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.

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.

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.

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.

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.

Immune selection of tumor cells in TCR ß-chain transgenic mice.

The concept of immunological surveillance implies that immunogenic variants of tumor cells arising in the organism can be recognized by the immune system. Tumor progression is provided by somatic evolution of tumor cells under the pressure of the immune system. The loss of MHC Class I molecules on the surface of tumor cells is one of the most known outcomes of immune selection. This study developed a model of immune selection based on the immune response of TCR 1d1 single ß-chain transgenic B10.D2(R101) (K(d)I(d)D(b)) mice to allogeneic EL4 (H-2(b)) thymoma cells. In wild-type B10.D2(R101) mice, immunization with EL4 cells induced a vigorous CTL response targeted to the H-2K(b) molecule and results in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in mixed-lymphocyte response assays and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, CD8(+) T-lymphocytes with endogenous ß-chains accumulated predominantly in the spleen of transgenic mice and only a small part of the T-lymphocytes expressing transgenic ß-chains became CD8(+)CD44(+)CD62L(-) effectors. Then, instead of a full elimination of tumor cells as in wild-type mice, a reproducible prolonged equilibrium phase and subsequent escape was observed in transgenic mice that resulted in death of 90% of the mice in 40-60 days after grafting. Prolonged exposure of tumor cells to the pressure of the immune system in transgenic mice in vivo resulted in a stable loss of H-2K(b) molecules on the EL4 cell surface. Genetic manipulation of the T-lymphocyte repertoire was sufficient to reproduce the classic pattern of interactions between tumor cells and the immune system, usually observed in reliable syngeneic models of anti-tumor immunity. This newly-developed model could be used in further studies of immunoregulatory circuits common for transplantational and anti-tumor immune responses.

MHC restriction and allogeneic immune responses.

Discovery of major histocompatability complex (MHC) restriction helped in the understanding of how T-lymphocytes recognize antigens on bacteria, viruses, and tumor cells. It was initially accepted that MHC restriction was a consequence of "adaptive differentiation" in the thymus; during differentiation, the forming repertoire of T-lymphocytes "learned" a low affinity for self MHC molecules via positive selection. This view was later countered by discovery of artifacts in underlying studies and the fact that adaptive differentiation could not explain direct allogeneic and allorestricted recognition phenomena. Data from experiments with TCR transgenic animals, individual MHC/peptide complex expression, and recipients of xenogenic thymus glands yielded evidence of an ability to adapt to microenvironment and a low specificity of positive selection. These facts led to an alternative interpretation of MHC restriction explained, in part, by specificity of a pool of effector cells activated by primary immunization. Details of this phenomenon were defined in studies that noted differential primary structures of peptides that bound various allelic forms of MHC molecules. Here, the T-lymphocyte repertoire formed in the thymus was a result, in part, of random rearrangement of germinal sequences of TCR gene fragments. Such pre-selected repertoires were inherently capable of reacting with different allelic forms of MHC molecules. In contrast, MHC molecules were characterized by significant intraspecies polymorphisms; negative and positive selections were aimed at adaptation of a pre-selected repertoire to a specific microenvironment in an individual. Via elimination of autoreactive clones and sparing of a broad spectrum of specificity to potential pathogens, selection in the thymus could be considered a life-long allogeneic reaction of a pre-selected repertoire to self MHC molecules resulting in tolerance to "self," increased responsiveness to foreign MHC molecules, and cross-reactivity of the mature T-lymphocyte repertoire to individual foreign peptides plus self MHC.

Intrathymic selection: new insight into tumor immunology.

Central tolerance to self-antigens is formed in the thymus where deletion of clones with high affinity to "self" takes place. Expression of peripheral antigens in the thymus has been implicated in T cell tolerance and autoimmunity. During the last years, it has been shown that medullary thymic epithelial cells (mTECs) are the unique cell type expressing a diverse range of tissue-specific antigens. Promiscuous gene expression is a cell autonomous property of thymic epithelial cells and is maintained during the entire period of thymic T cell output. The array of promiscuously expressed self-antigens was random and included well-known targets for cancer immunotherapy, such as alpha-fetoprotein, P1A, tyrosinase, and gp100. Gene expression in normal tissues may result in tolerance of high-avidity cytotoxic T lymphocyte (CTL), leaving behind low-avidity CTL that cannot provide effective immunity against tumors expressing the relevant target antigens. Thus, it may be evident that tumor vaccines that targeted the tumor-associated antigens should be inefficient due to the loss of high-avidity T cell clones capable to be stimulated. Stauss with colleagues have described a strategy to circumvent immunological tolerance that can be used to generate high-avidity CTL against self-proteins, including human tumor-associated antigens. In this strategy, the allorestricted repertoire of T cells from allogenic donor is used as a source of T cell clones with high avidity to tumor antigens of recipient for adoptive immunotherapy. Then, the T cell receptor (TCR) genes isolated from antigen-specific T cells can be exploited as generic therapeutic molecules for antigen-specific immunotherapy.

Avian adenovirus vector CELO-TK displays anticancer activity in human cancer cells and suppresses established murine melanoma tumors.

Avian adenovirus CELO is a novel adenovirus vector system with the advantages of efficient production, high virion stability, and the absence of crossreactivity with Ad5-neutralizing antibodies. In this study, we evaluated the anticancer efficacy of a CELO vector encoding the herpes simplex virus type 1 thymidine kinase, a prodrug-activating therapeutic gene. Vectors carrying the gene for HSV-tk or EGFP under the control of the HCMV promoter in place of the "nonessential" region of the CELO genome were constructed. Anticancer activity of the CELO-TK vector was studied in vitro, in human and murine tumor cells in cell culture, and in vivo, in established subcutaneous murine B16 melanoma tumors in C57BL/6 mice. The CELO-TK vector mediated delivery of functional HSV-tk to tumor cell lines in cell culture. Comparison of the CELO-TK vector to a first-generation human adenovirus type 5 vector Ad5-TK in cultured H1299 cells showed equal levels of functional activity at increasing multiplicities of infection with CELO-based vector. CELO vectors allowed for transduction and expression of EGFP and HSV-tk genes in subcutaneous melanoma tumors in C57BL/6 mice. Intratumoral injections of CELO-TK followed by ganciclovir administration resulted in suppression of tumor growth and significantly increased the median of survival. The results of the study demonstrated the efficacy of CELO vector as a vehicle for the delivery of prodrug-activating genes such as HSV-tk to tumor cells in vitro and in vivo.