Populations of enriched regulatory T cells and methods for producing same
Provided herein are populations of enriched ex vivo expanded umbilical cord blood-derived regulatory T cells. Also provided are methods of making and using the same.
1 . A method for producing an expanded population of human T regulatory (Treg) cells enriched for CXCR4 + Treg cells from a cryopreserved human umbilical cord blood unit, the method comprising:
(a) thawing the cryopreserved human umbilical cord blood unit;
(b) diluting and washing the thawed umbilical cord blood unit;
(c) isolating CD25+ Treg cells using double selection for CD25 + cell surface expression with a reagent that specifically binds to CD25;
(d) ex vivo expanding the isolated CD25 + Treg cells in a culture medium, in a gas permeable cultureware, in the presence of:
(1) an effective amount of interleukin-2 (IL-2);
(2) a reagent that specifically binds to CD3 and CD28; and
(3) magnetic microbeads coated with anti-CXCR4 antibody,
for up to 10 days or up to 12 days, wherein the culture medium is replaced about every 48 hours, to produce a CXCR4-enriched culture of CD25 + Treg cells; and
(e) harvesting the CD25 + CXCR4 + cells from the culture medium to produce an expanded population of human Treg cells enriched for CXCR4 + Treg cells.
2 . The method of claim 1 , wherein in step (d),
(1) the ex vivo expansion is initiated at day 0;
(2) the effective amount of IL-2 is added to the isolated CD25 + Treg cells at day 0; and
(3) the reagent that specifically binds to CD3 and CD28 is added to the isolated CD25 + Treg cells at day 0.
3 . The method of claim 1 , wherein the reagent that specifically binds to CD3 and CD28 is removed from the culture medium before the magnetic microbeads coated with anti-CXCR4 antibody are added to the culture medium.
4 . The method of claim 1 , wherein in step (d), the magnetic microbeads coated with anti-CXCR4 antibody are added to the culture medium 3 or 4 days after the ex vivo expansion is initiated.
5 . The method of claim 1 , wherein in step (d), the magnetic microbeads coated with anti-CXCR4 antibody are added to the culture medium for about 30 minutes before a double ferromagnetic column is used to harvest the CD25+ CXCR4 + Treg cells.
6 . The method of claim 1 , wherein step (d) takes place over 4 or 5 days.
7 . The method of claim 1 , wherein the reagent that specifically binds to CD25 is an anti-CD25 antibody or an antigen-binding fragment thereof.
8 . The method of claim 7 , wherein the CD25 + cells and the beads coated with anti-CD3 antibody and beads coated with anti-CD28 antibody are at a 1:1 ratio.
9 . The method of claim 1 , wherein the reagent that specifically binds to CD25 is conjugated to a solid support.
10 . The method of claim 9 , wherein the solid support is a magnetic microbead.
11 . The method of claim 1 , wherein the reagent that specifically binds to CD3 and CD28 comprises an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof.
12 . The method of claim 1 , wherein the reagent that specifically binds to CD3 and CD28 comprises beads coated with an anti-CD3 beads antibody or an antigen-binding fragment thereof and beads coated with anti-CD28 antibody or an antigen-binding fragment thereof.
13 . The method of claim 12 , wherein the beads coated with an anti-CD3 antibody and the beads coated with an anti-CD28 antibody are at a 1:1 ratio.
14 . The method of claim 1 , wherein the effective amount of IL-2 is about 1000 IU/ml.
15 . The method of claim 1 , wherein in step (d), IL-2 is added to the culture medium about every 48 hours.
16 . The method of claim 1 , wherein in step (e), about 1×10 6 CD25 + cells/ml are cultured.
17 . The method of claim 1 , wherein in step (e), the cells are initially cultured in gas-permeable cultureware that has a membrane surface area of 10 cm 2 .
18 . The method of claim 17 , wherein the culture is subsequently transferred to gas-permeable cultureware that has a membrane surface area of 100 cm 2 .
19 . The method of claim 1 , wherein in step (d), the culture is not rocked or agitated when the IL-2 is added.
20 . The method of claim 1 , wherein in step (a), the cryopreserved human umbilical cord blood unit is thawed in a single step in a water bath.
21 . The method of claim 1 , wherein step (b) does not comprise manual washing.
22 . The method of claim 1 , wherein step (b) takes place in a solution comprising PBS, EDTA, and about 0.5% human serum albumin.
23 . The method of claim 1 , wherein a double ferromagnetic column method is used in step (c) to isolate CD25 + Treg cells.
24 . The method of claim 1 , the method further comprising cryopreserving the expanded population of human Treg cells enriched for CXCR4 + Treg cells.
25 . The method of claim 1 , wherein the expanded population of human Treg cells enriched for CXCR4″ Treg cells is:
(i) ≥60% CD4 + CD25+;
(ii) ≥60% CD4 + CD25 + CXCR4 + ; and
(iii) ≤10% CD4 CD8 + , as measured by flow cytometry.
26 . An expanded population of human Treg cells enriched for CXCR4+Treg cells produced by the method of claim 1 , wherein the expanded population of human Treg cells comprises at least 60% CD4 + CD25 + cells and less than 10% CD4 − CD8 + cells.
27 . A method for producing an expanded population of human Treg cells enriched for CXCR4 + Treg cells from a cryopreserved human umbilical cord blood unit, the method comprising:
(a) thawing the cryopreserved human umbilical cord blood unit;
(b) diluting and washing the thawed umbilical cord blood unit;
(c) isolating Treg cells using double selection for CD25 + cell surface expression with a reagent that specifically binds to CD25;
(d) ex vivo expanding the isolated CD25 + Treg cells in a culture medium, in a gas permeable cultureware, wherein the ex vivo expansion step comprises:
(1) at day 0, adding beads coated with an anti-CD3antibody and beads coated with an anti-CD28 antibody to the culture medium;
(2) at day 2, adding about 1000 IU/ml IL-2 to the culture medium;
(3) at day 3 or 4, removing the beads coated with an anti-CD3 antibody and the beads coated with an anti-CD28 antibody from the culture medium and adding anti CXCR4 magnetic microbeads coated with anti-CXCR4 antibody to the culture medium; and
(4) at day 3 or 4, removing the magnetic microbeads coated with anti-CXCR4 antibody attached to CXCR4 + Treg cells from the culture medium, and adding fresh beads coated with an anti-CD3 antibody and beads coated with an anti-CD28 antibody to the CXCR4 + Treg cells,
wherein the ex vivo expansion takes place for up to 10 days or up to 12 days, wherein the culture medium is replaced about every 48 hours, to produce a CXCR4-enriched culture of CD25 + Treg cells; and
(e) harvesting the CD25 + CXCR4 + cells from the culture medium to produce an expanded population of human Treg cells enriched for CXCR4 + Treg cells.
28 . An expanded population of human Treg cells enriched for CXCR4 + Treg cells produced by the method of claim 27 , wherein the expanded population of human Treg cells comprises at least 60% CD4 + CD25 + cells and less than 10% CD4 − CD8 + cells.