Skip to main content
  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Cancer Immunology Essentials
    • Collections
      • COVID-19 & Cancer Resource Center
      • Toolbox: Coding and Computation
      • Toolbox: Signatures and Cells
      • "Best of" Collection
      • Editors' Picks
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • My Cart

Search

  • Advanced search
Cancer Immunology Research
Cancer Immunology Research
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
    • Reviewing
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • Meeting Abstracts
    • Cancer Immunology Essentials
    • Collections
      • COVID-19 & Cancer Resource Center
      • Toolbox: Coding and Computation
      • Toolbox: Signatures and Cells
      • "Best of" Collection
      • Editors' Picks
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Research Articles

CD28 Costimulatory Domain–Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function

Justin C. Boucher, Gongbo Li, Hiroshi Kotani, Maria L. Cabral, Dylan Morrissey, Sae Bom Lee, Kristen Spitler, Nolan J. Beatty, Estelle V. Cervantes, Bishwas Shrestha, Bin Yu, Aslamuzzaman Kazi, Xuefeng Wang, Said M. Sebti and Marco L. Davila
Justin C. Boucher
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gongbo Li
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hiroshi Kotani
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Hiroshi Kotani
Maria L. Cabral
2Department of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dylan Morrissey
3Morsani College of Medicine, University of South Florida Health, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sae Bom Lee
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
4Cancer Biology Ph.D. Program, University of South Florida, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kristen Spitler
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nolan J. Beatty
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
4Cancer Biology Ph.D. Program, University of South Florida, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Nolan J. Beatty
Estelle V. Cervantes
3Morsani College of Medicine, University of South Florida Health, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Bishwas Shrestha
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Bin Yu
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Aslamuzzaman Kazi
5Drug Discovery Program, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Xuefeng Wang
6Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Xuefeng Wang
Said M. Sebti
5Drug Discovery Program, H. Lee Moffitt Cancer Center, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marco L. Davila
1Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.
3Morsani College of Medicine, University of South Florida Health, Tampa, Florida.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Marco L. Davila
  • For correspondence: marco.davila@moffitt.org
DOI: 10.1158/2326-6066.CIR-20-0253 Published January 2021
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Additional Files
  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1.

    m1928z CAR T cells are less sensitive to antigen compared with m19hBBz. A, CAR T-cell numbers with the indicated domains in the bone marrow (BM) over time in vivo. Rag1−/− mice were i.v. injected with 1 × 106 CAR T cells. After 1, 2, 4, or 6 weeks, femurs were removed, and CAR T cells were counted by flow cytometry. B, Donor T cells in the BM after antigen challenge after 1 week. One week after Rag1−/− mice were i.v. injected with 1 × 106 CAR T cells, mice were challenged with 1 × 106 Eμ-ALL cells. One week after challenge, donor T cells in the femur were counted by flow cytometry. C–G, Rag1−/− mice were i.v. injected with m19dz, m19z, m1928z, or m19hBBz 1 × 106 CAR T cells. After 5 weeks, mice were challenged with 1 × 106 Eμ-ALL cells. One week after antigen challenge, CAR T (C) and donor (D) cells in the femur were counted by flow cytometry. CAR T cells were also analyzed for memory T-cell phenotype (E), PD-1 (F), and LAG3 (G). Data are representative of two independent experiments. For A–D, each group has 4 mice. Error bars, SD. *, P < 0.05; **, P < 0.01 by one-way ANOVA.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2.

    CD28 mutant CAR characterization. A, Schematic of CAR constructs. All included a 5′ long terminal repeat (LTR), CD8 signal peptide (black bar), scFv with a variable heavy chain connected with glycine–serine linker to a variable light chain (VH-G/S-VL), CD8 transmembrane and hinge domain, costimulatory and/or CD3ζ endodomain, glycine–serine linker (G/S), mCherry reporter, and 3′ LTR (33). B, CD28 mutant CAR transduction efficiency. A representative flow plot of CAR expression. FSC-A, forward-scatter area. C, CAR MFIs measured by mCherry. D and E, CD28 mutant CAR T-cell viability (D) and proliferation (E) compared with nonmutated CD28 CAR T cells prior to antigen stimulation. CAR T cells were counted after day 5 of production with trypan blue to determine viability and proliferation. For B and C, data are representative of 11 independent experiments. For D and E, each data point represents one CAR production. Error bars, SD.

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3.

    CD28 mutant CAR T-cell in vitro function. A–D, Cytokine production of CD28 mutant CAR T cells compared with m1928z and m19z CAR T cells. CAR T cells were stimulated with 3T3-mCD19 cells at a 10:1 E:T ratio. After 24 hours, supernatants were harvested, and IFNγ (A), IL6 (B), IL2 (C), and TNFα (D) were measured by Luminex. E and F, Cytotoxicity of CD28 mutant CAR T cells compared with nonmutated CD28 CAR T cells. CAR T cells were cocultured with 3T3-mCD19 at either 10:1 (E) or 1:6 (F) E:T ratios. The xCelligence RTCA system monitored cytotoxicity. Arrows indicate the time point of CAR T-cell addition. Data are representative of three independent experiments. Error bars, SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by one-way ANOVA.

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4.

    Mut06 CAR T cells support optimized in vivo function. C57BL/6 mice were injected i.p. with cyclophosphamide (300 mg/kg) 1 day prior to i.v. injection of 3 × 105 CAR T cells. Blood and bone marrow (BM) were collected at indicated time points and analyzed by flow cytometry. A, B-cell and CAR T-cell counts in the blood in weeks 1 to 6. B, B-cell and CAR T-cell percentage in BM after 8 weeks. C, CD28 mutant and nonmutated CAR T cells have similar memory phenotype counts at week 8. D, Mice with mut06 CAR T cells have better survival after Eμ-ALL challenge compared with m1928z CAR T cells. C57BL/6 mice were i.v. injected with 1 × 106 Eμ-ALL cells. On day 6, mice were injected i.p. with 300 mg/kg of cyclophosphamide. On day 7, mice were i.v. injected with 3 × 105 CAR T cells. Mice were then sacrificed after a 20% loss of body weight according to the IACUC-approved protocol. For A–C, each group has four mice and is representative of two independent experiments. For D, each group has eight mice. Error bars, SD. *, P < 0.05; **, P < 0.01 by one-way ANOVA.

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5.

    Mut06 CAR T cells remain sensitive to antigen in vivo. CAR T-cell phenotype after antigen challenge in vivo. Rag1−/− mice were injected with 1 × 106 CAR T cells. After 1 week, mice were challenged with 1 × 106 Eμ-ALL antigen. Following 1 week of antigen challenge, mice were sacrificed, and bone marrow was collected. A, CAR T-cell phenotypes were determined using flow cytometry. B, Mut06 CAR T-cell cytokine expression compared with m1928z CAR T cells. Following 1 week of antigen challenge, mice were sacrificed and splenocytes were collected. Splenocytes were then cocultured with 3T3-mCD19/mPD-L1 in the presence of 1× protein transport inhibitor. After 4 hours, cells were stained for intracellular cytokines and analyzed by flow cytometry. Each group has five mice. Data are representative of two independent experiments. Error bars, SD. *, P < 0.05; **, P < 0.01 by one-way ANOVA.

  • Figure 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 6.

    Mut06 CAR T cells have decreased expression of exhaustion-related transcription factors. A, Schematic of CD28 signaling. B, Mut06 CAR T-cell Nur77 expression compared with m1928z CAR T cells. Spleens were harvested from Nur77GFP mice, and T cells were isolated. CAR T cells were produced and stimulated for 24 hours with 3T3-mCD19 cells. GFP was measured by flow cytometry. NFAT (C) and AP1 (D) in mut06 CAR T cells compared with m1928z cells. Jurkat cells with NFAT or AP1 firefly luciferase reporters were transduced with CARs and stimulated with 3T3-mCD19 cells. After 24 hours, cells were lysed and supernatants were analyzed with a luminometer. E, Phospho(p)-NFAT in mut06 compared with m1928z CAR T cells. CAR T cells were stimulated for 24 hours by 3T3-mCD19 cells or immediately lysed for Western blot and probed for p-NFAT, p-LCK, and S6. B–E, Data are representative of three independent experiments. Error bars represent SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA.

  • Figure 7.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 7.

    Mut06 has less exhaustion-related gene expression and accessibility. A, RNA-seq heat map of 78 exhaustion-related T-cell genes. Cells were stimulated for 24 hours with 3T3-mCD19, and CAR T cells were sorted and lysed. RNA-seq was performed with 100-nt sequence reads using Illumina HiSeq 4000. B, RNA-seq heat map of 22 NFAT exhaustion-related T-cell genes (23, 33). C, Genome browser view of the Nfatc1, Tnf, Havcr2, Pdcd1, Nr4a1 (Nur77), Nr4a2, Lag3, Jun, and Fos loci after ATAC-seq. Cells were stimulated for 24 hours with 3T3-mCD19, and CAR T cells were sorted and lysed. Paired-end 42-bp sequencing reads generated by Illumina sequencing using NextSeq 500 were mapped to the genome using the BWA algorithm with default setting. Red stars denote the presence of the gene in ATAC-seq panel C.

Additional Files

  • Figures
  • Supplementary Data

    • Supplementary Data - Supplemental Tables and Figures
PreviousNext
Back to top
Cancer Immunology Research: 9 (1)
January 2021
Volume 9, Issue 1
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Editorial Board (PDF)

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Cancer Immunology Research article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
CD28 Costimulatory Domain–Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function
(Your Name) has forwarded a page to you from Cancer Immunology Research
(Your Name) thought you would be interested in this article in Cancer Immunology Research.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
CD28 Costimulatory Domain–Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function
Justin C. Boucher, Gongbo Li, Hiroshi Kotani, Maria L. Cabral, Dylan Morrissey, Sae Bom Lee, Kristen Spitler, Nolan J. Beatty, Estelle V. Cervantes, Bishwas Shrestha, Bin Yu, Aslamuzzaman Kazi, Xuefeng Wang, Said M. Sebti and Marco L. Davila
Cancer Immunol Res January 1 2021 (9) (1) 62-74; DOI: 10.1158/2326-6066.CIR-20-0253

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
CD28 Costimulatory Domain–Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function
Justin C. Boucher, Gongbo Li, Hiroshi Kotani, Maria L. Cabral, Dylan Morrissey, Sae Bom Lee, Kristen Spitler, Nolan J. Beatty, Estelle V. Cervantes, Bishwas Shrestha, Bin Yu, Aslamuzzaman Kazi, Xuefeng Wang, Said M. Sebti and Marco L. Davila
Cancer Immunol Res January 1 2021 (9) (1) 62-74; DOI: 10.1158/2326-6066.CIR-20-0253
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Authors' Disclosures
    • Authors' Contributions
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF
Advertisement

Related Articles

Cited By...

More in this TOC Section

  • Myeloid-Targeted CXCR2 Deletion Improves Antitumor Immunity
  • Metabolic Screening of CD8+ T Cells
  • Nutlin-3a: An Immune-Checkpoint Activator for NK Cells in Neuroblastoma
Show more Research Articles
  • Home
  • Alerts
  • Feedback
  • Privacy Policy
Facebook   Twitter   LinkedIn   YouTube   RSS

Articles

  • Online First
  • Current Issue
  • Past Issues
  • Cancer Immunology Essentials

Info for

  • Authors
  • Subscribers
  • Advertisers
  • Librarians

About Cancer Immunology Research

  • About the Journal
  • Editorial Board
  • Permissions
  • Submit a Manuscript
AACR logo

Copyright © 2021 by the American Association for Cancer Research.

Cancer Immunology Research
eISSN: 2326-6074
ISSN: 2326-6066

Advertisement