A Native Soluble Form of CTLA-4

doi:10.1006/cimm.2000.1649

Cellular Immunology

Volume 201, Issue 2, 1 May 2000, Pages 144-153

https://doi.org/10.1006/cimm.2000.1649 Get rights and content

Abstract

CTLA-4 is an immunoregulatory receptor expressed on the surface of activated T and B lymphocytes. The counterreceptors for CTLA-4 are the B7 family molecules. We describe alternatively spliced mRNAs expressed in hematolymphoid tissues of humans, mice, and rats that lack the transmembrane domain coded by exon 3 of the CTLA-4 gene. These alternate transcripts were detected by RT-PCR in B cells and resting T cells of both the CD4 and the CD8 phenotype. Activation of human blood mononuclear cells with PHA or anti-CD3 + anti-CD28 monoclonal antibodies appears to effect a decrease in the amount of the alternative transcript relative to the full-length transcript. Recombinant sCTLA-4 is a B7-binding protein and has immunomodulatory effects as measured by inhibition of the mixed leukocyte response. Human serum contains immunoreactive material consistent with a native soluble form of CTLA-4.

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A soluble isoform of cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) has been found in the serum of healthy individuals and alterations in its expression level have been linked with the development and progression of various cancers. Conventionally, soluble CTLA-4 (sCTLA-4) has been quantified by techniques such as ELISA, western blot, and flow cytometry, which however are time-consuming, highly expensive and require large sample volumes. Therefore, rapid, cost-effective and real-time monitoring of soluble CTLA-4 levels is much needed to facilitate timely diagnosis of a worsening disease and help patient selection for immunotherapeutic interventions in cancer. Here, for the first time, we report an ultrasensitive, highly selective electrochemical nanobody (NAb) based biosensor for the quantitative detection of soluble CTLA-4 employing poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) and gold nanoparticles modified electrode with attomole sensitivity. Incorporating nanomaterials with conductive polymers enhances the sensitivity of the electrochemical biosensor, while the nanobody's stability, specificity and ease of production make it a suitable choice as a bioreceptor. The proposed NAb-based sensor can detect sCTLA-4 from pure recombinant protein in a wide concentration range of 100 ag mL⁻¹– 500 μg mL⁻¹, with a limit of detection of 1.19 ag mL⁻¹ (+3σ of the blank signal). The sensor's relative standard deviation for reproducibility is less than 0.4% and has effective real sample analytics for cell culture supernatant with no significant difference with pure recombinant protein (p < 0.05). Our proposed nanobody based sensor exhibits stability for up to 2 weeks (<3% variation). Moreover, this nanobody-based sensor presents a future opportunity for quantitative, ultrasensitive, and economical biosensor development that can be adapted to monitor the immune landscape of cancer patients to provide a larger therapeutic window.
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Immune checkpoint inhibitors (ICIs) have shown unprecedented benefits in various adult cancers, and this success has prompted the exploration of ICI therapy even in childhood malignances. Although the use of ICIs as individual agents has achieved disappointing response rates, combinational therapies are likely to promise better results. However, only a subset of patients experienced prolonged clinical effects, thus suggesting the need to identify robust bio-markers that predict individual clinical response or resistance to ICI therapy as the main challenge. In this review, we focus on how the use of ICIs in adult cancers can be translated into pediatric malignances. We discuss the physiological mechanism of action of each IC, including PD-1, PD-L1 and CTLA-4 and the new emerging ones, LAG-3, TIM-3, TIGIT, B7-H3, BTLA and IDO-1, and evaluate their prognostic value in both adult and childhood tumors. Furthermore, we offer an overview of preclinical models and clinical trials currently under investigation to improve the effectiveness of cancer immunotherapies in these patients. Finally, we outline the main predictive factors that influence the efficacy of ICIs, in order to lay the basis for the development of a pan-cancer immunogenomic model, able to direct young patients towards more specific immunotherapy.
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^☆: This work was supported in part by grants from the Vince Lombardi Cancer Research Foundation (No. 225-25 to M.K.O.) and from the Department of Cardiothoracic Surgery.

^☆☆: The nucleotide and predicted amino acid sequence data reported in this paper have been submitted to the GenBank sequence database and have been assigned the following accession numbers: Mouse sCTLA-4, U90270; Rat sCTLA-4, U90271; Rat intron/exon boundaries, AF012711, AF012712, and AF012713; Human sCTLA-4, U90273.

³: To whom correspondence should be addressed at Transplant Research Laboratory, St. Luke's Medical Center, 2900 W. Oklahoma Ave., Milwaukee, WI 53215. Fax: (414)-647-1292. E-mail: [email protected].

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Regular ArticleA Native Soluble Form of CTLA-4☆,☆☆

Abstract

Immunity

J. Biol. Chem.

Cell

Cell

Cell

Cell

Immunity

Immunity

A new member of the immunoglobulin superfamily—CTLA-4

Nature

A differential molecular biology search for genes preferentially expressed in functional T lymphocytes: The CTLA genes

Immunol. Rev.

Regulation of CTLA-4 expression during T cell activation

J. Immunol.

Activated T cells can induce high levels of CTLA-4 expression on B cells

J. Immunol.

Human Ig superfamily CTLA-4 gene: chromosomal localization and identity of protein sequence between murine and human CTLA-4 cytoplasmic domains

Eur. J. Immunol.

CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location

J. Immunol.

Binding of the B cell activation antigen B7 to CD28 costimulates T cell proliferation and interleukin 2 mRNA accumulation

J. Exp. Med.

CTLA-4 is a second receptor for the B cell activation antigen B7

J. Exp. Med.

Coexpression and functional cooperativity of CTLA-4 and CD28 on activated T lymphocytes

J. Exp. Med.

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production

J. Immunol.

CD80 (B7) and CD86 (B70) provide similar costimulatory signals for T cell proliferation, cytokine production and generation of CTL

J. Immunol.

T-cell activation by the CD28 ligand B7 is required for cardiac allograft rejection in vivo

Proc. Natl. Acad. Sci. USA

Induction of alloantigen-specific hyporesponsiveness in human T-lymphocytes by blocking interaction of CD28 with its natural ligand B7/BB1

J. Exp. Med.

Regular Article
A Native Soluble Form of CTLA-4☆,☆☆