Clonal Composition of Human Multipotent Mesenchymal Stromal Cells: Application of Genetic Barcodes in Research.

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

[Modification of Cytotoxic Lymphocytes with T Cell Receptor Specific for Minor Histocompatibility Antigen ACC-1Y].

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative therapy for hematopoietic malignancies. The graft-derived donor lymphocytes are capable of eliminating the residual recipient malignant cells in the course of allogeneic immune response, thus decreasing the chances of a relapse of the disease. Foreign peptides of the recipient presented by the MHC molecules are able to elicit the immune response immunologically. These polymorphic peptides are known as minor histocompatibility antigens (MiHAs). MiHAs occur due to the nonsynonymous single nucleotide polymorphisms in human genome. Transfusion of T cells specific to MiHAs presented predominantly in the cells of hematopoietic origin will allow the targeted elimination of residual malignant clones avoiding undesirable damage to healthy tissues. To induce the immune response, the donor must be homozygous by the MiHA allele and the recipient must either be homozygous or heterozygous by the alternative MiHA allele. The therapeutic mismatch occurs in 25% of cases under the optimal frequency of allelic variants. Minor antigen ACC-1Y originates from polymorphism in the BCL-2A1 gene; its immunogenic mismatch occurrence approaches the theoretical maximum. In addition, BCL2A1 is overexpressed in cells of various lymphomas. ACC-1Y is presented on allele HLA-A*24:02, which is relatively frequent in the Russian population. Combination of these factors makes the minor antigen ACC-1Y a promising target for immunotherapy. Transfusion of donor CD8^(+) lymphocytes modified with transgenic MiHA-specific TCR is one of the promising methods of posttransplant leukemia therapy and relapse prophylaxis. We obtained a sequence of high-affinity ACC-1Y-specific TCR after the antigen-specific expansion of T cells derived from a healthy ACC-IY^(-/-) donor. We cloned this sequence into the lentiviral vector and obtained the assembled viral particles. Further, we transduced the CD8^(+) lymphocyte culture and demonstrated its antigen-specific cytotoxic activity. It is suggested that CD8^(+) lymphocytes modified by the described method could be potentially transferred to recipients as a therapy against relapse after allo-HSCT.

Development and investigation of recombinant immunotoxin protein 4D5scFv-mCherry-PE(40).

Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.