Applications of single-cell omics for chimeric antigen receptor T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy is a promising cancer treatment modality. The breakthroughs in CAR T cell therapy were, in part, possible with the help of cell analysis methods, such as single-cell analysis. Bulk analyses have provided invaluable information regarding the complex molecular dynamics of CAR T cells, but their results are an average of thousands of signals in CAR T or tumour cells. Since cancer is a heterogeneous disease where each minute detail of a subclone could change the outcome of the treatment, single-cell analysis could prove to be a powerful instrument in deciphering the secrets of tumour microenvironment for cancer immunotherapy. With the recent studies in all aspects of adoptive cell therapy making use of single-cell analysis, a comprehensive review of the recent preclinical and clinical findings in CAR T cell therapy was needed. Here, we categorized and summarized the key points of the studies in which single-cell analysis provided insights into the genomics, epigenomics, transcriptomics and proteomics as well as their respective multi-omics of CAR T cell therapy.

The effect of serum origin on cytokines induced killer cell expansion and function.

BACKGROUND: Cytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture. RESULTS: Cell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression. CONCLUSION: hPL at a concentration of 10% showed the best effect on CIK cell proliferation and function.

Advances in natural killer cell therapies for breast cancer.

Breast cancer (BC) is the most common cause of cancer death in women. According to the American Cancer Society's yearly cancer statistics, BC constituted almost 15% of all the newly diagnosed cancer cases in 2022 for both sexes. Metastatic disease occurs in 30% of patients with BC. The currently available treatments fail to cure metastatic BC, and the average survival time for patients with metastatic BC is approximately 2 years. Developing a treatment method that terminates cancer stem cells without harming healthy cells is the primary objective of novel therapeutics. Adoptive cell therapy is a branch of cancer immunotherapy that utilizes the immune cells to attack cancer cells. Natural killer (NK) cells are an essential component of innate immunity and are critical in destroying tumor cells without prior stimulation with antigens. With the advent of chimeric antigen receptors (CARs), the autologous or allogeneic use of NK/CAR-NK cell therapy has raised new hopes for treating patients with cancer. Here, we describe recent developments in NK and CAR-NK cell immunotherapy, including the biology and function of NK cells, clinical trials, different sources of NK cells and their future perspectives on BC.

Eosinophils in the tumor microenvironment: implications for cancer immunotherapy.

Despite being an integral part of the immune response in the tumor microenvironment (TME), few studies have mechanistically elucidated eosinophil functions in cancer outcomes. Eosinophils are a minor population of granulocytes that are mostly explored in asthma and allergic disorders. Their influence on primary and metastatic tumors, however, has recently come to light. Eosinophils' diverse armamentarium of mediators and receptors allows them to participate in innate and adaptive immunity, such as type 1 and type 2 immunity, and shape TME and tumor outcomes. Based on TME cells and cytokines, activated eosinophils drive other immune cells to ultimately promote or suppress tumor growth. Discovering exactly what conditions determine the pro-tumorigenic or anti-tumorigenic role of eosinophils allows us to take advantage of these signals and devise novel strategies to target cancer cells. Here, we first revisit eosinophil biology and differentiation as recognizing eosinophil mediators is crucial to their function in homeostatic and pathological conditions as well as tumor outcome. The bulk of our paper discusses eosinophil interactions with tumor cells, immune cells-including T cells, plasma cells, natural killer (NK) cells-and gut microbiota. Eosinophil mediators, such as IL-5, IL-33, granulocyte-macrophage colony-stimulating factor (GM-CSF), thymic stromal lymphopoietin (TSLP), and CCL11 also determine eosinophil behavior toward tumor cells. We then examine the implications of these findings for cancer immunotherapy approaches, including immune checkpoint blockade (ICB) therapy using immune checkpoint inhibitors (ICIs) and chimeric antigen receptor (CAR) T cell therapy. Eosinophils synergize with CAR T cells and ICB therapy to augment immunotherapies.

Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy.

BACKGROUND: The successful ex vivo expansion of T-cells in great numbers is the cornerstone of adoptive cell therapy. We aimed to achieve the most optimal T-cell expansion condition by comparing the expansion of T-cells at various seeding densities, IL-2 concentrations, and bead-to-cell ratios. we first expanded the peripheral blood mononuclear cells (PBMCs) of a healthy donor at a range of 20 to 500 IU/mL IL-2 concentrations, 125 × 103 to 1.5 × 106 cell/mL, and 1:10 to 10:1 B:C (Bead-to-cell) ratios and compared the results. We then expanded the PBMC of three healthy donors using the optimized conditions and examined the growth kinetics. On day 28, CD3, CD4, and CD8 expression of the cell populations were analyzed by flow cytometry. RESULTS: T-cells of the first donor showed greater expansion results in IL-2 concentrations higher than 50 IU/mL compared to 20 IU/mL (P = 0.02). A seeding density of 250 × 103 cell/mL was superior to higher or lower densities in expanding T-cells (P = 0.025). Also, we witnessed a direct correlation between the B:C ratio and T-cell expansion, in which, in 5:1 and 10:1 B:C ratios T-cell significantly expanded more than lower B:C ratios. The results of PBMC expansions of three healthy donors were similar in growth kinetics. In the optimized condition, 96-98% of the lymphocyte population expressed CD3. While the majority of these cells expressed CD8, the mean expression of CD4 in the donors was 19.3, 16.5, and 20.4%. CONCLUSIONS: Our methodology demonstrates an optimized culture condition for the production of large quantities of polyclonal T-cells, which could be useful for future clinical and research studies.

IL-35, a double-edged sword in cancer.

Interleukin 35 (IL-35), a cytokine mainly produced by regulatory T cells (Treg cells), is composed of an Epstein-Barr virus-induced gene 3 ß-chain and an IL-12 p35 α-chain. IL-35 causes tumorigenicity in cancer, protects cancer cells against apoptosis, and facilitates cancer progression. However, a few reports have referred to its contradictory roles in cancer prevention. Therefore, the exact purpose of this cytokine in cancer development has become a fundamental question that needs to be answered. In this review, we explain the structure of IL-35 and its receptors and their different signaling pathways. Finally, the function of IL-35 in some cancers and the possible application of this cytokine in approaches for cancer therapy have been discussed.

Report of a new six-panel flow cytometry marker for early differential diagnosis of APL from HLA-DR negative Non-APL leukemia.

Although AML-M3 (APL) and HLA-DR negative non-APL are characterized by negative HLA-DR antigen, they are different entities with similar morphology in some cases. The aim of this study is the precise, differential diagnosis of APL from HLA-DR negative non-APL by flow cytometry to narrow the diagnosis window. Bone marrow or blood samples of 580 AML patients were analyzed, and flow cytometry and molecular analysis were performed for the diagnosis of blood disorders. In 105 HLA-DR negative AML patients, expression of HLA-DR, CD33, CD117, CD11b, CD64, CD34, CD9 and myeloperoxidase staining pattern were evaluated. Fifty-six patients were diagnosed with APL, and 49 patients were diagnosed with HLA-DR negative non-APL. The APL blasts expressed CD33, CD117, CD64, and CD9 in 100%, 80.3%, 94.6%, and 100% of the cases, respectively. HLA-DR negative non-APL blasts expressed CD33, CD117, CD64 and CD9 in 75.5%, 59.1%, 32.6%, and 73.4% of the cases, respectively. APL cells were negative for HLA-DR, CD11b, and CD34 in 96.4%, 94.6%, and 91.0%, respectively. Blasts in HLA-DR negative non M3-AML were negative for CD11b, CD117, and CD34 in 77.5%, 40.9%, and 22.4%, respectively. We also investigated myeloperoxidase (MPO) staining pattern and found strong diffuse reaction in APL cells while HLA-DR negative non-APL cells showed focal positive reaction. In all of the APL patients, except for one, PML/RARA translocation was positive, and in another case with HLA-DR negative non-APL, PML/RARA and other translocations were not detected. The six-panel combination profile rapidly and specifically identifies APL from other HLA-DR negative AML.

CRISPR/Cas9 revitalizes adoptive T-cell therapy for cancer immunotherapy.

Cancer immunotherapy has gained attention as the supreme therapeutic modality for the treatment of various malignancies. Adoptive T-cell therapy (ACT) is one of the most distinctive modalities of this therapeutic approach, which seeks to harness the potential of combating cancer cells by using autologous or allogenic tumor-specific T-cells. However, a plethora of circumstances must be optimized to produce functional, durable, and efficient T-cells. Recently, the potential of ACT has been further realized by the introduction of novel gene-editing platforms such as the CRISPR/Cas9 system; this technique has been utilized to create T-cells furnished with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR) that have precise tumor antigen recognition, minimal side effects and treatment-related toxicities, robust proliferation and cytotoxicity, and nominal exhaustion. Here, we aim to review and categorize the recent breakthroughs of genetically modified TCR/CAR T-cells through CRISPR/Cas9 technology and address the pearls and pitfalls of each method. In addition, we investigate the latest ongoing clinical trials that are applying CRISPR-associated TCR/CAR T-cells for the treatment of cancers.

An overview of the recent findings of cell-based therapies for the treatment and management of COVID-19.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic taking the lives of millions. The virus itself not only invades and destroys the angiotensin-converting enzyme 2 (ACE2)-expressing cells of the lungs, kidneys, liver, etc. but also elicits a hyperinflammatory immune response, further damaging the tissue leading to acute respiratory distress syndrome (ARDS) and death. Although vaccines, as a prime example of active immunotherapy, have clearly disrupted the transmission of virus and reduced mortality, hospitalization, and burden of disease, other avenues of immunotherapy are also being explored. One such approach would be to adoptively transfer modified/unmodified immune cells to the critically ill. Here, we compiled and summarized the immunopathogenesis of SARS-CoV-2 and the recent preclinical and clinical data on the potential of cell-based therapies in the fight against COVID-19.

Induced Pluripotent Stem Cell Meets Severe Combined Immunodeficiency.

Severe combined immunodeficiency (SCID) is classified as a primary immunodeficiency, which is characterized by impaired T-lymphocytes differentiation. IL2RG, IL7Ralpha, JAK3, ADA, RAG1/RAG2, and DCLE1C (Artemis) are the most defective genes in SCID. The most recent SCID therapies are based on gene therapy (GT) of hematopoietic stem cells (HSC), which are faced with many challenges. The new studies in the field of stem cells have made great progress in overcoming the challenges ahead. In 2006, Yamanaka et al. achieved "reprogramming" technology by introducing four transcription factors known as Yamanaka factors, which generate induced pluripotent stem cells (iPSC) from somatic cells. It is possible to apply iPSC-derived HSC for transplantation in patients with abnormality or loss of function in specific cells or damaged tissue, such as T-cells and NK-cells in the context of SCID. The iPSC-based HSC transplantation in SCID and other hereditary disorders needs gene correction before transplantation. Furthermore, iPSC technology has been introduced as a promising tool in cellular-molecular disease modeling and drug discovery. In this article, we review iPSC-based GT and modeling for SCID disease and novel approaches of iPSC application in SCID.

Impact of various culture conditions on ex vivo expansion of polyclonal T cells for adoptive immunotherapy.

Currently, adoptive immunotherapy is considered as one of the leading treatments in cancer. Successful adoptive immunotherapy depends on producing large numbers of desired T cells ex vivo for infusion. This requires an effective protocol for maximum functional T-cell expansion while keeping the time and costs to a minimum. Current T-cell expansion protocols are diverse in their methodology, and a universal protocol of expansion is wanting. Also, new findings regarding T-cell biology, signaling, and activation have reshaped the strategies of T-cell propagation over the years, introducing new ways to expand T cells. Here, we reviewed different conditions for blood-derived polyclonal T-cell expansion so as to elucidate the influential factors of T-cell expansion and their efficacy.

A Review on Leukemia and iPSC Technology: Application in Novel Treatment and Future.

Leukemia is an uncontrollable growth of hematopoietic cells due to a mutation in DNA followed by cellular dysregulation and one or more chromosomal disorder that generally leads to a clonal abnormality. Theoretical and technical inability in early screening and distinguishing cancer, tumor tolerance to common treatment methods, repeated relapses of cancer after remission phase, heterogeneous chromosomal abnormality, and the side effects of current chemotherapies are some of challenges that we face with leukemia and other malignancies. Induced pluripotent stem cells (iPSC) opened a promising window to a wide range of diseases, including leukemia. Overcoming the barriers in leukemia is possible with iPSC technology. Induced hematopoietic stem cell transplantation (and gene therapy), induced cytotoxic T-lymphocytes and reprogrammed NK cells that strengthen the immune system, miRNAs, modeling approaches, and supportive cares are some aspects of the novel treatment based on iPSC technology.

Various Signaling Pathways in Multiple Myeloma Cells and Effects of Treatment on These Pathways.

Multiple myeloma (MM) results from malignancy in plasma cells and occurs at ages > 50 years. MM is the second most common hematologic malignancy after non-Hodgkin lymphoma, which constitutes 1% of all malignancies. Despite the great advances in the discovery of useful drugs for this disease such as dexamethasone and bortezomib, it is still an incurable malignancy owing to the development of drug resistance. The tumor cells develop resistance to apoptosis, resulting in greater cell survival, and, ultimately, develop drug resistance by changing the various signaling pathways involved in cell proliferation, survival, differentiation, and apoptosis. We have reviewed the different signaling pathways in MM cells. We reached the conclusion that the most important factor in the drug resistance in MM patients is caused by the bone marrow microenvironment with production of adhesion molecules and cytokines. Binding of tumor cells to stromal cells prompts cytokine production of stromal cells and launches various signaling pathways such as Janus-activated kinase/signal transduction and activator of transcription, Ras/Raf/MEK/mitogen-activated protein kinase, phosphatidyl inositol 3-kinase/AKT, and NF-KB, which ultimately lead to the high survival rate and drug resistance in tumor cells. Thus, combining various drugs such as bortezomib, dexamethasone, lenalidomide, and melphalan with compounds that are not common, including CTY387, LLL-12, OPB31121, CNTO328, OSI-906, FTY720, triptolide, and AV-65, could be one of the most effective treatments for these patients.

Comparison of outcomes in patients undergoing coronary bypass of patent versus restenosed bare metal stented coronary arteries.

It is unclear whether bypass of a patent stented artery affects clinical outcomes. We sought to compare the survival of patients who, as part of multisystem coronary artery bypass grafting, underwent revascularization of arteries with patent stents (<50% stenosis) or in-stent restenosis (>50% diameter stenosis). Of 550 consecutive patients with previously placed stents who underwent coronary artery bypass grafting from May 1995 to October 2003, we studied 399 who had only 1 stented vessel bypassed at surgery. Of these, 128 had coronary bypass to an artery with a patent stent and 271 had bypass to an artery with in-stent restenosis. Nonparametric survival estimates were obtained using the Kaplan-Meier method. A propensity-adjusted multivariate hazard model of group differences was generated using variables identified by bootstrap bagging. The unadjusted survival rate at 1 month, 1 year, and 5 years was 99.7%, 97.3%, and 89.1%, respectively, for the patent stent group and 96.6%, 93.9%, and 86.2%, respectively, for the in-stent restenosis group, a result of high early risk in the latter group. After adjusting for clinical variables, neither stent patency (p = 0.9) nor interval (p = 0.3) from stent placement was a risk factor, although advanced age, increased blood urea nitrogen, and preoperative atrial fibrillation were associated with poorer survival. In conclusion, survival after bypassing a patent stented coronary artery is comparable to that after bypassing a stented restenotic coronary artery.

Comparative Clinical and Histologic Assessments of Dental Implants Delivered with a Manual Torque Limiting Wrench Versus with an Electronically Controlled Torque Limiting Device.

The goal of this preclinical investigation was to evaluate the healing of tapered roughened surfaced dental implants that were delivered by either a manual torque limiting wrench or an electronically controlled torque limiting device. Three canines underwent bilateral extraction of third and fourth premolars and first molar. The extraction sites were allowed to heal for 2 months before two dental implants were placed bilaterally. All animals underwent a normal healing process. One animal was sacrificed at 1 month and the remaining two animals were sacrificed at 2 months to perform histologic evaluations including bone-to-implant contact (BIC) and soft tissue healing. The clinical stability and histologic osseointegration were similar when the results obtained with the manual torque limiting wrench were compared to those delivered by the electronically controlled torque limiting device. However, BIC and maintenance of the crestal bone level achieved appeared to be higher in the electronically controlled torque limiting device groups.

Carotid brachytherapy for in-stent restenosis.

Carotid stenting has emerged as an alternative revascularization modality to endarterectomy for the treatment of carotid artery disease. Restenosis of a carotid stent may be occasionally seen. Our experience in intravascular radiation therapy for coronary restenosis has provided us the opportunity to explore this treatment strategy for carotid restenosis. We report our initial experience with brachytherapy for the treatment of restenosis after carotid stenting.