Antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance

a technology of immune tolerance and antigen-presenting cells, which is applied in the field of cell-based pharmaceuticals, can solve the problems of inability to identify these apcs, and the mechanisms they use to induce tolerance, and the molecular mechanism used by immature dcs or other putative tolerogenic apcs to suppress t cell responses, and achieves low expression levels, high expression levels, and low expression levels. ido

Inactive Publication Date: 2006-12-28
MEDICAL COLLEGE OF GEORGIA RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0011] The present invention relies on the discovery that tolerance-inducing (suppressive) antigen-presenting cells (APCs) exhibit high levels of expression of the intracellular enzyme indoleamine-2,3-dioxygenase (IDO), and non-tolerance-inducing (non-suppressive or T-cell activating) APCs exhibit low levels of IDO expression. IDO is both a marker for the suppressive subset, and also the causal mechanism of suppression. Thus, the present invention describes the generation of enriched populations of tolerance-inducing APCs and their use as therapeutics, and the generation of enriched populations of non-suppressive APCs and their use as therapeutics. For example, APCs having high levels of IDO (IDO+), and exposed to antigens from a donor may be used to increase tolerance of a transplant recipient to the donor's tissue by presenting the donor's antigens on tolerance-inducing APCs. Conversely, APCs having low levels of IDO (IDOLO) may be used to enhance responses to neo-antigens from tumors and infectious agents.
[0015] In another aspect, the present invention comprises a method for increasing the number of tolerance-inducing antigen-presenting cells (APCs) in a subject comprising treating the subject to increase the production of antigen-presenting cells (APCs) or their precursors (APC progenitors) expressing levels of indoleamine 2,3-dioxygenase (IDO) enzyme activity sufficient to suppress proliferation of T cells (IDO+ APCs).

Problems solved by technology

However, in humans and other mammals (other than mice), the identity of these APCs, and the mechanisms they use to induce tolerance, remain elusive.
However, the molecular mechanism used by immature DCs or other putative tolerogenic APCs to suppress T cell responses is unclear.
Moreover, there is currently no way to identify or isolate tolerogenic APCs in vitro or in vivo, and thus, their use as therapeutic agents is still not available for most applications.
More fundamentally, the supposition that immature DCs are tolerogenic is based on an unproven and potentially flawed model of how APCs regulate T cell activation.
The presence of a mixed population of DCs in such preparations would explain why therapeutic immunization in cancer patients using DCs remains problematic, with most studies having only limited success (M. A. Morse and H. K. Lyerly, Curr. Opin. Mol. Ther., 2: 20 (2000)).
However, this approach inherently sacrifices efficient antigen presentation and co-stimulation due to the immaturity of the APCs, and risks delivering unwanted immunizing (non-tolerogenic) DCs as part of the heterogeneous DC population.

Method used

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  • Antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance
  • Antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance
  • Antigen-presenting cell populations and their use as reagents for enhancing or reducing immune tolerance

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example 1

Cell Culture

[0219] Human monocytes and lymphocytes were isolated as separate fractions by leukocytapheresis and counterflow elutriation (D. H. Munn et al., J. Exp. Med. 189, 1363-1372 (1999)). Monocytes (typically >95% purity) were cultured in 100 mm tissue culture petri dishes in RPMI-1640 medium with 10% newborn calf serum (Hyclone) and including penicillin / streptomycin and glutamine. Cultures received either MCSF (200 U / ml, Genetics Institute) on day 0, or GMCSF (50 ng / ml, R&D Systems)+IL4 (50 ng / ml, R&D Systems) on days 0, 2 and 4. For experiments where CCR6 expression was of interest, cultures received a single dose of GMCSF+IL4 (100 ng / ml each) on day 0, with no further supplementation. Loosely adherent dendritic cells (GMCSF+IL4) were harvested by gentle aspiration; adherent macrophages (MCSF) and non-dendritic APCs (GMCSF+IL4) were harvested with EDTA. Other cultures were conducted in serum-free medium (X-vivo 15; BioWhitaker, Walkersville, Md.) plus cytokines.

example 2

Production of Antibodies

[0220] All antibodies were obtained commercially except for polyclonal antiserum against human IDO which was manufactured as a work for hire by ZCB Inc., Hopkinton, Mass. All commercial antibodies and reagents were from BD Biosciences-Pharmingen (San Jose, Calif.) unless specified otherwise. For detection of cell surface antigens, DCs were triple-stained with anti-CD123-biotin (clone 7G3; it was found that clone 9F5 gave suboptimal results with dendritic cells) followed by streptavidin-perCP, plus anti-CD11c-allophycocyanin (clone S-HCL-3) or anti-CCR6-fluorescein (clone 53103.111, R&D systems, Minneapolis, Minn.). CCR6 results were also confirmed using a second anti-CCR6 antibody (clone 11A9; Pharmingen). For detection of IDO, cells were fixed and permeablized (Cytofix / Cytoperm), and then stained with rabbit anti-IDO antibody prepared against the peptide followed by polyerythrin-labeled anti-rabbit secondary antibody (Jackson Immunoresearch, West Grove Pa.)...

example 3

Regulation of IDO Expression During DC Maturation

[0222] In humans DCs, maturation has been associated with loss of tolerogenic activity (Dhodapkar, M. V., et al., J. Exp. Med., 193: 233-238 (2001)). The experiments described in FIG. 4 addressed the issue of whether DC maturation down-regulates IDO mediated suppressor activity. Monocyte-derived DCs were cultured for 7 days in X-vivo 15 medium with GMCSF+IL4 (non-adherent cell population,>95% IDO+, >95% CD123+). During the final two days, the cells were either (A) left as immature DCs (no additions); (B) matured using a cytokine cocktail comprising TNFα, IL1β, IL6 and PGE2 (Jonuleit, H. et al., Eur. J. Immunol., 27: 3135-3142 (1997)); or (C) matured using monocyte-conditioned medium (Reddy, A., et al., Blood 90: 3640-3546 (1997)). Each group was harvested and added to 5×105 allogeneic T cells in V-bottom 96 well microtiter wells in 200 μl medium (10% fetal calf serum in RPMI)). Differing numbers of DCs were added to a fixed number of...

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Abstract

The present invention is based on the discovery antigen-presenting cells (APCs) may be generated to have predetermined levels of expression of the intracellular enzyme, indoleamine 2,3-dioxygenase (IDO). Because expression of high levels of IDO is correlated with a reduced ability to stimulate T cell responses and an enhanced ability to induce immunologic tolerance, APCs having high levels of IDO may be used to increase tolerance in the immune system, as for example in transplant therapy or treatment of autoimmune disorders. For example, APCs having high levels of IDO, and expressing or loaded with at least one antigen from a donor tissue may be used to increase tolerance of the recipient to the donor's tissue. Alternatively, APCs having reduced levels of IDO expression and expressing or loaded with at least one antigen from a cancer or infectious pathogen may be used as vaccines to promote T cell responses and increase immunity.

Description

[0001] This application is a continuation of U.S. patent application Ser. No. 10 / 121,909, filed Apr. 12, 2002.[0002] The studies described herein were supported at least in part by Federal grants from the National Institutes of Health (NIH R01 HL60137; NIH R01 HL57930; NIH R01 A144219; NIH R21 AI49849; NIH R21 AI44759; and NIH K08 HL03395), the National Institutes of Health and National Cancer Institute (NIH / NCI / RAID) and the Mason Trust Foundation. Thus, the Federal government may have rights in this invention.FIELD OF THE INVENTION [0003] The invention relates to the use of cell-based pharmaceuticals, and more specifically, to the use of antigen-presenting cells (APCs) selected as comprising immunosuppressive APCs for inducing tolerance, or immunostimulatory APCs for inducing an increased immune response. As examples, immunosuppressive APCs may be used as transplant therapeutics, whereas preparations of immunostimulatory APCs may be used as anti-cancer or anti-viral vaccines. BACK...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68G01N33/574
CPCC12Q1/6886C12Q2600/118C12N5/064G01N33/573G01N33/564
Inventor MELLOR, ANDREWMUNN, DAVID
Owner MEDICAL COLLEGE OF GEORGIA RES INST
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