Characterization of gene expression in antibody secreting cells
Ontology highlight
ABSTRACT: The CD19 positive antibody secreting cells (ASC) in both bone marrow (BM) have the capacity to provide immune memory in addition to cells traditionally considered long-lived, the CD19-negative BM ASC. We performed flow cytometry (FCM) immunophenotyping, fluorescence-activated cell sorting (FACS) for cell subset isolation, ELISpot assays detecting the isotype of antibody secretion as well as antibodies against vaccine derived antigens, and comparative gene expression analyses of CD19- ASC, CD19+ ASC, CD20- B cells, and CD20+ B cells from BM. The findings may aid in the understanding of the differential cell subsets created through vaccination and lead to improved vaccine strategies and production. FACS sorted tissue B cells and antibody secreting cell subset gene expression.
Project description:Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observed in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.
Project description:Bone marrow plasma cells (BMPC) are the correlate of humoral immunity, consistently releasing antibodies into the bloodstream. It remains unclear if BMPC reflect different activation environments or maturation of their precursors. Here we define human BMPC heterogeneity and track the recruitment of antibody-secreting cells (ASC) from SARS-CoV-2 vaccine immune reactions to the bone marrow (BM). Trajectories based on single-cell transcriptomes and repertoires of peripheral and BM ASC reveal sequential colonisation of BMPC compartments. In activated B cells, IL-21 suppresses CD19 expression, indicating that CD19low-BMPC are derived from follicular, while CD19high-BMPC originate from extrafollicular immune reactions. In primary immune reactions, both CD19low- and CD19high-BMPC compartments are populated. In secondary immune reactions, most BMPC are recruited to CD19high-BMPC compartments, reflecting their origin from extrafollicular reactivations of memory B cells. A pattern also observed in vaccinated-convalescent individuals and upon diphtheria/tetanus/pertussis recall-vaccination. Thus, BMPC diversity reflects the evolution of a given humoral immune response.
Project description:Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibodies, some of which are present in high titers in a sustained, B cell-independent fashion consistent with their generation from long-lived plasma cells (LLPC). Active SLE displays high numbers of circulating antibody-secreting cells (ASC). Understanding the mechanisms of generation and survival of SLE ASC would contribute important insight into disease pathogenesis and novel targeted therapies. We studied the properties of SLE ASC through a systematic analysis of their phenotypic, molecular, structural, and functional features. Our results indicate that in active SLE, relative to healthy post-immunization responses, blood ASC contain a much larger fraction of newly generated mature CD19-CD138+ASC similar to bone marrow (BM) LLPC. SLE ASC were characterized by morphological and structural features of premature maturation. Additionally, SLE ASC express high levels of CXCR4 and CD138, and molecular programs consistent with increased longevity based on pro-survival and attenuated pro-apoptotic pathways.Notably, SLE ASC demonstrate autocrine production of APRIL and IL-10 and experience prolonged in vitro survival. Combined, our findings indicate that SLE ASC are endowed with enhanced peripheral maturation, survival and BM homing potential suggesting that these features likely underlie BM expansion of autoreactive PC.
Project description:Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibodies, some of which are present in high titers in a sustained, B cell-independent fashion consistent with their generation from long-lived plasma cells (LLPC). Active SLE displays high numbers of circulating antibody-secreting cells (ASC). Understanding the mechanisms of generation and survival of SLE ASC would contribute important insight into disease pathogenesis and novel targeted therapies. We studied the properties of SLE ASC through a systematic analysis of their phenotypic, molecular, structural, and functional features. Our results indicate that in active SLE, relative to healthy post-immunization responses, blood ASC contain a much larger fraction of newly generated mature CD19-CD138+ASC similar to bone marrow (BM) LLPC. SLE ASC were characterized by morphological and structural features of premature maturation. Additionally, SLE ASC express high levels of CXCR4 and CD138, and molecular programs consistent with increased longevity based on pro-survival and attenuated pro-apoptotic pathways.Notably, SLE ASC demonstrate autocrine production of APRIL and IL-10 and experience prolonged in vitro survival. Combined, our findings indicate that SLE ASC are endowed with enhanced peripheral maturation, survival and BM homing potential suggesting that these features likely underlie BM expansion of autoreactive PC.
Project description:Here we developed a protocol to extract total RNA from fixed and permeabilized cells that were intracellularly stained and FACS isolated. We applied these techniques to derive the transcriptome of influenza- and BCR isotype-specific antibody secreting cells or plasma cells. These data reveal that ASC isotypes have more distinct transcriptional signatures than previously appreciated.
Project description:Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASC) to long-lived plasma cells (LLPC). We provide single cell transcriptional resolution of 17,347 BM ASC from 5 healthy adults. Fifteen clusters were identified ranging from newly minted ASC (cluster 1) expressing MKI67 and high MHC Class II that progressed to late clusters 5-8 through intermediate clusters 2-4. Additional clusters included IgM-predominant ASC of likely extra-follicular origin; IFN-responsive; and high mitochondrial activity ASC. Late ASCs were distinguished by differences in G2M checkpoints, MTOR signaling, distinct metabolic pathways, CD38 expression, and utilization of TNF-receptor superfamily members. They mature through two distinct paths differentiated by the degree of TNF signaling through NFKB. This study provides the first single cell resolution atlas and molecular roadmap of LLPC maturation, thereby providing insight into differentiation trajectories and molecular regulation of these essential processes in the human BM microniche. This information enables investigation of the origin of protective and pathogenic antibodies in multiple diseases and development of new strategies targeted to the enhancement or depletion of the corresponding ASC.
Project description:Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASC) to long-lived plasma cells (LLPC). We provide single cell transcriptional resolution of 17,347 BM ASC from 5 healthy adults. Fifteen clusters were identified ranging from newly minted ASC (cluster 1) expressing MKI67 and high MHC Class II that progressed to late clusters 5-8 through intermediate clusters 2-4. Additional clusters included IgM-predominant ASC of likely extra-follicular origin; IFN-responsive; and high mitochondrial activity ASC. Late ASCs were distinguished by differences in G2M checkpoints, MTOR signaling, distinct metabolic pathways, CD38 expression, and utilization of TNF-receptor superfamily members. They mature through two distinct paths differentiated by the degree of TNF signaling through NFKB. This study provides the first single cell resolution atlas and molecular roadmap of LLPC maturation, thereby providing insight into differentiation trajectories and molecular regulation of these essential processes in the human BM microniche. This information enables investigation of the origin of protective and pathogenic antibodies in multiple diseases and development of new strategies targeted to the enhancement or depletion of the corresponding ASC.
Project description:Antibody secreting cells (ASC) circulate after vaccination and infection and migrate to the bone marrow (BM) where a subset known as long-lived plasma cells (LLPC) persist and secrete antibodies for a lifetime. The mechanisms of how circulating ASC become LLPC is not well elucidated. Here, we show that human early-minted blood ASCs have distinct morphology, transcriptomes, and epigenetics compared to human BM LLPC. Compared to blood ASC, BM LLPC have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. Additionally, LLPC acquire transcriptional and epigenetic differences in multiple cellular pathways that include apoptosis. Upregulation of the anti-apoptotic genes MCL1, BCL2, BCL-XL while simultaneously downregulation of pro-apoptotic genes HRK1, CASP3, and CASP8 occurs in LLPC. Consistent with the decrease in gene expression, pro-apoptotic gene loci are less accessible in LLPC. In contrast, anti-apoptotic gene expression is increased but is not always accompanied by changes in accessibility. Importantly, we show that early-minted blood ASCs undergo morphological and transcriptional changes that make these cells resemble ex vivo BM ASCs, suggesting that the BM microniche at least in part promotes LLPC maturation. Overall, our study demonstrates that early-minted blood ASC in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPC.
Project description:Antibody-secreting cells (ASC) circulate after vaccination and infection and migrate to the bone marrow (BM) where a subset known as long-lived plasma cells (LLPC) persist and secrete antibodies for a lifetime. The mechanisms of how circulating ASC become LLPC is not well elucidated. Here, we show that human early-minted blood ASCs have distinct morphology, transcriptomes, and epigenetics compared to human BM LLPC. Compared to blood ASC, BM LLPC have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. Additionally, LLPC acquire transcriptional and epigenetic differences in multiple cellular pathways that include apoptosis. Upregulation of the anti-apoptotic genes MCL1, BCL2, BCL-XL while simultaneously downregulation of pro-apoptotic genes HRK1, CASP3, and CASP8 occurs in LLPC. Consistent with the decrease in gene expression, pro-apoptotic gene loci are less accessible in LLPC. In contrast, anti-apoptotic gene expression is increased but is not always accompanied by changes in accessibility. Importantly, we show that early-minted blood ASCs undergo morphological and transcriptional changes that make these cells resemble ex vivo BM ASCs, suggesting that the BM microniche at least in part promotes LLPC maturation. Overall, our study demonstrates that early-minted blood ASC in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPC.
Project description:Human B-1 cells (CD20+CD27+CD43+CD38lo/int) and pre-plasmablast like cells (CD20+CD27hiCD38hi) are new antibody secreting cells identified in circulation. We used microarray to compare and contrast expressed genes between these two cell population