Spatiotemporal immune atlas of a clinical-grade gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. Here, we transplant a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and study the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells are uncommon in the porcine kidney cortex early after xenotransplantation and consist of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages express genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft is detectable. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression may be able to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

Highly Cross-Reactive and Protective Influenza A Virus H3N2 Hemagglutinin- and Neuraminidase-Specific Human Monoclonal Antibodies.

Due to antigenic drift and shift of influenza A viruses (IAV) and the tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to new strains of seasonal IAV and is at risk from viruses with pandemic potential for which limited or no immunity may exist. The genetic drift of H3N2 IAV is specifically pronounced, resulting in two distinct clades since 2014. Here, we demonstrate that immunization with a seasonal inactivated influenza vaccine (IIV) results in increased levels of H3N2 IAV-specific serum antibodies against hemagglutinin (HA) and neuraminidase (NA). Detailed analysis of the H3N2 B cell response indicated expansion of H3N2-specific peripheral blood plasmablasts 7 days after IIV immunization which expressed monoclonal antibodies (MAbs) with broad and potent antiviral activity against many H3N2 IAV strains as well as prophylactic and therapeutic activity in mice. These H3N2-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results demonstrate that IIV-induced H3N2 human MAbs can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IAV H3N2-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development. IMPORTANCE Influenza A virus (IAV) infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets within the influenza virus hemagglutinin and neuraminidase proteins. We have demonstrated that seasonal immunization with inactivated influenza vaccine (IIV) stimulates H3N2-specific monoclonal antibodies in humans that are broad and potent in their neutralization of virus in vitro. These antibodies also provide protection from H3N2 IAV in a mouse model of infection. Furthermore, they persist in the bone marrow, where they are expressed by long-lived antibody-producing plasma cells. This significantly demonstrates that seasonal IIV can induce a subset of H3N2-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

B cell and antibody targeting of transplant alloantigen epitopes is supported by both self and allo-recognition.

Donor-specific antibody (DSA) responses against human leukocyte antigen (HLA) proteins mismatched between kidney transplant donors and recipients cause allograft loss. Using single-cell, molecular, structural, and proteomic techniques, we profiled the HLA-specific (alloreactive) B cell response in kidney and blood of a transplant recipient with antibody-mediated rejection (AMR). We identified 14 distinct alloreactive B cell lineages, which spanned the rejected organ and blood and expressed high-affinity anti-donor HLA-specific B cell receptors, many of which were clonally linked to circulating DSA. The alloreactive B cell response was focused on exposed, solvent-accessible mismatched HLA residues, while also demonstrating extensive contacts with self-HLA residues. Consistent with structural evidence of self-recognition, measurable self-reactivity by donor-specific B cells was common and positively correlated with anti-donor affinity maturation. Thus, allo- and self-reactive signatures appeared to converge, suggesting that during AMR, the recognition of non-self and breaches of tolerance conspire to produce a pathogenic donor-specific adaptive response.

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.

A transcriptionally distinct subset of influenza-specific effector memory B cells predicts long-lived antibody responses to vaccination in humans.

Seasonal influenza vaccination elicits hemagglutinin (HA)-specific memory B (Bmem) cells, and although multiple Bmem cell populations have been characterized, considerable heterogeneity exists. We found that HA-specific human Bmem cells differed in the expression of surface marker FcRL5 and transcriptional factor T-bet. FcRL5+T-bet+ Bmem cells were transcriptionally similar to effector-like memory cells, while T-betnegFcRL5neg Bmem cells exhibited stem-like central memory properties. FcRL5+ Bmem cells did not express plasma-cell-commitment factors but did express transcriptional, epigenetic, metabolic, and functional programs that poised these cells for antibody production. Accordingly, HA+ T-bet+ Bmem cells at day 7 post-vaccination expressed intracellular immunoglobulin, and tonsil-derived FcRL5+ Bmem cells differentiated more rapidly into antibody-secreting cells (ASCs) in vitro. The T-bet+ Bmem cell response positively correlated with long-lived humoral immunity, and clonotypes from T-bet+ Bmem cells were represented in the secondary ASC response to repeat vaccination, suggesting that this effector-like population predicts influenza vaccine durability and recall potential.

Mixed Origins: HIV gp120-Specific Memory Develops from Pre-Existing Memory and Naive B Cells Following Vaccination in Humans.

The most potent and broad HIV envelope (Env)-specific antibodies often when reverted to their inferred germline versions representing the naive B cell receptor, fail to bind Env, suggesting that the initial responding B cell population not only exclusively comprises a naive population, but also a pre-existing cross-reactive antigen-experienced B cell pool that expands following Env exposure. Previously we isolated gp120-reactive monoclonal antibodies (mAbs) from participants in HVTN 105, an HIV vaccine trial. Using deep sequencing, focused on immunoglobulin G (IgG), IgA, and IgM, VH-lineage tracking, we identified four of these mAb lineages in pre-immune peripheral blood. We also looked through the ∼7 month postvaccination bone marrow, and interestingly, several of these lineages that were found in prevaccination blood were still persistent in the postvaccination bone marrow, including the CD138+ long-lived plasma cell compartment. The majority of the pre-immune lineage members included IgM, however, IgG and IgA members were also prevalent and exhibited somatic hypermutation. These results suggest that vaccine-induced gp120-specific antibody lineages originate from both naive and cross-reactive memory B cells. ClinicalTrials.gov NCT02207920.

Spatiotemporal immune atlas of the first clinical-grade, gene-edited pig-to-human kidney xenotransplant.

Pig-to-human xenotransplantation is rapidly approaching the clinical arena; however, it is unclear which immunomodulatory regimens will effectively control human immune responses to pig xenografts. We transplanted a gene-edited pig kidney into a brain-dead human recipient on pharmacologic immunosuppression and studied the human immune response to the xenograft using spatial transcriptomics and single-cell RNA sequencing. Human immune cells were uncommon in the porcine kidney cortex early after xenotransplantation and consisted of primarily myeloid cells. Both the porcine resident macrophages and human infiltrating macrophages expressed genes consistent with an alternatively activated, anti-inflammatory phenotype. No significant infiltration of human B or T cells into the porcine kidney xenograft was detected. Altogether, these findings provide proof of concept that conventional pharmacologic immunosuppression is sufficient to restrict infiltration of human immune cells into the xenograft early after compatible pig-to-human kidney xenotransplantation.

HLA-II-Associated HIV-1 Adaptation Decreases CD4+ T-Cell Responses in HIV-1 Vaccine Recipients.

Epitopes with evidence of HLA-II-associated adaptation induce poorly immunogenic CD4+ T-cell responses in HIV-positive (HIV+) individuals. Many such escaped CD4+ T-cell epitopes are encoded by HIV-1 vaccines being evaluated in clinical trials. Here, we assessed whether this viral adaptation adversely impacts CD4+ T-cell responses following HIV-1 vaccination, thereby representing escaped epitopes. When evaluated in separate peptide pools, vaccine-encoded adapted epitopes (AE) induced CD4+ T-cell responses less frequently than nonadapted epitopes (NAE). We also demonstrated that in a polyvalent vaccine, where both forms of the same epitope were encoded, AE were less immunogenic. NAE-specific CD4+ T cells had increased, albeit low, levels of interferon gamma (IFN-γ) cytokine production. Single-cell transcriptomic analyses showed that NAE-specific CD4+ T cells expressed interferon-related genes, while AE-specific CD4+ T cells resembled a Th2 phenotype. Importantly, the magnitude of NAE-specific CD4+ T-cell responses, but not that of AE-specific responses, was found to positively correlate with Env-specific antibodies in a vaccine efficacy trial. Together, these findings show that HLA-II-associated viral adaptation reduces CD4+ T-cell responses in HIV-1 vaccine recipients and suggest that vaccines encoding a significant number of AE may not provide optimal B-cell help for HIV-specific antibody production. IMPORTANCE Despite decades of research, an effective HIV-1 vaccine remains elusive. Vaccine strategies leading to the generation of broadly neutralizing antibodies are likely needed to provide the best opportunity of generating a protective immune response against HIV-1. Numerous studies have demonstrated that T-cell help is necessary for effective antibody generation. However, immunogen sequences from recent HIV-1 vaccine efficacy trials include CD4+ T-cell epitopes that have evidence of immune escape. Our study shows that these epitopes, termed adapted epitopes, elicit lower frequencies of CD4+ T-cell responses in recipients from multiple HIV-1 vaccine trials. Additionally, the counterparts to these epitopes, termed nonadapted epitopes, elicited CD4+ T-cell responses that correlated with Env-specific antibodies in one efficacy trial. These results suggest that vaccine-encoded adapted epitopes dampen CD4+ T-cell responses, potentially impacting both HIV-specific antibody production and efficacious vaccine efforts.

Neonatal Exposure to Commensal-Bacteria-Derived Antigens Directs Polysaccharide-Specific B-1 B Cell Repertoire Development.

B-1 B cells derive from a developmental program distinct from that of conventional B cells, through B cell receptor (BCR)-dependent positive selection of fetally derived precursors. Here, we used direct labeling of B cells reactive with the N-acetyl-D-glucosamine (GlcNAc)-containing Lancefield group A carbohydrate of Streptococcus pyogenes to study the effects of bacterial antigens on the emergent B-1 B cell clonal repertoire. The number, phenotype, and BCR clonotypes of GlcNAc-reactive B-1 B cells were modulated by neonatal exposure to heat-killed S. pyogenes bacteria. GlcNAc-reactive B-1 clonotypes and serum antibodies were reduced in germ-free mice compared with conventionally raised mice. Colonization of germ-free mice with a conventional microbiota promoted GlcNAc-reactive B-1 B cell development and concomitantly elicited clonally related IgA+ plasma cells in the small intestine. Thus, exposure to microbial antigens in early life determines the clonality of the mature B-1 B cell repertoire and ensuing antibody responses, with implications for vaccination approaches and schedules.

Persistence of HIV-1 Env-Specific Plasmablast Lineages in Plasma Cells after Vaccination in Humans.

Induction of persistent HIV-1 Envelope (Env) specific antibody (Ab) is a primary goal of HIV vaccine strategies; however, it is unclear whether HIV Env immunization in humans induces bone marrow plasma cells, the presumed source of long-lived systemic Ab. To define the features of Env-specific plasma cells after vaccination, samples were obtained from HVTN 105, a phase I trial testing the same gp120 protein immunogen, AIDSVAX B/E, used in RV144, along with a DNA immunogen in various prime and boost strategies. Boosting regimens that included AIDSVAX B/E induced robust peripheral blood plasmablast responses. The Env-specific immunoglobulin repertoire of the plasmablasts is dominated by VH1 gene usage and targeting of the V3 region. Numerous plasmablast-derived immunoglobulin lineages persisted in the bone marrow >8 months after immunization, including in the CD138+ long-lived plasma cell compartment. These findings identify a cellular linkage for the development of sustained Env-specific Abs following vaccination in humans.

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.

Broad and Protective Influenza B Virus Neuraminidase Antibodies in Humans after Vaccination and their Clonal Persistence as Plasma Cells.

Although most seasonal inactivated influenza vaccines (IIV) contain neuraminidase (NA), the extent and mechanisms of action of protective human NA-specific humoral responses induced by vaccination are poorly resolved. Due to the propensity of influenza virus for antigenic drift and shift and its tendency to elicit predominantly strain-specific antibodies, humanity remains susceptible to waves of new strains of seasonal viruses and is at risk from viruses with pandemic potential for which limited or no immunity may exist. Here we demonstrate that the use of IIV results in increased levels of influenza B virus (IBV) NA-specific serum antibodies. Detailed analysis of the IBV NA B cell response indicates concurrent expansion of IBV NA-specific peripheral blood plasmablasts 7 days after IIV immunization which express monoclonal antibodies with broad and potent antiviral activity against both IBV Victoria and Yamagata lineages and prophylactic and therapeutic activity in mice. These IBV NA-specific B cell clonal lineages persisted in CD138+ long-lived bone marrow plasma cells. These results represent the first demonstration that IIV-induced NA human antibodies can protect and treat influenza virus infection in vivo and suggest that IIV can induce a subset of IBV NA-specific B cells with broad protective potential, a feature that warrants further study for universal influenza vaccine development.IMPORTANCE Influenza virus infections continue to cause substantial morbidity and mortality despite the availability of seasonal vaccines. The extensive genetic variability in seasonal and potentially pandemic influenza strains necessitates new vaccine strategies that can induce universal protection by focusing the immune response on generating protective antibodies against conserved targets such as regions within the influenza neuraminidase protein. We have demonstrated that seasonal immunization stimulates neuraminidase-specific antibodies in humans that are broad and potent in their protection from influenza B virus when tested in mice. These antibodies further persist in the bone marrow, where they are expressed by long-lived antibody-producing cells, referred to here as plasma cells. The significance in our research is the demonstration that seasonal influenza immunization can induce a subset of neuraminidase-specific B cells with broad protective potential, a process that if further studied and enhanced could aid in the development of a universal influenza vaccine.

Visualizing nationwide variation in medicare Part D prescribing patterns.

BACKGROUND: To characterize the regional and national variation in prescribing patterns in the Medicare Part D program using dimensional reduction visualization methods. METHODS: Using publicly available Medicare Part D claims data, we identified and visualized regional and national provider prescribing profile variation with unsupervised clustering and t-distributed stochastic neighbor embedding (t-SNE) dimensional reduction techniques. Additionally, we examined differences between regionally representative prescribing patterns for major metropolitan areas. RESULTS: Distributions of prescribing volume and medication diversity were highly skewed among over 800,000 Medicare Part D providers. Medical specialties had characteristic prescribing patterns. Although the number of Medicare providers in each state was highly correlated with the number of Medicare Part D enrollees, some states were enriched for providers with > 10,000 prescription claims annually. Dimension-reduction, hierarchical clustering and t-SNE visualization of drug- or drug-class prescribing patterns revealed that providers cluster strongly based on specialty and sub-specialty, with large regional variations in prescribing patterns. Major metropolitan areas had distinct prescribing patterns that tended to group by major geographical divisions. CONCLUSIONS: This work demonstrates that unsupervised clustering, dimension-reduction and t-SNE visualization can be used to analyze and visualize variation in provider prescribing patterns on a national level across thousands of medications, revealing substantial prescribing variation both between and within specialties, regionally, and between major metropolitan areas. These methods offer an alternative system-wide and pattern-centric view of such data for hypothesis generation, visualization, and pattern identification.

Understanding B-cell activation and autoantibody repertoire selection in systemic lupus erythematosus: A B-cell immunomics approach.

Understanding antibody repertoires and in particular, the properties and fates of B cells expressing potentially pathogenic antibodies is critical to define the mechanisms underlying multiple immunological diseases including autoimmune and allergic conditions as well as transplant rejection. Moreover, an integrated knowledge of the antibody repertoires expressed by B cells and plasma cells (PC) of different functional properties and longevity is essential to develop new therapeutic strategies, better biomarkers for disease segmentation, and new assays to measure restoration of B-cell tolerance or, at least, of normal B-cell homeostasis. Reaching these goals, however, will require a more precise phenotypic, functional and molecular definition of B-cell and PC populations, and a comprehensive analysis of the antigenic reactivity of the antibodies they express. While traditionally hampered by technical and ethical limitations in human experimentation, new technological advances currently enable investigators to address these questions in a comprehensive fashion. In this review, we shall discuss these concepts as they apply to the study of Systemic Lupus Erythematosus.

A Highly Potent and Broadly Neutralizing H1 Influenza-Specific Human Monoclonal Antibody.

Influenza's propensity for antigenic drift and shift, and to elicit predominantly strain specific antibodies (Abs) leaves humanity susceptible to waves of new strains with pandemic potential for which limited or no immunity may exist. Subsequently new clinical interventions are needed. To identify hemagglutinin (HA) epitopes that if targeted may confer universally protective humoral immunity, we examined plasmablasts from a subject that was immunized with the seasonal influenza inactivated vaccine, and isolated a human monoclonal Ab (mAb), KPF1. KPF1 has broad and potent neutralizing activity against H1 influenza viruses, and recognized 83% of all H1 isolates tested, including the pandemic 1918 H1. Prophylactically, KPF1 treatment resulted in 100% survival of mice from lethal challenge with multiple H1 influenza strains and when given as late as 72 h after challenge with A/California/04/2009 H1N1, resulted in 80% survival. KPF1 recognizes a novel epitope in the HA globular head, which includes a highly conserved amino acid, between the Ca and Cb antigenic sites. Although recent HA stalk-specific mAbs have broader reactivity, their potency is substantially limited, suggesting that cocktails of broadly reactive and highly potent HA globular head-specific mAbs, like KPF1, may have greater clinical feasibility for the treatment of influenza infections.

Properties of healthcare teaming networks as a function of network construction algorithms.

Network models of healthcare systems can be used to examine how providers collaborate, communicate, refer patients to each other, and to map how patients traverse the network of providers. Most healthcare service network models have been constructed from patient claims data, using billing claims to link a patient with a specific provider in time. The data sets can be quite large (106-108 individual claims per year), making standard methods for network construction computationally challenging and thus requiring the use of alternate construction algorithms. While these alternate methods have seen increasing use in generating healthcare networks, there is little to no literature comparing the differences in the structural properties of the generated networks, which as we demonstrate, can be dramatically different. To address this issue, we compared the properties of healthcare networks constructed using different algorithms from 2013 Medicare Part B outpatient claims data. Three different algorithms were compared: binning, sliding frame, and trace-route. Unipartite networks linking either providers or healthcare organizations by shared patients were built using each method. We find that each algorithm produced networks with substantially different topological properties, as reflected by numbers of edges, network density, assortativity, clustering coefficients and other structural measures. Provider networks adhered to a power law, while organization networks were best fit by a power law with exponential cutoff. Censoring networks to exclude edges with less than 11 shared patients, a common de-identification practice for healthcare network data, markedly reduced edge numbers and network density, and greatly altered measures of vertex prominence such as the betweenness centrality. Data analysis identified patterns in the distance patients travel between network providers, and a striking set of teaming relationships between providers in the Northeast United States and Florida, likely due to seasonal residence patterns of Medicare beneficiaries. We conclude that the choice of network construction algorithm is critical for healthcare network analysis, and discuss the implications of our findings for selecting the algorithm best suited to the type of analysis to be performed.

Humoral Dysregulation Associated with Increased Systemic Inflammation among Injection Heroin Users.

BACKGROUND: Injection drug use is a growing major public health concern. Injection drug users (IDUs) have a higher incidence of co-morbidities including HIV, Hepatitis, and other infections. An effective humoral response is critical for optimal homeostasis and protection from infection; however, the impact of injection heroin use on humoral immunity is poorly understood. We hypothesized that IDUs have altered B cell and antibody profiles. METHODS AND FINDINGS: A comprehensive systems biology-based cross-sectional assessment of 130 peripheral blood B cell flow cytometry- and plasma- based features was performed on HIV-/Hepatitis C-, active heroin IDUs who participated in a syringe exchange program (n = 19) and healthy control subjects (n = 19). The IDU group had substantial polydrug use, with 89% reporting cocaine injection within the preceding month. IDUs exhibited a significant, 2-fold increase in total B cells compared to healthy subjects, which was associated with increased activated B cell subsets. Although plasma total IgG titers were similar between groups, IDUs had significantly higher IgG3 and IgG4, suggestive of chronic B cell activation. Total IgM was also increased in IDUs, as well as HIV Envelope-specific IgM, suggestive of increased HIV exposure. IDUs exhibited numerous features suggestive of systemic inflammation, including significantly increased plasma sCD40L, TNF-α, TGF-α, IL-8, and ceramide metabolites. Machine learning multivariate analysis distilled a set of 10 features that classified samples based on group with absolute accuracy. CONCLUSIONS: These results demonstrate broad alterations in the steady-state humoral profile of IDUs that are associated with increased systemic inflammation. Such dysregulation may impact the ability of IDUs to generate optimal responses to vaccination and infection, or lead to increased risk for inflammation-related co-morbidities, and should be considered when developing immune-based interventions for this growing population.

Expression of the Long Non-Coding RNA HOTAIR Correlates with Disease Progression in Bladder Cancer and Is Contained in Bladder Cancer Patient Urinary Exosomes.

Exosomes are 30-150nM membrane-bound secreted vesicles that are readily isolated from biological fluids such as urine (UEs). Exosomes contain proteins, micro RNA (miRNA), messenger RNA (mRNA), and long non-coding RNA (lncRNA) from their cells of origin. Although miRNA, protein and lncRNA have been isolated from serum as potential biomarkers for benign and malignant disease, it is unknown if lncRNAs in UEs from urothelial bladder cancer (UBC) patients can serve as biomarkers. lncRNAs are > 200 nucleotide long transcripts that do not encode protein and play critical roles in tumor biology. As the number of recognized tumor-associated lncRNAs continues to increase, there is a parallel need to include lncRNAs into biomarker discovery and therapeutic target algorithms. The lncRNA HOX transcript antisense RNA (HOTAIR) has been shown to facilitate tumor initiation and progression and is associated with poor prognosis in several cancers. The importance of HOTAIR in cancer biology has sparked interest in using HOTAIR as a biomarker and potential therapeutic target. Here we show HOTAIR and several tumor-associated lncRNAs are enriched in UEs from UBC patients with high-grade muscle-invasive disease (HGMI pT2-pT4). Knockdown of HOTAIR in UBC cell lines reduces in vitro migration and invasion. Importantly, loss of HOTAIR expression in UBC cell lines alters expression of epithelial-to-mesenchyme transition (EMT) genes including SNAI1, TWIST1, ZEB1, ZO1, MMP1 LAMB3, and LAMC2. Finally, we used RNA-sequencing to identify four additional lncRNAs enriched in UBC patient UEs. These data, suggest that UE-derived lncRNA may potentially serve as biomarkers and therapeutic targets.

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.