TECHNICAL FIELD The present invention relates to the development and differentiation of cells in a mammalian immune system. More particularly, the invention provides culture conditions for the in vitro maturation of precursor B cells. This is a valuable invention because it defines a method to induce development of antibody producing mature B-cells from early precursor cells.

BACKGROUND OF THE INVENTION

Significant health problems are faced in circumstance where immune function is compromised, such as where chemotherapy has destroyed a patient's peripheral blood cells, in radiation sickness, in organ transplant patients and in patients with a compromised immune system where Ig levels are subnormal, including patients with primary and secondary immune deficiencies, and premature infants that are highly susceptible to infections. These patients suffer problems from chronic and acute infections often caused by environmental pathogens found in many hospital settings. Supplementation of a compromised immune system with mature B cells will often restore at least partial normal immune system growth, differentiation and regulation.

Thus there is a need for a method to supplement a compromised immune system with mature B cells.

SUMMARY OF THE INVENTION

The present invention fills this need by providing for an in vitro method of inducing differentiation of precursor B cells (pre-B cells) to mature Immunoglobulin (Ig) -secreting B cells comprising contacting said precursor B cells with an aqueous medium comprised of IL-4 and a contact mediated signal provided by optimally activated CD4 ⁺ T cells.

The contact mediated signal is generally provided by incubating optimally activated CD4 ⁺ T cells, i.e., CD4+ T cells which have been activated for from 2-3 days, with the precursor B cells and the IL-4. The contact mediated signal can also be provided by cell membranes from optimally activated CD4 ⁺ T cells. The medium may also contain IL-7.

The present invention further provides for an in vitro method of inducing differentiation of precursor B cells (pre-B cells) to mature Immunoglobulin (Ig) -secreting B cells comprising contacting said precursor B cells with an aqueous medium comprised of IL-4, IL-7 and a contact mediated signal provided by non-optimally activated CD4 ⁺ T cells. An example of a non-optimally activated CD4 ⁺ T cell is a CD4 ⁺ T cell which has been activated for from 4-6 days.

The resulting mature B cells may be introduced into a host and in certain embodiments may be derived from the same host.

The present invention further provides for methods of reconstituting an impaired immune system of a human comprising the steps of: (a) isolating precursor B cells from a human; (b) maturing the precursor B cells to maturity by contacting the precursor B cells in an aqueous medium with an amount of IL-4 and contact mediated signal provided by optimally activated CD4 ⁺ T cells or membranes from an optimally activated CD4 ⁺ T cell effective to mature the precursor B cells; and (c) administering the mature B cells of step (b) to a human.

Alternatively, if non-optimally activated CD4 ⁺ T cells are used then IL-7 is added to the aqueous medium so as to produce mature Ig-secrering B cells.

The present invention further provides for in vivo methods of maturing precursor B cells in mammals by administering an effective amount of IL-4 and IL-7 sufficient to mature precursor B cells into mature B cells. The preferred mode involves human IL-4 and human and IL-7.

The present invention also provides a biological screening method to define the co-stimulating components needed to support growth and differentiation of early precursor B cells. The present

invention has allowed identification of the importance of activated CD4 ⁺ T cells or their membranes, in human B cell development. This in vitro assay can also be used to identify the factors and genes encoding co-stimulatory components.

The invention further discloses the therapeutic utility of IL-7 when combined with IL-4 for the in vivo maturation of pre-B cells.

DETAILED DESCRIPTION

Deficient humoral immunity is a problem in a number of situations, such as after bone marrow [BM] transplantation and in newborns, especially premature newborns, whose major cause of death are bacterial infections. In addition, agammaglobulinemias due to genetic defects can cause life long susceptibility to infections. We have now established a short-term culture system to induce growth and differentiation of plasma cells from pre-B cells in vitro. In addition, the clinical use of IL-4 and IL-7 are disclosed.

Mature resting B cells differentiate into Immunoglobulin [Ig] secreting cells and undergo Ig isotype switching in response to surface IgM [slgM] mediated signals in the presence of a contact mediated signal which is provided by CD4 ⁺ T cells via soluble and membrane bound factors. Cytokines are thought essential in B cell proliferation and differentiation, as well as in isotype switching. Under in vitro conditions, IL-4 is known to induce IgG4 and IgE switching, but additional signals provided by CD4 ⁺ T cells are required. Several other cytokines, such as IL-6, tumor necrosis factor (TNF)-α, TGF-β, IFN-α, and IFN-γ, are known to modulate IL-4-induced IgE synthesis in mature B cells. TGF-β also induces IgA switching.

Little is known about signal requirements for human pre-B cells to differentiate into mature B cells or into Ig secreting plasma cells. This is largely due to lack of knowledge of culture conditions that would induce pre-B cell differentiation in vitro. There has been successful work with murine B cell precursors which have been successfully cultured using the Whitlock-Witte culture system [Whitlock & Witte, Proc. Natl. Acad. Sci. 79: 3608-3602 (1982)].

The essential requirement of the Whitlock-Witte culture is the establishment of an adherent stromal cell microenvironment mimicking the bone marrow. Successful long-term culture of murine B cell precursors is dependent on the presence of stromal cells. In addition, murine pre-B cells were shown to undergo isotype switching and to differentiate into Ig secreting plasma cells when co-cultured with dendritic cells and activated T cells [Spalding & Griffen, Cell 44: 507-515 (1986)]. However, these culture systems have not been successful when human pre-B cells are studied.

Pre-B cells are classically characterized by lack of surface IgM (slgM") expression. Human pre-B cells were recently shown to spontaneously differentiate into IgM ⁺ cells when cultured in RPMI + 10% fetal calf serum in the absence of exogenous growth factors or cytokines, but no differentiation into Ig-secreting cells in these cultures was detected [Villablanca et al. }. Exp. Med. 172: 325-334 (1990)]. More recently, it was demonstrated that immature CD10 ⁺ , CD19 ⁺ , sIgM ⁺ B cells derived from fetal spleen or bone marrow (BM) are able to undergo isotype switching and differentiate into Ig-secreting cells in response to IL-4 in the presence of anti-CD40 monoclonal antibodies (mAb) or CD4 ⁺ T cells [Punnonen et al. }. Immunol. 148: 3398-3404 (1992)]. However, in those culture conditions slgM ^" pre-B cells failed to differentiate into Ig- secreting cells.

The present invention provides culture conditions permitting maturation of human pre-B cells. The work demonstrates that significant production of IgM, total IgG, IgG4 and IgE is detected when highly purified human slgM", CD19 ⁺ pre-B cells are co-cultured with optimally activated cloned CD4 ⁺ T cells in the presence of IL-4. An optimally activated CD4 ⁺ T cell is a CD4 ⁺ T cell which is able to produce a contact mediated signal needed for human slgM", CD19+ pre-B cells to develop into mature Ig secreting B cells in the presence of IL-4. Generally, the CD4 ⁺ T cells which are used are obtained 2-3 days after activation. When CD4 ⁺ T cells are used 4-6 days after activation, no Ig synthesis by the B cells is observed. However, if IL-7 is added to a culture containing IL-4 and CD4 ⁺ T cells which have been obtained 4-6 days after activation, the human slgM", CD19 ⁺ pre-B cells do develop

into Ig secreting mature B cells. This is distinguished from earlier work (Punnonen et al, 1992, supra) which disclosed that sIgM ⁺ fetal BM B cells underwent isotype switching and differentiation into Ig secreting cells in response to CD4 ⁺ T cells and IL-4 without IL-7.

Membrane preparations of activated CD4 ⁺ T cell clones will also induce IgM and IgG synthesis by pre-B cells in the presence of IL-4 indicating that the co-stimulatory signal required in pre-B cell differentiation is in a membrane-bound form.

Precursor B cells are surface IgM" and CD19 ⁺ . Classical definition of pre-B cells is that they have IgM in their cytoplasm (μ). By comparison, mature B cells are defined as expressing surface IgM and CD19". Pre-B cells can be obtained from bone marrow of either adults or fetuses.

In brief, pre-B cells are obtained from mammalian, e.g. human, bone marrow (BM). The marrow cells are extracted from the mammalian source using conventional techniques. These include extraction by needle or by surgical removal of bone material.

Details of obtaining bone marrow aspirate from a human patient are described in U.S. Patent Nos. 4,481,946 and 4,486,188. In brief prior to insertion of the needles into the bone marrow cavity, the cavity is washed with an anti-coagulation bath, e.g., heparin (2000 units). The bath comprises an intravenous solution (an isotonic saline electrolyte solution containing 4 units /cc heparin) which is introduced from a drip chamber by opening aspirator valves. Needles are inserted into the bone marrow cavity, and valve adjustments allow the entry of solution from the drip chamber followed by aspiration of marrow inducing the flow of marrow and sinusoidal blood.

The marrow cells are diluted with physiologically compatible buffer containing nutrients. Diluents include phosphate buffered saline (PBS) and RPMI-1640 from JRH Biosciences, Lenexa, KS which is preferred. The cells are repeatedly washed using low speed centrifugation. Red blood cells are lysed by osmotic shock using a hypotonic buffer solution. A preferred buffer and conditions are Tris-

buffered 0.83% NH4CI (pH 7.2) at room temperature for two minutes, after which the cells are washed twice. The intact cells are finally counted and resuspended in Yssel's medium [Yssel et al. }. Immunol. Methods, 72: 219 (1984)] supplemented with 10% fetal calf serum (FCS). Table 1 provides Yssel's medium.

TABLE 1 YSSEL'S MEDIUM

1. Iscove's modified Dulbecco's Medium (IMDM (JRH Biosciences 51-47178, 500 ml) (w/1-glutamine, w/25 mM HEPES, w/ Alpha thioglycerol).

2. Bovine serum albumin (Sigma, A2153 or <0.1ng/mg endotoxin detectable (Sigma A3675) to final concentration of 0.25% (w/v).

3. 2-aminoethanol (Ethanolamine: Sigma E0135, 100 ml) to final concentration of 1.8 μg per liter. 4. Transferrin (Apo form) (Pierce) liquid or powder final concentration 20 μg/ml.

5. Insulin (Sigma from bovine pancreas 1-5500, 1 gram) to final concentration of 5 μg/ml (dissolve insulin first in 5 ml 0.01N HC1 and add this to the medium). 6. Linoleic acid (Sigma L1376) to final concentration of 2 μg/ml.

7. Oleic acid (Sigma 03879) to final concentration of 2 μg/ml (Stock linoleic and oleic acid should be stored at -20°C under nitrogen to prevent oxidation of the unsarurated bond; make glass ampoules containing 5 mg of linoleic & 5 mg of oleic acid. For each preparation, dissolve the fatty acids in ethanol (100 μl) and add this mixture to the medium.

8. Palmitic acid (Sigma P-5917) to final concentration of 2 μg/ml [stock of palmitic acid in ethanol (20 mg/ml) and add the right concentration] (store at 4°C). 9. Penicillin / streptomycin (TRH cat. nr. 59-60277P, 100 ml) 5 ml in 500 ml.

From the remaining cells, the pre-B cells are obtained. The pre-B cells are isolated by monoclonal antibodies specific for pre-B cells. The desired slgM", CD19 ⁺ pre-B cells are then sorted using a fluorescence activated cell sorter (FACS) or magnetic beads according to standard procedures. The pre-B cells may be crudely enriched using a ficoll density gradient where the pre-B cells are separated from red blood cells

and granulocytes on a density gradient (1.077 gm/ml) spun at 800 x g for 20 minutes. The pre-B cells will be found at the interface. Alternatively, one can take advantage of the fact that the pre-B cells do not carry the following epitope markers CD3, CD4, CD8, CD14, CD33 or slgM. By passing the cells through binding columns which target these markers, the desired pre-B cells can be isolated from undesired cells. Finally the pre-B cells are isolated from the heterogeneous population of bone marrow cells using immunoaffinity methods which specifically target unique pre-B cell surface epitopes. Such epitopes include CD19 and CD10. Conditions which permit cell survival and binding between antibodies and the cells are known and a general reference explaining this art is Punnonen et al, }. Immunol. 148: 3398-3404 (1992).

The pre-B cells are cultured in the presence of optimally activated CD4 ⁺ T cells or membranes obtained from the activated T cells. The

CD4 ⁺ T cells are cloned from activated mammalian (human) peripheral blood cells (PBC). In brief, peripheral blood cells are obtained from standard blood drawing techniques. The cells are then activated by mitogenic lectins such as phytohemagglutinin (PHA), Concanavalin A (Con A), soluble antigens such as tetanus toxoid, alloantigens expressed in allogenic PBC, or Epstein Barr Virus (EBV) transformed in B cells. Next the cells are cloned according to standard procedures in the presence of a mixture of irradiated PBC and EBV transformed B cell line cells and PHA as described by Spits et al, }. Immunol. 128: 95-99 (1982).

The maturation of the pre-B cells takes place in the presence of contact mediated signal provided by optimally activated CD4 ⁺ T cells and IL-4. IL-7 can also be added to enhance differentiation of the pre-B cells into Ig-secreting cells. However, if the CD4 ⁺ T cells are not optimally activated, i.e., they have been activated for more than 4 days, then IL-7 must be added to the culture medium in order for the pre-B cells to differentiate into Ig-secreting cells. Both cytokines are available through conventional recombinant expression systems. For example, a suitable expression system for IL-4 is described in U.S. Pat. No. 5,017,691. The cloning and expression of IL-7 has been described by Goodwin

PNAS 86: 302-306 (1989) and Lupton, et al, }. Immunol. 3592-3601 (1990). Alternatively, these cytokines can be purchased from commercial vendors such as R & D Systems, Inc. Minneapolis, MN.

Maturation of the B cells can be monitored using a number of standard procedures. For example, one can measure the production of IgM, IgG, IgG4, IgE, or IgA using enzyme immunoassays. In brief, these methods involve standard immunoassays for the secretion of the several Ig classes known to be produced by mature B cells. Alternatively, surface molecules may be detected using analytical FACS methods.

The in vitro maturation of pre-B cells permits the re-infusion into a patient, preferably the same patient from which the pre-B cells originated. The cells are harvested from the nutrient medium and resuspended into a physiologically compatible medium such as standard isotonic infusion fluids. The cells are then re-infused directly into the patient using conventional intravenous equipment. Procedures follow those for autologous bone marrow transplantation, e.g., reviewed in Bombik, B.M., Strategy of Autologous Bone Marrow Transplantation, in Autologous Bone Marrow Transplantation and Solid Tumors, J.G. McVie, O. Dalesio and IE. Smith, eds., pp. 13-18 (1984); and Autologous Bone Marrow Transplantation, Dickey, Spitzer, and Jagannath (Eds.), The University of Texas, M.D. Anderson Hospital and Tumor Institute at Houston (1987).

For infusion, the total number of mature B cells are preferably between 1-8 x 10 ⁶ cells per kilogram of weight. The total liquid being infused should be between .75 and 1.5 liters for a mammal of 70 kg. Infusion of the entire mixture is typically carried out via a central venous catheter. As an optional step, the patient may receive supplemental therapy with a pharmaceutical preparation comprising the selected antibiotics.

From a practical perspective, one seeks to achieve maximum restoration of antibody producing B cells. Accordingly, a maximum number of B cells are desired for infusion. Pre-B cells represent approximately 1% of the cells in adult BM. Typically, no more than

5 x 10 ⁸ cells can be isolated from a standard BM sample. By growing the pre-B cells under present culture conditions the cells can be increased 3 to 6 fold after 7 days. Thus, 3 x 10 ⁹ cells will be available for infusion

from a single BM aspiration. The level of antibody production and/or B cell numbers are monitored in order to re-administer additional B cells as necessary.

Alternatively, there are patients which have a B cell deficiency who will benefit from the co-administration of IL-4 and IL-7. Such patients include those with hypogammaglobulinemias, newborns, patients after BM transplantations, or cancer after heavy chemo- or radiation therapy. Both cytokines are co-administered to these patients either simultaneously or in succession. For an average patient in need of in vivo maturation of B cells, a dosage range of about 0.5 to about 10 and preferably 1 to 5 μg/kg body weight of both IL-4 and IL-7 is co- administered on a daily basis until the level of B cells approaches normal.

EXAMPLES

The present invention can be illustrated by the following, non- limiting Examples. Unless otherwise specified, percentages given below for solids in solid mixtures, liquids in liquids, and solids in liquids are on a wt/wt, vol/vol and wt/vol basis, respectively.

1. Method for maturing B cells from precursor cells. A. Reagents.

The following reagents were used to isolate and mature human pre-B cells. Purified human recombinant IL-4 was provided by Schering-Plough Research (Kenilworth, NJ). Recombinant IL-7 was obtained from R & D Systems Inc. (Minneapolis, MN). Phycoerythrine (PE)-conjugated mAbs against CD19 as well as isotype control antibodies with irrelevant specifications, were obtained from Becton Dickinson (Mountain View, CA). FITC-conjugated anti-IgM mAb was obtained from Nordic Immunological Laboratories (Tilburg, the Netherlands).

B. Cell preparations.

Human fetal bone marrow (BM) samples were obtained from fetuses varying from 16 to 24 weeks of gestation. BM cells were obtained by flushing RPMI-1640 0RH Biosciences, Lenexa, KS) with a syringe into

the intramedullary cavities of the long bones. The cells were washed twice. Red blood cells were lysed by exposure to 2 ml of Tris-buffered 0.83% NH ₄ CI (pH 7.2) at room temperature for two minutes, after which the cells were washed twice. The cells were finally counted and resuspended in Yssel's medium [Yssel et al. }. Immunol. Methods, 72 : 219 (1984)] supplemented with 10% fetal calf serum [FCS].

B cell precursors were obtained from fetal BM using fluorescence- activated cell sorter, FACStar Plus® (Becton Dickinson). BM cells were first incubated with CD19-PE and slgM-FITC for 30 min at 4°C. FTTC- and PE-conjugated mAb with irrelevant specificities were used as negative controls. The cells were washed twice with PBS. Cells with the light scatter characteristics of lymphocytes were gated and the cells were sorted on the basis of fluorescence stainings. Re-analysis of the sorted cells indicated 97-100% purities.

C. Culture conditions.

The cells obtained were cultured in round-bottomed 96-well plates (Linbro, McLean, VA; Cat. No. 76-013-05) at 37°C in a humidified atmosphere containing 5% CO2 in 0.2 ml Yssel's medium supplemented with 10% FCS. The cells were cultured at a concentration of 5000 cells/well. All cultures were performed in triplicate. In co-culture experiments, the human CD4 ⁺ T cell clone B21 [Roncarolo et al, f. Exp. Med. 167 : 1523 (1988) and Vieira et. al, Proc. Natl. Acad. Sci, U.S.A. 88 : 1172-1176 (1991)] was added at the onset of the cultures at 5000 cells/well. B21 cells were obtained 2 days or 4-6 days after they had been activated by feeder cell mixture and PHA at 0.1 μg/ml as described by Gascan et. al, J. Exp. Med. 173 : 747-750 (1991) and Gascan et al, Eur. }. Immunol, 22 : 1133-1141 (1992). All cytokines used were added at the onset of the culture. IL-4 and IL-7 were added at a concentration of 8 μg/ml and 15 μg/ml, respectively.

D. Measurement of Ig production.

IgM, total IgG, IgA and IgE secretion were determined by ELISA as described previously by Pene et al, Proc. Natl. Acad. Sci, U.S.A. 85 : 8166- 8170 (1988) and Pene et al, }. Immunol. 141 : 1218-1224 (1988). IgG4

secretion was determined by ELISA as described by Punnonen et al, J. Immunology, 148 : 3398-3404 (1992). The sensitivities of IgM, total IgG, and IgA ELISAs were 0.5-1 ng/ml, and the sensitivities of IgG4 and IgE ELISAs were 0.2 ng/ml.

E. Results.

The highly purified pre-B cells were co-cultured in the presence of activated CD4 ⁺ T cells, IL-4 and/or IL-7. Significant production of IgM, total IgG, IgG4, and IgE in pre-B cell cultures was detected. Kinetic studies indicated that detectable levels of IgM and IgG in the supernatants of these cultures appeared at day 7, while IgE was detected at day 10. When the T cells were used 4-6 days after they had been activated, IL-7 was a specific requirement together with IL-4 of pre-B cells for maturation. When the T cells were used 2-3 days after activation, they were competent to mature the pre-B cells with only the addition of IL-4 and without the addition of IL-7.

To ensure that the production of immunoglobulins was not an artifact attributed to contaminating mature B cells, the production of Ig was measured at decreasing cell numbers. Ig production in pre-B cell cultures was detected at cell numbers as low as 100 B cells/well. Because Ig production was detected using such low pre-B cell numbers, and no slgM ^"1" cells among sorted slgM" pre-B cells could be detected by FACS- analysis, the possibility of contaminating sIgM ⁺ B cells as the cause of Ig synthesis in our pre-B cell cultures can be excluded.

In addition, the fact that the mature sIgM ⁺ , CD19 ⁺ B cells produce Ig when co-cultured with IL-4 and CD4 ⁺ cloned T cells 4-6 days after the T cells had been activated whereas slgM", CD19 ⁺ pre-B cells failed to do so also strongly argues against contaminating sIgM ⁺ cells as the cause of Ig synthesis detected, and indicates that the signals required for slgM ^"1" immature B cells to differentiate into Ig-secreting cells are not sufficient to mediate slgM" pre-B cell differentiation.

Supernatants of activated CD4 ⁺ T cell clone failed to induce pre-B cell differentiation regardless of whether IL-7 was present or not, suggesting that the co-stimulatory signal(s) provided by the T cells was

in membrane-bound form. The effect of T cell membranes on pre-B cell differentiation was studied to further investigate this possibility.

A CD4 ⁺ T cell clone was activated for two days by ConA, and the cells were disrupted and membrane preparations were prepared as described by Gascan et al, Eur. f. Immunol, 22 : 1133-1141(1992). The data indicate that these membrane preparations of CD4 ⁺ T cells induced IgM and IgG synthesis by pre-B cells in the presence of IL-4, indicating that the co-stimulatory signal required in pre-B cell differentiations, indeed, is in a membrane-bound form. This implies that the present culture system also can be used as a screening method to identify T-cell membrane associated molecules and the genes encoding these molecules.

2. Method for treating patients with mature B cells.

For a patient suffering from humoral immunodeficiency, an infusion of self-mature B cells is therapeutically beneficial. A bone marrow extract is obtained from the patient by aspiration. Pre-B cells are isolated from the marrow using the antibodies and cell sorting procedures provided in Example 1. The purified pre-B cells are then cultured and matured to antibody producing B cells according to the methods of Example 1. Mature antibody producing B cells are then harvested, suspended in physiological saline solution and re-infused. The number of circulating mature B cells and antibody production are then monitored using conventional techniques.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be recognized that certain changes and modifications may be practiced within the scope of the claims.