CD2 costimulation bridges potent CAR-induced cytolysis and durable persistence

Our plain-English summary of CD2 costimulation bridges potent CAR-induced cytolysis and durable persistence, by F. Liu, J.T. Keane, H.S. Lewis, et al (Journal for Immunotherapy of Cancer, 2026).

The study

• Authors: F. Liu, J.T. Keane, H.S. Lewis, T.R. King-Peoples, C.H. June, A.D. Posey
• Journal: Journal for Immunotherapy of Cancer
• Published: 2026;14(1)
• Cited by: 3 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Current second-generation CAR T cell products rely on CD28 or 4-1BB costimulatory domains, additions that respectively favor rapid cytolysis or long-term persistence, but rarely both. Preclinical modeling and retrospective analysis have linked CD2-CD58 engagement to superior preclinical and clinical responses, yet the direct contribution of CD2 intracellular signaling remains undefined.

Methods: We replaced the costimulatory domain of anti-mesothelin (SS1) and anti-TnMUC1 (5E5) CARs with the human CD2 cytoplasmic tail (CD2z) and benchmarked them against 28z and BBz formats. Transient mRNA expression was used to profile proximal Ca2+ flux and degranulation free of tonic viral signals; durable functional assays employed lentiviral CARs. Cytokine release, genome-wide transcriptional programs, and anti-tumor activity were assessed in vitro and in NSG xenograft models.

Results: CD2z CAR T cells degranulated as efficiently as other z-containing CARs and generated a Ca2+ flux signal intermediate to 28z and BBz CARs. Lentiviral CD2z CARs released a Th1-skewed cytokine panel and matched 28z cytolysis despite a lower acute cytokine release. Transcriptomic analysis characterized CD2z cells in an early effector-memory state: glycolytic, mTORC1, and TNFa-NF-κB hallmarks were upregulated, whereas exhaustion-up signatures were selectively depleted vs 28z. In vivo, a single CD2z infusion induced deep and durable tumor regressions over the 60-day observation period in subcutaneous mesothelin-positive mesothelioma and orthotopic TnMUC1-positive pancreatic tumor models, achieving tumor control comparable to 28z and more rapid early tumor clearance than BBz, while supporting peripheral T cell persistence similar to BBz.

Conclusions: The CD2 cytoplasmic tail, in combination with CD3z, delivers balanced costimulation that couples brisk tumor debulking to T cell persistence. CD2z therefore may provide a simple, versatile alternative to canonical CD28 and 4-1BB modules for next-generation CAR T therapies.

© Author(s) (or their employer(s)) 2026. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ Group.

In plain English

This was a laboratory study of chimeric antigen receptor (CAR) T cells, not a study of asbestos exposure, although a mesothelioma cell line was used as a tumor target. The authors replaced the usual CD28 or 4-1BB costimulatory part of anti-mesothelin (SS1) and anti-TnMUC1 (5E5) CARs with the 117-amino-acid human CD2 cytoplasmic tail (CD2z) and compared the three formats in human donor T cells and in NSG mice. With mRNA-expressed CARs, CD2z gave a calcium signal between that of 28z and BBz, and all zeta-containing CARs degranulated. Among lentivirally transduced cells, CD4+ T cells expressing SS1-CD2z released IFNg, IL-2 and TNFa above SS1-BBz and approaching SS1-28z. Separately, SS1-CD2z CAR T cells lysed AsPC-1, M108 and K562meso targets at a level matching SS1-28z. RNA sequencing showed CTLA4 about threefold lower and TOX2 50% lower than in 28z cells, with the exhausted-versus-memory module depleted (NES=-1.36, FDR=0.049). In M108 mesothelioma xenografts (8 mice per group), CD2z gave a median greater than 3 log tumor reduction, indistinguishable from 28z (p greater than 0.5) and better than BBz (p=0.0195).

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: preclinical laboratory study: in vitro experiments with human donor T cells and tumor cell lines plus NSG mouse xenograft experiments
• Who was studied: Human CD4+ and CD8+ T cells from healthy adult donors (2 to 3 biological donors per assay), human tumor cell lines (Jurkat E6-1, K562, SupT1, AsPC-1, Hs766T, M108 mesothelioma), and NSG mice, 8 per treatment group, at the University of Pennsylvania
• Exposure: CAR constructs in which the costimulatory domain was the human CD2 cytoplasmic tail (CD2z), benchmarked against CD28z (28z), 4-1BBz (BBz), zeta-only and signaling-deficient versions, using anti-mesothelin (SS1) and anti-TnMUC1 (5E5) binders; delivered by mRNA electroporation or lentiviral transduction
• Main results:
  • Degranulation and calcium flux with mRNA-expressed CARs: CD28z gave the largest sustained Ca2+ flux, 4-1BBz almost none, and CD2z an intermediate peak
  • Only zeta-containing CARs degranulated when exposed to mesothelin-expressing K562 cells
  • Cytokines from lentiviral SS1 CAR T cells, CD4+ subset: SS1-CD2z released IFNg, IL-2 and TNFa at levels higher than SS1-BBz and approaching SS1-28z
  • Cytokines from lentiviral SS1 CAR T cells, CD8+ subset: CD2z output resembled BBz more than 28z
  • Cytotoxicity of SS1-CD2z CAR T cells (subset not specified) against mesothelin-positive AsPC-1, M108 mesothelioma and K562meso targets at 1:1 effector-to-target ratio: trend toward better killing than SS1-BBz, matching SS1-28z
  • Transcriptomic comparison of CD2z with 28z CAR T cells: CTLA4 about threefold lower, TOX2 50% lower, TOX and LAG3 unchanged
  • Gene set enrichment, CD2z versus 28z: the exhausted-versus-memory CD8 T-cell UP module was significantly depleted (NES=-1.36, FDR=0.049)
  • M108 mesothelioma xenografts, 8 mice per group: CD2z achieved a median greater than 3 log reduction in viable tumor burden, superior to BBz, non-transduced cells and vehicle (p=0.0195 and 0.0034) and indistinguishable from 28z (p greater than 0.5)
  • Individual mice in the M108 model: all CD2z-treated mice had at least 2 log bioluminescence reductions and 6 of 8 reached background signal during the 60-day window
  • Peripheral persistence in the mesothelin model, day 21: higher CD4+ and CD8+ T cell frequencies with CD2z or BBz, reduced with 28z and non-transduced cells
  • TnMUC1 (5E5) CAR T cells against Hs766T pancreatic cancer cells in vitro: CD2z produced IFNg, IL-2, TNFa, IL-6 and GM-CSF at significantly lower levels than 28z or BBz
  • Orthotopic Hs766T pancreatic tumor model at day 56: greater tumor burden reduction with CD2z than BBz (p less than 0.001) and 28z (p less than 0.05)
  • Cytotoxicity of all three 5E5 CAR formats against antigen-positive Jurkat cells at 1:1 outperformed non-transduced controls (p=0.0003 for each), with no significant differences detected among CD2z, 28z and BBz
• The authors' conclusion: The authors concluded that the CD2 cytoplasmic domain acts as a costimulatory module that augments zeta-driven cytotoxicity and durable persistence in CAR T cells, and that this may translate into improved tumor control relative to CD28 and 4-1BB, a hypothesis requiring further head-to-head testing
• Limitations the authors state:
  • Reliance on NSG xenograft mice, which lack myeloid and stromal compartments, and a small donor pool
  • In vivo studies ran only 56 to 60 days and could not capture very late relapse or differential long-term efficacy
  • Antigen-density thresholds for activation and killing were not systematically mapped
  • Several in vitro assays used only two donors and were reported descriptively, without formal hypothesis testing

Topics

Medical terms in this study, explained in our Lexicon: Mesothelin, Cytokine, Mesothelioma.

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