Inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) analyses of genetic diversity in Mehr cotton cultivar and its crossing progenies

Cotton is cultivated in Iran with diploid and tetraploid forms and hybridization is a means to increase the genetic diversity and obtain new elite cultivars in this crop. This present study considered the molecular genetic diversity in Mehr (Gossypium hirsutum) cotton cultivar and its crossing progenies. 21 of 30 random amplified polymorphic DNA (RAPD) primers produced 220 reproducible bands with average of 10.47 bands per primer and 80.12% of polymorphism. OPR02 primer showed the highest number of effective allele (Ne), Shannon index (I) and genetic diversity (H). Some of the cultivars had specific bands, for example the F1 progeny of Mehr X No. 200, Mehr parental genotype and Mehr X Belilzovar hybrid genotype. Results show that some RAPD bands were present in the F1 progenies, but absent in the parental genotypes. Some others were present in the parental genotypes, but were absent in their hybrids. The highest values of genetic diversity in RAPD markers were obtained in Mehr X Sindose and Mehr X Belilzovar hybrids. Nine of ten inter simple sequence repeats (ISSR) primers used produced 113 reproducible bands with average of 54.35% polymorphism. UBC834 locus revealed the highest number of Ne, I and H values. Also, some ISSR bands occurred only in the parental genotype while some others occurred only in the hybrid genotypes. The highest values of genetic diversity in ISSR markers were obtained in Mehr X Sindose and Mehr X Belilzovar hybrids.Key words: Cotton, genetic diversity, random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR)

Targeted knockdown of Tim3 by short hairpin RNAs improves the function of anti-mesothelin CAR T cells

T-cell immunoglobulin mucin 3 (Tim3) is an immune checkpoint receptor that plays a central role in chimeric antigen receptor (CAR) T cell exhaustion within the tumor microenvironment. This study was aimed to evaluate the effects of targeted-knockdown of Tim3 on the antitumor function of anti-mesothelin (MSLN)-CAR T cells. To knockdown Tim3 expression, three different shRNA sequences specific to different segments of the human Tim3 gene were designed and co-inserted with an anti-MSLN-CAR transgene into lentiviral vectors. To investigate the efficacy of Tim3 targeting in T cells, expression of Tim3 was assessed before and after antigen stimulation. Afterwards, cytotoxic effects, proliferative response and cytokine production of MSLN-CAR T cells and Tim3-targeted MSLN-CAR T cells were analyzed. Our results showed that activation of T cells and MSLN-CAR T cells led to up-regulation of Tim3. Tim3 knockdown significantly decreased its expression in different groups of MSLN-CAR T cells. Tim3 knockdown significantly improved cytotoxic function, cytokine production and proliferation capacity of MSLN-CAR T cells. Our findings indicate that targeted knockdown of Tim3 allows tumor-infiltrating CAR T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby altering the tumor microenvironment from immunosuppressive to immunosupportive via mitigated Tim3 signaling

Construction and functional characterization of a fully human anti-CD19 chimeric antigen receptor (huCAR)-expressing primary human TÂ cells

Although remarkable results have been attained by adoptively transferring T cells expressing fully murine and/or humanized anti-CD19 chimeric antigen receptors (CARs) to treat B cell malignancies, evidence of human anti-mouse immune responses against CARs provides a rationale for the development of less immunogenic CARs. By developing a fully human CAR (huCAR), these human anti-mouse immune responses are likely eliminated. This, perhaps, not only increases the persistence of anti-CD19 CAR T cells�thereby reducing the risk of tumor relapse�but also facilitates administration of multiple, temporally separated doses of CAR T cells to the same recipient. To these ends, we have designed and constructed a second-generation fully human anti-CD19 CAR (or huCAR19) containing a fully human single-chain variable fragment (ScFv) fused with a CD8a hinge, a 4-1BB transmembrane domain and intracellular T cell signaling domains of 4-1BB and CD3z. T cells expressing this CAR specifically recognized and lysed CD19 + target cells produced cytokines and proliferated in vitro. Moreover, cell volume data revealed that our huCAR construct cannot induce antigen-independent tonic signaling in the absence of cognate antigen. Considering our results, our anti-CD19 huCAR may overcome issues of transgene immunogenicity that plague trials utilizing CARs containing mouse-derived ScFvs. These results suggest that this huCAR19 be safely and effectively applied for adaptive T cell immunotherapy in clinical practice. © 2018 Wiley Periodicals, Inc

Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR) Expressing T Cell

Chimeric antigen receptor (CAR) T cell therapy is considered as an encouraging approach for the treatment of hematological malignancies. However, its efficacy in solid tumors has not been satisfying, mainly in the immunosuppressive network of the tumor microenvironment and paucity of appropriate target antigens. Mesothelin (MSLN) is a tumor-associated antigen (TAA) expressed in numerous types of solid tumors such as gastrointestinal, ovarian, and pancreatic tumors. Owing to high expression in tumor cells and low expression in normal tissues, MSLN-targeted therapies like monoclonal antibodies have been previously developed. In the present study, a CAR T cell harboring the second-generation of a fully human anti-MSLN-CAR construct containing CD3 zeta and 4-1BB signaling domains was produced and it was functionally evaluated against an MSLN-expressing cell line. The findings showed potent, specific proliferation, cytotoxic activity, and interleukin (IL)-2, Tumor necrosis factor-(TNF) alpha, and Interferon-(IFN) gamma production in an antigen-dependent manner. Cytotoxic activity was shown in effector-to-target ratio from 1:1 to 20:1, but the most adequate efficacy was observed in the ratio of 10:1. Non-specific activity against MSLN negative cell line was not observed. Our data demonstrated that primary human T cells expressing fully human MSLN-CAR construct are effective against MSLN-expressing cell lines in vitro, suggesting this MSLN-CAR construct as a potential therapeutic tool in a clinical setting

Construction and Functional Characterization of a Fully Human Anti-mesothelin Chimeric Antigen Receptor (CAR) Expressing T Cell

Chimeric antigen receptor (CAR) T cell therapy is considered as an encouraging approach for the treatment of hematological malignancies. However, its efficacy in solid tumors has not been satisfying, mainly in the immunosuppressive network of the tumor microenvironment and paucity of appropriate target antigens. Mesothelin (MSLN) is a tumor-associated antigen (TAA) expressed in numerous types of solid tumors such as gastrointestinal, ovarian, and pancreatic tumors. Owing to high expression in tumor cells and low expression in normal tissues, MSLN-targeted therapies like monoclonal antibodies have been previously developed. In the present study, a CAR T cell harboring the second-generation of a fully human anti-MSLN-CAR construct containing CD3ζ and 4-1BB signaling domains was produced and it was functionally evaluated against an MSLN-expressing cell line. The findings showed potent, specific proliferation, cytotoxic activity, and interleukin (IL)-2, Tumor necrosis factor-(TNF) α, and Interferon-(IFN) γ production in an antigen-dependent manner. Cytotoxic activity was shown in effector-to-target ratio from 1:1 to 20:1, but the most adequate efficacy was observed in the ratio of 10:1. Non-specific activity against MSLN negative cell line was not observed. Our data demonstrated that primary human T cells expressing fully human MSLN-CAR construct are effective against MSLN-expressing cell lines in vitro, suggesting this MSLN-CAR construct as a potential therapeutic tool in a clinical setting