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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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TNF induction of Tregs in humans and summary finding of TNFR2 agonist and antagonist activity.(a) In a small double-blinded, placebo-controlled trial of human subjects, BCG treatment induces TNF and shortly thereafter a surge of Tregs appears in the treated subject. (b) In contrast, in the same double-blinded trial, placebo treatment induces neither TNF nor Tregs in the circulation. (c). BCG treatment of a human subject taking chronic Enbrel (etanercept), an anti-TNF antibody, reveals neither TNF nor Treg induction. This finding supports endogenous TNF as being necessary for Treg expansion in humans. (d) After expansion, the TNFR2 agonist is better than TNFR2 antagonist at proliferating and yielding more phenotypically homogeneous Tregs (CD4+ CD25hi FOXP3+ CTLA4+ TNFR2+ CD45RO+ CD62L+ CD127−, HLA-DRhi CCR5− CCR7− CXCR3− ICOS−), with higher suppression capacity for CD8+ cells, and lower cytokine-producing capability. The TNF2 antagonist was able to suppress Treg expansion.
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f6: TNF induction of Tregs in humans and summary finding of TNFR2 agonist and antagonist activity.(a) In a small double-blinded, placebo-controlled trial of human subjects, BCG treatment induces TNF and shortly thereafter a surge of Tregs appears in the treated subject. (b) In contrast, in the same double-blinded trial, placebo treatment induces neither TNF nor Tregs in the circulation. (c). BCG treatment of a human subject taking chronic Enbrel (etanercept), an anti-TNF antibody, reveals neither TNF nor Treg induction. This finding supports endogenous TNF as being necessary for Treg expansion in humans. (d) After expansion, the TNFR2 agonist is better than TNFR2 antagonist at proliferating and yielding more phenotypically homogeneous Tregs (CD4+ CD25hi FOXP3+ CTLA4+ TNFR2+ CD45RO+ CD62L+ CD127−, HLA-DRhi CCR5− CCR7− CXCR3− ICOS−), with higher suppression capacity for CD8+ cells, and lower cytokine-producing capability. The TNF2 antagonist was able to suppress Treg expansion.

Mentions: The small proof-of-principle, double-blind placebo-controlled clinical trial enrolled two human subjects. One subject received repeat BCG injections (1.6–3.2 × 106 cfu/injection) and one placebo subject received repeat saline, twice, 4-weeks apart. Both were monitored weekly for 20-weeks to study the pharmacokinetics of TNF and Treg induction. After each BCG injection, TNF induced Tregs in a bi-modal fashion with slightly delayed kinetics (Fig. 6a, left panels). After 20 weeks of observation, saline injections induced neither TNF nor Tregs (Fig. 6b, center panels). The total CD4+ cell counts did not change in the BCG and placebo patients other than in the percentages of CD4 + CD25hiFOXP3+ cells (data not shown). Because TNF is not the only cytokine stimulated by BCG injection, we also sought to determine whether TNF was necessary and sufficient for induction of Tregs. During routine care, a rheumatoid arthritis subject on Enbrel (etanercept), an anti-TNF antibody, was administered a BCG vaccine for protection from tuberculosis. The same tight weekly TNF and Treg monitoring revealed that Enbrel blocked both endogenous secretion of TNF and induction of Tregs (Fig. 6c, right panels). This in vivo evidence supports endogenous TNF as being obligatory for Treg induction in humans.


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

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

TNF induction of Tregs in humans and summary finding of TNFR2 agonist and antagonist activity.(a) In a small double-blinded, placebo-controlled trial of human subjects, BCG treatment induces TNF and shortly thereafter a surge of Tregs appears in the treated subject. (b) In contrast, in the same double-blinded trial, placebo treatment induces neither TNF nor Tregs in the circulation. (c). BCG treatment of a human subject taking chronic Enbrel (etanercept), an anti-TNF antibody, reveals neither TNF nor Treg induction. This finding supports endogenous TNF as being necessary for Treg expansion in humans. (d) After expansion, the TNFR2 agonist is better than TNFR2 antagonist at proliferating and yielding more phenotypically homogeneous Tregs (CD4+ CD25hi FOXP3+ CTLA4+ TNFR2+ CD45RO+ CD62L+ CD127−, HLA-DRhi CCR5− CCR7− CXCR3− ICOS−), with higher suppression capacity for CD8+ cells, and lower cytokine-producing capability. The TNF2 antagonist was able to suppress Treg expansion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: TNF induction of Tregs in humans and summary finding of TNFR2 agonist and antagonist activity.(a) In a small double-blinded, placebo-controlled trial of human subjects, BCG treatment induces TNF and shortly thereafter a surge of Tregs appears in the treated subject. (b) In contrast, in the same double-blinded trial, placebo treatment induces neither TNF nor Tregs in the circulation. (c). BCG treatment of a human subject taking chronic Enbrel (etanercept), an anti-TNF antibody, reveals neither TNF nor Treg induction. This finding supports endogenous TNF as being necessary for Treg expansion in humans. (d) After expansion, the TNFR2 agonist is better than TNFR2 antagonist at proliferating and yielding more phenotypically homogeneous Tregs (CD4+ CD25hi FOXP3+ CTLA4+ TNFR2+ CD45RO+ CD62L+ CD127−, HLA-DRhi CCR5− CCR7− CXCR3− ICOS−), with higher suppression capacity for CD8+ cells, and lower cytokine-producing capability. The TNF2 antagonist was able to suppress Treg expansion.
Mentions: The small proof-of-principle, double-blind placebo-controlled clinical trial enrolled two human subjects. One subject received repeat BCG injections (1.6–3.2 × 106 cfu/injection) and one placebo subject received repeat saline, twice, 4-weeks apart. Both were monitored weekly for 20-weeks to study the pharmacokinetics of TNF and Treg induction. After each BCG injection, TNF induced Tregs in a bi-modal fashion with slightly delayed kinetics (Fig. 6a, left panels). After 20 weeks of observation, saline injections induced neither TNF nor Tregs (Fig. 6b, center panels). The total CD4+ cell counts did not change in the BCG and placebo patients other than in the percentages of CD4 + CD25hiFOXP3+ cells (data not shown). Because TNF is not the only cytokine stimulated by BCG injection, we also sought to determine whether TNF was necessary and sufficient for induction of Tregs. During routine care, a rheumatoid arthritis subject on Enbrel (etanercept), an anti-TNF antibody, was administered a BCG vaccine for protection from tuberculosis. The same tight weekly TNF and Treg monitoring revealed that Enbrel blocked both endogenous secretion of TNF and induction of Tregs (Fig. 6c, right panels). This in vivo evidence supports endogenous TNF as being obligatory for Treg induction in humans.

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