CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters

Nishikawa, Tetsushi; Takeuchi, Arata; Machiyama, Hiroaki; Wakamatsu, Ei; Nishijima, Hitoshi; Furuhata, Masae; Toyota, Hiroko; Nishi, Wataru; Matsushima, Ryohei; Yoshida, Yosuke; Harada, Kazutoshi; Yokosuka, Tadashi

doi:10.1038/s42003-025-08906-y

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Published: 29 October 2025

CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters

Communications Biology volume 8, Article number: 1506 (2025) Cite this article

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Abstract

Various improvements have been made to chimeric antigen receptor (CAR)-T cells to enhance their antitumor effects and expand their indications to various cancer types. Although second-generation CARs directly induce second signals in a linear pathway via their own costimulatory domain such as CD28, CAR-T cells themselves express costimulatory receptors, which induce additional second signals in parallel. To clarify the differences between these two types of second signals, we here reveal CD28-mediated signalosomes using high-resolution imaging. CAR constructed using CD3ζ and CD28 (CD28ζ.CAR)-T cells demonstrate intense and persistent accumulation of the CD28 downstream kinase protein kinase C θ (PKCθ) at CAR signalosomes, and endogenous CD28 on CAR-T cells prolongs the CD28-CAR association. This assembling of PKCθ is correlated with IL-2 production and in vivo tumor suppression by CAR-T cells. These results indicate that the development and improvement of CARs requires analysis of intrinsic T-cell responses, which can be effectively assessed from signalosome dynamics evaluated by molecular imaging.

Introduction

Chimeric antigen receptor (CAR)-T cell therapy targeting specific antigens on tumor cells has been used in a variety of tumors over the last decade as an evolution of adoptive immunotherapy¹. The first-generation CAR, which consists of a single-chain variable fragment (scFv) fused with the intracellular region of CD3ζ, is the basic component of CARs², but these types of CAR-T cells disappear rapidly in vivo and have limited clinical efficacy. To overcome these issues, several second-generation CARs have been developed that exhibit potent in vivo antitumor activity through the tandem linkage of the intracellular region of a CD3ζ with those of costimulatory molecules such as CD28³ and 4-1BB⁴. However, some issues remain to be resolved, including the possibility of relapse after remission^5,6 and limited efficacy against solid tumors⁷, among others. In order to obtain effective cytotoxic activity over a long period, new designs of CARs continue to be investigated by combining costimulatory molecules as an intracellular region^8,9,10.

Effective T-cell activation to induce an appropriate immune response requires not only the first signaling via T-cell receptors (TCRs) bound to major histocompatibility complexes (MHCs) with antigen peptides but also the second signaling from costimulatory receptors through ligand bindings¹¹. The second-generation human (h) CD19.CARs, which possess both cytoplasmic regions of CD3ζ and a costimulatory receptor, simultaneously transduce first and second signaling by binding to hCD19. CAR-T cells also express their own endogenous costimulatory receptors; therefore, an additional second signaling may be transduced in parallel with the second signaling through the costimulatory domains incorporated into the CAR, when tumor cells or professional antigen-presenting cells (APCs) express certain ligands for costimulatory receptors on CAR-T cells. The representative T-cell costimulatory receptor CD28 binds to CD80 and CD86 expressed on APCs, and CD28-mediated signaling is indispensable for cell proliferation, survival, metabolism, and cytokine production^12,13,14. Particularly in B-cell malignancies that naturally express CD80 and/or CD86, such as large B-cell lymphoma (LBCL)¹⁵, the intrinsic second signal via endogenous CD28 on CAR-T cells may affect antitumor activity. Although a previous report demonstrated that CD28-mediated intrinsic costimulatory signaling on CAR-T cells enhanced antitumor activity in a case of the B-cell maturation antigen (BCMA) CAR bearing the 4-1BB cytoplasmic region (4-1BBζ.CAR)¹⁶, to our knowledge, there are still no sufficient reports comparing CD28 signaling via its cytoplasmic domain incorporated into a CAR and endogenous CD28 expressed on a CAR-T cell itself.

In the recognition of tumor antigens on APCs or tumor cells, a T cell shows a characteristic rearrangement of signaling molecules at the T cell–APC/tumor cell interface, called an “immunological synapse”¹⁷. High-resolution imaging has revealed its precise structure, where 20 to 30 TCRs are assembled together with their downstream molecules, known as a “TCR microcluster” as an early signalosome for T-cell activation^18,19, and these signalosomes show centripetal movement to form a central supramolecular activation cluster (cSMAC). At an immunological synapse, CD28 exhibits similar clustering along with TCRs, functioning as TCR-CD28 microclusters to transduce crosstalk or additional signaling via CD28 downstream^20,21. Through biochemical analyses, phosphatidylinositol 3-kinase (PI3K) is recognized as the well-known effector molecule working downstream of CD28, contributing to various aspects of CD28 biology, including cell cycle progression and metabolic improvement. Our previous molecular imaging depicted obvious enhancement of PI3K accumulated at TCR microclusters by binding of CD28 to its ligands. Furthermore, we previously demonstrated that protein kinase C θ (PKCθ)^22,23, another candidate effector molecule downstream of CD28, strongly colocalizes with CD28 throughout T-cell activation via an immunological synapse²⁰. This finding is consistent with the fact that expression of PKCθ is strongly correlated with IL-2 production by T cells, and that the phenotype of CD28-deficient mice closely resembles that of gene-deficient mice in PKCθ or further downstream molecules such as CARMA1 and Rltpr^24,25,26,27.

Here, we show the contributions of intrinsic and extrinsic CD28 signaling in both first- and second-generation hCD19.CARs using molecular imaging of CD28-mediated costimulatory signaling. Compared with PI3K, PKCθ specifically, intensively, and persistently formed clusters at hCD19 ζ.CARs collaborating with CD28 and at solo hCD19 CD28ζ.CARs. The behavior of PKCθ had less effects on in vitro cytotoxicity but apparently correlated with cytokine production and in vivo tumor suppression of hCD19 CAR-T cells. We found that endogenous CD28 signaling overcame the lack of CD28-mediated signaling in hCD19 ζ.CAR. These results suggest the functional benefits of costimulatory signaling in all generations of CAR-T cells in clinical applications.

Results

CD28-CD80 binding recruits PKCθ at CD28-TCR microclusters and enhances IL-2 production without an increment in cytotoxicity

PI3K is widely known to be involved in both mitogenic and cell survival signaling as an upstream molecule in the PI3K-Akt-mTOR pathway^28,29 and as a key contributing molecule in CD28 signaling. Therefore, we first examined the behavior of PI3K triggered by CD28-CD80 binding to confirm accumulation of the catalytic subunit of type I PI3K (p110δ) at the T cell–APC interface. We prepared splenic CD8⁺ T cells from OT-I TCR transgenic (OT-I-Tg) [specific for ovalbumin 257–264 (OVA_257-264) loaded on H-2K^b] Rag2-deficient (Rag2⁻^/⁻) mice, and retrovirally transduced by HaloTag-tagged CD28 (CD28-HaloTag) plus enhanced green fluorescent protein-tagged p110δ (p110δ-EGFP). We then conjugated these T cells with OVA_257-264-prepulsed EL-4 cells that were not transduced or transduced by CD80 and imaged them in real time using confocal microscopy. In the presence of CD80, CD28 accumulated at the T cell–APC interface, and p110δ was shown to accumulate densely at the same interface within 5 min and to fade out 20 min after conjugation (Fig. 1a, b).

**Fig. 1: CD28-CD80 binding recruits PKCθ at an immunological synapse to enhance cell functions in primary T cells.**

PKCθ, another candidate effector molecule downstream of CD28, is activated by TCR-mediated solo and CD28-mediated stimulation and is known to contribute to both T-cell proliferation and IL-2 production via activation of the NF-κB signaling pathway^24,30. Next, we conjugated OT-I-Tg T cells expressing CD28-HaloTag and PKCθ-EGFP with EL-4 cells not transduced or transduced by CD80 to image the accumulation of PKCθ at the T cell–APC interface. As previously reported²⁰, in the presence of CD80, OT-I-Tg T cells also demonstrated that PKCθ remained at the interface for more than 20 min after conjugation (Fig. 1c, d). To examine more precise patterns of CD28 and PKCθ distributed at the interface, we imaged the same cells settled on a Supported Lipid Bilayer (SLB) that contained glycosylphosphatidylinositol-anchored H-2K^b (H-2K^b–GPI) and ICAM-1 plus CD80. The density of the CD80 molecule was adjusted to an equivalent or lower level compared with conventional APCs²⁰. OT-I-Tg T cells expressing CD28-HaloTag and PKCθ-EGFP settled onto an OVA_257-264-pulsed SLB. In the presence of CD80, CD28 was initially translocated together with TCRs to form TCR-CD28 microclusters^20,21; simultaneously, an effector molecule downstream of CD28, PKCθ, densely colocalized with those clusters (Fig. 1e, left, and Fig. 1f). After maximum expansion of a T cell, all three molecules then migrated toward the center of an immunological synapse and formed a cSMAC. At the cSMAC, CD28 clusters were dimly colocalized with TCRs but strongly merged with PKCθ in an annular form surrounding dense TCR clusters²⁰ (Fig. 1e, right). Next, to confirm the contribution of CD28 signaling to T-cell activation, we conjugated OT-I-Tg T cells with OVA_257-264-pulsed EL-4 cells not transduced or transduced by CD80 and evaluated their activation status by measurement of IL-2 or using an in vitro cytotoxic assay. T cells produced more IL-2 in the presence of CD28-CD80 binding (Fig. 1g) but less increased cytotoxicity under the same conditions (Fig. 1h). These data indicated that CD28-mediated costimulatory signaling via association with PKCθ contributes to the T-cell response, and that, although IL-2 production is efficiently increased by antigen-specific stimulation, there is no enhancement of cytotoxic function, which may be regulated by TCR signaling alone.

hCD19 CD28ζ.CAR recruits PKCθ at CAR microclusters and enhances IL-2 production without an increment in cytotoxicity

hCD19 CAR-T cells are known to form “CAR microclusters” by binding to CD19. We first imaged the behavior of CARs by using a high-resolution total internal reflection fluorescence (TIRF) microscopy. Splenic CD3⁺ cells from C57BL/6 mice were transduced by EGFP-tagged hCD19 ζ. or CD28ζ.CAR and settled onto SLBs expressing both hCD19– and ICAM-1–GPI³¹. We confirmed the clustering of CARs at a CAR-SLB interface and centripetal movement or these CAR microclusters toward the center of an immunological synapse, as shown by conventional TCR microclusters (Fig. 2a, and Supplementary Movies 1, 2). We next examined whether PKCθ, a responsive molecule for CD28 signaling, is imaged at the hCD19 CD28ζ.CAR microclusters via association with the cytoplasmic domain of CD28, which is incorporated into the CAR. CD3⁺ T cells expressing HaloTag-tagged hCD19 ζ., CD28ζ., or 4-1BBζ.CAR and PKCθ-EGFP were conjugated with EL-4 cells expressing hCD19 and real-time imaged by confocal microscopy. The accumulation of PKCθ at the CAR-T cell–APC interface was more strongly detected in CD28ζ.CAR-T cells than in other CAR-T cells (Fig. 2b, c). Subsequently, we imaged more accurate distributions of CARs and PKCθ in the immunological synapse. CD28ζ.CAR-T cells showed enhanced colocalization of PKCθ at CAR microclusters compared with ζ. or 4-1BBζ.CAR-T cells (Fig. 2d, left, and Fig. 2e). PKCθ persistently stayed at CD28ζ.CAR microclusters, and when the CD28ζ.CAR microclusters were aggregated to form a cSMAC, PKCθ finally accumulated at the center of the immunological synapse (Fig. 2d, right, and Fig. 2f). Next, to evaluate the biological responses of ζ., CD28ζ., or 4-1BBζ.CAR-T cells (Supplementary Fig. 1), we measured the concentration of IL-2 in supernatant when each cell was stimulated by EL-4 cells expressing hCD19. We found that both CD28ζ. and 4-1BBζ.CAR-T cells produced much more IL-2 than ζ.CAR-T cells (Fig. 2g). However, all three CAR-T cells exhibited similar cytotoxicity against hCD19⁺ target tumor cells (Fig. 2h), as shown in primary cytotoxic T cells that equally killed target cells without or with CD28-CD80 binding (Fig. 1h). These results demonstrate that, correlating with PKCθ recruitment to CAR, CD28-mediated intrinsic signaling via the CD28 cytoplasmic region in CD28.ζ CAR is partially important for CAR-T cell functions, as shown by endogenous CD28 signaling in T cells but is less effective in cytotoxicity in vitro.

**Fig. 2: CD28ζ.CAR effectively introduces translocation of PKCθ at an immunological synapse and CAR microclusters correlating with T-cell responses.**

Endogenous CD28 on T cells and CD28ζ.CAR together form microclusters

Activation of hCD19 CD28ζ.CAR-T cells was cooperatively transduced through the first signals via CD3ζ and the second signals via CD28 within the CAR following CAR-hCD19 binding. Although endogenous CD28 is also expressed on CAR-T cells, it is not clear how endogenous CD28 contributes to costimulatory functions in those CAR-T cells. Therefore, we next examined the behavior of both CAR and CD28. Accumulation of CAR at the CAR-T cell–EL-4 cell interface was detected on EL-4 cells without CD80 expression, whereas that of CD28 was limited to transduction of CD80 in EL-4 cells as an APC, and there was no difference between ζ. and CD28ζ.CAR (Fig. 3a, b). Consistent with the T cell–EL-4 cell conjugation assay, endogenous CD28 and hCD19 CAR were shown to form microclusters at an immunological synapse by binding to CD80 and hCD19, respectively (Fig. 1e, left, and Fig. 2c, left). Therefore, we next examined the structural relationship between CD28 and hCD19 CAR microclusters at the CAR-T cell–SLB interface and found that CD28 formed microclusters clearly colocalized with CARs (CAR-CD28 microclusters) when CD80–GPI was additionally introduced to an SLB expressing hCD19– and ICAM-1–GPI (Fig. 3c, left). CD28ζ.CAR, which contains the cytoplasmic region of CD28, formed CAR-endogenous CD28 microclusters in a similar way to ζ.CAR (Fig. 3d). After maximal spreading of CAR-T cells, both CAR and CD28 microclusters began centripetal transmigration and accumulated at the center of the immunological synapse. Although both ζ.CAR and CD28ζ.CAR were simply consolidated at the center, forming a conventional cSMAC in the absence of CD80–GPI, they constructed a bull’s eye or ring-shaped structure further colocalized with CD28 in the presence of CD80–GPI (Fig. 3c, right). These specific patterns were imaged in more than half of cases (Fig. 3e, and Supplementary Fig. 2). These results confirmed that CAR-endogenous CD28 microclusters were imaged in the presence of CD28-CD80 binding and that even after transmigration to the center of immunological synapse, the CAR forms characteristic structures in response to the CD28-CD80 binding, similar to conventional TCR-CD28 assembly.

**Fig. 3: Extrinsic CD28 on CAR-T cells translocates at CAR microclusters dependent on binding to CD80.**

Translocation of PKCθ at CAR microclusters is increased by CD28-CD80 binding, cooperatively with cytokine production via enhanced CD28 signaling

We have clearly shown that PKCθ accumulated at the T cell–EL-4 cell interface or TCR microclusters in the presence of endogenous CD28-CD80 binding or when CD28 was constructed into CARs, even in the absence of CD28-CD80 binding. These findings indicate that parallel costimulatory signaling from endogenous CD28 and CD28 incorporated into CARs will sometimes be simultaneously transduced. Based on this hypothesis, we evaluated the difference in behavior of PKCθ between the two types of CD28-mediated signaling. hCD19 CAR-T cells were conjugated with non-transduced or CD80-transduced EL-4 cells expressing hCD19. PKCθ was clearly shown to accumulate at the interface between ζ.CAR-T cells and EL-4 cells expressing CD80, and CD28ζ.CAR-T cells and EL-4 cells regardless of CD80 expression (Fig. 4a, b). In order to compare the behavior of PKCθ in two types of CD28 signaling—either direct signaling from CD28ζ.CAR or parallel signaling from CD28 intrinsically expressed on primary T cells—PKCθ in CAR-T cells on the SLB was imaged. PKCθ accumulated at CAR microclusters to the same extent in ζ.CAR on CD80-expressing SLBs and in CD28ζ.CAR on SLBs not expressing CD80, and PKCθ further accumulated at CD28ζ.CAR microclusters at a higher concentration in the presence of CD80 on SLBs (Fig. 4c, left, Fig. 4d, and Supplementary Movies 3–6). Even after CAR microclusters reached the center of an immunological synapse, PKCθ remained at CD28 and CD28ζ.CAR clusters, reflecting the tight correlation between CD28 and PKCθ in localization and possible CD28-mediated costimulatory signaling (Fig. 4c, right, Fig. 4e, and Supplementary Fig. 3a).

**Fig. 4: CD28-mediated second signaling is required for cytokine production but not for enhancement of cytotoxicity.**

We next examined the biological contribution of the two types of CD28 signaling in CAR-T cells. AND-TCR-expressing T-cell hybridoma (2D12) cells were introduced by ζ. or CD28ζ.CAR, stimulated by CD80-transduced or not transduced EL-4 cells without or with hCD19 expression and examined for phosphorylation of both Erk and the CD3ζ cytoplasmic region within the CARs. The phosphorylation status of Erk was increased to a greater extent in CAR-T cells stimulated by EL-4 cells expressing CD80 than in cells stimulated by EL-4 cells not expressing CD80, and was maintained at a higher level in CD28ζ.CAR T cells than in ζ.CAR-T cells stimulated by EL-4 cells not expressing CD80 (Fig. 4f). Phosphorylation of CD3ζ was slightly enhanced by CD28-CD80 binding and, as previously reported, CD28ζ.CAR was phosphorylated at basal levels without hCD19 stimulation³² and further phosphorylated by the addition of CAR-hCD19 binding (Fig. 4f). We next confirmed CD28-mediated costimulation, which was comparably introduced via endogenous CD28 expressed on T cells and the cytoplasmic region of CD28 incorporated into CARs (Figs. 1g, 2f). To precisely compare the contribution of these CD28-mediated signals to IL-2 production, CD4⁺ or CD8⁺ T cells were introduced by ζ. or CD28ζ.CAR at the same expression level and stimulated by hCD19-transduced EL-4 cells (Supplementary Fig. 3b, c) not expressing or expressing CD80 at a low (CD80^Lo) or high (CD80^Hi) level according to their CD80 expression (Supplementary Fig. 3c). We demonstrated that IL-2 production was increased in both CD4⁺ and CD8⁺ CAR-T cells in a CD28 signaling-dependent manner (Fig. 4g). The production of IFNγ, which exhibits an antitumor immune response, also tended to increase by CD28-mediated costimulation (Fig. 4h). However, costimulatory signaling via CD28 incorporated into the CAR and endogenously expressed on the CAR-T cell itself did not affect in vitro cytotoxicity, even if costimulatory signaling was simultaneously derived from CD28 in the CAR and from endogenous CD28 (Fig. 4i). These results indicate that CD28-mediated costimulatory signaling distinctively contributes to CAR-T cell function, particularly in cytokine production, but not cytotoxicity in vitro, which is correlated with enhanced accumulation of PKCθ at CAR microclusters.

CD28-mediated signaling increases antitumor effects by CAR-T cells in vivo

Second-generation CARs are known to have a number of advantages over first generations when used clinically, indicating that second signals via CD28 or 4-1BB incorporated into CARs may be effective in vivo and important for antitumor effects. Therefore, we examined the potential requirement of second signals for long-term tumor suppression in vivo and then evaluated the importance of CD28 signaling from CD28ζ.CARs or endogenous CD28 in a tumor-bearing mouse model (Fig. 5a). The volume of CD80-transduced EL-4 cells was reduced to a greater degree than that of CD80-negative cells 14 days after CAR-T cell transplantation, and CD28ζ.CAR-T cells inhibited the growth of both CD80-positive and CD80-negative EL-4 cells more effectively than ζ.CAR-T cells (Fig. 5b, and Supplementary Fig. 4a). In mice transplanted by CD80-transfected EL-4 cells with CD28ζ.CAR-T cells, the growth of EL-4 cells was significantly suppressed (Fig. 5b), and these antitumor effects correlated with survival curves in tumor-bearing mice (Fig. 5c). To elucidate the mechanism by which CD28ζ.CAR-T cells effectively suppress the growth of CD80-expressing EL-4 cells in vivo, we examined the number of CD28ζ.CAR-T cells infiltrating tumor tissues in histology. CAR-T cells in tumors were increased in number to a greater extent when EL-4 cells expressed CD80. Furthermore, the number of tumor-infiltrating CD28ζ.CAR-T cells was clearly increased in CD80-expressing EL-4 cells compared with EL-4 cells not expressing CD80, and in CD28ζ.CAR-T cells compared with ζ.CAR-T cells (Fig. 5d, e). Next, we isolated tumor-infiltrating lymphocytes (TILs) from tumor tissue and examined the characteristics of CAR-T cells among those TILs. CD8⁺ CAR-T cells highly expressing TNFα or IFNγ were increased more in CD28ζ.CAR-T cells infiltrating CD80-expressing tumors than in ζ.CAR-T cells in tumors without CD80 expression (Fig. 5f, g, and Supplementary Fig. 4b). Frequency of the terminally exhausted T (Tex) cell, which is characterized by TIM-3 and lack of Ly108 expression^33,34,35, in PD-1⁺ effector CD8⁺ T cells was higher in CD28ζ.CAR-T cells infiltrating CD80-negative tumors than in ζ. or CD28ζ.CAR-T cells in CD80-expressing tumors (Fig. 5h and Supplementary Fig. 4c). Although CD28-mediated costimulatory signaling was less effective in cytotoxicity in vitro, these data indicate that CD28-mediated signaling could support the tumoricidal function of CAR-T cells in vivo by direct signaling from the cytoplasmic region of CD28 in CD28ζ.CAR or by parallel signaling from CD28 endogenously expressed on CAR-T cells. The latter CD28-mediated signaling may be sufficient or more effective in tumor suppression in vivo.

**Fig. 5: CD28-mediated direct and parallel signaling in hCD19 CAR-T cells is effective in suppressing tumor growth.**

Discussion

In this report, we examined the importance of costimulatory signaling from CD28 in the function of CAR-T cells by using our advanced imaging system and biochemical and physiological analyses, to compare the intrinsic signals from cytoplasmic region of CD28 incorporated into hCD19 CAR with extrinsic signals from endogenous CD28 expressed on CAR-T cells. hCD19 CD28ζ.CAR and CD28 expressed on ζ.CAR-T cells were found to effectively recruit the responsible signaling molecule, PKCθ, to CAR microclusters by CAR-hCD19 and CD28-CD80 binding, respectively. The accumulation of PKCθ at microclusters and at the plasma membrane is known to reflect the activation of nuclear factor kappa B (NF-κB), which correlates with cytokine production and tumor suppression in vivo. Therefore, we evaluated CD28-mediated costimulation in CAR-T cells and conventional T cells.

In various articles on CD28 costimulatory signaling, PI3K has been shown to be the predominant downstream effector molecule. A regulatory subunit of PI3K, p85α, binds to the YMNM motif in the intracellular region of CD28, and a catalytic unit, p110δ, is then recruited to p85α. This heterodimeric lipid kinase contributes to the production of phosphatidylinositol 3,4,5-triphosphate (PIP3), which induces activation of the Akt/mTOR pathway, promoting cell proliferation and survival^36,37. The T cell–APC interface becomes a location for PI3K to remain at the plasma membrane³⁸. In our imaging examinations, accumulation of p110δ was detected at the T cell–APC interface and p110δ remained there for 20 min after T cell–APC conjugation. As another candidate molecule downstream of CD28, PKCθ is known to associate with the plasma membrane through its C1 domain, which has specific affinity for diacylglycerol (DAG) produced by phospholipase Cγ1 (PLCγ1) at the T-cell activation interface and to play a crucial role in T-cell activation by facilitating the nuclear translocation and transcriptional activation of NF-κB³⁹. Because PLCγ1 can be activated by a solo signal via TCR, PKCθ translocates to the T cell–APC interface even in the absence of CD28-CD80 binding⁴⁰. However, such TCR solo signaling translocates PKCθ to the T cell–APC interface only temporarily, and our imaging analysis showed that accumulation of PKCθ was limited to a short period at low intensity in the absence of CD28-mediated signaling. In contrast, binding to CD80 allows CD28 to recruit PKCθ at the T cell–APC interface for a longer period and at higher intensity. We have demonstrated a mechanism for the extended association between CD28 and PKCθ, in which the kinase Lck interacts with the proline-rich motif of PKCθ via the SH3 domain of Lck, and the SH2 domain of Lck binds to the tyrosine motif of the CD28 cytoplasm^22,23.

The similarities and differences between the signaling pathways of TCR and CAR signaling are being comprehensively elucidated by biochemical methods and, more recently, by protein mass spectrometry. The two leading signaling molecules, zeta-chain-associated protein kinase 70 (Zap70) and SH2-domain-containing leukocyte protein of 76 kDa (SLP-76), are translocated into clusters of TCRs generated by TCR-MHCp binding and form signaling units. We defined these functional clusters as a signalosome or “TCR microcluster.” By binding to CAR ligands expressed on tumor cells, CARs also form their own signalosomes, cooperating with some representative molecules in the downstream of TCR; however, differences in the contents of signalosomes between TCR and CAR have been reported. Some reports note that a much greater number of antigens on target tumor cells is required for CAR-T cells to be able to induce activation signaling compared with that of MHCp recognition by a TCR^41,42. As for the composition of CAR signalosomes, the transmembrane adapter protein, linker for activation of T cells (LAT), is either not phosphorylated or is very weakly phosphorylated after crosslinking of CAR⁴³. In contrast to these differences, Zap70 is reported to accumulate at CAR microclusters, and the kinetics of synapse formation in CAR-T cells is comparable to that of T cells^44,45. These results suggest that analyzing the clustering and spatiotemporal behavior of CARs using molecular imaging may provide an objective and digital way to estimate CAR-T cell activation.

In our experiments, after CAR-hCD19 binding, the accumulation of PKCθ at hCD19 CAR microclusters was induced to a greater extent in CD28ζ.CAR-T cells than in ζ.CAR- or 4-1BBζ.CAR-T cells. Compared with such intrinsic CD28 signaling, parallel extrinsic CD28 signaling via CD28 expressed on CAR-T cells comparably introduced costimulatory signaling and led to dense and prolonged accumulation of PKCθ at CAR microclusters. In contrast, Lck has been shown to behave differently at the immunological synapse between TCR and ζ.CAR stimulation⁴⁶. The transmembrane of hCD19 CD28ζ.CAR used here is derived from CD28; therefore, the distance from the plasma membrane to the Lck-binding motif in CD28ζ.CAR is almost the same as that of endogenous CD28 on T cells. In contrast, because CD3ζ is tandemly linked to the end of the cytoplasmic region of CD28 in the CD28ζ.CAR, the distance from the plasma membrane to the ITAMs in CD28ζ.CAR is different from that of endogenous CD3ζ. Such differences may explain the unusual association of Lck with CD28ζ.CAR, CD28, and CD3ζ. Since the distances from the plasma membrane to the cytoplasmic motifs that contribute to signaling may affect downstream signaling, when we design a new CAR, it is better to consider those structural factors that affect signal transduction in CAR and endogenous receptors expressed on T cells.

By using high-resolution imaging with SLBs, we clearly demonstrated the subtle but dynamic behavior of signalosomes constructed within an immunological synapse. When T cells adhere to APCs or antigen-presenting SLBs, TCR microclusters are sequentially formed at the nascent contact regions of the adhesion surface and then migrate toward the center of the adhesion surface and become internalized^19,47. This spatiotemporal behavior of each receptor could be explained by the outcomes of cytoskeletal rearrangement, including the assembling of actin filaments (F-actin) and dynein, a molecular motor on microtubules^19,48,49. Even if CD28-mediated signaling is absent in CAR-T cells, ζ.CAR microclusters exhibit centripetal movement with similar kinetics to TCR microclusters. Through more detailed analyses, we noted a small difference between ζ.CAR and CD28ζ.CAR. Whereas ζ.CARs were tightly clustered at cSMAC, CD28ζ.CARs were widely distributed, forming an annular shape. This finding implies that CD28ζ.CAR microclusters were delayed or inhibited from centripetal movement, which is supported by CD28 function in cytoskeletal remodeling by association with F-actin and filamin-A⁵⁰.

TCR signaling diverts to a variety of downstream signals. A certain T-cell function may require more specific signals than others¹¹, but it is difficult to clearly determine the extent to which individual signaling pathways are needed, including costimulatory signaling^51,52. T-cell responses that result in cell differentiation and transcription of effector molecules typically require multiple complicated signaling pathways. However, cytotoxic activity is a fundamental function of innate immune cells like NK cells. Reorientation of microtubule-organizing center (MTOC) to the immune synapse by DAG production and Ca²⁺ influx might be sufficient for proper exocytosis of the lytic granule^53,54,55. Consistent with a previous report⁵⁶, in vitro cytotoxic activity was not increased by CD28-mediated second signaling in our experiments. In contrast, in an in vivo tumor-bearing mouse model, suppression of tumor growth and prolonged survival were observed in the group in which CD28 signaling was transmitted intrinsically via CD28ζ.CAR or extrinsically or in parallel via endogenous CD28 expressed on CAR-T cells. As previously reported, both IL-2 and IFNγ production in vitro are enhanced by the addition of CAR intrinsic CD28 signaling⁵⁷. Furthermore, in both ζ.CAR- and CD28ζ.CAR-T cells, cytokine production was enhanced by extrinsic CD28 signaling in proportion to the degree of CD80 expression on tumor cells^56,58. As a previous report showed that TILs in solid tumors, which highly expressed CD80/CD86, were increased in number⁵⁹, our immunohistochemical examinations revealed that the number of CAR-T cells infiltrating tumor tissue was affected by intrinsic or extrinsic CD28 signaling. These results may explain why we could not find a correlation between in vitro killing and in vivo tumor suppression. We have not examined the production of any other cytokines, but inflammatory cytokines involuntarily produced by CAR-T cells will cause cytokine release syndrome (CRS). Signaling from endogenous CD28 expressed on CAR-T cells may also contribute to the physiological behavior and response of CAR-T cells^16,60, so the clinical outcome of CAR-T cell therapy may vary depending on the expression level of CD80 and CD86 in the tumor environment, including oncogenic inflammation and regional lymph nodes and the tumor itself. Currently, CAR-T cell therapy is being actively challenged not only in various cancer types but also in non-malignant diseases⁶¹, and costimulatory signaling must be controlled more precisely to suppress toxicity such as CRS and to achieve long-term remission.

In this article, we examined the importance of CD28-mediated costimulatory signaling in CAR-T cells from the viewpoint of molecular dynamics and T-cell biology. Without costimulatory signaling from intrinsic CD28 within a CAR itself, endogenous CD28 expressed on CAR-T cells induces extrinsic costimulatory signals comparable to the intrinsic signals from CD28ζ.CAR and compensates for the loss of signaling in ζ.CAR-T cells. Moreover, those extrinsic signals further enhanced the activation of CD28ζ.CAR-T cells. We suggest here the importance of considering not only the costimulatory domain within CARs but also the costimulatory and coinhibitory receptor intrinsically expressed on CAR-T cells, particularly when designing new constructs of CARs and examining CAR-T cell functions. The strength of extrinsic CD28 signaling may be significantly affected by cell surface expression of CD80/CD86 on tumor cells, APCs, and other parenchymal cells in the tumor microenvironment; therefore, the contribution of intrinsic and extrinsic CD28 signaling to CAR-T cell function in vivo should be further investigated.

Methods

Reagents

Antibodies and reagents were purchased from the following suppliers: PE-anti-I-A/I-E (M5/114.15.2), PE-anti-CD4 (RM4-5), PE-anti-CD8α (53-6.7), PE-anti-CD80 (16-10A1), APC-anti-CD28 (37.51), anti-IL-2 (JES6-1A12) and biotin-labeled anti-IL-2 (JES6-5H4) from eBioscience; PE-anti-H-2K^b (AF6-88.5) and mouse anti-pCD3ζ (K25-407.69) from BD Bioscience; PE-anti-human CD19 (HIB19), Brilliant Violet 785 labeled-anti-CD8α (53-6.7), PE-anti-TNFα (MP6-XT22), APC-anti-IFNγ (XMG1.2), PE/Cyanine7-anti-PD-1 (29F.1A12), APC-anti-TIM-3 (RMT3-23), PE-anti-Ly108 (330-AJ) and Alexa Fluor 647-streptavidin from BioLegend; mouse anti-CD3ζ (6B10.2) from Santa Cruz Biotechnology Inc.; anti-IFNγ (RA-6A2), biotin-anti-IFNγ (XMG1.2), rabbit anti-Erk (137F5), rabbit anti-pErk (D13.14.4E), HRP-anti-rabbit IgG polyclonal Abs, and HRP-anti-mouse IgG polyclonal Abs from Cell Signaling Technology; DyLight 650 and 549 labeling kits and AlexaFluor488-conjugated anti-rabbit IgG antibody from Thermo Fisher Scientific; biotin-anti-human IgG Fcγ from Jackson ImmunoResearch; HaloTag (HT) STELLA Fluor 650 and TMR ligands from Promega; OVA_257–264 (SIINFEKL) peptides and its variant Q4H7 (SIIQFEHL) from GenScript; and rabbit anti-GFP antibody from MBL. A B-cell hybridoma producing anti-CD28 (PV-1) was provided by R. Abe (Tokyo University of Science, Chiba, Japan); anti-CD3ζ (145-2c11) by J. Bluestone (University of California, California, USA), anti-TCRβ (H57-597) by R. T. Kubo (Cytel Corp., California, USA); anti-ICAM-1 (YN1/1.7.4) by M. L. Dustin (University of Oxford, Oxford, UK).

Mice and cells

OT-I TCR-Tg Rag2^−/− mice were provided by Dr. W. Heath (University of Melbourne, Melbourne, Australia); Rag2⁻^/⁻ Strain B6(Cg)-Rag2^tm1.1Cgn/J mice by Dr. F. Alt (Boston Children’s Hospital, Boston, MA); C57BL/6 was purchased from CLEA Japan Inc. Mice were maintained in specific-pathogen-free conditions with a 12-h light/dark cycle at 22 °C and controlled humidity (60 ± 10%) at Tokyo Medical University. All experiments were performed on 8- to 12-week-old age- and sex-matched mice. Experimental and control animals were co-housed. All experiments were performed in accordance with a protocol approved by the Animal Care and Use Committee of Tokyo Medical University (R3-009, R4-002, R5-059). Mice were humanely euthanized by cervical dislocation once they reached endpoints, such as reaching 2000 mm³ in tumor volume or loss of weight/mobility/body condition and severe neurological disabilities. PLAT-E, the retrovirus packaging cell line, was provided by G. Nolan (Stanford University, Stanford, CA, USA). BHK, the EL-4 cell line, was purchased from ATCC. The T-cell hybridoma expressing the AND-TCR (AND-TCR T cell hybridoma, 2D12) was established by cell fusion of activated AND-TCR-Tg CD4⁺ T cells with lymphoma cell line BW5147²⁰.

Plasmid construction

Mouse CD80, HaloTag-tagged mouse CD28, and EGFP-tagged mouse p110δ, PKCθ, CD28 and Renilla luciferase (RLuc) 8 were generated by polymerase chain reaction (PCR) and subcloned into the retroviral vector pMXs (kindly provided by T. Kitamura, University of Tokyo, Tokyo, Japan)⁶². pMXs-RLuc8 was constructed by PCR using Yellow Nano-lanterns (kindly provided by Dr. Y. Okada, Riken, Japan) as a template⁶³. hCD19 fragments were amplified from Jurkat cells. The fragments of ζ. and CD28ζ.CAR, composed of anti-hCD19 antibody scFv (clone: FMC63), hinge of IgG4, CH3 domain of IgG1, transmembrane domain of hCD28 and cytoplasmic signaling domain of hCD28 or h4-1BB and/or hCD3ζ, were provided by M. K. Brenner (Baylor College of Medicine, Houston, TX, USA). EGFP- or HaloTag-tagged ζ., CD28ζ., or 4-1BBζ.CAR genes were generated and subcloned into pMXs or pMCs (provided by T. Kitamura, University of Tokyo, Tokyo, Japan)⁶².

Primary cell culture and transduction

Lipofectamine 2000 (Invitrogen) was used to transiently transduce retroviral vectors into packaging cells (PLAT-E), and the culture supernatant was collected. The supernatants were concentrated 40- to 80-fold by centrifugation at 8000 g for 12 h. OT-I TCR-Tg CD8⁺ T cells were purified from OT-I TCR-Tg Rag2⁻^/⁻ mice and stimulated with 100 nM Q4H7 and irradiated spleen cells from B6 mice, or with plate-bound anti-CD3ζ and anti-CD28 antibodies. One day after stimulation, the cells were suspended in retroviral supernatant with 10 μg/mL polybrene (Sigma-Aldrich) and 200 U/mL recombinant mouse IL-2 (Peprotech) and centrifuged at 1000×g for 90 min at 37 °C. On day 3, the cells for microscopy were sorted to obtain populations with homogeneous fluorescence intensity. Cells were maintained in RPMI 1640 medium (Sigma-Aldrich) containing 10% fetal calf serum (FCS) (Thermo Fisher Scientific) and mouse IL-2.

Microscopy

Cells expressing proteins tagged with GFP and HaloTag stained by DyLight 650-labeled H57 Fab and/or TMR- or Stella650-labeled HaloTag ligand were allowed to settle onto a supported lipid bilayer (SLB). A confocal laser scanning microscope (TCS SP8, Leica Microsystems) consisting of a ×63 oil-immersion objective, a highly sensitive HyD detector, and 488-, 561-, and 633-nm laser lines was used. LAS X software (Leica, Germany) was used for image acquisition. TIRF images were acquired under a conventional inverted microscope (Ti-E, Nikon, Tokyo, Japan) consisting of a ×100 oil-immersion TIRF lens, fiber-coupled 488 nm and 640 nm lasers, and two scientific complementary metal oxide semiconductor (sCMOS) cameras (Hamamatsu). Simultaneously emitted green and infrared fluorescence from 488 nm and 640 nm lasers, respectively, were split using image splitting optics (W-VIEW GEMINI-2C, Hamamatsu), and detected by each sCMOS camera without the time-lag between two cameras. NIS-elements software (RRID:SCR_014329) was used for image acquisition. ImageJ software (NIH, Bethesda, MD, USA, RRID:SCR_003070) was used for image processing and final figure preparation.

Planar bilayers

The purification and fluorescent labeling of GPI-anchored proteins was established according to published protocols¹⁷. The mouse MHC class I molecule H-2K^b with a GPI anchor (H-2K^b–GPI), mouse ICAM-1 with a GPI anchor (ICAM-1–GPI), and mouse CD80 with a GPI anchor (CD80–GPI) were purified from transfected Chinese hamster ovary and baby hamster kidney cells, respectively, and were incorporated into dioleoyl phosphatidylcholine liposomes (Avanti Polar Lipids). BHK cells (ATCC) highly expressing hCD19–GPI were established. hCD19–GPI were purified from the lysates by affinity column with anti-hCD19 antibody (4G7, Bio X cell). The expression level of each GPI-anchored protein on the planar bilayer was quantified using silica beads with a diameter of 5 μm (Bangs Laboratories, SS05N)⁶⁴. The densities were calculated based on the standard beads, Quantum FITC-5 MESF (Bangs Laboratories), and adjusted to the approximate concentration by comparison with natural APCs: H-2K^b, 200 molecules/μm²; ICAM-1, 150/μm²; CD80, 28–69/μm², and hCD19, 50–150/μm². Planar bilayers were prepared by mixing GPI-anchored proteins, dropping onto clean glass (40-mm glass coverslips, Biotechs), and overlaying with a clean cover glass (Fisherbrand, Circles; size 12 mm) for 30 min. The planar bilayers were then blocked with 5% nonfat dried milk (Cell Signaling Technology, 9999S) in PBS for 30 min at 37 °C, the cover glass was removed, and the slide left to stand in the assay medium (Hepes-buffered saline, Sigma-Aldrich, H3375-250G) containing 1% FCS (Thermo Fisher Scientific, 10270106), 2 mM MgCl₂, and 1 mM CaCl₂ in a flow cell chamber system (Bioptechs). For OT-I TCR-Tg CD8⁺ T cells, the planar bilayers were loaded with 10 μM OVA_257-264 in citrate buffer (pH 4.5) for 24 h at 37 °C before blocking.

Image processing

The size and fluorescence intensity of each region were examined in all images and measured using ImageJ. Fluorescence intensities were measured based on raw imaging data with the following formula: ([intensity of fluorescence at each spot on a diagram] − [minimal intensity of each fluorescence on the entire line]) / ([mean intensity of each fluorescence on the entire line] − [minimal intensity of each fluorescence along the entire line])⁶⁴. Pearson correlation coefficients (PCCs) were subsequently calculated from each fold intensity. One PCC value was defined as the average value of the correlation coefficients of each microcluster on the two diagonal lines of one cell.

T cell-APC conjugation assay

Non-transduced or CD80-transduced EL-4 cells were prepulsed with 100 nM OVA_257-264 overnight at 37 °C. OT-I TCR-Tg CD8⁺ T cells, both CD28-HaloTag and p100δ-EGFP or PKCθ-EGFP, were cultured with those EL-4 cells. Primary CD3⁺ T cells expressing both ζ.- or CD28ζ.-, or 4-1BBζ.CAR-HaloTag and PKCθ-EGFP were cultured with EL-4 cells expressing hCD19. Primary CD3⁺ T cells expressing ζ.- or CD28ζ.CAR-HaloTag, and CD28- or PKCθ-EGFP were cultured with non-transduced or CD80-transduced EL-4 cells expressing hCD19. The conjugates were visualized by confocal microscopy.

Western blotting

A total of 1 × 10⁶ 2D12 cells expressing ζ.- or CD28ζ.CAR-EGFP were stimulated with 1 × 10⁶ non-transduced or/and hCD19-transduced or/and CD80-transduced EL-4 cells. The cells were lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate). Whole cell lysates (WCLs) were blotted with anti-CD3ζ (1:500), anti-pCD3ζ (1:1000), anti-Erk (1:1000), or anti-pErk (1:1000) as a first antibody and HRP-anti-rabbit IgG polyclonal Abs (1:10,000) or HRP-mouse-IgG polyclonal Abs (1:10,000) as a second antibody. Band intensity was calculated using ImageJ.

Flow cytometry

Cells were stained with antibodies for cell surface molecules in HBSS. A cell sorter, SH800S (Sony), was used for cell isolation, and cell analyzers, FACS Canto II (BD) and Guava easyCyte (Merck), were used for analysis. Data were depicted using FlowJo software (Tree Star, Ashland, OR, USA).

T-cell stimulation assay

A total of 5 × 10⁴ OT-I TCR-Tg CD8⁺ T cells were stimulated with 5 × 10⁴ non-transduced or CD80-transduced EL-4 cells with 1 μM OVA_257-264 for 10 h. Then, 5 × 10⁴ primary ζ., CD28ζ., or 4-1BBζ.CAR-T cells were stimulated with 5 × 10⁴ EL-4 cells expressing hCD19 for 6 or 18 h. Similarly, 5 × 10⁴ primary ζ. or CD28ζ.CAR-T cells were stimulated with 5 × 10⁴ non-transduced, hCD19-transduced, or hCD19- and CD80-transduced EL-4 cells for 6 or 18 h. The concentrations of IL-2 and IFNγ were measured in the supernatant using ELISA. All experiments were performed in triplicate.

CTL killing assay

At the indicated E/T ratios, 5 × 10⁴ OT-I TCR-Tg CD8⁺ T cells were stimulated with 5 × 10⁴ non-transduced or CD80-transduced EL-4 cells with 1 μM OVA_257-264 for 10 h. Similarly, 5 × 10⁴ primary ζ., CD28ζ., or 4-1BBζ.CAR-T cells were stimulated with 5 × 10⁴ EL-4 cells expressing hCD19 for 18 h, and 5 × 10⁴ primary ζ. or CD28ζ.CAR-T cells were stimulated with 5 × 10⁴ non-transduced, hCD19-transduced, or hCD19- and CD80-transduced EL-4 cells for 18 h. All EL-4 cells were transduced with RLuc8. After treatment with coelenterazine, an RLuc8 substrate (FUJIFILM Wako), the intensity of RLuc8 luminescence in live target cells was measured using a lumino image analyzer, ImageQuant LAS4000 mini (GE Healthcare). All experiments were performed in triplicate.

In vivo tumorigenicity assay

A total of 5 × 10⁵ non-transduced or CD80-transduced EL-4 cells expressing hCD19 in 100 μL of PBS were subcutaneously inoculated in the dorsal region of 7- to 11-week-old female Rag2^−/− mice. Tumors were allowed to grow for 7 days before treatment. Mice received an intravenous injection of 100 μL of PBS containing 2 × 10⁶ CD3⁺ GFP⁺ ζ.CAR-transduced, CD28ζ.CAR-transduced, or non-transduced T cells into the ophthalmic venous plexus. Tumor volume was measured using digital calipers and calculated using the equation: volume = length × width × width/2. For survival studies, the endpoint was established at tumor volume ≥2000 mm³, and surviving mice were terminated at 50 days after CAR-T cell transfer.

Fluorescent immunohistochemistry

Tumor specimens were fixed with 4% paraformaldehyde. After fixation, specimens were equilibrated gradually with 10%, 20%, and 30% sucrose in PBS at 4 °C, embedded in O.C.T. compound (Sakura Finetech), sectioned at a thickness of 10 μm, air-dried for 30 min, fixed with ice-cold acetone for 15 min, and subjected to immunohistochemistry. The tissue sections were treated with blocking solution with 1% BSA containing PBS for 30 min and then incubated with rabbit anti-GFP antibody and AlexaFluor 488-conjugated anti-rabbit IgG antibody diluted with PBS supplemented with 1 h at room temperature in a humidified chamber. After washing 3 times with PBS supplemented with 0.02% Tween-20 (PBS-T), the slides were mounted using Permafluor mountant (Thermo Fisher Scientific) and observed by confocal microscopy.

Analysis of TILs

Tumor-bearing mice were generated following the method, in vivo tumorigenicity assay. At 3 weeks after the inoculation of CAR-T cells, tumor tissues were resected from the mice, and TILs were isolated by the tumor dissociation Kit (Miltenyi Biotec, 130-096-730) with gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec). The lymphocyte fraction was further enriched from the cell suspension by Percoll (Cytiva, 17089101) and stained with antibodies for cell surface molecules. For intracellular staining, TILs were once stimulated by PMA and ionomycin with BD GolgiStop (BD Bioscience, 554724) for 4 h, stained for cell surface molecules, then fixed, permeabilized by a fixation/permeabilization solution kit (BD Bioscience), stained with anti-TNFα or anti-IFNγ and analyzed by Symphony A1 (BD Bioscience). Data analysis was performed by FlowJo software (BD Bioscience).

Statistics and reproducibility

Data are presented as the mean ± standard deviation (SD). Statistical analysis was performed by the unpaired t-test, one-way analysis of variance (ANOVA), or log-rank test using GraphPad Prism. p-values < 0.05 were considered statistically significant. Reproducibility, including biological independent sample sizes and replicates, is stated in each figure legend.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the conclusions are available within the article and its supplementary information. The source data behind the graphs in the paper can be found in Supplementary Data. Source data are provided with this paper.

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Acknowledgements

We thank Dr. Malcolm K Brenner and Dr. Maksim Mamonkin for the CAR vector, Dr. Toshio Kitamura (The University of Tokyo) for retroviral vectors pMXs and pMCs, and Mai Kozuka for secretarial assistance. This work was supported by JSPS KAKENHI (JP25113725, JP15H01194, JP16H06501, JP17H03600, JP19K22545, JP20H03536, JP23H02775, JP23H04790 to T.Y.), PRESTO from JSI (U1114011 to T.Y.), the Takeda Science Foundation (to T.Y.), and the Naito Foundation (4465-135 to T.Y.).

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Authors and Affiliations

Department of Immunology, Tokyo Medical University, Tokyo, Japan
Tetsushi Nishikawa, Arata Takeuchi, Hiroaki Machiyama, Ei Wakamatsu, Hitoshi Nishijima, Masae Furuhata, Hiroko Toyota, Wataru Nishi, Ryohei Matsushima, Yosuke Yoshida & Tadashi Yokosuka
Department of Dermatology, Tokyo Medical University, Tokyo, Japan
Tetsushi Nishikawa & Kazutoshi Harada
Department of Thoracic Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Wataru Nishi & Ryohei Matsushima
Department of Nephrology, Tokyo Medical University, Tokyo, Japan
Yosuke Yoshida

Authors

Tetsushi Nishikawa
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Arata Takeuchi
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Hiroaki Machiyama
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Ei Wakamatsu
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Hitoshi Nishijima
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Masae Furuhata
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Hiroko Toyota
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Wataru Nishi
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Ryohei Matsushima
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Yosuke Yoshida
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Kazutoshi Harada
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Tadashi Yokosuka
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Contributions

T.N., A.T. and T.Y. designed the research; T.N., A.T., H.M., E.W., H.N., M.F., H.T., W.N., R.M., Y.Y. and T.Y. performed the research; T.N., A.T. and H.M. analyzed the data; A.T., K.H. and T.Y. supervised the research; and T.N. and T.Y. wrote the paper.

Corresponding authors

Correspondence to Arata Takeuchi or Tadashi Yokosuka.

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The authors declare no competing interests.

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Communications Biology thanks Nicholas Gascoigne and Prasad Adusumilli for their contribution to the peer review of this work. Primary handling editors: Yuting Ma and Mengtan Xing.

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Nishikawa, T., Takeuchi, A., Machiyama, H. et al. CD28-mediated linear and parallel costimulatory signaling cooperatively regulate CAR-T cell functions via CAR-CD28 microclusters. Commun Biol 8, 1506 (2025). https://doi.org/10.1038/s42003-025-08906-y

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Received: 13 December 2024
Accepted: 17 September 2025
Published: 29 October 2025
Version of record: 29 October 2025
DOI: https://doi.org/10.1038/s42003-025-08906-y