High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays

Pira, Giuseppina Li; Ivaldi, Federico; Bottone, Laura; Manca, Fabrizio

doi:10.1016/j.jim.2007.06.012

Cited by 17 publications

(25 citation statements)

References 38 publications

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“…The cell-ELISA method, based on an assay originally designed for mouse splenocytes in 96-well plates (12), has been described in detail for 384-well plates (8). The adoption of 1,536-well plates required extensive automation of all procedures for antigen dispensing (proteins or peptides), for liquid and cell handling, and for plate development and scanning.…”

Section: Methodsmentioning

confidence: 99%

“…Pentamers were from Proimmune, Oxford, United Kingdom. Protein antigens, derived from different opportunistic pathogens or whole inactivated pathogen bodies, have been described previously (6,7,8,9). Pathogens used as whole bodies or pathogens from which proteins were derived included the following: Candida albicans, Aspergillus fumigatus, Aspergillus niger, Cryptococcus neoformans, Toxoplasma gondii, Mycobacterium bovis ("Mycobacterium tuberculosis var.…”

Section: Methodsmentioning

confidence: 99%

“…A miniaturized assay based on 384-well plates instead of conventional 96-well plates or 5-ml tubes was developed in our laboratory and named cell-enzyme-linked immunosorbent assay (cell-ELISA) (8). This method was based on a previous observation that lymphocyte cultures can be run in 96-well plates in which the wells had been precoated with an anticytokine antibody.…”

mentioning

confidence: 99%

“…The cytokine secreted by antigen-specific T cells was captured by the solid-phase antibody, and the plate was developed as a conventional ELISA plate (11). Miniaturization from 96-to 384-well plates (8) resulted in a reduction of tested lymphocytes from 2 ϫ 10 5 to 5 ϫ 10 5 down to 5 ϫ 10 4 per single antigen, corresponding to a scaling-down factor of 4-to 10-fold. In this report, we describe further miniaturization in 1,536-well plates by seeding 10 4 PBMCs per well, meaning a reduction in cell number by a factor of 20-to 50-fold over conventional assays.…”

mentioning

confidence: 99%

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Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

Pira

Ivaldi

Dentone

et al. 2008

Clin Vaccine Immunol

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The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-l volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-␥) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 10 4 PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20-to 50-fold fewer PBMCs than other analytical methods.Numerous methods are currently available to enumerate antigen-specific T cells and to measure their functions, most of them being based on flow cytometric analysis. Such methods have been compared and critically discussed in recent reviews (4, 17). A common feature of these methods is the requirement, on average, of 2 ϫ 10 5 to 5 ϫ 10 5 peripheral blood mononuclear cells (PBMCs) to test a single antigen. This imposes a limitation on the analytical antigenic breadth, which depends on the number of available PBMCs. In the case of lymphopenic subjects, the population that most frequently needs to be monitored for immunocompetence, this is particularly crucial. Also, the small volumes of pediatric blood samples may result in inadequate numbers of PBMCs for extended analysis of specific T-cell immunity. By the same token, screening of large peptide panels for epitope mapping on PBMCs from healthy donors (blood donors or vaccinees) is also limited to a few hundred peptides (16) instead of the thousands of peptides needed to cover several immunodominant proteins (14). Therefore, we felt the need to develop a miniaturized assay that makes use of limited numbers of PBMCs, thereby enhancing our screening power.A miniaturized assay based on 384-well plates instead of conventional 96-well plates or 5-ml tubes was developed in our laboratory and named cell-enzyme-linked immunosorbent assay (cell-ELISA) (8). This method was based on a previous observation that lymphocyte cultures can be run in 96-well plates in which the wells had been precoated with an anticytokine antibody. The cytokine secreted by antigen-specific T cells was captured by the solid-phase antibody, and the plate was de...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

Pira

Ivaldi

Dentone

et al. 2008

Clin Vaccine Immunol

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“…Tissue biopsies also typically yield less than 10 6 cells, with certain samples, such as cytobrushes or cerebrospinal fluids, containing only ∼10 4 -10 5 cells (8,9). Microscale assays that use microtiter plates or spotted arrays of proteins/ peptides have provided alternative means to enumerate antigenspecific T cells from limited numbers of cells but have not enabled routine cloning of identified cells (10)(11)(12). More efficient processes for recovering T cells for additional characterization would improve knowledge about the phenotypic and functional diversity of T-cell responses across anatomical sites and patient populations.…”

mentioning

confidence: 99%

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving

Varadarajan

Kwon

Law

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The nature of certain clinical samples (tissue biopsies, fluids) or the subjects themselves (pediatric subjects, neonates) often constrain the number of cells available to evaluate the breadth of functional T-cell responses to infections or therapeutic interventions. The methods most commonly used to assess this functional diversity ex vivo and to recover specific cells to expand in vitro usually require more than 10 6 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 10 4 –10 5 single cells from blood or mucosal tissues using dense arrays of subnanoliter wells. The approach combines on-chip imaging cytometry with a technique for capturing secreted proteins—called “microengraving”—to enumerate antigen-specific responses by single T cells in a manner comparable to conventional assays such as ELISpot and intracellular cytokine staining. Unlike those assays, however, the individual cells identified can be recovered readily by micromanipulation for further characterization in vitro. Applying this method to assess HIV-specific T-cell responses demonstrates that it is possible to establish clonal CD8 + T-cell lines that represent the most abundant specificities present in circulation using 100- to 1,000-fold fewer cells than traditional approaches require and without extensive genotypic analysis a priori. This rapid (<24 h), efficient, and inexpensive process should improve the comparative study of human T-cell immunology across ages and anatomic compartments.

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Pathogen specific T-lymphocytes for the reconstitution of the immunocompromised host

Pira¹,

Kapp

Manca

et al. 2009

Current Opinion in Immunology

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High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays

Cited by 17 publications

References 38 publications

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving

Pathogen specific T-lymphocytes for the reconstitution of the immunocompromised host

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High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays

Cited by 17 publications

References 38 publications

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

Evaluation of Antigen-Specific T-Cell Responses with a Miniaturized and Automated Method

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 + T cells using microengraving

Pathogen specific T-lymphocytes for the reconstitution of the immunocompromised host

Contact Info

Product

Resources

About

Rapid, efficient functional characterization and recovery of HIV-specific human CD8 ⁺ T cells using microengraving