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Background: Chimeric antigen receptor (CAR)-T therapy has demonstrated sustained clinical remission in numerous hematologic malignancies and has expanded to encompass solid tumors and autoimmune diseases. While progress is being made in establishing optimal culture conditions for CAR-T cells, the identification of the most effective cytokine for promoting their persistence in vitro remains elusive., Methods: Here, we employed scRNA-seq (single-cell RNA sequencing) analysis to investigate the potential alterations in biological processes within CAR-T cells following exposure to cytokines (IL-2, IL-12, and IL-21) and antigens. Transcriptomic changes in diverse CAR-T groups were compared following various treatments, with a focus on epigenetic modifications, metabolic shifts, cellular senescence, and exhaustion., Results: Our study reveals that CAR-T cells treated with antigen, IL-2, and IL-12 exhibit signs of exhaustion and senescence, whereas those treated with IL-21 do not display these characteristics. The activities of glycolysis and epigenetic changes were significantly increased by treatments with antigens, IL-2, and IL-12, while IL-21 treatment maintained the oxidative phosphorylation (OXPHOS) of CAR-T cells., Conclusions: Our findings suggest that IL-21 may play a role in preventing senescence and could be utilized in combination with other strategies, such as IL-2 and IL-12, for CAR-T culture., 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 Elsevier Ltd. All rights reserved.)

Herpes zoster (HZ), or shingles, is caused by reactivation of the varicella-zoster virus (VZV), which remains latent in the sensory ganglia until immunity wanes with age. The representative HZ vaccine, Shingrix is efficacious but causes side effects due to vaccine adjuvants. Therefore, the development of highly efficacious vaccines with minimal side effects is required. We developed chimeric adenovirus vector (ChimAd)-based HZ vaccine candidates encoding the VZV glycoprotein E (gE). These candidates include ChimAd-tPAgE, in which the signal peptide is replaced with tissue plasminogen activator (tPA), and ChimAd-WTgE, which retains the original signal peptide. C57BL/6 mice were immunized with VZV-vaccine candidates, and cellular and humoral immune responses were evaluated using interferon-γ ELISPOT and ELISA. The ChimAd-based HZ vaccines induced high levels of gE-specific antibodies and cell-mediated immunity. ChimAd-tPAgE (optimal dose: 1 × 10 7 IFU) elicited a more robust gE-specific T-cell response than Shingrix and Zostavax, showing potential as HZ prophylactic vaccines., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests.Sun Ok Jang, Jae Hyun Lee, Yong-Ju Chung, Hyun-Seok Oh, Myeongcheol Shin, Soo-Ok Kim, Sun Pyo Hong reports financial support was provided by Ministry of Food and Drug Safety. Kim, S. HS, Kim S, Jang S, Kim H, Lee S has patent licensed to GeneMatrix, Inc. If there are other authors, they 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.)

Background: Lymphocyte migration plays a key role in the development of acute graft-versus-host disease (aGVHD). Blocking lymphocyte migration by targeting chemokine receptors, such as CXCR3, may be a promising strategy for preventing and treating aGVHD. Our previous studies have shown that short-term CXCR3 antagonist treatment combined with cyclosporine A alleviated aGVHD. However, the effect of long-term AMG487 treatment on aGVHD survival has not been thoroughly investigated., Methods: A murine aGVHD model was used to examine the expression of CXCR3 in donor T cells. The effects of short- and long-term AMG487 treatment on aGVHD survival were assessed. The infiltration of donor T cells into the liver and spleen tissues and the activation of donor T cells in splenic tissues were also examined., Results: CXCR3 was consistently highly expressed in donor T cells in a murine aGVHD model. Long-term AMG487 treatment, but not short-term, improved survival and aGVHD outcomes (p < 0.05). Furthermore, long-term AMG487 administration reduced the number of donor T cells in the liver but increased the number of donor T cells in the spleen (p < 0.05). Long-term AMG487 treatment also inhibited donor T cell activation in the spleen (p < 0.05)., Conclusion: This study demonstrates that long-term AMG487 treatment has a potential therapeutic effect on aGVHD and could be used as a novel therapy., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children. Typical therapy for CNS tumors in children involves a combination of surgery, radiation, and chemotherapy. While upfront therapy is effective for many high-grade tumors, therapy at the time of relapse remains limited. Furthermore, for diffuse intrinsic pontine glioma (DIPG) and diffuse midline glioma (DMG), there are currently no curative therapies. Chimeric antigen receptor T (CAR T) cell therapy is a promising novel treatment avenue for these tumors. Here, we review the preclinical evidence for CAR T cell use in pediatric brain tumors, the preliminary clinical experience of CNS CAR T cell trials, toxicity associated with systemic and locoregional CAR T cell therapy for CNS tumors, challenges in disease response evaluation with CAR T cell therapy, and the knowledge gained from correlative biologic studies from these trials in the pediatric and young adult population., Competing Interests: Declarations Competing interests Dr. Vitanza is chair of the Scientific Board for Brain Child Bio. Otherwise, the authors have no financial or nonfinancial interests that are directly or indirectly related to the work submitted for publication., (© 2024. The Author(s).)

Background: Glycosylation is a complex and fundamental metabolic biosynthetic process orchestrated by multiple glycosyltransferases (GT) and glycosidases enzymes. Functions of GT have been extensively examined in multiple human diseases. Our study investigated the potential role of GT genes in T-cell mediated rejection (TCMR) and possible prediction of graft loss of kidney transplantation., Methods: We downloaded the microarray datasets and GT genes from the GEO and the HUGO Gene Nomenclature Committee (HGNC) databases, respectively. Differentially expressed GT genes (DE-GTGs) were obtained by differential expression and Venn analysis. A TCMR diagnostic model was developed based on the hub DE-GTGs using LASSO regression and XGboost machine learning algorithms. In addition, a predictive model for graft survival was constructed by univariate Cox and LASSO Cox regression analysis., Results: We have obtained 15 DE-GTGs. Both GO and KEGG analyses showed that the DE-GTGs were mainly involved in the glycoprotein biosynthetic process. The TCMR diagnostic model exhibited high diagnostic potential with generally highly correlated accuracies [aera under the curve (AUC) of 0.83]. The immune characteristics analysis revealed that higher levels of immune cell infiltration and immune responses were observed in the high-risk group than in the low-risk group. In particular, the Kaplan-Meier survival analysis revealed that renal grafts in the high-risk group have poor prognostic outcomes than the low-risk group. The predictive AUC values of 1-, 2- and 3-year graft survival were 0.76, 0.81, and 0.70, respectively., Conclusion: Our results indicated that GT genes could be used for diagnosis of TCMR and prediction of graft loss in kidney transplantation. These results provide new perspectives and tools for diagnosing, treating and predicting kidney transplant-related diseases., Competing Interests: Declaration of competing interest The authors have declared that no competing interests exist., (Copyright © 2024. Published by Elsevier B.V.)

Balanced immune responses in the eyes are crucial to preserve vision. The ocular immune system has long been considered distinct, owing to the so-called 'immune privilege' of its component tissues. More recently, intravital imaging and transcriptomic techniques have reshaped scientific understanding of the ocular immune landscape, such as revealing the specialization of immune cell populations in the various tissues of the eye. As knowledge of the phenotypes of corneal and retinal immune cells has evolved, links to both the systemic immune system, and the central and peripheral nervous systems, have been identified. Using intravital imaging, T cells have recently been found to reside in, and actively patrol, the healthy human cornea. Disease-associated retinal microglia with links to retinal degeneration have also been identified. This Review provides an updated guide to the ocular immune system, highlighting current knowledge of the immune cells that are present in steady-state and specific diseased ocular tissues, as well as evidence for their relationship to systemic disease. In addition, we discuss emerging intravital imaging techniques that can be used to visualize immune cell morphology and dynamics in living human eyes and how these could be applied to advance understanding of the human immune system., Competing Interests: Competing interests: M.W., H.R.C, L.E.D and S.N.M have submitted an Australian Provisional Patent Application (2023901150) relating to the imaging method described in this article (intellectual property owned by institution). E.L.F. declares no competing interests., (© 2024. Springer Nature Limited.)

Introduction: CVID is the commonest and most symptomatic primary immune deficiency of adulthood. NHLs are the most prevalent malignancies in CVID. The cross-talk between tumor cells and immune cells may be an important risk factor in lymphomagenesis., Areas Covered: The present review highlights immune cell, genetic and histopathological alterations in the CVID-associated NHLs., Expert Opinion: CVID patients exhibit some notable immune defects that may predispose to lymphomas. T/NK cell defects including reduced T cells, naïve CD4+T cells, T regs, and Th17 cells, increased CD8+T cells with reduced T cell proliferative and cytokine responses and reduced iNKT and NK cell count and cytotoxicity. B cell defects include increased transitional and CD21 low B cells, clonal IgH gene rearrangements, and increased BCMA levels. Increase in IL-9, sCD30 levels, and upregulation of BAFF-BAFFR signaling are associated with lymphomas in CVID. Increased expression of PFTK1 , duplication of ORC4L , germline defects in TACI , NFKB1 , and PIK3CD , and somatic mutations in NOTCH2 and MYD88 are reported in CVID-associated lymphomas. Upregulation of PD-L1-PD-1 pathway may also promote lymphomagenesis in CVID. These abnormalities need to be explored as prognostic or predictive markers of CVID-associated NHLs by large multicentric studies.

Substantial progress in understanding T cell signalling, particularly with respect to T cell co-receptors such as the co-stimulatory receptor CD28, has been made in recent years. This knowledge has been instrumental in the development of innovative immunotherapies for patients with cancer, including immune checkpoint blockade antibodies, adoptive cell therapies, tumour-targeted immunostimulatory antibodies, and immunostimulatory small-molecule drugs that regulate T cell activation. Following the failed clinical trial of a CD28 superagonist antibody in 2006, targeted CD28 agonism has re-emerged as a technologically viable and clinically promising strategy for cancer immunotherapy. In this Review, we explore recent insights into the molecular functions and regulation of CD28. We describe how CD28 is central to the success of current cancer immunotherapies and examine how new questions arising from studies of CD28 as a clinical target have enhanced our understanding of its biological role and may guide the development of future therapeutic strategies in oncology., Competing Interests: Competing interests: M.T.L. is the chairman of the strategic advisory board and has ownership interest in iRepertoire, Inc; he is a member of the scientific advisory boards of KSQ, Inc.; BioCytics; Zai Laboratories; Appel Sauce, Studios; and the Ludwig Institute. S.H.O. declares no competing interests. D.S. is a full-time employee and shareholder of Regeneron Pharmaceuticals, Inc., (© 2024. Springer Nature Limited.)

Approximately 5%-10% of fractures go on to delayed healing and nonunion, posing significant clinical, economic, and social challenges. Current treatment methods involving open bone harvesting and grafting are associated with considerable pain and potential morbidity at the donor site. Hence, there is growing interest in minimally invasive approaches such as bone marrow aspirate concentrate (BMAC), which contains mesenchymal stromal cells (MSCs), macrophages (Mφ), and T cells. However, the use of cultured or activated cells for treatment is not yet FDA-approved in the United States, necessitating further exploration of optimal cell types and proportions for effective bone formation. As our understanding of osteoimmunology advances, it has become apparent that factors from anti-inflammatory Mφ (M2) promote bone formation by MSCs. Additionally, M2 Mφ promote T helper 2 (Th2) cells and Treg cells, both of which enhance bone formation. In this study, we investigated the interactions among MSCs, Mφ, and T cells in bone formation and explored the potential of subsets of BMAC. Coculture experiments were conducted using primary MSCs, Mφ, and CD4+ T cells at specific ratios. Our results indicate that nonactivated T cells had no direct influence on osteogenesis by MSCs, while coculturing MSCs with Mφ and T cells at a ratio of 1:5:10 positively impacted bone formation. Furthermore, higher numbers of T cells led to increased M2 polarization and a higher proportion of Th2 cells in the early stages of coculture. These findings suggest the potential for enhancing bone formation by adjusting immune and mesenchymal cell ratios in BMAC. By understanding the interactions and effects of immune cells on bone formation, we can develop more effective strategies and protocols for treating bone defects and nonunions. Further studies are needed to investigate these interactions in vivo and explore additional factors influencing MSC-based therapies., (© 2024 Wiley Periodicals LLC.)

T cells are key mediators of alloresponse during liver transplantation (LTx). However, the dynamics of donor-reactive T-cell clones in peripheral blood during a clinical T-cell-mediated rejection (TCMR) episode remain unknown. Here, we collected serial peripheral blood mononuclear cell samples spanning from pre-LTx to 1 year after LTx and available biopsies during the TCMR episodes from 26 rejecting patients, and serial peripheral blood mononuclear cell samples were collected from 96 nonrejectors. Immunophenotypic and repertoire analyses were integrated on T cells from rejectors, and they were longitudinally compared to nonrejected patients. Donor-reactive T-cell clone was identified and tracked by cross-matching with the mappable donor-reactive T-cell receptor repertoire of each donor-recipient pair in 9 rejectors and 5 nonrejectors. Before transplantation, the naive T-cell percentage and T-cell receptor repertoire diversity of rejectors was comparable to that of healthy control, but it was reduced in nonrejectors. After transplantation, the naïve T-cell percentages decreased, and T-cell receptor repertoires were skewed in rejectors; the phenomenon was not observed in nonrejectors. Alloreactive clones increased in proportion in the peripheral blood of rejectors before TCMR for weeks. The increase was accompanied by the naïve T-cell decline and memory T-cell increase and acquired an activated phenotype. Intragraft alloreactive clone tracking in pre-LTx and post-LTx peripheral blood mononuclear cell samples revealed that the pretransplant naïve T cells were significant contributors to the donor-reactive clones, and they temporarily increased in proportion and subsequently reduced in blood at the beginning of TCMR. Together, our findings offer an insight into the dynamic and origin of alloreactive T cells in clinical LTx TCMR cases and may facilitate disease prediction and management., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

The promising results seen in the treatment of refractory hematologic malignancies with tisagenlecleucel (Kymriah), the pioneering Chimeric Antigen Receptor (CAR) T-cell immunotherapy, has stimulated a fast growth in research and clinical testing of novel CAR-T constructs, targets, and various generations of CAR T-cells. Pharmacists may receive inquiries about active clinical trials or may be contributing to the care of patients participating in these studies. This briefing discusses the on-going clinical trials that explore novel CAR T-cell immunotherapies in pediatric refractory malignancies of hematologic and solid organ origins. It can be valuable in assisting practitioners in navigating the rapidly evolving field of CAR T-cell immunotherapies., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Tick-borne encephalitis virus (TBEV) infection may cause acute central nervous system inflammation varying in clinical manifestations and severity. A possible correlation of TBEV-specific antibody and cell-mediated immune responses, shortly after infection, with clinical manifestations, severity and long-term outcome has been poorly investigated. In a cohort of thirty early tick-borne encephalitis (TBE) patients, we assessed the magnitude, specificity and functional properties of TBEV-specific T-cell and antibody responses. These responses early during disease were assessed in view of clinical manifestations, severity and long-term outcome. TBEV-specific T-cell responses to C, E, NS1, and NS5 proteins were significantly lower in patients with severe acute illness than in patients with mild TBE. Lower T-cell responses to E, NS1, and NS5 proteins also correlated with the development of meningoencephalomyelitis. Virus-specific antibody titres early after infection did not correlate with disease severity, clinical manifestations, or long-term outcome in this study, possibly due to the small number of patients of which matching serum and peripheral blood mononuclear cells were available. The findings suggest that virus-specific T cells afford a certain degree of protection against the development of severe TBEV-induced disease.

Adoptive immunotherapy shows great promise as a treatment for cancer and other diseases. Recent evidence suggests that the therapeutic efficacy of these cell-based therapies can be enhanced by the enrichment of less-differentiated T cell subpopulations in the therapeutic product, giving rise to a need for advanced manufacturing technologies capable of enriching these subpopulations through regulation of T cell differentiation. Studies have shown that modifying certain critical process control parameters, such as cytokines, metabolites, amino acids, and culture environment, can effectively manipulate T cell differentiation in ex vivo cultures. Advanced process analytical technologies (PATs) are crucial for monitoring these parameters and the assessment of T cell differentiation during culture. In this review, we examine such critical process parameters and PATs, with an emphasis on their impact on enriching less-differentiated T cell population. We also discuss the limitations of current technologies and advocate for further efforts from the community to establish more stringent critical process parameters (CPPs) and develop more at-line/online PATs that are specific to T cell differentiation. These advancements will be essential to enable the manufacturing of more efficacious adoptive immunotherapy products., 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 The Authors. Published by Elsevier Inc. All rights reserved.)

Scaling up the manufacture of cell therapies can be complex and challenging. Maintaining critical quality attributes of the cell product during its final formulation and fill-finish into multiple containers can be especially difficult and laborious. Here, we tested the automated Finia™ Fill and Finish System to efficiently scale up the formulation and fill-finish of a T cell product, and then assessed cell quality and product consistency across different sub-lots filled during this expanded process. We found that this automated system could be effectively scaled to 4 times its singular capacity in a 2-h time interval, with variation in cell number and product volume less than 12% across all containers. Analysis of the different sub-lots of the final product revealed high cell viability and consistent T cell phenotype, with a high proportion of effector memory and central memory T cells and low expression of T cell senescence and exhaustion markers. The functionality of the T cell product was compared by measuring cytokine response after restimulation, with secreted levels of effector cytokines like IFN-γ and TNF-α being similar across the different sub-lots. Collectively, these results show that automation can scale up the formulation and fill-finish of a cell manufacturing process while maintaining the phenotype and functionality of the cell product. Better understanding of how to maintain product uniformity and quality during final manufacturing is important to the further scale-up and development of successful cell therapies., Competing Interests: Declaration of competing interest All authors are paid employees of Charles River Laboratories Inc. or Terumo BCT, as indicated by the author affiliations. The authors report no other conflict of interest beyond these respective employments., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The utilization of anti-CD3/CD28 magnetic beads for T cell expansion in vitro has been investigated for adoptive cell transfer therapy. However, the impact of the CD3/CD28 antibody ratio on T cell differentiation and function remains incompletely elucidated. This study seeks to address this knowledge gap. To begin with, CD3 antibodies with a relatively low avidity for Jurkat cells (Kd = 13.55 nM) and CD28 antibodies with a relatively high avidity (Kd = 5.79 nM) were prepared. Afterwards, anti-CD3/CD28 antibodies with different mass ratios were attached to magnetic beads to examine the impacts of different antibody ratios on T cell capture, and proliferation. The research demonstrated that the most significant expansion of T cells was stimulated by the anti-CD3/CD28 magnetic beads with a mass ratio of 2:1 for CD3 antibodies and CD28 antibodies. Moreover, CD25 and PD1 expression of expanded T cells increased and then decreased, with lower CD25 and PD1 expression in the later stages of expansion indicating that T cells were not depleted. These T cells, which are massively expanded in vitro and have excellent expansion potential, can be infused back into the patient to treat tumor patients. This study shows that altering the ratio of anti-CD3/CD28 antibodies can control the strength of T cell stimulation, thereby leading to the improvement of T cell activation. This discovery can be utilized as a guide for the creation of other T cell stimulation approaches, which is beneficial for the further development of tumor immunotherapy technology., (© 2024 International Union of Biochemistry and Molecular Biology.)

Therapeutics for eradicating hepatitis B virus (HBV) infection are still limited and current nucleos(t)ide analogs (NAs) and interferon are effective in controlling viral replication and improving liver health, but they cannot completely eradicate the hepatitis B virus and only a very small number of patients are cured of it. The TCR-like antibodies recognizing viral peptides presented on human leukocyte antigens (HLA) provide possible tools for targeting and eliminating HBV-infected hepatocytes. Here, we generated three TCR-like antibodies targeting three different HLA-A2.1-presented peptides derived from HBV core and surface proteins. Bispecific antibodies (BsAbs) were developed by fuzing variable fragments of these TCR-like mAbs with an anti-CD3ϵ antibody. Our data demonstrate that the BsAbs could act as T cell engagers, effectively redirecting and activating T cells to target HBV-infected hepatocytes in vitro and in vivo . In HBV-persistent mice expressing human HLA-A2.1, two infusions of BsAbs induced marked and sustained suppression in serum HBsAg levels and also reduced the numbers of HBV-positive hepatocytes. These findings highlighted the therapeutic potential of TCR-like BsAbs as a new strategy to cure hepatitis B.

Amyotrophic lateral sclerosis (ALS) represents a rare and potentially fatal neurodegenerative disease. Diverse T-cell subsets could potentially exert diametrically opposite impacts upon ALS development. A two-sample Mendelian randomization (MR) analysis was performed to investigate the correlation between 244 T-cell subsets and ALS risk. Genetic instrumental variables were procured from a standard genome-wide association study (GWAS) that encompassed 244 T-cell subsets in 3757 individuals of European lineage. ALS-related data were collected from a GWAS comprising 20,806 ALS instances and 59,804 European control participants. Multiple sensitivity analyses were performed to verify the robustness of the significant results. Reverse MR analysis was used for delineating the effects of ALS on the characteristics of T-cells. After multiple comparison corrections, 24 out of the 244 subtypes demonstrated a potential association with ALS risk. Significantly, 75% of these associations encompassed the expression of the CD3 on diverse T-cell subtypes, revealing a highly consistent inverse relation to ALS risk. The proportion of T regulatory cells (Tregs) in CD4+ T cells and secreting Tregs in CD4+ T cells demonstrated negative associations with the risk of ALS. CCR7 expression on naive CD4+ T cells and CCR7 expression on naive CD8+ T cells showed positive associations with ALS risk. Certain T-cell subsets, particularly those identified by CD3 expression on terminally differentiated CD8+ T cells, proportions of Tregs, and CCR7 expression, indicated an association with ALS risk. These findings harmonize with and extend previous observational studies investigating the involvement of T lymphocyte subset-induced immunological processes in ALS., Competing Interests: Declarations. Ethics Approval and Consent to Participate: Not applicable Consent for Publication: Not applicable Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA technology has emerged as a promising route to accelerated manufacture of sequence agnostic vaccines. For activity, DNA vaccines must be protected and delivered to the correct antigen presenting cells. However, the physicochemical properties of the vector must be carefully tuned to enhance interaction with immune cells and generate sufficient immune response for disease protection. In this study, we have engineered a range of polymer-based nanocarriers based on the poly(beta-amino ester) (PBAE) polycation platform to investigate the role that surface poly(ethylene glycol) (PEG) density has on pDNA encapsulation, formulation properties and gene transfectability both in vitro and in vivo. We achieved this by synthesising a non-PEGylated and PEGylated PBAE and produced formulations containing these PBAEs, and mixed polyplexes to tune surface PEG density. All polymers and co-formulations produced small polyplex nanoparticles with almost complete encapsulation of the cargo in all cases. Despite high gene transfection in HEK293T cells, only the fully PEGylated and mixed formulations displayed significantly higher expression of the reporter gene than the negative control in dendritic cells. Further in vivo studies with a bivalent SARS-CoV-2 pDNA vaccine revealed that only the mixed formulation led to strong antigen specific T-cell responses, however this did not translate into the presence of serum antibodies indicating the need for further studies into improving immunisation with polymer delivery systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cameron Alexander, Pratik Gurnani report financial support was provided by the Wellcome Trust. Cameron Alexander reports financial support was provided by The Royal Society. Hulya Bayraktutan reports financial support was provided by the Turkish Ministry of National Education. Peter Symonds, A Brentville, Lindy Durrant reports financial support was provided by Scancell Ltd. Peter Symonds, Victoria A Brentville, Lindy Durrant report a relationship with Scancell Ltd that includes: employment and equity or stocks. Other 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chimeric antigen receptor (CAR) T-cell therapy is highly effective for treating blood cancers such as B-cell malignancies, however, its effectiveness as an approach to treat solid tumors remains to be further explored. Here, we focused on the development of CAR-T cell therapies targeting tropomyosin-related kinase receptor B (TRKB), a highly expressed protein that is significantly associated with tumor progression, malignancy, and drug resistance in multiple forms of aggressive solid tumors. To achieve this, we screened brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NTF4) ligand-based CAR-T cells for their efficiency in targeting the TRKB receptor in the context of solid tumors, particularly hepatocellular carcinoma and pancreatic cancer. We demonstrated that TRKB is overexpressed not only in hepatocellular carcinoma and pancreatic carcinoma cell lines but also in cancer stem-like cells (CSCs). Notably, BDNF-CAR T and NTF4-CAR T cells could not only effectively target and kill TRKB-expressing pan-cancer cell lines in a dose-dependent manner but also effectively kill CSCs. We also performed in vivo studies to show that NTF4-CAR T cells have a better potential to inhibit the tumor growth of hepatocellular carcinoma xenografts in mice, compared with BDNF-CAR T cells. Taken together, our findings suggest that CAR-T targeting TRKB may be a promising approach for developing novel therapies to treat solid cancers., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Per- and polyfluoroalkyl substances (PFAS) are anthropogenic organofluorine compounds that persist indefinitely in the environment and bioaccumulate throughout all trophic levels. Biomonitoring efforts have detected multiple PFAS in the serum of most people. Immune suppression has been among the most consistent effects of exposure to PFAS. PFAS often co-occur as mixtures in the environment, however, few studies have examined immunosuppression of PFAS mixtures or determined whether PFAS exposure affects immune function in the context of infection. In this study, mixtures containing two or four different PFAS and a mouse model of infection with influenza A virus (IAV) were used to assess immunotoxicity of PFAS mixtures. PFAS were administered via the drinking water as either a binary mixture of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) or quaternary mixture of PFOS, PFOA, perfluorohexane sulfonate (PFHxS), and perfluorononanoic acid (PFNA). The results indicated that the binary mixture affected the T-cell response, while the quaternary mixture affected the B-cell response to infection. These findings indicate that the immunomodulatory effects of PFAS mixtures are not simply additive, and that the sensitivity of immune responses to PFAS varies by cell type and mixture. The study also demonstrates the importance of studying adverse health effects of PFAS mixtures.

Cutaneous T-cell lymphoma is characterized by malignant T cells proliferating in a unique tumor microenvironment dominated by keratinocytes (KCs). Skin colonization and infection by Staphylococcus aureus are a common cause of morbidity and are suspected of fueling disease activity. In this study, we show that expression of HLA-DRs, high-affinity receptors for staphylococcal enterotoxins (SEs), by KCs correlates with IFN-γ expression in the tumor microenvironment. Importantly, IFN-γ induces HLA-DR, SE binding, and SE presentation by KCs to malignant T cells from patients with Sézary syndrome and malignant and nonmalignant T-cell lines derived from patients with Sézary syndrome and mycosis fungoides. Likewise, preincubation of KCs with supernatant from patient-derived SE-producing S aureus triggers proliferation in malignant T cells and cytokine release (including IL-2), when cultured with nonmalignant T cells. This is inhibited by pretreatment with engineered bacteriophage S aureus-specific endolysins. Furthermore, alteration in the HLA-DR-binding sites of SE type A and small interfering RNA-mediated knockdown of Jak3 and IL-2Rγ block induction of malignant T-cell proliferation. In conclusion, we show that upon exposure to patient-derived S aureus and SE, KCs stimulate IL-2Rγ/Jak3-dependent proliferation of malignant and nonmalignant T cells in an environment with nonmalignant T cells. These findings suggest that KCs in the tumor microenvironment play a key role in S aureus-mediated disease activity in cutaneous T-cell lymphoma., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by RVF virus (RVFV). RVFV infections in humans are usually asymptomatic or associated with mild febrile illness, although more severe cases of haemorrhagic disease and encephalitis with high mortality also occur. Currently, there are no licensed human vaccines available. The safety and efficacy of a genetically engineered four-segmented RVFV variant (hRVFV-4s) as a potential live-attenuated human vaccine has been tested successfully in mice, ruminants, and marmosets though the correlates of protection of this vaccine are still largely unknown. In the present study, we have assessed hRVFV-4s-induced humoral and cellular immunity in a mouse model of RVFV infection. Our results confirm that a single dose of hRVFV-4s is highly efficient in protecting naïve mice from developing severe disease following intraperitoneal challenge with a highly virulent RVFV strain and data show that virus neutralizing (VN) serum antibody titres in a prime-boost regimen are significantly higher compared to the single dose. Subsequently, VN antibodies from prime-boost-vaccinated recipients were shown to be protective when transferred to naïve mice. In addition, hRVFV-4s vaccination induced a significant virus-specific T cell response as shown by IFN-γ ELISpot assay, though these T cells did not provide significant protection upon passive transfer to naïve recipient mice. Collectively, this study highlights hRVFV-4s-induced VN antibodies as a major correlate of protection against lethal RVFV infection.

Adoptive cell transfer (ACT) using neoantigen-specific T cells is an effective immunotherapeutic strategy. However, the difficult isolation of neoantigen-specific T cells limits the clinical application of ACT. Here, we propose a method to prepare neoantigen-reactive T cells (NRT) for ACT following immunization with a tumor lysate-loaded dendritic cell (DC) vaccine. We show that the DC vaccine not only induces a neoantigen-reactive immune response in lung cancer-bearing mice in vivo, but also facilitate NRT cell preparation in vitro. Adoptive transfer of the NRTs as combinatorial therapy into DC vaccine-immunized, LL/2 tumor-bearing mice allows infiltration of the infused NRTs, as well as the enrichment of neoantigen reactive, non-ACT/NRT T cells into the tumor microenvironment with the function of these neoantigen-reactive T-cell receptors validated in vitro. In summary, we propose a method for preparing NRTs that increases ACT efficacy and paves the way to the design of personalized immunotherapies., Competing Interests: Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

The T cell receptor (TCR), despite its importance, is underutilized in single-cell analysis, with gene expression features solely driving current strategies. Here, we argue for a TCR-first approach, more suited toward T cell repertoires. To this end, we curated a large T cell atlas from 12 prominent human studies, containing in total 500,000 T cells spanning multiple diseases, including melanoma, head and neck cancer, blood cancer, and lung transplantation. Here, we identified severe limitations in cell-type annotation using unsupervised approaches and propose a more robust standard using a semi-supervised method or the TCR arrangement. We showcase the utility of a TCR-first approach through application of the STEGO.R tool for the identification of treatment-related dynamics and previously unknown public T cell clusters with potential antigen-specific properties. Thus, the paradigm shift to a TCR-first can highlight overlooked key T cell features that have the potential for improvements in immunotherapy and diagnostics.

Marburg virus (MARV) is a zoonotic virus that can infect humans and non-human primates (NHPs) and lead to a fatal Marburg hemorrhagic fever (MHF), while there is no approved vaccine or antiviral treatment for MHF. The nucleic acid vaccine has unique advantages, including fast and simple preparation, easy to follow the virus mutation situation, and less adverse reactions. Therefore, we constructed the DNA and mRNA candidate vaccines based on codon-optimized MARV glycoprotein sequence, and evaluated the immune effect in mice through ELISA, ELISpot, and Flow cytometry. After the second booster immunization, both of the candidate vaccines induced strong humoral immune response, enhanced T cell response, and elicited neutralizing antibodies. Notably, DNA candidate vaccine induced stronger humoral immune response, while mRNA candidate vaccine elicited higher levels of IFN-γ and IL-4. In addition, transcriptome analysis revealed that the candidate vaccines activated immune response related pathways. Our study shed new light on the nucleic acid vaccines for MARV and further confirmed the potential of nucleic acid vaccine for future MHF prevention and control., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Over the past decade, chimeric antigen receptor (CAR)-T cell therapy has emerged as a revolutionary immunotherapeutic approach to combat cancer. This therapy constructs a CAR on the surface of T cells through genetic engineering techniques. The CAR is formed from a combination of antibody-derived or ligand-derived domains and T-cell receptor (TCR) domains. This enables T cells to specifically bind to and activate against tumor cells. However, the efficacy of CAR-T cells in solid tumors remains inconclusive due to several challenges such as poor tumor trafficking, infiltration, and the immunosuppressive tumor microenvironment (TME). In response, CAR natural killer (CAR-NK) and CAR macrophages (CAR-M) have been developed as complementary strategies for solid tumors. CAR-NK cells do not require HLA compatibility, demonstrate reduced toxicity, and are thus seen as potential substitutes for CAR-T cells. Furthermore, CAR-M immunotherapy is also being researched and has shown phagocytic capabilities and tumor-antigen presentation. This study discusses the features, advantages, and limitations of CAR-T, CAR-NK, and CAR-M cells in the treatment of solid tumors and suggests prospective solutions for enhancing the efficacy of CAR host-cell-based immunotherapy., Competing Interests: Declarations. Conflict of interest: 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., (© 2024. The Author(s).)

Various public health measures have contained outbreaks of SARS-CoV-2, but concerns remain over the possibility of future surges. Improvements in broadening the vaccine response can stifle new and nascent infections. In this study, we tested the effects of different adjuvant combinations on the immunization of mice with the receptor-binding domain (RBD)-containing the S1-subunit of the spike protein (S1 protein) from SARS-CoV-2 to induce a robust humoral and cellular immune response. We showed that subcutaneous immunization of S1 protein co-delivered with IL-15 and TLR-ligands (MALP-2, poly I: C, and CpG) or with IL-12 and GM-CSF in DOTAP, or Alum induced significantly high titers of durable antibodies, predominantly IgG1, IgG2a, and IgG2b, that could bind to RBD, S1-subunit, and the full-length ectodomain of SARS-CoV-2 spike protein in sera compared to the immunization with S1 protein alone in both B6 wild-type (WT) and the K18-hACE2 transgenic mice. In addition, immunization with S1 protein co-delivered with IL-15 and TLR-ligands induced antibody responses against S1 protein in aged mice, and sera from younger mice reduced plaque formation of live SARS-CoV-2, and had effective binding to S1 protein from ten different variants of SARS-CoV-2, including Omicron (B.1.1.529), and greater neutralization activity as early as day 21 post-immunization measured by inhibition of RBD binding to hACE2 than sera from mice immunized with S1 protein alone or co-delivered with Alum. We also identified antibody-binding epitopes using 18-mer peptides with 9-residue overlaps from the S1 protein. CD8 + T-cell responses specific to RBD and S1 protein peptide pools were observed up to day 200 post-immunization by tetramer staining. These data show the efficacy of specific immunologically targeted adjuvants for increasing S1 protein immunogenicity in mice and can contribute to more effective vaccines., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Human immune cells are under constant evolutionary pressure, primarily through their role as first line of defence against pathogens. Most studies on immune adaptation are, however, based on protein-coding genes without considering their cellular context. Here, using data from the Human Cell Atlas, we infer the gene adaptation rate of the human immune landscape at cellular resolution. We find abundant cell types, like progenitor cells during development and adult cells in barrier tissues, to harbour significantly increased adaptation rates. We confirm the adaptation of tissue-resident T and NK cells in the adult lung located in compartments directly facing external challenges, such as respiratory pathogens. Analysing human iPSC-derived macrophages responding to various challenges, we find adaptation in early immune responses. Together, our study suggests host benefits to control pathogen spread at early stages of infection, providing a retrospect of forces that shaped the complexity, architecture, and function of the human body., Competing Interests: Competing interests: H.H. is a co-founder and shareholder of Omniscope, a member of the Scientific Advisory Board of Nanostring and MiRXES, and a consultant to Moderna and Singularity. J.C.N. is a consultant to Omniscope. The remaining authors declare no competing interests., (© 2024. The Author(s).)

The telomerase RNA component (Terc) constitutes a non-coding RNA critical for telomerase function, commonly associated with aging and pivotal in immunomodulation during inflammation. Our study unveils heightened susceptibility to pneumonia caused by Staphylococcus aureus (S. aureus ) in Terc knockout ( Terc ko/ko ) mice compared to both young and old infected counterparts. The exacerbated infection in Terc ko/ko mice correlates with heightened inflammation, manifested by elevated interleukin-1β (IL-1β) levels and activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome within the lung. Employing mRNA sequencing methods alongside in vitro analysis of alveolar macrophages (AMs) and T cells, our study elucidates a compelling correlation between Terc ko/ko , inflammation, and impaired T cell functionality. Terc deletion results in compromised T cell function, characterized by dysregulation of the T cell receptor and absence of CD247, potentially compromising the host's capacity to mount an effective immune response against S. aureus . This investigation provides insights into the intricate mechanisms governing increased vulnerability to severe pneumonia in the context of Terc deficiency, which might also contribute to aging-related pathologies, while also highlighting the influence of Terc on T cell function., Competing Interests: YR, FH, AH, TL, SN, BL, SD No competing interests declared, (© 2024, Reisser et al.)

Members of the vanin gene family include VNN1, VNN2, and VNN3 in humans. Although the functions of vanins have been widely examined in myeloid cells, their expression and functions have not been clarified in T lymphocytes. This study aimed to elucidate the significance of Vanin-2 (VNN2) on human peripheral blood T lymphocytes and study its expression in systemic lupus erythematosus (SLE). The differential expression of Vanins was analyzed by bioinformatics. VNN2 expressions in peripheral blood T-cell subsets were analyzed by single-cell RNA sequencing data and flow cytometry. Changes of VNN2 expression before and after T-cell activation were further clarified by western blot. The function of VNN2+ cells was studied by granzyme B (GZMB) and perforin detection. Changes in VNN2+ proportions in T-cell subsets of patients with SLE were further analyzed. In the present study, only VNN2 among vanins showed distinguishable expression in T cells. VNN2+ percentages were higher in CD8+ T cells those in CD4+ T cells. VNN2+ T cells were with a higher memory T-cell composition. VNN2 expression was significantly increased after T-cell stimulation. VNN2+ T cells had higher levels of GZMB and perforin secretion than VNN2- T cells. Clinically, VNN2+ percentages in T cells of patients with SLE were upregulated. Together, these data suggested that VNN2 is expressed in peripheral blood T cells characterized more GZMB and perforin secretion, and increased VNN2+ T cells in patients with SLE could reflect altered T-cell functions in vivo., Competing Interests: Conflict of interest statement. The authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The treatment of multiple myeloma has changed dramatically in recent years, with huge strides forward made in the field. Chimeric antigen receptor T-cell therapy targeting the B cell maturation antigen (BCMA) is now widely approved in relapsed refractory patients and is moving into earlier treatment lines. In this review, we discuss the evidence underpinning current regulatory approvals and consider mechanisms through which CAR-T cell efficacy could be improved. These include tackling BCMA-loss, harnessing the immunosuppressive tumour microenvironment, manufacturing concerns including the potential role of other cellular sources, safety issues such as cytokine release syndrome and neurotoxicity, and optimal patient selection., Competing Interests: Competing interests: DS: none; DM: Employment: Johnson and Johnson; JH: none., (© 2024. The Author(s).)

Hidradenitis suppurativa (HS) is a chronic, debilitating inflammatory skin disease with a poorly understood immunopathogenesis. Here, we report that HS lesional skin is characterized by the expansion of innate lymphocytes and T cells expressing CD2, an essential activation receptor and adhesion molecule. Lymphocytes expressing elevated CD2 predominated with unique spatial distribution throughout the epidermis and hypodermis in the HS lesion. CD2 + cells were mainly innate lymphocytes expressing the NK cell marker, CD56, and CD4 + T cells. Importantly, these CD2 + cells interacted with CD58 (LFA3) expressing epidermal keratinocytes and fibroblasts in the hypodermis. Granzyme A bright NKT cells (CD2 + CD3 + CD56 bright ) clustered with α-SMA expressing fibroblasts juxtaposed to epithelialized tunnels and fibrotic regions of the hypodermis. Whereas NK cells (CD2 + CD56 dim ) were perforin + , granzymes A + and B + , and enriched adjacent to hyperplastic follicular epidermis and tunnels of HS showing presence of apoptotic cells. The cytokines IL-12, IL-15, and IL-18, which enhance NK cell maturation and function were significantly elevated in HS. Ex vivo HS skin explant cultures treated with CD2:CD58 interaction-blocking anti-CD2 monoclonal antibody attenuated secretion of inflammatory cytokines/chemokines and suppressed inflammatory gene signature. Additionally, CD2:CD58 blockade altered miRNAs involved in NK/NKT differentiation and/or function. In summary, we show that a cellular network of heterogenous NKT and NK cell populations drives inflammation and is critical in the pathobiology of HS, including tunnel formation and fibrosis. Finally, CD2 blockade is a viable immunotherapeutic approach for the effective management of HS., Competing Interests: Competing interests statement:E.B. and D.B. are co-founders of ITB-MED. Patent application number: PCT/US2023/022364.

The coronavirus disease 2019 (COVID-19) pandemic has reminded us of human infections with the H7N9 virus and has raised questions related to the clinical and molecular pathophysiological diversity between the two diseases. Here, we performed a proteomic approach on sera samples from patients with H7N9-virus or SARS-CoV-2-virus infection and healthy controls. Compared to SARS-CoV-2, H7N9-virus infection caused elevated neutrophil concentrations, T cell exhaustion, and increased cytokine/interleukin secretion. Cell-type deconvolution and temporal analysis revealed that T cells and neutrophils could regulate the core immunological trajectory and influence the prognosis of patients with severe H7N9-virus infection. Elevated tissue-enhanced proteins combined with alterations of clinical biochemical indexes suggested that H7N9 infection induced more severe inflammatory organ injury and dysfunction in the liver and intestine. Further mechanical analysis revealed that the high concentration of neutrophils might impact the intestinal enterocyte cells through cytokine-receptor interaction, leading to intestinal damage in patients with H7N9-virus infection., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immune checkpoint blockade (ICB) therapy, while promising for cancer treatment, faces challenges like unexpected side effects and limited objective responses. Here, we develop an in vivo gene-editing strategy for improving ICB cancer therapy in a lastingly effective manner. The approach uses a conductive hydrogel-based electroporation system to enable nucleofection of programmed cell death protein 1 (PD1) targeted CRISPR-Cas9 DNAs into T-cells directly within the lymph nodes, and subsequently produces PD1-deficient T-cells to combat tumor growth, metastasis and recurrence in different melanoma models in mice. Following in vivo gene editing, animals show enhanced cellular and humoral immune responses along with multi-fold increases of effector T-cells infiltration to the solid tumors, preventing tumor recurrence and prolonging their survival. These findings provide a proof-of-concept for direct in vivo T-cell engineering via localized gene-editing for enhanced cancer immunotherapy, and also unlock the possibilities of using this method to treat more complex human diseases., Competing Interests: Competing interests: The authors declare the following competing interests: P.S., J.Q., Y.W., and X.H. are listed as inventors on a patent application, titled “Micro-electroporation based drug delivery system, methods for use and fabrication thereof” (US Priority No. 17/804,118), filed by the City University of Hong Kong. The other authors declare no competing interests., (© 2024. The Author(s).)

IQGAP1 is a multi-functional scaffold protein. However, its role in B cell development and function is unknown. Here, we show IQGAP1 as an essential scaffold that regulates early B cell development and function. Iqgap1 -/- mice contained significantly increased numbers of B220 + B, B220 + IgM - pro/pre-B, and B220 Low IgM + immature-B cells in the bone marrow. In the spleens of the Iqgap1 -/- mice, newly formed and follicular B cell numbers were increased, while the marginal zone B cell numbers were significantly reduced. Lack of IQGAP1 reduced T-dependent and T-independent humoral responses. Mechanistically, the lack of IQGAP1 considerably decreased the phosphorylation of Mek1/2, Erk1/2, and Jnk1/2. B cells from Iqgap1 -/- mice failed to suppress IL-7R-mediated activation of Stat5a/b, an essential step for cell-cycle exit and initiate light-chain recombination, reducing RS rearrangement frequency. Our study provides the first evidence that IQGAP1-based signalosome is necessary for the development and functions of B cells., Competing Interests: Declarations. Conflict of interest: The authors declare that this study was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Ethics approval: All mice used in this study were utilized responsibly, and all protocols were approved by the institutional IACUC committee at the Medical College of Wisconsin (MCW), Milwaukee, WI. Author consent: All the authors fully agree to be part of the manuscript and its content. Both authors have participated in collecting, analyzing, and preparing this manuscript., (© 2024. The Author(s).)

Aims: The distinct functions of immune cells in atherosclerosis have been mostly defined by pre-clinical mouse studies. Contrastingly, the immune cell composition of human atherosclerotic plaques and their contribution to disease progression are only poorly understood. It remains uncertain whether genetic animal models allow for valuable translational approaches., Methods and Results: Single-cell RNA-sequencing (scRNA-seq) was performed to define the immune cell landscape in human carotid atherosclerotic plaques. The human immune cell repertoire demonstrated an unexpectedly high heterogeneity and was dominated by cells of the T-cell lineage, a finding confirmed by immunohistochemistry. Bioinformatical integration with 7 mouse scRNA-seq data sets from adventitial and atherosclerotic vascular tissue revealed a total of 51 identities of cell types and differentiation states, of which some were only poorly conserved between species and exclusively found in humans. Locations, frequencies, and transcriptional programmes of immune cells in mouse models did not resemble the immune cell landscape in human carotid atherosclerosis. In contrast to standard mouse models of atherosclerosis, human plaque leucocytes were dominated by several T-cell phenotypes with transcriptional hallmarks of T-cell activation and memory formation, T-cell receptor, and pro-inflammatory signalling. Only mice at the age of 22 months partially resembled the activated T-cell phenotype. In a validation cohort of 43 patients undergoing carotid endarterectomy, the abundance of activated immune cell subsets in the plaque defined by multi-colour flow cytometry associated with the extent of clinical atherosclerosis., Conclusion: Integrative scRNA-seq reveals a substantial difference in the immune cell composition of murine and human carotid atherosclerosis-a finding that questions the translational value of standard mouse models for adaptive immune cell studies. Clinical associations suggest a specific role for T-cell driven (auto-)immunity in human plaque formation and instability., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Purpose: The intention of this work is to probe the role of senescence-related gene CD161 in extranodal NK/T cell lymphoma (ENKTL)., Methods: This study used H 2 O 2 to establish three distinct in vitro oxidative stress aging models (NKL, SNT-8, and YT). Western blotting was employed to assess the levels of two iconic aging proteins, MMP1 and P53, and flow cytometry was utilized to investigate cell cycle and the expressions of CD4, CD8, and CD161. Cell viability was evaluated via the CCK-8 assay. The transcriptome analysis assessed the differential gene expression between the control and aging group of NKL. In vivo, we established a BALB/c mice aging tumor model. After 15 days, the mice were euthanized to harvest tumors. ELISA was employed to measure aging indicators in the mouse tissues. Flow cytometry was utilized to assess the levels of CD4, CD8, and CD161 in tumor samples. Hematoxylin-eosin (HE) staining was performed to evaluate the structure and cellular morphology of the tumor tissue., Results: In the NKL, SNT-8 and YT aging models, the levels of MMP1 and P53 proteins were significantly increased. Flow cytometry results indicated that all three cell types exhibited marked arrest in the G1 phase. Compared with the control group, the expressions of CD4 and CD161 in the aging group were significantly increased, while the expression of CD8 was decreased. Transcriptome analysis revealed 2,843 differentially expressed genes (DEGs) between the control and aging groups, with 2,060 up-regulated and 783 down-regulated genes identified. Following CD161 knockdown, cell viability of three cell types in the aging group was significantly reduced compared to the control group. The G1 phase of the cells was significantly interrupted. The expressions of CD4 and CD161 were significantly increased, and the expression of CD8 was decreased. However, in the aging + si-CD161 group, a partial alleviation of oxidative stress was observed with a reduction in CD161 expression levels. Animal experiments demonstrated that knockout of CD161 can inhibit tumor progression and partially mitigate oxidative stress., Conclusions: CD161 may inhibit ENKTL tumor development by regulating cell cycle and T-cell phenotype., Competing Interests: Declarations. Ethics approval and consent to participate: Each animal experimental procedure gained approval from Animal Ethics Committee of the Second Hospital of Jilin University. The experimental protocol was performed in accordance with the relevant guidelines and regulations of the Basel Declaration. The study is reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Consent for publication: Not applicable. Competing interests: The authors declare that they have no conflict of interest., (© 2024. The Author(s).)

Objective: To study the crosstalk between maternal immune cells and the developing embryo by investigating the immunogenic properties of human blastocyst spent media (SM) on dendritic cells., Methods: In this prospective multicenter experimental study, human preimplantation embryo spent media were collected after blastocyst formation, grouped based on successful or unsuccessful implantation, and analyzed by protein array or used to stimulate monocyte derived dendritic cells (moDC). The immunomodulatory properties of SM on moDC were investigated by analyzing changes in phenotype, cytokine secretion, indoleamine 2,3-dioxygenase (IDO) activity, and ability to activate T cells., Results: A plethora of cytokines and growth factors secreted from preimplantation embryos was detected. Exposure to embryo SM altered the phenotype of moDC in a manner dependent on the implantation outcome. Specifically, SM from non-implanted embryos increased the expression of co-stimulatory molecules and activation markers on moDC. Furthermore, SM treated dendritic cells secreted low levels of cytokines and growth factors and were able to stimulate naïve T cells. Activation of IDO was decreased in moDC after stimulation with SM., Conclusions: Our findings show that human preimplantation embryos secrete an abundance of molecules with the ability to significantly affect and even regulate immune cells in their environment., Competing Interests: Declarations. Ethics approval and consent to participate: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the ethics committee of the Medical University of Innsbruck, Austria (EK Nr AN2014-0312 343/4.6) and the ethics committee of “Land Vorarlberg” (EK-2-9/2020). Signed informed consent was obtained from all subjects involved in the study. Consent for publication: Not applicable. Animal research: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The non-polymorphic HLA-E molecule offers opportunities for new universal immunotherapeutic approaches to chronic infectious diseases. Chronic Hepatitis B virus (HBV) infection is driven in part by T cell dysfunction due to elevated levels of the HBV envelope (Env) protein hepatitis B surface antigen (HBsAg). Here we report the characterization of three genotypic variants of an HLA-E-binding HBsAg peptide, Env 371-379, identified through bioinformatic predictions and verified by biochemical and cellular assays. Using a soluble affinity-enhanced T cell receptor (TCR) (a09b08)-anti-CD3 bispecific molecule to probe HLA-E presentation of the Env 371-379 peptides, we demonstrate that only the most stable Env 371-379 variant, L6I, elicits functional responses to a09b08-anti-CD3-redirected polyclonal T cells co-cultured with targets expressing endogenous HBsAg. Furthermore, HLA-E-Env 371-379 L6I-specific CD8 + T cells are detectable in HBV-naïve donors and people with chronic HBV after in vitro priming. In conclusion, we provide evidence for HLA-E-mediated HBV Env peptide presentation, and highlight the effect of viral mutations on the stability and targetability of pHLA-E molecules., Competing Interests: Competing interests: G.M., R.L.P., R.K., V.I., R.J.S., M.M.C., V.K., R.P., A.J., J.D., T.H., W.B., G.P., K.O., D.K., A.S., C.B., R.R., C.P., T.G., A.T., A.W., M.D., D.G., M.H., R.J.C., K.E.A., L.D., A.K., S.L., M.S., and L.F.G. are or were employees of Immunocore Ltd., (© 2024. The Author(s).)

Three-dimensional (3D) cell culture has revolutionized life sciences, particularly in organoid technologies. Traditional bioscaffold materials, however, complicate the detachment of tumor organoids and hamper the routine use of organoid-immune cell cocultures. Here, we show an acoustic virtual 3D scaffold (AV-Scaf) method to achieve 3D tumor organoid culture, enabling a direct-interacting tumor organoid-immune cell coculture system. The self-organization process of tumor cells is facilitated by a vortex acoustic field, which enables the cell bioassembly and ion channel activation. This approach can significantly enhance the influx of calcium ions, thereby accelerating intercellular interactions of cellular assemblies. We established scaffold-free melanoma and breast cancer organoids using AV-Scaf and cocultured melanoma organoids with T cells. We found that our coculture system resulted in a high activation state of T cells, characterized by notable up-regulation of granzyme B (2.82 to 17.5%) and interferon-γ (1.36 to 16%). AV-Scaf offers an efficient method for tumor organoid-immune cell studies, advancing cancer research and immunotherapy development.

The intricate tumor microenvironment presents formidable obstacles to the efficacy of adoptive T cell therapy in the management of solid tumors by limiting the infiltration and inducing exhaustion of the transferred T cells. Here, we developed a bacterial-based adjuvant approach that augments the efficacy of adoptive T-cell therapy for solid tumor treatment. Our study reveals that intratumor injection of E. coli MG1655 normalizes tumor vasculatures and reprograms tumor-associated macrophages into M1 phenotype that produce abundant CCL5, together facilitating tumor infiltration of adoptively transferred T cells. The depletion of tumor-associated macrophages or CCL5 neutralization in vivo leads to the significantly decreased solid tumor infiltration of adoptive T cells in the presence of bacteriotherapy. This combinatorial therapy, consisting of E. coli adjuvant and adoptive T-cell therapy, effectively eradicates early-stage melanoma and inhibits the progression of pancreatic tumors. Notably, this dual strategy also strengthened the distal tumor control capabilities of adoptive T-cell therapy through the induction of in situ tumor vaccination. This dual therapeutic approach involving bacterial therapy targeting the interior of solid tumors and adoptive T-cell therapy attacking the tumor periphery exhibits potent therapeutic efficacy in achieving the eradication of advanced-stage tumors, including melanoma and hepatocellular carcinoma, by converging attacks from both inside and outside the tumor tissues., Competing Interests: Competing interests: Z.G. is the co-founder of Zenomics Inc., ZCapsule Inc., and µZen Pharma Co., Ltd. The other authors declare no competing interests., (© 2024. The Author(s).)

The application of chimeric antigen receptor (CAR) T cell therapy in solid tumors is hindered by life-threatening toxicities resulting from on-target, off-tumor killing of nonmalignant cells that express low levels of the target antigen. Mesothelin (MSLN) has been identified as a target antigen for CAR T cell treatment of mesothelioma, lung, ovarian, and other cancers because of its high expression on tumor cells and limited expression on mesothelial cells. However, fatal off-tumor toxicity of high-affinity MSLN-targeting CAR T cells has been reported in multiple clinical trials. In this study, we constructed CARs using mutant variants of a single-domain nanobody that bind both human and mouse MSLN with a wide range of affinities and examined tumor responses and their toxicities from on-target, off-tumor interactions in mouse models. CAR T cells with low nanomolar affinity (equilibrium dissociation constant, KD) exhibited profound systemic expansion with no apparent infiltration into the tumor. With a gradual reduction of CAR affinity toward the micromolar KD, the expansion of CAR T cells became more restricted to tumors. Our preclinical studies demonstrated that high-affinity MSLN CARs were associated with fatal on-target, off-tumor toxicity and that affinity-tuned CARs rendered T cells more selective for MSLN-high tumors.

Human cytomegalovirus (CMV) causes a common congenital infection leading to long-term neurological impairments including brain, cochlear, and ocular pathology. Infection of newborn mice with murine (M)CMV is an established model of neuropathology caused by congenital CMV infection, with recent work suggesting that brain pathology may be driven by immune responses. In the eye, however, CMV retinitis is thought to result from virus-driven necrosis in the absence of T cell responses. We found that MCMV infection of newborn mice recapitulates human eye disease after congenital CMV infection, including focal chorioretinitis, inflamed vasculature, and disrupted blood-retinal barriers. Moreover, infection drove extensive T cell infiltration of the retina and marked gliosis. Blocking immune responses generally, or via targeting the chemokine receptor CXCR3, did not exacerbate retinal disease but instead prevented pathology despite retinal MCMV infection. Thus, our data establish this model for studies of congenital retinal disease and show that the immune system drives pathology in the neonatal eye after MCMV infection.

Immune evasion by tumors is promoted by low T cell infiltration, ineffective T cell activity directed against the tumor, and reduced tumor antigen presentation. The TET2 DNA dioxygenase gene is frequently mutated in hematopoietic malignancies and loss of TET enzymatic activity is found in a variety of solid tumors. We showed previously that vitamin C (VC), a cofactor of TET2, enhances tumor-associated T cell recruitment and checkpoint inhibitor therapy responses in a TET2-dependent manner. Using single-cell RNA sequencing (scRNA-seq) analysis performed on B16-OVA melanoma tumors, we have shown here that an additional function for TET2 in tumors is to promote expression of certain antigen presentation machinery genes, which is potently enhanced by VC. Consistently, VC promoted antigen presentation in cell-based and tumor assays in a TET2-dependent manner. Quantifying intercellular signaling from the scRNA-seq dataset showed that T cell-derived IFN-γ-induced signaling within the tumor and tumor microenvironment requires tumor-associated TET2 expression, which is enhanced by VC treatment. Analysis of patient tumor samples indicated that TET activity directly correlates with antigen presentation gene expression and with patient outcomes. Our results demonstrate the importance of tumor-associated TET2 activity as a critical mediator of tumor immunity, which is augmented by high-dose VC therapy.

Calcium signaling plays a crucial role in the activation of T lymphocytes. However, modulating calcium levels to control T cell activation in vivo remains a challenge. In this study, we investigate T cell activation using 12-myristate 13-acetate (PMA)-encapsulated CaCO 3 nanoparticles. We find that anti-PD-1 antibody-conjugated CaCO 3 nanoparticles can be internalized by T cells via receptor-mediated endocytosis and then gradually release calcium. This results in an increase in cytosolic calcium, which triggers the activation of NFAT and NF-κB pathways, especially when the surface of the CaCO 3 nanoparticles is loaded with PMA. Animal studies demonstrate that the PMA-loaded calcium nanoparticles enhance the activation and proliferation of cytotoxic T cells, leading to improved tumor suppression without additional toxicity. When tested in metastatic tumor models, T cells loaded with the calcium nanoparticles prior to adoptive cell transfer control tumor growth better, resulting in prolonged animal survival. Our approach offers an alternative T cell activation strategy to potentiate immunotherapy by targeting a fundamental signaling pathway., Competing Interests: Competing interests: The University of Georgia has filed a patent application on the technology and intellectual property reported here for which W.Y., Z.C., and J.X. are inventors. The remaining authors declare no competing interests., (© 2024. The Author(s).)

In systemic lupus erythematosus, immune complexes deposited in the kidney vasculature represent a potent inflammatory trigger with a high potential to progress to glomerulonephritis and organ failure. These immune complexes can be recognized by multiple effector cells via complement and Fcγ receptors. The transcriptome of CD16-bearing NK cells has been documented in kidneys from patients with SLE. In this study, we show that NK cells accumulate in the kidney in response to immune complex deposition and modulate the behavior of local T cells. Depletion of NK cells transiently ameliorated disease, suggesting NK cells may play a role in lupus nephritis and other immune complex-mediated conditions., Competing Interests: The authors have no competing interests to declare., (Copyright: © 2024 De la Cruz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Oncofetal splice variants of extracellular matrix (ECM) proteins present a unique group of target antigens for the immunotherapy of pediatric cancers. However, limited data is available if these splice variants can be targeted with T cells expressing chimeric antigen receptors (CARs)., Methods: To determine the expression of the oncofetal version of tenascin C (TNC) encoding the C domain (C.TNC) in pediatric brain and solid tumors, we used quantitative reverse transcription PCR and immunohistochemistry. Genetically modified T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo., Results: We demonstrate that C.TNC is expressed on a protein level in pediatric tumors, including diffuse intrinsic pontine glioma, osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma. We generate C.TNC-CAR T cells and establish that these recognize and kill C.TNC-positive tumor cells. However, their antitumor activity in vivo is limited. To improve the effector function of C.TNC-CAR T cells, we design a leucine zipper-based chimeric cytokine receptor that activates interleukin-18 signaling pathways (Zip18R). Expression of Zip18R in C.TNC-CAR T cells improves their ability to secrete cytokines and expand in repeat stimulation assays. C.TNC-CAR.Zip18R T cells also have significantly greater antitumor activity in vivo compared with unmodified C.TNC-CAR T cells., Conclusions: Our study identifies the C domain of the ECM protein TNC as a promising CAR T-cell therapy for pediatric solid tumors and brain tumors. While we focus here on pediatric cancer, our work has relevance to a broad range of adult cancers that express C.TNC., Competing Interests: Competing interests: EW, SL, JW, TIS, GK, JZ, and SG have patent applications in the fields of cell or gene therapy for cancer. MB, GK, and SG are coinventors on a patent application for the developed Zip receptor technology. SG is a member of the Scientific Advisory Board of Be Biopharma and CARGO, and the Data and Safety Monitoring Board (DSMB) of Immatics and has received honoraria from TESSA Therapeutics within the last year. The other authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

NKG2D ligands (NKG2DLs) are broadly expressed in cancer. To target these, we describe an adaptor chimeric antigen receptor (CAR) termed NKG2D/Dap10-12. Herein, T cells are engineered to co-express NKG2D with a fusion protein that comprises Dap10 joined to a Dap12 endodomain. NKG2D/Dap10-12 T cells elicit compelling efficacy, eradicating or controlling NKG2DL-expressing tumors in several established xenograft models. Importantly, durable responses, long-term survival, and rejection of tumor re-challenge are reproducibly achieved. Efficacy is markedly superior to a clinical stage CAR analog, comprising an NKG2D-CD3ζ fusion. Structure-function analysis using an extended CAR panel demonstrates that potency is dependent on membrane proximity of signaling units, high NKG2D cell surface expression, adaptor structure, provision of exogenous Dap10, and inclusion of one rather than three immune tyrosine activation motifs per signaling unit. Potent therapeutic impact of NKG2D/Dap10-12 T cells is also underpinned by enhanced oxidative phosphorylation, reduced senescence, and transcriptomic re-programming for increased ribosomal biogenesis., Competing Interests: Declaration of interests J.M. is CSO, scientific founder, and shareholder of Leucid Bio. M.G., F.K., C.M.H., R.M., C.B., C.T., and D.M.D. are employees of Leucid Bio while P.D. is a former Leucid Bio employee. D.L.-Y. is undertaking a PhD studentship funded by Leucid Bio and has acted as a consultant for Leucid Bio. J.M., D.M.D., D.L.-Y., and F.K. are co-inventors on patent filings in relation to adaptor CAR technology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immune checkpoint blockade therapy has revolutionized cancer treatment, but resistance remains prevalent, often due to dysfunctional tumor-infiltrating lymphocytes. A key contributor to this dysfunction is mitochondrial dysfunction, characterized by defective oxidative phosphorylation, impaired adaptation, and depolarization, which promotes T cell exhaustion and severely compromises antitumor efficacy. This review summarizes recent advances in restoring the function of exhausted T cells through mitochondria-targeted strategies, such as metabolic remodeling, enhanced biogenesis, and regulation of antioxidant and reactive oxygen species, with the aim of reversing the state of T cell exhaustion and improving the response to immunotherapy. A deeper understanding of the role of mitochondria in T cell exhaustion lays the foundation for the development of novel mitochondria-targeted therapies and opens a new chapter in cancer immunotherapy., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Glioblastoma is the most common lethal primary brain tumor, urging evaluation of new treatment options. Chimeric antigen receptor (CAR)-T cells targeting B7 homolog 3 (B7-H3) are promising because of the overexpression of B7-H3 on glioblastoma cells but not on healthy brain tissue. Nanobody-based (nano)CARs are gaining increasing attention as promising alternatives to classical single-chain variable fragment-based (scFv)CARs, because of their single-domain nature and low immunogenicity. Still, B7-H3 nanoCAR-T cells have not been extensively studied in glioblastoma., Methods: B7-H3 nanoCAR- and scFvCAR-T cells were developed and evaluated in human glioblastoma models. NanoCAR-T cells targeting an irrelevant antigen served as control. T cell activation, cytokine secretion and killing capacity were evaluated in vitro using ELISA, live cell imaging and flow cytometry. Antigen-specific killing was assessed by generating B7-H3 knock-out cells using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-genome editing. The tumor tracing capacity of the B7-H3 nanobody was first evaluated in vivo using nuclear imaging. Then, the therapeutic potential of the nanoCAR-T cells was evaluated in a xenograft glioblastoma model., Results: We showed that B7-H3 nanoCAR-T cells were most efficient in lysing B7-H3 pos glioblastoma cells in vitro. Lack of glioblastoma killing by control nanoCAR-T cells and lack of B7-H3 neg glioblastoma killing by B7-H3 nanoCAR-T cells showed antigen-specificity. We showed in vivo tumor targeting capacity of the B7-H3 nanobody-used for the nanoCAR design-in nuclear imaging experiments. Evaluation of the nanoCAR-T cells in vivo showed tumor control in mice treated with B7-H3 nanoCAR-T cells in contrast to progressive disease in mice treated with control nanoCAR-T cells. However, we observed limiting toxicity in mice treated with B7-H3 nanoCAR-T cells and showed that the B7-H3 nanoCAR-T cells are activated even by low levels of mouse B7-H3 expression., Conclusions: B7-H3 nanoCAR-T cells showed promise for glioblastoma therapy following in vitro characterization, but limiting in vivo toxicity was observed. Off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells was identified as a potential cause for this toxicity, warranting caution when using highly sensitive nanoCAR-T cells, recognizing the low-level expression of B7-H3 on healthy tissue., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)