Dendritic cells; methods

Details Dendritic cells; methods Dendritic cells; methods

2001-09-18

2003-12-09

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C435S372000, C435S325000, C435S070400, C424S093210, C530S351000

Reexamination Certificate

active

06660523

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to methods of making and using certain defined subsets of dendritic cells, more particularly, to methods of in vitro production of a subset of dendritic cells which produce large amounts of interferon.
BACKGROUND
The circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid and myeloid cell lineages. See, e.g., Rapaport (1987)
Introduction to Hematology
(2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987)
Blood: Textbook of Hematology,
Little, Brown and Co., Boston, Mass.; and Paul (ed. 1993)
Fundamental Immunology
(3d ed.) Raven Press, N.Y.
Dendritic cells (DCs) are the most potent of antigen-presenting cells. See, e.g., Paul (ed. 1993)
Fundamental Immunology
3d ed., Raven Press, NY. Antigen presentation refers to the cellular events in which a proteinaceous antigen is taken up, processed by antigen presenting cells (APC), and then recognized to initiate an immune response. The most active antigen presenting cells have been characterized as the macrophages (which are direct developmental products from monocytes), dendritic cells, and certain B cells. DCs are highly responsive to inflammatory stimuli such as bacterial lipopolysaccharides (LPS) and cytokines such as tumor necrosis factor alpha (TNF&agr;). The presence of cytokines and LPS can induce a series of phenotypic and functional changes in DC that are collectively referred to as maturation. See, e.g., Banchereau and Schmitt
Dendritic Cells in Fundamental and Clinical Immunology
Plenum Press, NY.
Dendritic cells can be classified into various categories, including: interstitial dendritic cells of the heart, kidney, gut, and lung; Langerhans cells in the skin and mucous membranes; interdigitating dendritic cells in the thymic medulla and secondary lymphoid tissue; and blood and lymph dendritic cells. Although dendritic cells in each of these compartments are CD45+ leukocytes that apparently arise from bone marrow, they may exhibit differences that relate to maturation state and microenvironment. Maturational changes in DCs include, e.g., silencing of antigen uptake by endocytosis, upregulation of surface molecules related to T cell activation, and active production of a number of cytokines including TNF&agr; and IL-12. Upon local accumulation of TNF&agr;, DCs migrate to the T cell areas of secondary lymphoid organs to activate antigen specific T cells.
Many factors have been identified which influence the differentiation process of precursor cells, or regulate the physiology or migration properties of specific cell types. See, e.g., Mire-Sluis and Thorpe (1998)
Cytokines
Academic Press, San Diego; Thomson (ed. 1998)
The Cytokine Handbook
(3d ed.) Academic Press, San Diego; Metcalf and Nicola (1995)
The Hematopoietic Colony Stimulating Factors
Cambridge University Press; and Aggarwal and Gutterman (1991)
Human Cytokines
Blackwell. These factors provide yet unrecognized biological activities, e.g., on different untested cell types.
However, dendritic cells are poorly characterized, both in terms of responses to soluble factors, and many of their functions and mechanisms of action. The absence of knowledge about the physiological properties and responses of these cells limits their understanding. Thus, medical conditions where regulation, development, or physiology of dendritic cells is unusual remain unmanageable. The present invention addresses these issues.
SUMMARY OF THE INVENTION
The present invention is based, in part, upon the surprising discovery of conditions which result in large numbers of viable type I IFN producing cells, or pDC2 cells. The invention provides methods comprising contacting CD34
++
CD45RA
−
early haematopoietic progenitor cells with an effective amount of FLT3 ligand ex vivo, thereby inducing differentiation of the cells to IFN producing DC. Typically, the effective amount is at least 70 ng/ml; the contacting is for at least 15 days; the IFN producing DC produce at least 5000 pg IFN per 20,000 cells over 24 h after viral stimulation; the early progenitor cells expand at least about 10 fold; and/or the IFN producing cells number at least 2.5 million. In a preferred embodiment, the contacting is with TPO, and the early progenitor cells expand at least 30 fold. In other embodiments, the early progenitor cells expand at least 100 fold; or after the expansion, at least 3% of the resulting cell culture is IFN producing DC; or the IFN producing DC accumulate in 24 h at least 40,000 pg IFN per 20,000 cells after viral stimulation.
In other embodiments, the invention provides methods of producing IPC comprising contacting IPC precursors with an effective amount of a combination of both FLT3 Ligand and TPO. Preferably, the contacting is for at least 13 days; the precursors are CD34
++
CD45RA
−
early haematopoietic progenitor cells; the IPC accumulate in 24 h at least 5000 pg IFN per 20,000 IPC after viral stimulation; and/or the IPC number at least 1×10
7
cells. Typically, the contacting is ex vivo.
In yet another embodiment, the invention provides populations of at least 3×10
6
viable IPC derived from a single individual, e.g., at least 7, 10, or 15×10
6
cells. Preferably, cells are cultured in the presence of both FLT3 Ligand and TPO to produce the IPC, e.g., in vitro for at least 14 days where the FLT3 ligand is at least 70 ng/ml; and/or the TPO is at least 70 ng/ml. Typically, the IPC are CD34
−
CD45RA
++
CD4
+
IL-3R&agr;
++
cells.
DETAILED DESCRIPTION OF THE INVENTION
Outline
I. General
A. IPC
B. Developmental pathway
II. Producing IPC
A. FLT3 Ligand
B. thrombopoietin (TPO)
C. Other Molecules
III. Uses
I. General
Natural Interferon-&agr; producing cells (IPC) are key effector cells in anti-viral innate immunity. These cells produce up to 1000 times more IFN-&agr; than other blood cell types in response to viral stimulation. IPCs also have the capacity to become dendritic cells, which are key antigen presenting cells in the induction of T cell mediate immune responses.
Upon viral stimulation, the natural IFN-&agr;/&bgr; producing cells (IPCs, also known as pre-DC2) in human blood and peripheral lymphoid tissues rapidly produce very large amounts of IFN-&agr;/&bgr;. After performing this innate anti-viral immune response, IPCs can differentiate into dendritic cells and strongly stimulate T cell mediated adaptive immune responses. Using four-color immunofluorescence flow cytometry, the developmental pathway has been mapped herein to pre-DC2/IPCs from CD34
+
heamatopoietic stem cells in human fetal liver, bone marrow, and cord blood. At least four developmental stages have been identified, including CD34
++
CD45RA
−
early progenitor cells, CD34
++
CD45RA
+
late progenitor cells, CD34
+
CD45RA
++
CD4
+
IL-3R&agr;
++
pro-DC2, and CD34
−
CD45RA
++
CD4
+
IL-3R&agr;
++
pre-DC2/IPCs. Pro-DC2s already have acquired the capacity to produce large amounts of IFN-&agr;/&bgr; upon viral stimulation and to differentiate into DCs in culture with IL-3 and CD40-Ligand. The expression of pre-T cell receptor (TCR) alpha chain mRNA by both pro-DC2 and pre-DC2 supports the lymphoid origin of the pre-DC2/IPC lineage. CD34
++
CD45RA
−
early progenitor cells did not have the capacity to produce large amounts of IFN-&agr;/&bgr; in response to viral stimulation, however they can be induced to undergo clonal expansion and differentiation into IPCs/Pre-DC2 in culture with FLT3-Ligand.
Dendritic cells (DCs) represent heterogeneous populations of heamatopoietic-derived cells that display potent ability to induce primary T cell activation, polarization, and in certain circumstances tolerance. See Sousa, et al. (1999)
Curr. Op. Immunol.
11:392-399; Sallusto and Lanzavecchia (1999)
J. Exp. Med.
189:611-614; Banchereau and Steinman (1998)
Nature
392:245-252; Cella, et al.

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