Reverse TCR repertoire evolution toward dominant low-affinity clones during chronic CMV infection.

Adaptive evolution is a key feature of T cell immunity. During acute immune responses, T cells harboring high-affinity T cell antigen receptors (TCRs) are preferentially expanded, but whether affinity maturation by clonal selection continues through the course of chronic infections remains unresolved. Here we investigated the evolution of the TCR repertoire and its affinity during the course of infection with cytomegalovirus, which elicits large T cell populations in humans and mice. Using single-cell and bulk TCR sequencing and structural affinity analyses of cytomegalovirus-specific T cells, and through the generation and in vivo monitoring of defined TCR repertoires, we found that the immunodominance of high-affinity T cell clones declined during the chronic infection phase, likely due to cellular senescence. These data showed that under conditions of chronic antigen exposure, low-affinity TCRs preferentially expanded within the TCR repertoire, with implications for immunotherapeutic strategies.

Breast cancer is marked by specific, Public T-cell receptor CDR3 regions shared by mice and humans.

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires.

Sequence and structural variation in a human genome uncovered by short-read, massively parallel ligation sequencing using two-base encoding.

We describe the genome sequencing of an anonymous individual of African origin using a novel ligation-based sequencing assay that enables a unique form of error correction that improves the raw accuracy of the aligned reads to >99.9%, allowing us to accurately call SNPs with as few as two reads per allele. We collected several billion mate-paired reads yielding approximately 18x haploid coverage of aligned sequence and close to 300x clone coverage. Over 98% of the reference genome is covered with at least one uniquely placed read, and 99.65% is spanned by at least one uniquely placed mate-paired clone. We identify over 3.8 million SNPs, 19% of which are novel. Mate-paired data are used to physically resolve haplotype phases of nearly two-thirds of the genotypes obtained and produce phased segments of up to 215 kb. We detect 226,529 intra-read indels, 5590 indels between mate-paired reads, 91 inversions, and four gene fusions. We use a novel approach for detecting indels between mate-paired reads that are smaller than the standard deviation of the insert size of the library and discover deletions in common with those detected with our intra-read approach. Dozens of mutations previously described in OMIM and hundreds of nonsynonymous single-nucleotide and structural variants in genes previously implicated in disease are identified in this individual. There is more genetic variation in the human genome still to be uncovered, and we provide guidance for future surveys in populations and cancer biopsies.

Maternal plasma DNA analysis with massively parallel sequencing by ligation for noninvasive prenatal diagnosis of trisomy 21.

BACKGROUND: Noninvasive prenatal diagnosis of trisomy 21 (T21) has recently been shown to be achievable by massively parallel sequencing of maternal plasma on a sequencing-by-synthesis platform. The quantification of several other human chromosomes, including chromosomes 18 and 13, has been shown to be less precise, however, with quantitative biases related to the chromosomal GC content. METHODS: Maternal plasma DNA from 10 euploid and 5 T21 pregnancies was sequenced with a sequencing-by-ligation approach. We calculated the genomic representations (GRs) of sequenced reads from each chromosome and their associated measurement CVs and compared the GRs of chromosome 21 (chr21) for the euploid and T21 pregnancies. RESULTS: We obtained a median of 12 x 10(6) unique reads (21% of the total reads) per sample. The GRs deviated from those expected for some chromosomes but in a manner different from that previously reported for the sequencing-by-synthesis approach. Measurements of the GRs for chromosomes 18 and 13 were less precise than for chr21. z Scores of the GR of chr21 were increased in the T21 pregnancies, compared with the euploid pregnancies. CONCLUSIONS: Massively parallel sequencing-by-ligation of maternal plasma DNA was effective in identifying T21 fetuses noninvasively. The quantitative biases observed among the GRs of certain chromosomes were more likely based on analytical factors than biological factors. Further research is needed to enhance the precision for measuring for the representations of chromosomes 18 and 13.

Antitumor Potency of an Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy, Lisocabtagene Maraleucel in Combination With Ibrutinib or Acalabrutinib.

Chimeric antigen receptor (CAR) T-cell therapy is a promising treatment for patients with CD19 B-cell malignancies. Combination strategies that improve CAR T-cell potency, limit tumor environment-mediated immune dysfunction, and directly reduce tumor burden may increase the potential for durable clinical benefit of CAR T-cell therapy. Lisocabtagene maraleucel (liso-cel) is a product therapy candidate being tested in patients with relapsed/refractory non-Hodgkin lymphoma or chronic lymphocytic leukemia. This study assessed the in vitro and in vivo functionality of CAR T cells transduced to express the anti-CD19 CAR of liso-cel in combination with ibrutinib or acalabrutinib. In prolonged stimulation assays, the presence of ibrutinib or acalabrutinib improved the CAR T-cell effector function. RNA-Seq analysis and surface marker profiling of these CAR T cells treated with ibrutinib but not acalabrutinib revealed gene expression changes consistent with skewing toward a memory-like, type 1 T-helper, Bruton tyrosine kinase phenotype. Ibrutinib or acalabrutinib improved CD19 tumor clearance and prolonged survival of tumor-bearing mice when used in combination with CAR T cells. A combination of the defined cell product therapy candidate, liso-cel, with ibrutinib or acalabrutinib is an attractive approach that may potentiate the promising clinical responses already achieved in CD19 B-cell malignancies with each of these single agents.

Mosaic deletion patterns of the human antibody heavy chain gene locus shown by Bayesian haplotyping.

Analysis of antibody repertoires by high-throughput sequencing is of major importance in understanding adaptive immune responses. Our knowledge of variations in the genomic loci encoding immunoglobulin genes is incomplete, resulting in conflicting VDJ gene assignments and biased genotype and haplotype inference. Haplotypes can be inferred using IGHJ6 heterozygosity, observed in one third of the people. Here, we propose a robust novel method for determining VDJ haplotypes by adapting a Bayesian framework. Our method extends haplotype inference to IGHD- and IGHV-based analysis, enabling inference of deletions and copy number variations in the entire population. To test this method, we generated a multi-individual data set of naive B-cell repertoires, and found allele usage bias, as well as a mosaic, tiled pattern of deleted IGHD and IGHV genes. The inferred haplotypes may have clinical implications for genetic disease predispositions. Our findings expand the knowledge that can be extracted from antibody repertoire sequencing data.

Anti-B-cell Maturation Antigen Chimeric Antigen Receptor T cell Function against Multiple Myeloma Is Enhanced in the Presence of Lenalidomide.

Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T cells have shown promising clinical responses in patients with relapsed/refractory multiple myeloma. Lenalidomide, an immunomodulatory drug, potentiates T cell functionality, drives antimyeloma activity, and alters the suppressive microenvironment; these properties may effectively combine with anti-BCMA CAR T cells to enhance function. Using an anti-BCMA CAR T, we demonstrated that lenalidomide enhances CAR T cell function in a concentration-dependent manner. Lenalidomide increased CAR T effector cytokine production, particularly under low CAR stimulation or in the presence of inhibitory ligand programmed cell death 1 ligand 1. Notably, lenalidomide also enhanced CAR T cytokine production, cytolytic activity, and activation profile relative to untreated CAR T cells in chronic stimulation assays. This unique potentiation of both short-term CAR T activity and long-term functionality during chronic stimulation prompted investigation of the molecular profile of lenalidomide-treated CAR T cells. Signatures from RNA sequencing and assay for transposase-accessible chromatin using sequencing indicated that pathways associated with T-helper 1 response, cytokine production, T cell activation, cell-cycle control, and cytoskeletal remodeling were altered with lenalidomide. Finally, study of lenalidomide and anti-BCMA CAR T cells in a murine, disseminated, multiple myeloma model indicated that lenalidomide increased CAR T cell counts in blood and significantly prolonged animal survival. In summary, preclinical studies demonstrated that lenalidomide potentiated CAR T activity in vivo in low-antigen or suppressive environments and delayed onset of functional exhaustion. These results support further investigation of lenalidomide and anti-BCMA CAR T cells in the clinic.

Exploration of immunoglobulin transcriptomes from mice immunized with three-finger toxins and phospholipases A2 from the Central American coral snake, Micrurus nigrocinctus.

Snakebite envenomings represent a neglected public health issue in many parts of the rural tropical world. Animal-derived antivenoms have existed for more than a hundred years and are effective in neutralizing snake venom toxins when timely administered. However, the low immunogenicity of many small but potent snake venom toxins represents a challenge for obtaining a balanced immune response against the medically relevant components of the venom. Here, we employ high-throughput sequencing of the immunoglobulin (Ig) transcriptome of mice immunized with a three-finger toxin and a phospholipase A2 from the venom of the Central American coral snake, Micrurus nigrocinctus. Although exploratory in nature, our indicate results showed that only low frequencies of mRNA encoding IgG isotypes, the most relevant isotype for therapeutic purposes, were present in splenocytes of five mice immunized with 6 doses of the two types of toxins over 90 days. Furthermore, analysis of Ig heavy chain transcripts showed that no particular combination of variable (V) and joining (J) gene segments had been selected in the immunization process, as would be expected after a strong humoral immune response to a single antigen. Combined with the titration of toxin-specific antibodies in the sera of immunized mice, these data support the low immunogenicity of three-finger toxins and phospholipases A2 found in M. nigrocinctusvenoms, and highlight the need for future studies analyzing the complexity of antibody responses to toxins at the molecular level.

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.

Polymorphism discovery in high-throughput resequenced microarray-enriched human genomic loci.

Identifying genetic variants and mutations that underlie human diseases requires development of robust, cost-effective tools for routine resequencing of regions of interest in the human genome. Here, we demonstrate that coupling Applied Biosystems SOLiD system-sequencing platform with microarray capture of targeted regions provides an efficient and robust method for high-coverage resequencing and polymorphism discovery in human protein-coding exons.

Direct control of neurogenesis by selector factors in the fly eye: regulation of atonal by Ey and So.

During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of ;eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. We show here that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. We find that these transcription factors interact in vitro and provide indirect evidence that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation.

Nicastrin controls aspects of photoreceptor neuron specification and differentiation in the Drosophila eye.

Nicastrin is a component of the Notch signaling pathway involved in proteolytic release of the Notch receptor intracellular domain. It has been postulated that intracellular Notch is required within the nucleus of fly eye progenitor cells to enhance (pro-neural enhancement) and then repress (lateral inhibition) transcription of pro-neural genes. We present here an analysis of Nicastrin function during eye development and find that Nicastrin is essential to early photoreceptor neuron development. Nicastrin mutant tissue displays neuronal loss or hyperplasia; these phenotypes can be rescued by targeted expression of an intracellular form of Notch. Thus, nuclear translocation of Notch and its direct regulation of gene expression appear to be critical to pro-neural enhancement as well as lateral inhibition. In addition, we show that Nicastrin as well as Notch are required to maintain normal R-cell morphology, because the nuclei of mutant photoreceptor neurons cannot maintain their proper position. Thus, Notch signaling plays a role, not only in cell fate specification, but also in differentiation of photoreceptor neurons.

Fly SIX-type homeodomain proteins Sine oculis and Optix partner with different cofactors during eye development.

Two members from the Six class of homeobox transcription factors, Sine oculis (SO) and Optix, function during development of the fly visual system. Differences in gain-of-function phenotypes and gene expression suggest that these related factors play distinct roles in the formation of the fly eye. However, the molecular nature of their functional differences remains unclear. In this study, we report the identification of two novel factors that participate in specific partnerships with Sine oculis and Optix during photoreceptor neurons formation and in eye progenitor cells. This work shows that different cofactors likely mediate unique functions of Sine oculis and Optix during the development of the fly eye and that the repeated requirement for SO function at multiple stages of eye development reflects the activity of different SO-cofactor complexes.