+

WO1993025661A1 - Clones heterogenes de cellules t specifiques contre le peptide proteolipidique 139-151 - Google Patents

Clones heterogenes de cellules t specifiques contre le peptide proteolipidique 139-151 Download PDF

Info

Publication number
WO1993025661A1
WO1993025661A1 PCT/US1993/005647 US9305647W WO9325661A1 WO 1993025661 A1 WO1993025661 A1 WO 1993025661A1 US 9305647 W US9305647 W US 9305647W WO 9325661 A1 WO9325661 A1 WO 9325661A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
peptide
plp
clones
seq
Prior art date
Application number
PCT/US1993/005647
Other languages
English (en)
Inventor
Vijay Kuchroo
Marjorie B. Lees
Original Assignee
President And Fellows Of Harvard College
Eunice Kennedy Shriver Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by President And Fellows Of Harvard College, Eunice Kennedy Shriver Center filed Critical President And Fellows Of Harvard College
Publication of WO1993025661A1 publication Critical patent/WO1993025661A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • MS multiple sclerosis
  • CNS myelin is the target of an immune attack, but the precise mechanisms that lead to myelin breakdown remain to be elucidated.
  • the 2 major proteins of CNS myelin are proteolipid protein (PLP) and myelin basic protein (MBP) , and both we been implicated in the principal animal
  • EAE can be induced in mice by synthetic PLP peptides, providing unequivocal evidence that PLP is encephalitogenic (Tuohy, V.K. , et al. , J. Immunol. r
  • EAE can be adoptively transferred by PLP-specific T cell lines and clones which i show no cross-reactivity to MBP (Satoh, J. et al., J.
  • the present invention relates to heterogeneous T cell clones which react in vitro with a 13 amino acid long PLP peptide 139-151 (SEQ ID NO: 1).
  • a T cell clone of the present invention is produced by immunization of a mammal with PLP peptide 139-151 (SEQ ID NO: 1) . Immunization is followed by harvesting T cells from the immunized mammal and maintaining the harvested cells under conditions appropriate for cell growth. The T cells from the im u- nized mammal are then stimulated in vitro with PLP peptide 139-151 (SEQ ID NO: 1) , thereby causing the population of PLP peptide-specific T cell clones to proliferate.
  • the T cells are cloned by diluting the cell population, followed by plating at a density likely to result in separated individual cells.
  • a T cell clone of the present invention reacts in vitro with the whole PLP molecule, the PLP peptide 139-151 (SEQ ID NO: 1) , and may also react with a truncated PLP peptide 139-151 (SEQ ID NO: 1) which con ⁇ sists of an amino acid sequence that is less than the amino acid sequence of the intact PLP peptide 139-151 (SEQ ID NO: 1) .
  • the present invention more particularly relates to a PLP peptide 139-151-specific T cell clone which expresses one of a variety of T cell receptor V ⁇ regions including V ⁇ l7, V ⁇ lO, V ⁇ 2, V ⁇ 6 and other V ⁇ regions.
  • the present invention relates to a T cell clone selected from the group of PLP peptide 139-151-specific T cell clones consisting of: 2E5, 7A5, SPL1.1, SJ.A2, 5B6 and 4E3.
  • the present invention also relates to a PLP peptide antigen in which residue 144 is altered.
  • the altered PLP peptide antigen is PLP peptide 139-151 (SEQ ID NO: 1) in which amino acid residue 144 is altered (i.e., is not the tryptophan residue which is present in PLP 139-151 (SEQ ID NO: 1) .
  • the PLP peptide antigen includes a portion, but not all, of the amino acid sequence of PLP peptide antigen residues 139-151 (SEQ ID NO: 1) , and is at least six amino acids long. Alteration of residue 144 can be accomplished by, but is not limited to, the substitution of the native amino acid at position 144 with alanine.
  • the present invention also relates to PLP peptide 139-151-specific T cell hybridomas which are produced by fusing the PLP peptide 139-151-specific T cell clones of the present invention with T cell tumor lines, thereby producing stable T cell hybridomas which express the same T cell receptors as the parent PLP peptide-specific T cell clones with which they were fused.
  • PLP peptide-specific T cell hybridomas of the present invention express one or more of a variety of T cell receptor V ⁇ regions, including V ⁇ l7, V ⁇ lO, V ⁇ 2, V ⁇ 6 and other V ⁇ regions.
  • a T cell hybridoma of the present inven ⁇ tion expresses the same T cell receptor as that expressed by one member of a panel of T cell clones consisting of 2E5, 7A5, SPL1.1, SJ.A2, 5B6 and 4E3.
  • a battery of truncated peptide fragments of an autoimmuno ⁇ genic peptide is provided. These truncated peptides are used to characterize the reactivity of T cell clones or hybridomas, and are also used to more precisely define which portions of the peptide are able to stimulate or react with T cells involved in eliciting autoimmune dis ⁇ ease.
  • the present invention is a method for producing a heterogeneous set of peptide-spe ⁇ cific T cell clones. In this method, a potential auto ⁇ immunogenic protein is assessed to determine which peptide portion(s) cause T cell-mediated autoimmune disease in a mammal.
  • T cell-mediated autoimmune disease occurs in the immunized mammal as a result.
  • T cells are harvested from the mammal and those which express T cell receptors specific for (i.e., those which recognize) the peptide portion used to immu ⁇ nize the animal (the peptide portion of interest) are identified and isolated.
  • the harvested T cells can be re-exposed to the antigenic peptide m vitro.
  • T cell clones selected in this manner can be used individually or as a set of heterogeneous, peptide antigen-specific T cell clones to design or screen potential therapeutic agents for their effectiveness against the specific T cell-medi ⁇ ated events involved in autoimmune disease.
  • the present invention also relates to a method for designing therapeutic agents against the specific T cell- mediated events involved in autoimmune disease.
  • This method involves exposing T cell clones of the present invention to each of the different truncated peptides in order to determine which portion(s) of the peptide is (are) necessary for eliciting a T cell response.
  • the individual T cell clones are then used to immunize a mammal in order to determine which T cell characteristics correlate with adoptive transfer of disease.
  • This infor ⁇ mation can then be used to design or choose highly specif- ic therapeutic agents which block or interfere with only those T cell events which are associated with autoimmune disease.
  • Non-limiting examples of such agents include antibodies directed only to the specific V ⁇ regions in ⁇ volved in disease, and antigen analogues which interfere with native autoimmunogen-induced T cell activation.
  • the present invention is a method of screening potential therapeutic agents for their effectiveness against the specific T cell-mediated events involved in T cell-mediated autoimmune disease.
  • a T cell clone of the present invention is used to produce a T cell hybridoma that expresses the same PLP peptide-specific T cell receptors as the T cell clone from which it derives. This is accomplished by fusing a PLP peptide-specific T cell .clone with cells from an immortal T cell line, thereby producing a T cell hybridoma expressing PLP peptide specific T cell receptors. The hybridoma cells are then combined with the agent to be assessed and the autoimmunogenic peptide.
  • the effect of the agent on T cell activation by the autoimmunogenic peptide is determined.
  • the occurrence of T cell activa ⁇ tion by the autoimmunogenic peptide to a lesser extent in the presence of the agent being assessed than in the absence of the agent is indicative of an effective thera ⁇ Guideic agent (i.e., one which can reduce T cell activa- tion) .
  • Figure la is a graphic representation of the results of experiments demonstrating that lymph node cells from mice immunized with PLP peptide 139-151 (SEQ ID NO: 1) proliferated when exposed to the immunizing peptide, but showed little or no response to truncated PLP peptide 141- 149 (SEQ ID NO: 5) or 141-150 (SEQ ID NO: 6) .
  • Figure lb is a graphic demonstration that lymph node cells from mice immunized with PLP peptide 141-150 (SEQ ID NO: 6) showed a significant response when exposed to that peptide and to PLP peptide 141-149 (SEQ ID NO: 5) , and they showed an even greater response to PLP peptide 139- 151 (SEQ ID NO: 1) .
  • Figure lc is a graphic demonstration that lymph node cells from mice immunized with PLP peptide 141-149 (SEQ ID NO: 5) showed a significant response when exposed to that peptide, or to PLP peptide 141-150 (SEQ ID NO: 6) , and they showed an even greater response to PLP peptide 139- 151 (SEQ ID NO: 1) .
  • Figure 2 is a graphic representation of the results of experiments demonstrating that PLP peptide 139-151 (SEQ ID NO: 1) cannot activate all of the PLP peptide 139-151- specific T cells if a single base change is made at posi- tion 144, but that changes at any of the other amino acid positions do not completely abolish the peptide's ability to activate PLP peptide 139-151-specific T cells.
  • This invention is based on the production and charac- terization of multiple, heterogeneous, PLP peptide 139- 151-specific encephalitogenic T cell clones. This inven ⁇ tion is further based on the demonstration that not all PLP peptide 139-151-specific T cell clones are encephali ⁇ togenic. This invention is further based on the unexpect- ed discovery that immunization with a 13 amino acid en ⁇ cephalitogenic peptide results in the generation of a heterogeneous population of T cells which express a vari ⁇ ety of T cell receptor V ⁇ regions and range from highly encephalitic to nonencephalitic.
  • This invention is also based on the surprising discovery that a single amino acid residue within the encephalitogenic PLP peptide 139-151 (SEQ ID NO: 1) is critical for activation of all of the different PLP peptide 139-151-specific T cells examined.
  • SEQ ID NO: 1 The availability of these T cell clones, both encephalito- genic and nonencephalitogenic, now makes it possible to study the surface phenotype and encephalitogenic charac ⁇ teristics of autoimmune-activated T cells, and to relate these characteristics to their encephalitic potential. Analyses of TCR usage by MBP-specific T cell clones in mice (Acha-Orbea, et al. , Cell. 5 ⁇ :263 ((1988)), rats
  • MBP-induced EAE in mice and rats has been sup ⁇ pressed with anti-TCR antibody reagents and by TCR-based peptide therapy (Zamvil, S.S., and L. Steinman, Annu.
  • the present invention provides a heterogeneous panel of T cell clones and hybridomas specific for a single 13-mer peptide which could be used to design and screen specific pharmaceuticals. Furthermore, because of the diversity of the clones and hybridomas, the cell lines of the present invention provide us with a situation comparable to that of in vivo. In addition, the altered PLP peptides and the PLP peptide-specific hybridomas provide novel reagents for assaying antagonists of PLP peptide-specific T cell activation. In one embodiment, the present invention is a method for producing heterogeneous autoreactive peptide-specific T cell clones.
  • a potential autoimmunogenic protein is assessed to determine which peptide portion(s) have potential to cause autoimmune disease in a mammal. This is done by immunizing a mammal with a portion of the autoimmunogenic protein, and assess ⁇ ing whether T cell-mediated autoimmune disease occurs in the immunized mammal as a result. After sufficient time has elapsed to induce T cell-mediated autoimmune disease in the mammal, T cells are harvested from the mammal and those which express T cell receptors specific for (i.e., those which recognize the peptide portion used to immunize the animal (the peptide portion of interest) are identi ⁇ fied and isolated.
  • the harvested T cells can be re- exposed to the antigenic peptide .in vitro.
  • Peptide-spe ⁇ cific T cell clones of the present invention are then isolated on the basis of their ability to recognize the immunizing peptide.
  • T cell clones selected in this manner can be used individually or as a set of heterogeneous, peptide antigen-specific T cell clones to design or screen potential therapeutic agents for their effectiveness against the specific T cell-mediated events involved in autoimmune disease.
  • Novel PLP peptide 139-151-specific T cell clones were derived as specified in Examples 1 and 5.
  • the six PLP peptide 139-151-specific T cell clones showed five differ ⁇ ent reactivity patterns to a panel of truncated and over- lapping forms of the encephalitogenic peptide.
  • Clone 5B6 reacts with truncated peptides that overlap at only two residues (144 and 145) , suggesting that peptide 139-151 (SEQ ID NO: 1) may contain more than one major histocom- patibility complex (MHC) binding site.
  • MHC major histocom- patibility complex
  • peptides may bind to the MHC molecule in only a limited way, and forma ⁇ tion of the epitope may depend upon the conformation of the peptide within the cleft of the Class II MHC molecule. Therefore, epitope diversity could be generated from discontinuous amino acid residues.
  • PLP residues 144-146 also appear to be critical for responsiveness by clone 4E3, and residues 144-148 for clone 7A5.
  • the T cell clones of the present invention were used to produce T cell hybridomas, each of which expresses the same T cell receptor as the T cell clone from which it was derived. Each T cell hybridoma reacts with the same specificity as the T cell clone from which it was derived.
  • T cell hybridomas of the present invention were exposed to a panel of alanine-substituted PLP peptide 139- 151, peptides in which alanine substitution was made at a position other than position 144 elicit a variety of responses from among the T cell hybridomas.
  • alanine substitution at position 144 abolishes PLP peptide-induced T cell activation in all 5 PLP pep ⁇ tide-specific T cell hybridomas examined.
  • the T cell clones showed variable abilities to cause autoimmune disease when they were adoptively transferred into syngeneic mice. At least two clones induced EAE in naive mice, while for one clone, pretreatment of the recipients with irradiation and pertussis led to more complete disease expression. One clone induced evidence of disease in only 1 of 14 recipients, and one clone was essentially nonencephalitogenic, even after pretreatment.
  • the present invention pro ⁇ vides a method for designing therapeutic agents against the specific T cell-mediated events involved in autoimmune disease.
  • This method first involves exposing the T cell clones of the present invention to each of the different truncated peptides in order to determine which portion(s) of the peptide is (are) necessary for eliciting a T cell response.
  • T cell clones are isolated from the animals exhibiting autoimmune disease, and their surface pheno- types and other characteristics determined. The individu ⁇ al T cell clones are then administered to mice in order to determine which T cell characteristics correlate with adoptive transfer of disease. This information can then be used to design new specific therapeutic agents or identify existing agents which block or interfere with those T cell events which are associated with autoimmune disease.
  • Non-limiting examples of such agents include antibodies directed only to the specific V ⁇ regions in ⁇ volved in disease, and autoim unogen analogues which interfere with native autoimmunogen-induced T cell activa ⁇ tion.
  • Peptide autoimmunogen analogue antagonists of autoimmunogen-induced T cell activation can be designed, for example, by creating synthetic peptides which are very similar to the peptide im unogen except for an alteration in a single amino acid residue.
  • a set of such synthetic peptides can be synthesized so that each member of the set represents an alteration at a different amino acid resi ⁇ due.
  • the autoreactive peptide-specific T cell clones of the present invention can then be exposed to the synthetic peptides, and the extent to which T cell activation occurs can be measured. If it can be determined that only a small number of amino acid residues is responsible for T . cell activation, therapeutic peptide analogues of the native autoimmunogen can be designed wherein alterations have been made at the critical amino acid residue(s) . Such analogues could potentially interfere with autoimmu ⁇ nogenic peptide-specific T cell activation when adminis ⁇ tered to a mammal.
  • a set of synthetic PLP peptide 139-151 analogues was made by substituting alanine for each amino acid residue of the peptide, in turn.
  • These 13 synthetic analogues are iden ⁇ tified as: A139 (SEQ ID NO: 10), A140 SEQ ID NO: 11), A141 (SEQ ID NO: 12), A142 (SEQ ID NO: 13), A143 (SEQ ID NO: 14), A144 (SEQ ID NO: 15), A145 (SEQ ID NO: 16), A146 (SEQ ID NO: 17), A147 (SEQ ID NO: 18), A148 (SEQ ID NO: 19), A149 (SEQ ID NO: 20), A150 (SEQ ID NO: 21), A151 (SEQ ID NO: 22) (Example 9 and Figure 2) .
  • the present invention is a ' method of screening potential therapeutic agents for their effectiveness against the specific T cell-mediated events involved in autoimmune disease.
  • a T cell clone of the present invention is used to produce a T cell hybridoma that expresses the same PLP peptide-specif- ic T cell receptor as the T cell clone from which it was derived. This is accomplished by fusing a PLP peptide- specific T cell clone with cells from an immortal T cell line, thereby producing a T cell hybridoma expressing PLP peptide specific T cell receptors.
  • Example 9 and Figure 2 show the response of 5 different PLP peptide 139-151-specific T cell hybridomas to alanine-substituted PLP peptide analogues. These results demonstrate that the peptides in which alanine substitution was made at a position other than position 144 elicit a variety of responses from among the T cell hybridoma ⁇ .
  • a battery of truncated peptide fragments of an autoimmuno ⁇ genic peptide is provided. These truncated peptides are used to characterize the T cell response to the intact peptide, and are also used to more precisely define which portions of the peptide are able to stimulate or react with T cells involved in eliciting autoimmune disease
  • T cell clone cells were incubated with anti-TCR- ⁇ / ⁇ and various anti-TCR V ⁇ -spe ⁇ cific monoclonal antibodies, washed, and then stained with an appropriate FITC-labeled secondary antibody. Cells were analyzed with a cell sorter, and at least 10,000 cells were counted in each assay. Using seven different anti-TCR V ⁇ -specific monoclonal antibodies, three TCR V ⁇ were identified by flow cytometry: V ⁇ 2 in SPL1.1, V ⁇ 6 in 5B6, and V ⁇ l7a in 2E5.
  • TCR V ⁇ usage by a long term PLP 139-151 (SEQ ID NO: 1) peptide-specific T cell line (SJL.T) derived from a single mouse. This T cell line expressed several different TCR V ⁇ (V ⁇ 2, 20%; V ⁇ 6, 12%; V ⁇ l7a, 25%), each of which had been identified on the clones.
  • TCR V ⁇ usage was analyzed by Northern blotting. Total RNA (25 ⁇ g) from each of the PLP peptide-specific T cell clones was electrophoresed and transferred. The blots were then probed with various 32 P-labeled V ⁇ cDNA fragments. This analysis showed that RNA from clones SPL1.1 and 2E5 specifically hybridized with the V ⁇ 2 and V ⁇ l7 probes respectively, thus confirming the flow cyto- metric data. Clones 7A5 and SJ.A2 hybridized with the V ⁇ lO probe, and not with any other probes tested. TCR V ⁇ lO has recently been cloned and sequenced from the T cell clone 7A5.
  • Anti-Ly6c monoclonal antibody had no effect on antigen-mediated proliferative responses of most clones, but augmented antigen-specific proliferation of clones 7A5, SJ.A2 by a factor of 2 (see Table 6; experi ⁇ ment 2) .
  • the experiments described in Example 8 were conducted to determine whether tl-e clones induce EAE.
  • the clone cells showed variable encephalitogenic potencies when they were adoptively transferred into syngeneic mice. At least two clones induced EAE in naive mice, whereas for one clone, pretreatment of the recipients with irradiation and pertussis vaccine led to more complete disease expres ⁇ sion.
  • One clone only induced evidence of disease in 1 of 14 recipients, and one clone was essentially nonencephal- itogenic, even after pretreatment.
  • the PLP peptide 139-151 (SEQ ID NO: 1) was substituted with alanine (A) at each residue.
  • A alanine
  • the ala- nine-substituted peptides were then used to activate T cell hybridomas in order to determine the TCR and the MHC contact residues.
  • T cell hybridomas were made of five of the PLP-specific T cell clones described above, resulting in immortal cell lines expressing the different TCRs expressed by the PLP-specific T cell clones described above. These hybridomas can be activated by exposure to an antigen which is recognized by the T-cell clone from which the T cell hybridoma derived.
  • the hybridomas were incubated with the wild type PLP peptide 139-151 (SEQ ID NO: 1) and the alanine substituted peptides.
  • the experi- mer . 1 data shown in Figure 2 demonstrate that the hybri- do ⁇ .. i can not be activated when the substitution (trypto- phan - alanine) is made at the position 144, suggesting that the tryptophan at position 144 is critical for acti- vation of all the hybridomas. Further analysis suggests that the tryptophan at position 144 is a dominant TCR binding residue.
  • mice Female SJL/J(H-2 S ) mice were purchased from the following materials and methods.
  • anti-CD4 GK-1.5
  • anti-CD8 TIB105
  • anti- Thy-1.1 HO.22.1
  • anti-Thy-1.2 HO.13.4
  • Anti-CD3 145-2C11
  • anti-TcR- ⁇ / ⁇ H57.597 antibodies were obtained from Drs. Jeffrey Bluestone (University of Chica ⁇ go) and Ralph Kubo (National Jewish Center for Immunology and Respiratory medicine, Denver, CO) , respectively. All antibodies were used as culture supernatants.
  • the peptides used in the present study included murine PLP residues 139-151 (HSLGKWLGHPDKF) (SEQ ID NO: 1), 141-150 (LGKWLGHPDK) (SEQ ID NO: 6), 141-149 (LGKWLGHPD) (SEQ ID NO: 5) and 103-116 (YKTTICGKGLSATV) (SEQ ID NO: 23).
  • Proteolipid protein (PLP) was obtained from a washed total lipid extract of bovine white matter (Folch, J., et al., Biol. Chem..
  • MBP MBP was purified from bovine brain as described by Deibler, et al. (Diebler, G.E., et al- » Prep. Biochem. 2:139-165 (1972)), and keyhole-limpet hemocyanin (KLH) was obtained from Calbiochem (LaJolla, CA.).
  • mice were immunized subcutaneously in both abdom- inal flanks on day 0 with 100 ⁇ g of PLP peptide, 50 ⁇ g of Mvcobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI) in 200 ⁇ l of an emulsion of equal volumes of water and incomplete Freund's adjuvant (Difco).
  • PLP peptide 50 ⁇ g of Mvcobacterium tuberculosis H37RA (Difco Laboratories, Detroit, MI) in 200 ⁇ l of an emulsion of equal volumes of water and incomplete Freund's adjuvant (Difco).
  • Each mouse was also injected intravenously on day 0 and on day 3 with 0.75 x 10 10 Bordetella pertussis bacilli (pertussis vaccine, lot No. WF 262, Massachusetts Public Health Biologies Laboratories, Boston, MA) .
  • Inguinal lymph node cells from 8- to 10-week-old female SJL mice were obtained 8 days after immunization with PLP peptide 139-151 (SEQ ID NO: 1).
  • the lymph node cells were stimulated in vitro with 10-30 ⁇ g/ml of the immunizing peptide for 3 days.
  • Live lymphoblasts were separated by Ficoll-Hypaque density gradient centrifuga- tion and maintained in Dulbecco's minimum essential medium (DMEM) containing 10% human T cell growth factor (TCGF; Advanced Biotechnologies, Columbia, MD) or murine T cell growth promoter (TCGP; American Biotechnologies, Cam- bridge, MA).
  • DMEM Dulbecco's minimum essential medium
  • Clones were maintained by restimulation every 10-14 days with irradiated spleen cells and 10- 30 ⁇ g/ml of 139-151 (SEQ ID NO: 1) PLP peptide, and fed with DMEM containing 10% human interleukin-2, every other day. Cultures were expanded when cell densities exceeded 10 6 /ml.
  • lymph node cells were obtained 8 days after immunization with 100 ⁇ g peptide in complete Freund's adjuvant per mouse. Lymph node cells (3 x 10 s cells/well) were incubated with the indicated concentrations of anti ⁇ gen for 72 hours, [ 3 H]-thymidine was added and incubation continued for another 16-18 hours. The plates were har ⁇ vested and counted using liquid scintillation techniques, as described above.
  • mice that survived an initial attack were observed regularly and followed for 135 days prior to sacrifice.
  • Brains and spinal cords were fixed in 10% phosphate-buffered formalin, and paraffin-embedded sec ⁇ tions were stained with luxol fast blue-hematoxylin and eosin for light microscopy. Histological disease was quantified by counting the number of inflammatory foci in meninges and parenchyma as previously described (Sobel, R.A. , et al. , J. Immunol.. 112:2393-2401 (1984); Sobel, R.A.,, et ai « i J. Neuropathol. Exp. Neurol.. 49:468-479 (1990)) .
  • Samples containing at least 1 x 10 6 clone or line cells were incubated on ice for 30 minutes with 20-50 ⁇ l of hybridoma culture supernatant and washed 2 or 3 times with phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin and 0.1% NaN 3 . These samples were then incubated for 30 minutes with 25 ⁇ l of the appropri ⁇ ate fluorescein isothiocyanate (FITC)-conjugated second antibody reagent.
  • PBS phosphate-buffered saline
  • FITC fluorescein isothiocyanate
  • Second-step reagents included a 1:50 dilution of FITC-conjugated rabbit antihamster immunoglob- ulin, goat antirat immunoglobulin or goat antimouse immu ⁇ noglobulm (Cooper Biomedicals, Organon Tecknica, Malvern, PA) . After the final incubation, samples were washed twice, fixed in 1% paraformaldehyde and analyzed on a fluorescence-activated cell sorter (FACS) II or EPICS V cell sorter.
  • FACS fluorescence-activated cell sorter
  • T cell line specific for a small antigenic peptide (PLP peptide 139-151 (SEQ ID NO: 1)) comprises a heteroge ⁇ neous opulation of T cells, different members of which react with dif rent portions of PLP peptide 139-151 (SEQ ID NO: 1) .
  • T cell clones were derived from a single T cell line (SPL) specific for PLP peptide 139-151 (SEQ ID NO: 1) . Of 672 wells seeded, 8 clones arose. Of these, 4 rapidly growing clones (2E5, 4E3, 5B6 and 7A5) were selected for further characterization of their surface phenotype.
  • Samples containing approximately 1 X 10 8 cells were incu ⁇ bated for 30 minutes with 50 ⁇ l of antibody-containing culture supernatent. The cells were washed and then incubated with a 1:50 dilution of an appropriate FITC- labeled second antibody. After the final incubation, the cells were washed, fixed in paraformaldehyde and analyzed on a FACS II cell sorter. Phenotypic analysis of the line SPL and the 4 T cell clones showed that they express Thy- 1.2, CD4, CD3 and TcR- ⁇ / ⁇ but do not express Thy-1.1 and CD8.
  • T Cell line SPL and all of the T cell clones showed a strong proliferative response to the whole PLP molecule and to PLP peptide 139-151 (SEQ ID NO: 1) , but not to PLP peptide 103-116 or to other irrelevant antigens, MBP and KLH (Table 1) .
  • the response of the T cell line and T cell clones to 2 truncated PLP peptides was evalu ⁇ ated.
  • T cell clones (5 x 10 4 ) were cultured with 5 x 10 5 syngeneic irradiated spleen cells and 30 ⁇ g/ml of the indicated antigen for 72 hours; 1 ⁇ Ci of [ 3 H]-thymidine was added for the last 16-20 hours of culture.
  • b Stimulation index Mean cpm in test wells (with antigen)/ background cpm. ⁇ Background counts varied between 400 and 1200 cpm. Data are presented as stimulation index from three repli ⁇ cates; SD ⁇ 20%. None of the PLP peptides stimulated the 2 T cell clones specific for KLH (data not shown) .
  • Example 2 Disease Induction by T Cell Line and Clones This next Example describes the results of experi ⁇ ments designed to determine the encephalitogenic potential of each of the 4 T cell clones described above.
  • the data show that T cell clones of multiple specificities can be obtained from animals immunized with the encephalito ⁇ genic PLP peptide 139-151 (SEQ ID NO: 1) , and that some, but not all of the clones are encephalitogenic.
  • the T cell line and clones were transferred intravenously into SJL mice 3 days after specific activation with PLP peptide 139-151 (SEQ ID NO: 1).
  • the SPL line and 3 of the T cell clones (2E5, 5B6 and 7A5) induced clinical disease in SJL mice (Table 2) .
  • the mean day of onset of the disease for the different T cell clones varied between 6 and 11 days.
  • the affected animals showed weight loss, followed by tail weakness and ascending paralysis, leading to complete hindleg paralysis.
  • mice injected with clone 7A5 displayed typical signs of EAE
  • animals injected with clone 4E3 which has a similar fine specificity pattern (Table 1)
  • failed to induce disease when observed up to 60-70 days Table 2 .
  • the clone 7A5 induced disease only in animals that were pretreated with irradiation (390 rad) and pertussis, whereas the other cell lines (SPL, 2E5 and 5B6) did not require this type of manipulation to induce disease.
  • N/A Not applicable, ND - not determined.
  • mice were injected subcutaneously with 100 ⁇ g of 4 different syn ⁇ thetic PLP peptides in complete Freund's adjuvant with pertussis (Table 3) .
  • Peptide 139-151 (SEQ ID NO: 1) induced severe clinical disease in 9 out of 10 animals tested.
  • the truncated peptide 141-150 (SEQ ID NO: 6) also induced clinical disease in 10 out of 14 mice tested.
  • 4 out of the 5 mice developed clinical disease by day Il ⁇ ls and 2 of these mice died by day 14.
  • mice were observed for more than 6 months but the sharp relapses and favour ⁇ sions that were observed with peptide 141-150 (SEQ ID NO: 6) were not observed with this peptide.
  • Table 3 Comparison of the ability of truncated PLP peptides to induce EAE a
  • mice immunized with PLP peptide 139-151 proliferated to the immunizing peptide, but showed little or no response to the truncated peptides 141-150 (SEQ ID NO: 6) or 141-149 (SEQ ID NO: 5) .
  • mice immunized with the truncated peptide showed a significant response to the truncated peptide, and an even stronger response to peptide 139-151 (SEQ ID NO: 1) .
  • Inguinal lymph node cells were obtained from groups of 2 SJL mice, 8 days following immunization with each of the following PLP peptides: PLP 139-151 (SEQ ID NO: 1), 141-150 (SEQ ID NO: 6) , 141-149 (SEQ ID NO: 5) and 103-116 (SEQ ID NO: 23) .
  • the proliferative response of the lymph node cells to each of the peptides was ascertained.
  • Lymph node cells from mice immunized with PLP peptide 139-151 showed a strong response to the immunizing 139-151 (SEQ ID NO: 1) peptide (>45 times above back- ground) , but little or no response to the truncated pep ⁇ tides (141-150 (SEQ ID NO: 6) and 141-149 (SEQ ID NO: 5)) or to the control peptide 103-116 (SEQ ID NO: 23) (Fig. la) .
  • mice immunized with the truncated peptides 141-150 (SEQ ID NO: 6) or 141-149 (SEQ ID NO: 5) showed no cross-reactivi ⁇ ty to the control PLP peptide 103-116 (SEQ ID NO: 23), thus establishing the specificity of the response.
  • SJL mice immunized with the control PLP peptide 103-116 (SEQ ID NO: 23) did not show a significant response to this peptide or to the encephalitogenic SJL peptides described above (data not shown) .
  • the following materials and methods were used in Examples 5 through 8.
  • mice used in this study were either purchased from the Jackson Laboratory (Bar Harbor, ME) or were bred at the Harvard Medical School Animal Facility (Cambridge, MA) . SJL mice were obtained at 5 to 8 weeks of age and were immunized between 8 and 14 weeks of age. The animals were maintained in accordance with the guidelines of the Committee on Animals of the Harvard Medical School, the Institutional Use and Care of Animals Committee of the
  • CD4-producing hybridoma (GK 1.5) was a gift from Dr. Frank Fitch (University of Chicago, Chicago, IL) .
  • Anti-CD3 (145-2C11) and anti-Ly6c (144-4B11) were obtained from Dr. Jeffrey Bluestone (Ben May Institute, University of Chica- go), and anti-TCR- ⁇ / ⁇ (H57.597) was obtained from Dr. Ralph Kubo (National Jewish Center for Immunology and Respiratory Medicine, Denver, CO).
  • the following anti-TCR V ⁇ -specific monoclonal antibodies were used: hybridoma cell lines producing anti-V ⁇ 3 (KJ25a) and anti-V ⁇ l7a (KJ23a) were gifts of Drs.
  • T cell clones against this peptide show signif ⁇ icant reactivity to the intact PLP molecule (Table 4) and T cells generated against the intact PLP molecule cross- react with the serine-sub ⁇ tituted PLP peptide (Van der Veen, R.C., J.L. Trotter, W.F. Hickey, and J.A. Kapp, J. Neuroimmunol.. -26:139 (1990)). Furthermore, six of the eight truncated peptides tested do not contain residue 140 (Table 5) . Therefore, it is unlikely that the serine substitution plays a role in the heterogeneity of the response. Peptides were synthesized by Dr. Z. Lu in the laboratory of Dr. Richard A.
  • Laursen (Boston University, Boston, MA) either by the method of Houghten (Houghten, R.A., General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen- antibody interaction at the level of individual amino acids, Proc. Natl. Acad. Sci. USA. 12:5131 (1985)) or on a Milligen model 9050 synthesizer using Milligen PAL resins and FMOC chemistry. By both methods, peptides with C- terminal amides were obtained. Peptides were >90% pure, as determined by high pressure liquid chromatography.
  • the PLP peptides used were: 139-151 (HSLGKWLGHPDKF) (SEQ ID NO: 1); 139-145 (HSLGKWL) (SEQ ID NO: 2); 141-146 (LGKWLG) (SEQ ID NO: 3); 141-148 (LGKWLGHP) (SEQ ID NO: 4); 141-149 (LGKWLGHPD) (SEQ ID NO: 5) ; 141-150 (LGKWLGHPDK) (SEQ ID NO: 6); 144-151 (WLGHPDKF) (SEQ ID NO: 7); 145-150 (LGHPDK) ; 145-151 (LGHPDKF) (SEQ ID NO: 8) ; and 103-116 (YKTTICGKGLSATV) (SEQ ID NO: 23).
  • PLP was obtained from a washed total lipid extract of bovine white matter (Folch, J. , et al. , J. Biol. Chem.. 266:497, (1957)) and lipid was removed by chromatography on a Sephadex LH60 column (Biz- zozero, O.A. , et al--* J. Chro atogr.. 227:33 (1982)).
  • MBP was purified from bovine brain as described by Deibler, et al. f (Deibler, G.E., et al. , Prep. Biochem.. 2:139, (1972)) and keyhole-limpet hemocyanin (KLH) was obtained from Calbiochem (LaJolla, CA) .
  • T cell lines were independently developed from pooled inguinal lymph node cells of three to five PLP peptide 139-151-immunized (SEQ ID NO: 1) female SJL mice. Each mouse was immunized subcutaneously in the flanks with 150 ⁇ g of PLP peptide 139-151 (SEQ ID NO: 1) emulsified in CFA (Sigma Chemical Co., St. Louis, MO). The lymph node cells were obtained 8 days after immunization and were stimulated in vitro with 10 to 30 ⁇ g/ml of the immunizing peptide for 3 days in (Dulbecco's Modified Eagles Medium (DMEM) .
  • DMEM Dulbecco's Modified Eagles Medium
  • Live lymphoblasts were separated by Ficoll-Hy- paque density gradient centrifugation and maintained in DMEM containing 10% human T cell growth factor (TCGF; Advanced Biotechnologies, Columbia, MD) or murine T cell growth promoter (TCGP; American Biotechnologies, Cam- bridge, MA) .
  • TCGF human T cell growth factor
  • TCGP murine T cell growth promoter
  • Bulk cultures were restimulated three more times with the peptide in the presence of irradiated syngeneic spleen cells at approximately 10-day intervals. The two lines obtained were then cloned at 1 cell/well. Cloned cells were gradually expanded from 96- to 24-well plates. Cultures were fed every 48 to 72 hours with complete DMEM supplemented with 10% TCGF or TCGP and 10% fetal calf serum.
  • Clones were maintained by restimulation every 10 to 20 days with irradiated spleen cells and 10 to 30 ⁇ g/ml of PLP peptide 139-151 (SEQ ID NO: 1), and fed with DMEM containing 10% TCGF or TCGP every other day. Cultures were expanded when cell densities exceeded 10 6 cells/ml.
  • a KLH-specific T cell clone (SK1.F9) was derived from SJL mice for use as a control.
  • Samples containing at least 1 x 10 6 clone cells were incubated on ice for 30 minutes with 20 to 50 ⁇ l of hybri ⁇ doma culture supernatant and washed two or three times with PBS containing 0.1% bovine serum albumin and 0.1% NaN 3 . These samples were then incubated for 30 minutes with 25 ⁇ l of the appropriate fluorescein isothiocyanate (FITC)-conjugated second antibody reagent.
  • FITC fluorescein isothiocyanate
  • Second anti ⁇ body reagents included a 1/50 dilution of FITC-conjugated rabbit anti-hamster immunoglobulin (for anti-CD3, anti-TCR o ⁇ , and anti-TCR V ⁇ 3) , goat anti-rat immunoglobulin (for anti-CD4, anti-CD8, anti-TCR V ⁇ 4, anti-V ⁇ 6, anti-V ⁇ 2, anti-V ⁇ 7, and anti- 1 • 14) , and goat anti-mouse immunoglobin (for anti-Thy 1.1, anti-Thy 1.2, and anti-V ⁇ l7) (all from Cooper Biomedicals, Organon Tecknica, Malvern, PA) .
  • RNA samples 25 ⁇ g were electrophoresed into formaldehyde gels, transferred to Nitroplus 2000 membranes (Micron Separation, Westboro,
  • TCR V ⁇ cDNA probes V ⁇ 2, V ⁇ lO, and V ⁇ l7 were used. Plas id-containing fragments of TCR V ⁇ lO were generously provided by Dr. David Ferrick (University of California, Davis, CA) ; purified V ⁇ 2 was obtained from Dr. Arun Foe- tadar (LaJolla Institute of Allergy and Immunology, La- Jolla, CA) ; and V ⁇ l7 fragments were obtained from Dr. Ed Palmer (National Jewish Center for immunology and Respira ⁇ tory Medicine) .
  • Example 5 Derivation and Specificity of T Cell Clones Shown in this Example is the derivation of six PLP peptide 139-151-specific T cell clones, four of which were previously described in Example 1. In addition, this Example further demonstrates the heterogeneity of the six PLP peptide 139-151-specific T cell clones.
  • Clones were derived from two different T cell lines specific for PLP peptide 139-151 (SEQ ID NO: 1). Of 969 wells seeded, 10 clones arose and 6 rapidly growing clones were selected for further characterization. Clones 2E5, 4E3, 5B6, 7A5, and SPL1.1 were obtained from one T cell line, whereas clone SJ.A2 was obtained from a second T cell line.
  • Phenotypic analysis showed that all clones express Thy 1.2, CD4, CD3, and TCR- ⁇ , but lack Thy-1.1 and CD8.
  • Samples containing approximately 1 x 10° cells were incubated for 30 minutes with 50 ⁇ l of antibody- containing culture supernatant. The cells were washed and then incubated with a 1/40 dilution of an appropriate FITC-labeled second antibody. After the final incubation, the cells were washed, fixed in paraformaldehyde and analyzed on a Profile II 11 cell sorter.
  • prolifera ⁇ tive responses were tested with whole PLP, PLP peptide 139-151 (SEQ ID NO: 1), PLP peptide 103-116 (SEQ ID NO: 23), bovine MBP, and KLH (Table 4). All PLP peptide- derived clones tested showed specific proliferative re- sponses to PLP and PLP peptide 139-151 (SEQ ID NO: 1) , but not to the irrelevant antigens, PLP peptide 103-116 (SEQ ID NO: 23) , bovine MBP, or KLH.
  • T cell clones (5 x 10 4 cells) were cultured with 5 x 10 5 syngeneic irradiated SC and Ag for 72 hours: 1 ⁇ Ci of [ 3 H]TdR was added for the last 16 to 20 hours of culture. Data are presented as mean cpm of three replicates. SD were ⁇ 20%. Italicized values indicate at least threefold increase of counts over background.
  • c PLP and MBP were of bovine origin.
  • d Peptides were synthesized based on the mouse PLP sequence but serine was substituted for cysteine at residue 140.
  • Clone 4E3 reacted with five of the truncated peptides; clone 7A5 reacted with four of the truncated peptides; and clones SPL1.1 and SJ.A2 responded to the same three truncated peptides (141-148 (SEQ ID NO: 4), 141-149 (SEQ ID NO: 5), and 141-150 (SEQ ID NO:6)).
  • Clone 5B6 responded to six truncated peptides, including peptide 139-145 (SEQ ID NO: 2) , to which no other clone responded.
  • T cell clones (5 x 10 4 cells) were cultured with 5 x 10 5 syngeneic irradiated SC and the indicated peptide for 72 hours; 1 ⁇ Ci of [ 3 H]TdR was added for the last 16 to 20 hours of culture. Data is presented as mean cpm of .three replicates; SD were ⁇ 20%. Italicized values indicate at least threefold increase of counts over background.
  • D Peptides were synthesized based on the PLP sequence, but serine was substituted for cysteine at residue 140. Peptide concentrations were tested between 0.1 and 100 ⁇ g/ml. Maximal proliferative responses were obtained using peptide concentrations of either 10 or 100 ⁇ g/ml.
  • the six PLP peptide 139-151-specific clones showed five different reactivity patterns to the panel of trun ⁇ cated and overlapping forms of the encephalitogenic pep ⁇ tide.
  • the results with clone 5B6 suggest that peptide 139-151 (SEQ ID NO: 1) may contain more than one major histocompatibility complex (MHC)-binding site in that this clone reacts with truncated peptides that overlap at only two residues (144 and 145) . These residues may be re ⁇ quired for TCR binding, whereas the ends of the peptide may each be able to bind to the IA molecule.
  • MHC major histocompatibility complex
  • peptides may bind to the MHC molecule in only a limited way and the formation of the epitope may depend on the conformation of the peptide within the cleft of the class II MHC molecule (Kurata, A., and J.A. Berzofsky, J. Immu ⁇ nol.. 144:4526 (1990)).
  • Clone 2E5 may be an example of this type of conformationally-dependent recognition in that it reacts only with the parent peptide, and not with any of the truncated peptides. Therefore, in contrast to the previous example, epitope diversity could be generated from discontiguous residues.
  • PLP residues 144-146 also appear to be critical for responsiveness by clone 4E3 and residues 144-148 for clone 7A5.
  • the combined data . from the present study suggest that the PLP residue 144 is essential for activation of at least five of the six clones.
  • T cell clones 4E3 and 7A5 to respond to peptide 145-151 (SEQ ID NO: 8) (Table 5 and Figure 2).
  • Clones 4E3 and 5B6 both react to a short 6-mer peptide, PLP 141-146 (SEQ ID NO: 3) .
  • the 8-mer peptide PLP 141- 148 contains the shortest epitope recog ⁇ nized by clones SPL1.1 and SJ.A2, yet these clones are distinguishable based on the relative degree of reactivity with PLP peptide 141-150 (SEQ ID NO: 6).
  • the T cell clones of the present invention were used to produce T cell hybridomas, each of which expresses the same T cell receptor as the T cell clone from which it was derived. Each T cell hybridoma reacts with the same specificity as the T cell clone from which it was derived.
  • T cell hybridomas of the present invention were exposed to a panel of alanine-substituted PLP peptide 139-151, peptides in which alanine substitution was made at a position other than position 144 elicit a variety of responses from among the T cell hybridomas.
  • alanine substi- tution at position 144 abolishes PLP peptide-induced T cell activation in all 5 PLP peptide-specific T cell hybridomas examined.
  • T cell clone cells were incubated with anti-TCR-o/ ⁇ and various anti-TCR V ⁇ -specific monoclonal antibodies, washed, and then stained with an appropriate FITC-labeled secondary antibody. Cells were analyzed with a cell sorter, and at least 10,000 cells were counted in each assay. Using seven different anti-TCR V ⁇ -specific mono ⁇ clonal antibodies, three TCR V ⁇ were identified by flow cyto etry: V ⁇ 2 in SPL1.1, V ⁇ 6 in 5B6, and V ⁇ l7a in 2E5. None of the other clones reacted with monoclonal antibod ⁇ ies to these V ⁇ or to monoclonal antibodies to V ⁇ 3, V ⁇ 4, V ⁇ 7, or V ⁇ l4.
  • TCR V ⁇ usage by a long term PLP 139- 151 SEQ ID NO: 1
  • PLP 139- 151 SEQ ID NO: 1
  • This T cell line expressed several different TCR V ⁇ (V ⁇ 2, 20%; V ⁇ 6, 12%; V ⁇ l7a, 25%), each of which had been identified on the clones.
  • TCR V ⁇ usage was analyzed by Northern blotting.
  • Total RNA (25 ⁇ g) from each of the PLP peptide-specific T cell clones was electrophoresed and transferred. The blots were then probed with various 32 P-labeled V ⁇ frag ⁇ ments. This analysis showed that RNA from clones SPL1.1 and 2E5 specifically hybridized with the V ⁇ 2 and V ⁇ l7 probes, respectively, thus confirming the flow cytometric data.
  • Clones 7A5 and SJ.A2 hybridized with the V ⁇ lO probe, and not with any other probes tested.
  • TCR V ⁇ lO has recently been cloned and sequenced from the T cell clone 7A5.
  • the anti-CD8 monoclonal antibody failed to affect proliferative responses.
  • the responses of all of the clones were at least partially blocked by anti-IA s but not by anti-IA b antibody, suggesting that the encephalitogenic peptide is recognized in the context of IA S (Table 6) .
  • Anti-Ly6c monoclonal antibody had no effect on antigen- mediated proliferative responses of most clones, but augmented antigen-specific proliferation of clones 7A5 and SJ.A2 by a factor of 2 (Table 6, experiment 2) .
  • T cell clones (5 x 10 4 cells) were cultured with 5 x 10 5 syngeneic irradiated SC and 10 ⁇ g/ml of PLP peptide 139-151. + indicates Ag present; - indicates Ag not added.
  • b mAb either as as ⁇ ites (1/100 dilution) or culture super- natants (1/10 dilution), were added at the initiation of culture. Data is presented as mean cpm of three replicates: SD were ⁇ 20%. Italicized values indicate reduction by at least 33% of positive control values.
  • c - indicates mAb not added.
  • Clone 7A5 induced a low incidence of mild disease in untreated mice, but pretreatment resulted in severe disease in most of the mice (Table 7) .
  • Clone 4E3 did not induce either clinical or histologic EAE during a 60- day observation period, even after injection with 10 7 activated cells.
  • clinical and histologic features were similar to those observed after active immunization with PLP peptide 139- 151 (SEQ ID NO: 1) , and have been previously detailed above.
  • the clone cells showed variable encepha ⁇ litogenic potencies when they were adoptively transferred into syngeneic mice. At least two clones induced EAE in naive mice, whereas for one clone, pretreatment of the recipients with irradiation and pertussis vaccine led to more complete disease expression. One clone only induced evidence of disease in 1 of 14 recipients, and one clone was essentially nonencephalitogenic, even after pretreat- ment.
  • the mechanisms by which the secondary manipulations affect expression of EAE are not clear, but it is known that low doses of body irradiation can inactivate or circumvent regulatory (suppressor) circuits (Eardley, D.D., and R.K. Gershon, J. EXP. Med.. 142:524 (1975)).
  • mice were either untreated or were treated with 390 rad irradiation and injected i.v. with 10 9 B. pertussis bacilli 30 minutes before injection of T cell clones.
  • c Histologic EAE was considered present if at least two eningeal or parenchymal inflammatory cuffs were pres-ent in central nervous system tissues.
  • the PLP peptide 139-151 (SEQ ID NO: 1) was substituted with alanine (A) at each residue.
  • the alanine substituted peptides were then used to activate T cell hybridomas, to determine the T cell receptor (TcR) and the major histo ⁇ compatibility complex (MHC) contact residues.
  • T cell hybridomas were made of five of the PLP-specific T cell clones, resulting in immortal cell lines expressing the different TCRs expressed by the PLP-specific T cell clones described above. These hybridomas can be activated by exposure to an antigen recognized by the TCR expressed by the T-cell clone from which it derived.
  • the hybridomas were incubated with the wild type PLP peptide 139-151 (SEQ ID NO: 1) and the alanine substituted peptides. 16-18 hours later, supernatants were harvested and tested for the presence of IL-2/IL4 on HT-2 cell line. Proliferative response of HT-2 cells is shown as mean cpm of [ 3 H]-thymi- dine incorporation in triplicate wells. The data demon ⁇ strate that the hybridomas can not be activated when the substitution (tryptophan - alanine) is made at the posi ⁇ tion 144, suggesting that the tryptophan at position 144 is critical for activation of all the hybridomas. Further analysis suggests that the W at position 144 is a dominant TCR binding residue.
  • Tyr Lys Thr Thr lie Cys Gly Lys Gly Leu Ser Ala Thr Val

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Rheumatology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hematology (AREA)
  • Rehabilitation Therapy (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Diabetes (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention est fondée sur la découverte qu'une portion peptidique de l'acide aminé treize d'une protéine auto-immunogénique déclenche chez une souris immunisée une réponse des cellules T hétérogènes. Des clones hétérogènes des cellules T qui réagissent in vitro avec le peptide auto-immunogénique long de l'acide aminé treize ont été isolés et caractérisés. En outre, la présente invention se fonde sur la découverte qu'un seul résidu aminoacide est critique pour l'activation d'une batterie hétérogène de clones de cellules T spécifiques contre un seul peptide auto-immunogénique 13-mer. Par ailleurs, des hybridomes de cellules T ont été réalisés, dont chacun exprime un récepteur des cellules T qui est le même que le récepteur des cellules T exprimé par l'un des clones des cellules T de la présente invention. Les clones et les hybridomes des cellules T de la présente invention constituent une batterie hétérogène de clones et d'hybridomes de cellules T qui peuvent s'utiliser pour tester l'efficacité de produits pharmaceutiques contre la maladie auto-immune induite par les cellules T.
PCT/US1993/005647 1992-06-10 1993-06-10 Clones heterogenes de cellules t specifiques contre le peptide proteolipidique 139-151 WO1993025661A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US89670492A 1992-06-10 1992-06-10
US07/896,704 1992-06-10

Publications (1)

Publication Number Publication Date
WO1993025661A1 true WO1993025661A1 (fr) 1993-12-23

Family

ID=25406679

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/005647 WO1993025661A1 (fr) 1992-06-10 1993-06-10 Clones heterogenes de cellules t specifiques contre le peptide proteolipidique 139-151

Country Status (1)

Country Link
WO (1) WO1993025661A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012737A3 (fr) * 1994-10-25 1996-10-10 Immulogic Pharma Corp Compositions et traitement pour la sclerose en plaques
EP0825870A1 (fr) * 1995-04-20 1998-03-04 BRIGHAM & WOMEN'S HOSPITAL Modulation de sequences cytokiniques de clones de lymphocytes t humains autoreactifs
WO1999021982A1 (fr) * 1997-10-27 1999-05-06 Hunan Medical University Gene m6b1 humain

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
R. SOBEL ET AL.: "Myelin proteolipid protein PLP-specific T cell clones in SJL-J mice multiple functional epitopes heterogeneous T cell receptor TcR V-beta usage and induction of experimental allergic encephalomyelitis EAE.", THE FASEB JOURNAL, vol. 5, no. 6, 1991, pages A1678 *
R. SOBEL ET AL.: "The immunopathology of acute experimental allergic encephalomyelitis induced with myelin proteolipid protein. T cell receptors in inflammatory lesions.", JOURNAL OF IMMUNOLOGY, vol. 149, no. 4, 15 August 1992 (1992-08-15), BALTIMORE MD, USA, pages 1444 - 1451 *
V. KUCHROO ET AL.: "Characterization of myelin proteolipid protein-specific T cell clones in SJL mice.", JOURNAL OF NEUROCHEMISTRY, vol. 57 (SUP), 1991, NEW YORK, USA, pages S58 *
V. KUCHROO ET AL.: "Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of melin proteolipid protein. Fine specificity and T cell receptor V beta usage.", JOURNAL OF IMMUNOLOGY, vol. 148, no. 12, 15 June 1992 (1992-06-15), BALTIMORE MD, USA, pages 3776 - 3782 *
V. KUCHROO ET AL.: "Induction of experimental allergic encephalomyelitis by myelin proteolipid protein-specific T cell clones and synthetic peptides.", PATHOBIOLOGY, vol. 59, no. 5, 1991, BASEL, SWITZERLAND, pages 305 - 312 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996012737A3 (fr) * 1994-10-25 1996-10-10 Immulogic Pharma Corp Compositions et traitement pour la sclerose en plaques
EP0825870A1 (fr) * 1995-04-20 1998-03-04 BRIGHAM & WOMEN'S HOSPITAL Modulation de sequences cytokiniques de clones de lymphocytes t humains autoreactifs
EP0825870A4 (fr) * 1995-04-20 2002-09-25 Brigham & Womens Hospital Modulation de sequences cytokiniques de clones de lymphocytes t humains autoreactifs
WO1999021982A1 (fr) * 1997-10-27 1999-05-06 Hunan Medical University Gene m6b1 humain

Similar Documents

Publication Publication Date Title
Kuchroo et al. Experimental allergic encephalomyelitis mediated by cloned T cells specific for a synthetic peptide of myelin proteolipid protein. Fine specificity and T cell receptor V beta usage.
Kuchroo et al. Induction of experimental allergic encephalomyelitis by myelin proteolipid-protein-specific T cell clones and synthetic peptides
AU652540B2 (en) T cell receptor peptides as therapeutics for autoimmune and malignant disease
Markovic-Plese et al. T cell recognition of immunodominant and cryptic proteolipid protein epitopes in humans.
Martin et al. Immunological aspects of demyelinating diseases
Vanderlugt et al. Pathologic role and temporal appearance of newly emerging autoepitopes in relapsing experimental autoimmune encephalomyelitis
US5614192A (en) T cell receptor peptides as therapeutics for immune-related disease
RU2138512C1 (ru) Вакцина для профилактики или лечения опосредованной т-клетками патологии или нерегулируемой репликации клонами т-клеток, способ выделения вакцины, способ диагностирования или прогнозирования восприимчивости к ревматоидному артриту или рассеянному склерозу, способ профилактики или лечения ревматоидного артрита или рассеянного склероза и содержащий последовательность sgdqggne пептид, являющийся агентом для обнаружения, профилактики или лечения рассеянного склероза
Correale et al. Isolation and characterization of autoreactive moteolioid protein‐peptide specific T‐cell clones from multiple sclerosis patients
Merryman et al. Characterization of a new, potent, immunopathogenic epitope in S-antigen that elicits T cells expressing V beta 8 and V alpha 2-like genes.
Oshima et al. Autoimmune T cell recognition of human acetylcholine receptor: the sites of T cell recognition in myasthenia gravis on the extracellular part of the α subunit
EP0587735B1 (fr) Peptides recepteurs de lymphocytes t utilises comme agents therapeutiques contre des maladies du systeme immunitaire
Chou et al. Specificity of human T cell clones reactive to immunodominant epitopes of myelin basic protein
Abromson-Leeman et al. Experimental autoimmune encephalomyelitis-resistant mice have highly encephalitogenic myelin basic protein (MBP)-specific T cell clones that recognize a MBP peptide with high affinity for MHC class II.
KR20210041559A (ko) 다발경화증에서의 면역우세 단백질 및 단편
EP0667908B1 (fr) Chaine alpha de receptor de lymphocytes t soluble, et derives utilises comme agents prophylactiques et therapeutiques de maladies auto-immunes
WO1996040778A1 (fr) PEPTIDES Vβ5 DE RECEPTEURS DE LYMPHOCYTES T
WO1993025661A1 (fr) Clones heterogenes de cellules t specifiques contre le peptide proteolipidique 139-151
Clark et al. Shared T‐cell receptor gene usage in experimental allergic neuritis and encephalomyelitis
Kellermann et al. TSH receptor sequences recognized by CD4+ T cells in Graves' disease patients and healthy controls
Richert Human T-cell recognition of myelin basic protein peptides
JPH07242566A (ja) 免疫抑制剤
Wang et al. CD4+ T cell repertoire on the ε subunit of muscle acetylcholine receptor in myasthenia gravis
JPH10501210A (ja) 糖尿病に対するペプチドおよび方法
WO1999027957A1 (fr) Vaccination et procede de lutte contre la sclerose en plaques au moyen de peptides tcr vbeta specifiques

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载