TCR-Engineered Lymphocytes Targeting NY-ESO-1: In Vitro Assessment of Cytotoxicity against Tumors.

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.

Exploring TCR-like CAR-Engineered Lymphocyte Cytotoxicity against MAGE-A4.

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.

In Vitro Model of Suppression of the Alloantigen Response by Tolerogenic Dendritic Cells Transfected with Personalized DNA Constructs Encoding HLA Epitopes.

BACKGROUND: A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. METHODS: The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). RESULTS: A significant increase in the amounts of FoxP3+ CD4+CD25+ cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. CONCLUSIONS: The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.

Dendritic Cells Transfected with MHC Antigenic Determinants of CBA Mice Induce Antigen-Specific Tolerance in C57Bl/6 Mice.

BACKGROUND: Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. METHODS: IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. RESULTS: We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejection and GVHD but through distinct nonspecific and antigen-specific mechanisms, respectively. Discussion. We provide data that the novel approach for DCs antigen loading using DNA constructs encoding distinct homologous determinants derived from major histocompatibility complex genes is effective in antigen-specific suppression of transplantation reactions. Such an approach eliminates the necessity of donor material use and may be useful in immunosuppressive therapy side effects prevention.

Comparison of CD4+CD25hiFoxP3+ Treg Induction by pIL-10-Transfected Dendritic Cells in Different Mouse Strains.

Tolerogenic dendritic cells (tolDCs) and T-regulatory cells (Tregs) are involved in maintaining tolerance to self-antigens and foreign antigens. The cells are used as therapeutic tools for inducing tolerance to transplanted organs or tissues. We investigated the possibility of inducing Tregs in splenocyte cultures using DCs transfected with a DNA construct encoding mouse interleukin-10 (DCpIL-10). DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4 and electroporated with a plasmid encoding mouse IL-10. Furthermore, DCpIL-10 was cocultured with syngeneic splenocytes. The CD4+CD25hiFoxP3+ Treg frequency, IL-10 expression, and inhibition of the mixed lymphocyte reaction were evaluated. C57Bl/6 and CBA mice differ in their initial frequency of CD4+CD25hiFoxP3+ Tregs and baseline IL-10 production. Also, the effectiveness of CD4+CD25hiFoxP3+ Treg upregulation by tolDCpIL-10 was different. In this study, DCpIL-10 from C57Bl/6 mice induced CD4+CD25hiFoxP3+ Tregs in syngenic splenocytes, which was accompanied by an increase in the IL-10 production and a decrease in the proliferation of splenocytes in response to the alloantigen. DCpIL-10 may be used to induce CD4+CD25hiFoxP3+ Tregs and the regulatory potential of splenocytes.

Cytotoxic Activity and Memory T Cell Subset Distribution of in vitro-Stimulated CD8+ T Cells Specific for HER2/neu Epitopes.

Minimal residual disease remaining after resection of primary tumors can lead to tumor recurrence and metastasis, increasing mortality and morbidity rates among cancer patients. Thus, there is a need for new technologies for recognition and elimination of single cancer cells remaining in a patient's body after radiation therapy, chemotherapy, or surgical resection. Effector CD8+ T cells, also commonly known as cytotoxic T lymphocytes (CTLs), play a key role in antitumor cellular immunity and, when properly activated, are able to effectively destroy tumor cells. The aims of this study were to obtain CD8+ CTLs specific for the HER2/neu epitopes E75 and E88 and to assess the cytotoxic activity and composition of these cells in terms of the distribution of memory T-cell subsets. We obtained HER2-specific CD8+ T cells and assessed T cell subset distribution among them including naive T cells (TN), central memory T cells (TCM), effector memory T cells (TEM), stem cell-like memory T cells (TSCM) and terminally-differentiated T cells (TEMRA) via eight-color flow cytometry. HER2-specific CTLs were largely (~40-50%) represented by TSCM cells, a population capable of mounting pronounced antitumor immune responses due to a combination of effector function and self-maintenance. In comparison with activated peripheral blood mononuclear cells (PBMCs) and bulk CD8+ T cells, HER2-specific CTLs exhibited greater cytotoxicity against the HER2-expressing human breast adenocarcinoma cell line MCF-7 and produced higher levels of IFN-γ in response to tumor cells. We also showed the presence of HER2-specific CTLs in healthy individuals and increase in them in HER2-positive breast cancer patients. Collectively, our results suggest that HER2-specific CD8+ T cells isolated using this approach could be used for adoptive T-cell transfer to eliminate tumor cells and prevent metastasis and relapse in patients with HER2-overexpressing cancers.

Transfection of bone marrow derived cells with immunoregulatory proteins.

In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins.

Polymorphisms in the tumor necrosis factor receptor genes affect the expression levels of membrane-bound type I and type II receptors.

The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFα receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNF α receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP -609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP -1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14(+) monocytes compared to individuals with the GC genotype. The frequency differences in the CD3(+) and CD19(+) cells expressing TNFRII in relation to SNP -1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP -3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14(+) cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI -609GT + TNFRII -3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNF α receptors and TNF α -mediated signaling.