The identification of the mechanisms of lrba deficiency dependent defects in regulatory t-cell function

Primary immunodeficiencies are a very diverse group of disorders characterized by recurring infections. Loss of function mutations in the gene encoding LRBA (lipopolysaccharide-responsive beige like anchor protein) were recently identified to cause PID which is associated with autoimmunity. Several studies have shown that the LRBA molecule regulates CTLA-4 cell surface expression. Besides CTLA-4, LRBA has been claimed to have an effect on the vesicular trafficking of epidermal growth factor receptor (EGFR) and cell surface expression of FasL in the plasma membrane. In this study, the main objective was to discover the defects caused by LRBA deficiency in T-cells. For this purpose, the Jurkat leukemic T-cell line was used as the model cell line and the CRISPR/Cas9 system was used to knock-out (KO) this protein in the Jurkat cell line. The differentially expressed proteins on the surface of WT and KO Jurkat cells were assessed by the means of cell surface biotinylation coupled with mass spectrometry (MS) analysis. CD3, CD4, PVRIG, NOTCH3, CD1d, and Sema7A were found to be downregulated and CD53, CD148, CD154, CD134, A33, and CD70 to be upregulated in LRBA negative cell lines. We identify the decrease in CD3 and CD4 seemed to be most significant and relevant to the loss of LRBA. As expected, endogenous CTLA-4 expression was also decreased in our LRBA KO cell lines. Additionally, CTLA-4 protein overexpression could not rescue CTLA4 expression to WT levels in LRBA KO Jurkat iv cell lines indicating the dominant phenotype obtained by LRBA loss. This study identifies alternative targets of the LRBA protein and delineates the mechanism of surface CTLA4 expression, potentially identifying new targets that can be used to cure immunodeficiencies resulting from LRBA loss

Nanobodies as molecular imaging probes

Camelidae derived single-domain antibodies (sdAbs), commonly known as nanobodies (Nbs), are the smallest antibody fragments with full antigen-binding capacity. Owing to their desirable properties such as small size, high specificity, strong affinity, excellent stability, and modularity, nanobodies are on their way to overtake conventional antibodies in terms of popularity. To date, a broad range of nanobodies have been generated against different molecular targets with applications spanning basic research, diagnostics, and therapeutics. In the field of molecular imaging, nanobody-based probes have emerged as a powerful tool. Radioactive or fluorescently labeled nanobodies are now used to detect and track many targets in different biological systems using imaging techniques. In this review, we provide an overview of the use of nanobodies as molecular probes. Additionally, we discuss current techniques for the generation, conjugation, and intracellular delivery of nanobodies

Co2+, Fe2+, and Ni2+: modifiers for photocatalytic deposition of highly active Pt on graphene-based supports

This study focuses on photocatalytic syntheses, in which transition metal ions (Co2+, Fe2+, or Ni2+), as the hole scavengers and surface modifiers of partially reduced graphene oxide, PRGO, were utilized to photoreduce Pt4+. A pulsed UV reactor was used to illuminate the precursors. The electrostatic interaction between the metal ions (Co2+, Fe2+, or Ni2+) and the oxygen functional groups on PRGO was the main parameter, proposed to be the reason controlling Pt4+ reduction and Pt structure and activity. The alternative assumption in managing the oxidation states of Pt was the variation in the oxidation rates of hole scavengers. Pt electrocatalysts' structural and electrochemical characteristics revealed that utilizing the cobalt-based hole scavenger caused a dominant growth phase of Pt particles at preferred positions on PRGO, with metallic states and improved electrocatalytic activities (ECSA value of 191 m2·g-1 for Co2+ vs 141 m2·g-1 and 127 m2·g-1 for Fe2+ and Ni2+, respectively). Density functional theory (DFT) calculation, on the other hand, suggested that the greater affinity of cobalt and iron ions to oxygen groups could detach more "O"from the graphene plane. Based on the DFT results, less "O"groups in the vicinity of Pt particles gave an amorphous morphology to Pt and facilitated the hydrogen oxidation reaction (HOR)

Engineering antigen‐specific NK cell lines against the melanoma‐associated antigen tyrosinase via TCR gene transfer

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T‐lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL‐TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off‐target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen‐specific TCR complex on NK cell lines NK‐92 and YTS, demonstrated by using a TCR against the HLA‐A2‐restricted tyrosinase‐derived melanoma epitope, Tyr368‐377. Most importantly, the introduction of a TCR complex to NK cell lines enables MHC‐restricted, antigen‐specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of “mispairing” in TCR gene therapy