The interaction between the costimulatory molecules CD80/86 and CD28 contributed to CD4+ T lymphocyte activation in flounder (Paralichthys olivaceus).

CD28 and CD80/86 are crucial co-stimulatory molecules for the T cell activation. Previous study illustrated that CD28 and CD80/86 present on T cells and antigen-presenting cells in flounder (Paralichthys olivaceus), respectively. The co-stimulatory molecules were closely associated with cell immunity. In this paper, recombinant protein of flounder CD80/86 (rCD80/86) and phytohemagglutinin (PHA) were added to peripheral blood leukocytes (PBLs) in vitro. Lymphocytes were significantly proliferated with CFSE staining, and the proportion of CD4+ and CD28+ lymphocytes significantly increased. In the meantime, genes related to the CD28-CD80/86 signaling pathway or T cell markers were significantly upregulated (p < 0.05). For further study, the interaction between CD80/86 and CD28 was confirmed. The plasmid of CD28 (pCD28-FLAG and pVN-CD28) or CD80/86 (pVC-CD80/86) was successfully constructed. In addition, pVN-ΔCD28 without the conserved motif "TFPPPF" was constructed. The results showed that bands of pCD28-FLAG bound to rCD80/86 were detected by both anti-FLAG and anti-CD80/86. pVN-CD28 complemented to pVC-CD80/86 showing positive fluorescent signals, and pVN-ΔCD28 failed to combine with pVC-CD80/86. The motif "TFPPPF" in CD28 played a crucial role in this linkage. These results indicate that CD28 and CD80/86 molecules interact with each other, and their binding may modulate T lymphocytes immune response in flounder. This study proved the existence of CD28-CD80/86 signaling pathway in flounder.

Costimulatory receptors in the channel catfish: CD28 family members and their ligands.

The CD28-B7 interaction is required to deliver a second signal necessary for T-cell activation. Additional membrane receptors of the CD28 and B7 families are also involved in immune checkpoints that positively or negatively regulate leukocyte activation, in particular T lymphocytes. BTLA is an inhibitory receptor that belongs to a third receptor family. Fish orthologs exist only for some of these genes, and the potential interactions between the corresponding ligands remain mostly unclear. In this work, we focused on the channel catfish (Ictalurus punctatus), a long-standing model for fish immunology, to analyze these co-stimulatory and co-inhibitory receptors. We identified one copy of cd28, ctla4, cd80/86, b7h1/dc, b7h3, b7h4, b7h5, two btla, and four b7h7 genes. Catfish CD28 contains the highly conserved mammalian cytoplasmic motif for PI3K and GRB2 recruitment, however this motif is absent in cyprinids. Fish CTLA4 share a C-terminal putative GRB2-binding site but lacks the mammalian PI3K/GRB2-binding motif. While critical V-domain residues for human CD80 or CD86 binding to CD28/CTLA4 show low conservation in fish CD80/86, C-domain residues are highly conserved, underscoring their significance. Catfish B7H1/DC had a long intracytoplasmic domain with a P-loop-NTPase domain that is absent in mammalian sequences, while the lack of NLS motif in fish B7H4 suggests this protein may not regulate cell growth when expressed intracellularly. Finally, there is a notable expansion of fish B7H7s, which likely play diverse roles in leukocyte regulation. Overall, our work contributes to a better understanding of fish leukocyte co-stimulatory and co-inhibitory receptors.

Disruption of SUV39H1-Mediated H3K9 Methylation Sustains CAR T-cell Function.

Suboptimal functional persistence limits the efficacy of adoptive T-cell therapies. CD28-based chimeric antigen receptors (CAR) impart potent effector function to T cells but with a limited lifespan. We show here that the genetic disruption of SUV39H1, which encodes a histone-3, lysine-9 methyl-transferase, enhances the early expansion, long-term persistence, and overall antitumor efficacy of human CAR T cells in leukemia and prostate cancer models. Persisting SUV39H1-edited CAR T cells demonstrate improved expansion and tumor rejection upon multiple rechallenges. Transcriptional and genome accessibility profiling of repeatedly challenged CAR T cells shows improved expression and accessibility of memory transcription factors in SUV39H1-edited CAR T cells. SUV39H1 editing also reduces expression of inhibitory receptors and limits exhaustion in CAR T cells that have undergone multiple rechallenges. Our findings thus demonstrate the potential of epigenetic programming of CAR T cells to balance their function and persistence for improved adoptive cell therapies. SIGNIFICANCE: T cells engineered with CD28-based CARs possess robust effector function and antigen sensitivity but are hampered by limited persistence, which may result in tumor relapse. We report an epigenetic strategy involving disruption of the SUV39H1-mediated histone-silencing program that promotes the functional persistence of CD28-based CAR T cells. See related article by López-Cobo et al., p. 120. This article is featured in Selected Articles from This Issue, p. 5.

Eribulin promotes proliferation of CD8+ T cells and potentiates T cell-mediated anti-tumor activity against triple-negative breast cancer cells.

PURPOSE: Chemotherapeutic agents exert immunomodulatory effects on triple-negative breast cancer (TNBC) cells and immune cells. Eribulin favorably affects the immunological status of patients with breast cancer. However, the effects of eribulin on the immune cells remain unexplored. The aim of this study was to investigate the effects of eribulin on immune cells. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors and mouse splenocytes were stimulated with anti-CD3 and anti-CD28 antibodies. The effects of eribulin and paclitaxel on cell proliferation and differentiation status were analyzed using flow cytometry. RNA sequencing was performed to assess alterations in gene expression in CD8+ T cells following eribulin and paclitaxel treatment. Using TNBC cell lines (MDA-MB-231, Hs578T, and MDA-MB-157), the anti-tumor activity of CD3/CD28-stimulated T cells combined with eribulin or paclitaxel was evaluated. RESULTS: Eribulin did not affect CD3/CD28-stimulated PBMCs proliferation. However, eribulin significantly decreased the CD4/CD8 ratio in T cells, indicating that eribulin facilitates CD8+ T cell proliferation. Furthermore, eribulin significantly increased the frequency of less differentiated CD45RA+, CCR7+, and TCF1+ subsets of CD8+ T cells. RNA sequencing revealed that eribulin enhanced the expression of gene sets related to cell proliferation and immune responses. Moreover, eribulin augmented the anti-tumor effects of CD3/CD28-stimulated T cells against TNBC cells. These results were not observed in experiments using paclitaxel. CONCLUSIONS: Eribulin promoted CD8+ T cell proliferation, repressed effector T cell differentiation, and harnessed T cell-mediated anti-tumor effects. These mechanisms may be one of the cues that eribulin can improve the immunological status of tumor-bearing hosts.

CAR T-cell Design-dependent Remodeling of the Brain Tumor Immune Microenvironment Modulates Tumor-associated Macrophages and Anti-glioma Activity.

Understanding the intricate dynamics between adoptively transferred immune cells and the brain tumor immune microenvironment (TIME) is crucial for the development of effective T cell-based immunotherapies. In this study, we investigated the influence of the TIME and chimeric antigen receptor (CAR) design on the anti-glioma activity of B7-H3-specific CAR T-cells. Using an immunocompetent glioma model, we evaluated a panel of seven fully murine B7-H3 CARs with variations in transmembrane, costimulatory, and activation domains. We then investigated changes in the TIME following CAR T-cell therapy using high-dimensional flow cytometry and single-cell RNA sequencing. Our results show that five out of six B7-H3 CARs with single costimulatory domains demonstrated robust functionality in vitro. However, these CARs had significantly varied levels of antitumor activity in vivo. To enhance therapeutic effectiveness and persistence, we incorporated 41BB and CD28 costimulation through transgenic expression of 41BBL on CD28-based CAR T-cells. This CAR design was associated with significantly improved anti-glioma efficacy in vitro but did not result in similar improvements in vivo. Analysis of the TIME revealed that CAR T-cell therapy influenced the composition of the TIME, with the recruitment and activation of distinct macrophage and endogenous T-cell subsets crucial for successful antitumor responses. Indeed, complete brain macrophage depletion using a CSF1R inhibitor abrogated CAR T-cell antitumor activity. In sum, our study highlights the critical role of CAR design and its modulation of the TIME in mediating the efficacy of adoptive immunotherapy for high-grade glioma. SIGNIFICANCE: CAR T-cell immunotherapies hold great potential for treating brain cancers; however, they are hindered by a challenging immune environment that dampens their effectiveness. In this study, we show that the CAR design influences the makeup of the immune environment in brain tumors, underscoring the need to target specific immune components to improve CAR T-cell performance, and highlighting the significance of using models with functional immune systems to optimize this therapy.

Temporal patterns of gene expression in response to inoculation with a virulent Anaplasma phagocytophilum strain in sheep.

The aim of this study was to characterize the gene expression of host immune- and cellular responses to a Norwegian virulent strain of Anaplasma phagocytophilum, the cause of tick-borne fever in sheep. Ten sheep were intravenously inoculated with a live virulent strain of A. phagocytophilum. Clinical-, observational-, hematological data as well as bacterial load, flow cytometric cell count data from peripheral blood mononuclear cells and host's gene expression post infection was analysed. The transcriptomic data were assessed for pre-set time points over the course of 22 days following the inoculation. Briefly, all inoculated sheep responded with clinical signs of infection 3 days post inoculation and onwards with maximum bacterial load observed on day 6, consistent with tick-borne fever. On days, 3-8, the innate immune responses and effector processes such as IFN1 signaling pathways and cytokine mediated signaling pathways were observed. Several pathways associated with the adaptive immune responses, namely T-cell activation, humoral immune responses, B-cell activation, and T- and B-cell differentiation dominated on the days of 8, 10 and 14. Flow-cytometric analysis of the PBMCs showed a reduction in CD4+CD25+ cells on day 10 and 14 post-inoculation and a skewed CD4:CD8 ratio indicating a reduced activation and proliferation of CD4-T-cells. The genes of important co-stimulatory molecules such as CD28 and CD40LG, important in T- and B-cell activation and proliferation, did not significantly change or experienced downregulation throughout the study. The absence of upregulation of several co-stimulatory molecules might be one possible explanation for the low activation and proliferation of CD4-T-cells during A. phagocytophilum infection, indicating a suboptimal CD4-T-cell response. The upregulation of T-BET, EOMES and IFN-γ on days 8-14 post inoculation, indicates a favoured CD4 Th1- and CD8-response. The dynamics and interaction between CD4+CD25+ and co-stimulatory molecules such as CD28, CD80, CD40 and CD40LG during infection with A. phagocytophilum in sheep needs further investigation in the future.

Releasing the brake: CTLA-4 loss turbocharges CAR T cells.

Immune checkpoint receptor-induced T cell dysfunction is a major cause of CAR T cell treatment failure. In this issue, Agarwal et al. report that CRISPR/Cas9 deletion of CTLA4, but not PDCD1 or CTLA4 and PDCD1, enhances CD28 signaling, restoring fitness and antitumor function of CAR T cells, including those derived from patients who failed CAR T cell therapy.

CD28/PD1 co-expression: dual impact on CD8+ T cells in peripheral blood and tumor tissue, and its significance in NSCLC patients' survival and ICB response.

BACKGROUND: Immune checkpoint blockade (ICB) has significantly prolonged survival of non-small cell lung cancer (NSCLC) patients, although most patients develop mechanisms of resistance. Recently single-cell RNA-sequencing (scRNA-Seq) revealed a huge T-cell phenotypic and (dys)functional state variability. Accordingly, T-cell exhaustion is recognized as a functional adaptation, with a dynamic progression from a long-lived "pre-exhausted stem-like progenitor" to a "terminally exhausted" state. In this scenario it is crucial to understand the complex interplay between co-stimulatory and inhibitory molecules in CD8+ T-cell functionality. METHODS: To gain a baseline landscape of the composition, functional states, and transcriptomic signatures predictive of prognosis, we analyzed CD8+ T-cell subsets characterized by the presence/absence of PD1 and CD28 from periphery, adjacent non-tumor tissue and tumor site of a cohort of treatment-naïve NSCLC patients, by integrated multiparametric flow cytometry, targeted multi-omic scRNA-seq analyses, and computational pipelines. RESULTS: Despite the increased PD1 levels, an improved PD1+CD28+ T-cell polyfunctionality was observed with the transition from periphery to tumor site, associated with lack of TIGIT, TIM-3 and LAG-3, but not with Ag-experienced-marker CD11a. Differently from CD28+ T cells, the increased PD1 levels in the tumor were associated with reduced functionality in PD1+CD28- T cells. CD11ahigh, although expressed only in a small fraction of this subset, still sustained its functionality. Absence of TIGIT, TIM-3 and CTLA-4, alone or combined, was beneficial to CD28- T cells. Notably, we observed distinct TRM phenotypes in the different districts, with CD28+ T cells more capable of producing TGFß in the periphery, potentially contributing to elevated CD103 levels. In contrast CD28- TRM mainly produced CXCL13 within the tumor. ScRNA-seq revealed 5 different clusters for each of the two subsets, with distinctive transcriptional profiles in the three districts. By interrogating the TCGA dataset of patients with lung adenocarcinoma (LUAD) and metastatic NSCLC treated with atezolizumab, we found signatures of heterogeneous TRM and "pre-exhausted" long-lived effector memory CD8+ T cells associated with improved response to ICB only in the presence of CD28. CONCLUSIONS: Our findings identify signatures able to stratify survival of LUAD patients and predict ICB response in advanced NSCLC. CD28 is advocated as a key determinant in the signatures identified, in both periphery and tumor site, thus likely providing feasible biomarkers of ICB response.

Exploration of the association between the single-nucleotide polymorphism of co-stimulatory system and rheumatoid arthritis.

Introduction: The human leukocyte antigen (HLA) has been linked to the majority of autoimmune diseases (ADs). However, non-HLA genes may be risk factors for ADs. A number of genes encoding proteins involved in regulating T-cell and B-cell function have been identified as rheumatoid arthritis (RA) susceptibility genes. Methods: In this study, we investigated the association between RA and single-nucleotide polymorphisms (SNPs) of co-stimulatory or co-inhibitory molecules in 124 RA cases and 100 healthy controls without immune-related diseases [including tumor necrosis factor superfamily member 4 (TNFSF4), CD28, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and programmed cell death protein 1 (PDCD1)]. Results: The results showed that there were 13 SNPs associated with RA, including rs181758110 of TNFSF4 (CC vs. CT, p = 0.038); rs3181096 of CD28 (TT vs. CC + CT, p = 0.035; CC vs. TT, p = 0.047); rs11571315 (TT vs. CT, p = 0.045), rs733618 (CC vs. TT + CT, p = 0.043), rs4553808 (AA vs. AG vs. GG, p = 0.035), rs11571316 (GG vs. AG vs. AA, p = 0.048; GG vs. AG + AA, p = 0.026; GG vs. AG, p = 0.014), rs16840252 (CC vs. CT vs. TT, p = 0.007; CC vs. CT, p = 0.011), rs5742909 (CC vs. CT vs. TT, p = 0.040), and rs11571319 of CTLA4 (GG vs. AG vs. AA, p < 0.001; GG vs. AG + AA, p = 0.048; AA vs. GG + AG, p = 0.001; GG vs. AA, p = 0.008; GG vs. AG, p ≤ 0.001); and rs10204525 (TT vs. CT + CC, p = 0.024; TT vs. CT, p = 0.021), rs2227982 (AA vs. GG, p = 0.047), rs36084323 (TT vs. CT vs. CC, p = 0.022; TT vs. CT + CC, p = 0.013; CC vs. TT + CT, p = 0.048; TT vs. CC, p = 0.008), and rs5839828 of PDCD1 (DEL vs. DEL/G vs. GG, p = 0.014; DEL vs. DEL/G + GG, p = 0.014; GG vs. DEL + DEL/G, p = 0.025; DEL vs. GG, p = 0.007). Discussion: Consequently, these SNPs may play an important role in immune regulation, and further research into the role of these SNPs of immune regulatory genes in the pathogenesis of RA is required.

c-Kit signaling potentiates CAR T cell efficacy in solid tumors by CD28- and IL-2-independent co-stimulation.

The limited efficacy of chimeric antigen receptor (CAR) T cell therapy for solid tumors necessitates engineering strategies that promote functional persistence in an immunosuppressive environment. Herein, we use c-Kit signaling, a physiological pathway associated with stemness in hematopoietic progenitor cells (T cells lose expression of c-Kit during differentiation). CAR T cells with intracellular expression, but no cell-surface receptor expression, of the c-Kit D816V mutation (KITv) have upregulated STAT phosphorylation, antigen activation-dependent proliferation and CD28- and interleukin-2-independent and interferon-γ-mediated co-stimulation, augmenting the cytotoxicity of first-generation CAR T cells. This translates to enhanced survival, including in transforming growth factor-ß-rich and low-antigen-expressing solid tumor models. KITv CAR T cells have equivalent or better in vivo efficacy than second-generation CAR T cells and are susceptible to tyrosine kinase inhibitors (safety switch). When combined with CD28 co-stimulation, KITv co-stimulation functions as a third signal, enhancing efficacy and providing a potent approach to treat solid tumors.

Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cells.

IL-17-producing antigen-specific human T cells elicit potent antitumor activity in mice. Yet, refinement of this approach is needed to position it for clinical use. While activation signal strength regulates IL-17 production by CD4+ T cells, the degree to which T cell antigen receptor (TCR) and costimulation signal strength influences Th17 immunity remains unknown. We discovered that decreasing TCR/costimulation signal strength by incremental reduction of αCD3/costimulation beads progressively altered Th17 phenotype. Moreover, Th17 cells stimulated with αCD3/inducible costimulator (ICOS) beads produced more IL-17A, IFNγ, IL-2, and IL-22 than those stimulated with αCD3/CD28 beads. Compared with Th17 cells stimulated with the standard, strong signal strength (three beads per T cell), Th17 cells propagated with 30-fold fewer αCD3/ICOS beads were less reliant on glucose and favored the central carbon pathway for bioenergetics, marked by abundant intracellular phosphoenolpyruvate (PEP). Importantly, Th17 cells stimulated with weak αCD3/ICOS beads and redirected with a chimeric antigen receptor that recognizes mesothelin were more effective at clearing human mesothelioma. Less effective CAR Th17 cells generated with high αCD3/ICOS beads were rescued by overexpressing phosphoenolpyruvate carboxykinase 1 (PCK1), a PEP regulator. Thus, Th17 therapy can be improved by using fewer activation beads during manufacturing, a finding that is cost effective and directly translatable to patients.

Investigating combination benefit of PD1 and LAG3 co-blockade using an engineered cellular bioassay.

LAG3 and PD1 are both immunomodulatory receptor that act by inhibiting activation of T cells, producing a more immunosuppressive environment. Even with the recent clinical success of PD1 and LAG3 co-blockade, signal transduction downstream of LAG3 remains largely unknown. We have leveraged an engineered Jurkat (T cell) and Raji (APC) co-culture system to assess simultaneous blockade of PD1 and LAG3 pathways using antibodies. RNA-Seq analysis of cell pellets individually treated with LAG3 or PD1 antibodies revealed modest immune activation however, 5-fold more genes were upregulated upon combination treatment. There were increases in costimulatory genes like CD28, CD5, CD6 as well as intracellular signaling molecules like LCP2 and ITK. Given the role of ERK in immune activation of T cells, pERK levels of Jurkat cells in the assay were evaluated. A very modest activation of pERK was observed with anti-LAG3 compared to anti-PD1 but a combination treatment resulted in prolonged ERK phosphorylation. Treatment of Jurkat cells with a commercial phosphatase inhibitor NSC87877 which can impact many phosphatases resulted in immune activation, measured by increased IL2 levels, only in the presence of LAG3. When NSC87877 was combined with the PD1 antibody, it could phenocopy combination benefit of PD1 and LAG3 blocking antibodies. CD28 has a recognized role in PD1 signaling but the impact on LAG3 signaling remains unknown. CD28 knockout in Jurkat cells affected overall IL-2 response of both LAG3 and PD1 antibody treatment but still retained combination benefit. Taken together this reductionist system highlights differences in downstream effects of LAG3 and PD1 blockade and we believe that the assay may have further utility to dissect convergence of both signaling pathways and augment studies in primary cells.

Generation of reporter mice for detecting the transcriptional activity of nuclear factor of activated T cells.

Nuclear factor of activated T cells (NFAT) is a transcription factor essential for immunological and other biological responses. To develop analyzing system for NFAT activity in vitro and in vivo, we generated reporter mouse lines introduced with NFAT-driven enhanced green fluorescent protein (EGFP) expressing gene construct. Six tandem repeats of -286 to -265 of the human IL2 gene to which NFAT binds in association with its co-transcription factor, activator protein (AP)-1, was conjunct with thymidine kinase minimum promoter and following EGFP coding sequence. Upon introduction of the resulting reporter cassette into C57BL/6 fertilized eggs, the transgenic mice were obtained. Among 7 transgene-positive mice in 110 mice bone, 2 mice showed the designated reporter mouse character. Thus, the EGFP fluorescence of CD4+ and CD8+ T cells in these mice was enhanced by stimulation through CD3 and CD28. Each of phorbol 12-myristate 13-acetate (PMA) and ionomycin (IOM) stimulation weakly but their combined stimulation strongly enhanced EGFP expression. The stimulation-induced EGFP upregulation was also observed following T cell subset differentiation in a different manner. The EGFP induction by PMA + IOM stimulation was more potent than that by CD3/CD28 stimulation in helper T (Th)1, Th2, Th9, and regulatory T cells, while both stimulation conditions displayed the equivalent EGFP induction in Th17 cells. Our NFAT reporter mouse lines are useful for analyzing stimulation-induced transcriptional activation mediated by NFAT in cooperation with AP-1 in T cells.

CD28-signaling can be partially compensated in CD28-knockout mice but is essential for virus elimination in a murine model of multiple sclerosis.

The intracerebral infection of mice with Theiler's murine encephalomyelitis virus (TMEV) represents a well-established animal model for multiple sclerosis (MS). Because CD28 is the main co-stimulatory molecule for the activation of T cells, we wanted to investigate its impact on the course of the virus infection as well as on a potential development of autoimmunity as seen in susceptible mouse strains for TMEV. In the present study, 5 weeks old mice on a C57BL/6 background with conventional or tamoxifen-induced, conditional CD28-knockout were infected intracerebrally with TMEV-BeAn. In the acute phase at 14 days post TMEV-infection (dpi), both CD28-knockout strains showed virus spread within the central nervous system (CNS) as an uncommon finding in C57BL/6 mice, accompanied by histopathological changes such as reduced microglial activation. In addition, the conditional, tamoxifen-induced CD28-knockout was associated with acute clinical deterioration and weight loss, which limited the observation period for this mouse strain to 14 dpi. In the chronic phase (42 and 147 dpi) of TMEV-infection, surprisingly only 33% of conventional CD28-knockout mice showed chronic TMEV-infection with loss of motor function concomitant with increased spinal cord inflammation, characterized by T- and B cell infiltration, microglial activation and astrogliosis at 33-42 dpi. Therefore, the clinical outcome largely depends on the time point of the CD28-knockout during development of the immune system. Whereas a fatal clinical outcome can already be observed in the early phase during TMEV-infection for conditional, tamoxifen-induced CD28-knockout mice, only one third of conventional CD28-knockout mice develop clinical symptoms later, accompanied by ongoing inflammation and an inability to clear the virus. However, the development of autoimmunity could not be observed in this C57BL/6 TMEV model irrespective of the time point of CD28 deletion.

An immune checkpoint-based signature predicts prognosis and chemotherapy response for patients with small cell lung cancer.

BACKGROUND: Therapeutic options for small cell lung cancer (SCLC), a particularly lethal malignancy, remain limited. Members of the B7-CD28 family are compelling targets for immune checkpoint blockade strategies, which involve activating, inhibiting, and fine-tuning the T cell immune response. However, their clinical features and significance have not been explored comprehensively. METHOD: We enrolled 228 patients with an initial diagnosis of SCLC, including 77 cases from Cbioportal and a validation cohort of 151 cases with qPCR data. Kaplan-Meier analysis and LASSO Cox model were used to identify a signature based on the B7-CD28 family, which was applied for accurate prediction of chemotherapy benefit and prognosis for SCLC patients. In addition, we applied bioinformatics analysis to explore potential signature-related molecular mechanisms and the immune landscape. RESULTS: The mutation profiles of healthy tissues and SCLC tissues were distinct. A signature consisting of seven genes (CD86, ICOSLG, CD276, CD28, CTLA-4, PDCD1, and TMIGD2) was identified and applied to group patients based on risk level (high-risk and low-risk), producing two groups for which survival outcomes differed significantly (HR = 3.81, 95% CI: 2.16-6.74, P < 0.001). The immune checkpoint-based signature accurately predicted patient outcomes for the selected training and validation sets. Notably, low-risk patients were more likely to benefit from chemotherapy and showed greater immune activation. Additionally, time-dependent ROC curves and C-index analysis confirmed that the immune checkpoint-based signature has excellent predictive power for prognosis and chemotherapy benefit compared to clinically recognized parameters. Finally, multivariate analysis confirmed the identified signature as an independent risk factor for prognosis and chemotherapeutic response. CONCLUSION: We systematically obtained a comprehensive molecular profile for B7-CD28 family members in SCLC patients, from which we produced a reliable and robust prognostic immune checkpoint-based signature with the potential to improve prognostic stratification and therapy strategies for SCLC patients.

Inclusion of 4-1BB Costimulation Enhances Selectivity and Functionality of IL13Rα2-Targeted Chimeric Antigen Receptor T Cells.

Chimeric antigen receptor (CAR) T cell immunotherapy is emerging as a powerful strategy for cancer therapy; however, an important safety consideration is the potential for off-tumor recognition of normal tissue. This is particularly important as ligand-based CARs are optimized for clinical translation. Our group has developed and clinically translated an IL13(E12Y) ligand-based CAR targeting the cancer antigen IL13Rα2 for treatment of glioblastoma (GBM). There remains limited understanding of how IL13-ligand CAR design impacts the activity and selectivity for the intended tumor-associated target IL13Rα2 versus the more ubiquitous unintended target IL13Rα1. In this study, we functionally compared IL13(E12Y)-CARs incorporating different intracellular signaling domains, including first-generation CD3ζ-containing CARs (IL13ζ), second-generation 4-1BB (CD137)-containing or CD28-containing CARs (IL13-BBζ or IL13-28ζ), and third-generation CARs containing both 4-1BB and CD28 (IL13-28BBζ). In vitro coculture assays at high tumor burden establish that second-generation IL13-BBζ or IL13-28ζ outperform first-generation IL13ζ and third-generation IL13-28BBζ CAR designs, with IL13-BBζ providing superior CAR proliferation and in vivo antitumor potency in human xenograft mouse models. IL13-28ζ displayed a lower threshold for antigen recognition, resulting in higher off-target IL13Rα1 reactivity both in vitro and in vivo. Syngeneic mouse models of GBM also demonstrate safety and antitumor potency of murine IL13-BBζ CAR T cells delivered systemically after lymphodepletion. These findings support the use of IL13-BBζ CARs for greater selective recognition of IL13Rα2 over IL13Rα1, higher proliferative potential, and superior antitumor responsiveness. This study exemplifies the potential of modulating factors outside the antigen targeting domain of a CAR to improve selective tumor recognition. Significance: This study reveals how modulating CAR design outside the antigen targeting domain improves selective tumor recognition. Specifically, this work shows improved specificity, persistence, and efficacy of 4-1BB-based IL13-ligand CARs. Human clinical trials evaluating IL13-41BB-CAR T cells are ongoing, supporting the clinical significance of these findings.

Human VH-based chimeric antigen receptor T cells targeting glypican 3 eliminate tumors in preclinical models of HCC.

BACKGROUND AND AIMS: Efficacy of chimeric antigen receptor (CAR) T cells for treating solid tumors, including HCC, remains a challenge. Nanobodies are emerging building blocks of CAR T cells due to their small size and high expression. Membrane proximal sites have been shown as attractive epitopes of CAR T cells. However, current CAR formats are not tailored toward nanobodies or targeting membrane distal epitopes. APPROACH AND RESULTS: Using hYP7 Fv (membrane proximal) and HN3 VH nanobody (membrane distal) as GPC3 targeting elements, we sought to determine how hinges and transmembrane portions of varying structures and sizes affect CAR T-cell function. We generated multiple permutations of CAR T cells containing CD8, CD28, IgG4, and Fc domains. We show that engineered HN3 CAR T cells can be improved by 2 independent, synergistic changes in the hinge and transmembrane domains. The T cells expressing the HN3 CAR which contains the hinge region of IgG4 and the CD28 transmembrane domain (HN3-IgG4H-CD28TM) exhibited high cytotoxic activity and caused complete HCC tumor eradication in immunodeficient mice. HN3-IgG4H-CD28TM CAR T cells were enriched for cytotoxic-memory CD8+ T cells and NFAT signals, and reduced ß catenin levels in HCC cells. CONCLUSION: Our findings indicate that altering the hinge and transmembrane domains of a nanobody-based CAR targeting a distal GPC3 epitope, in contrast to a membrane proximal epitope, lead to robust T-cell signaling and induce swift and durable eradication of HCC tumors.

The signaling and the metabolic differences of various CAR T cell designs.

Chimeric antigen receptor (CAR) T cell therapy is introduced as an effective, rapidly evolving therapeutic to treat cancer, especially cancers derived from hematological cells, such as B cells. CAR T cell gene constructs combine a tumor-targeting device coupled to the T cell receptor (TCR) zeta chain domain with different signaling domains such as domains derived from CD28 or 4-1BB (CD137). The incorporation of each specific co-stimulatory domain targets the immunometabolic pathways of CAR T cells as well as other signaling pathways. Defining the immunometabolic and signaling pathways by which CAR T cells become and remain active, survive, and eliminate their targets may represent a huge step forward in this relatively young research field as the CAR gene can be tailored to gain optimal function also for solid tumors with elaborate immunosuppression and protective stroma. There is a close relationship between different signaling domains applied in CAR T cells, and difficult to evaluate the benefit from different tested CAR gene constructs. In this review, we attempt to collect the latest findings regarding the CAR T cell signaling pathways that affect immunometabolic pathways.

In silico engineering a CD80 variant with increased affinity to CTLA-4 and decreased affinity to CD28 for optimized cancer immunotherapy.

CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.

Structural dynamics of the N-terminal SH2 domain of PI3K in its free and CD28-bound states.

Protein conformational changes with fluctuations are fundamental aspects of protein-protein interactions (PPIs); understanding these motions is required for the rational design of PPI-regulating compounds. Src homology 2 (SH2) domains are commonly found in adapter proteins involved in signal transduction and specifically bind to consensus motifs of proteins containing phosphorylated tyrosine (pY). Here, we analysed the interaction between the N-terminal SH2 domain (nSH2) of the regulatory subunit in phosphoinositide 3-kinase (PI3K) and the cytoplasmic region of the T-cell co-receptor, CD28, using NMR and molecular dynamics (MD) simulations. First, we assigned the backbone signals of nSH2 on 1 H-15 N heteronuclear single quantum coherence spectra in the absence or presence of the CD28 phosphopeptide, SDpYMNMTPRRPG. Chemical shift perturbation experiments revealed allosteric changes at the BC loop and the C-terminal region of nSH2 upon CD28 binding. NMR relaxation experiments showed a conformational exchange associated with CD28 binding in these regions. The conformational stabilisation of the C-terminal region correlated with the regulation of PI3K catalytic function. Further, using 19 F- and 31 P-labelled CD28 phosphopeptide, we analysed the structural dynamics of CD28 and demonstrated that the aromatic ring of the pY residue fluctuated between multiple conformations upon nSH2 binding. Our MD simulations largely explained the NMR results and the structural dynamics of nSH2 and CD28 in both bound and unbound states. Notably, in addition to its major conformation, we detected a minor conformation of nSH2 in the CD28 bound state that may explain the allosteric conformational change in the BC loop.