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Tumor evasion has recently been associated with a novel member of the B7 family, HERV-H LTR-associating 2 (HHLA2), which is mostly overexpressed in PDL-1 neg tumors. HHLA2 can either induce a costimulation signal when bound to CD28H or inhibit it by binding to KIR3DL3 on T- and NK cells. Given the broad distribution of CD28H expression on NK cells and its role, we compared two monoclonal antibodies targeting this novel NK-cell engager in this study. We show that targeting CD28H at a specific epitope not only strongly activates Ca 2+ flux but also results in NK-cell activation. CD28H-activated NK cells further display increased cytotoxic activity against hematopoietic cell lines and bypass HHLA2 and HLA-E inhibitory signals. Additionally, scRNA-seq analysis of clear cell renal cancer cells revealed that HHLA2 + clear cell renal cancer cell tumors were infiltrated with CD28H + NK cells, which could be targeted by finely chosen anti-CD28H Abs., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Multiple factors in the design of a chimeric antigen receptor (CAR) influence CAR T-cell activity, with costimulatory signals being a key component. Yet, the impact of costimulatory domains on the downstream signaling and subsequent functionality of CAR-engineered natural killer (NK) cells remains largely unexplored. Here, we evaluated the impact of various costimulatory domains on CAR-NK cell activity, using a CD70-targeting CAR. We found that CD28, a costimulatory molecule not inherently present in mature NK cells, significantly enhanced the antitumor efficacy and long-term cytotoxicity of CAR-NK cells both in vitro and in multiple xenograft models of hematologic and solid tumors. Mechanistically, we showed that CD28 linked to CD3ζ creates a platform that recruits critical kinases, such as lymphocyte-specific protein tyrosine kinase (LCK) and zeta-chain-associated protein kinase 70 (ZAP70), initiating a signaling cascade that enhances CAR-NK cell function. Our study provides insights into how CD28 costimulation enhances CAR-NK cell function and supports its incorporation in NK-based CARs for cancer immunotherapy. Significance: We demonstrated that incorporation of the T-cell-centric costimulatory molecule CD28, which is normally absent in mature natural killer (NK) cells, into the chimeric antigen receptor (CAR) construct recruits key kinases including lymphocyte-specific protein tyrosine kinase and zeta-chain-associated protein kinase 70 and results in enhanced CAR-NK cell persistence and sustained antitumor cytotoxicity., (©2024 American Association for Cancer Research.)

The CD4 T cell, when engineered with a chimeric antigen receptor (CAR) containing specific intracellular domains, has been transformed into a zero-order drug-delivery platform. This introduces the capability of prolonged, disease-specific engineered protein biologics production, at the disease site. Experimental findings demonstrate that CD4 T cells offer a solution when modified with a CAR that includes 4-1BB but excludes CD28 intracellular domain. In this configuration, they achieve ~3X transduction efficiency of CD8 T cells, ~2X expansion rates, generating ~5X more biologic, and exhibit minimal cytolytic activity. Cumulatively, this addresses two main hurdles in the translation of cell-based drug delivery: scaling the production of engineered T cell ex vivo and generating sufficient biologics in vivo. When programmed to induce IFNβ upon engaging the target antigen, the CD4 T cells outperforms CD8 T cells, effectively suppressing cancer cell growth in vitro and in vivo. In summary, this platform enables precise targeting of disease sites with engineered protein-based therapeutics while minimizing healthy tissue exposure. Leveraging CD4 T cells' persistence could enhance disease management by reducing drug administration frequency, addressing critical challenges in cell-based therapy., Competing Interests: Competing interests statement:Bhatnagar P., Radhakrishnan H. (SRI International). Primary T-cell Biofactory chassis and delivery of immunostimulatory proteins. PCT Appl. No. PCT/US2023/073035. (Aug 29, 2023). Research reported in this publication was supported in part by the National Institute of Biomedical Imaging and Bioengineering (DP2EB024245: NIH Director’s New Innovator Award Program (https://commonfund.nih.gov/newinnovator); the National Cancer Institute (R21CA236640, R33CA247739, R21CA288900, R61CA281785) of the NIH; and the Defense Advanced Research Projects Agency (DARPA) (D19AP00024: DARPA Young Faculty Award (https://www.darpa.mil/work-with-us/for-universities/young-faculty-award).

CAR T cell therapy for AML remains limited due to the lack of a proper target without on-target off-tumor toxicity. TIM3 is a promising target due to its high expression on AML cells and absence in most normal hematopoietic cells. Previous reports have shown that each CAR component impacts CAR functionality. Here, we optimized TIM-3 targeting CAR T cells for AML therapy. We generated CARs targeting TIM3 with two different non-signaling domains: an IgG2-CH3 spacer with CD28 transmembrane domain (CH3/CD28) and a CD8α spacer with CD8α transmembrane domain (CD8/CD8), and evaluated their characteristics and function. Incorporating the non-signaling CH3/CD28 domain resulted in unstable CAR expression in anti-TIM3 CAR T cells, leading to lower surface CAR expression over time and reduced cytotoxic function compared to anti-TIM3 CARs with the CD8/CD8 domain. Both types of anti-TIM3 CAR T cells transiently exhibited fratricide, which subsided overtime, and both CAR T cells achieved substantial T cell expansion. To further optimize the design, we explored the effects of different costimulatory domains. Compared with CD28 costimulation, 4-1BB and CD27 combined with a CD8/CD8 non-signaling domain showed higher cytokine secretion, superior antitumor activity, and enhanced T-cell persistence after repeated antigen exposure. These findings emphasize the impact of the optimal design of CAR constructs that provide efficient function. In the context of anti-TIM3 CAR T cells, using a CD8α spacer and transmembrane domain with TNFR-based costimulation is a promising CAR design to improve anti-TIM3 CAR T cell function for AML therapy., Competing Interests: Declaration of Competing Interest The authors have nothing to declare., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Expansion of CD4 + CD28 null T-lymphocytes is common in chronic heart failure (CHF) patients. Its ability to produce high levels of proinflammatory cytokines is probably the key role of these cells in CHF. IL-10 is a candidate for limiting CD4 + CD28 null T-lymphocyte responses, whereas tumour necrosis factor (TNF) is the cytokine most closely involved in the loss of CD28 expression. Serum levels of TNF and IL-10 were measured in 65 CHF patients (mean age, 65.2 ± 13.84 years). Patients with an IL-10/TNF ratio ≥1 had significantly lower levels of CD4 + CD28 null T-lymphocytes than those with a ratio <1. In vitro, IL-10 reduced the frequency of proliferative CD4 + CD28 null T-lymphocytes stimulated with anti-CD3. Pre-treatment with IL-10 before anti-CD3 stimulation was required for the cytokine to inhibit TNF production by CD4 + CD28 null T-lymphocytes. In addition to the previously described effect of IL-10 on HLA-DR and ICAM-1 expression, LFA-3 protein and mRNA levels were reduced in the presence of the cytokine in monocytes. IL-10 inhibition on CD4 + CD28 null T-lymphocytes may be mediated by a reduction in HLA class II and LFA-3 expression because blocking interactions with these costimulators has similar effects to those of IL-10 treatment. Moreover, costimulation through CD2/LFA-3 interaction is enough to induce proliferation and cytokine production in CD4 + CD28 null T-lymphocytes., (© 2024 John Wiley & Sons Ltd.)

Introduction: Immunotherapy has revolutionized cancer treatment, and Chimeric Antigen Receptor T cell therapy (CAR-T) is a groundbreaking approach. Traditional second-generation CAR-T therapies have achieved remarkable success in hematological malignancies, but there is still room for improvement, particularly in developing new targeting strategies. To address this limitation, engineering T cells with multi-target universal CARs (UniCARs) based on monomeric streptavidin has emerged as a versatile approach in the field of anti-tumor immunotherapy. However, no studies have been conducted on the importance of the intracellular signaling domains of such CARs and their impact on efficiency and specificity., Method: Here, we developed second-generation and third-generation UniCARs based on an extracellular domain comprising an affinity-enhanced monomeric streptavidin, in addition to CD28 and 4-1BB co-stimulatory intracellular domains. These UniCAR structures rely on a biotinylated intermediary, such as an antibody, for recognizing target antigens. In co-culture assays, we performed a functional comparison between the third-generation UniCAR construct and two second-generation UniCAR variants, each incorporating either the CD28 or 4-1BB as co-stimulatory domain., Results: We observed that components in culture media could inhibit the binding of biotinylated antibodies to monomeric streptavidin-CARs, potentially compromising their efficacy. Furthermore, third-generation UniCAR-T cells showed robust cytolytic activity against cancer cell lines upon exposure to specific biotinylated antibodies like anti-CD19 and anti-CD20, underscoring their capability for multi-targeting. Importantly, when assessing engineered UniCAR-T cell activation upon encountering their target cells, third-generation UniCAR-T cells exhibited significantly enhanced specificity compared to second-generation CAR-T cells., Discussion: First, optimizing culture conditions would be essential before deploying UniCAR-T cells clinically. Moreover, we propose that third-generation UniCAR-T cells are excellent candidates for preclinical research due to their high specificity and multi-target anti-tumor cytotoxicity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gallego-Valle, Pérez-Fernández, Rosales-Magallares, Gil-Manso, Castellá, Gonzalez-Navarro, Correa-Rocha, Juan and Pion.)

Programming cell signaling during T-cell activation represents a simple strategy for improving the potency of therapeutic T-cell products. Stim-R technology (Lyell Immunopharma) is a customizable, degradable synthetic cell biomimetic that emulates physiologic, cell-like presentation of signal molecules to control T-cell activation. A breadth of Stim-R formulations with different anti-CD3/anti-CD28 (αCD3/αCD28) antibody densities and stoichiometries were screened for their effects on multiple metrics of T-cell function. We identified an optimized formulation that produced receptor tyrosine kinase-like orphan receptor 1 (ROR1)-targeted chimeric antigen receptor (CAR) T cells with enhanced persistence and polyfunctionality in vitro, as assessed in repeat-stimulation assays, compared with a benchmark product generated using a conventional T-cell-activating reagent. In transcriptomic analyses, CAR T cells activated with Stim-R technology showed downregulation of exhaustion-associated gene sets and retained a unique subset of stem-like cells with effector-associated gene signatures following repeated exposure to tumor cells. Compared with the benchmark product, CAR T cells activated using the optimized Stim-R technology formulation exhibited higher peak expansion, prolonged persistence, and improved tumor control in a solid tumor xenograft model. Enhancing T-cell products with Stim-R technology during T-cell activation may help improve therapeutic efficacy against solid tumors., (© 2024. The Author(s).)

In recent years, Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past, the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study, we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally, we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications.

Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4 + T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4 + T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4 + T-cell function., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Cytomegalovirus (CMV) infection has a life-long impact on the immune system, particularly on memory T cells. However, the effect of early life CMV infection on the phenotype and functionality of T cells in infants and especially longitudinal changes occurring during childhood have not been explored in detail. The phenotype and functionality of peripheral blood CD8+ and CD4+ T cells from children infected with CMV in early life (< 6 months of age) was analyzed using high-dimensional flow cytometry. Samples from CMV IgG-seropositive (CMV+) children were collected at 6 months and 6 years of age and compared to samples from CMV-seronegative (CMV-) children. Early life CMV infection caused multiple alterations within T cells. These include downregulation of CD28 expression and upregulation of CD57 expression within both CD27+ early and CD27- late effector memory CD8+ and CD4+ T-cells at 6 months of age. Of these changes, only alterations within the highly differentiated late effector memory compartment persisted at the age of 6 years. Early life CMV-infection has a distinct impact on developing CD8+ and CD4+ memory T cell compartments. It appears to induce both temporary as well as longer-lasting alterations, which may affect the functionality of the immune system throughout life., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Cyclosporin A (CSA) and tacrolimus (TAC) suppress T-cell activation and subsequent proliferation by inhibiting calcineurin. Though they have the same target, CSA and TAC have quite different molecular structures, indicating quantitative and/or qualitative differences in their effects., Objective: CD28 is a costimulatory molecule that enhances T-cell activation. It has also been shown to attenuate calcineurin inhibitors. In this study, we compared the CD28-mediated resistance of CD4+ T cells to those calcineurin inhibitors and tried to predict CD28's impact on infectious diseases., Methods: CD4+ T-cell proliferation was induced with anti-CD3 mAb in the presence or absence of anti-CD28 mAb in vitro. CSA or TAC was added at various concentrations, and the half-maximal inhibitory concentration on CD4+ T-cell proliferation was determined. Effects of lipopolysaccharide (LPS) on dendritic cells (DCs) and CD4+ T-cell proliferation were also evaluated in vitro., Results: Anti-CD28 mAb conferred CD4+ T cells with resistance to both CSA and TAC, and CD28's effect on the latter was approximately twice that on the former. LPS induced expression of CD28 ligands CD80/86 on DCs. The addition of LPS to culture containing DCs seemed to make CD4+ T cells slightly resistant to TAC but not to CSA. However, its effect on the former was very weak under our experimental conditions., Conclusions: CD28 attenuated TAC more strongly than CSA. Although LPS did not demonstrate strong enough resistance in our in vitro model, TAC might maintain a better antibacterial immune response than CSA in clinical use.

T-cell therapies based on chimeric antigen receptor (CAR) targeting of a tumor-specific antigen offer hope for patients with relapsed or refractory cancers. CAR hinge and transmembrane regions link antigen recognition domains to intracellular signal transduction domains. Here, we apply biophysical methods to characterize the structure and dynamic properties of the CD28 CAR hinge (CD28H) used in an FDA-approved CD19 CAR for the treatment of B-lineage leukemia/lymphoma. By using nuclear Overhauser effect spectroscopy (NOESY), which detects even transiently occupied structural motifs, we observed otherwise elusive local structural elements amidst overall disorder in CD28H, including a conformational switch from a native β-strand to a 3 10 -helix and polyproline II helix-like structure. These local structural motifs contribute to an overall loosely formed extended geometry that could be captured by NOESY data. All FDA-approved CARs use prolines in the hinge region, which we find in CD28, and previously in CD8α, isomerize to promote structural plasticity and dynamics. These local structural elements may function in recognition and signaling events and constrain the spacing between the transmembrane and antigen recognition domains. Our study thus demonstrates a method for detecting local and transient structure within intrinsically disordered systems and moreover, our CD28H findings may inform future CAR design., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Improving chimeric antigen receptor (CAR)-T cell therapeutic outcomes and expanding its applicability to solid tumors requires further refinement of CAR-T cells. We previously reported that CAR-T cells bearing a herpes virus entry mediator (HVEM)-derived co-stimulatory signal domain (CSSD) (HVEM-CAR-T cells) exhibit superior functions and characteristics. Here, we conducted comparative analyses to evaluate the impact of different CSSDs on CAR-T cell exhaustion. The results indicated that HVEM-CAR-T cells had significantly lower frequencies of exhausted cells and exhibited the highest proliferation rates upon antigenic stimulation. Furthermore, proliferation inhibition by programmed cell death ligand 1 was stronger in CAR-T cells bearing CD28-derived CSSD (CD28-CAR-T cells) whereas it was weaker in HVEM-CAR-T. Additionally, HVEM-CAR-T cells maintained a low exhaustion level even after antigen-dependent proliferation and exhibited potent killing activities, suggesting that HVEM-CAR-T cells might be less prone to early exhaustion. Analysis of CAR localization on the cell surface revealed that CAR formed clusters in CD28-CAR-T cells whereas uniformly distributed in HVEM-CAR-T cells. Analysis of CD3ζ phosphorylation indicated that CAR-dependent tonic signals were strongly sustained in CD28-CAR-T cells whereas they were significantly weaker in HVEM-CAR-T cells. Collectively, these results suggest that the HVEM-derived CSSD is useful for generating CAR-T cells with exhaustion-resistant properties, which could be effective against solid tumors.

This study investigates the effects of Daphnes Cortex and its processed products on the differentiation of Th17/Treg cells in SD rats with type Ⅱ collagen-induced arthritis(CIA).Sixty-four SD rats were randomly divided into the normal group(normal),model group(model),fried Daphne giraldii Nitsche low-dose and high-dose groups(FDGN-L group, FDGN-H group),raw D. giraldii Nitsche low-dose and high-dose groups(RDGN-L group, RDGN-H group),daphnetin group(DAPH group),and tripterygium glycosides group(GTW group).Except for the normal group, the CIA model was immunized on the seventh day after the first immunization, and was gavaged for 28 days after the second immunization.After sampling, the inflammation of articular synovial membrane in CIA rats was observed by hematoxylin-eosin(HE)staining; the levels of transforming growth factor-β(TGF-β),interferon-γ(IFN-γ),interleukin(IL)-2,IL-4,and IL-10 in serum were detected by enzyme-linked immunosorbent assay(ELISA); real-time reverse transcription-PCR(qRT-PCR)and Western blot were used to detect the mRNA and protein expressions of cluster of differentiation(CD) 80(B7-1),CD 86(B7-2),CD28,and cytotoxic T lymphocyte-associated antigen 4(CTLA-4)in the synovial membrane of rats; flow cytometry was used to detect the proportion of Th17 and Treg cells in the synovial membrane of rats.The results showed that compared with the normal group, the joint synovial inflammation of rats in the model group was significantly aggravated, the arthritis index was significantly increased, and the immune organ index was increased(P&lt;0.01).Compared with the model group, each drug administration group could improve the joint inflammation of rats to varying degrees, reduce the arthritis index, inhibit synovial hyperplasia, and reduce the immune organ index; compared with the model group, the serum levels of IL-2 and IFN-γ in each drug administration group were significantly decreased(P&lt;0.01),TGF-β,IL-4,and IL-10 were significantly increased(P&lt;0.01),the mRNA and protein expressions of B7-1 and CTLA-4 in the synovial membrane were significantly increased(P&lt;0.01),and the proportion of Th17 cells and Treg cells in the joint tissue was significantly decreased(P&lt;0.01).In conclusion, Daphnes Cortex inhibits the expression of Th17 cells in CIA rats and promotes the expression of Treg cells by regulating the B7/CD28/CTLA-4 pathway and the balance of Th17/Treg, thereby treating rheumatoid arthritis.

Background Aims: Vγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain., Methods: Specifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium., Results: We found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity., Conclusions: This study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement., Competing Interests: Declaration of Competing Interest HEH is a cofounder with equity in Allovir and Marker Therapeutics; has equity in Fresh Wind Biotechnologies, CoRegen and March Biosciences; has served on advisory boards for Tessa Therapeutics, Marker Therapeutics and Kiadis and has received research funding from Tessa Therapeutics and Kuur Therapeutics. MKB is a cofounder with equity in Allovir, Marker Therapeutics and March Biosciences; has served on advisory boards for Tessa Therapeutics, Marker Therapeutics, Allogene, Walking Fish, Turnstone Biologics, Tscan, Bluebird Bio, Adaptimmune Therapeutics, Adintus, Onkimure, Triumvira Immunologics, Allogene, Bellicum Pharmaceuticals, Memgen, LLC and Coya; has received royalties from Takeda, Bellicum and Maker Therapeutics and has stock with Turnstone, Allogene, Walking Fish, AlloVir and Tscan. MM is a cofounder of March Biosciences and serves on advisory boards for March Biosciences and NKILT Therapeutics., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

CD8 + T cells must persist and function in diverse tumor microenvironments to exert their effects. Thus, understanding common underlying expression programs could better inform the next generation of immunotherapies. We apply a generalizable matrix factorization algorithm that recovers both shared and context-specific expression programs from diverse datasets to a single-cell RNA sequencing (scRNA-seq) compendium of 33,161 CD8 + T cells from 132 patients with seven human cancers. Our meta-single-cell analyses uncover a pan-cancer T cell dysfunction program that predicts clinical non-response to checkpoint blockade in melanoma and highlights CXCR6 as a pan-cancer marker of chronically activated T cells. Cxcr6 is trans-activated by AP-1 and repressed by TCF1. Using mouse models, we show that Cxcr6 deletion in CD8 + T cells increases apoptosis of PD1 + TIM3 + cells, dampens CD28 signaling, and compromises tumor growth control. Our study uncovers a TCF1:CXCR6 axis that counterbalances PD1-mediated suppression of CD8 + cell responses and is essential for effective anti-tumor immunity., Competing Interests: Declaration of interests A.C.A. is a member of the SAB for Tizona Therapeutics, Trishula Therapeutics, Compass Therapeutics, ExcepGen, and Zumutor Biologics, which have interests in cancer immunotherapy. A.C.A. is also a paid consultant for iTeos Therapeutics and Larkspur Biosciences. A.C.A.’s interests were reviewed and managed by the Brigham and Women’s Hospital and Partners Healthcare in accordance with their conflict-of-interest policies. O.R.-R. is an employee of Genentech. A.R. is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas, and was an SAB member of Thermo Fisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics, and Asimov. From August 1, 2020, A.R. is an employee of Genentech and has equity in Roche. When at the Broad, A.R.’s interests were reviewed and managed by the Broad Institute, MIT and HHMI in accordance with their conflict-of-interest policies. A provisional patent application was filed including work in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Surface expression of programmed death-ligand 1 (PD-L1) is mainly observed on antigen presenting cells (APC) such as monocytes or dendritic cells (DCs). Our results showing a high expression of PD-L1 on human naïve CD4 + effector T-cells (TEFFs) and CD4 + regulatory T cells (TREGs) after activation with human DCs, allow us to propose a new role for PD-L1 and its ligands and their potential impact on new signaling pathways. Indeed, expression of PD-L1 on activated CD4 + T cells could allow cis interaction with its ligands such as PD-1 and CD80, thus disrupting interactions with other signaling receptors, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or CD28, which interact with CD80. The ability to compete with hypothetical configuration modifications that may cause a change in affinity/avidity for the trans and cis interactions between these proteins expressed on T cells and/or DCs is discussed. As the study of cancer is strongly influenced by the role of the PD-L1/PD-1 pathway and CD4 + T cells, new interactions, cis and/or trans, between TEFFs, TREGs and tumor cells are also proposed. The presence of PD-L1 on activated CD4 + T cells could influence the quality of the cytotoxic T lymphocyte response during priming to provide other help signals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

This study aimed to develop synthetic Claudin18.2 (CLDN18.2) chimeric antigen receptor (CAR)-T (CAR-T) cells as a treatment for advanced gastric cancer using lentiviral vector genetic engineering technology that targets the CLDN18.2 antigen and simultaneously overcomes the immunosuppressive environment caused by programmed cell death protein 1 (PD-1). Synthetic CAR T cells are a promising approach in cancer immunotherapy but face many challenges in solid tumors. One of the major problems is immunosuppression caused by PD-1. CLDN18.2, a gastric-specific membrane protein, is considered a potential therapeutic target for gastric and other cancers. In our study, CLDN18.2 CAR was a second-generation CAR with inducible T-cell costimulatory (CD278), and CLDN18.2-PD1/CD28 CAR was a third-generation CAR, wherein the synthetic PD1/CD28 chimeric-switch receptor (CSR) was added to the second-generation CAR. In vitro, we detected the secretion levels of different cytokines and the killing ability of CAR-T cells. We found that the secretion of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) secreted by three types of CAR-T cells was increased, and the killing ability against CLDN18.2-positive GC cells was enhanced. In vivo, we established a xenograft GC model and observed the antitumor effects and off-target toxicity of CAR-T cells. These results support that synthetic anti-CLDN18.2 CAR-T cells have antitumor effect and anti-CLDN18.2-PD1/CD28 CAR could provide a promising design strategy to improve the efficacy of CAR-T cells in advanced gastric cancer., (© 2024. The Author(s).)

The limited expansion ability and functional inactivation of T cells within the solid tumor microenvironment are major problems faced during in the application of using tumor-infiltrating lymphocytes (TILs) in vivo. We sought to determine whether TILs carrying a PD-1-CD28-enhanced receptor and CD19 CAR could overcome this limitation and mediate tumor regression. First, anti-tumor effects of PD-1-CD28-enhanced receptor or CD19 CAR modified NY-ESO-1-TCR-T cells to mimic the TILs function (hereafter "PD-1-CD28-TCR-T" or "CD19 CAR-TCR-T" cells, respectively) were tested using the NY-ESO-1 over-expressed tumor cell line in vitro and in a tumor-bearing model. Furthermore, the safety and anti-tumor ability of S-TILs (TILs modified through transduction with a plasmid encoding the PD-1-CD28-T2A-CD19 CAR) were evaluated in vivo. PD-1-CD28-TCR-T cells showed a formidable anti-tumor ability that was not subject to PD-1/PD-L1 signaling in vivo. CD19 CAR-TCR-T cells stimulated with CD19 + B cells exhibited powerful expansion and anti-tumor abilities both in vitro and in vivo. Three patients with refractory solid tumors received S-TILs infusion. No treatment-related mortality was observed, and none of the patients experienced serious side effects. One patient with melanoma achieved a partial response, and two patients with colon or kidney cancer achieved long-term stable disease following S-TILs therapy. To the best of our knowledge, this is the first study describing the safety and efficacy of the adoptive transfer of autologous S-TILs to control disease in patients with advanced cancers, suggesting that S-TILs may be a promising alternative therapy for cancer., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies., (© 2024 Wiley‐VCH GmbH.)

Abstract: Effective T-cell responses not only require the engagement of T-cell receptors (TCRs; "signal 1"), but also the availability of costimulatory signals ("signal 2"). T-cell bispecific antibodies (TCBs) deliver a robust signal 1 by engaging the TCR signaling component CD3ε, while simultaneously binding to tumor antigens. The CD20-TCB glofitamab redirects T cells to CD20-expressing malignant B cells. Although glofitamab exhibits strong single-agent efficacy, adding costimulatory signaling may enhance the depth and durability of T-cell-mediated tumor cell killing. We developed a bispecific CD19-targeted CD28 agonist (CD19-CD28), RG6333, to enhance the efficacy of glofitamab and similar TCBs by delivering signal 2 to tumor-infiltrating T cells. CD19-CD28 distinguishes itself from the superagonistic antibody TGN1412, because its activity requires the simultaneous presence of a TCR signal and CD19 target binding. This is achieved through its engineered format incorporating a mutated Fc region with abolished FcγR and C1q binding, CD28 monovalency, and a moderate CD28 binding affinity. In combination with glofitamab, CD19-CD28 strongly increased T-cell effector functions in ex vivo assays using peripheral blood mononuclear cells and spleen samples derived from patients with lymphoma and enhanced glofitamab-mediated regression of aggressive lymphomas in humanized mice. Notably, the triple combination of glofitamab with CD19-CD28 with the costimulatory 4-1BB agonist, CD19-4-1BBL, offered substantially improved long-term tumor control over glofitamab monotherapy and respective duplet combinations. Our findings highlight CD19-CD28 as a safe and highly efficacious off-the-shelf combination partner for glofitamab, similar TCBs, and other costimulatory agonists. CD19-CD28 is currently in a phase 1 clinical trial in combination with glofitamab. This trial was registered at www.clinicaltrials.gov as #NCT05219513., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Chimeric antigen receptor (CAR)-T cell therapy shows impressive efficacy treating hematologic malignancies but requires further optimization in solid tumors. Here, we developed a TMIGD2 optimized potent/persistent (TOP) CAR that incorporated the costimulatory domain of TMIGD2, a T and NK cell costimulator, and monoclonal antibodies targeting the IgV domain of B7-H3, an immune checkpoint expressed on solid tumors and tumor vasculature. Comparing second- and third-generation B7-H3 CARs containing TMIGD2, CD28, and/or 4-1BB costimulatory domains revealed superior antitumor responses in B7-H3.TMIGD2 and B7-H3.CD28.4-1BB CAR-T cells in vitro. Comparing these two constructs using in vivo orthotopic human cancer models demonstrated that B7-H3.TMIGD2 CAR-T cells had equivalent or superior antitumor activity, survival, expansion, and persistence. Mechanistically, B7-H3.TMIGD2 CAR-T cells maintained mitochondrial metabolism; produced less cytokines; and established fewer exhausted cells, more central memory cells, and a larger CD8/CD4 T cell ratio. These studies demonstrate that the TOP CAR with TMIGD2 costimulation offered distinct benefits from CD28.41BB costimulation and is effective against solid tumors.

Background: For many years it has been postulated that the immune system controls the progress of multiple myeloma (MM). However, the phenotypes of T cells in MM remain to be elucidated. In this study, we compared the phenotypes of T cells, which were obtained from the peripheral blood, in MM patients with those in healthy donors (HD). The expression of CCR7, CD57, CD28, HLA-DR, CD38, CD45RA, and CD45RO were assessed on T cells from MM patients and HDs using multicolor flow cytometry (MFC)., Methods: For this study, 17 newly diagnosed MM patients were selected, and 20 healthy people were selected as a control group. MFC was used to detect the markers on T cells., Results: We detected significant increases in the expression levels of HLA-DR, CD38, and CD57on CD8+ T cells, significant decreases in the expression levels of CD28 and CD45RA on CD8+ T cells, and a decrease of CD4+ effec-tor T cells in MM patients, compared to the HD group., Conclusions: Our study shows that the accumulation of peripheral CD8+CD57+T cells, CD8+CD38high T cells, and CD8+HLA-DR+CD38high T cells is reflective of an ongoing antitumor T cell response and a progressive immune dysfunction in MM. During chemotherapy, the recovery of immune function can be monitored by detecting the proportion of activated molecules of T lymphocytes.

Chimeric antigen receptor T cells have dramatically improved the treatment of hematologic malignancies. T cell antigen receptor (TCR)-based cell therapies are yet to achieve comparable outcomes. Importantly, chimeric antigen receptors not only target selected antigens but also reprogram T cell functions through the co-stimulatory pathways that they engage upon antigen recognition. We show here that a fusion receptor comprising the CD80 ectodomain and the 4-1BB cytoplasmic domain, termed 80BB, acts as both a ligand and a receptor to engage the CD28 and 4-1BB pathways, thereby increasing the antitumor potency of human leukocyte antigen-independent TCR (HIT) receptor- or TCR-engineered T cells and tumor-infiltrating lymphocytes. Furthermore, 80BB serves as a switch receptor that provides agonistic 4-1BB co-stimulation upon its ligation by the inhibitory CTLA4 molecule. By combining multiple co-stimulatory features in a single antigen-agnostic synthetic receptor, 80BB is a promising tool to sustain CD3-dependent T cell responses in a wide range of targeted immunotherapies., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

Accumulating evidence supports the active involvement of vascular inflammation in atherosclerosis pathogenesis. Vascular inflammatory events within atherosclerotic plaques are predominated by innate antigen-presenting cells (APCs), including dendritic cells, macrophages, and adaptive immune cells such as T lymphocytes. The interaction between APCs and T cells is essential for the initiation and progression of vascular inflammation during atherosclerosis formation. B7-CD28 family members that provide either costimulatory or coinhibitory signals to T cells are important mediators of the cross-talk between APCs and T cells. The balance of different functional members of the B7-CD28 family shapes T cell responses during inflammation. Recent studies from both mouse and preclinical models have shown that targeting costimulatory molecules on APCs and T cells may be effective in treating vascular inflammatory diseases, especially atherosclerosis. In this review, we summarize recent advances in understanding how APC and T cells are involved in the pathogenesis of atherosclerosis by focusing on B7-CD28 family members and provide insight into the immunotherapeutic potential of targeting B7-CD28 family members in atherosclerosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Adoptive cellular therapy using chimeric antigen receptors (CARs) has revolutionized our treatment of relapsed B cell malignancies and is currently being integrated into standard therapy. The impact of selecting specific T cell subsets for CAR transduction remains under investigation. Previous studies demonstrated that effector T cells derived from naive, rather than central memory T cells mediate more potent antitumor effects. Here, we investigate a method to skew CAR transduction toward naive T cells without physical cell sorting. Viral-mediated CAR transduction requires ex vivo T cell activation, traditionally achieved using antibody-mediated strategies. CD81 is a T cell costimulatory molecule that when combined with CD3 and CD28 enhances naive T cell activation. We interrogate the effect of CD81 costimulation on resultant CAR transduction. We identify that upon CD81-mediated activation, naive T cells lose their identifying surface phenotype and switch to a memory phenotype. By prelabeling naive T cells and tracking them through T cell activation and CAR transduction, we document that CD81 costimulation enhanced naive T cell activation and resultantly generated a CAR T cell product enriched with naive-derived CAR T cells., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Introduction: Atrial fibrillation (AF) represents the most common cardiac arrhythmia in daily clinical practice and substantially impacts affected patients by elevation of both morbidity and mortality. Previous investigations proved that inflammatory processes are closely linked to this multifactorial pathogenesis-especially autoreactive CD4 + CD28 null T cells received in-depth attention., Purpose: Consequently, a potential pathophysiological pathway of the impact of CD4 + CD28null T lymphocytes on the development and progression AF can be outlined., Conclusion: Considering the available data in the literature, it needs to be assumed that CD4 + CD28 null T lymphocytes are mainly involved in the development of AF and disease progression. Of utmost importance, it can be considered as the result of a T-cell-mediated auto-immune reaction among myocardial tissue. However, mechanisms which recruit CD4 + CD28 null cells in cardiac tissue remain unclear and need further investigation., (© 2021. The Author(s).)

Activation of co-stimulatory pathways in cytotoxic T lymphocytes expressing chimeric antigen receptors (CARs) have proven to boost effector activity, tumor rejection and long-term T cell persistence. When using antigen-specific T cell receptors (TCR) instead of CARs, the lack of co-stimulatory signals hampers robust antitumoral response, hence limiting clinical efficacy. In solid tumors, tumor stroma poses an additional hurdle through hindrance of infiltration and active inhibition. Our project aimed at generating chimeric co-stimulatory switch proteins (CSP) consisting of intracellular co-stimulatory domains (ICD) fused to extracellular protein domains (ECD) for which ligands are expressed in solid tumors. The ECD of CD40L was selected for combination with the ICD from the CD28 protein. With this approach, it was expected to not only provide co-stimulation and strengthen the TCR signaling, but also, through the CD40L ECD, facilitate the activation of tumor-resident antigen-presenting cells (APCs), modulate activation of tumor endothelium and induce TCR-MHC independent apoptotic effect on tumor cells. Since CD28 and CD40L belong to different classes of transmembrane proteins (type I and type II, respectively), creating a chimeric protein presented a structural and functional challenge. We present solutions to this challenge describing different CSP formats that were successfully expressed in human T cells along with an antigen-specific TCR. The level of surface expression of the CSPs depended on their distinct design and the state of T cell activation. In particular, CSPs were upregulated by TCR stimulation and downregulated following interaction with CD40 on target cells. Ligation of the CSP in the context of TCR-stimulation modulated intracellular signaling cascades and led to improved TCR-induced cytokine secretion and cytotoxicity. Moreover, the CD40L ECD exhibited activity as evidenced by effective maturation and activation of B cells and DCs. CD40L:CD28 CSPs are a new type of switch proteins designed to exert dual beneficial antitumor effect by acting directly on the gene-modified T cells and simultaneously on tumor cells and tumor-supporting cells of the TME. The observed effects suggest that they constitute a promising tool to be included in the engineering process of T cells to endow them with complementary features for improved performance in the tumor milieu., Competing Interests: EN declares financial relationship due to patent WO2017/162797. The remaining author(s) declare(s) that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Olguín-Contreras, Mendler, Popowicz, Hu and Noessner.)

Because of its size, anatomical similarities, and now also accessibility to genetic manipulations, pigs are used as animal models for human diseases and immune system development. However, expression and function of CD28, the most important costimulatory receptor expressed by T cells, so far is poorly understood in this species. Using a newly generated mAb (mAb 3D11) with specificity for pig CD28, we detected CD28 on CD8 + and CD4 + αβ T cells. Among γδ T cells, CD28 expression was restricted to a small CD2 + subpopulation of phenotypically naive cells. Functionally, CD28 ligation with mAb 3D11-costimulated porcine T cells, enhanced proliferation and cytokine secretion in vitro. We used a second, likewise newly generated but superagonistic, anti-CD28 mAb (CD28-SA; mAb 4D12) to test the function of CD28 on porcine T cells in a pilot study in vivo. Injection of the CD28-SA into pigs in vivo showed a very similar dose-response relationship as in humans (i.e., 100 µg/kg body weight [BW]) of CD28-SA induced a cytokine release syndrome that was avoided at a dose of 10 µg/kg BW and below. The data further suggest that low-dose (10 µg/kg BW) CD28-SA infusion was sufficient to increase the proportion of Foxp3 + regulatory T cells among CD4 + T cells in vivo. The pig is thus a suitable animal model for testing novel immunotherapeutics. Moreover, data from our pilot study in pigs further suggest that low-dose CD28-SA infusion might allow for selective expansion of CD4 + regulatory T cells in humans., (Copyright © 2021 by The American Association of Immunologists, Inc.)

CD28 is well known as a critical T-cell costimulatory receptor involved in T cell activation by binding to its ligands. In this study, CD28 was cloned, and its expression profiles were characterized in flounder ( Paralichthys olivaceus ); variations of CD28+ cells after being stimulated with different types of antigens and the function of the CD28 costimulatory pathway on T-cell activation were investigated in vitro . fCD28 consists of four exons and three introns, and the full-length cDNA of fCD28 was 675-bp encoded 224 amino acids. The conserved motif ( 121 TFPPPF 126 ) binding to the CD80/86 ligand exists in the Ig-superfamily homology domain. The high expression of fCD28 is in gills, PBLs, head kidney, and spleen. CD28+ cells were co-localized with CD4+ T lymphocytes but not on IgM+ B lymphocyte cells. Moreover, the expression of CD28 was significantly varied in flounder after being stimulated by keyhole limpet hemocyanin (KLH) at both the transcriptional and cellular levels, while no significant differences were observed between lipopolysaccharide (LPS) stimulation and the control group. Notably, treatment of PBLs cultured in vitro with CD28 molecule-specific antibody (anti-CD28 Abs) and PHA produced more cell colonies and stimulated the proliferation of cultured leukocytes compared to PHA stimulation alone and the control group, and a higher level of IL-2 was detected in the culture medium. Meanwhile, anti-CD28 Abs increased the percent of CD28+ cells (10.41 ± 1.35%), CD4+ T lymphocytes (18.32 ± 2.15%), and CD28+/CD4+ double-positive cells (6.24 ± 1.52%). This effect also resulted in significant variations in the genes of cell membrane-bound molecules, cytokines, and related signaling pathways in cultured leukocytes, with significant changes in the genes of interleukin-2 (IL-2) and nuclear factor of activated T cells (NFAT) in the early stages of culture, and the expression of other molecules increased over time. These results proved the localization of the CD28 molecule on T lymphocytes in flounder, and anti-CD28 may act as the B7 ligand involved in T cell activation after antigen stimulation. These data provide a basis for a more in-depth study of the mechanism of the CD28 costimulatory pathway in T cell activation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Xing, Liu, Tang, Sheng, Chi and Zhan.)

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition., (© 2021. The Author(s).)

To avoid graft rejection, the hematopoietic stem cells with matched classical human leukocyte antigen (HLA) alleles are the primary choice for clinical allogeneic transplantation. However, even if the fully HLA-matched hematopoietic stem cells are used for transplantation, some patients still have poor prognosis after hematopoietic stem cell transplantation (HSCT), suggesting that the HLA system was not the only determinant of the outcomes of HSCT. In this study, we investigated whether the single-nucleotide polymorphisms (SNPs) of the co-stimulatory genes within non-HLA regions were related to the outcomes of HSCT. The genomic DNAs of 163 patients who had acute leukemia and received HSCT and their respective donors were collected for analysis. Thirty-four SNPs located in the four co-stimulatory genes including cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD28, tumor necrosis factor ligand superfamily 4 (TNFSF4), and programmed cell death protein 1 (PDCD1) were selected to explore their relationship with the adverse outcomes after transplantation, including mortality, cytomegalovirus infection, graft-versus-host disease, and relapse. Our results revealed that nine SNPs in the CTLA4 gene, five SNPs in the PDCD1 gene, two SNPs in the TNFSF4 gene, and four SNPs in the CD28 gene were significantly associated with the occurrence of adverse outcomes post-HSCT. These SNPs may play important roles in immune response to allografts post-HSCT and can be the targets for developing strategy to identify appropriate donors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Chang, Wang, Lin, Hsu, Wang and Tseng.)

The marketing authorization of tisagenlecleucel, a 2nd generation of CD19-directed CAR T-cells, containing the 4-1 BB co-stimulatory domain, in 2017 in USA and in 2018 in EU, has revolutionized the therapeutic strategy in advanced B-cell acute lymphoblastic leukemia (B-ALL) in children, adolescents and young adults (AYAs) with relapsed or refractory disease. This innovative treatment, based on a "living drug", has shown very impressive short-term responses. However, safety profile and complex logistics require high expertise centers and tight collaborations between addressing and treating centers. Current research is exploring the possibility to move to first line ALL with high-risk features and/or first high-risk relapse. More efficient CAR T-cells products, are still lacking to counteract the escape mechanisms already described. Moreover, to define the bridge-to-CAR time for each patient remains a challenge to obtain optimal disease burden allowing expansion and persistence of CAR T-cells. Also difficult is to identify patients who will benefit from further therapy after infusion, such as allogeneic HSCT or may be immuno-modulatory treatment. Finally, CAR T-cells directed against T-ALL are only in their beginning but require more complex engineering process to avoid T- cell immune-deficiency or fratricide., (Copyright © 2021 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.)

Coronavirus disease 2019 (COVID-19), a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes infectious disease, and manifests in a wide range of symptoms from asymptomatic to severe illness and even death. Severity of infection is related to many risk factors, including aging and an array of underlying conditions, such as diabetes, hypertension, chronic obstructive pulmonary disease (COPD), and cancer. It remains poorly understood how these conditions influence the severity of COVID-19. Expansion of the CD28 null senescent T-cell populations, a common phenomenon in aging and several chronic inflammatory conditions, is associated with higher morbidity and mortality rates in COVID-19. Here, we summarize the potential mechanisms whereby CD28 null cells drive adverse outcomes in disease and predispose patients to devastating COVID-19, and discuss possible treatments for individuals with high counts of CD28 null senescent T-cells.

Co-inhibitory B7-CD28 family member proteins negatively regulate T cell responses and are extensively involved in tumor immune evasion. Blockade of classical CTLA-4 (cytotoxic T lymphocyte-associated antigen-4) and PD-1 (programmed cell death protein-1) checkpoint pathways have become the cornerstone of anti-cancer immunotherapy. New inhibitory checkpoint proteins such as B7-H3, B7-H4, and BTLA (B and T lymphocyte attenuator) are being discovered and investigated for their potential in anti-cancer immunotherapy. In addition, soluble forms of these molecules also exist in sera of healthy individuals and elevated levels are found in chronic infections, autoimmune diseases, and cancers. Soluble forms are generated by proteolytic shedding or alternative splicing. Elevated circulating levels of these inhibitory soluble checkpoint molecules in cancer have been correlated with advance stage, metastatic status, and prognosis which underscore their broader involvement in immune regulation. In addition to their potential as biomarker, understanding their mechanism of production, biological activity, and pathological interactions may also pave the way for their clinical use as a therapeutic target. Here we review these aspects of soluble checkpoint molecules and elucidate on their potential for anti-cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Khan, Arooj and Wang.)

The inflammatory activity of staphylococcal enterotoxin B (SEB) relies on its capacity to trigger polyclonal T-cell activation by binding both T-cell receptor (TCR) and costimulatory receptor CD28 on T cells and MHC class II and B7 molecules on antigen presenting cells (APC). Previous studies highlighted that SEB may bind TCR and CD28 molecules independently of MHC class II, yet the relative contribution of these interactions to the pro-inflammatory function of SEB remained unclear. Here, we show that binding to MHC class II is dispensable for the inflammatory activity of SEB, whereas binding to TCR, CD28 and B7 molecules is pivotal, in both human primary T cells and Jurkat T cell lines. In particular, our finding is that binding of SEB to B7 molecules suffices to trigger both TCR- and CD28-mediated inflammatory signalling. We also provide evidence that, by strengthening the interaction between CD28 and B7, SEB favours the recruitment of the TCR into the immunological synapse, thus inducing lethal inflammatory signalling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kunkl, Amormino, Caristi, Tedeschi, Fiorillo, Levy, Popugailo, Kaempfer and Tuosto.)

Stem cell-like memory T (Tscm) cells are a subset of memory T cells that have characteristics of stem cells. The characteristics of Tscm cells in patients with rheumatoid arthritis (RA) are not well known. The percentage of CD4 + and CD8 + Tscm cells in PBMCs and synovial fluid mononuclear cells was measured. After confirming the stem cell nature of Tscm cells, we examined their pathogenicity in RA patients and healthy controls (HCs) by assessing T cell activation markers and cytokine secretion after stimulation with anti-CD3/CD28 beads and/or IL-6. Finally, RNA transcriptome patterns in Tscm cells from RA patients were compared with those in HCs. In this study, the percentage of CD4 + and CD8 + Tscm cells in total T cells was significantly higher in RA patients than in HCs. Tscm cells self-proliferated and differentiated into memory and effector T cell subsets when stimulated. Compared with Tscm cells from HCs, Tscm cells from RA patients were more easily activated by anti-CD3/CD28 beads augmented by IL-6. Transcriptome analyses revealed that Tscm cells from RA patients showed a pattern distinct from those in HCs; RA-specific transcriptome patterns were not completely resolved in RA patients in complete clinical remission. In conclusion, Tscm cells from RA patients show a transcriptionally distinct pattern and are easily activated to produce inflammatory cytokines when stimulated by TCRs in the presence of IL-6. Tscm cells can be a continuous source of pathogenicity in RA., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via β2-gp1 (β2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4 + and CD8 + cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The adaptor protein TNFR-associated factor 3 (TRAF3) is required for in vivo T cell effector functions and for normal TCR/CD28 signaling. TRAF3-mediated enhancement of TCR function requires engagement of both CD3 and CD28, but the molecular mechanisms underlying how TRAF3 interacts with and impacts TCR/CD28-mediated complexes to enhance their signaling remains an important knowledge gap. We investigated how TRAF3 is recruited to, and regulates, CD28 as a TCR costimulator. Direct association with known signaling motifs in CD28 was dispensable for TRAF3 recruitment; rather, TRAF3 associated with the CD28-interacting protein linker of activated T cells (LAT) in human and mouse T cells. TRAF3-LAT association required the TRAF3 TRAF-C domain and a newly identified TRAF2/3 binding motif in LAT. TRAF3 inhibited function of the LAT-associated negative regulatory protein Dok1, which is phosphorylated at an inhibitory tyrosine residue by the tyrosine kinase breast tumor kinase (Brk/PTK6). TRAF3 regulated Brk activation in T cells, limiting the association of protein tyrosine phosphatase 1B (PTP1B) with the LAT complex. In TRAF3-deficient cells, LAT complex-associated PTP1B was associated with dephosphorylation of Brk at an activating tyrosine residue, potentially reducing its ability to inhibit Dok1. Consistent with these findings, inhibiting PTP1B activity in TRAF3-deficient T cells rescued basal and TCR/CD28-mediated activation of Src family kinases. These results reveal a new mechanism for promotion of TCR/CD28-mediated signaling through restraint of negative regulation of LAT by TRAF3, enhancing the understanding of regulation of the TCR complex., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Clinical use of various forms of immunotherapeutic drugs in glioblastoma (GBM), has highlighted severe T cell dysfunction such as exhaustion in GBM patients. However, reversing T cell exhaustion using immune checkpoint inhibitors in GBM clinical trials has not shown significant overall survival benefit. Phenotypically, CD8 + T cells with downregulated CD28 coreceptors, low CD27 expression, increased CD57 expression, and telomere shortening are classified as senescent T cells. These senescent T cells are normally seen as part of aging and also in many forms of solid cancers. Absence of CD28 on T cells leads to several functional irregularities including reduced TCR diversity, incomplete activation of T cells, and defects in Ag-induced proliferation. In the context of GBM, presence and/or function of these CD8 + CD28 - T cells is unknown. In this clinical correlative study, we investigated the effect of aging as well as tumor microenvironment on CD8 + T cell phenotype as an indicator of its function in GBM patients. We systematically analyzed and describe a large population of CD8 + CD28 - T cells in both the blood and tumor-infiltrating lymphocytes of GBM patients. We found that phenotypically these CD8 + CD28 - T cells represent a distinct population compared with exhausted T cells. Comparative transcriptomic and pathway analysis of CD8 + CD28 - T cell populations in GBM patients revealed that tumor microenvironment might be influencing several immune related pathways and thus further exaggerating the age associated immune dysfunction in this patient population., (Copyright © 2021 The Authors.)

Chimeric antigen receptor (CAR) is generated by fusing a cancer-specific antibody's antigen recognition site with costimulatory molecules such as CD28 and CD3ζ. T cells transduced with CAR recognizes cancer-specific antigens and kill cancer cells. The effect of CD19-targeted CAR T cells on B-cell hematologic cancer is surprising and has already been approved in many countries including Japan. More targets for several kinds of cancers are being searched now. We have also shown that CAR T cells specific for activated integrin β7 are highly effective for multiple myeloma in pre-clinical tests.

T cell-based adoptive cell transfer therapy is now clinically used to fight cancer with CD19-targeting chimeric antigen receptor T cells. The use of other T cell-based immunotherapies relying on antigen-specific T cells, genetically modified or not, is expanding in various neoplastic diseases. T cell manufacturing has evolved through sophisticated processes to produce T cells with improved therapeutic potential. Clinical-grade manufacturing processes associated with these therapies must meet pharmaceutical requirements and therefore be standardized. Here, we focus on the use of cytokines to expand minimally differentiated T cells, as well as their standardization and harmonization in research and clinical settings., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lymphocyte cytosolic protein 2 (LCP2) is one of the SLP-76 family of adapters, which are critical intermediates in signal cascades downstream of several receptors. LCP2 regulates immunoreceptor signaling (such as T-cell receptors) and is also required for integrin signaling in neutrophils and platelets. However, the role of LCP2 in the tumor microenvironment is still unknown. In this study, we found a significant increase of mRNA and protein expression of LCP2 in metastatic skin cutaneous melanoma compared to normal skin. The upregulation of LCP2 was associated with good overall survival of patients with metastatic skin cutaneous melanoma, who received pharmacotherapy and radiation. GSEA signaling pathways analysis showed that LCP2 was involved in multiple pathways of immune response and correlation analysis revealed LCP2 was positively correlated with molecules in TCR signaling and 11 immune checkpoints, while LCP2 negatively correlated with 2 immune checkpoints in the metastatic skin cutaneous melanoma. According to the different expressions of LCP2, high LCP2 expression was positively correlated with more tumor-infiltrating CD8 + T cells. Furthermore, Kaplan-Meier plot indicated that LCP2 acted as a prognostic biomarker for progression-free survival of patients with metastatic skin cutaneous melanoma receiving anti-PD1 immunotherapy. In conclusion, our results integrated both the expression and function of LCP2 in melanoma using multiple tools, shedding light on the potential role of LCP2 in melanoma, and suggesting LCP2 serves as a prognostic biomarker and therapeutic target in anti-tumor immunity.

Following infusion of the anti-CD28 superagonist monoclonal antibody TGN1412, three of six previously healthy, young male recipients developed gastrointestinal irritability associated with increased expression of 'gut-homing' integrin β7 on peripheral blood αβT cells. This subset of patients with intestinal symptoms also displayed a striking and persistent expansion of putative Vδ2 + γδT cells in the circulation which declined over a 2-year period following drug infusion, concordant with subsiding gut symptoms. These data demonstrate that TGN1412-induced gastrointestinal symptoms were associated with dysregulation of the 'gut-homing' pool of blood αβ and γδT cells, induced directly by the antibody and/or arising from the subsequent cytokine storm.

CD28 expression is generally considered to be T lymphocyte specific. We have previously shown CD28 mRNA expression in M-CSF-dependent anti-inflammatory monocyte-derived macrophages (M-MØ), and now demonstrate that CD28 cell surface expression is higher in M-MØ than in GM-CSF-dependent macrophages, and that macrophage CD28 expression is regulated by MAFB and activin A. In vivo, CD28 was found in tumor-associated macrophages and, to a lower extent, in pro-inflammatory synovial fluid macrophages from rheumatoid arthritis patients. Analysis of mouse macrophages confirmed Cd28 expression in bone-marrow derived M-MØ. Indeed, anti-CD28 antibodies triggered ERK1/2 phosphorylation in mouse M-MØ. At the functional level, Cd28KO M-MØ exhibited a significantly higher capacity to activate the OVA-specific proliferation of OT-II CD4 + T cells than WT M-MØ, as well as enhanced LPS-induced IL-6 production. Besides, the Cd28KO M-MØ transcriptome was significantly different from WT M-MØ regarding the expression IFN response, inflammatory response, and TGF-β signaling related gene sets. Therefore, defective CD28 expression in mouse macrophages associates to changes in gene expression profile, what might contribute to the altered functionality displayed by Cd28KO M-MØ. Thus, CD28 expression appears as a hallmark of anti-inflammatory macrophages and might be a target for immunotherapy., (© 2020 Wiley-VCH GmbH.)

Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN-γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin-and-lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4 + T cells whereas MAL expression is diminished on central memory- and almost lost on effector memory T cells. MAL - T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL + T cells are naïve and MAL - T cells memory subtypes. Further, resting MAL - T cells harbor a larger pool of Ser59- and Tyr394- double phosphorylated lymphocyte-specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation-induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

TGN1412, a superagonist monoclonal antibody targeting CD28, caused cytokine storm in six healthy volunteers in a first-in-man study in 2006. Despite clinical improvement and termination of the cytokine release syndrome within days, anemia persisted in all patients with hemoglobin reaching baseline levels as much as 6 months later. Granulocytic dysplasia continued for 20 days in association with increased expression of CD69 and IL-4, but reduced IL-10; with resolution, this profile reversed to higher IL-10 expression and counter-balanced circannual cycling of IL-4 and IL-10 thereafter over 7 months. Along with immune cell subset and cytokine correlates monitored over 2 years, these observations offer unique insights into the expected changes in myelopoiesis and natural resolution in otherwise healthy young individuals in response to acute inflammation and cytokine storm in the absence of concomitant infection or comorbidity.

Human Cytomegalovirus (CMV) infection is associated with atherosclerosis, higher cardiovascular disease (CVD) risk, and an increase in memory T-cells (T mem ). T-cells have also been implicated in CVD, independently of CMV infection. To better understand the CMV-associated CVD risk, we examined the association between CMV (IgG) serostatus and central aortic (carotid-to-femoral) pulse wave velocity (cfPWV), an early, independent predictor of CVD. We also investigated if such an association might be reflected by the distribution of T mem and/or other T-cell subsets. Methods: Healthy older volunteers (60-93 years) underwent routine clinical and laboratory evaluation, including assessment of cfPWV in eligible participants. Flow-cytometry was used to assess proportions of memory T-cells, CD28 null T-cells, and CMV-specific T-cells. The following associations were examined; CMV serostatus/cfPWV, CMV serostatus/proportion of T mem , proportion of T mem /cfPWV, CD28 null T-cells/cfPWV, and CMV-specific T-cells/cfPWV. Linear regression models were used to adjust for age, sex, socioeconomic status, smoking, waist-to-hip ratio, cholesterol, and blood pressure as required. Results: Statistically significant positive associations were found (P-values for the fully adjusted models are given); CMV serostatus/cfPWV in men (P ≤ 0.01) but not in women, CMV serostatus/proportions of CD4 T mem in men (P ≤ 0.05) but not in women; proportions of CD4 T mem /cfPWV among CMV seropositive (CMV+) people (P ≤ 0.05) but not CMV seronegative (CMV-) people. Conclusion: CMV infection increases the CVD risk of older men by increasing cfPWV. This may be mediated in part by increased proportions of CD4 T mem , higher numbers of which are found in CMV+ older people and more so among men than women. Given the high prevalence of CMV worldwide, our findings point to a significant global health issue. Novel strategies to mitigate the increased CVD risk associated with CMV may be required., Competing Interests: Competing Interests: Frances Kirkham, Aalia Bano, Amanda M. Simanek, Alejandra Pera, Bernhard Reus, Stefano Caserta, Helen E Smith, Kevin A Davies, Chakravarthi Rajkumar, have no disclosures to make/declare no conflict of interest. Florian Kern is co-inventor and co-owner of a patent describing the use of protein-spanning, overlapping peptide pools for detecting antigen-specific T-cells as applied in this study (WO2001063286A2)., (© The author(s).)