Concurrent detection of secreted products from human lymphocytes by microengraving: Cytokines and antigen-reactive antibodies

Bradshaw, Elizabeth M.; Kent, Sally C.; Tripuraneni, Vinay; Orban, Tihamer; Ploegh, Hidde L.; Hafler, David A.; Love, J. Christopher

doi:10.1016/j.clim.2008.06.009

Cited by 77 publications

(63 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The arrangement of captured antibodies on the resulting microarray matches the arrangement of cells in the microwells. This printing process can be repeated with the same set of cells to produce multiple replicate antibody microarrays [supporting information (SI) Scheme S1a] (11,12).…”

Section: Resultsmentioning

confidence: 99%

Profiling antibody responses by multiparametric analysis of primary B cells

Story

Papa

et al. 2008

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

Self Cite

View full text Add to dashboard Cite

Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (Ϸ10 3 -10 4 ). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigenspecific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases.affinity ͉ immunomics ͉ microengraving ͉ soft lithography ͉ vaccines

show abstract

Section: Resultsmentioning

confidence: 99%

Profiling antibody responses by multiparametric analysis of primary B cells

Story

Papa

et al. 2008

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

Self Cite

View full text Add to dashboard Cite

show abstract

“…In the absence of a direct assay to measure both functions (cytolysis and secretion), it has remained unclear to what extent cells exhibit both responses concurrently when encountering an APC. We have previously shown that the elastomeric array of microwells used here can also detect the frequencies and rates of secretion of multiple cytokines by a process called microengraving (26,27). According to this method, a glass slide coated with antibodies specific for .…”

Section: Detection Of Hiv-specific Cytotoxic Effector T Cells From CLmentioning

confidence: 99%

A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis

Varadarajan¹,

Jülg²,

Yamanaka³

et al. 2011

J. Clin. Invest.

Self Cite

143

136

View full text Add to dashboard Cite

CD8 + T cells are a key component of the adaptive immune response to viral infection. An inadequate CD8 +T cell response is thought to be partly responsible for the persistent chronic infection that arises following infection with HIV. It is therefore critical to identify ways to define what constitutes an adequate or inadequate response. IFN-γ production has been used as a measure of T cell function, but the relationship between cytokine production and the ability of a cell to lyse virus-infected cells is not clear. Moreover, the ability to assess multiple CD8 + T cell functions with single-cell resolution using freshly isolated blood samples, and subsequently to recover these cells for further functional analyses, has not been achieved. As described here, to address this need, we have developed a high-throughput, automated assay in 125-pl microwells to simultaneously evaluate the ability of thousands of individual CD8 + T cells from HIV-infected patients to mediate lysis and to produce cytokines. This concurrent, direct analysis enabled us to investigate the correlation between immediate cytotoxic activity and short-term cytokine secretion. The majority of in vivo primed, circulating HIV-specific CD8 + T cells were discordant for cytolysis and cytokine secretion, notably IFN-γ, when encountering cognate antigen presented on defined numbers of cells. Our approach should facilitate determination of signatures of functional variance among individual effector CD8 + T cells, including those from mucosal samples and those induced by vaccines.

show abstract

“…In the earlier mentioned human T1D study, an insulin-specific, PLN-isolated T cell clone was not found in the autologous spleen (21), suggesting that no expansion or retention of insulin-specific T cells was taking place. In contrast, autoreactive B cells from mouse and human diabetic pancreas infiltrates have been identified in the spleen of NOD mice and blood of diabetic patients, respectively (27,28). B cells have been shown to be efficient autoantigen-specific APCs in the PLNs and the spleen of NOD mice, as they express high levels of costimulatory molecules (29,30).…”

Section: T Ype 1 Diabetes (T1d) Is An Autoimmune Disease Defined By Tmentioning

confidence: 99%

TCR Bias of In Vivo Expanded T Cells in Pancreatic Islets and Spleen at the Onset in Human Type 1 Diabetes

Codina-Busqueta

Scholz

Torres

et al. 2011

The Journal of Immunology

View full text Add to dashboard Cite

Autoreactive T cells, responsible for the destruction of pancreatic β cells in type 1 diabetes, are known to have a skewed TCR repertoire in the NOD mouse. To define the autoreactive T cell repertoire in human diabetes, we searched for intraislet monoclonal expansions from a recent onset in human pancreas to then trace them down to the patient’s peripheral blood and spleen. Islet infiltration was diverse, but five monoclonal TCR β-chain variable expansions were detected for Vβ1, Vβ7, Vβ11, Vβ17, and Vβ22 families. To identify any sequence bias in the TCRs from intrapancreatic T cells, we analyzed 139 different CDR3 sequences. We observed amino acid preferences in the NDN region that suggested a skewed TCR repertoire within infiltrating T cells. The monoclonal expanded TCR sequences contained amino acid combinations that fit the observed bias. Using these CDR3 sequences as a marker, we traced some of these expansions in the spleen. There, we identified a Vβ22 monoclonal expansion with identical CDR3 sequence to that found in the islets within a polyclonal TCR β-chain variable repertoire. The same Vβ22 TCR was detected in the patient’s PBMCs, making a cross talk between the pancreas and spleen that was reflected in peripheral blood evident. No other pancreatic monoclonal expansions were found in peripheral blood or the spleen, suggesting that the Vβ22 clone may have expanded or accumulated in situ by an autoantigen present in both the spleen and pancreas. Thus, the patient’s spleen might be contributing to disease perpetuation by expanding or retaining some autoreactive T cells.

show abstract

Concurrent detection of secreted products from human lymphocytes by microengraving: Cytokines and antigen-reactive antibodies

Cited by 77 publications

References 33 publications

Profiling antibody responses by multiparametric analysis of primary B cells

Profiling antibody responses by multiparametric analysis of primary B cells

A high-throughput single-cell analysis of human CD8+ T cell functions reveals discordance for cytokine secretion and cytolysis

TCR Bias of In Vivo Expanded T Cells in Pancreatic Islets and Spleen at the Onset in Human Type 1 Diabetes

Contact Info

Product

Resources

About