Regulatory T cells; methods

Details Regulatory T cells; methods Regulatory T cells; methods

2001-10-03

2003-12-30

Chan, Christina (Department: 1644)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

C435S325000, C435S383000, C435S375000, C435S377000, C435S405000

Reexamination Certificate

active

06670146

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to methods of preparing immune cells in a mammal. These cells, designated regulatory T cells, produce IL-10 and can suppress antigen specific immune responses.
BACKGROUND
Interleukin-10 is a cytokine which was originally characterized by its activities in suppressing production of Th1 cytokines. See, e.g., de Vries and de Waal Malefyt (eds. 1995)
Interleukin
-10 Landes Co., Austin, Tex.; and Fowler and Powrie (1999)
Springer Semin. Immunopathol.
21(3):287-294. Th1 cells are implicated in the induction of pathology in transplantation and autoimmune contexts.
Suppression of immunological function finds utility in many different contexts. See, e.g., Paul (ed. 1998)
Fundamental Immunology
4th ed., Raven Press, NY. In particular, allogeneic immunity is important in a transplantation context, due largely to its extraordinary strength. As organ and tissue transplants become more common in medical contexts, the ability to minimize problems from tissue rejection exhibit larger economic advantages. In addition, means to minimize adverse autoimmune conditions, to block certain responses to particulate antigens, e.g., bacterial and parasitic, and to minimize the reaction to certain soluble antigens, both protein and allergens, will be significant advances for therapeutic purposes.
The lack of fully effective therapeutics to minimize or eliminate tissue rejection, graft vs. host disease, autoimmunity, or these other immunological responses leads to many problems. The present invention addresses and provides solutions to many of these problems.
SUMMARY OF THE INVENTION
The present invention is based, in part, upon the surprising discovery of methods to improve the efficiency, yield, and/or purity of regulatory T cell populations.
The present invention provides methods comprising contacting a naive T cell with a stimulatory signal and an appropriate amount of a combination of Vitamin D3 and Dexamethasone, wherein the contacting results in differentiation to a regulatory T cell. In certain embodiments, the regulatory T cell produces essentially only the cytokine IL-10; stimulatory signal is activation with an antigen or anti-CD3, e.g., where stimulation is with: antigen, e.g., HLA, and antigen presenting cells; or anti-CD3 and anti-CD28. Particularly, the contacting may be in vitro, and/or repeated two times. Typically, the amount of Vitamin D3 is about 4 times as much as Dexamethasone, and may be at least 10
−9
M; and/or at least 4×10
−9
M, respectively, and preferably at least 4×10
−8
M; and/or at least 10
−8
M, respectively.
In other embodiments, the regulatory cell suppresses the response to a defined antigen; or the contacting occurs in the presence of an antagonist of IL-4, of IFN-&ggr;, and/or IL-12. For example, the response may be a pathology inducing response; or the contacting may occur in the presence of at least two of the antagonists. Typically, the regulatory T cells produce: at least 100 ng of IL-10 per 10
6
cells; less than 1 ng of IL-4 per 10
6
cells; less than 30 pg IL-5 per 10
6
cells; and/or less than 30 pg IFN-&ggr; per 10
6
cells. The invention also provides populations of cells made by the described methods.
Further methods include those which further comprise administering the regulatory T cell to an animal with specific antigen. Preferably, the regulatory T cell and the antigen are administered simultaneously; the animal exhibits signs or symptoms of an inflammatory or autoimmune pathology; or the administering results in suppression of an inflammatory or autoimmune pathology.
Yet other methods are provided, e.g., comprising administering regulatory T cells specific for an exogenous antigen with the antigen to an animal undergoing an inflammatory or autoimmune pathology. In some embodiments: the exogenous antigen is ovalbumin; or the regulatory T cells and antigen are administered simultaneously. Preferably, the administering results in suppression of the pathology.
DETAILED DESCRIPTION OF THE INVENTION
OUTLINE
I. General
II. Regulatory T Cells
A. Vitamin D and Dexamethasone
B. Properties
III. Uses
I. General
T-regulatory cells have an important role in peripheral tolerance, but it has been difficult to isolate a homogeneous population of cells with suppressive activity in vitro and to define their mechanism of action. A CD4
+
T-regulatory cell subset has been described which is able to suppress antigen-specific immune responses in vitro and in vivo. See, e.g., U.S. Ser. No. 07/846,208, filed Mar. 4, 1992; U.S. Ser. No. 08/643,810, filed May 6, 1996; and Groux, et al. (1997)
Nature
389:737-742; each of which is incorporated herein by reference. These T cells appeared to arise after antigenic stimulation with antigen presenting cells, in the presence of exogenously added IL-10. These cells had regulatory capacity in that they inhibited the development of inflammatory bowel disease as well as T cell proliferation in vitro.
The cells are useful in that they exhibit a bystander effect, e.g., suppressing immune responses in other cells exposed to the same antigen. Thus, administration of regulatory T cells can suppress the induction of a response upon stimulation or exposure of antigen to these other cells. This property is important in the context of, e.g., suppression of response to transplantation antigens. If regulatory T cells specific for the donor antigens are available and administered to a recipient, the tissue rejection response may be suppressed. Conversely, in a bone marrow transplant, the graft immune response to host antigens may be suppressed.
Since the early methods were described, similar culture conditions have not given rise to homogeneous IL-10-producing cells. The cultures are always contaminated with IL-4-producing Th2 cells, possibly as a result of the poor growth capacity of the regulatory T cells. Type 1 T-regulatory (Tr1) cells are defined, in part, by their unique cytokine profile: they produce high levels of IL-10, significant levels of TGF-&bgr;, IL-5, and IFN-&ggr;, but no significant amounts of IL-4 or IL-2.
Many different ways were tested for isolating such homogeneous populations of CD4+ T cells, producing essentially only IL-10 and very low amounts of other tested cytokines. Immunosuppressive drugs were evaluated for their capacity to support production of such cells. To this end it was shown that culturing antigen specific CD4+ T cells with antigen and antigen presenting cells (APC) with a combination of the immunosuppressive agents Vitamin D3 (VitD3) and Dexamethazone (Dex) gives rise to a homogeneous population of IL-10-producing cells, producing little to no IL-4, IL-5, or IFN-&ggr;. Similarly, culturing antigen specific CD4+ T cells with antigen and T cell mitogens anti-CD3 and anti-CD28 (e.g., without APC) with a combination of VitD3 and Dex did likewise.
VitD3 on its own appears to drive the development of Th2 cells producing high levels of IL-4 and IL-5 and reduced IFN-&ggr;. Dex alone partially inhibited both IL-4 and IL-5 and IFN-&ggr;, and enhanced the production of IL-10. However, the cells proliferated only poorly and still produced some IL-4. The combination of both drugs led to an enhancement of IL-10 producing cells, even in the complete absence of IL-4. These resulting T cells are regulatory T cells, with properties which distinguish them from the Tr1 cells described above. In particular, these regulatory T cells do not produce IL-5 and are not IL-4 dependent.
However, the production of these IL-10 producing regulatory T cells and their proliferation is further enhanced by neutralization of IL-4, IFN-&ggr;, and IL-12 in the same cultures with neutralizing mAbs to eliminate small numbers of Th1 or Th2 cells from developing. The process is independent of IL-4 and partially dependent on IL-10 (and in some cases TGF-&bgr;). This allows the isolation of large numbers of relatively homogeneous, antigen-specific, (predominantly only) IL-10 producing cells (since the treatment does not inhibit the prolif

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