Development of microanalysis tools for characterization of the humoral response to infections diseases

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (p. 127-134).Antibodies are higher order protein structures produced by a subset of lymphocytes (B cells) in the immune system for protection against pathogenic species. These homodimers of heterodimers form highly specific interactions with their cognate antigens and hence have become very important for the development of prophylactic or therapeutic agents against different disease pathogens. A key step in the development of human monoclonal antibodies as therapeutics is identification of candidate antibodies either by direct screening of human antibody repertoires or by filtering through combinatorial libraries of human variable genes using display technologies. Combinatorial libraries of human variable genes afford the flexibility to pursue many targets of interest, but often result in the selection of low affinity antibodies or unnatural heavy and light chain pairings that would have been selected against in vivo. With direct screening of the human B cell repertoire, the challenge is how to efficiently isolate clones of interest. Presented in this thesis is a high-throughput, integrated, single-cell methodology based on microengraving that allows the rapid recovery of antigen-specific human B cells. Microengraving is an analytical technique wherein secreted molecules from individual cells seeded into a dense array of subnanoliter wells are captured on the surface of a glass slide, generating a microarray from which desirable cells can be identified and recovered. Combined with high resolution epifluorescence microscopy and single-cell RT-PCR, we have developed assays for the simultaneous profiling of surface-expressed phenotypes of primary antibodyproducing cells, as well as functional characteristics of their secreted antibodies and germline variable gene usage. Using clinical samples from HIV- and West Nile virus-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies from both plasmablast/ plasma cell and memory B cell populations, and is ideal for the detailed characterization of cells from anatomical sites where sample sizes are often limited and disease pathophysiology is poorly understood (e,g. gut tissue, bone marrow).by Adebola O. Ogunniyi.Ph.D

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

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 106 cells. Here we present a process to identify antigen-specific responses efficiently ex vivo from 104–105 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 antigenspecific 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

Profiling Human Antibody Responses by Integrated Single-Cell Analysis

Comprehensive characterization of the antigen-specific B cells induced during infections or following vaccination would facilitate the discovery of novel antibodies and inform how interventions shape protective humoral responses. The analysis of human B cells and their antibodies has been performed using flow cytometry to evaluate memory B cells and expanded plasmablasts, while microtechnologies have also provided a useful tool to examine plasmablasts/plasma cells after vaccination. Here we present an integrated analytical platform, using arrays of subnanoliter wells (nanowells), for constructing detailed profiles for human B cells comprising the immunophenotypes of these cells, the distribution of isotypes of the secreted antibodies, the specificity and relative affinity for defined antigens, and for a subset of cells, the genes encoding the heavy and light chains. The approach combines on-chip image cytometry, microengraving, and single-cell RT-PCR. Using clinical samples from HIV-infected subjects, we demonstrate that the method can identify antigen-specific neutralizing antibodies, is compatible with both plasmablasts/plasma cells and activated memory B cells, and is well-suited for characterizing the limited numbers of B cells isolated from tissue biopsies (e.g., colon biopsies). The technology should facilitate detailed analyses of human humoral responses for evaluating vaccines and their ability to raise protective antibody responses across multiple anatomical compartments

Illustration of microengraving.

<p>Arrays of nanowells with dimensions of 50 µm×50 µm×50 µm were used for microengraving. Spleen or cervical lymph nodes cells were loaded in the nanowells. Cells in the nanowells were imaged using an automated epifluorescence microscope. Micrograving is performed by hybridizing nanowells with capture slides containing anti-mouse Ig for 2 hrs at 37°C with 5% CO₂. After incubation, nanowells containing intact live cells and capture slides were separated. A mixture of antibodies containing IgG1-Alexa Fluor 488, B6 SG lysate-Alexa Fluor 594 and <i>Aec1Aec2</i> SG lysate-Alexa Fluor 555 were added to the capture slides. Micrographs of microarrays were generating by scanning using a Genepix 4200AL microarray scanner.</p

Frequency of IgG1.

<p>Enumeration of IgG1-secretion cells from arrays of nanowells occupied by single cells from the spleens and cervical lymph nodes of C57BL/6 (n = 4) and C57BL/6.NOD-<i>Aec1Aec2</i> mice (n = 4). Data extracted from the image processing using Genepix software were used to identify the appropriate signals. The data were correlated with the nanowell image data in which nanowells contained a single cell positive for both Calcein (live cells) and CD19. The frequency was determined by using the ratio of positive IgG1 signal from wells with single cells and the total number of wells with single cells. *p<0.05 by unpaired t test. NS: not significant.</p

Characterization of cells from the cervical lymph nodes of C57BL/6 and C57BL/6.NOD-<i>Aec1Aec2</i> mice using microengraving.

<p><b>A.</b> Representative micrographs of live cells from C57BL/6 cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, C57BL/6 (B6) salivary glands proteins labeled with Alexa Fluor 594 and C57BL/6.NOD-<i>Aec1Aec2</i> (SjS) salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features (arrows) (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. <b>B.</b> Representative micrographs of live cells from C57BL/6.NOD-<i>Aec1Aec2</i> cervical lymph nodes (n = 4) in nanowells labeled with Calcein (live cells), CD19-FITC and CD4-Cy7. Micrographs of matching microarray showing detection signals for IgG1-Alexa Fluor 488, B6 salivary glands proteins labeled with Alexa Fluor 594 and SjS salivary glands proteins labeled with Alexa Fluor 555. The last vertical panel illustrates the close-up features pointed by the arrows (Live cell: CD19FITC, IgG1: IgG1-488 signal, B6 gland: signal of antibody binding to salivary gland proteins isolated from B6 mice. SjS gland: signal of antibody binding to salivary proteins isolated from SjS mice. All experiments were repeated at least twice for consistency.</p

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

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

The distribution of polysaccharide-specific antibody-secreting and memory B cells against the four polysaccharides.

<p>The number of PS-specific ASCs (A) and MBCs (B) specific for each antigen at days 0, 7 and 28 post-vaccination are depicted. The radius of each circle in the center panel is proportional to the number of PS-specific ASCs or MBCs detected, and the antigen specificity is indicated by the color of each circle. The antigen distribution for all ASCs and MBCs within each vaccine-group are depicted in pie charts. Numbers in the center of the pie charts indicate the total numbers of ASCs or MBCs detected. The distribution of antigen specificity was compared using a 2XN Fisher’s test.</p