Drug – bio-affecting and body treating compositions – Lymphokine – Interferon
Reexamination Certificate
2001-08-14
2004-06-08
Spector, Lorraine (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interferon
C424S085200, C424S198100, C435S002000
Reexamination Certificate
active
06746670
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to methods of preparing immune cells in a mammal. These cells, designated Tr1 cells, are a subset of regulatory T cells which 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.; etc.
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 autoimmune conditions, to block certain responses to particulate antigens, e.g., bacterial and parasitic, and to minimize 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, 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 or yields of making Tr1 cell populations. The present invention provides methods comprising contacting T cell precursor with an appropriate amount of interferon-&agr; (IFN-&agr;), wherein the contacting induces differentiation to a Tr1 cell. In certain embodiments, the Tr1 cell is characterized by CD4 expression, high level of IL-10 production, significant levels of TGF-&bgr; or IFN-&ggr; production, and/or little or no IL-4 or IL-2. In preferred embodiments, the high level of IL-10 production is at least 6000 pg in 1 ml for 10
6
cells in 48 h; the significant level of TGF-&bgr; production is at least 600 pg in 1 ml for 10
6
cells in 48 h; the significant level of IFN-&ggr; production is at least 1000 pg in 1 ml for 10
6
cells in 48 h; the little or no IL-4 is less than 200 pg in 1 ml for 10
6
cell in 48 h; and/or the little or no IL-2 is less than 200 pg in 1 ml for 10
6
cell in 48 h. In other embodiments, the Tr1 cell: has a reduced proliferative potential in response to polyclonal activation; and/or suppresses response to alloantigens by responder T cells.
In yet other methods, the Tr1 cells suppress antigen-specific activation of naive autologous T cells; the suppressed response to alloantigens is mediated by IL-10 and/or TGF-&bgr;; the T cell precursors are: CD4+; and/or cord blood leucocytes, including a peripheral blood T cell. In many embodiments, the contacting is in combination with an appropriate amount of IL-10 and/or with an antigen. Preferred antigens used in the method will be alloantigens. Methods are also provided wherein the Tr1 cells are further proliferated in IL-15 or the Tr1 cells are further tested for antigen specificity.
In another embodiment, the invention provides methods for increasing the number of Tr1 cells, comprising contacting cells with IL-15 and allowing growth, thereby resulting in an increase in the number of the Tr1 cells. In certain embodiments, the allowing growth is culturing for at least 2 days; the increase is at least 3 fold; or the cells are Tr1 cells. Often, the contacting is in a tissue culture plate; or the Tr1 cells are specifically anergic to alloantigen.
DETAILED DESCRIPTION OF THE INVENTION
OUTLINE
I. General
II. Tr1 Cells
A. IFN-&agr; and Differentiation
B. Properties
C. IL-15 Cultures
III. Uses
I. General
T-regulatory cells have an important role in peripheral tolerance, but it has been difficult to isolate 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. Pat. No. 6,277,635; and Groux, et al. (1997)
Nature
389:737-742; each of which is incorporated herein by reference. 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; and IFN-&ggr;, but no IL-4 or IL-2. Herein, it is investigated whether in vitro differentiation of human Tr1 cells from naive CD4
+
T cells is regulated by cytokines. It is shown that in cord blood T cells, IFN-&agr; induces differentiation of a population of cells with a Tr1-like profile of cytokine production. In contrast, with peripheral blood T cells, both exogenous IL-10 and IFN-&agr; were required for differentiation of Tr1 cells. Cultures with Tr1 cells had a reduced proliferative capacity in response to polyclonal activation, and a suppressed response to alloantigens. Suppression of the alloantigen response was mediated in part by IL-10 and TGF-&bgr;. The present invention is based, in part, on the definition of conditions for in vitro differentiation of human Tr1 cells. This will facilitate further characterization of this unique T-cell subset and enable their clinical use as cellular therapy to induce tolerance to foreign proteins, e.g., alloantigens.
The qualitative characteristics of immune responses are regulated by T-cell subsets through their production of distinctive cytokines. Two well-characterized T-cell subsets are the Th1 cells that, via production of IFN-&ggr;, promote cell-mediated responses against bacteria, and Th2 cells that, by producing IL-4, IL-5, and IL-13, promote antibody production, and the anti-parasite mast cell and eosinophil responses. Abbas, et al. (1996)
Nature
383:787-793. Both T helper subsets originate from a naive T-cell precursor, whose differentiation is influenced by both the manner and the environment in which it is initially stimulated. Variables known to influence the development of T-cell subsets include the affinity of the TCR for antigen (Constant and Bottomly (1997)
Ann. Rev. Immunol.
15:297-322), the duration of the interaction between TCR and antigen (lezzi, et al. (1999)
Eur. J. Immunol.
29:4092-4101), and differential co-stimulation by APCs (McAdam, et al. (1998)
Immunol. Rev.
165:231-247). However, the best defined differentiating factors are the cytokines present upon T cell activation. Thus, it is clear that the presence of IL-12 during priming favors the development of Th1 cells, whereas IL-4 favors the development of Th2 cells. O'Garra (1998)
Immunity
8:275-283; Romagnani (1991)
Immunol. Today
12:256-257; and Romagnani (1997)
Immunol. Today
18:263-266.
Evidence has been provided for the existence of a CD4
+
T cell subset which has a profile of cytokine production distinctive from classical Th1 or Th2 cells. Groux, et al. (1997)
Nature
389:737-742. Activation of human or mouse CD4
+
T cells in the presence of IL-10 resulted in T-cell clones which produced high levels of IL-10, significant amounts of IFN-&ggr;, TGF-&bgr;, and IL-5, but no IL-4 or IL-2. Groux, et al. (1997)
Nature
389:737-742. In addition, kinetic studies demonstrated that these T-cell clones produced IL-10 much more rapidly than did Th1 or Th2 clones. Bacchetta, et al. (1994)
J. Exp. Med.
179:493-502. Importantly, antigen-specific activation of naive autologous T cells was blocked by these T cell clones, and this inhibition was partially mediated by IL-10 and TGF-&bgr;. This novel CD4
+
T-cell subset has been designated T-regulatory type 1 (Tr1) cells. Both human and mouse Tr1 clones display a low proliferative capacity under standard culture conditions, and this is likely a major reason why Tr1 cells had not been previously isolated.
In addition, in a murine model of inflammatory bowel disease (IBD) in SCID mice, co-transfer of Tr1 clones together with pathogenic CD4
+
CD45RB
hi
T cells prev
de Waal Malefyt Rene
Levings Megan K.
Roncarolo Maria Grazia
Andres Janet L.
Brody Tom
Ching Edwin P.
Schering Corporation
Spector Lorraine
LandOfFree
Regulatory T cells; methods does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Regulatory T cells; methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Regulatory T cells; methods will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3344761