Cells For Adoptive Immunotherapy Research Articles

Bioceramics Enhance the Anti-Tumor Activity of Immune Cells in Adoptive Immunotherapy.

Recent research has focused on immunotherapy with no side effects as an innovative medical treatment for cancer. However, typical drugs for immunotherapy are very expensive. Here, we propose the use of immunoceramics that activate immune cells by contact with their surface. Previous studies demonstrated that polymers, including the phenylboronic acid group, could activate lymphocytes. This activation may be due to the interaction between the sugar chains in cells and the OH group in B(OH)3 formed via the dissociation of the BO2 group. We have clarified that boron-containing apatite (BAp) activated lymphocytes in vitro. In this study, we fabricated the ceramic surfaces using the CaO-P2O5-SiO2-B2O3 system (CPSB ceramics) containing BAp as a main crystalline phase. The results of the in vitro evaluation indicated that killer T cells in splenocytes cocultured with the CPSB ceramics were more numerous than in splenocytes cocultured on a control surface. The results of the in vivo evaluation indicated that the CPSB ceramics significantly inhibited tumor growth when CD8-positive T cells were cultured on individual ceramics and subsequently injected into tumor-bearing mice. The present CPSB ceramics are expected to be a valuable biomaterial for immunotherapy.

NKG2A genetic deletion promotes human primary NK cell anti-tumor responses better than an anti-NKG2A monoclonal antibody

NK cells eliminate infected or cancer cells via their cytotoxic capacity. NKG2A is an inhibitory receptor on NK cells and cancer cells often overexpress its ligand HLA-E to evade NK cell surveillance. Given the successes of immune checkpoint blockade in cancer therapy, NKG2A is an interesting novel target. However, anti-NKG2A antibodies have shown limited clinical response. In the pursuit of enhancing NK cell-mediated anti-tumor responses, we devised a Cas9-based strategy to delete KLRC1, encoding NKG2A, in human primary NK cells. Our approach involved electroporation of KLRC1-targeting Cas9-ribonucleoprotein resulting in effective ablation of NKG2A expression. Compared to anti-NKG2A antibody blockade, NKG2A-knockout NK cells exhibited enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors. NKG2A-deficient NK cells overcame inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells. We validated this efficacy across multiple cell lines, a xenograft mouse model, and primary human leukemic cells. Combining NKG2A knockout with antibody-coating of tumor cells further enhanced cytotoxicity through ADCC. Thus, we provide a comprehensive comparison of inhibition of the NKG2A pathway using genetic ablation and antibodies and provide novel insight in the observed differences molecular mechanisms, which can be translated to enhance adoptive NK cell immunotherapy.

Feasibility of Using Adoptive Cell Immunotherapy to Target Differentiation of CD4+T Cells and Co-culture of DC-CIK to Kill Tumor in Vitro

Feasibility of Using Adoptive Cell Immunotherapy to Target Differentiation of CD4+T Cells and Co-culture of DC-CIK to Kill Tumor in Vitro

OS-113 Adoptive natural killer cell immunotherapy can increase the recurrence-free survival following transarterialchemoembolization in patients with intermediate stage hepatocellular carcinoma

OS-113 Adoptive natural killer cell immunotherapy can increase the recurrence-free survival following transarterialchemoembolization in patients with intermediate stage hepatocellular carcinoma

Natural killer cell-related anti-tumour adoptive cell immunotherapy.

Chimeric antigen receptor- (CAR-)modified T cells have been successfully used to treat blood cancer. With the improved research on anti-tumour adoptive cell therapy, researchers have focused on immune cells other than T lymphocytes. Natural killer (NK) cells have received widespread attention as barriers to natural immunity. Compared to T lymphocyte-related adoptive cell therapy, the use of NK cells to treat tumours does not cause graft-versus-host disease, significantly improving immunity. Moreover, NK cells have more sources than T cells, and the related modified cells are less expensive. NK cells function through several pathways in anti-tumour mechanisms. Currently, many anti-tumour clinical trials have used NK cell-related adoptive cell therapies. In this review, we have summarized the recent progress in NK cell-related adoptive cellular immunotherapy for tumour treatment and propose the current challenges faced by CAR-NK cell therapy.

Cancer biotherapy: review and prospect.

Malignant tumors pose a grave threat to the quality of human life. The prevalence of malignant tumors in China is steadily rising. Presently, clinical interventions encompass surgery, radiotherapy, and pharmaceutical therapy in isolation or combination. Nonetheless, these modalities fail to completely eradicate malignant tumor cells, frequently leading to metastasis and recurrence. Conversely, tumor biotherapy has emerged as an encouraging fourth approach in preventing and managing malignant tumors owing to its safety, efficacy, and minimal adverse effects. Currently, a range of tumor biotherapy techniques are employed, including gene therapy, tumor vaccines, monoclonal antibody therapy, cancer stem cell therapy, cytokine therapy, and adoptive cellular immunotherapy. This study aims to comprehensively review the latest developments in biological treatments for malignant tumors.

Venetoclax acts as an immunometabolic modulator to potentiate adoptive NK cell immunotherapy against leukemia

Natural killer (NK) cell-based immunotherapy holds promise for cancer treatment; however, its efficacy remains limited, necessitating the development of alternative strategies. Here, we report that venetoclax, an FDA-approved BCL-2 inhibitor, directly activates NK cells, enhancing their cytotoxicity against acute myeloid leukemia (AML) both invitro and invivo, likely independent of BCL-2 inhibition. Through comprehensive approaches, including bulk and single-cell RNA sequencing, avidity measurement, and functional assays, we demonstrate that venetoclax increases the avidity of NK cells to AML cells and promotes lytic granule polarization during immunological synapse (IS) formation. Notably, we identify a distinct CD161lowCD218b+ NK cellsubpopulation that exhibits remarkable sensitivity to venetoclax treatment. Furthermore, venetoclax promotes mitochondrial respiration and ATP synthesis via the NF-κB pathway, thereby facilitating IS formation in NK cells. Collectively, our findings establish venetoclax as a multifaceted immunometabolic modulator of NK cell function and provide a promising strategy for augmenting NK cell-based cancer immunotherapy.

Redirecting NK cells to the lymph nodes to augment their lymphoma-targeting capacity

CAR-NK cells can induce remission in lymphoma patients. We speculate that the full potential of adoptive NK cell immunotherapy against lymphoma is restricted by their poor lymph node (LN) homing capacity. Here, we have utilized a clinically approved transfection method with the aim of redirecting NK cells to LNs. Electroporation of ex vivo expanded NK cells with mRNAs coding for CCR7, CXCR5, and CD62L resulted in increased in vitro migration towards chemokines and mouse LN-derived supernatant. Following infusion into SCID/Beige mice, modified NK cells showed enhanced LN homing. Importantly, lymphoma patient-derived NK cells were equally well expanded and engineered as healthy donor NK cells, highlighting their translational potential. Additionally, the introduction of high-affinity CD16, together with the homing molecules, also augmented their ADCC capacity against autologous lymphoma cells. Hence, genetic engineering can be utilized to enhance NK cell LN homing. The homing concept may synergize with CAR- or monoclonal/bi-/tri-specific antibody-based approaches.

Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4- subset that has a TH1/cytolytic profile, and a CD4+ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4- iNKT cells limited growth of CD1d+ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4+ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4+ iNKT cells. Immunotherapeutic CD4+ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4+ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4+ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

Efficacy and safety of bispecific antibodies vs. immune checkpoint blockade combination therapy in cancer: a real-world comparison.

Emerging tumor immunotherapy methods encompass bispecific antibodies (BSABs), immune checkpoint inhibitors (ICIs), and adoptive cell immunotherapy. BSABs belong to the antibody family that can specifically recognize two different antigens or epitopes on the same antigen. These antibodies demonstrate superior clinical efficacy than monoclonal antibodies, indicating their role as a promising tumor immunotherapy option. Immune checkpoints are also important in tumor immunotherapy. Programmed cell death protein-1 (PD-1) is a widely acknowledged immune checkpoint target with effective anti-tumor activity. PD-1 inhibitors have demonstrated notable therapeutic efficacy in treating hematological and solid tumors; however, more than 50% of patients undergoing this treatment exhibit a poor response. However, ICI-based combination therapies (ICI combination therapies) have been demonstrated to synergistically increase anti-tumor effects and immune response rates. In this review, we compare the clinical efficacy and side effects of BSABs and ICI combination therapies in real-world tumor immunotherapy, aiming to provide evidence-based approaches for clinical research and personalized tumor diagnosis and treatment.

XELOX (capecitabine plus oxaliplatin) plus bevacizumab (anti-VEGF-A antibody) with or without adoptive cell immunotherapy in the treatment of patients with previously untreated metastatic colorectal cancer: a multicenter, open-label, randomized, controlled, phase 3 trial

Fluoropyrimidine-based combination chemotherapy plus targeted therapy is the standard initial treatment for unresectable metastatic colorectal cancer (mCRC), but the prognosis remains poor. This phase 3 trial (ClinicalTrials.gov: NCT03950154) assessed the efficacy and adverse events (AEs) of the combination of PD-1 blockade-activated DC-CIK (PD1-T) cells with XELOX plus bevacizumab as a first-line therapy in patients with mCRC. A total of 202 participants were enrolled and randomly assigned in a 1:1 ratio to receive either first-line XELOX plus bevacizumab (the control group, n = 102) or the same regimen plus autologous PD1-T cell immunotherapy (the immunotherapy group, n = 100) every 21 days for up to 6 cycles, followed by maintenance treatment with capecitabine and bevacizumab. The main endpoint of the trial was progression-free survival (PFS). The median follow-up was 19.5 months. Median PFS was 14.8 months (95% CI, 11.6–18.0) for the immunotherapy group compared with 9.9 months (8.0–11.8) for the control group (hazard ratio [HR], 0.60 [95% CI, 0.40–0.88]; p = 0.009). Median overall survival (OS) was not reached for the immunotherapy group and 25.6 months (95% CI, 18.3–32.8) for the control group (HR, 0.57 [95% CI, 0.33–0.98]; p = 0.043). Grade 3 or higher AEs occurred in 20.0% of patients in the immunotherapy group and 23.5% in the control groups, with no toxicity-associated deaths reported. The addition of PD1-T cells to first-line XELOX plus bevacizumab demonstrates significant clinical improvement of PFS and OS with well tolerability in patients with previously untreated mCRC.

Cytokine-induced killer (CIK) cells, successes and challenges: report on the first international conference dedicated to the clinical translation of this unique adoptive cell immunotherapy

On August 30, 2023, experts from Germany and abroad met to discuss the successes and challenges of cytokine-induced killer cell (CIK) therapy, that recently celebrated its 30th anniversary providing treatment for cancer. This first virtual conference was hosted by CIO Bonn, a certified Comprehensive Cancer Center (CCC) funded by German Cancer Aid (DKH). In addition to keynote speakers involved in CIK cell clinical trials or optimized preclinical models to improve this adoptive cell immunotherapy, more than 100 attendees from around the world also participated in this event. Initiatives to establish the International Society of CIK Cells (ISCC) and a stronger CIK cell network guiding preclinical research and future clinical trials were also announced.

Engineering of CD34+ progenitor-derived natural killer cells with higher-affinity CD16a for enhanced antibody-dependent cellular cytotoxicity

Background aimsNatural killer (NK) cell transfer is a promising cellular immunotherapy for cancer. Previously, we developed a robust method to generate large NK cell numbers from CD34+ hematopoietic stem and progenitor cells (HSPCs), which exhibit strong anti-tumor activity. However, since these cells express low levels of the Fc receptor CD16a in vitro, antibody-dependent cellular cytotoxicity (ADCC) by these cells is limited. To broaden clinical applicability of our HSPC-NK cells toward less NK-sensitive malignancies, we aimed to improve ADCC through CD16a transduction. MethodsUsing wildtype and S197P mutant greater-affinity (both with V158) CD16a retroviral transgenes (i.e., a cleavable and noncleavable CD16a upon stimulation), we generated CD16a HSPC-transduced NK cells, with CD34+ cells isolated from umbilical cord blood (UCB) or peripheral blood after G-CSF stem cell mobilization (MPB). CD16a expressing NK cells were enriched using flow cytometry–based cell sorting. Subsequently, phenotypic analyses and functional assays were performed to investigate natural cytotoxicity and ADCC activity. ResultsMean transduction efficiency was 34% for UCB-derived HSPCs and 20% for MPB-derived HSPCs, which was enriched by flow cytometry-based cell sorting to >90% for both conditions. Expression of the transgene remained stable during the entire NK expansion cell generation process. Proliferation and differentiation of HSPCs were not hampered by the transduction process, resulting in effectively differentiated CD56+ NK cells after 5 weeks. Activation of the HSPC-derived NK cells resulted in significant shedding of wildtype CD16a transcribed from the endogenous gene, but not of the noncleavable mutant CD16a protein expressed from the transduced construct. The mean increase of CD107+IFNγ+ expressing NK cells after inducing ADCC was tenfold in enriched noncleavable CD16a HSPC-NK cells. Killing capacity of CD16a-transduced NK cells was significantly improved after addition of a tumor-targeting antibody in tumor cell lines and primary B-cell leukemia and lymphoma cells compared to unmodified HSPC-NK cells. ConclusionsTogether, these data demonstrate that the applicability of adoptive NK cell immunotherapy may be broadened to less NK-sensitive malignancies by upregulation of CD16a expression in combination with the use of tumor-targeting monoclonal antibodies.

Development of Novel Invariant TCR-Anchored Bispecific Engagers to Enhance Anti-Cancer Activity of iNKT Cells

iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. iNKT cells are characterised by an invariant TCRVa24Ja18 chain nearly always pairing with a TCRVb11 chain (iTCR). The ability of allogeneic iNKT to protect from aGVHD makes them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. Indeed, CAR-iNKT show promise in pre-clinical models of blood cancer as well as early clinical development against B cell lymphoma and outperform CAR-T counterparts. Here we aimed to enhance the modularity of adoptive iNKT cell immunotherapy for blood cancers by designing a bispecific iNKT cell engager (biNTe) in which one arm would engage the iTCR while the other arm would engage tumour antigen(s) of interest. As a proof-of-principle we designed and synthesised a mono- and a bi-valent biNTe, one each, against the clinically validated myeloma target BCMA with purities of 90% and 70% respectively. We focused on the bivalent biNTe which we first used to demonstrate its iNKT cell binding specificity. Indeed, in flow-cytometry assays, biNTe followed by fluorescent anti-human Fc Ab, stained purified iNKT and but not T cells while in the presence of the same iTCR-binding mAb used to generate the biNTes, binding of the bivalent biNTe on iNKT cells was abrogated. This shows the iTCR engaging specificity of the bivalent biNTe. To demonstrate BCMA specificity of the other arm of the biNTe, we used it to stain iNKT cells, followed by staining with biotinylated soluble BCMA and fluorescently labelled streptavidin. This, compared to staining without soluble BCMA, showed strong staining of iNKT cells thus validating the BCMA specificity of the bivalent biNTe. Next, we set up co-cultures of pre-expanded iNKT against the BCMA-expressing and -knockout myeloma cell line MM1.S in the presence of varying concentrations of the biNTe. While, as assessed by a flow-cytometric cytotoxicity assay, only background killing of myeloma cells was observed in the absence of biNTe or when BCMA ko cell were used as targets, in the presence of biNTe, BCMA+ MM1S cells were killed, in a dose dependent manner (Figure 1A). Consistent with iNKT specificity of the biNTe similar co-cultures involving T cells showed only background killing of MM1S cells (Figure 1B). The same results were obtained using the BCMA-expressing myeloma cell line H929 and its corresponding BCMA knockout. The overall estimated EC50 for MM1.S and H929 cells were 6.56 and 3.4 nM respectively. Using the EC50 concentrations we show that biNTe very effectively kill and almost eliminate patient-derived bone marrow myeloma plasma cells (n=3). To test whether engagement of the iTCR by biNTe would impact its ability to engage with CD1d, we co-cultured iNKT and MM1.S cells transduced with CD1D in the presence of biNTe and the activating, iNKT-specific, CD1d-binding glycolipid ligand a-galactosylceramide (aGalCer). We found that compared to no biNTe control and at biNTE concentrations lower than the EC50, killing of myeloma targets cells was consistently increased. This suggests that iTCR retains its ability to engage CD1d and the efficacy of future biNTe-based immunotherapy could be enhanced by aGalCer. While in vivo validation experiments are ongoing, we conclude that biNTe constitute a novel therapeutic approach with notable pre-clinical anti-myeloma activity. We envisage that they can be used in conjunction with other therapeutic modalities such as CAR and iTCR to enhance the modularity and the efficacy of iNKT cells as an ‘off -the-shelf’ platform for the treatment of myeloma, other blood or solid tumour cancers.

Immune Function of Chimeric Antigen Receptor T Cells Quantitatively Assessed Via Molecular Imaging Flow Cytometry

Chimeric antigen receptor (CAR) T cells are an adoptive cell immunotherapy genetically synthesized to reprogram T cells for antigen recognition and intracellular signaling to target tumor cells for destruction. A sensitive and reproducible assay to evaluate CAR T cell function is essential to research and develop novel therapies, optimize manufacturing, and evaluate efficacy in the clinical and non-clinical settings. However, conventional methods, such as cytotoxicity assays and in vitro cytokine production, require a long cell culture processing time with a less quantitative and more time-consuming variability. In the present study, we analyzed the change in localization of CAR molecules immediately following antigen recognition using molecular imaging flow cytometry (MI-FCM) to establish a quantitative method to evaluate the immune functionality of CAR T cells, which is correlated with cytotoxic activity. Anti-CD19 CAR (FMC63-28z) was constructed using anti-CD19 scFv (FMC63) based on the method described by Kochenderfer et al. (J Immunol, 2009). CAR T cells were generated by isolating human peripheral blood mononuclear cells from nine healthy donors (21-69 years old). They were then stimulated on plates pre-coated with CD3 and CD28 antibodies in ALys505N-7 medium containing 700 IU/mL IL-2, transfected with anti-CD19 CAR using a lentivirus on day 3, and subsequently expanded on plates pre-coated with CD3 and CD28 antibodies for maximum to 18 days. Second, each CAR T cell was stimulated with or without CD19 antigen, as well as the K562 cell line stably transfected with CD19 (K562-CD19), fixed with paraformaldehyde, and immunostained with anti-CAR, CD4, and CD8 antibodies. We defined the decrease in the CAR molecule area of expression on the cell surface as concentrated CAR. The existence and number of spots of concentrated CAR on CD4- or CD8-positive CAR T cells were analyzed using MI-FCM. The concentration of superficial CAR antigens on some CAR T cells was observed in every lot from the nine healthy donors when they were stimulated with K562-CD19 cells or CD19 antigen. In contrast, this was not observed in the unstimulated controls. When each lot of CAR T cells was stimulated with CD19 antigen and then quantitatively assessed using MI-FCM, the proportion of cells with concentrated CAR among the CD4-positive and CD8-positive CAR T cells was 83.1 ± 11.7% and 81.3 ± 8.1%, respectively, while the proportion of cells with a single spot fraction of concentrated CAR was 31.2 ± 4.0% and 33.8 ± 6.1%, respectively. Third, the cytotoxic activity of each lot of CAR T cells co-cultured with K562-CD19 cells transfected with the firefly luciferase reporter gene for 15 h was evaluated using a luciferase-mediated bioluminescence imaging assay. Importantly, the proportion of single-spot concentrated CAR among CD8-positive CAR T cells was very strongly positively correlated with killing activity (R = 0.916; 95% CI: 0.644-0.982), while the proportion of single-spot concentrated CAR among CD4-positive CAR T cells was significantly positive correlated (R = 0.693; 95% CI: 0.053-0.930). These results suggest that quantifying the CAR concentration in CAR T cells using MI-FCM could be used to evaluate their immune function.

Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells.

Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.

Adoptive cell immunotherapy for breast cancer: harnessing the power of immune cells.

Breast cancer is the most prevalent malignant neoplasm worldwide, necessitating the development of novel therapeutic strategies owing to the limitations posed by conventional treatment modalities. Immunotherapy is an innovative approach that has demonstrated significant efficacy in modulating a patient's innate immune system to combat tumor cells. In the era of precision medicine, adoptive immunotherapy for breast cancer has garnered widespread attention as an emerging treatment strategy, primarily encompassing cellular therapies such as tumor-infiltrating lymphocyte therapy, chimeric antigen receptor T/natural killer/M cell therapy, T cell receptor gene-engineered T cell therapy, lymphokine-activated killer cell therapy, cytokine-induced killer cell therapy, natural killer cell therapy, and γδ T cell therapy, among others. This treatment paradigm is based on the principles of immune memory and antigen specificity, involving the collection, processing, and expansion of the patient's immune cells, followed by their reintroduction into the patient's body to activate the immune system and prevent tumor recurrence and metastasis. Currently, multiple clinical trials are assessing the feasibility, effectiveness, and safety of adoptive immunotherapy in breast cancer. However, this therapeutic approach faces challenges associated with tumor heterogeneity, immune evasion, and treatment safety. This review comprehensively summarizes the latest advancements in adoptive immunotherapy for breast cancer and discusses future research directions and prospects, offering valuable guidance and insights into breast cancer immunotherapy.

The prospect of universal coronavirus immunity: characterization of reciprocal and non-reciprocal T cell responses against SARS-CoV2 and common human coronaviruses.

T cell immunity plays a central role in clinical outcomes of Coronavirus Infectious Disease 2019 (COVID-19) and T cell-focused vaccination or cellular immunotherapy might provide enhanced protection for some immunocompromised patients. Pre-existing T cell memory recognizing SARS-CoV-2 antigens antedating COVID-19 infection or vaccination, may have developed as an imprint of prior infections with endemic non-SARS human coronaviruses (hCoVs) OC43, HKU1, 229E, NL63, pathogens of "common cold". In turn, SARS-CoV-2-primed T cells may recognize emerging variants or other hCoV viruses and modulate the course of subsequent hCoV infections. Cross-immunity between hCoVs and SARS-CoV-2 has not been well characterized. Here, we systematically investigated T cell responses against the immunodominant SARS-CoV-2 spike, nucleocapsid and membrane proteins and corresponding antigens from α- and β-hCoVs among vaccinated, convalescent, and unexposed subjects. Broad T cell immunity against all tested SARS-CoV-2 antigens emerged in COVID-19 survivors. In convalescent and in vaccinated individuals, SARS-CoV-2 spike-specific T cells reliably recognized most SARS-CoV-2 variants, however cross-reactivity against the omicron variant was reduced by approximately 47%. Responses against spike, nucleocapsid and membrane antigens from endemic hCoVs were significantly more extensive in COVID-19 survivors than in unexposed subjects and displayed cross-reactivity between α- and β-hCoVs. In some, non-SARS hCoV-specific T cells demonstrated a prominent non-reciprocal cross-reactivity with SARS-CoV-2 antigens, whereas a distinct anti-SARS-CoV-2 immunological repertoire emerged post-COVID-19, with relatively limited cross-recognition of non-SARS hCoVs. Based on this cross-reactivity pattern, we established a strategy for in-vitro expansion of universal anti-hCoV T cells for adoptive immunotherapy. Overall, these results have implications for the future design of universal vaccines and cell-based immune therapies against SARS- and non-SARS-CoVs.

Unlocking the potential of Tregs: innovations in CAR technology.

Regulatory T cells (Tregs) adoptive immunotherapy is emerging as a viable treatment option for both autoimmune and alloimmune diseases. However, numerous challenges remain, including limitations related to cell number, availability of target-specific cells, stability, purity, homing ability, and safety concerns. To address these challenges, cell engineering strategies have emerged as promising solutions. Indeed, it has become feasible to increase Treg numbers or enhance their stability through Foxp3 overexpression, post-translational modifications, or demethylation of the Treg-specific demethylated region (TSDR). Specificity can be engineered by the addition of chimeric antigen receptors (CARs), with new techniques designed to fine-tune specificity (tandem chimeric antigen receptors, universal chimeric antigen receptors, synNotch chimeric antigen receptors). The introduction of B-cell targeting antibody receptor (BAR) Tregs has paved the way for effective regulation of B cells and plasma cells. In addition, other constructs have emerged to enhance Tregs activation and function, such as optimized chimeric antigen receptors constructs and the use of armour proteins. Chimeric antigen receptor expression can also be better regulated to limit tonic signaling. Furthermore, various opportunities exist for enhancing the homing capabilities of CAR-Tregs to improve therapy outcomes. Many of these genetic modifications have already been explored for conventional CAR-T therapy but need to be further considered for CAR-Tregs therapies. This review highlights innovative CAR-engineering strategies that have the potential to precisely and efficiently manage immune responses in autoimmune diseases and improve transplant outcomes. As these strategies are further explored and optimized, CAR-Treg therapies may emerge as powerful tools for immune intervention.

Reversing T Cell Exhaustion by Converting Membrane PD-1 to Its Soluble form in Jurkat Cells; Applying The CRISPR/Cas9 Exon Skipping Strategy.

T-cells express two functional forms of the programmed cell death protein 1 (PD-1): membrane (mPD-1) and soluble (sPD-1). The binding of mPD-1 and its ligand (PD-L1) on tumor cells could lead activated lymphocytes toward exhaustion. Selective deletion of the transmembrane domain via alternative splicing of exon-3 in PD-1 mRNA could generate sPD-1. Overexpression of sPD-1 could disrupt the mPD-1/PD-L1 interaction in tumor-specific T cells. We investigated the effect of secreted sPD-1 from pooled engineered and non-engineered T cell supernatant on survival and proliferation of lymphocytes in the tumor microenvironment (TME). In this experimental study, we designed two sgRNA sequences upstream and downstream of exon-3 in the PDCD1 gene. The lentiCRISPRv2 puro vector was used to clone the dual sgRNAs and produce lentiviral particles to transduce Jurkat T cells. Analysis assays were used to clarify the change in PD-1 expression pattern in the pooled (engineered and non-engineered) Jurkat cells. Co-culture conditions were established with PD-L1+ cancer cells and lymphocytes. CRISPR/Cas9 could delete exon-3 of the PDCD1 gene in the engineered cells based on the tracking of indels by decomposition (TIDE) and interference of CRISPR edit (ICE) sequencing analysis reports. Our results showed a 12% reduction in mPD-1 positive cell population after CRISPR manipulation and increment in sPD-1 concentration in the supernatant. The increased sPD-1 confirmed its positive effect on proliferation of lymphocytes co-cultured with PDL1+ cancer cells. The survival percent of lymphocytes co-cultured with the pooled cells supernatant was 12.5% more than the control. The CRISPR/Cas9 exon skipping approach could be used in adoptive cell immunotherapies to change PD-1 expression patterns and overcome exhaustion.