Potent Activation of Human T Cells by mRNA Encoding Constitutively Active CD40

Members of the type 1 TNFR family, which plays an essential role in T cell immunity, draw increasing attention in the field of cancer immunotherapy. In particular, 4-1BB and OX40 receptors that are mainly expressed by T cells are known to exert multiple immunostimulatory effects and are extensively explored in preclinical studies and clinical trials, evaluating different approaches for adoptive T cell therapy for cancer (1–3).

CD40 is another prominent member of the type 1 TNFR family that is primarily expressed by professional APCs and plays a pivotal role in cell-mediated immunity. Like other family members, physiological CD40 signaling requires engagement with its trimeric cell surface CD40L (CD40L, CD154), which is naturally expressed on activated T cells. The concomitant induction of receptor homo-oligomerization is followed by the recruitment of adaptor TNFR-associated factor (TRAF) proteins and activation of the NF-κB, p38 MAPK, or JNK/SAPK pathways. This outcome can be experimentally mimicked by CD40 ligation with agonistic anti-CD40 Abs or soluble CD40L. Agonist-mediated CD40 activation has been exploited for enhancing antitumor immunity independently of CD4 T cell help in numerous experimental settings as well as in clinical studies (4–7). An alternative approach for inducing CD40 signaling in vivo at will uses a lipid-permeant dimerizing drug capable of cross-linking genetically engineered CD40 activation domains, either alone or when fused with the MyD88 adaptor protein. This design was reported to improve the immunostimulatory properties of mouse and human dendritic cells (DCs) (8–10) and, most recently, also of chimeric Ag receptor (CAR)–modified human T cells (11).

Recently, we were capable of expressing a constitutively active CD40 (caCD40) configuration that was based on a spontaneously homo-oligomerizing CD40 signaling domain (12). To achieve this, we exploited the GCN4 yeast transcriptional activator, which contains a leucine zipper DNA-binding motif known to induce homophilic interactions. We assembled and expressed three derivatives of this motif that preferably self-organize to form homodimers, trimers, or tetramers and incorporated these at the membrane-proximal portion of the CD40 intracellular domain lacking the entire extracellular region. In our early experiments, the trimer appeared superior to the other conformers in its ability to activate the NF-κB signaling pathway and was chosen for further investigation. In human monocyte-derived DCs and different cell lines, the electroporation of mRNA encoding caCD40 upregulated a panel of surface costimulatory molecules and induced the secretion of proinflammatory cytokines, and the same was true for bone marrow–derived mouse DCs (12).

A growing number of studies, most of which have been performed in mice, suggest that CD40 is functionally expressed also by T cells. The direct T cell stimulatory capacity of CD40 was manifested in a wide range of effects, including differentiation, memory formation, improvement of functional avidity, upregulating antiapoptotic signals and decreasing proapoptotic ones, rescue from exhaustion, and acquisition of resistance to regulatory T cell–mediated suppression (13–23). Yet, other studies failed to confirm these observations (24–27), and the immunological role played by T cell–expressed CD40 under physiological conditions is still elusive.

The adoptive transfer of naturally occurring antitumor T cells, such as tumor-infiltrating lymphocytes (TILs) or gene-modified T cells expressing tumor-specific TCR or CARs, is widely explored today for cancer immunotherapy. During the past several years, we have been developing a series of genetic adjuvants designed to enhance the function and survival of adoptively transferred T cells, aiming to improve their antitumor reactivity. These adjuvants include constitutively active TLR4 (caTLR4) devoid of its extracellular ligand binding domain (28) and membrane-anchored cytokines, so far tested with IL-2, IL-12, and IL-15 (29). Each of these adjuvants alone could exert diverse immunostimulatory effects in electroporated human CD8 and CD4 T cells and antimelanoma TILs. Yet, some combinations of caTLR4 with these membrane cytokines displayed striking synergy, which was manifested, for example, in the induction of IFN-γ production and in the upregulation of activation molecules such as CD25 and CD69 (28, 29).

In this study, we present evidence that following mRNA electroporation, caCD40 induces multiple costimulatory effects in human CD8 and CD4 T cells and in selected TILs (sTILs) and nonselected TILs. Furthermore, caCD40 can cooperate and, at times, synergize with caTLR4 in exerting some of these effects.

The following mouse mAbs against human Ags were used: CD8a -PerCP-Cy5.5/eFluor 450, CD25-APC, CD137 (4-1BB)-PE, CD134 (OX40)-FITC, CD69-PE-Cy7, CD28-FITC, CD279 (PD-1)-PerCP-eFluor 710, CTLA-4–PerCP-eFluor 710, CD127-FITC, IFN-γ–FITC, CD107A (LAMP)-FITC, and isotype controls were all from eBioscience (San Diego, CA); CD8-PE-Cy7, CD107a–Pacific Blue, TNF-α–Pacific Blue, GM-CSF–APC, CEACAM-I-PE, and their isotype controls were from BioLegend (San Diego, CA). The mAb OKT3 (anti-human CD3 ε chain) was isolated from hybridoma supernatant. Recombinant human soluble CD40L/TRAP (rhsCD40L) was from BioVision (Milpitas, CA)

The human melanoma cell lines 624mel (HLA-A2+), M579, M579-A2 (a stable transfectant of M579 expressing HLA-A2), and M171 and M425 (both HLA-A2⁻) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mmol/l l-glutamine, and combined antibiotics. TIL425 is a bulk CD8 TIL culture prepared from a melanoma patient at the Sharett Institute of Oncology, Hadassah Hebrew University Hospital, (Jerusalem, Israel) (28). Young (y) TIL14, yTIL52, yTIL144, and yTIL412 (also a bulk CD8 TIL culture) were prepared from melanoma patients at the Ella Lemelbaum Institute for Immuno-Oncology, Sheba Medical Center (Ramat Gan, Israel). Human lymphocytes were cultured in complete RPMI 1640 medium supplemented with 10% heat-inactivated human AB serum (Sigma-Aldrich, Saint Louis, MO) or FCS, 300 and 6000 IU/ml recombinant human IL-2 (rhIL-2; Chiron, Amsterdam, the Netherlands) for PBMCs and TIL cultures, respectively, 2 mmol/l l-glutamine, 1 mmol/l sodium pyruvate, 1% nonessential amino acids, 25 mM HEPES, 50 μM 2-ME, and combined antibiotics.

The study on sTILs was approved by the Institutional Review Board (no. 383-23.12.05; Hadassah Hebrew University Hospital). Generation of primary melanoma and unselected, yTIL cultures at the Ella Lemelbaum Institute for Immuno-Oncology, Sheba Medical Center Hospital at Tel HaShomer was performed as part of clinical adoptive transfer protocols, which were approved by the Israel Ministry of Health (Approval no. 3518/2004, https://clinicaltrials.gov, Identifier NCT00287131). All patients and healthy donors gave their informed consent prior to initiation of melanoma and lymphocyte cell cultures. Fresh or thawed PBLs from healthy donors or antimelanoma TILs were cultured in the presence of 300 or 6000 IU/ml rhIL-2, respectively. In some experiments, T cells from healthy donors were separated after 24 h rest (without IL-2) by positive selection to CD4 and CD8 subsets using magnetic beads (BD Biosciences, San Jose, CA). To activate T cells and achieve efficient mRNA transfection, T cells were grown for 72 h in the presence of plate-bound anti-CD3 (OKT3) and 0.5 μg/ml soluble anti-CD28 mAbs and 100 IU/ml rhIL-2. Stimulated T cells were then cultured for another 24 h in fresh medium in the absence of Abs or cytokines to stop stimulation.

The cloning of monomeric CD40, trimeric caCD40, and the control H-2K^b–based trimeric construct, in which the intracellular portion of CD40 has been replaced with that of the mouse MHC class I (MHC-I) H-2K^b H chain, has been described in detail previously (12), as has the cloning of caTLR4 (28). For expressing native human CD40 (nCD40) (GenBank accession no. NM_001250), the extracellular portion of CD40 was cloned by RT-PCR using mRNA prepared from the human monocytic cell line THP-1 with the forward primer 5′-GGT CTA GAC TCG CTA TGG TTC GTC TGC C-3′ and the reverse primer 5′-AAC AGG ATC CCG AAG ATG GGG-3′. The resulting fragment and the DNA stretch encoding the intracellular portion of monomeric CD40 were inserted in a single step into the pGEM4Z/EGFP/A64 vector [kindly provided by Dr. E. Gilboa (30), University of Miami] following removal of the EGFP gene to create the intact transcription unit. The cloning of caTLR4 has been described previously (12, 31, 32).

Template plasmids were linearized with SpeI. Transcription and capping reactions were carried out using an AmpliCap-Max T7 High Yield Message Maker Kit (Epicentre, Madison, WI). The mRNA product was purified by DNase I digestion, followed by LiCl precipitation, according to the manufacturer’s instructions. The quality of the mRNA product was assessed by agarose gel electrophoresis, and concentration was determined by spectrophotometric analysis. Purified mRNA was stored at −80°C in small aliquots.

Electroporation was performed with ECM 830 Square Wave Electroporation System (Harvard Apparatus BTX, Holliston, MA) at LV mode, single pulse, 500 V, 1 ms; or with a Gene Pulser Xcell Electroporation System (Bio-Rad Laboratories, Hercules, CA) using a square-wave pulse, 500 V, 1 ms in cold 2-mm cuvettes as follows: stimulated PBLs and TILs were washed twice with Opti-MEM medium (Life Technologies, Grand Island, NY) and resuspended in Opti-MEM at a final concentration of 3 × 10⁷/ml. For electroporation, 0.1 to 0.4 ml prechilled cells (5 min on ice) were mixed with the required amount of in vitro–transcribed mRNA. In transfection experiments involving more than one mRNA species, the appropriate amount of irrelevant mRNA was cointroduced into T cells to normalize for the total amount of exogenous mRNA.

Cells were harvested, washed once with cold FACS buffer (PBS with 1% FCS and 0.1% sodium azide), and incubated for 30 min at 4°C in the dark with the respective Ab conjugate at a concentration recommended by the manufacturer. Cells were washed again with 4 ml of FACS buffer, resuspended in 0.3 ml of PBS with 0.1% sodium azide, and subjected to flow cytometry using FACSCalibur or FACSAria II (Becton Dickinson, San Jose, CA). Data were analyzed by BD LSR II (BD) and FCS Express software (DeNovo Software, Los Angeles, CA).

IFN-γ in the growth medium was monitored with a commercial ELISA kit (R&D Systems, Minneapolis, MN). For assaying antimelanoma response, 24 h postelectroporation TILs at 1–3 × 10⁶ cells per well were washed and cocultured in complete medium with the respective melanoma target cells at an E:T ratio of 1:1 for 24 h. At 1-d intervals, cells were washed and fixed in 1% formaldehyde. T cell reactivity was evaluated by flow cytometry analysis for CD107a and intracellular staining for IFN-γ, TNF-α, and GM-CSF, following the addition of Brefeldin A (eBioscience) at 10 μg/ml for the last 4 h of coculture.

Cells were plated in 96-well plates at 5:1 E:T ratio, and growth medium was harvested after 5–12 h. Lactate dehydrogenase (LDH) cytotoxicity assay was then performed using a commercial kit (BioVision).

Statistical significance was determined by the Kruskal–Wallis test. Data sets denoted with separate letters represent statistically significant differences.

We first evaluated the contribution of caCD40, alone or in combination with caTLR4, on the activation state of PBL-derived human T cells. To establish the effect on activation molecules, CD8 and CD4 cells were analyzed for CD25, 4-1BB, CEACAM-I, CTLA-4, and CD62L. (Fig. 1A, 1B). Interestingly, in both T cell subsets, caCD40 was moderately superior to caTLR4 in the induction of 4-1BB, and the reverse was true for CD25, although cooperativity between the two adjuvants was relatively mild in both subsets (Fig. 1B). The expression of CD62L by naive or central memory T cells is required for entering secondary lymphoid organs and is downregulated upon T cell activation. Indeed, the expression of caCD40 and, to a lesser extent, of caTLR4 led to reduction in cell surface CD62L as expected (Fig. 1C). Although some positive effect on the level of CEACAM-1, a cell adhesion molecule involved in T cell inhibition, was observed for the combination of caCD40 plus caTLR4, the level of the inhibitory receptor CTLA-4 remained practically unchanged for both subsets.

Next we assessed the effect of caCD40 on the expression of activation markers by selected and nonselected antimelanoma TILs. In sTIL412, selected for recognition of the HLA-A2–binding MART-1_26–35 peptide (33) and exclusively comprising CD8 T cells, caCD40 alone upregulated 4-1BB, OX40, and CD25 (Fig. 2A). The same was also true for nonselected yTIL14 (only CD8 T cells, data not shown) testing 4-1BB and CD25, in which strong cooperativity with caTLR4 was observed 24 and 48 h posttransfection (Fig. 2B). We then examined the effects of the same mRNAs on yTIL52 (70% CD8, 30% CD4 T cells), another clinical nonselected TIL preparation, adding CD28, PD-1, and CTLA-4 to the analysis. In accord with the previous observations, caCD40 alone induced a marked increase in the expression of CD25, CD28, 4-1BB, and OX40 but not of PD-1 or CTLA-4 (Fig. 3A). This increase was further augmented by the codelivery of caTLR4 only for CD25, likely owing to the striking degree of activation elicited by caCD40 alone. Pronounced elevation of 4-1BB and OX40 was also evident at 48 h (Fig. 3B).

Despite reports on the regulated expression of CD40 by some human T cells (e.g., Refs. 19 and 34), we could never detect CD40 on the plasma membrane of our TIL cultures nor on T cells isolated from PBLs (see Fig. 4B for yTIL14 and N. Levin, unpublished observations). We wanted on to confirm that human T cells are indeed equipped with the machinery required for CD40 signaling and that the enhancing effects exerted by caCD40 in PBL-derived T cells (Fig. 1) and TILs (Figs. 2, 3) could indeed be attributed to this machinery and were not triggered by an unrelated component of the caCD40 construct. To this end we assembled the gene encoding intact nCD40 and verified that it is properly expressed at the cell surface following electroporation (Fig. 4B). To induce activation via nCD40, we used recombinant soluble CD40L. In yTIL14, neither the expression of nCD40 alone nor incubation of these cells with CD40L had any effect on CD25 or 4-1BB, whereas the combination of nCD40 and CD40L enhanced both markers (Fig. 4B). Interestingly, whereas nCD40–CD40L-mediated enhancement of 4-1BB, OX40, or CD127 (the α-chain of the IL-7 receptor) was comparable to that induced by trimeric caCD40, the effect of the latter on CD25 was clearly more pronounced. The inability of the trimeric Kb (Kb-trimer) construct to exert a discernible effect on any of these markers (Fig. 4C–E) further rules out any artifact created by the homophilic interactions induced by GCN4. Notably, in this experiment, caTLR4 strongly cooperated with CD40 in the upregulation of CD25, and 4-1BB (Fig. 4C) only mildly coopereated with regard to CD127 (Fig. 4D) and had practically no additional effect on OX40 (Fig. 4E).

Clear differences in the magnitude of the effects exerted by caCD40 and caTLR4 mRNA on the level of CD25, 4-1BB, and OX40 in the panel of TIL cultures we have examined prompted us to determine the actual reproducibility of our results. To this end, we compiled all flow cytometry analyses performed on yTIL14, yTIL52, yTIL144, and two sTIL412s (18 altogether) 24 h postelectroporation and applied the Kruskal–Wallis and Mann–Whitney nonparametric test to determine statistical significance between the different mRNAs (Fig. 5). Indeed, clear patterns of response of the TIL preparations to the introduced mRNA emerge, which apply for both single- and double-staining with the respective Abs. The double-staining experiments included in this analysis indicate that the observed effects have been exerted on the same cells. The combination of caCD40 with caTLR4 was superior to each of the two alone in the upregulation of CD25 and to caTLR4 alone in all other flow cytometry analyses, whereas the difference between caCD40 plus caTLR4 to caCD40 alone was evident in all other analyses except for OX40 but did not reach statistical significance of p < 0.001.

IFN-γ is an important proinflammatory cytokine that also plays a key role in the anticancer immune response (35). We examined the effect of caCD40 and caTLR4 mRNA on the level and kinetics of IFN-γ secretion by young and selected antimelanoma TILs (Fig. 6). In sTIL425 (Fig. 6A), caTLR4, which induced the secretion of several hundred picograms per milliliter, exerted a clear synergistic effect with caCD40, increasing IFN-γ secretion from ∼5000 pg/ml, observed for the latter when delivered alone, to close to 15,000 pg/ml within the first 24 h posttransfection. Marked synergy with caCD40 was also exhibited in transfected yTIL14 cells (Fig. 6B).

The massive induction of IFN-γ indeed attests to the potency of these genes. However, in the clinical setting, they may turn into a “double-edged sword,” inducing an uncontrollable cytokine storm regardless of actual encounters with target cells. To follow the kinetics of IFN-γ secretion after mRNA transfection of sTIL425, growth medium of transfectants was collected every 24 h and subjected to IFN-γ ELISA, replenishing the cells with fresh medium. No IFN-γ secretion could be detected later than 24 h posttransfection (data not shown). This result is in agreement with the pattern and kinetics of IFN-γ, TNF-α, and GM-CSF production by caTLR4 (28), demonstrating that spontaneous cytokine secretion resulting from the mere expression of the introduced mRNA completely wanes within the first 24 h of expression.

Having established the immunostimulatory effects exerted by caCD40 alone or with the caTLR4 on peripheral blood CD4 and CD8 T cells and sTILs and yTILs, we went on to assess their actual effects on the antitumor reactivity of TILs. To achieve this, we took advantage of the availability of the autologous 425 melanoma cells (M425), which can serve as targets for sTIL425. sTIL425 cells have been transfected with mRNA encoding caCD40 and caTLR4. Three days posttransfection, when no spontaneous secretion of IFN-γ was evident, we cocultured the transfected TILs with their autologous melanoma (M425), with an HLA-A2–matched melanoma (M624), with HLA-I–mismatched melanomas (M579), or with no target cells. The results show clear target cell–specific enhancement in the production of IFN-γ and TNF-α, which is also accompanied by an elevation in the degranulation marker CD107a (Fig. 7A). No cooperative effect was observed in this experiment. Of note, some enhancement of these cytokines and CD107a could also be observed in response to the allogeneic melanoma cell lines, which could reflect a certain degree of cross-reactivity with M425 Ags.

We proceeded to assess the actual in vitro killing of target melanoma cells by mRNA-transfected TILs. To this end we examined the ability of caCD40 and caTLR4 mRNA, alone or combined, to enhance the ability of yTIL144 to kill autologous M144 melanoma cells using the LDH cytotoxicity assay (Fig. 7B). Indeed, when delivered alone, both caCD40 and caTLR4 mRNA could enhance target cell killing 2 d posttransfection, and this effect was further augmented by their combination. Notably, at 4 d posttransfection, enhancement could still be detected but only for the combination of both mRNAs.

Most protocols explored today for cancer ACT use either T cells naturally recognizing shared or individual tumor Ags, which are derived from the pre-existing lymphocyte pool of the patient (either at the tumor site or in blood) or polyclonal T cells genetically modified to express tumor-specific TCRs or CARs.

A growing number of reports from trials employing both routes reveal an unprecedentedly high rate of clinical response, including complete remission in patients that are refractory to all other treatments (36, 37). T cell exhaustion, which is manifested in the downregulation of effector mechanisms and low persistence of the transferred T cells, is one critical factor limiting the clinical efficacy and the broader use of ACT (38). Patient preconditioning for allowing the successful acceptance of the huge number of transferred T cells and the concurrent removal of opposing regulatory T cells is an integral step of most clinical protocols today. This is a harsh procedure, which often leads to exclusion of patients from treatment or their dropout following its initiation (39). Enhancing in vivo survival and tumoricidal activity of the introduced T cells could be translated into a marked reduction in the number of cells required for an effective treatment and, potentially, obviate the need in preconditioning. Alleviating these functional barriers is mandatory for improving the clinical outcome of all current approaches for ACT. Toward the accomplishment of this goal, we have been developing new genetic adjuvants designed to be expressed in the therapeutic T cells and to enhance functional properties that are associated with better performance in vivo.

To this end we have electroporated human T cells with mRNA encoding caCD40 alone or with caTLR4, examining phenotypic and functional markers known to correlate with clinical efficacy of ACT. We have shown that caCD40 alone could markedly upregulate the T cell activation and costimulatory molecules 4-1BB, OX40, CD28, CD25, and CD127. This upregulation was demonstrated in sTILs, yTILs, and CD8 and CD4 T cells isolated from PBLs, in which, among the latter, the effects recorded for CD8 T cells were generally more pronounced than for CD4 T cells. In most of the experiments, cotransfection with caTLR4 further enhanced the observed effects, displaying cooperativity and, at times, synergy between the gene products.

Whereas the expression of inhibitory molecules such as PD-1 and CTLA-4 negatively correlates with clinical outcome, their upregulation is a normal step in T cell activation and progression of the T cell response. In most of our experiments in TILs and PBL-derived T cells, the level of PD-1 or CTLA-4 remained unchanged or was, at best, only marginally elevated (Fig. 3A). CD62L (L-selectin) is an adhesion molecule that allows naive and central memory T cells entry to secondary lymphoid organs via HEV (40) and is downregulated in activated T cells. Indeed, the introduction of caCD40 repeatedly led to a discernible decrease in cell surface CD62L, providing additional evidence for T cell activation. However, in both CD4 and CD8 T cells, coexpression of caTLR4 reduced this effect. This is indeed an unexpected observation, which may be related to the demonstration by Cui et al. (41) of the dual function of TLR4. These authors showed that two TLR4 ligands, LPS and MPLA, differentially regulated effector and memory CD8 T cell differentiation. Whereas LPS boosted the generation of central memory cells expressing more CD62L, MPLA promoted terminal differentiation characterized by downregulation of CD62L.

T cell–enhancing effects mediated by caCD40 were comparable to those induced by ligation of nCD40 expressed exogenously by soluble CD40L (Fig. 4). Furthermore, the Kb-trimeric construct, which harbors all components of the caCD40 construct except for the CD40 signaling moiety, did not produce any of these effects. These findings argue that the signals transmitted by caCD40 are indeed produced by the intracellular domain of CD40 and that human T cells possess the machinery required for processing these signals. Our inability to detect native CD40 on any of the human T cell preparations we have tested (Fig. 4B and N. Levin, unpublished observations) suggests that CD40 expression by T cells may be more limited to mouse T cells and is not a general phenomenon.

IFN-γ is an essential component of the antitumor T cell response and its secretion by activated T cells correlates with tumor regression (42, 43). The antitumor effects exerted by IFN-γ include the inhibition of tumor cell division, sensitization of tumor cells to apoptosis, induction of antiangiogenic cues, and upregulation of MHC-I and MHC class II on tumor cells, rendering them more vulnerable to T cell attack (44–48). IFN-γ, therefore, serves in the clinical setting employing TILs as a predictor of their tumoricidal activity (49, 50). Indeed, in this work we have shown that the transfection of caCD40 alone enhanced massive IFN-γ secretion, whereas caCD40 cotransfection with caTLR4 synergized with caCD40 in enhancing IFN-γ secretion. In the absence of Ag, this effect has never lasted more than 24 h (data not shown).

Perhaps the most important observation regarding the actual enhancement of the antitumor activity of transfected T cells was the demonstration that caCD40 mRNA robustly boosted the antimelanoma activity of human TILs 3 d posttransfection. This enhancement was manifested in the increase in IFN-γ, TNF-α, and surface expression of the degranulation marker CD107a in antimelanoma TILs in the presence of autologous but not mismatched melanoma (Fig. 7A) and in target cell killing (Fig. 7B). The lack of spontaneous activation and the unresponsiveness to an HLA-I–mismatched tumor at these time points suggest the induction of a prolonged presensitized state. This outcome is manifested in an elevated response to TCR-mediated stimuli, which is evident long after the initial burst of cytokine production had ceased.

This striking improvement of the selective antitumor response holds promise for potential clinical implementation of caCD40, alone or as a component of an adjuvant mixture. Noteworthy, the GCN4-based platform is instrumental for creating additional constitutively active TNFRs, such as 4-1BB, OX-40, CD27, and GITR that could be used in T cell ACT. Work along this direction has already been initiated.

Although the various cells employed throughout this project, including human PBL-derived CD4 and CD8 T cells, yTILs, and sTILs, all clearly responded to caCD40 and its combinations with caTLR4, the actual pattern and magnitude of response differed. In fact, in some cases this was also true for different preparations of cells from the same source, likely reflecting different experimental conditions that may affect the basal activation status of the tested cells.

In a recent publication (11), Foster et al. have demonstrated the regulated expansion and survival of CAR-modified human T cells following the cross-linking of a MyD88/CD40 signaling device by a cell-permeable dimerizing agent. Our design, in contrast, does not require any external reagents and, in addition, induces massive upregulation of the key costimulatory molecules 4-1BB and OX40 while sustaining considerable enhancement of target cell killing at least 4 d postelectroporation.

In summary, in this study, we put forward oligomeric CD40 as an entirely new genetic adjuvant that exerts a broad range of stimulatory effects on human T cells in a constitutive manner, which can be further enhanced by caTLR4. This genetic platform can be efficiently implemented via mRNA electroporation, offering a powerful tool with potentially diverse applications in adoptive cell therapy.

We thank Adi Sharabi-Nov for help with statistical analysis.

The authors have no financial conflicts of interest.