T cells are crucial players in adaptive anti-cancer immunity. The gene modification of T cells with tumor antigen-specific T cell receptors (TCRs) was a milestone in personalized cancer ...immunotherapy. TCR is a heterodimer (either α/β or γ/δ) able to recognize a peptide antigen in a complex with self-MHC molecules. Although traditional concepts assume that an α- and β-chain contribute equally to antigen recognition, mounting data reveal that certain receptors possess chain centricity, i.e., one hemi-chain TCR dominates antigen recognition and dictates its specificity. Chain-centric TCRs are currently poorly understood in terms of their origin and the functional T cell subsets that express them. In addition, the ratio of α- and β-chain-centric TCRs, as well as the exact proportion of chain-centric TCRs in the native repertoire, is generally still unknown today. In this review, we provide a retrospective analysis of studies that evidence chain-centric TCRs, propose patterns of their generation, and discuss the potential applications of such receptors in T cell gene modification for adoptive cancer immunotherapy.
Adoptive T-cell therapy (ACT) is successfully applied in cancer treatment; however, its efficiency can be limited by a low viability, short persistence time, and loss of functional activity of ...T-cells after adoptive transfer. The search for novel immunomodulators that can improve the viability, expansion, and functions of T-cells after their infusion with the minimal side effects could contribute to the development of more efficient and safe ACT strategies. Recombinant human cyclophilin A (rhCypA) is of particular interest in this respect, as it exhibits pleiotropic immunomodulatory activity and stimulates both innate and adaptive anti-tumor immunity. Here, we evaluated the effect of rhCypA on the efficacy of ACT in the mouse EL4 lymphoma model. Lymphocytes from transgenic 1D1a mice with an inborn pool of EL4-specific T-cells were used as a source of tumor-specific T-cells for ACT. In models of immunocompetent and immunodeficient transgenic mice, the course (3 days) rhCypA administration was shown to significantly stimulate EL4 rejection and prolong the overall survival of tumor-bearing mice after adoptive transfer of lowered doses of transgenic 1D1a cells. Our studies showed that rhCypA significantly improved the efficacy of ACT by enhancing the effector functions of tumor-specific cytotoxic T-cells. These findings open up the prospects for the development of innovative strategies of adoptive T-cell immunotherapy for cancer using rhCypA as an alternative to existing cytokine therapies.
Different regimens of low-dose chemotherapy (LDC) are currently being actively developed and introduced into clinical practice. Along with its obvious advantages compared to conventional chemotherapy ...(low toxicity, prevention of drug resistance), LDC could also stimulate anti-tumor immune responses in a patient by activating effectors of innate and adaptive immunity and diminishing tumor-associated immunosuppression. As non-myeloablative, LDC could be successfully combined with different anti-cancer immunotherapeutic strategies, including immunoregulatory cytokines. Secreted cyclophilin A (CypA) is of particular interest in this respect. Previously, we showed that recombinant human CypA (rhCypA) had pleiotropic immunostimulatory activity and anti-tumor effects. Thus, rhCypA could be potentially proposed as a perspective component of combined therapy with LDC.PURPOSEDifferent regimens of low-dose chemotherapy (LDC) are currently being actively developed and introduced into clinical practice. Along with its obvious advantages compared to conventional chemotherapy (low toxicity, prevention of drug resistance), LDC could also stimulate anti-tumor immune responses in a patient by activating effectors of innate and adaptive immunity and diminishing tumor-associated immunosuppression. As non-myeloablative, LDC could be successfully combined with different anti-cancer immunotherapeutic strategies, including immunoregulatory cytokines. Secreted cyclophilin A (CypA) is of particular interest in this respect. Previously, we showed that recombinant human CypA (rhCypA) had pleiotropic immunostimulatory activity and anti-tumor effects. Thus, rhCypA could be potentially proposed as a perspective component of combined therapy with LDC.In this work, we evaluated the anti-tumor effects of rhCypA combined with low doses of cyclophosphamide, doxorubicin, dacarbazine, and paclitaxel in the experimental mouse tumor models of melanoma B16 and lymphoma EL4 in vivo.METHODSIn this work, we evaluated the anti-tumor effects of rhCypA combined with low doses of cyclophosphamide, doxorubicin, dacarbazine, and paclitaxel in the experimental mouse tumor models of melanoma B16 and lymphoma EL4 in vivo.Synergic and potentiating effects of rhCypA combined with LDC were shown in these studies. Furthermore, as a monotherapeutic agent and a component of combined chemoimmunotherapy, rhCypA was shown to modulate the immune tumor microenvironment by enhancing tumor infiltration with macrophages, NK cells, and T cells. It was also found that rhCypA stimulated both systemic and local anti-tumor immune responses.RESULTSSynergic and potentiating effects of rhCypA combined with LDC were shown in these studies. Furthermore, as a monotherapeutic agent and a component of combined chemoimmunotherapy, rhCypA was shown to modulate the immune tumor microenvironment by enhancing tumor infiltration with macrophages, NK cells, and T cells. It was also found that rhCypA stimulated both systemic and local anti-tumor immune responses.RhCypA could be potentially proposed as a perspective component of the combined cancer chemoimmunotherapy.CONCLUSIONRhCypA could be potentially proposed as a perspective component of the combined cancer chemoimmunotherapy.
A T cell receptor (TCR) consists of α- and β-chains. Accumulating evidence suggests that some TCRs possess chain centricity, i.e., either of the hemi-chains can dominate in antigen recognition and ...dictate the TCR's specificity. The introduction of TCRα/β into naive lymphocytes generates antigen-specific T cells that are ready to perform their functions. Transgenesis of the dominant active TCRα creates transgenic animals with improved anti-tumor immune control, and adoptive immunotherapy with TCRα-transduced T cells provides resistance to infections. However, the potential detrimental effects of the dominant hemi-chain TCR's expression in transgenic animals have not been well investigated. Here, we analyzed, in detail, the functional status of the immune system of recently generated 1D1a transgenic mice expressing the dominant active TCRα specific to the H2-K
molecule. In their age dynamics, neither autoimmunity due to the random pairing of transgenic TCRα with endogenous TCRβ variants nor significant disturbances in systemic homeostasis were detected in these mice. Although the specific immune response was considerably enhanced in 1D1a mice, responses to third-party alloantigens were not compromised, indicating that the expression of dominant active TCRα did not limit immune reactivity in transgenic mice. Our data suggest that TCRα transgene expression could delay thymic involution and maintain TCRβ repertoire diversity in old transgenic mice. The detected changes in the systemic homeostasis in 1D1a transgenic mice, which are minor and primarily transient, may indicate variations in the ontogeny of wild-type and transgenic mouse lines.
The synthesis of phosphorescent transition metal complexes with emission in the near infrared (NIR) region, appreciable quantum yields, compatibility with physiological media and suitability for ...application as effective oxygen sensors in biological systems is still a challenge in the field of synthetic organometallic chemistry. In addition to the sufficiently high requirements in photophysics, they also have to meet strict conditions for biocompatibility and selectivity of sensory responses in a multicomponent biological medium. Building on our previous research, we have synthesized three novel iridium Ir(N^C)
2
(N^N)
+
complexes (N^C - metalated ligands based on benzothienyl-phenanthridine core; N^N - pyridine-triazole diimine chelate), with their periphery decorated with 1, 2 or 3 relatively small "double-tail" oligo(ethylene glycol) (OEG) {-C(O)NHCH(CH
2
OC
2
H
4
OC
2
H
4
OCH
3
)
2
} functions. All these complexes display desirable photophysical characteristics: excitation at the high energy limit of the "window of transparency", NIR emission at
ca.
710-720 nm, rather high quantum yields (13-15% in degassed methanol solution) and strong lifetime responses to variations in oxygen concentration. However, the study of their behavior in model biological systems (human serum albumin (HSA) and fetal bovine serum (FBS) solutions) by gel permeation chromatography and absorption/luminescence spectroscopy showed that only the complex bearing OEG functions at all three chelate ligands is compatible with the requirements of imaging experiments,
i.e.
it is water soluble and bleaching stable, has low toxicity and its sensory response is insensitive to the presence of the components of biological media, temperature, and pH. The sensing properties of this molecular probe were studied in detail and test imaging experiments in phosphorescence lifetime imaging (PLIM) mode were carried out on cancer cell cultures. We demonstrate that this probe is accumulated in the cell cytoplasm and exhibits low cytotoxicity at concentrations up to 125 μM. Phosphorescence lifetime imaging microscopy (PLIM) of live mouse colorectal cancer cells (CT26) and human oral squamous carcinoma (SCC-4) stained with the probe has shown lifetimes in the 1.35-2.51 μs interval with pronounced responses to oxygen under hypoxic conditions, thus providing the basis for qualitative detection of hypoxia and semi-quantitative oxygen concentration measurements.
NIR emitting Ir(
iii
) complexes decorated with oligo(ethylene glycol) were used to assess the degree of hypoxia in biosamples.
In this work, we developed the method of preparative production of recombinant human cyclophilin A (rhCypA) in Escherichia coli. The full-length cDNA encoding the gene of human CypA (CYPA) was ...amplified by RT-PCR from the total RNA of human T cell lymphoma Jurkat. The nucleotide sequence of CYPA was optimized to provide highly effective translation in E. coli. Recombinant CYPA DNA was cloned into the pET22b(+) vector, and the resulted expression plasmid was used to transform E. coli strain BL21(DE3)Gold. The recombinant producer strain of E. coli produced soluble rhCypA in the bacterial cytoplasm. The synthesis efficiency of rhCypA was up to 50% of the total cell protein allowing to produce rhCypA in the amount of 1 g per liter of the culture. We also developed the method for rhCypA purification, consisting of a single-step tandem anion exchange chromatography on DEAE- and Q-Sepharose columns. The protein purity was 95% according to electrophoresis (SDS-PAGE), and its contamination with endotoxin did not exceed 0.05 ng per 1 mg of the protein that met the requirements of European pharmacopoeia for injectable preparations. The produced recombinant protein exhibited functional features of native CypA, i.e., isomerase activity and chemokine activity as assessed by stimulation of migration of mouse bone marrow hematopoietic stem cells in vivo. The generated producer strain of E. coli is a super-producer and could be used for large-scale experimental studies of rhCypA and in its preclinical and clinical trials as a drug.
The current coronavirus disease (COVID-19) pandemic remains one of the most serious public health problems. Increasing evidence shows that infection by severe acute respiratory syndrome coronavirus-2 ...(SARS-CoV-2) causes a very complex and multifaceted disease that requires detailed study. Nevertheless, experimental research on COVID-19 remains challenging due to the lack of appropriate animal models. Herein, we report novel humanized mice with Cre-dependent expression of hACE2, the main entry receptor of SARS-CoV-2. These mice carry
hACE2
and
GFP
transgenes floxed by the STOP cassette, allowing them to be used as breeders for the creation of animals with tissue-specific coexpression of hACE2 and GFP. Moreover, inducible expression of hACE2 makes this line biosafe, whereas coexpression with GFP simplifies the detection of transgene-expressing cells. In our study, we tested our line by crossing with Ubi-Cre mice, characterized by tamoxifen-dependent ubiquitous activation of Cre recombinase. After tamoxifen administration, the copy number of the STOP cassette was decreased, and the offspring expressed
hACE2
and
GFP
, confirming the efficiency of our system. We believe that our model can be a useful tool for studying COVID-19 pathogenesis because the selective expression of hACE2 can shed light on the roles of different tissues in SARS-CoV-2-associated complications. Obviously, it can also be used for preclinical trials of antiviral drugs and new vaccines.
Graphic abstract
Complications after vaccination, lack of vaccines against certain infections, and the emergence of antibiotic-resistant microorganisms point to the need for alternative ways of protection and ...treatment of infectious diseases. Here, we proposed a therapeutic approach to control salmonellosis based on adoptive cell therapy. We showed that the T cell receptor (TCR) repertoire of salmonella-specific memory cells contains 20% of TCR variants with the dominant-active α-chain. Transduction of intact T lymphocytes with the dominant salmonella-specific TCRα led to their enhanced in vitro proliferation in response to salmonella. Adoptive transfer of transduced T cells resulted in a significant decrease in bacterial loads in mice infected with salmonella before or after the adoptive transfer. We demonstrated that adoptive immunotherapy based on T cells, transduced with dominant-specific TCRα could be successfully applied for treatment and prevention of infectious diseases and represent a useful addition to vaccination and existing therapeutic strategies.
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•A regular TCR repertoire of memory T cells contains alpha-chain-centric TCRs•Dominant-active TCRα, paired with random TCRβ, recognizes specific microbial antigens•Adoptive immunotherapy could be applied for treatment of infections
Immunology; Microbiology
The melanoma origin of cell lines obtained from the axillary lymph node (mel Kas, mel Pet, and mel Lap from patients with a verified diagnosis) was confirmed by the detection of the Melan A ...melanocyte marker expression. A hyperdiploid (2n+) for the mel Kas line; near-diploid (2n), and in some cells near-tertaploid (4n), and even hypo-octaploid (8n) set (172–179 chromosomes) in the mel Pet cell line; and a hypotetraploid (4n−) for the mel Lap line were detected by karyotypic analysis. All three cell lines are tumorigenic; however, mel Pet demonstrates tumor growth in Balb/c nude mice only in the presence of matrigel. All three lines showed a high expression of TUBB3 and PD-L1 markers, while ERa was low (minimum for mel Pet). Significant differences in the expression level were shown for the Cyt molecular marker. In the transplantation of cells to Balb/c nude mice, a stable expression level is observed only for TUBB3. For the rest of the markers, a decrease in their expression level of varying degrees was noted when the cells were growing in solid tumors in vivo. Mutations were detected in oncogenes (BRAF, EZH2, KIT, KRAS, NRAS, ROS1) and tumor suppressor genes (CDKN2A, FAT4, KMT2C, LRP1B, PTEN, PTPRB, TP53). The detailed characterization of the cell lines makes them valuable for various scientific and regulatory experiments, particularly those involving preclinical data on antiproliferative drugs for malignant melanoma or investigations into melanoma cell properties and progression.
Many reports have documented the role of INS (insulin) as growth factors in a variety of cancers. Epidemiological studies revealed that INS therapy causes increased mortality in multiple myeloma (MM) ...patients with pre-existing or steroid-induced type 2 diabetes. However, there is limited experimental evidence of this association. In the present study, the dual effect of INS on the viability of myeloma RPMI8226 and lymphoblastoid IM9 cells was revealed. In serum-containing medium exogenous INS serves as a growth factor, whereas INS decreases the number of cells under serum-free medium. In the last case, the main mechanism of decreasing the cell population is apoptosis through up-regulation of Cas-3 and downregulation of Bcl-2 expression. INS has also been shown to be involved in the regulation of necrotic cell death.
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