Characteristics of premanufacture CD8+T cells determine CAR-T efficacy in patients with diffuse large B-cell lymphoma.

Although chimeric antigen receptor (CAR) T cells have become an important treatment option for patients with relapsed/refractory B-cell malignancies, more than 60% of patients with diffuse large B-cell lymphoma (DLBCL) treated with CAR-T cell therapies fail to achieve a durable response. To reveal changes in CAR-T cell therapy and identify response biomarkers, we conducted a retrospective analysis of pre-manufacture source T cells and CAR-T cell products and their association with outcome in 58 patients with r/rDLBCL who received tandem CD19/CD20 CAR-T cell therapy. We performed bulk RNA-Seq, single-cell RNA-Seq, and paired T cell receptor sequencing on CAR-T cell products and pre-manufacture T cells from DLBCL patients. We note that a CD8+ stem cell-like memory T cell population with a higher proportion and enhanced activating capacity of the CAR-T cell products was key to achieving durable clinical response. By analysing autologously-derived, pre-manufacture T cells, our data suggest that heterogeneity in the cellular and molecular features of pre-manufacture T cells contribute to the variation in efficacy after CAR-T cell therapy in DLBCL. The differences in anti-tumour efficacy of CAR-T cells among patients with different clinical outcomes appear to be due to the loss of CCR7 gene expression, coupled with increased expression of activation- and inhibitor-related genes in the CD8+ naïve-T cell populations among the apheresis T cells from patients with a poor molecular response. These findings significantly advance our understanding of the underlying molecular determinants of pre-manufacture T cell function.

CD58 loss in tumor cells confers functional impairment of CAR T cells.

Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in treating a variety of hematologic malignancies, but resistance to this treatment in some patients limited its wider application. Using an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screening, we identified and validated loss of CD58 conferred immune evasion from CAR T cells in vitro and in vivo. CD58 is a ligand of the T-cell costimulatory molecule CD2, and CD58 mutation or downregulated expression is common in hematological tumors. We found that disruption of CD58 in tumor cells induced the formation of suboptimal immunological synapse (IS) with CAR T cells, which conferred functional impairment of CAR T cells, including the attenuation of cell expansion, degranulation, cytokine secretion, and cytotoxicity. In summary, we describe a potential mechanism of tumor-intrinsic resistance to CAR T-cell therapy and suggest that this mechanism may be leveraged for developing therapeutic strategies to overcome resistance to CAR T-cell therapy in B-cell malignancies.

Identification of NOXA as a pivotal regulator of resistance to CAR T-cell therapy in B-cell malignancies.

Despite the remarkable success of chimeric antigen receptor (CAR) T-cell therapy for treating hematologic malignancies, resistance and recurrence still occur, while the markers or mechanisms underlying this resistance remain poorly understood. Here, via an unbiased genome-wide CRISPR/Cas9 screening, we identified loss of NOXA, a B-cell lymphoma 2 (BCL2) family protein in B-cell malignancies, as a pivotal regulator of resistance to CAR T-cell therapy by impairing apoptosis of tumor cells both in vitro and in vivo. Notably, low NOXA expression in tumor samples was correlated with worse survival in a tandem CD19/20 CAR T clinical trial in relapsed/refractory B-cell lymphoma. In contrast, pharmacological augmentation of NOXA expression by histone deacetylase (HDAC) inhibitors dramatically sensitized cancer cells to CAR T cell-mediated clearance in vitro and in vivo. Our work revealed the essentiality of NOXA in resistance to CAR T-cell therapy and suggested NOXA as a predictive marker for response and survival in patients receiving CAR T-cell transfusions. Pharmacological targeting of NOXA might provide an innovative therapeutic strategy to enhance CAR T-cell therapy.

Inducing immunogenic cell death in immuno-oncological therapies.

Immunotherapy has revolutionized cancer treatment and substantially improved patient outcomes with respect to multiple types of tumors. However, most patients cannot benefit from such therapies, mainly due to the intrinsic low immunogenicity of cancer cells (CCs) that allows them to escape recognition by immune cells of the body. Immunogenic cell death (ICD), which is a form of regulated cell death, engages in a complex dialogue between dying CCs and immune cells in the tumor microenvironment (TME), ultimately evoking the damage-associated molecular pattern (DAMP) signals to activate tumor-specific immunity. The ICD inducers mediate the death of CCs and improve both antigenicity and adjuvanticity. At the same time, they reprogram TME with a "cold-warm-hot" immune status, ultimately amplifying and sustaining dendritic cell- and T cell-dependent innate sensing as well as the antitumor immune responses. In this review, we discuss how to stimulate ICD based upon the biological properties of CCs that have evolved under diverse stress conditions. Additionally, we highlight how this dynamic interaction contributes to priming tumor immunogenicity, thereby boosting anticancer immune responses. We believe that a deep understanding of these ICD processes will provide a framework for evaluating its vital role in cancer immunotherapy.

Low-dose decitabine priming endows CAR T cells with enhanced and persistent antitumour potential via epigenetic reprogramming.

Insufficient eradication capacity and dysfunction are common occurrences in T cells that characterize cancer immunotherapy failure. De novo DNA methylation promotes T cell exhaustion, whereas methylation inhibition enhances T cell rejuvenation in vivo. Decitabine, a DNA methyltransferase inhibitor approved for clinical use, may provide a means of modifying exhaustion-associated DNA methylation programmes. Herein, anti-tumour activities, cytokine production, and proliferation are enhanced in decitabine-treated chimeric antigen receptor T (dCAR T) cells both in vitro and in vivo. Additionally, dCAR T cells can eradicate bulky tumours at a low-dose and establish effective recall responses upon tumour rechallenge. Antigen-expressing tumour cells trigger higher expression levels of memory-, proliferation- and cytokine production-associated genes in dCAR T cells. Tumour-infiltrating dCAR T cells retain a relatively high expression of memory-related genes and low expression of exhaustion-related genes in vivo. In vitro administration of decitabine may represent an option for the generation of CAR T cells with improved anti-tumour properties.

Clinical development of CAR T cell therapy in China: 2020 update.

Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in the treatment of hematological malignancies. In recent years, fast-growing CAR T clinical trials have actively explored their potential application scenarios. According to the data from the clinicaltrials.gov website, China became the country with the most registered CAR T trials in September 2017. As of June 30, 2020, the number of registered CAR T trials in China has reached 357. In addition, as many as 150 other CAR T trials have been registered on ChiCTR. Although CAR T therapy is flourishing in China, there are still some problems that cannot be ignored. In this review, we aim to systematically summarize the clinical practice of CAR T-cell therapy in China. This review will provide an informative reference for colleagues in the field, and a better understanding of the history and current situation will help us more reasonably conduct research and promote cooperation.

Adaptive T cell immunotherapy in cancer.

Impaired tumor-specific effector T cells contribute to tumor progression and unfavorable clinical outcomes. As a compensatory T cell-dependent cancer immunoediting strategy, adoptive T cell therapy (ACT) has achieved encouraging therapeutic results, and this strategy is now on the center stage of cancer treatment and research. ACT involves the ex vivo stimulation and expansion of tumor-infiltrating lymphocytes (TILs) with inherent tumor reactivity or T cells that have been genetically modified to express the cognate chimeric antigen receptor or T cell receptor (CAR/TCR), followed by the passive transfer of these cells into a lymphodepleted host. Primed T cells must provide highly efficient and long-lasting immune defense against transformed cells during ACT. Anin-depth understanding of the basic mechanisms of these living drugs can help us improve upon current strategies and design better next-generation T cell-based immunotherapies. From this perspective, we provide an overview of current developments in different ACT strategies, with a focus on frontier clinical trials that offer a proof of principle. Meanwhile, insights into the determinants of ACT are discussed, which will lead to more rational, potent and widespread applications in the future.

The model of cytokine release syndrome in CAR T-cell treatment for B-cell non-Hodgkin lymphoma.

Chimeric antigen receptor T (CAR T) cell therapy has demonstrated efficacy in the treatment of haematologic malignancies. However, the accompanying adverse events, the most common of which is cytokine release syndrome (CRS), substantially limit its wide application. Due to its unique physiological characteristics, CRS in CAR T-cell treatment for B-cell non-Hodgkin lymphoma (B-NHL) may exhibit some special features. Although existing guidelines had greatly promoted the recognition and management of CRS, many recommendations are not fully applicable to B-NHL. Therefore, it is imperative to identify responses that are specific to CRS observed following CAR T treatment for B-NHL. Based on underlying biological processes and known pathophysiological mechanisms, we tentatively propose a new model to illustrate the occurrence and evolution of CAR T-cell-therapy-related CRS in B-NHL. In this model, tumour burden and bone marrow suppression are considered determinants of CRS. Novel phenomena after CAR T-cell infusion (such as local inflammatory response) are further identified. The proposed model will help us better understand the basic biology of CRS and recognize and manage it more rationally.

Multi-antigen-targeted chimeric antigen receptor T cells for cancer therapy.

The approval of two chimeric antigen receptor-modified T cell types by the US Food and Drug Administration (FDA) for the treatment of hematologic malignancies is a milestone in immunotherapy; however, the application of CAR-T cells has been limited by antigen escape and on-target, off-tumor toxicities. Therefore, it may be a potentially effective strategy to select appropriate targets and to combine multi-antigen-targeted CAR-T cells with "OR", "AND" and "NOT" Boolean logic gates. We summarize the current limitations of CAR-T cells as well as the efficacy and safety of logic-gated CAR-T cells in antitumor therapy. This review will help to explore more optimized strategies to expand the CAR-T cell therapeutic window.

PD-1 silencing impairs the anti-tumor function of chimeric antigen receptor modified T cells by inhibiting proliferation activity.

BACKGROUND: Blocking programmed death-1 (PD-1) is considered to be a promising strategy to improve T cell function, and this is being explored in many ongoing clinical trials. In fact, our knowledge about PD-1 is primarily based on the results of short-term experiments or observations, but how long-lasting PD-1 blockade can affect T cell function remains unclear. METHODS: We planned to use shRNA-based gene knockdown technology to mimic long-lasting PD-1 blockade. We constructed PD-1 steadily blocked chimeric antigen receptor modified T (CAR-T) cells, and with these cells we can clearly study the effects of PD-1 knockdown on T cell function. The anti-tumor function, proliferation ability and differentiation status of PD-1 silenced CAR-T cells were studied by in vitro and animal experiments. RESULTS: According to short-term in vitro results, it was reconfirmed that the resistance to programmed death-ligand 1 (PD-L1)-mediated immunosuppression could be enhanced by PD-1 blockade. However, better anti-tumor function was not presented by PD-1 blocked CAR-T cells in vitro or in vivo experiments. It was found that PD-1 knockdownmight impair the anti-tumor potential of CAR-T cells because it inhibited T cells' proliferation activity. In addition, we observed that PD-1 blockade would accelerate T cells' early differentiation and prevent effector T cells from differentiating into effect memory T cells, and this might be the reason for the limited proliferation of PD-1 silenced CAR-T cells. CONCLUSION: These results suggest that PD-1 might play an important role in maintaining the proper proliferation and differentiation of T cells, and PD-1 silencing would impair T cells' anti-tumor function by inhibiting their proliferation activity.

Target selection for CAR-T therapy.

Chimeric antigen receptor-modified T (CAR-T) cells have achieved significant success in the treatment of several hematological malignancies. However, the translation of the existing achievements into the treatment of other tumors, especially solid tumors, is not smooth. In addition to the optimization of CAR structures, preparation, and clinical protocols, rational selecting and utilizing the targets was more pivotal. In this review, the criteria for target selection and some new strategies for targets utilization were summarized and discussed. This systematic review will help researchers better understand how the efficacy and safety of CAR-T treatment would be affected by targets and thus more rationally select targets and conduct clinical trials.

Collagen scaffold microenvironments modulate cell lineage commitment for differentiation of bone marrow cells into regulatory dendritic cells.

The microenvironment plays a pivotal role for cell survival and functional regulation, and directs the cell fate determination. The biological functions of DCs have been extensively investigated to date. However, the influences of the microenvironment on the differentiation of bone marrow cells (BMCs) into dendritic cells (DCs) are not well defined. Here, we established a 3D collagen scaffold microenvironment to investigate whether such 3D collagen scaffolds could provide a favourable niche for BMCs to differentiate into specialised DCs. We found that BMCs embedded in the 3D collagen scaffold differentiated into a distinct subset of DC, exhibiting high expression of CD11b and low expression of CD11c, co-stimulator (CD40, CD80, CD83, and CD86) and MHC-II molecules compared to those grown in 2D culture. DCs cultured in the 3D collagen scaffold possessed weak antigen uptake ability and inhibited T-cell proliferation in vitro; in addition, they exhibited potent immunoregulatory function to alleviate allo-delay type hypersensitivity when transferred in vivo. Thus, DCs differentiated in the 3D collagen scaffold were defined as regulatory DCs, indicating that collagen scaffold microenvironments probably play an important role in modulating the lineage commitment of DCs and therefore might be applied as a promising tool for generation of specialised DCs.

Spotlight on chimeric antigen receptor engineered T cell research and clinical trials in China.

T cell mediated adoptive immune response has been characterized as the key to anti-tumor immunity. Scientists around the world including in China, have been trying to harness the power of T cells against tumors for decades. Recently, the biosynthetic chimeric antigen receptor engineered T cell (CAR-T) strategy was developed and exhibited encouraging clinical efficacy, especially in hematological malignancies. Chimeric antigen receptor research reports began in 2009 in China according to our PubMed search results. Clinical trials have been ongoing in China since 2013 according to the trial registrations on clinicaltrials. gov.. After years of assiduous efforts, research and clinical scientists in China have made their own achievements in the CAR-T therapy field. In this review, we aim to highlight CAR-T research and clinical trials in China, to provide an informative reference for colleagues in the field.

The miR-20-Rest-Wnt signaling axis regulates neural progenitor cell differentiation.

Increasing evidence suggests that three dimensional (3-D) cell cultures are an improvement over traditional two dimensional (2-D) cell cultures. Current researches have extensively focused on the study of utilizing biomaterial-based 3-D culture systems to study and direct stem-cell fate both in vitro and in vivo. Here in our study, we screened the differential expression patterns of miRNAs between 2-D cultured and 3-D cultured NPCs using microarray analysis. Among these differentially expressed miRNAs, miR-20 was found to increase during differentiation of NPCs. Specifically, the facilitative effect on neural differentiation of miR-20 is mediated, at least in part by directly target the Rest gene, which is essential for preventing neural differentiation and maintaining NPCs self-renewal. Furthermore, the expression of miR-20 was decreased when the WNT pathway was inhibited by knock down of ß-catenin or by exogenous Dkk protein, whereas it increased when the WNT pathway was activated by exogenous Wnt3a protein. Overall, miR-20, Rest and Wnt signaling are suggested to be involved in a regulatory circuit that can modulate the neural differention of NPCs. This novel regulatory circuit provides additional insight into how microRNAs interact with signaling molecules during neural differentiation of NPCs, allowing for fine-tuning of intricate cellular processes.

Functionalized collagen scaffold implantation and cAMP administration collectively facilitate spinal cord regeneration.

Previous studies have demonstrated that several mechanisms, including numerous inhibitory molecules, weak neurotrophic stimulation and deficient intrinsic regenerative responses, collectively contribute to the failure of mature spinal cord axon regeneration. Thus, combinatorial therapies targeting multiple mechanisms have attracted much attention. In the present study, a porous collagen scaffold was used to support neuronal attachment and bridge axonal regeneration. The scaffold was specifically functionalized using neutralizing proteins (CBD-EphA4LBD, CBD-PlexinB1LBD and NEP1-40) and collagen-binding neurotrophic factors (CBD-BDNF and CBD-NT3) to simultaneously antagonize myelin inhibitory molecules (ephrinB3, Sema4D and Nogo) and exert neurotrophic protection and stimulation. Cerebellar granular neurons cultured on the functionalized collagen scaffold promoted neurite outgrowth in the presence of myelin. Furthermore, a full combinatorial treatment comprising functionalized scaffold implantation and cAMP administration was developed to evaluate the synergistic repair ability in a rat T10 complete removal spinal cord injury model. The results showed that full combinatorial therapy exhibited the greatest advantage in reducing the volume of cavitation, facilitating axonal regeneration, and promoting neuronal generation. The newborn neurons generated in the lesion area could form the neuronal relay and enhance the locomotion recovery after severe spinal cord injury. STATEMENT OF SIGNIFICANCE: A porous collagen scaffold was specifically functionalized with neutralizing proteins and neurotrophic factors to antagonize the myelin inhibitory molecules and exert neurotrophic protection and stimulation for spinal cord regeneration. Cerebellar granular neurons seeded on the functionalized collagen scaffold showed enhanced neurite outgrowth ability in vitro. The functionalized scaffold implantation combined with cAMP administration exhibited synergistic repair ability for rat T10 complete spinal cord transection injury.

Functionalized Collagen Scaffold Neutralizing the Myelin-Inhibitory Molecules Promoted Neurites Outgrowth in Vitro and Facilitated Spinal Cord Regeneration in Vivo.

Research has demonstrated that many myelin-associated inhibitory molecules jointly contribute to the failure of adult spinal cord regeneration. Therapies comprehensively targeting the multiple inhibitory nature of the injured spinal cord are being concerned. Here, two collagen-binding proteins, CBD-EphA4LBD and CBD-PlexinB1LBD, were constructed, respectively, to neutralize the axon guidance molecules ephrinB3 and sema4D that inhibit the regeneration of nerve fibers. The two neutralizing proteins have proven their ability to specifically bind collagen and to continuously release from collagen scaffolds. They could also promote neurites outgrowth of cerebellar granular neurons and dorsal root ganglion neurons in vitro. Subsequently, the functionalized collagen scaffolds by physically absorbing NEP1-40 and immobilizing CBD-EphA4LBD and CBD-PlexinB1LBD were transplanted into a rat T10 complete spinal cord transection model. Our results showed that rats that received the treatment of transplanting the functionalized collagen scaffold exhibited great advantage on axonal regeneration and locomotion recovery after spinal cord injury.

The importance of three-dimensional scaffold structure on stemness maintenance of mouse embryonic stem cells.

Revealing the mechanisms of cell fate regulation is important for scientific research and stem cell-based therapy. The traditional two-dimensional (2D) cultured mES cells are in a very different 2D niche from the in vivo equivalent-inner cell mass (ICM). Because the cell fate decision could be regulated by many cues which could be impacted by geometry, the traditional 2D culture system would hamper us from understanding the in vivo situations correctly. Three-dimensional (3D) scaffold was believed to provide a 3D environment closed to the in vivo one. In this work, three different scaffolds were prepared for cell culture. Several characters of mES cells were changed under 3D scaffolds culture compared to 2D, and these changes were mainly due to the alteration in geometry but not the matrix. The self-renewal of mES cells was promoted by the introducing of dimensionality. The stemness maintenance of mES was supported by all three 3D scaffolds without feeder cells in the long-time culture. Our findings demonstrated that the stemness maintenance of mES cells was promoted by the 3D geometry of scaffolds and this would provide a promising platform for ES cell research.

The miR-7 identified from collagen biomaterial-based three-dimensional cultured cells regulates neural stem cell differentiation.

Increasing evidence suggests that three-dimensional (3D) cultures provide more appropriate microenvironments to control stem cell response compared with traditional two-dimensional (2D) cultures. However, the molecular mechanism involved in 3D cultured stem cells is not well known. Several microRNAs whose target genes involved in the regulation of self-renewal and differentiation of stem cells were found to be downregulated in 3D cultured PA-1 cells. Among them, miR-7 was predicted to target Kruppel-like factor 4 (Klf4), a key gene for self-renewal of neural stem cells (NSCs). We showed that the differentiation of NSCs was inhibited in 3D collagen scaffolds compared with 2D cultured cells. The quantitative real-time PCR (qPCR) analysis indicated that the expression of miR-7 and Klf4 changed significantly in 2D cultures, whereas the expression stability of miR-7 and Klf4 was detected in 3D cultures. Using luciferase assay and western blot, Klf4 was identified as a target of miR-7 indicating that miR-7 plays a critical role in maintaining the self-renewal capacity through a Klf4-dependent mechanism in 3D cultured cells. Thus, the collagen scaffold-based 3D cell cultures may provide a platform to reveal the regulatory mechanism of cell regulators, which are difficult to find in traditional 2D cell cultures.