WO2005124346A1

WO2005124346A1 - 4-1bb receptors are expressed on regulatory t-cells

Info

Publication number: WO2005124346A1
Application number: PCT/US2005/016665
Authority: WO
Inventors: Guoxing Zheng; Aoshuang Chen
Original assignee: University of Illinois at Urbana Champaign; University of Illinois System
Current assignee: University of Illinois at Urbana Champaign; University of Illinois System
Priority date: 2004-05-17
Filing date: 2005-05-12
Publication date: 2005-12-29
Anticipated expiration: 2006-11-17
Also published as: US7592313B2; US20070292431A1

Abstract

The thymus-derived CD4+CD25+ T cells belong to a subset of regulatory T cells potentially capable of suppressing the proliferation of pathogenic effector T cells. Intriguingly, these suppressor cells are themselves anergic, proliferating poorly to mitogenic stimulation in culture. The inventors have found that the 4-1BB co-stimulator receptor, best known for promoting the proliferation and survival of CD8+ T cells, also induces the proliferation of the CD4+CD25+ regulatory T cells both in culture and in vivo. The proliferating CD4+CD25+ T cells produce no detectable IL-2, suggesting that 4-1BB costimulation of these cells does not involve IL-2 production. The 4-1BB-expanded CD4+CD25+ T cells are functional, as they remain suppressive to other T cells in co-culture. These results support the notion that the peripheral expansion of the CD4+CD25+ T cells is controlled in part by co-stimulation.

Description

4-1BB RECEPTORS ARE EXPRESSED ON REGULATORY T-

CELLS

[01] This invention was made using funds from the U.S. government, National Institutes of Health Grant CA92243. The government therefore retains certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

[02] This invention is related to the area of immunology. In particular, it relates to regulation of CD4⁺CD25⁺ regulatory T cells.

BACKGROUND OF THE INVENTION

[03] The immune system operates on the principle of checks and balances. For T cells, there is evidence that their activities may be controlled in part by subsets of T cells specialized in regulation. The thymic-derived CD4⁺CD25⁺ T cells belong to one of these subsets. Unlike conventional T cells that function as effector cells during an immune reaction, the CD4⁺CD25⁺ T cells act to suppress the activation of conventional T cells and in doing so, actively maintain immunologic tolerance in the periphery (reviewed in references^1"3). Recent studies indicate that the CD4⁺CD25⁺ T cells may play important roles in the prevention of certain auto- immune and allo- immune diseases^4"11.

[04] Given the importance of these regulatory cells, it is critical to understand the mechanisms controlling their activation and expansion in the periphery. Intriguingly, freshly isolated CD4⁺CD25⁺ T cells are anergic, proliferating poorly in response to TCR stimulation in vitro. How these cells expand in vivo in response to physiological stimuli has yet to be determined. Recently it has been shown that CD4⁺CD25⁺ T cells carrying a transgenic TCR can undergo clonal expansion in vivo in response to immunization with a specific antigen, and that the dynamics of the regulatory cell population depends largely on local environment^12"14. In addition to antigen presentation, co-stimulation by activated dendritic cells (DC) is likely to be a key factor affecting the dynamics of CD4⁺CD25 T cells proliferation, as it has been shown that the CD4⁺CD25⁺ T cells can be expanded in vivo with bone manow- derived, antigen-presenting DC, in a manner that is dependent on B7 expression¹⁴. Interestingly, deficiency in B7 only reduces, but does not entirely eliminate, the potency of DC, suggesting that additional co-stimulators other than B7 may also play a role¹⁴. Such a role of additional co-stimulators is consistent with previous evidence that the development of IL-10-secreting, respiratory regulatory T cells depends on ICOS, not B7¹⁵.

[05] 4- IBB (CD 137) is a membrane receptor protein of the TNFR superfamily. Like CD28, 4-1BB is a potent co-stimulator, known to promote CD4⁺ and CD8⁺ T cell activation and survival (reviewed in reference¹⁶). Unlike CD28, which competes for B7 with another receptor, CTLA-4¹⁷, 4-1BB binds monogamously to a single ligand, 4-1BBL, thereby allowing more straightforward determination. Furthermore, previous studies have indicated that 4- IBB gene expression is upregulated in CD4⁺CD25⁺ T cells¹⁸' ¹⁹.

[06] There is a continuing need in the art for methods of obtaining CD4⁺CD25⁺ T cells and overcoming their anergy. If reasonable amounts of CD4⁺CD25⁺ T cells were available, they could be used inter alia for drug screening and therapy of immune- related diseases.

SUMMARY OF THE INVENTION

[07] A first embodiment of the invention provides a method of stimulating the proliferation of CD4⁺CD25⁺ regulatory T cells. One or more CD4⁺CD25⁺ regulatory T cells are contacted in vitro with an effective amount of soluble 4-1BBL. Proliferation of the cell(s) is thereby stimulated, forming an expanded CD4⁺CD25⁺ regulatory T cell population. [08] A second emoodimerit of fee invention provides a kit for testing candidate therapeutic agents. The kit contains an in vitro expanded population of CD4⁺CD25⁺ regulatory T cells. It also contains a plurality of identical vessels.

[09] A third embodiment of the invention provides a method for testing substances as candidate therapeutic agents. A test substance is contacted with an in vitro expanded population of CD4+CD25+ regulatory T cells. The proliferative index of the population is determined. A test substance is identified as a candidate therapeutic agent if the test substance increases the proliferative index relative to a control population which has not been contacted with a test substance.

[10] According to another aspect of the invention a method of stimulating the proliferation of CD4+CD25+ regulatory T cells in a mammal is provided. An effective amount of soluble 4-1BBL is administered to a mammal in need of immune suppression or immune toleration. Proliferation of CD4+CD25+ regulatory T cells is thereby stimulated.

[11] Another aspect of the invention is another method of stimulating the proliferation of CD4+CD25+ regulatory T cells. CD4+CD25+ regulatory T cells are contacted in vitro with an effective amount of reagent cells comprising 4-lBBL-Fc on their surfaces. Proliferation of said CD4+CD25+ regulatory T cells is thereby stimulated.

[12] Another embodiment of the invention provides another method of stimulating the proliferation of CD4+CD25+ regulatory T cells in a mammal. An effective amount of reagent cells comprising 4-lBBL-Fc on their surfaces is adininistered to a mammal in need of immune suppression or immune toleration. Proliferation of CD4+CD25+ regulatory T cells is thereby stimulated.

[13] Still another embodiment of the invention is an isolated population of reagent cells for stimulating CD4+CD25+ regulatory T cells. The reagent cells comprise 4-lBBL-Fc and OX40L-Fc on their surfaces. [14] An additional embødMenϊ of" the invention is an isolated population of reagent cells for stimulating CD4+CD25+ regulatory T cells. The reagent cells comprise 4-1BBL- Fc and ICOSL-Fc on their surfaces.

[15] Yet another embodiment of the invention is an isolated population of reagent cells for stimulating CD4+CD25+ regulatory T cells. The reagent cells comprise 4-lBBL-Fc and anti-CD3 monoclonal antibody on their surfaces.

[16] These and other embodiments, which will be apparent to those of skill in the art upon reading the specification, provide the art with reagents and methods for tolerizing or suppressing immune responses.

BRIEF DESCRIPTION OF THE DRAWINGS

[17] Figs. 1 A-ID. demonstrate that the 4-lBB receptor was present on CD4⁺CD25⁺ T cells. (Figs. 1A - C) Freshly-isolated bulk spleen cells were depleted of MHC it cells by magnet cell sorting (Miltenyi Biotec) and stained with biotin-conjugated anti-CD25 mAb/APC-conjugated streptavidin, FITC-conjugated anti-CD4 mAb, and a specific reagent for 4-lBB, in the presence of anti-CD 16/CD32 mAb as Fc blocker. The stained cells were analyzed for 4-lBB expression by multi-color flow cytometry. (Fig. 1A) CD4⁺CD25^" and CD4⁺CD25⁺ cells were identified and gated. (Fig. IB) Comparative analysis of 4-lBB-specific signals from the two gated cell populations of BALB/c origin. The cells were stained with either PE-conjugated anti-4-lBB mAb (top) or 4-lBBL-Fc/PE-conjugated goat anti-human Fcγ Ab (bottom). (Fig. 1C) Similar analysis of 4-lBB-specific signals in gated cells of DO 11.10 origin. The cells were stained with PE-conjugated anti-4-lBB mAb. (Fig. ID) 4-lBB staining of activated BALB/c CD4⁺CD25^" and CD4⁺CD25⁺ T cells. Purified BALB/c splenic CD4⁺CD25⁺ and CD4⁺CD25^"T cells were stimulated, respectively, in culture for two days with plate-bound anti-CD3 mAb (bound at 8 μg/ml) and murine IL-2 (100 U/ml). The cells were stained with PE-conjugated anti-4-lBB mAb and analyzed by flow cytometry. Solid line, 4-lBB-specific staining; dashed line, isotype-matched control staining. Data in each panel are representative of two or three independent experiments. [18] Figs. 2A and 2B* s ow 'that¹ signaling of 4-lBB augments CD4+CD25+ T cell proliferation. (Fig. 2A) PRO-IAd cells, non-painted (dashed line) or painted with 30 μg/ml of 4-lBBL-Fc (solid line), were stained with PE-conjugated anti-mouse 4- 1BBL mAb and analyzed by flow cytometry. (Fig. 2B) CD4+CD25+ T cells were stimulated with plate-bound anti-CD3 mAb (bound at 8 μg/ml) and PRO-IAd cells, either non-painted or painted with indicated Fc protein, in the absence or presence of anti-4-lBB blocking mAb (5 μg per well). The cells were pulsed with 1 μCi of 3H- thymidine per well at 48 h and harvested at 64 h; 3H-thymidine incorporation was then determined. Data are representative of three independent experiments.

[19] Fig. 3 demonstrates that soluble 4-lBB ligand co-stimulates CD4+CD25+ T cell proliferation. CD4+CD25+ T cells were stimulated with indicated concentrations of plate-bound anti-CD3 mAb, either alone (filled bar) or in combination with 4 μg of soluble 4-lBBL-Fc (open bar). Cell proliferation was determined as described in Figs. 2A and 2B. Data are representative of three independent experiments.

[20] Figs. 4A-4D demonstrate that 4-lBB ligand-coated CD4⁺CD25⁺ T cells exhibited enhanced proliferative activity in vivo. CFSE-labeled DO 11.10 TCR transgenic CD4⁺CD25⁺ T cells (99% pure), treated either with a human IgG control Fc protein (Figs. 4A & 4C) or with 4-lBBL-Fc (Figs. 4B & 4D), were injected contralaterally into the left and right footpad, respectively, of syngeneic BALB/c mice (Materials and Methods). At day 4, cells were recovered from the draining (popliteal) LNs and stained with APC-conjugated anti-CD4 mAb (RM4-5), PE-conjugated anti-DOll.10 mAb (K 1-26), and 7-AAD. The cells were analyzed by four-color flow cytometry. (Figs. 4A & 4B) Donor cell population (CD4 and KJ1-26 double positive, circled) was identified and gated on as a small fraction of total CD4⁺ T cells. (Figs. 4C & 4D) Proliferation (decrement of CFSE fluorescence) and death (7-AAD positive stain) of the gated cells were analyzed by quadrant plots. Cell counts in each quadrant are indicated. Data are representative of three independent experiments. The increased counts of proliferating cells (lower left quadrant) produced in the 4-lBBL-Fc-tτeated groups (Fig. 4D), as compared to the counts produced in the control-treated groups (Fig. 4C), is statistically significant by one-tailed Student t test (p < 0.01). [21] Fig. 5 demonstrates that 4 fflB-co-stimulated, proliferating CD4+CD25+ T cells do not produce IL-2. CD4+CD25+ or CD4+CD25- T cells (in 200 μl) were stimulated (in duplicate) with plate-bound anti-CD3 (bounded at 8 μg/ml for CD4+CD25+ cells and 4 μg/ml for CD4+CD25- cells) and soluble 4-lBBL-Fc (4 μg per well). At 48 h, 50 μl of conditioned medium was taken from each of the duplicate wells and combined, and cytokine content in the medium was analyzed with Cytometric Bead Array (BD Biosciences). In parallel, the remaining cultures were pulsed with 3H- thyrnidine (1 μCi per well), and the proliferation of the cytokine-producing T cells was assessed as described in Figs. 2A and 2B. Data are representative of two independent experiments.

[22] Fig. 6 demonstrates that 4-lBB-co-stimulated CD4⁺CD25⁺ T cells remained suppressive. DO 11.10 CD4⁺CD25⁺ or CD4⁺CD25^" T cells that had proliferated under 4- IBB co-stimulation for three days were harvested and tested as suppressor in co- culture, together with freshly isolated DO11.10 CD4 CD25^" T cells as responder, mitomycin C-treated DO11.10 spleen cells as stimulator, and the OVA_323-339 antigen peptide (0.1 μg/ml). The proliferation of the responder was determined by the ³H- thyrnidine incorporation method described in the description of Fig. 2. Data are representative of two independent experiments.

[23] Fig. 7 shows that 4- IBB co-stimulation augmented antigen-specific proliferation of DO11.10 CD4⁺CD25⁺ T cells. DO11.10 splenic CD4⁺CD25⁺ T cells were stimulated with indicated concentrations of the OVA₃₂₃.₃₃₉ peptide antigen, presented by the PRO-IAd artificial APC that were generated by painting with 4-lBBL-Fc (open bar) or human IgG as control Fc protein (filled bar). Cell proliferation was determined as described in Figure 2. Data are representative of two independent experiments.

[24] Figs. 8A-8D show that CD4⁺CD25⁺ T cells proliferated more vigorously in vivo in response to artificial APC presenting 4-lBBL-Fc. CFSE-labeled DO11.10 TCR transgenic CD4⁺CD25⁺ T cells (98% pure) were pre-mixed with PRO-IAd cells painted with human IgG (Fig. 8A & 8C) or the same PRO-IAd cells painted with 4- lBBL-Fc (Fig. 8B & 8D), and the two different cell mixtures were injected counter- laterally into the left and right footpads, respectively, of BALB/c mice. At day 4, donor cell were recwereu- -arid analyzed by four-color flow cytometry. Similarly, donor cell population (CD4 and KJ1-26 double positive, circled) was gated on (Fig. 8A & 8B) and proliferation (decrement of CFSE fluorescence) and death (7-AAD positive stain) of the gated cells were analyzed by quadrant plots (Fig. 8C & 8D). Cell count is indicated for each quadrant. Data are representative of four independent experiments. Compared to the control (Fig. 8C), the increase in the counts of proliferating cells (lower left quadrant) caused by 4-lBBL-Fc-painted APC (Fig. 8D) is statistically significant by one-tailed Student t test (p < 0.025).

DETAILED DESCRIPTION OF THE INVENTION

[25] It is a discovery of the present inventors that freshly isolated CD4⁺CD25⁺ T cells constitutively express low levels of the 4-lBB receptor protein at the cell surface and that the signaling of this receptor by 4-1BBL can significantly augment the proliferation of these cells in culture and in vivo. In addition, the 4-lBB-expanded cells remain suppressive to other T cells. Hence, 4-lBB plays a role in the expansion of functional CD4⁺CD25⁺ T cells.

[26] A proliferative index is a measurement of the proliferation of cells. This can be measured by any means known in the art. For example, cells can be tested at different times to measure the rate of incorporation of various metabolites, such as nucleotides, amino, acids, sugars, etc., into macromolecules. Cells can be simply counted at different times to observe an increase in cell or colony number. Any means known in the art for determining the rate of proliferation of a population of cells can be used.

[27] Stimulatory molecules according to the present invention are any that engage the 4- 1BB receptor molecule and cause it to signal other cellular components resulting in cellular proliferation. The 4-lBB ligand (4-1BBL) can be used in a soluble form, in a dimeric form attached to an immunoglobulin Fc region, or attached to a cell surface, such as an antigen presenting cell. A protein form or a nucleotide encoding the protein form may be delivered to the CD4⁺CD25⁺ T cells. The ligand can be from the same species as the cells to maximize activity and minimize adverse antigenic events. One such ligand is a human ligand having the sequence: MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACP

WAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLID

GPLSWYSDPGLAGVSLTGGLSYKEDTKELWAKAGVYYVFFQLELRRWAGE

GSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAG

QRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE

(SEQ ID NO: 1). A polynucleotide encoding this ligand may have any of the possible sequences which encode it. One such sequence is:

ATGGAATACGCCTCTGACGCTTCACTGGACCCCGAAGCCCCGTGGCCTCC CGCGCCCCGCGCTCGCGCCTGCCGCGTACTGCCTTGGGCCCTGGTCGCGG GGCTGCTGCTGCTGCTGCTGCTCGCTGCCGCCTGCGCCGTCTTCCTCGCCT GCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGC CCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCT CTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTC TGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGG CGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTG GTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCG GCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACC TGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTG GACCTGCCACCCGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCA GGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTC ACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCAC AGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTT CACCGAGGTCGGAA (SEQ ID NO: 2).

A murine form of the protein has a sequence:

MDQHTLDVEDTADARHPAGTSCPSDAALLRDTGLLADAALLSDTVRPTNAA LPTDAAYPAVNVRDREAAWPPALNFCSRHPKLYGLVALVLLLLIAACVPIFTR TEPRPALTITTSPNLGTRENNADQVTPVSfflGCPNTTQQGSPVFAKLLAKNQAS LCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFT NTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLL LKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE (SEQ ID NO: 3).

It can be encoded by a DNA sequence:

ATGGACCAGCACACACTTGATGTGGAGGATACCGCGGATGCCAGACATCC AGCAGGTACTTCGTGCCCCTCGGATGCGGCGCTCCTCAGAGATACCGGGC TCCTCGCGGACGCTGCGCTCCTCTCAGATACTGTGCGCCCCACAAATGCCG CGCTCCCCACGGATGCTGCCTACCCTGCGGTTAATGTTCGGGATCGCGAG GCCGCGTGGCCGCCTGCACTGAACTTCTGTTCCCGCCACCCAAAGCTCTAT GGCCTAGTCGCTTTGGTTTTGCTGCTTCTGATCGCCGCCTGTGTTCCTATCT TCACCCGCACCGAGCCTCGGCCAGCGCTCACAATCACCACCTCGCCCAAC CTGGGTACCCGAGAGAATAATGCAGACCAGGTCACCCCTGTTTCCCACAT TGGCTGCCCCAACACTACACAACAGGGCTCTCCTGTGTTCGCCAAGCTACT GGCTAAAAACCAAGCATCGTTGTGCAATACAACTCTGAACTGGCACAGCC AAGATGGAGCTGGGAGCTCATACCTATCTCAAGGTCTGAGGTACGAAGAA GACAAAAAGGAGTTGGTGGTAGACAGTCCCGGGCTCTACTACGTATTTTT GGAACTGAAGCTCAGTCCAACATTCACAAACACAGGCCACAAGGTGCAG GGCTGGGTCTCTCTTGTTTTGCAAGCAAAGCCTCAGGTAGATGACTTTGAC AACTTGGCCCTGACAGTGGAACTGTTCCCTTGCTCCATGGAGAACAAGTT AGTGGACCGTTCCTGGAGTCAACTGTTGCTCCTGAAGGCTGGCCACCGCCT CAGTGTGGGTCTGAGGGCTTATCTGCATGGAGCCCAGGATGCATACAGAG ACTGGGAGCTGTCTTATCCCAACACCACCAGCTTTGGACTCTTTCTTGTGA AACCCGACAACCCATGGGAA (SEQ ID NO: 4)

Allelic variants can be used as well. The criterion for suitable protein molecules is that they effectively signal CD4⁺CD25⁺ regulatory T cells via the 4- IBB receptor.

[28] Regulatory T cells can be isolated by use of antibodies specific for the CD4 and CD25 cell surface antigens. Any technique can be used including immunoaffinity chromatography, fluorescence activated cell sorting (FACS), immunoprecipitation, etc. Single regulatory T cells can be obtained by dilution or dividing of a population of such cells to an extent that at least some of the subpopulations (aliquots) have a single cell in them. Some of the subpopulations may have more and some may have less. After expansion, subpopulations that started from a single cell will be homogeneous or monoclonal. Homogeneity can be determined by determination of cell surface markers, for example. The FACS technique can be useful in this determination.

[29] Packaging for cells which have been expanded can be in a divided or undivided container or vessel. Undivided vessels include bottles, flasks, tubes, etc. Divided vessels incfude assemblies of multiple cuvettes or tubes, microtiter plates, divided Petri dishes, etc. Vessels may be insulated to maintain a desired lower than ambient temperature during shipment. Dry ice, wet ice, or liquid nitrogen may be used to maintain a lower than ambient temperature for preservation of cell viability. Cells may be frozen or lyophilized for storage or shipment. Techniques for storing and shipping mammalian cells are well known in the art.

[30] Kits may be made using the cells of the present invention. They may comprise other components which are useful for performing assays. These may be, for example, particular assay reagents, microtiter plates, instructions for performing assays or for growing the cells. The reagents may be for growing the cells in vitro or for assaying the cells for proliferative index. Exemplary of such reagents are labeled amino acids or nucleotides.

[31] In vitro expanded populations of CD4⁺CD25 regulatory T cells are cells that have increased in cell number by at least 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 50-fold, 100-fold, 1000-fold, 10,000-fold or 100,000-fold. The manner of stimulating the cells to proliferate in vitro can lead to a particular subpopulation of cells having particular marker expression and function. Thus, for example, populations stimulated with 4-1BBL will express the 4- IBB receptor on their surface. These cells remain anergic even after expansion.

[32] In vitro expanded populations of CD4 CD25 regulatory T cells can be used for testing substances for their ability to stimulate proliferation of the cells. Substances which are able to stimulate the proliferation are candidate therapeutic agents for treating human auto-immune diseases, such as graft versus host disease, Hashimoto's thyroiditis, pernicious anemia, Addison's disease, diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, multiple sclerosis, myasthenia gravis, systemic sclerosis (scleroderma), Reiter's syndrome, and Grave's disease. Immune-mediated diseases in other mammals may also be treated according to the present invention. A common form of immune disease in dogs and cats, for example, is immune mediated hemolytic anemia. Other immune mediated diseases that can be treated in mammals other than humans include but are not limited to host versus graft disease, immune-mediated thryroiditis, Cobalamin deficiency, Addison's disease or hypoadrenocorticism, diabetes mellitus, Rheumatoid arthritis, immune-mediated arthropathies, including SLE, Dermatomyositis, Sjorgen's Syndrome, and immune-mediated keratoconjunctival sicca. Substances can be further checked using other types of assays or other animal model tests. These same diseases can be treated by administration of in vitro expanded populations of CD4⁺CD25⁺ regulatory T cells according to the invention. Similarly, these same diseases can be treated with soluble 4-lBBL-Fc, or other 4-1BBL constructs. Cells which are "painted" with 4-lBBL-Fc can also be delivered to a mammal in need thereof to stimulate in vivo proliferation of CD4⁺CD25⁺ regulatory T cells.

[33] Suitable substances for testing for stimulatory activity are any natural or synthetic products, including products of libraries of compounds and peptides. The substances can be those previously known to have a biological activity or a therapeutic activity, or the substances can have non previously known activities. Libraries of compounds are available for testing commercially or can be made by the ordinary artisan.

[34] Painted cells can have additional ligands painted on their surfaces. These include any ligands that stimulate CD4⁺CD25⁺ regulatory T cells directly or ligands that stimulate accessory cells which stimulate CD4⁺CD25⁺ regulatory T cells. Suitable ligands in addition to 4-1BBL include without limitation OX40L, ICOSL, anti-CD28 antibodies, CTLA-4, and anti-CD3 antibodies.

[35] Although enhanced in vivo expansion of the 4-lBBL-Fc-treated CD4⁺CD25⁺ T cells is evident in our study, it is far less extensive than that demonstrated previously in animals immunized with either a soluble antigen or mature DC loaded with an antigen^12"14. The sub-optimal amount of OVA antigen applied in our experiments may have attributed to the result. In addition, 4-lBB alone may be insufficient to bring about the full force of co-stimulation as in vivo irnmunizations did. In those latter experiments, host DC activated upon encountering a large dose of immunizing antigen, or donor DC activated prior to transfusion, are likely to express not one, but a spectrum of co-stimulators. As these co-stimulators may engage several receptors on the CD4⁺CD25⁺ T cells, such as 4-lBB, OX40, and CD28 and/or CTLA-4¹⁸' ¹⁹, their combined effect could he greater than what 4- IBB alone could produce. It will be interesting to test whether multiple recombinant co-stimulators can indeed elicit stronger functional outputs from CD4⁺CD25⁺ T cells.

[36] In vivo expansion of CD4 CD25 T cells can be used for controlling harmful autoimmune and allo-immune reactions. Other means for expanding CD4⁺CD25⁺ T cells (other than using a co-stimlatory molecule) can be used in conjunction with 4-1BBL, such as antigen immunization¹²' ¹³, infusion of antigen-presenting dendritic cells (DC)¹⁴, and various other methods for ex vivo and in vivo expansion⁹' ^20"22.

[37] The above disclosure generally describes the present mvention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE 1 — Materials and methods Mice, peptide, and cell line

[38] BALB/c and DO 11.10 [BALB/c-TgN(DO11.10)10Loh] mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The animals were maintained in a pathogen-free facility and used in accordance with institutional guidelines for animal care. The OVA_323-339 antigen peptide was synthesized by Princeton Biomolecules (Langhorne, PA). The PRO-IAd cell line was a gift from Jim Miller of the University of Chicago and maintained as previously described .

Antibodies

[39] PE-conjugated hamster anti-mouse 4-lBB mAb (17B5), PE-conjugated rat anti-mouse 4-1BBL mAb (TKS-1), and isotype controls were purchased from eBioscience (San Diego, CA). Non-conjugated 4-lBB-blocking mAb (17B5) was also purchased from eBioscience and dialyzed in PBS before use. PE-conjugated goat anti-human Fc_γ was pu iasβd from -T41e» iacksόή ImmunoResearch Laboratories (West Grove, PA). Rat anti-mouse CD3 mAb (TK3) was purchased from Serotec Ltd. (Oxford, UK). FJTC- conjugated rat anti-mouse 4- IBB mAb (1AH2), FITC-conjugated rat anti-mouse CD4 mAb (GK1.5), APC-conjugated rat anti-mouse CD4 mAb (RM4-5), biotin-conjugated rat anti-mouse CD25 mAb (7D4), PE- or APC-conjugated streptavidin, rat anti-mouse CD16/CD32 mAb (2.4G2), and various isotype-matched controls were purchased from BD Biosciences (San Jose, CA). PE-conjugated mouse anti-DOll.10 mAb (KJ1-26) was purchased from Caltag Laboratories (Burlingame, CA).

T cell purification

[40] CD4 T cells were prepared from the spleens or lymph nodes of BALB/c or DOl 1.10 mice and enriched via negative selection by magnetic cell sorting (Miltenyi Biotec, Auburn, CA), as per manufacturer's protocols. The cells were blocked with anti- mouse CD16/CD32 mAb, immunostained with FITC-conjugated anti-mouse CD4 mAb and biotin-conjugated anti-mouse CD25 mAb/PE-conjugated streptavidin, and then sorted by a FACSDiVa cell sorter (BD Biosciences). CD4⁺CD25⁺ and CD4⁺CD25^" cells were separately collected; purity of the preparations was typically > 98 %. For some experiments, the CD4⁺CD25⁺ and CD4⁺CD25^" cells were purified by magnetic cell sorting, using the CD4⁺CD25⁺ regulatory T cell isolation kit from Miltenyi Biotec, as per manufacturer's protocol. To obtain highly purified cells, the MS column step was repeated three times before the CD4⁺CD25⁺ cells were finally collected. The purity of such cell preparations were typically > 98 %, whereas the yield was reduced by about 40 %.

Cell painting

[41] PRO-IAd artificial accessory cells were inactivated by incubation at 37° C for 1 h with 100 μg/ml of mitomycin C (Sigma-Aldrich Corp., St. Louis, MO), at 2 x 10⁶ cells/ml in DMEM/2 % FCS. The cells were thoroughly washed and incubated at 37° C for 30 min with pahnitated protein A²⁴, at 30 μg/10⁶ cells/ml in DMEM/0.1 % BSA. The cells were washed and incubated at 4° C for 30 min with Fc_γi-derivatized mouse 4- 1BBL (4-lBBL-Fc)²⁵ or Fc_γl-derivatized human CD28 (CD28-Fc)²⁴, at 30 μg/10⁶ cells/ml in DMEM/0.1 % BSA. The cells were thoroughly washed and suspended in RPMI 1164/10 % FCS/15 mM HEPES/50 μM β-ME (R10 medium).

Proliferation assay

[42] 0.25 - 1 x 10⁵ T cells (per well) were cultured in R10 medium in U-bottomed 96-well plates in duplicates or triplicates, along with 2.5 - 5 x 10⁴ PRO-IAd cells (per well) painted with 4-lBBL-Fc or 4 μg of soluble 4-lBBL-Fc (per well). For Ab blocking, anti-4-lBB mAb (17B5) and isotype-matched control were each used at 5 μg per well. For assays involving the DO 11.10 TCR transgenic T cells, the OVA_323-339 peptide was added to the wells at 0.1 - 100 μg/ml, in conjunction with the PRO-IAd cells. For assays involving BALB/c T cells, rat anti-mouse CD3 mAb (TK3) was used as a polyclonal mitogen. The mAb was diluted to 1 - 8 μg/ml in PBS and bound to U-bottomed 96-well plates (50 μl per well) at 37 °C for at least 6 h before the cells were plated. The cultures were pulsed at 48 h with ³H-thymidine (1 μCi per well) and harvested at 64 h with a Tomtec MACH III cell harvester (Hamden, CT). ³H- thymidine incorporation was analyzed with a MicroBeta liquid scintillation counter (Wallac Oy, Turku, Finland).

Adoptive transfer

[43] CD4⁺CD25⁺ T cells were purified from the spleens of DOl 1.10 mice as described earlier, washed three times in PBS, thoroughly re-suspended at 2.5 x 10⁶ cells/ml in PBS, and then stained with CFSE (Molecular Probes, Eugene, OR) at 1 μM for 5 min at room temperature. The reaction was terminated by adding FCS to 10 %, followed by two washes with DMEM/10 % FCS/50 μM β-ME. For co-stimulator treatment, CFSE-stained cells were re-suspended in DMEM/0.1 % BSA/50 μM β-ME and incubated at 4 °C for 20 min with 4-lBBL-Fc or human IgG (30 μg protein/1 x 10⁷ cells/ml). The cells were washed twice in DMEM and injected into the hind leg footpads of two or more BALB/c mice (2.5 - 5 x 10⁵ cells in 30 μl of DMEM per footpad). 4-lBBL-Fc-treated cells were injected at the right footpads; human IgG (control Fc)-treated cells, left footpads. To prime the injected donor cells, 30 min prior to cell transfer, the OVA_323-339 peptide (0.08 μg in 30 μl of PBS per footpad) was injected into the right and left footpads of the recipient mice. The recipient mice were maintained in the dark for four days and then sacrificed. Cell suspensions were prepared from the right (4-lBBL-Fc-treated) and left (human IgG-treated) popliteal LNs; cells from each treatment group were pooled. The pooled cells were then immunostained for CD4 and the DOl 1.10 TCR with APC-conjugated anti-mouse CD4 mAb and PE-conjugated anti-DOl l.10 mAb, respectively, in the presence of anti-mouse CD16/CD32 mAb as Fc blocker. Immediately before FACS analysis, the cells were additionally stained for DNA with 7-AAD (Molecular Probes, Eugene, OR). Four-color FACS was performed on a FACSCalibur. The donor cells were gated as a DOl 1.10 TCR and CD4 double-positive population, and the viability (7- AAD negativity) and cell division (CFSE decrement) of this gated population were analyzed with the Cellquest software.

Co-adoptive transfer

[44] CFSE-labeled DOl 1.10 CD4⁺CD25⁺ T cells prepared as described before were premixed with antigen-pulsed and 4-lBBL-painted PRO-IAd cells and injected in the form of a cell mixture into the footpad of BALB/c mice. For preparation of the PRO- IAd cells for co-adoptive transfer, the cells were inactivated with mitomycin C as described before and incubated with the OVA_323-339 peptide (80 μM) at 37 °C for 1 h, at 10⁷ cells/ml in DMEM/0.1 % BSA. Palmitated protein A was then added to the cells at 30 μg/ml and the incubation was continued for another 50 min at 37 °C, followed by 10 min incubation at 45 °C. The cells were washed and incubated at 4 °C for 20 min with 4-lBBL-Fc or, as control, human IgG, at 30 μg/10⁶ cells/ml in DMEM/0.1 % BSA. The cells were washed twice in DMEM and combined with the T cells at 1:1 ratio. Footpad injection (two or more mice per experiment) and FACS analysis were performed essentially as described before, with the mixed cells containing the human ϊgG'-pa ned PRO-IAd cells injected (0.5 - 1 x 10⁶ total cells per foot) into the left footpads (control) and an equal number of mixed cells containing the 4-lBBL-painted PRO-IAd cells injected into the right footpads (test).

Suppression assay

[45] DO 11.10 CD4⁺CD25⁺ or CD4⁺CD25^" T cells were stimulated to proliferation with mitomycin C-inactivated, 4-lBBL-Fc-painted PRO-IAd cells and OVA_323-339 (10 μg/ml) in U-bottomed 96-well plates and R10 medium. The cells were harvested three days later and thoroughly washed in R10 medium. Live cells (T cells) were counted and re-seeded as suppressor (0.025 x 10⁶ cells/well) in U-bottomed 96-well plates in R10 medium, together with freshly isolated DOl 1.10 CD4⁺CD25^" T cells as responder (0.05 x 10⁶ cells/well), DOl 1.10 accessory cells as stimulator (0.05 x 10⁶ cells/well), and OVA_323-339 (0.1 μg/ml). The accessory cells were obtained from bulk spleen cells depleted of CD4⁺ and CD8⁺ cells by magnetic cell sorting (Miltenyi Biotec) and chemically inactivated with mitomycin C before use. The proliferation of the responder T cells was determined by the ³H-thymidine incorporation method described before.

4-lBBL-Fc construct

[46] An expression construct for 4-lBBL-Fc was assembled via splicing PCR, in several -steps. A coding sequence for the extracellular domain of murine 4-1BBL was synthesized using primers F (5 - GTGCCACGCCTCGAGCGCACCGAGCCTCGGCC-3'; SEQ ID NO: 5) and G (5'- T ACTGGA TCCTCA TTCCCA TGGGTTGT CGGG-3'; SEQ ID NO: 6), along with a plasmid containing murine 4-1BBL cDNA (a generous gift from Dr. Lieping Chen) as template. The resulting PCR product was digested with BamHI and Xhol, and subcloned into the conesponding sites of pBluescript, to generate pm4-lBBL/BT. In turn, the murine 4-IBBL extracellular domain sequence was mobilized from pm4- BBLBT with NM, fiiled-τtϊ with Klenow, digested with Xhol, and subcloned into a derivative of pEE14 that contains human Fcγj sequence upstream of the conesponding site, to generate pm4-lBBL-Fc EE14.

[47] The murine TNFSF9 gene sequence segment contained in the 4-lBBL-Fc construct is:

[48] CGCACCGAGCCTCGGCCAGCGCTCACAATCACCACCTCGCCCAACCTGGG TACCCGAGAGAATAATGCAGACCAGGTCACCCCTGTTTCCCACATTGGCT GCCCCAACACTACACAACAGGGCTCTCCTGTGTTCGCCAAGCTACTGGCT AAAAACCAAGCATCGTTGTGCAATACAACTCTGAACTGGCACAGCCAAGA TGGAGCTGGGAGCTCATACCTATCTCAAGGTCTGAGGTACGAAGAAGACA AAAAGGAGTTGGTGGTAGACAGTCCCGGGCTCTACTACGTATTTTTGGAA CTGAAGCTCAGTCCAACATTCACAAACACAGGCCACAAGGTGCAGGGCTG GGTCTCTCTTGTTTTGCAAGCAAAGCCTCAGGTAGATGACTTTGACAACTT GGCCCTGACAGTGGAACTGTTCCCTTGCTCCATGGAGAACAAGTTAGTGG ACCGTTCCTGGAGTCAACTGTTGCTCCTGAAGGCTGGCCACCGCCTCAGTG TGGGTCTGAGGGCTTATCTGCATGGAGCCCAGGATGCATACAGAGACTGG GAGCTGTCTTATCCCAACACCACCAGCTTTGGACTCTTTCTTGTGAAACCC GACAACCCATGGGAA (SEQ ID NO: 7)

[49] The murine TNFSF9 protein sequence segment contained in the 4-lBBL-Fc construct is:

[50] RTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKNQ ASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSP TFlT^TGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQ LLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE (SEQ ID NO: 8) EXAMPLE' 2 Signaling of- 4 IBB augmented the proliferation of CD4⁺CD25⁺ T cells

[51] Previously, Gavin et al.¹⁸ and McHugh et al.¹⁹ detected constitutive expression of the 4- IBB gene in mouse CD4+CD25+ T cells using DNA microanay. To confirm the actual presence of the 4-lBB protein on the cell surface, we isolated spleen cells from normal BALB/c mice and their congenics carrying a rearranged TCR transgene (DOl 1.10), and immunostained the cells with hamster anti-mouse 4-lBB antibody (17B5), together with antibodies specific for CD4 and CD25. Using multi-color flow cytometry, we were able to identify and gate on CD4+CD25+ and CD4+CD25- T cells and examine the 4-lBB-specific stains simultaneously in both subsets (Figure 1A). The CD4+CD25- subset served as an internal control, since naϊve CD4+CD25- T cells are known to express little 4-lBB¹⁶. Indeed, in the freshly isolated spleen cells of BALB/c or DOl 1.10 origins, 4-lBB was detected only in the CD4+CD25+ subset, but not in the CD4+CD25- subset (Figure IB &1C). However, the 4-lBB stain in the CD4+CD25+ subset was rather weak, suggesting a low-level presence of the 4-lBB protein on the cell surface. This observation was confirmed with the use of a second anti-4-lBB mAb (1 AH2) of different species origin (rat) (data not shown), and with the use of a ligand of 4-lBB, 4-lBBL-Fc²⁵, as the 4-lBB-specific staining reagent (Figure IB).

[52] To investigate whether the level of 4-lBB expression in the CD4+CD25+ T cells changes after T cell activation, splenic CD4+CD25+ T cells were isolated and stimulated for two days in culture with anti-CD3 mAb and IL-2. The cells so activated were again stained with anti-4-lBB mAb. Flow cytometric analysis of these cells detected considerably stronger staining than what had been detected in the freshly isolated same cells (Figure ID), suggesting that the presence of 4-lBB on the cell surface is up-regulated after cell activation.

[53] To determine whether the low-level presence of 4-lBB on CD4⁺CD25⁺ T cells is functionally significant, we next probed the cells with a ligand of 4-lBB, 4-1BBL- ¹ Fc²⁵. To that end, 4-lBBL-Fc was painted onto an artificial accessory cell line, PRO- IAd (Figure 2A). Splenic CD4⁺CD25⁺ T cells isolated from BALB/c mice were stimulated in culture with plated-bound anti-CD3 mAb, in combination with the PRO- lAd eeils. As expected^*, Erie e⁾D4⁺CD25⁺ T cells displayed a typical anergic phenotype and proliferated poorly in response to anti-CD3 stimulation alone (Figure 2B). However, they proliferated actively when co-stimulated by PRO-IAd cells painted with 4-lBBL-Fc, but not with a control Fc protein (CD28-Fc), indicating that the cells were sensitive to 4-1BBL co-stimulation. The activity was blocked by anti-4-lBB mAb, confirming that it was specifically mediated by the 4- IBB receptor. These results establish that the low-level expression of 4- IBB on CD4⁺CD25⁺ T cells is sufficient to render the cells sensitive to 4-1BBL co-stimulation, and that such co- stimulation can potently drive the cells to proliferation. Additional experiments with CD4⁺CD25⁺ T cells isolated from the lymph nodes showed that 4-lBBL-Fc could also co-stimulate these cells to proliferate, as expected (data not shown).

[54] To probe the effect of 4- IBB engagement on antigen-specific proliferation, the PRO- IAd cells, which neo-express the MHC II molecules I-A^d molecules on the cell surface ²³, were further tested as antigen presenting cells (APC) for CD4+CD25+ T cells bearing the DO 11.10 transgenic TCR. When painted with 4-lBBL-Fc and loaded with the cognate OVA323-339 peptide antigen, the artificial APC vigorously stimulated the proliferation of the antigen-specific CD4+CD25+ T cells over a range of antigen doses (Figure 7). In contrast, the same APC showed only marginal stimulatory activity when painted with a control Fc-containing protein (human IgG). This result confirms that 4- IBB co-stimulation can promote antigen-specific proliferation of CD4+CD25+ T cells.

[55] 4- IBB binds 4- IBB ligand with high affinity²⁶' ²⁷. Although both exist predominantly as membrane proteins, the high affinity interaction between the two allows the receptor to be triggered by soluble forms of 4- IBB ligand as well, as has been observed in conventional T cells²⁸. To determine whether this may also be the case for CD4⁺CD25⁺ regulatory T cells, we applied soluble 4-lBBL-Fc directly to cell proliferation assays, without invoking artificial accessory cells to present the ligand. The proliferation of CD4⁺CD25⁺ T cells over a range of anti-CD3 stimulation was consistently augmented by the addition of soluble 4-lBBL-Fc (Figure 3), suggesting that the 4-lBB receptor on these cells can be triggered directly by a soluble ligand. This' finding eftab έd us 'to 'directly "coat" CD4⁺CD25⁺ T cells with soluble 4-1BBL- Fc for in vivo studies.

[56] To determine whether 4-lBB could co-stimulate antigen-specific proliferation of CD4⁺CD25⁺ T cells in vivo, TCR transgenic CD4⁺CD25⁺ T cells were isolated from the spleens of DOl 1.10 mice and labeled with CFSE to allow subsequent analysis of cell division by FACS²⁹. The labeled cells were incubated at 4 °C with 4-lBBL-Fc to allow binding of the protein to the 4- IBB receptor on the cell surface; unbound ligand was subsequently removed by washing. The treated cells were adoptively transfened via footpad injection into syngeneic BALB/c mice. As control, an equal number of mock-treated T cells (incubated with human IgG as control Fc proteins) were injected into the contralateral footpads of the same animals. To provide specific antigen stimulation to the donor cells, the recipient mice were also injected at the footpads with OVA_323-339, a cognate antigen peptide recognized by the DOl 1.10 TCR, 30 min prior to cell transfusion. Four days after priming, draining LNs (popliteal) were isolated from the recipient mice and pooled from each group separately (test or control). Total cellular content of each group of pooled LNs was recovered and analyzed in its entirety by FACS to assure that all LN cells were accounted for.

[57] Preliminary experiments established that the proliferation of donor CD4⁺CD25⁺ T cells in recipient animals is antigen dose-dependent (data not shown), consistent with previous reports by others^12'14, i our hand, donor cell proliferation could not be detected at the antigen dose below 0.03 μg per footpad. Proliferation became obvious as the antigen dose increased; at 0.4 μg per footpad, nearly all the donor cells proliferated. To limit the antigen dose to a sub-optimal level where the effect of co- stimulation would be most obvious, recipient animals were injected with only 0.08 μg of peptide per footpad.

[58] 4-lBBL-Fc augmented in vivo expansion of the donor cell population under this antigen-limiting condition. Four-color FACS showed that the counts of donor cells, identifiable by their CD4+KJ1-26+ (DO11.10 TCR-specific) status, were increased by an average (three experiments) of 2-fold in the test group relative to that in the control group (Figure 4A & 4B). CFSE analysis further showed that in both groups the donor cell population Was¹ divided rnto two major fractions - the dividing-cell fraction (with incrementally-decreased CFSE stain, lower left quadrant) and non-dividing-cell fraction (with highest CFSE stain, lower right quadrant) (Figure 4C & 4D). While the counts of non-dividing cells were relatively even across the test and control groups, that of dividing cells were increased by an average (three experiments) of 2.5 fold in the test group. Taken together, the analyses indicated that the donor cell population in the test group was expanded primarily by augmented cell proliferation.

[59] To further demonstrate such augmented proliferation in a setting more closely simulating the physiological priming of T cells, a co-adoptive transfer experiment was performed where the donor T cells were transfused together with PRO-IAd cells as artificial APC. The PRO-IAd cells used were pre-loaded with OVA323-339 and painted with 4-lBBL-Fc. The resulting artificial APC were capable of stimulating the proliferation of DO 11.10 CD4+CD25+ T cells as demonstrated earlier in vitro (Figure 7). In parallel, the same PRO-IAd cells loaded with antigen but painted with human IgG were used as control. Footpad injection and FACS analysis were performed essentially as described before, except the donor T cells were pre-formulated with the artificial APC (1:1) and administered as a cell mixture. The result showed that the donor T cells co-transfened with the 4-lBBL-Fc-painted APC proliferated more vigorously as compared to those co-transfened with the control APC (Figure 8). Again, on average (four experiments), there was a 4-fold increase in the counts of proliferating donor T cells that was directly attributable to the presence of 4-lBBL-Fc on APC. This result agrees well with the earlier result obtained from the 4-lBBL-Fc- coated donor T cells

EXAMPLE 3 — 4- IBB co-stimulation did not involve IL-2 production

[60] Activated T cells are known to secrete IL-2 that in turn initiates an autocrine loop for the continued expansion and survival of the proliferating population. However, this has not been the case for the CD4⁺CD25⁺ regulatory T cell subset expanded in culture, including those expanded under anti-CD28 co-stimulation^{21, 30}. To determine whether 4- IBB co-stimulation in particular can result in IL-2 secretion, we stimulated the CD4⁺CD25⁺ T cells with anti-CD3 mAb and soluble 4-lBBL-Fc. 48 h after stift la i, a small ortio of conditioned medium was taken from the culture and analyzed for IL-2, together with a penal of other cytokines (IL-4, JL-5, ENTF-γ, and TNF-α). To directly conelate cytokine secretion with cell proliferation, the rest of the same culture was also pulsed with ³H-thymidine and assayed for proliferation. No IL- 2 was detected in proliferating CD4⁺CD25⁺ T cell cultures (Figure 5). In contrast, IL- 2 secretion was evident in parallel CD4⁺CD25^" T cell cultures that were stimulated to proliferation at a similar level. Like CD4⁺CD25^" T cells, however, CD4⁺CD25⁺ T cells did secrete increased amounts of IFN-γ and TNF-α during proliferation, albeit at considerably lower levels than that by CD4⁺CD25^" T cells. IL-4 and IL-5 were present only at marginal levels in both CD4⁺CD25⁺ and CD4⁺CD25^" T cell cultures and were not significantly increased upon proliferation (data not shown). These results suggest that, like anti-CD28 co-stimulation, the mechanism of 4-lBB co- stimulation in the context of CD4⁺CD25⁺ T cells may not involve IL-2 production.

EXAMPLE 4~4-lBB-expanded CD4⁺CD25⁺ T cells remained suppressive

[61] The CD4+CD25+ regulatory T cells are known to be able to suppress the proliferation of conventional T cells, which can be demonstrated by the well-established co-culture assay³¹. Such activity is generally limited toward weakly, but not strongly, stimulated T cells^31"34, with physiological implication that it might function primarily to raise the threshold of autoimmune reactions³³. Having established the augmenting effect of 4- 1BB co-stimulation on cell proliferation, we sought to determine whether the CD4+CD25+ T cells remained suppressive after proliferation.

[62] To that end, DOl 1.10 CD4+CD25+ T cells were first co-stimulated to proliferation with 4-lBBL-Fc-painted PRO-IAd cells and a high dose of OVA323-339 (10 μg/ml; Figure 3). Three days later, the proliferating cells were harvested and their suppressive activity was subsequently assessed by the co-culture assay against DOl 1.10 CD4+CD25- T cells, in the presence of spleen-derived accessory cells and a low dose of OVA323-339 (0.1 μg/ml). As depicted in Figure 6, the CD4+CD25+ T cells, which ceased to proliferate under this latter condition, were able to inhibit the p^'rolrfer'ation of Λ* 0BTOD25- T cells, indicating that the CD4+CD25+ T cells remained suppressive after proliferating under 4- IBB co-stimulation. In contrast, DOl 1.10 CD4+CD25- T cells pre-expanded identically in the same experiment did not exhibit such suppressive activity, suggesting that 4- IBB co-stimulation could not confer the suppressive activity to conventional T cells.

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The disclosure of each reference cited is expressly incorporated herein.

Claims

We claim: 1. A method of stimulating the proliferation of CD4⁺CD25⁺ regulatory T cells, comprising: contacting one or more CD4⁺CD25⁺ regulatory T cells in vitro with an effective amount of soluble 4-lBBL, whereby proliferation of said cell(s) is stimulated and an expanded CD4⁺CD25⁺ regulatory T cell population is formed.

2. The method of claim 1 wherein one CD4+CD25+ regulatory T cell is contacted, and the expanded CD4⁺CD25⁺ regulatory T cell population is monoclonal.

3. The method of claim 1 wherein prior to the step of contacting the CD4⁺CD25⁺ regulatory T cells are diluted or divided to provide at least one aliquot comprising one CD4⁺CD25⁺ regulatory T cell.

4. The method of claim 1 wherein the expanded CD4⁺CD25⁺ regulatory T cell population is packaged in a package that is sufficient to maintain viability of the expanded CD4⁺CD25⁺ regulatory T cell population.

5. The method of claim 1 wherein the expanded CD4⁺CD25⁺ regulatory T cell population is diluted or divided into subpopulations and packaged.

6. The method of claim 1 wherein the expanded CD4⁺CD25⁺ regulatory T cell population is lyophylized.

7. The method of claim 4 wherein the the expanded CD4⁺CD25⁺ regulatory T cell population is lyophylized.

8. An in vitro expanded population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 1.

9. An in vitro expanded population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 2.

10. An ZR vitro expanded population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 3.

11. An in vitro expanded population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 5, wherein the diluted or divided subpopulations are in a plurality of identical vessels.

12. A kit for testing candidate therapeutic agents comprising: an in vitro expande population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 1; and a plurality of identical vessels.

13. The kit of claim 12 wherein the cells are frozen.

14. The kit of claim 12 wherein the cells are lyophilized.

15. A method for testing substances as candidate therapeutic agents comprising: contacting a test substance with an in vitro expanded population of CD4⁺CD25⁺ regulatory T cells; determining a proliferative index of the population; identifying a test substance as a candidate therapeutic agent if the test substance increases the proliferative index relative to a control population which has not been contacted with a test substance.

16. The method of claim 13 wherein the proliferative index is measured by incorporation of a labeled metabolite.

17. The method of claim 13 wherein the proliferative index is measured by counting cells.

18. The method of claim 1 further comprising the step of: administering said CD4⁺CD25⁺ regulatory T cells to a mammal, whereby an immune response in the mammal is diminished.

19. The method of claim 2 wherein the CD4⁺CD25⁺ regulatory T cells were obtained from the mammal.

20. The method of claim 1 wherein the soluble 4-lBBL is a fusion protein with IgG- Fc.

21. The method of claim 18 wherein the mammal has a natural or induced autoimmune disease.

22. The method of claim 18 wherein the mammal is a human.

23. The method of claim 22 wherein the mammal is a transplant recipient.

24. The method of claim 22 wherein the mammal has graft versus host disease.

25. The method of claim 18 wherein the human has Hashimoto's thyroiditis .

26. The method of claim 22 wherein the mammal has pernicious anemia.

27. The method of claim 22 wherein the mammal has Addison's disease.

28. The method of claim 22 wherein the mammal has diabetes.

29. The method of claim 22 wherein the mammal has rheumatoid arthritis.

30. The method of claim 22 wherein the mammal has systemic lupus erythematosus.

31. The method of claim 22 wherein the mammal has dermatomyositis.

32. The method of claim 22 wherein the mammal has Sjogren's syndrome.

33. The method of claim 18 wherein the human has multiple sclerosis.

34. The method of claim 22 wherein the mammal has myasthenia gravis.

35. The method of claim 18 wherein the human has systemic sclerosis (scleroderma).

36. The method of claim 18 wherein the human has Reiter's syndrome.

37. The method of claim 18 wherein the human has Grave's disease.

38. The method of claim 1 wherein OX40L is also contacted with said cells.

39. The method of claim 1 wherein ICOSL is also contacted with said cells.

40. The method of claim 1 wherein anti-CD3 monoclonal antibody is also contacted with said cells.

41. A method of stimulating the proliferation of CD4⁺CD25⁺ regulatory T cells in a mammal, comprising: administering an effective amount of soluble 4-lBBL to a mammal in need of immune suppression or immune toleration, whereby proliferation of CD4⁺CD25⁺ regulatory T cells is stimulated.

42. The method of claim 41 wherein the soluble 4-lBBL is a fusion protein with IgG- Fc.

43. The method of claim 41 wherein the mammal is a human.

44. The method of claim 41 wherein the mammal has a natural or induced autoimmune disease.

45. The method of claim 43 wherein the mammal is a transplant recipient.

46. The method of claim 43 wherein the mammal has graft versus host disease.

47. The method of claim 43 wherein the mammal has Hashimoto's thyroiditis.

48. The method of claim 43 wherein the mammal has pernicious anemia.

49. The method of claim 43 wherein the mammal has Addison's disease.

50. The method of claim 43 wherein the mammal has diabetes.

51. The method of claim 43 wherein the mammal has rheumatoid arthritis.

52. The method of claim 43 wherein the mammal has systemic lupus erythematosus.

53. The method of claim 43 wherein the mammal has dermatomyositis.

54. The method of claim 43 wherein the mammal has Sjogren's syndrome.

55. The method of claim 43 wherein the mammal has multiple sclerosis.

56. The method of claim ^'43 wherein the mammal has myasthenia gravis.

57. The method of claim 43 wherein the mammal has systemic sclerosis (scleroderma).

58. The method of claim 43 wherein the mammal has Reiter's syndrome.

59. The method of claim 43 wherein the mammal has Grave's disease.

60. The method of claim 41 wherein OX40L is also administered to the mammal.

61. The method of claim 41 wherein ICOSL is also administered to the mammal.

62. The method of claim 41 wherein anti-CD3 monoclonal antibody is also administered to the mammal.

63. A method of stimulating the proliferation of CD4⁺CD25⁺ regulatory T cells, comprising: contacting CD4⁺CD25⁺ regulatory T cells in vitro with an effective amount of reagent cells comprising 4-lBBL-Fc on their surfaces, whereby proliferation of said CD4⁺CD25⁺ regulatory T cells is stimulated.

64. The method of claim 63 further comprising the step of: administering said CD4⁺CD25⁺ regulatory T cells to a mammal, whereby an immune response in the mammal is diminished.

65. A method of stimulating the proliferation of CD4⁺CD25 regulatory T cells in a mammal, comprising: administering an effective amount of reagent cells comprising 4-lBBL-Fc on their surfaces to a mammal in need of immune suppression or immune toleration, whereby proliferation of CD4⁺CD25⁺ regulatory T cells is stimulated.

66. The method of claim 64 or 65 wherein the mammal is a human.

67. The method of claim 63 wherein the reagent cells are antigen presenting cells.

68. The method of claim 65 wherein the reagent cells are antigen presenting cells.

69. The method of claim 64 wherein the CD4⁺CD25⁺ regulatory T cells were obtained from the mammal.

70. The method of claim 64 or 65 wherem the mammal has a natural or induced autoimmune disease.

71. The method of claim 66 wherein the mammal is a transplant recipient.

72. The method of claim 66 wherein the mammal has graft versus host disease.

73. The method of claim 66 wherein the mammal has Hashimoto's thyroiditis.

74. The method of claim 66 wherein the mammal has pernicious anemia.

75. The method of claim 66 wherein the mammal has Addison's disease.

76. The method of claim 66 wherein the mammal has diabetes.

77. The method of claim 66 wherein the mammal has rheumatoid arthritis.

78. The method of claim 66 wherein the mammal has systemic lupus erythematosus.

79. The method of claim 66 wherein the mammal has dermatomyositis.

80. The method of claim 66 wherein the mammal has Sjogren's syndrome.

81. The method of claim 66 wherein the mammal has multiple sclerosis.

82. The method of claim 66 wherein the mammal has myasthenia gravis.

83. The method of claim 66 wherein the mammal has systemic sclerosis (scleroderma).

84. The method of claim 66 wherein the mammal has Reiter's syndrome.

85. The method of claim 66 wherein the mammal has Grave's disease.

86. The method of claim 63 wherein said reagent cells further comprise OX40L on their surfaces.

87. The method of claim 63 wherein said reagent cells further comprise ICOSL on their surfaces.

88. The method of claim 63 wherein said reagent cells further comprises anti-CD3 monoclonal antibody on their surfaces.

89. The method of claim 65 wherein said reagent cells further comprise OX40L on their surfaces .

90. The method of claim 65 wherein said reagent cells further comprise ICOSL on their surfaces.

91. The method of claim 65 wherein said reagent cells further comprises anti-CD3 monoclonal antibody on their surfaces.

92. The method of claim 63 wherein not more than one CD4+CD25+ regulatory T cell is contacted.

93. The method of claim 63 wherein prior to the step of contacting the CD4⁺CD25⁺ regulatory T cells are diluted or divided to provide at least one aliquot comprising not more than one CD4⁺CD25⁺ regulatory T cell.

94. The method of claim 63 wherein the expanded CD4⁺CD25⁺ regulatory T cell population is packaged in a package that is sufficient to maintain viability of the expanded CD4⁺CD25⁺ regulatory T cell population.

95. The method of claim 63 wherein the expanded CD4⁺CD25⁺ regulatory T cell population is diluted or divided into subpopulations and packaged.

96. The method of claim 63 wherein the expanded CD4⁺CD25⁺ regulatory T cell population is lyophylized.

97. The method of claim 95 wherein the expanded CD4⁺CD25⁺ regulatory T cell population is lyophylized.

98. An in vitro expanded population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 63.

99. An in vitro expanded population of CD4 CD25 regulatory T cells made by the process of claim 92.

100. An in vitro expanded population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 93.

101. An in vitro expanded population of CD4⁺CD25⁺ regulatory T cells made by the process of claim 95, wherein the diluted or divided subpopulations are in a plurality of identical vessels.

102. An isolated population of reagent cells for sthnulating CD4⁺CD25⁺ regulatory T cells, wherein said reagent cells comprise 4-lBBL-Fc and OX40L-Fc on their surfaces.

103. An isolated population of reagent cells for stimulating CD4⁺CD25⁺ regulatory T cells, wherein said reagent cells comprise 4-lBBL-Fc and ICOSL-Fc on their surfaces.

104. An isolated population of reagent cells for stimulating CD4⁺CD25⁺ regulatory T cells, wherein said reagent cells comprise 4-lBBL-Fc and anti-CD3 monoclonal antibody on their surfaces.

105. The population of any of claims 102 to 104 wherein the reagent cells are antigen presenting cells (APCs).

106. The population of claim 102 wherein said reagent cells further comprise anti-CD3 monoclonal antibody on their surfaces.

107. The population of claim 102 wherein said reagent cells further comprise anti-CD3 monoclonal antibody and ICOSL-Fc on their surfaces.

108. The population of claim 103 wherein said reagent cells further comprise anti-CD3 monoclonal antibody on their surfaces.

109. The population of claim 108 wherein said reagent cells further comprise OX40L on their surfaces.