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Homogeneous expansion of human T-regulatory cells via tumor necrosis factor receptor 2.

Okubo Y, Mera T, Wang L, Faustman DL - Sci Rep (2013)

Bottom Line: In in vitro studies, a TNFR2 agonist was found superior to standard methods in proliferating human T(regs) into a phenotypically homogeneous population consisting of 14 cell surface markers.The TNFR2 agonist-expanded T(regs) also were functionally superior in suppressing a key T(reg) target cell, cytotoxic T-lymphocytes.Targeting the TNFR2 receptor during ex vivo expansion is a new means for producing homogeneous and potent human T(regs) for clinical opportunities.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Massachusetts General Hospital and Harvard Medical School, Rm 3602, MGH-East, Bldg 149, 13th Street, Boston, MA 02129.

ABSTRACT
T-regulatory cells (T(regs)) are a rare lymphocyte subtype that shows promise for treating infectious disease, allergy, graft-versus-host disease, autoimmunity, and asthma. Clinical applications of T(regs) have not been fully realized because standard methods of expansion ex vivo produce heterogeneous progeny consisting of mixed populations of CD4 + T cells. Heterogeneous progeny are risky for human clinical trials and face significant regulatory hurdles. With the goal of producing homogeneous T(regs), we developed a novel expansion protocol targeting tumor necrosis factor receptors (TNFR) on T(regs). In in vitro studies, a TNFR2 agonist was found superior to standard methods in proliferating human T(regs) into a phenotypically homogeneous population consisting of 14 cell surface markers. The TNFR2 agonist-expanded T(regs) also were functionally superior in suppressing a key T(reg) target cell, cytotoxic T-lymphocytes. Targeting the TNFR2 receptor during ex vivo expansion is a new means for producing homogeneous and potent human T(regs) for clinical opportunities.

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TNFR2 agonist versus antagonist on expansion of Treg population.(a) Protocol for purifying CD4 + CD25hi cells from CD4+ cells from fresh blood and expanding for 16 days by incubation in 96 well round-bottom plate (2 × 104 cells/well) with anti-CD3 and anti-CD28 antibodies, human IL-2, and rapamycin. (b) Representative CD25 and FOXP3 flow diagrams of CD4+ cells before versus after CD25hi purification and expansion, indicating purity of populations. (c) Cell counts of purified Tregs, by treatment group, reveal that TNFR2 agonist induced more expansion than any other group. (**P < 0.01, by paired t-test.) The TNFR2 antagonist suppresses expansion versus standard treatment. Data in (c) are samples from 10 subjects.
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f3: TNFR2 agonist versus antagonist on expansion of Treg population.(a) Protocol for purifying CD4 + CD25hi cells from CD4+ cells from fresh blood and expanding for 16 days by incubation in 96 well round-bottom plate (2 × 104 cells/well) with anti-CD3 and anti-CD28 antibodies, human IL-2, and rapamycin. (b) Representative CD25 and FOXP3 flow diagrams of CD4+ cells before versus after CD25hi purification and expansion, indicating purity of populations. (c) Cell counts of purified Tregs, by treatment group, reveal that TNFR2 agonist induced more expansion than any other group. (**P < 0.01, by paired t-test.) The TNFR2 antagonist suppresses expansion versus standard treatment. Data in (c) are samples from 10 subjects.

Mentions: We separated fresh human blood to obtain pure CD4+ and CD25hi co-expressing Tregs (Fig. 3). We purified and expanded these Tregsin vitro using another standard method of Treg expansion, anti-CD3 plus anti-CD28 plus IL-2 for 16 days with or without TNF, TNFR2 antagonist, or TNFR2 agonist (Fig. 3a), then rested them overnight before counting. We added rapamycin (until day 7) because it selectively expands the highest number of Tregs with greatest capacity for suppressing CD8+ cells40414243. This process successfully produced CD4+CD25hi Tregs (Fig. 3b). We assessed Treg expansion by treatment group: standard treatment, treatment with TNF, TNFR2 agonist, or TNFR2 antagonist. The TNFR2 agonist outperformed every other group, expanding Treg numbers at least twofold higher than standard treatment or antagonist treatment (Fig. 3c). Because rapamycin is known to inhibit proliferation, we examined the effects of treatment without rapamycin, yet found similarly opposing effects between agonist versus antagonist treatment, albeit at smaller mean absolute values (Supplementary Fig. 3). The yields of expanded cells tended to be less without rapamycin, but the agonist still expanded Tregs.


Homogeneous expansion of human T-regulatory cells via tumor necrosis factor receptor 2.

Okubo Y, Mera T, Wang L, Faustman DL - Sci Rep (2013)

TNFR2 agonist versus antagonist on expansion of Treg population.(a) Protocol for purifying CD4 + CD25hi cells from CD4+ cells from fresh blood and expanding for 16 days by incubation in 96 well round-bottom plate (2 × 104 cells/well) with anti-CD3 and anti-CD28 antibodies, human IL-2, and rapamycin. (b) Representative CD25 and FOXP3 flow diagrams of CD4+ cells before versus after CD25hi purification and expansion, indicating purity of populations. (c) Cell counts of purified Tregs, by treatment group, reveal that TNFR2 agonist induced more expansion than any other group. (**P < 0.01, by paired t-test.) The TNFR2 antagonist suppresses expansion versus standard treatment. Data in (c) are samples from 10 subjects.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818650&req=5

f3: TNFR2 agonist versus antagonist on expansion of Treg population.(a) Protocol for purifying CD4 + CD25hi cells from CD4+ cells from fresh blood and expanding for 16 days by incubation in 96 well round-bottom plate (2 × 104 cells/well) with anti-CD3 and anti-CD28 antibodies, human IL-2, and rapamycin. (b) Representative CD25 and FOXP3 flow diagrams of CD4+ cells before versus after CD25hi purification and expansion, indicating purity of populations. (c) Cell counts of purified Tregs, by treatment group, reveal that TNFR2 agonist induced more expansion than any other group. (**P < 0.01, by paired t-test.) The TNFR2 antagonist suppresses expansion versus standard treatment. Data in (c) are samples from 10 subjects.
Mentions: We separated fresh human blood to obtain pure CD4+ and CD25hi co-expressing Tregs (Fig. 3). We purified and expanded these Tregsin vitro using another standard method of Treg expansion, anti-CD3 plus anti-CD28 plus IL-2 for 16 days with or without TNF, TNFR2 antagonist, or TNFR2 agonist (Fig. 3a), then rested them overnight before counting. We added rapamycin (until day 7) because it selectively expands the highest number of Tregs with greatest capacity for suppressing CD8+ cells40414243. This process successfully produced CD4+CD25hi Tregs (Fig. 3b). We assessed Treg expansion by treatment group: standard treatment, treatment with TNF, TNFR2 agonist, or TNFR2 antagonist. The TNFR2 agonist outperformed every other group, expanding Treg numbers at least twofold higher than standard treatment or antagonist treatment (Fig. 3c). Because rapamycin is known to inhibit proliferation, we examined the effects of treatment without rapamycin, yet found similarly opposing effects between agonist versus antagonist treatment, albeit at smaller mean absolute values (Supplementary Fig. 3). The yields of expanded cells tended to be less without rapamycin, but the agonist still expanded Tregs.

Bottom Line: In in vitro studies, a TNFR2 agonist was found superior to standard methods in proliferating human T(regs) into a phenotypically homogeneous population consisting of 14 cell surface markers.The TNFR2 agonist-expanded T(regs) also were functionally superior in suppressing a key T(reg) target cell, cytotoxic T-lymphocytes.Targeting the TNFR2 receptor during ex vivo expansion is a new means for producing homogeneous and potent human T(regs) for clinical opportunities.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Massachusetts General Hospital and Harvard Medical School, Rm 3602, MGH-East, Bldg 149, 13th Street, Boston, MA 02129.

ABSTRACT
T-regulatory cells (T(regs)) are a rare lymphocyte subtype that shows promise for treating infectious disease, allergy, graft-versus-host disease, autoimmunity, and asthma. Clinical applications of T(regs) have not been fully realized because standard methods of expansion ex vivo produce heterogeneous progeny consisting of mixed populations of CD4 + T cells. Heterogeneous progeny are risky for human clinical trials and face significant regulatory hurdles. With the goal of producing homogeneous T(regs), we developed a novel expansion protocol targeting tumor necrosis factor receptors (TNFR) on T(regs). In in vitro studies, a TNFR2 agonist was found superior to standard methods in proliferating human T(regs) into a phenotypically homogeneous population consisting of 14 cell surface markers. The TNFR2 agonist-expanded T(regs) also were functionally superior in suppressing a key T(reg) target cell, cytotoxic T-lymphocytes. Targeting the TNFR2 receptor during ex vivo expansion is a new means for producing homogeneous and potent human T(regs) for clinical opportunities.

Show MeSH
Related in: MedlinePlus