Methods of expanding and assessing B cells and using expanded B cells to treat disease
Provided herein are methods of expanding B cells, and in particularly B10 cells capable of producing IL-10, ex vivo. The methods include incubation of harvested B cells in the presence of IL-21. Compositions comprising the ex vivo expanded B cells and methods of using the expanded B cell-containing compositions to treat diseases or conditions are also provided. Methods of assessing B10 cell function in a subject are also provided.
1. A method of expanding B10 cells capable of producing IL-10 ex vivo comprising culturing polyclonal B cells harvested from a subject with IL-21 and feeder cells expressing a CD40 agonist and a B cell survival promoter and isolating the polyclonal B10 cells.
2. The method of claim 1 , further comprising first culturing the B cells on feeder cells expressing a CD40 agonist and BAFF (BLyS) in the presence of IL-4.
3. A method of expanding B cells ex vivo comprising:
a) Culturing polyclonal B cells harvested from a subject on feeder cells expressing a CD40 agonist and a B cell survival promoter in the presence of IL-4;
b) Culturing the B cells from step (a) on feeder cells expressing a CD40 agonist and a B cell survival promoter in the presence of IL-21;
c) Isolating the B cells from step (b); and
d) Isolating the polyclonal B10 cells from the B cells of step (c).
4. The method of claim 3 , wherein the feeder cells further express at least one of VCAM-1, CD24, IL-7, Mst1, Tslp and CD44.
5. The method of claim 3 , wherein the feeder cells express at least two fold less of CD99, CXCR7, Dlk1, Jag1, Notch1 or TGFBI than control feeder cells.
6. The method of claim 3 , wherein the B cells are harvested from the blood, spleen, peritoneal cavity, lymph nodes, bone marrow, site of autoimmune disease, site of inflammation or tissue undergoing transplant rejection of the subject.
7. The method of claim 3 , wherein the B cells are isolated by removal of non-B cells and selection for cell surface immunoglobulin (IgM, IgD, IgA, or IgE) prior to use in the method.
8. The method of claim 3 , wherein the CD40 agonist is CD154, a fragment of CD154, or antibody, aptamer or polypeptide, or fragment thereof reactive with CD40.
9. The method of claim 3 , wherein the B cell survival promoter is selected from at least one of feeder cells, BAFF (BLyS), BAFF fragments, APRIL, CD22 ligand, CD22 monoclonal antibody, or fragments thereof.
10. The method of claim 3 , wherein the feeder cells are fibroblast, endothelial cells, epithelial cells, keratinocytes, melanocytes, or other mesenchymal or stromal cells.
11. The method of claim 3 , wherein the culturing step with IL-4 is a three to ten day culture period and the culturing step with IL-21 is a four to eight day culture period.
12. The method of claim 3 , wherein the isolated B10 cells are more than 50% B10 cells capable of producing IL-10.
13. The method of claim 3 , wherein the number of B10 cells is expanded by at least 5,000 fold relative to the number of B cells harvested from the subject.
14. The method of claim 1 , wherein the B10 cells are isolated by selecting for cells expressing a cell surface marker selected from the group consisting of CD1d, CD5, CD24, CD27 and combinations thereof.
15. The method of claim 3 , wherein the B10 cells are isolated by selecting for cells expressing a cell surface marker selected from the group consisting of CD1d, CD5, CD24, CD27 and combinations thereof.
16. The method of claim 1 , wherein the B cells used in the method represent the total B cells.
17. The method of claim 3 , wherein the B cells used in the method represent the total B cells.
18. The method of claim 1 , wherein the B cells are isolated by removal of non-B cells and selection for cell surface IgM prior to use in the method.