Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection

Martinuzzi, Emanuela; Scotto, Matthieu; Énée, Emmanuelle; Brezar, Vedran; Ribeil, Jean‐Antoine; Endert, Peter Van; Mallone, Roberto

doi:10.1016/j.jim.2008.01.003

Cited by 23 publications

(33 citation statements)

References 34 publications

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“…Martinuzzi and colleagues [54] reported superior PBMC recoveries in samples frozen in AIM-V® medium (Invitrogen, Carlsbad, CA, USA) compared to human AB serum, both containing 10% DMSO. After thawing, the IFN-g ELISPOT signal against CD8 + T cell viral peptides titrated at minimal concentrations did not differ significantly, but AIM-V® freezing medium allowed an average recovery of 17% more cells (P < 0·04) [54]. Interestingly, AIM-V® medium contains HSA, which may explain its superior performance.…”

Section: Freezing Mediamentioning

confidence: 99%

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society

Mallone

Mannering

Brooks-Worrell

et al. 2010

Clinical and Experimental Immunology

220

200

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Summary Autoimmune T cell responses directed against insulin‐producing β cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D ‘immune staging’ and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays. To this end, we review data relevant to critical parameters in peripheral blood mononuclear cell (PBMC) isolation, (cryo)preservation, distribution and usage for detecting antigen‐specific T cell responses. Based on these data, we propose recommendations on processing blood samples for T cell assays and identify gaps in knowledge that need to be addressed. These recommendations may be relevant not only for the analysis of T cell responses in autoimmune disease, but also in cancer and infectious disease, particularly in the context of clinical trials.

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Section: Freezing Mediamentioning

confidence: 99%

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society

Mallone

Mannering

Brooks-Worrell

et al. 2010

Clinical and Experimental Immunology

220

200

View full text Add to dashboard Cite

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“…IL-7 (0.5 ng/mL; R&D Systems) was added along with maturation stimuli. 17 When used, peptide Ags (0.1-10M) were added on day 1 with maturation stimuli. On day 2 (48 hours after start of culture), nonadherent cells were collected, washed, and analyzed.…”

Section: Acdc Stimulation On Human Pbmcsmentioning

confidence: 99%

“…Mouse GM-CSF and IL-4 (R&D Systems) were added as for human acDCs with or without KLH (0.1 g/mL); LPS (10 ng/mL) was added at day 1 and ELISPOT assays performed on day 2, as described previously. 15,17 …”

Section: Acdc Stimulation On Mouse Bloodmentioning

confidence: 99%

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acDCs enhance human antigen–specific T-cell responses

Martinuzzi

Afonso

Gagnerault

et al. 2011

Blood

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Detection of human Ag-specific T cells is limited by sensitivity and blood requirements. As dendritic cells (DCs) can potently stimulate T cells, we hypothesized that their induction in PBMCs in situ could link Ag processing and presentation to Ag-specific T-cell activation. To this end, unfractionated PBMCs (fresh or frozen) or whole blood were incubated for 48 hours with protein or peptide Ag together with different DC-activating agents to rapidly and sequentially induce, pulse, and mature DCs. DC activation was therefore lined up with Ag recognition by neighboring T cells, thus telescoping the sequential steps of T-cell activation. Efficient processing of protein Ags made prior knowledge of epitopes and HLA restrictions dispensable. While reducing stimulation time, manipulation and blood requirements, in situ DC induction specifically amplified Ag-specific T-cell responses (cytokine secretion, proliferation, CD137/CD154 up-regulation, and binding of peptide-HLA multimers). IL-1␤, although released by DCs, was also secreted in an Ag-specific fashion, thus providing an indirect biomarker of T-cell responses. These accelerated cocultured DC (acDC) assays offered a sensitive means with which to evaluate T-cell responses to viral and melanoma Ag vaccination, and may therefore find application for immune monitoring in viral, tumor, autoimmune, and transplantation settings. (Blood. 2011;118(8):2128-2137) IntroductionDespite the central role of T cells in immune responses to foreign or self-Ags, routine immune diagnosis and monitoring relies largely, if not exclusively, on the measurement of Abs. However, Abs do not always mediate or reflect the underlying pathology and may be less informative when the immune process is predominantly T-cell mediated. The sole clinical application of Ag-specific T-cell assays to date has been in the diagnosis of Mycobacterium tuberculosis infection. 1 Moreover, T-cell monitoring is required to evaluate immune modulation therapies aimed at boosting viral or tumor-specific immunity, 2 or at quenching immunity against self-3,4 or transplanted 5 tissues. T-cell-screening tools to assess the immunogenic potential of vaccines 6 or of replacement proteins (eg, coagulation factors) 7 are also required.The lack of routine human T-cell assays is mainly because of the very low frequency (0.1%-0.001%) 8 of T cells specific for a given Ag in blood. Although these cells are sometimes detectable ex vivo, their rarity challenges the sensitivity of technologies such as ELISPOT and flow cytometry. The frequency of these cells may be augmented by preliminary expansion steps, 9 but these require additional time and manipulation.Epitope peptides that bind to HLA molecules for presentation and recognition by the T-cell receptor are frequently used to elicit T-cell responses because they do not require processing by APCs. While bypassing this initial limiting step for T-cell activation, epitopes nevertheless first need to be identified as binding to specific HLA molecules, and they stimulate a limited reper...

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“…ϩ T-cells, we have recently proposed an isletspecific CD8 ϩ T-cell ␥-interferon (IFN-␥) ELISpot (ISL8Spot) assay (18,29). The ISL8Spot employs HLA-A2-restricted ␤-cell epitopes derived from preproinsulin (PPI) (30), GAD, insulinoma-associated protein 2 (IA-2) (17), and IGRP (31), which have been mostly identified by proteasome digestion and DNA immunization strategies (32).…”

Section: For Cd8mentioning

confidence: 99%

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

et al. 2008

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OBJECTIVE-Islet-reactive CD8ϩ T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown. RESEARCH DESIGN AND METHODS-We took advantage of a recently validated islet-specific CD8ϩ T-cell ␥-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2 ϩ adult type 1 diabetic patients close to diagnosis and at a second time point 7-16 months later. RESULTS-CD8ϩ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P ϭ 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60 -67 to 20% (P Ͻ 0.02). The previously subdominant IA-2 206 -214 and IGRP [265][266][267][268][269][270][271][272][273] peptides were newly targeted, thus becoming the immunodominant epitopes. CONCLUSIONS-Shifts both in frequency and in immunodominance of CD8ϩ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements. Diabetes 57:1312-1320, 2008

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Serum-free culture medium and IL-7 costimulation increase the sensitivity of ELISpot detection

Cited by 23 publications

References 34 publications

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society

acDCs enhance human antigen–specific T-cell responses

The Frequency and Immunodominance of Islet-Specific CD8+ T-cell Responses Change after Type 1 Diabetes Diagnosis and Treatment

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