Recombinant Human Cyclophilin A in Combination with Adoptive T-cell Therapy Improves the Efficacy of Cancer Immunotherapy in Experimental Models in vivo.

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.

Eyebox enlargement in holographic AR glasses.

Despite the high output characteristics of holographic optical elements (HOEs), there are still no affordable holographic AR glasses that combine qualities such as a wide field of view (FOV) and a large eyebox (EB). In this study, we propose an architecture for holographic augmented reality glasses that covers both needs. Our solution is based on the combination of an axial HOE with a directional holographic diffuser (DHD) illuminated by a projector. A transparent-type DHD redirects the light from the projector, increasing the angular aperture of the image beams and providing a large EB. A reflection-type axial HOE redirects the light, transforming the spherical beams into parallel ones and providing a wide FOV for the system. The main feature of our system is the coincidence of the DHD position with the planar intermediate image of the axial HOE. This unique condition makes the system free of off-axial aberrations and ensures high output characteristics. The proposed system has a horizontal FOV of 60 deg and an EB width of 10 mm. We used modeling and a prototype to prove our investigations.

T Cell Receptor Chain Centricity: The Phenomenon and Potential Applications in Cancer Immunotherapy.

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/ß or γ/δ) able to recognize a peptide antigen in a complex with self-MHC molecules. Although traditional concepts assume that an α- and ß-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 ß-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.

Cyclophilin A as a Pro-Inflammatory Factor Exhibits Embryotoxic and Teratogenic Effects during Fetal Organogenesis.

The precise balance of Th1, Th2, and Th17 cytokines is a key factor in successful pregnancy and normal embryonic development. However, to date, not all humoral factors that regulate and influence physiological pregnancy have been completely studied. Our data here pointed out cyclophilin A (CypA) as the adverse pro-inflammatory factor negatively affecting fetal development and associated with pregnancy complications. In different mouse models in vivo, we demonstrated dramatic embryotoxicity and teratogenicity of increased CypA levels during pregnancy. Using generated transgenic models, we showed that CypA overexpression in fetal tissues induced the death of all transgenic fetuses and complete miscarriage. Administration of recombinant human CypA in a high dose to pregnant females during fetal organogenesis (6.5-11.5 dpc) exhibited teratogenic effects, causing severe defects in the brain and bone development that could lead to malformations and postnatal behavioral and cognitive disorders in the offspring. Embryotoxic and teratogenic effects could be mediated by CypA-induced up-regulation of M1 macrophage polarization via activation of the STAT1/3 signaling pathways. Here, we propose secreted CypA as a novel marker of complicated pregnancy and a therapeutic target for the correction of pregnancy complications.

Physiological and Functional Effects of Dominant Active TCRα Expression in Transgenic Mice.

A T cell receptor (TCR) consists of α- and ß-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/ß 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-Kb molecule. In their age dynamics, neither autoimmunity due to the random pairing of transgenic TCRα with endogenous TCRß 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 TCRß 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.

Preparative Production and Purification of Recombinant Human Cyclophilin A.

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.

Pupil replication waveguide system for autostereoscopic imaging with a wide field of view.

Augmented reality head-up displays (HUDs) require virtual-object distance matching to the real scene along an adequate field of view (FoV). At the same time, pupil-replication-based waveguide systems provide a wide FoV while affording compact HUDs. To provide 3D imaging and enable virtual-object distance matching in such waveguide systems, we propose a time-sequential autostereoscopic imaging architecture using a synchronized multi-view picture generation and eyebox formation units. Our simulation setup to validate the system feasibility yields an FoV of 15° × 7.5° with clear crosstalk-less images with a resolution of 60 pix/deg for each eye. Our proof-of-concept prototype with reduced specs yields results that are consistent with the simulation in terms of the viewing-zone formation. Thus, viewing zones for the left and right eyes in plane of the eyebox can be clearly observed. Finally, we discuss how the initial distance of the virtual image can be set for quantified fatigue-free 3D imaging, and an FoV can be further extended in such types of waveguide systems by varying parameters of the eyebox formation unit.

Novel transgenic mice with Cre-dependent co-expression of GFP and human ACE2: a safe tool for study of COVID-19 pathogenesis.

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.

Pharmacokinetic Parameters of Recombinant Human Cyclophilin A in Mice.

BACKGROUND AND OBJECTIVE: Cyclophilin A (CypA) is an isomerase that functions as a chaperone, housekeeping protein, and cyclosporine A (CsA) ligand. Secreted CypA is a proinflammatory factor, chemoattractant, immune regulator, and factor of antitumor immunity. Experimental data suggest clinical applications of recombinant human CypA (rhCypA) as a biotherapeutic for cancer immunotherapy, stimulation of tissue regeneration, treatment of brain pathologies, and as a supportive treatment for CsA-based therapies. The objective of this study is to analyze the pharmacokinetics of rhCypA in a mouse model. METHODS: rhCypA was isotope-labeled with 125I and injected intraperitoneally (i.p.) or subcutaneously (s/c) into female mice as a single dose of 100 µg per mouse, equivalent to the estimated first-in-human dose. Analysis of 125I-rhCypA biodistribution and excretion was performed by direct radiometry of the blood, viscera, and urine of mice 0.5-72 h following its administration. RESULTS: rhCypA showed rapid and even tissue-organ distribution, with the highest tropism (fT = 1.56) and accumulation (maximum concentration, Cmax = 137-167 µg/g) in the kidneys, its primary excretory organ. rhCypA showed the lowest tropism to the bone marrow and the brain (fT = 0.07) but the longest retention in these organs [mean retention time (MRT) = 25-28 h]. CONCLUSION: This study identified promising target organs for rhCypA's potential therapeutic effects. The mode of rhCypA accumulation and retention in organs could be primarily due to the expression of its receptors in them. For the first time, rhCypA was shown to cross the blood-brain barrier and accumulate in the brain. These rhCypA pharmacokinetic data could be extrapolated to humans as preliminary data for possible clinical trials.

Immunoregulatory cyclophilin a improves low-dose chemotherapy with a modulation of the immune tumor microenvironment in experimental models of melanoma B16 and lymphoma EL4 in vivo.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: RhCypA could be potentially proposed as a perspective component of the combined cancer chemoimmunotherapy.

Unique features of the TCR repertoire of reactivated memory T cells in the experimental mouse tumor model.

T cell engineering with T cell receptors (TCR) specific to tumor antigens has become a breakthrough towards personalized cancer adoptive cell immunotherapy. However, the search for therapeutic TCRs is often challenging, and effective strategies are strongly required for the identification and enrichment of tumor-specific T cells that express TCRs with superior functional characteristics. Using an experimental mouse tumor model, we studied sequential changes in TCR repertoire features of T cells involved in the primary and secondary immune responses to allogeneic tumor antigens. In-depth bioinformatics analysis of TCR repertoires showed differences in reactivated memory T cells compared to primarily activated effectors. After cognate antigen re-encounter, memory cells were enriched with clonotypes that express α-chain TCR with high potential cross-reactivity and enhanced strength of interaction with both MHC and docked peptides. Our findings suggest that functionally true memory T cells could be a better source of therapeutic TCRs for adoptive cell therapy. No marked changes were observed in the physicochemical characteristics of TCRß in reactivated memory clonotypes, indicative of the dominant role of TCRα in the secondary allogeneic immune response. The results of this study could further contribute to the development of TCR-modified T cell products based on the phenomenon of TCR chain centricity.

Secreted cyclophilin A is non-genotoxic but acts as a tumor promoter.

Chronic inflammation is associated with malignant transformation and creates the microenvironment for tumor progression. Cyclophilin A (CypA) is one of the major pro-inflammatory mediators that accumulates and persists in the site of inflammation in high doses over time. According to multiomics analyses of transformed cells, CypA is widely recognized as a pro-oncogenic factor. Vast experimental data define the functions of intracellular CypA in carcinogenesis, but findings on the role of its secreted form in tumor formation and progression are scarce. In the studies here, we exploit short-term in vitro and in vivo tests to directly evaluate the mutagenic, recombinogenic, and blastomogenic effects, as well as the promoter activity of recombinant human CypA (rhCypA), an analogue of secreted CypA. Our findings showed that rhCypA had no genotoxicity and, thus, was neither involved in nor influenced the initiation stage of carcinogenesis. At high doses, rhCypA could disrupt gap junctions in rat liver epithelial IAR-2 cells in vitro by decreasing the expression of connexins 26 and 43 in these cells and inhibit A549 cell adhesion. These data suggested that rhCypA could contribute to epithelial-mesenchymal transition in malignant cells. The research presented here elucidated the role of secreted CypA in carcinogenesis, revealing that it is not a tumor initiator but can act as a tumor promoter at high concentrations.

Safety evaluation of the mouse TCRα - transduced T cell product in preclinical models in vivo and in vitro.

Adoptive cell therapy (ACT) based on TCR- or CAR-T cells has become an efficient immunotherapeutic approach for the treatment of various diseases, including cancer. Previously, we developed a novel strategy for generating therapeutic T cell products based on chain-centric TCRs, in which either α- or ß-chain dominates in cognate antigen recognition. To assess the suitability of our experimental approach for the clinical application and predict its possible adverse effects, in studies here, we evaluated the safety of the experimental TCRα-modified T cell product in mouse preclinical models. Our data showed no tumorigenic or mutagenic activity in vitro of TCRα-transduced T cells, indicating no genotoxicity of viral vectors used for the generation of the experimental T cell product. Adoptive transfer of TCRα-engineered T cells in a wide dose range didn`t disturb the host homeostasis and exhibited no acute toxicity or immunotoxicity in vivo. Based on pharmacokinetics and pharmacodynamics analysis here, modified T cells rapidly penetrated and distributed in many viscera after infusion. Histological evaluations revealed no pathological changes in organs caused by T cells accumulation, indicating the absence of non-specific off-target activity or cross-reactivity of the therapeutic TCRα. Studies here provide valuable information on the potential safety of TCRα-T cell based ACT that could be extrapolated to possible effects in a human host.

Generation of TCRα-transduced T cells for adoptive transfer therapy of salmonellosis in mice.

Adoptive transfer therapy has great potential to treat diseases such as cancer as well as autoimmune and infectious diseases. Identification of chain-centric T cell receptors (TCRs) with the dominant-active antigen-specific α-chains (TCRα) can significantly improve the efficacy of adoptive cell therapy while reducing time, labor, and costs of generation of TCR-modified antigen-specific T cells. This protocol describes how to generate salmonella-specific TCRα-modified mouse T cells by retroviral transduction and evaluate their functional activity in vivo in the mouse model of salmonellosis. For complete details on the use and execution of this protocol, please refer to Kalinina et al. (2020).

Cyclophilin A is a factor of antitumor defense in the early stages of tumor development.

Cyclophilin A (CypA) is a pro-inflammatory factor with multiple immunomodulating effects. Here, we investigated the effects of recombinant human CypA (rhCypA) as a factor of antitumor host defense. Our results demonstrated that rhCypA dramatically inhibited the growth of murine transplantable tumors (mammary adenocarcinoma Ca755, melanoma B16, Lewis lung carcinoma (LLC), and cervical cancer CC-5). In the B16 model, rhCypA effects were observed only when tumor cells were transplanted at the significantly reduced injection dose, indicating that antitumor properties of rhCypA are more effective at the initial stages of cancer development. Antitumor effect of rhCypA in the CC-5 model was comparable to the action of 5-fluorouracil (5FU), and rhCypA administration prevented 5FU - induced leukopenia in the blood of tumor-bearing mice. In the LLC model, rhCypA injection before but not after tumor resection significantly suppressed the formation of post-surgical metastases. RhCypA exhibited no direct cytotoxic effects in vitro on human leukemia cells (K-562, HL-60, KG-1), indicating that rhCypA antitumor action could be mediated by its immunomodulating activity. In the B16 model, rhCypA had no impact on tumor angiogenesis and gene expression of several MMPs, endogenous CypA, and CD147, which play a crucial role in cancer progression. However, in this model, rhCypA stimulated gene expression of MMPs 8, 9, and 12 that could contribute to malignancy growth inhibition. Here, our findings pointed out CypA as one of the factors of antitumor host defense that can effectively control the initial stages of tumor and metastases formation by regulating the action of MMPs and changing the tumor microenvironment.

Adoptive Immunotherapy Based on Chain-Centric TCRs in Treatment of Infectious Diseases.

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.