Understanding heterogeneity of human bone marrow plasma cell maturation and survival pathways by single-cell analyses.

Human bone marrow (BM) plasma cells are heterogeneous, ranging from newly arrived antibody-secreting cells (ASCs) to long-lived plasma cells (LLPCs). We provide single-cell transcriptional resolution of 17,347 BM ASCs from five healthy adults. Fifteen clusters are identified ranging from newly minted ASCs (cluster 1) expressing MKI67 and high major histocompatibility complex (MHC) class II that progress to late clusters 5-8 through intermediate clusters 2-4. Additional ASC clusters include the following: immunoglobulin (Ig) M predominant (likely of extra-follicular origin), interferon responsive, and high mitochondrial activity. Late ASCs are distinguished by G2M checkpoints, mammalian target of rapamycin (mTOR) signaling, distinct metabolic pathways, CD38 expression, utilization of tumor necrosis factor (TNF)-receptor superfamily members, and two distinct maturation pathways involving TNF signaling through nuclear factor κB (NF-κB). This study provides a single-cell atlas and molecular roadmap of LLPC maturation trajectories essential in the BM microniche. Altogether, understanding BM ASC heterogeneity in health and disease enables development of new strategies to enhance protective ASCs and to deplete pathogenic ones.

Single-cell analysis of human nasal mucosal IgE antibody secreting cells reveals a newly minted phenotype.

Immunoglobulin (Ig) E is central to the pathogenesis of allergic conditions, including allergic fungal rhinosinusitis. However, little is known about IgE antibody secreting cells (ASCs). We performed single-cell RNA sequencing from cluster of differentiation (CD)19+ and CD19- ASCs of nasal polyps from patients with allergic fungal rhinosinusitis (n = 3). Nasal polyps were highly enriched in CD19+ ASCs. Class-switched IgG and IgA ASCs were dominant (95.8%), whereas IgE ASCs were rare (2%) and found only in the CD19+ compartment. Through Ig gene repertoire analysis, IgE ASCs shared clones with IgD-CD27- "double-negative" B cells, IgD+CD27+ unswitched memory B cells, and IgD-CD27+ switched memory B cells, suggesting ontogeny from both IgD+ and memory B cells. Transcriptionally, mucosal IgE ASCs upregulate pathways related to antigen presentation, chemotaxis, B cell receptor stimulation, and survival compared with non-IgE ASCs. Additionally, IgE ASCs have a higher expression of genes encoding lysosomal-associated protein transmembrane 5 (LAPTM5) and CD23, as well as upregulation of CD74 (receptor for macrophage inhibitory factor), store-operated Calcium entry-associated regulatory factor (SARAF), and B cell activating factor receptor (BAFFR), which resemble an early minted ASC phenotype. Overall, these findings reinforce the paradigm that human ex vivo mucosal IgE ASCs have a more immature plasma cell phenotype than other class-switched mucosal ASCs and suggest unique functional roles for mucosal IgE ASCs in concert with Ig secretion.

B-1 plasma cells require non-cognate CD4 T cell help to generate a unique repertoire of natural IgM.

Evolutionarily conserved, "natural" (n)IgM is broadly reactive to both self and foreign antigens. Its selective deficiency leads to increases in autoimmune diseases and infections. In mice, nIgM is secreted independent of microbial exposure to bone marrow (BM) and spleen B-1 cell-derived plasma cells (B-1PC), generating the majority of nIgM, or by B-1 cells that remain non-terminally differentiated (B-1sec). Thus, it has been assumed that the nIgM repertoire is broadly reflective of the repertoire of body cavity B-1 cells. Studies here reveal, however, that B-1PC generate a distinct, oligoclonal nIgM repertoire, characterized by short CDR3 variable immunoglobulin heavy chain regions, 7-8 amino acids in length, some public, many arising from convergent rearrangements, while specificities previously associated with nIgM were generated by a population of IgM-secreting B-1 (B-1sec). BM, but not spleen B-1PC, or B-1sec also required the presence of TCRαß CD4 T cells for their development from fetal precursors. Together, the studies identify important previously unknown characteristics of the nIgM pool.

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa.

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Differential transcriptome and development of human peripheral plasma cell subsets.

Human antibody-secreting cells (ASCs) triggered by immunization are globally recognized as CD19loCD38hiCD27hi. Yet, different vaccines give rise to antibody responses of different longevity, suggesting ASC populations are heterogeneous. We define circulating-ASC heterogeneity in vaccine responses using multicolor flow cytometry, morphology, VH repertoire, and RNA transcriptome analysis. We also tested differential survival using a human cell-free system that mimics the bone marrow (BM) microniche. In peripheral blood, we identified 3 CD19+ and 2 CD19- ASC subsets. All subsets contributed to the vaccine-specific responses and were characterized by in vivo proliferation and activation. The VH repertoire demonstrated strong oligoclonality with extensive interconnectivity among the 5 subsets and switched memory B cells. Transcriptome analysis showed separation of CD19+ and CD19- subsets that included pathways such as cell cycle, hypoxia, TNF-α, and unfolded protein response. They also demonstrated similar long-term in vitro survival after 48 days. In summary, vaccine-induced ASCs with different surface markers (CD19 and CD138) are derived from shared proliferative precursors yet express distinctive transcriptomes. Equal survival indicates that all ASC compartments are endowed with long-lived potential. Accordingly, in vivo survival of peripheral long-lived plasma cells may be determined in part by their homing and residence in the BM microniche.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

Robust Antitumor Responses Result from Local Chemotherapy and CTLA-4 Blockade.

Clinical responses to immunotherapy have been associated with augmentation of preexisting immune responses, manifested by heightened inflammation in the tumor microenvironment. However, many tumors have a noninflamed microenvironment, and response rates to immunotherapy in melanoma have been <50%. We approached this problem by utilizing immunotherapy (CTLA-4 blockade) combined with chemotherapy to induce local inflammation. In murine models of melanoma and prostate cancer, the combination of chemotherapy and CTLA-4 blockade induced a shift in the cellular composition of the tumor microenvironment, with infiltrating CD8+ and CD4+ T cells increasing the CD8/Foxp3 T-cell ratio. These changes were associated with improved survival of the mice. To translate these findings into a clinical setting, 26 patients with advanced melanoma were treated locally by isolated limb infusion with the nitrogen mustard alkylating agent melphalan followed by systemic administration of CTLA-4 blocking antibody (ipilimumab) in a phase II trial. This combination of local chemotherapy with systemic checkpoint blockade inhibitor resulted in a response rate of 85% at 3 months (62% complete and 23% partial response rate) and a 58% progression-free survival at 1 year. The clinical response was associated with increased T-cell infiltration, similar to that seen in the murine models. Together, our findings suggest that local chemotherapy combined with checkpoint blockade-based immunotherapy results in a durable response to cancer therapy. Cancer Immunol Res; 6(2); 189-200. ©2018 AACR.

TCR Repertoire Intratumor Heterogeneity in Localized Lung Adenocarcinomas: An Association with Predicted Neoantigen Heterogeneity and Postsurgical Recurrence.

Genomic intratumor heterogeneity (ITH) may be associated with postsurgical relapse of localized lung adenocarcinomas. Recently, mutations, through generation of neoantigens, were shown to alter tumor immunogenicity through T-cell responses. Here, we performed sequencing of the T-cell receptor (TCR) in 45 tumor regions from 11 localized lung adenocarcinomas and observed substantial intratumor differences in T-cell density and clonality with the majority of T-cell clones restricted to individual tumor regions. TCR ITH positively correlated with predicted neoantigen ITH, suggesting that spatial differences in the T-cell repertoire may be driven by distinct neoantigens in different tumor regions. Finally, a higher degree of TCR ITH was associated with an increased risk of postsurgical relapse and shorter disease-free survival, suggesting a potential clinical significance of T-cell repertoire heterogeneity.Significance: The present study provides insights into the ITH of the T-cell repertoire in localized lung adenocarcinomas and its potential biological and clinical impact. The results suggest that T-cell repertoire ITH may be tightly associated to genomic ITH and disease relapse. Cancer Discov; 7(10); 1088-97. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow.

Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19(-)CD38(hi)CD138(+) subset was morphologically distinct, differentially expressed PC-associated genes, and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for more than 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19(-)CD38(hi)CD138(+) PCs in the BM. Finally, we found that CD19(-)CD38(hi)CD138(+) PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and probably represents the B cell response's "historical record" of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum.

Elucidation of seventeen human peripheral blood B-cell subsets and quantification of the tetanus response using a density-based method for the automated identification of cell populations in multidimensional flow cytometry data.

BACKGROUND: Advances in multiparameter flow cytometry (FCM) now allow for the independent detection of larger numbers of fluorochromes on individual cells, generating data with increasingly higher dimensionality. The increased complexity of these data has made it difficult to identify cell populations from high-dimensional FCM data using traditional manual gating strategies based on single-color or two-color displays. METHODS: To address this challenge, we developed a novel program, FLOCK (FLOw Clustering without K), that uses a density-based clustering approach to algorithmically identify biologically relevant cell populations from multiple samples in an unbiased fashion, thereby eliminating operator-dependent variability. RESULTS: FLOCK was used to objectively identify seventeen distinct B-cell subsets in a human peripheral blood sample and to identify and quantify novel plasmablast subsets responding transiently to tetanus and other vaccinations in peripheral blood. FLOCK has been implemented in the publically available Immunology Database and Analysis Portal-ImmPort (http://www.immport.org)-for open use by the immunology research community. CONCLUSIONS: FLOCK is able to identify cell subsets in experiments that use multiparameter FCM through an objective, automated computational approach. The use of algorithms like FLOCK for FCM data analysis obviates the need for subjective and labor-intensive manual gating to identify and quantify cell subsets. Novel populations identified by these computational approaches can serve as hypotheses for further experimental study.

The expression of prodynorphin gene is down-regulated by activation with lipopolysaccharide in U-937 macrophage cells.

Dynorphins are endogenous kappa opioid peptides widely distributed in the central nervous system. A number of recent reports have established their roles in modulating immunological functions. In this study, we investigated the expression of prodynorphin (PDYN) gene, which encodes the precursor of dynorphin peptides, in human macrophage U-937 cell line. PDYN mRNA was expressed at a detectable level, measured with a standard RT-PCR method in U-937 cells, but not in Jurkat and Raji cells, which are human T and B lymphocytes, respectively. Further analyses with RT-PCR assays by using primers covering each exon of the PDYN gene showed that U-937 cells expressed the adult brain-type PDYN mRNA. Most interestingly, activation of U-937 cells with lipopolysaccharide (LPS) led to a decrease in PDYN mRNA levels. This decrease was dependent on both the concentration of LPS and the duration of LPS treatment. In order to test the role of transcription in LPS-mediated down-regulation of PDYN gene expression in U937 cells, the proximal PDYN gene promoter with a length of either approximately 300 base pairs or approximately 900 base pairs was cloned and inserted upstream of luciferase reporter gene. Results from transient transfections and dual luciferase assays showed that PDYN gene promoter activity was decreased by LPS treatment. Taken together, our results suggested the U-937 cells expressed adult brain-type PDYN mRNA which was down-regulated by activation of the cells with LPS due to an inhibition of PDYN gene transcription.