Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion

Keli L. Hippen; Scott N. Furlan; Rahul Roychoudhuri; Ena Wang; Yigang Zhang; Mark J. Osborn; Sarah C. Merkel; Sophia Hani; Margaret L. MacMillan; Frank Cichocki; Jeffrey S. Miller; John E. Wagner; Nicholas P. Restifo; Leslie S. Kean; Bruce R. Blazar

doi:10.1016/j.jcyt.2021.02.118

Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion

Keli L. Hippen, Scott N. Furlan, Rahul Roychoudhuri, Ena Wang, Yigang Zhang, Mark J. Osborn, Sarah C. Merkel, Sophia Hani, Margaret L. MacMillan, Frank Cichocki, Jeffrey S. Miller, John E. Wagner, Nicholas P. Restifo, Leslie S. Kean, Bruce R. Blazar

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Background aims: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 10⁶ cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of ‘off the shelf’ Treg. Results: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. Discussion: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile—desirable properties that support the possibility of off-the-shelf Treg therapeutics.

Original language	English (US)
Pages (from-to)	704-714
Number of pages	11
Journal	Cytotherapy
Volume	23
Issue number	8
DOIs	https://doi.org/10.1016/j.jcyt.2021.02.118
State	Published - Aug 2021

Bibliographical note

Funding Information:
This work was supported in part by research grants from the Children's Cancer Research Fund (KLH); Leukemia and Lymphoma Translational Research (BRB; grant no. R6029–07); National Institutes of Health (NIH) (MLM and KLH; grant no. R01 HL114512-01; BRB; grant nos. R37 AI34495, R01 HL11879 and P01 AI056299; and LSK; grant nos. 2U19 AI051731 and 2R01 HL095791); National Heart, Lung, and Blood Institute (JEW, JSM and KLH; grant no. N01HB037164); and National Cancer Institute (BRB, JEW and JSM; grant no. P01 CA067493). This work was supported in part by an NIH Clinical and Translational Science Award to the University of Minnesota (8UL1TR000114) and an NIH P30 grant using the shared Flow Cytometry Core resource from the Masonic Cancer Center, University of Minnesota (CA77598). Conception and design of the study: KLH, NPR and BRB. Acquisition of data: KLH, RR, SCM and SH. Analysis and interpretation of data: YZ, EW and SNF. Drafting or revising the manuscript: KLH, SNF, LSK and BRB. All authors have approved the final article. The authors wish to acknowledge Drs Bruce Levine, James Riley and Carl June (University of Pennsylvania Abramson Family Cancer Center Research Institute) for providing the aAPCs (KT64/86) used to expand the CD4 Teffs and tTregs.

Funding Information:
BRB is a founder of Tmunity Therapeutics, serves as an advisor for and receives research support from BlueRock Therapeutics and, along with KLH, holds patents for the production and use of Tregs for clinical trials. LSK is on the scientific advisory board for HiFiBio and reports research funding from Bristol Myers Squibb, Kymab Limited, Magenta Therapeutics, BlueBird Bio and Regeneron Pharmaceuticals; consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gilead Sciences and Takeda Pharmaceuticals; the patent “Method to prevent relapse after transplant,” which is pending; and the patent “Method to prevent GVHD after transplant,” with royalties paid.

Funding Information:
This work was supported in part by research grants from the Children's Cancer Research Fund (KLH); Leukemia and Lymphoma Translational Research (BRB; grant no. R6029–07); National Institutes of Health (NIH) (MLM and KLH; grant no. R01 HL114512-01; BRB; grant nos. R37 AI34495, R01 HL11879 and P01 AI056299; and LSK; grant nos. 2U19 AI051731 and 2R01 HL095791); National Heart, Lung, and Blood Institute (JEW, JSM and KLH; grant no. N01HB037164); and National Cancer Institute (BRB, JEW and JSM; grant no. P01 CA067493). This work was supported in part by an NIH Clinical and Translational Science Award to the University of Minnesota (8UL1TR000114) and an NIH P30 grant using the shared Flow Cytometry Core resource from the Masonic Cancer Center, University of Minnesota (CA77598).

Publisher Copyright:
© 2021 International Society for Cell & Gene Therapy

Keywords

Treg
cGMP production
cell therapy
graft-versus-host disease
regulatory T cell

PubMed: MeSH publication types

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

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10.1016/j.jcyt.2021.02.118

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Hippen, K. L., Furlan, S. N., Roychoudhuri, R., Wang, E., Zhang, Y., Osborn, M. J., Merkel, S. C., Hani, S., MacMillan, M. L., Cichocki, F., Miller, J. S., Wagner, J. E., Restifo, N. P., Kean, L. S., & Blazar, B. R. (2021). Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion. Cytotherapy, 23(8), 704-714. https://doi.org/10.1016/j.jcyt.2021.02.118

Hippen, KL, Furlan, SN, Roychoudhuri, R, Wang, E, Zhang, Y, Osborn, MJ, Merkel, SC, Hani, S, MacMillan, ML , Cichocki, F , Miller, JS , Wagner, JE, Restifo, NP, Kean, LS & Blazar, BR 2021, 'Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion', Cytotherapy, vol. 23, no. 8, pp. 704-714. https://doi.org/10.1016/j.jcyt.2021.02.118

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abstract = "Background aims: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of {\textquoteleft}off the shelf{\textquoteright} Treg. Results: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. Discussion: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile—desirable properties that support the possibility of off-the-shelf Treg therapeutics.",

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author = "Hippen, {Keli L.} and Furlan, {Scott N.} and Rahul Roychoudhuri and Ena Wang and Yigang Zhang and Osborn, {Mark J.} and Merkel, {Sarah C.} and Sophia Hani and MacMillan, {Margaret L.} and Frank Cichocki and Miller, {Jeffrey S.} and Wagner, {John E.} and Restifo, {Nicholas P.} and Kean, {Leslie S.} and Blazar, {Bruce R.}",

note = "Funding Information: This work was supported in part by research grants from the Children's Cancer Research Fund (KLH); Leukemia and Lymphoma Translational Research (BRB; grant no. R6029–07); National Institutes of Health (NIH) (MLM and KLH; grant no. R01 HL114512-01; BRB; grant nos. R37 AI34495, R01 HL11879 and P01 AI056299; and LSK; grant nos. 2U19 AI051731 and 2R01 HL095791); National Heart, Lung, and Blood Institute (JEW, JSM and KLH; grant no. N01HB037164); and National Cancer Institute (BRB, JEW and JSM; grant no. P01 CA067493). This work was supported in part by an NIH Clinical and Translational Science Award to the University of Minnesota (8UL1TR000114) and an NIH P30 grant using the shared Flow Cytometry Core resource from the Masonic Cancer Center, University of Minnesota (CA77598). Conception and design of the study: KLH, NPR and BRB. Acquisition of data: KLH, RR, SCM and SH. Analysis and interpretation of data: YZ, EW and SNF. Drafting or revising the manuscript: KLH, SNF, LSK and BRB. All authors have approved the final article. The authors wish to acknowledge Drs Bruce Levine, James Riley and Carl June (University of Pennsylvania Abramson Family Cancer Center Research Institute) for providing the aAPCs (KT64/86) used to expand the CD4 Teffs and tTregs. Funding Information: BRB is a founder of Tmunity Therapeutics, serves as an advisor for and receives research support from BlueRock Therapeutics and, along with KLH, holds patents for the production and use of Tregs for clinical trials. LSK is on the scientific advisory board for HiFiBio and reports research funding from Bristol Myers Squibb, Kymab Limited, Magenta Therapeutics, BlueBird Bio and Regeneron Pharmaceuticals; consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gilead Sciences and Takeda Pharmaceuticals; the patent “Method to prevent relapse after transplant,” which is pending; and the patent “Method to prevent GVHD after transplant,” with royalties paid. Funding Information: This work was supported in part by research grants from the Children's Cancer Research Fund (KLH); Leukemia and Lymphoma Translational Research (BRB; grant no. R6029–07); National Institutes of Health (NIH) (MLM and KLH; grant no. R01 HL114512-01; BRB; grant nos. R37 AI34495, R01 HL11879 and P01 AI056299; and LSK; grant nos. 2U19 AI051731 and 2R01 HL095791); National Heart, Lung, and Blood Institute (JEW, JSM and KLH; grant no. N01HB037164); and National Cancer Institute (BRB, JEW and JSM; grant no. P01 CA067493). This work was supported in part by an NIH Clinical and Translational Science Award to the University of Minnesota (8UL1TR000114) and an NIH P30 grant using the shared Flow Cytometry Core resource from the Masonic Cancer Center, University of Minnesota (CA77598). Publisher Copyright: {\textcopyright} 2021 International Society for Cell & Gene Therapy",

year = "2021",

month = aug,

doi = "10.1016/j.jcyt.2021.02.118",

language = "English (US)",

volume = "23",

pages = "704--714",

journal = "Cytotherapy",

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TY - JOUR

T1 - Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion

AU - Hippen, Keli L.

AU - Furlan, Scott N.

AU - Roychoudhuri, Rahul

AU - Wang, Ena

AU - Zhang, Yigang

AU - Osborn, Mark J.

AU - Merkel, Sarah C.

AU - Hani, Sophia

AU - MacMillan, Margaret L.

AU - Cichocki, Frank

AU - Miller, Jeffrey S.

AU - Wagner, John E.

AU - Restifo, Nicholas P.

AU - Kean, Leslie S.

AU - Blazar, Bruce R.

N1 - Funding Information: This work was supported in part by research grants from the Children's Cancer Research Fund (KLH); Leukemia and Lymphoma Translational Research (BRB; grant no. R6029–07); National Institutes of Health (NIH) (MLM and KLH; grant no. R01 HL114512-01; BRB; grant nos. R37 AI34495, R01 HL11879 and P01 AI056299; and LSK; grant nos. 2U19 AI051731 and 2R01 HL095791); National Heart, Lung, and Blood Institute (JEW, JSM and KLH; grant no. N01HB037164); and National Cancer Institute (BRB, JEW and JSM; grant no. P01 CA067493). This work was supported in part by an NIH Clinical and Translational Science Award to the University of Minnesota (8UL1TR000114) and an NIH P30 grant using the shared Flow Cytometry Core resource from the Masonic Cancer Center, University of Minnesota (CA77598). Conception and design of the study: KLH, NPR and BRB. Acquisition of data: KLH, RR, SCM and SH. Analysis and interpretation of data: YZ, EW and SNF. Drafting or revising the manuscript: KLH, SNF, LSK and BRB. All authors have approved the final article. The authors wish to acknowledge Drs Bruce Levine, James Riley and Carl June (University of Pennsylvania Abramson Family Cancer Center Research Institute) for providing the aAPCs (KT64/86) used to expand the CD4 Teffs and tTregs. Funding Information: BRB is a founder of Tmunity Therapeutics, serves as an advisor for and receives research support from BlueRock Therapeutics and, along with KLH, holds patents for the production and use of Tregs for clinical trials. LSK is on the scientific advisory board for HiFiBio and reports research funding from Bristol Myers Squibb, Kymab Limited, Magenta Therapeutics, BlueBird Bio and Regeneron Pharmaceuticals; consulting fees from Equillium, FortySeven Inc, Novartis Inc, EMD Serono, Gilead Sciences and Takeda Pharmaceuticals; the patent “Method to prevent relapse after transplant,” which is pending; and the patent “Method to prevent GVHD after transplant,” with royalties paid. Funding Information: This work was supported in part by research grants from the Children's Cancer Research Fund (KLH); Leukemia and Lymphoma Translational Research (BRB; grant no. R6029–07); National Institutes of Health (NIH) (MLM and KLH; grant no. R01 HL114512-01; BRB; grant nos. R37 AI34495, R01 HL11879 and P01 AI056299; and LSK; grant nos. 2U19 AI051731 and 2R01 HL095791); National Heart, Lung, and Blood Institute (JEW, JSM and KLH; grant no. N01HB037164); and National Cancer Institute (BRB, JEW and JSM; grant no. P01 CA067493). This work was supported in part by an NIH Clinical and Translational Science Award to the University of Minnesota (8UL1TR000114) and an NIH P30 grant using the shared Flow Cytometry Core resource from the Masonic Cancer Center, University of Minnesota (CA77598). Publisher Copyright: © 2021 International Society for Cell & Gene Therapy

PY - 2021/8

Y1 - 2021/8

N2 - Background aims: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of ‘off the shelf’ Treg. Results: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. Discussion: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile—desirable properties that support the possibility of off-the-shelf Treg therapeutics.

AB - Background aims: Adoptive transfer of suppressive CD4+CD25+ thymic regulatory T cells (tTregs) can control auto- and alloimmune responses but typically requires in vitro expansion to reach the target cell number for efficacy. Although the adoptive transfer of expanded tTregs purified from umbilical cord blood ameliorates graft-versus-host disease in patients receiving hematopoietic stem cell transplantation for lymphohematopoietic malignancy, individual Treg products of 100 × 106 cells/kg are manufactured over an extended 19-day time period using a process that yields variable products and is both laborious and costly. These limitations could be overcome with the availability of ‘off the shelf’ Treg. Results: Previously, the authors reported a repetitive restimulation expansion protocol that maintains Treg phenotype (CD4+25++127-Foxp3+), potentially providing hundreds to thousands of patient infusions. However, repetitive stimulation of effector T cells induces a well-defined program of exhaustion that leads to reduced T-cell survival and function. Unexpectedly, the authors found that multiply stimulated human tTregs do not develop an exhaustion signature and instead maintain their Treg gene expression pattern. The authors also found that tTregs expanded with one or two rounds of stimulation and tTregs expanded with three or five rounds of stimulation preferentially express distinct subsets of a group of five transcription factors that lock in Treg Foxp3expression, Treg stability and suppressor function. Multiply restimulated Tregs also had increased transcripts characteristic of T follicular regulatory cells, a Treg subset. Discussion: These data demonstrate that repetitively expanded human tTregs have a Treg-locking transcription factor with stable FoxP3 and without the classical T-cell exhaustion gene expression profile—desirable properties that support the possibility of off-the-shelf Treg therapeutics.

KW - Treg

KW - cGMP production

KW - cell therapy

KW - graft-versus-host disease

KW - regulatory T cell

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ER -

Multiply restimulated human thymic regulatory T cells express distinct signature regulatory T-cell transcription factors without evidence of exhaustion

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