B2M gene knockout in HEK293T cells by non-viral delivery of CRISPR-Cas9 for the induction of universal cell

Abstract Allogeneic stem cells have been introduced as a potential approach to generate grafts in regenerative medicine. But the clinical usage of them is limited due to the risk of immune rejection that is induced by the incompatibility of human leukocyte antigens (HLAs) between donors and recipients. To overcome this limitation, we knocked out the β2 microglobulin (B2M) gene which is crucial for HLA class I expression, using CRISPR/Cas9 approach. Non-viral transfer of two gRNAs targeting exon 1 and intron 1 in B2M gene caused in large and predictable deletion in region between two loci, which was defined by sequencing and polymerase chain reaction (PCR). Furthermore, results revealed that roughly 11.11% and 22.22% of the GFP expressing cells reflecting a homozygous and heterozygous pattern of genomic modifications. We demonstrated that the dual guide RNA approach is a simple and efficient method for gene disruption. Significantly, these engineered hypoimmunogenic cells could be proposed as universal cells that are not distinguishable to the recipient immune system in cell therapy and transplantation.

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Human Leukocyte Antigens I and II Haplotypes Associated With Human Papillomavirus 16-Positive Invasive Cervical Cancer in Mexican Women

International Journal of Gynecological Cancer ◽

10.1111/igc.0b013e3181a83cf4 ◽

2009 ◽

Vol 19 (6) ◽

pp. 1099-1106 ◽

Cited By ~ 9

Author(s):

Dulce M. Hernández-Hernández ◽

Ricardo M. Cerda-Flores ◽

Teresa Juárez-Cedillo ◽

Julio Granados-Arriola ◽

Gilberto Vargas-Alarcón ◽

...

Keyword(s):

Cervical Cancer ◽

Human Papillomavirus ◽

Odds Ratio ◽

Human Leukocyte ◽

Hla Class I ◽

Class I ◽

Human Leukocyte Antigens ◽

Chain Reaction ◽

Leukocyte Antigens ◽

Polymerase Chain

Infection with human papillomavirus (HPV), mainly HPV type 16, is the major etiologic factor associated with cervical cancer (CC), but HPV infection alone is not sufficient for progression of precursor lesions. Host genetic susceptibility may lead to abnormal immune response resulting from virus persistence. Several studies have suggested a possible association with specific human leukocyte antigen (HLA) class I and II alleles and CC, but results are not consistent. The association of genetic HLA class I (A and B) and HLA class II (DR*B1 and DQ*B1) haplotypes with HPV16-positive CC (n = 104) and base population controls (n = 104) was evaluated in this Mexican population study. Sequence-specific primer HLA genes were determined by polymerase chain reaction (PCR)-based methods in peripheral blood cell counts (PCR sequence-specific oligonucleotides). The cervical swabs of 208 women were tested for HPV16 by Hybrid Capture II. Allele and haplotype HLA frequencies, Hardy-Weinberg tests, and a haplotype homogeneity test were estimated using the Arlequin software v. 3.01. Odds ratio (OR) was calculated to compare cases and control women. Consistent associations across other studies in women with CC and infected by HPV16 were observed for HLA-DRB1*15 (OR, 3.9; 95% CI, 1.6-10.2) and the haplotype DRB1*15 DQB1*0602 (OR, 4.1; 95% CI, 1.4-12.7) compared with control women. The HLA-A2-B44-DR4-DQ*0302, HLA-A24-B35-DR16-DQ*0301, and HLA-A2-B40-DR4-DQ*0302 haplotypes showed a positive association with CC (OR, >1), whereas HLA-A2-B39-DR4-DQ*0302, HLA-A24-B35-DR4-DQ*0302, and HLA-A68-B40-DR4-DQ*0302 showed a negative association (OR, <1). These results support the hypothesis that some HLA class I and II haplotypes could be involved with susceptibility for developing CC.Abbreviations:Cervical Cancer-CC, confidence interval-CI, human leukocyte antigens-HLA, human papillomavirus-HPV, odds ratio-OR, polymerase chain reaction-PCR, relative risk-RR, relative light units-RLU, ribonucleic acid-RNA, sequence-sensitive oligonucleotide-SSO

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Selective deletion of human leukocyte antigens protects stem cell-derived islets from immune rejection

Cell Reports ◽

10.1016/j.celrep.2021.109538 ◽

2021 ◽

Vol 36 (7) ◽

pp. 109538

Author(s):

Audrey V. Parent ◽

Gaetano Faleo ◽

Jessica Chavez ◽

Michael Saxton ◽

David I. Berrios ◽

...

Keyword(s):

Stem Cell ◽

Human Leukocyte ◽

Human Leukocyte Antigens ◽

Immune Rejection ◽

Leukocyte Antigens

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Extração de DNA a partir de sangue humano coagulado para aplicação nas técnicas de genotipagem de antígenos leucocitários humanos e de receptores semelhantes à imunoglobulina

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822009000600008 ◽

2009 ◽

Vol 42 (6) ◽

pp. 651-656 ◽

Cited By ~ 8

Author(s):

Daniela Maira Cardozo ◽

Gláucia Andréia Guelsin ◽

Samaia Laface Clementino ◽

Fabiano Cavalcante de Melo ◽

Marco Antônio Braga ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Killer Cell ◽

Human Leukocyte ◽

Reaction Sequence ◽

Chain Reaction ◽

Specific Sequence ◽

Salting Out ◽

Leukocyte Antigens ◽

Polymerase Chain ◽

Made In

O objetivo deste estudo foi padronizar uma metodologia de extração de DNA de alta qualidade a partir de amostras de sangue coagulado. Quarenta e oito amostras de sangue humano coagulado foram utilizadas para a extração de DNA pelo kit comercial EZ-DNA® (Biological Industries, Beit Haemek, Israel), pelo kit de coluna Neoscience® (One Lambda Inc., San Diego, CA) e pelo método modificado de salting out. Apenas o método de salting out foi capaz de extrair altas concentrações de DNA (média, 180ng/µL), as quais foram medidas pelo detector de fluorescência Qubit® (Invitrogen, USA). Este método permitiu a amplificação dos genes HLA (human leukocyte antigens) pela tecnologia PCR-SSO (polymerase chain reaction - specific sequence of oligonucleotides) Luminex, a qual exige DNA de boa qualidade, e de genes KIR (killer cell immunoglobulin-like receptors) pela técnica made in house PCR-SSP (polymerase chain reaction-sequence specific of primers), a qual demanda uma concentração específica de DNA (10ng/µL). Concluímos que a técnica de salting out modificada foi muito eficiente, simples e rápida para a extração de DNA de amostras de sangue humano coagulado, com o objetivo de realizar a genotipagem de genes HLA e KIR.

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Human leukocyte antigens (HLA) class I (Bg) on red cells studied with monoclonal antibodies

Immunohematology ◽

10.21307/immunohematology-2019-1041 ◽

2020 ◽

Vol 6 (3) ◽

pp. 53-58

Author(s):

Carolyn M. Giles

Keyword(s):

Monoclonal Antibodies ◽

Human Leukocyte ◽

Hla Class I ◽

Red Cells ◽

Class I ◽

Human Leukocyte Antigens ◽

Leukocyte Antigens

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Generation of HLA Universal Megakaryocytes and Platelets by Genetic Engineering

Frontiers in Immunology ◽

10.3389/fimmu.2021.768458 ◽

2021 ◽

Vol 12 ◽

Author(s):

Constanca Figueiredo ◽

Rainer Blasczyk

Keyword(s):

Genetic Engineering ◽

New Technologies ◽

Human Leukocyte ◽

Hla Class I ◽

Genetically Engineered ◽

Leukocyte Antigens ◽

Cell Sources ◽

Technical Advances

Patelet transfusion refractoriness remains a relevant hurdle in the treatment of severe alloimmunized thrombocytopenic patients. Antibodies specific for the human leukocyte antigens (HLA) class I are considered the major immunological cause for PLT transfusion refractoriness. Due to the insufficient availability of HLA-matched PLTs, the development of new technologies is highly desirable to provide an adequate management of thrombocytopenia in immunized patients. Blood pharming is a promising strategy not only to generate an alternative to donor blood products, but it may offer the possibility to optimize the therapeutic effect of the produced blood cells by genetic modification. Recently, enormous technical advances in the field of in vitro production of megakaryocytes (MKs) and PLTs have been achieved by combining progresses made at different levels including identification of suitable cell sources, cell pharming technologies, bioreactors and application of genetic engineering tools. In particular, use of RNA interference, TALEN and CRISPR/Cas9 nucleases or nickases has allowed for the generation of HLA universal PLTs with the potential to survive under refractoriness conditions. Genetically engineered HLA-silenced MKs and PLTs were shown to be functional and to have the capability to survive cell- and antibody-mediated cytotoxicity using in vitro and in vivo models. This review is focused on the methods to generate in vitro genetically engineered MKs and PLTs with the capacity to evade allogeneic immune responses.

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SNP analysis reveals estrogen receptor 1 (<i>ESR1</i>) gene variants associated with laying traits in quails

Archives Animal Breeding ◽

10.5194/aab-58-441-2015 ◽

2015 ◽

Vol 58 (2) ◽

pp. 441-444 ◽

Cited By ~ 1

Author(s):

Y. Wu ◽

A. L. Pan ◽

J. S. Pi ◽

Y. J. Pu ◽

J. P. Du ◽

...

Keyword(s):

Estrogen Receptor ◽

Snp Analysis ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Esr1 Gene ◽

Intron 1 ◽

Egg Number ◽

Exon 1 ◽

Polymerase Chain ◽

Estrogen Receptor 1

Abstract. In this study, the estrogen receptor 1 (ESR1) gene was studied as a candidate gene for laying traits of two quail populations (the yellow-feather quail and chestnut-feather quail). Five pairs of primers were designed to detect single-nucleotide polymorphisms (SNPs) of exon 1, 2, 4, 8 and intron 1 of the ESR1 gene by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and sequencing methods. Only the products amplified from exon 8 displayed polymorphism. The results showed one novel variation: a variation in exon 8 of ESR1 gene (g.91C > T, KC977991 and KC977992). It was associated with some laying traits in two quail populations including egg weight, the age of first egg and egg number at 20 weeks. And the CC genotype was associated with superior egg number at 20 weeks. Therefore, we speculated that the variation in exon 8 of ESR1 gene may have an effect on laying traits in the abovementioned quail populations.

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Monospecific and Polyreactive Monoclonal Antibodies against Human Leukocyte Antigen-E: Diagnostic and Therapeutic Relevance

Monoclonal Antibodies ◽

10.5772/intechopen.95235 ◽

2021 ◽

Author(s):

Mepur H. Ravindranath ◽

Fatiha E.L. Hilali

Keyword(s):

Amino Acid ◽

Nk Cells ◽

Major Histocompatibility Complex Gene ◽

Human Leukocyte ◽

Hla Class I ◽

Amino Acid Sequences ◽

Leukocyte Antigens ◽

Hla Class I Molecules ◽

Specific Localization ◽

High Degree

A monoclonal antibody (mAb) binds to an antigen recognizing an epitope (a sequence of amino acids). A protein antigen may carry amino acid sequence unique to that antigen as well as sequences found in other proteins. Human leukocyte antigens (HLA), a family of proteins expressed by the Major Histocompatibility Complex gene family represent a special case, in that it displays a high degree of polymorphism. Every HLA molecule possesses both specific (private) epitopes and epitopes shared (public) with other HLA class Ia and class Ib molecules. HLA-E is overexpressed in cancer cells more than any other HLA Class I molecules. Therefore specific localization of HLA-E with mAbs is pivotal for developing targeted therapy against cancer. However, the commercially available mAbs for immunodiagnosis are polyreactive. We have developed anti-HLA-E mAbs and distinguished monospecific from polyreactive mAbs using Luminex multiplex single antigen bead (SAB) assay. HLA-E-binding of monospecific-mAbs was also inhibited by E-restricted epitopes. The amino acid sequences in the region of the epitopes bind to CD94/NKG2A receptors on CD8+ T cells and NK cells and block their antitumor functions. Monospecific-HLA-E mAbs recognizing the epitopes sequences can interfere with the binding to restore the anti-tumor efficacy of NK cells. Also, monospecific-mAbs augment the proliferation of CD4-/CD+ cytotoxic T-lymphocytes. Therefore, anti-HLA-E monospecific-mAb can serve as a double-edged sword for eliminating tumor cells.

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Spliced HLA bound peptides; a Black-Swan event in Immunology

10.22541/au.160976778.87618567/v1 ◽

2021 ◽

Author(s):

Pouya Faridi ◽

Mohammadreza .Dorvash ◽

Anthony Purcell

Keyword(s):

Global Analysis ◽

Critical Role ◽

Human Leukocyte ◽

Hla Class I ◽

Peptide Ligands ◽

Considerable Proportion ◽

Immune Recognition ◽

Leukocyte Antigens ◽

Long Time ◽

Hla Molecules

Peptides that bind to and are presented on the cell surface by Human Leukocyte Antigens (HLA) molecules play a critical role in adaptive immunity. For a long time, it was believed all of the HLA bound peptides were generated through simple proteolysis of linear sequences of cellular proteins, and therefore, are templated in the genome and proteome. However, evidence for untemplated peptide ligands of HLA molecules has accumulated over the last two decades, with a recent global analysis of HLA-bound peptides suggesting that a considerable proportion of HLA bound peptides are potentially generated through splicing/fusion of discontinuous peptide segments from one or two distinct proteins. In this review, we will review recent discoveries and debates on the contribution of spliced peptides to the HLA class I immunopeptidome, consider biochemical rules for splicing, and the potential role of these spliced peptides in immune recognition.

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Distribution of HLA-B Locus Antigens in Patients with Psoriatic Arthritis in Bangladesh

Bangladesh Journal of Medical Microbiology ◽

10.3329/bjmm.v12i2.51691 ◽

2018 ◽

Vol 12 (2) ◽

pp. 14-20

Author(s):

Devolina Bhowmik ◽

Md Ruhul Amin Miah ◽

Shirin Tarafder ◽

Ahmed Abu Saleh ◽

Manash Chandra Sarker

Keyword(s):

Psoriatic Arthritis ◽

Human Leukocyte ◽

Healthy Controls ◽

Chain Reaction ◽

Specific Primers ◽

Disease Manifestation ◽

B Locus ◽

Leukocyte Antigens ◽

Healthy Donors ◽

Polymerase Chain

This study was designed to investigate the distribution of HLA-B locus antigens in patients with psoriatic arthritis (PsA) in Bangladesh and identify HLA markers related to disease manifestation in PsA. HLA-B typing was carried out by polymerase chain reaction (PCR) with sequence specific primers in a group of 50 consecutive PsA patients. The reports of HLA-B locus antigens typing were collected from 50 ages and sex matched unrelated healthy donors as controls. A total of 17 HLA-B locus antigens were determined in both patients and controls. The most common antigen was B*15 (34%) followed by B*07 (26%), B*27 (24%), B*38 (20%). Human leukocyte antigens B*07, B*27 and B*38 alleles were found to be significantly prevalent in PsA patients compared with healthy controls. We found a statistically significant association between spondylitis pattern and the presence of HLA-B*27. PsA in Bangladeshi patients seems to be associated with the presence of B*07, B*27 and B*38 alleles. Bangladesh J Med Microbiol 2018; 12 (2): 14-20

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