-3"), respectively, were lyophilized, resuspended at 2mM in water and stored at - 80°C. These peptides are known to be presented by HLA-A2 molecules (See co- pending application, U.S. Serial No. 08/217,187, filed March 24, 1994, the contents of which are incorporated herein by reference). Isolation of CD8β+ Cells Blood mononuclear cells from patient LB373, whose tumor cells present HLA- A2-I- and express MAGE-3, were isolated by density gradient centrifugation and stored at -80°C in medium containing 10% DMSO. Blood mononuclear cells were thawed and labelled with mAb 1A3.3 coupled to fluorescein. The CD8B + lymphocytes were sorted on an ATC 3000 flow cytometer, and seeded at 1000 cells/well in 96 N-bottom microwells in 25 ml of medium. The final volume included 100 μl Iscove's medium supplemented with 10% human serum, asparagine-arginine- glutamine, 10 U/ml of r-hu-IL-2 and 10 ng/ml of r-hu-IL-7. Stimulation of CD8β* lymphocytes T2 cells, which present HLA-A2, but have an antigen processing defect which results in an increased capacity to present exogenous peptides (Cerundolo et al., Nature 345:449 (1990)), were resuspended in Iscove's medium without serum + 1/3 (v/v) anti human class I mab W6/32 culture supernatant, incubated at 4°C for 30 minutes, and irradiated with 100 Gy from a cesium source. The cells were then centrifuged and the pellet was resuspended in X-Vivo medium with 2.5 μg/ml human B2-microglobulin and lOO g/ml of either LB-373-1, LB-373-2, or LB-373-3 peptide. Cells were incubated for at least 1 hour at 37°C, then centrifuged, and resuspended in Iscove's medium supplemented with 10% human serum. On day zero, 3,000 T2 cells preincubated with LB-373-1, LB-373-2, or LB- 373-3 peptide were added to microwells containing the CD8β + cells obtained from step (B). On day 7, the microcultures were restimulated by addition of 100 μl of fresh medium containing 10 U/ml of r-hu-IL-2 and 10 ng/ml of r-hu-IL-7, 6,000 LB373- PBLs preincubated with LB-373-1, LB-373-2, or LB-373-3 peptide. These were prepared, as described, supra. On day 15, the CD8β + cells were transferred into flat-bottom microwells and restimulated by the addition of 100 μl of fresh medium containing 10 U/ml of r-hu- IL-2 and 10 ng/ml of r-hu-IL-7, 6,000 LB373-PBLs preincubated with LB-373-1, LB- 373-2, or LB-373-3 peptide. These were prepared, as described, supra. On day 16, because of strong proliferation, microcultures were divided in two (2/3 and 1/3). D. Lytic Activity of CD8β + lymphocytes On day 21, the lymphocytes were tested for their lytic activity on HLA-A2 + target cells preincubated with or without one of the Mage-3 peptides. On day 23, the microcultures (1/3) were restimulated by the addition of 100 μl of fresh medium containing 10 U/ml of r-hu-IL2 and 10 ng/ml of r-hu-IL7, 25,000 LB373-PBL preincubated with LB-373-1 peptide. "Positive" microcultures were then restimulated weekly by the addition of different allogeneic HLA-A2 + and MAGE-3 + irradiated melanoma cells, LG2-EBN feeder cells, IL-2 (50 U/ml) and IL4 (± 5 U/ml). The lymphocytes from the different microcultures were tested for their lytic activity on HLA-A2 target cells preincubated with or without one of the MAGE-3 peptides. In 22 microcultures out of 864, the lymphocytes had higher lytic activity on a HLA-A2 + target preincubated with Phe-Leu-Tip-Gly-Pro-Arg-Ala-Leu-Nal than on the same target without peptides. "Positive" microcultures were then restimulated weekly with different HLA-A2 + stimulator cells. The following table shows the lytic activity of some of the "CTL clones". 17.01.94 day 33 24.01.94 day 40 10.02.94 day 57 The results reported in the Table demonstrate that three CTL lines shown were able to recognize the peptide LB-373-1 when presented by HLA-A2 + targets, and thus demonstrate the operability of the subject invention. It should be kept in mind that the order of steps is not critical. For example, while it may be preferred to combine the APCs with peptide prior to contact to the CD8β + cells, it is also possible to combine the two cell types prior to adding the peptide. Similarly, one may add proliferation stimulating agents to the cells before or after they are combined. Further, the culture conditions may vary. While the examples show the use of serum free medium for culturing BMCs, any appropriate medium can be used. Other possible variations on the specific examples given herein will be apparent to the skilled artisan, and need not be elaborated upon here. SEQUENCE LISTING (1) GENERAL INFORMATION (i) APPLICANT: Coulie, Pierre; van der Bruggen, Pierre; Boon-Falleur, Thierry; (ii) TTTLE OF INVENTION: Method for Producing Cytolytic T Cells Specific for a Complex of Peptide and MHC-Molecule (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Felfe & Lynch (B) STREET: 805 Third Avenue (C) CITY: New York City (D) STATE: New York (E) COUNTRY: USA (F) ZIP: 10022 (v) COMPUTER READABLE FORM; (A) MEDIUM TYPE: Diskette, 5.25 inch, 500 kb storage (B) COMPUTER: IBM (C) OPERATING SYSTEM: PC/DOS (D) SOFTWARE: Wordperfect (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 08/261,541 (B) FILING DATE: 17-June-1994 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION (A) NAME: Bauer, John A. (B) REGISTRATION NUMBER: 32,554 (C) REFERENCE/DOCKET NUMBER: LUD 5328 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 212-688-9200 (B) TELEFAX: 212-838-3884 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acids (C) TYPE OF STRAND: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr (2) INFORMAΗON FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acids (C) TYPE OF STRAND: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Phe-Leu-Trp-Gly-Pro-Arg-Ala-Leu-Val (2) INFORMAΗON FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acids (C) TYPE OF STRAND: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPΗON: SEQ ID NO: 3: Ala-Leu-Ser-Arg-Lys-Val-Ala-Glu-Leu-Val (2) INFORMAΗON FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acids (C) TYPE OF STRAND: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Ala-Leu-Val-Glu-Thr-Ser-Tyr-Val-Lys-Val

Claims: Claims 1. Method of obtaining a cytolytic T cell subpopulation specific for a peptide/MHC complex, comprising: (a) contacting a sample consisting of blood mononuclear cells with said peptide to bind said peptide directly to MHC molecules on the surfaces of said blood mononuclear cells; (b) contacting said sample to a population of CD8β + cells, under conditions favoring stimulation of any CD8β + cells specific to complexes of said peptide and any MHC molecule to which it binds; and (c) obtaining a cytolytic T cell subpopulation specific for said peptide/MHC complex obtained from step (b). 2. Method of claim 1, wherein said peptide has a length from 9 to 12 amino acids. 3. Method of claim 2, wherein said peptide has a length of 9 amino acids. 4. Method of claim 1, wherein said MHC molecule is HLA-Al. 5. Method of claim 4, wherein said peptide is Glu-Ala-Asp-Pro-Thr-Gly- His-Ser-Tyr. 6. Method of claim 1, wherein said MHC molecule is HLA-A2. 7. Method of claim 6, wherein said peptide is Phe-Leu-Trp-Gly-Pro-Arg- Ala-Leu-Val. 8. Method of claim 6, wherein said peptide is Ala-Leu-Ser-Arg-Lys-Val- Ala-Glu-Leu-Val. 9. Method of claim 6, wherein said peptide is Ala-Leu-Val-Glu-Thr-Ser- Tyr-Val-Lys-Val. 10. The method of claim 1, further comprising contacting said blood mononuclear cells with β2-microglobulin prior to contact to said CD8β + cells. 11. The method of claim 1, further comprising contacting said blood mononuclear cells with said CD8β + cells in a serum free medium. 12. The method of claim 1, further comprising irradiating said blood mononuclear cells prior to contact to said CD8β + cells. 13. The method of claim 1, further comprising contacting said CD8β + cells with an agent which stimulates proliferation of said CD8β + cells. 14. The method of claim 13, wherein said agent is interleukin-2. 15. The method of claim 1, further comprising adding an additional quantity of said peptide to a culture containing said sample of blood mononuclear cells obtained from step(a) and said population of CD8β + cells of step (b) following contact of said sample of blood mononuclear cells obtained from step (a) to said population of CD8β + of step (b). 16. The method of claim 15, further comprising adding a CD8β + proliferation stimulating agent to said culture. 17. The method of claim 15, wherein said agent is interleukin 2. 18. A method for identifying a peptide/MHC complex capable of generating a cytolytic T cell response comprising: (a) contacting a sample consisting of blood mononuclear cells with said peptide to bind said peptide directly to MHC molecules on the surface of said blood mononuclear cells, (b) contacting said sample to a population of CD8β + cells, under conditions favoring stimulation of any T CD8β + cells specific to complexes of said peptide and any MHC molecule to which it binds, and (c) determining whether a cytolytic T cell response is generated. 19. Method of claim 18, wherein said peptide has a length from 9 to 12 amino acids. 20. Method of claim 19, wherein said peptide has a length of 9 amino acids. 21. The method of claim 18, further comprising contacting said blood mononuclear cells with β2-microglobulin prior to contact to said CD8β + cells. 22. The method of claim 18, further comprising contacting said blood mononuclear cells with said CD8β + cells in a serum free medium. 23. The method of claim 18, further comprising irradiating said blood mononuclear cells prior to contact to said CD8β + cells. 24. The method of claim 18, further comprising contacting said CD8β + cells with an agent which stimulates proliferation of said CD8β + cells. 25. The method of claim 24, wherein said agent is interleukin-2. 26. The method of claim 18, further comprising adding an additional quantity of said peptide to a culture containing said sample of blood mononuclear cells obtained from step(a) and said population of CD8β + cells of step (b) following contact of said sample of blood mononuclear cells obtained from step (a) to said population of CD8β + of step (b). 27. The method of claim 26, further comprising adding a CD8β + proliferation stimulating agent to said culture. 28. The method of claim 27, wherein said agent is interleukin 2.

Inventors: BOON-FALLEUR THIERRY, COULIE PIERRE, VAN DER BRUGGEN PIERRE, BOON-FALLEUR, THIERRY, COULIE, PIERRE, VAN DER BRUGGEN, PIERRE
Applicants: LUDWIG INST CANCER RES, LUDWIG INSTITUTE FOR CANCER RESEARCH
Application year: 1995
Main classification: G01N33
IPC codes: C12N5, G01N33