Nitya Nair

combinatorial tetramer staining and mass cytometry analysis facilitate t -cell epitope mapping and characterization

It is currently not possible to predict which epitopes will be recognized by T cells in different individuals. This is a barrier to the thorough analysis and understanding of T-cell responses after vaccination or infection. Here, by combining mass cytometry with combinatorial peptide–MHC tetramer staining, we have developed a method allowing the rapid and simultaneous identification and characterization of T cells specific for many epitopes. We use this to screen up to 109 different peptide–MHC tetramers in a single human blood sample, while still retaining at least 23 labels to analyze other markers of T-cell phenotype and function. Among 77 candidate rotavirus epitopes, we identified six T-cell epitopes restricted to human leukocyte antigen (HLA)-A*0201 in the blood of healthy individuals. T cells specific for epitopes in the rotavirus VP3 protein displayed a distinct phenotype and were present at high frequencies in intestinal epithelium. This approach should be useful for the comprehensive analysis of T-cell responses to infectious diseases or vaccines.

Innate immune response to homologous rotavirus infection in the small intestinal villous epithelium at single-cell resolution

“Bulk” measurements of antiviral innate immune responses from pooled cells yield averaged signals and do not reveal underlying signaling heterogeneity in infected and bystander single cells. We examined such heterogeneity in the small intestine during rotavirus (RV) infection. Murine RV EW robustly activated type I IFNs and several antiviral genes (IFN-stimulated genes) in the intestine by bulk analysis, the source of induced IFNs primarily being hematopoietic cells. Flow cytometry and microfluidics-based single-cell multiplex RT-PCR allowed dissection of IFN responses in single RV-infected and bystander intestinal epithelial cells (IECs). EW replicates in IEC subsets differing in their basal type I IFN transcription and induces IRF3-dependent and IRF3-augmented transcription, but not NF-κB–dependent or type I IFN transcripts. Bystander cells did not display enhanced type I IFN transcription but had elevated levels of certain IFN-stimulated genes, presumably in response to exogenous IFNs secreted from immune cells. Comparison of IRF3 and NF-κB induction in STAT1−/− mice revealed that murine but not simian RRV mediated accumulation of IkB-α protein and decreased transcription of NF-κB–dependent genes. RRV replication was significantly rescued in IFN types I and II, as well as STAT1 (IFN types I, II, and III) deficient mice in contrast to EW, which was only modestly sensitive to IFNs I and II. Resolution of “averaged” innate immune responses in single IECs thus revealed unexpected heterogeneity in both the induction and subversion of early host antiviral immunity, which modulated host range.

Mass cytometry as a platform for the discovery of cellular biomarkers to guide effective rheumatic disease therapy

The development of biomarkers for autoimmune diseases has been hampered by a lack of understanding of disease etiopathogenesis and of the mechanisms underlying the induction and maintenance of inflammation, which involves complex activation dynamics of diverse cell types. The heterogeneous nature and suboptimal clinical response to treatment observed in many autoimmune syndromes highlight the need to develop improved strategies to predict patient outcome to therapy and personalize patient care. Mass cytometry, using CyTOF®, is an advanced technology that facilitates multiparametric, phenotypic analysis of immune cells at single-cell resolution. In this review, we outline the capabilities of mass cytometry and illustrate the potential of this technology to enhance the discovery of cellular biomarkers for rheumatoid arthritis, a prototypical autoimmune disease.

High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry.

In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus double-layered particles with a metal isotope and characterized B cells specific to the rotavirus VP6 major structural protein. We describe the heterogeneity of the intestinal B cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B cell subsets in the intestine and blood, and revealed that IgM+ memory B cells (MBCs) and naïve B cells were phenotypically related as were CD27− MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B cell subsets in immune donors, including naïve B cells, with phenotypes representative of the overall B cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state.

VP4- and VP7-specific antibodies mediate heterotypic immunity to rotavirus in humans

Individual human VP4- and VP7-binding monoclonal antibodies mediate serotype cross-reactive neutralizing immunity to rotaviruses. Investigating heterotypic immunity to rotavirus There are many serotypes of rotavirus, and currently available vaccines include only one or a handful of different strains. However, these vaccines are generally able to induce cross-reactive immunity that prevents diarrheal disease in young children. To get a better understanding of heterotypic immunity, Nair et al. isolated rotavirus-specific B cells from human intestinal tissue and cloned the antibodies for functional analyses. They discovered that heterotypic immunity was often derived from antibodies targeting the VP5* stalk epitope. Although only a small number of antibodies were studied, these results suggest that vaccines focused on the VP5* region may be better able to induce broadly protective immunity to rotavirus. Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell–sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.