Insights into human early-life antibody-secreting cells via FlowLITE: A high-throughput assay to characterize and quantify antibody isotypes using flow cytometry. Restricted; Files Only
Hahm, Justin (Spring 2025)
Abstract
B lymphocytes are essential cells of the adaptive immune system that differentiate into antibody-secreting cells (ASCs) or plasma cells. Different cell receptors and soluble molecules recognize distinct structural forms of the antibody, organized into isotypes/subclasses, playing unique, sometimes opposing, effector functions to protect against infections and maintain homeostasis. These antibodies can also participate in disease pathology, including autoimmune diseases such as inflammatory bowel disease (IBD) in the gut. Thus, quantifying and characterizing the various isotypes of antibodies can provide crucial insights into disease pathology. We developed a novel technology called FlowLITE, a flow cytometry-based bead assay to measure antibody LIght chain and isoType Expression. FlowLITE is a modular assay, offering flexibility for multiplexing and high-throughput quantification of antibodies, including the ability to report the absolute numbers of antibody molecules detected in samples.
We applied FlowLITE to profile antibody secretion at birth. To understand how to generate ASCs in early life, we cultured mature B cells found in the spleen. Surprisingly, we found that BCR stimulation inhibited the formation of ASCs whereas TLR9 stimulation resulted in the formation of cells (8.8%) with a canonical plasma cell phenotype (CD38hi CD319hi). Utilizing FlowLITE, we found persistent natural IgA1 secretion and IgA2 class switching with TLR9 stimulation, suggesting that these mature cells may be programmed for mucosal sites. We confirmed cell surface expression for chemokine receptor CCR6 to the gut.
Thus, we analyzed human prenatal intestine with high-dimensional flow cytometry to characterize B cell development and other major immune lineages across gestational ages, from 82 to 145 days post conception (dpc). We found B cells, defined as CD19+ cells, emerge even at 82 dpc, rapidly increasing over gestation (~2% to ~15% of total immune cells at 82 dpc and 145 dpc, respectively). Surprisingly, CD34+ progenitors are present at 82 dpc before an astonishing robust acceleration of B cell maturation. By 145 dpc, over 70% of CD19+ B cells have differentiated into mature B cells (CD20+IgM+IgD+). Thus, there is a diversity of human plasma cells in early life that could mature locally and function to prevent gastrointestinal infections and support a healthy gut microbiome.
Table of Contents
Introduction 1
Figure 1. B lymphocytes are critical cells of the adaptive immune system that differentiate into antibody-secreting cells, or plasma cells, upon activation 1
Figure 2. Unique receptors for Antibody Secreting Cells in fetal life revealed by heavy chain gene expression 3
Methods 5
Sample acquisition and processing 5
B cell culture 6
Flow cytometry staining 7
FlowLITE 7
Chapter 1. FlowLITE: A flow cytometry-based bead assay to measure antibody Light chain and isotype Expression. 8
Figure 3. Cell STAR Protocols Graphical Abstract 8
Figure 4. Schematic of 8 bead global detection assay FlowLITE 9
Figure 5. Saturation of individual bead species by anti-human isotype mouse IgG1 antibodies 14
Figure 6. Analysis of calibration curve for 3 bead FlowLITE assay (IgM, pan-IgG, and pan-IgA) 22
Figure 7. Dilution series and quantification of healthy human serum 25
Figure 8. Application of FlowLITE in 8 bead assay and quantification of IgG subclasses 26
Figure 9. Reporting absolute count of antibody molecules with QuantiBrite 26
Chapter 2. Signaling Requirements to Generate Antibody Secreting Cells at Birth 31
Figure 10. BCR signaling inhibits ASC differentiation in early life spleen cells 31
Figure 11. FlowLITE 8 isotype profiling of supernatant culture conditions 32
Figure 12. Gene expression data from prenatal B cells demonstrate chemokine receptor expression across multiple tissues 33
Chapter 3. Local Maturation of Immune Cells in the Human Gut During Gestation Defines Mucosal Immunity at Birth 34
Figure 13. CD19+ B cells are present as early as 82 dpc in fetal intestine 35
Figure 14. Higher expression of CD45 and loss of CD19 in gut B cells demonstrates progression in maturation. Maturation is marked with a gain in CD20 at 117dpc 35
Figure 15. Maturation in the fetal ileum is accelerated compared to other hematopoietic organs at 145dpc. Notably, sites such as the fBM and fL are predominantly immature 36
Figure 16. The percentage of myeloid cells decreases over gestational time to ~30% of immune cells, while lymphocytes become the dominant population 37
Discussion 37
References 42
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File download under embargo until 22 May 2027 | 2025-04-08 14:35:10 -0400 | File download under embargo until 22 May 2027 |
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