Immunotherapies that block inhibitory checkpoint receptors on T cells have transformed the clinical care of cancer patients 1 . However, whether the T cell response to checkpoint blockade relies on reinvigoration of pre-existing tumor infiltrating T cells (TILs) or on recruitment of novel T cells remains unclear 2 – 4 . Here, we performed paired single-cell RNA (scRNA) and T cell receptor (TCR)- sequencing on 79,046 cells from site-matched tumors from patients with basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) pre- and post-anti-PD-1 therapy. Tracking TCR clones and transcriptional phenotypes revealed a coupling of tumor-recognition, clonal expansion, and T cell dysfunction marked by clonal expansions of CD8 + CD39 + T cells, which co-expressed markers of chronic T cell activation and exhaustion. However, this expansion did not derive from pre-existing TIL clones; rather, it was comprised of novel clonotypes not previously observed in the same tumor. Clonal replacement of T cells was preferentially observed in exhausted CD8 + T cells and evident in BCC and SCC patients. These results demonstrate that pre-existing tumor-specific T cells may have limited reinvigoration capacity, and that the T cell response to checkpoint blockade derives from a distinct repertoire of T cell clones that may have just recently entered the tumor.

CD4 + T cells are critical to fighting pathogens, but a comprehensive analysis of human T cell specificities is hindered by the diversity of HLA alleles (>20,000) and the complexity of many pathogen genomes. We previously described GLIPH, an algorithm to cluster T cell receptors (TCRs) that recognize the same epitope and to predict their HLA restriction, but this method loses efficiency and accuracy when analyzing >10,000 TCRs. Here we describe an improved algorithm, GLIPH2, that can process millions of TCR sequences. We used GLIPH2 to analyze 19,044 unique TCRβsequences from 58 individuals latently infected with Mycobacterium tuberculosis (Mtb) and to group them according to their specificity. To identify the epitopes targeted by clusters of Mtb -specific T cells, we carried out a screen of 3,724 distinct proteins covering 95% of Mtb protein-coding genes using artificial antigen presenting cells (aAPC) and reporter T cells. We found that at least five PPE (Pro-Pro-Glu) proteins are targets for T cell recognition in Mtb .

Circoviruses are known to infect birds and pigs and can cause a wide range of severe symptoms with significant economic impact. Using viral metagenomics, we identified circovirus-like DNA sequences and characterized 15 circular viral DNA genomes in stool samples from humans in Pakistan, Nigeria, Tunisia, and the United States and from wild chimpanzees. Distinct genomic features and phylogenetic analysis indicate that some viral genomes were part of a previously unrecognized genus in the Circoviridae family we tentatively named "Cyclovirus" whose genetic diversity is comparable to that of all the known species in the Circovirus genus. Circoviridae detection in the stools of U.S. adults was limited to porcine circoviruses which were also found in most U.S. pork products. To determine whether the divergent cycloviruses found in non-U.S. human stools were of dietary origin, we genetically compared them to the cycloviruses in muscle tissue samples of commonly eaten farm animals in Pakistan and Nigeria. Limited genetic overlap between cycloviruses in human stool samples and local cow, goat, sheep, camel, and chicken meat samples indicated that the majority of the 25 Cyclovirus species identified might be human viruses. We show that the genetic diversity of small circular DNA viral genomes in various mammals, including humans, is significantly larger than previously recognized, and frequent exposure through meat consumption and contact with animal or human feces provides ample opportunities for cyclovirus transmission. Determining the role of cycloviruses, found in 7 to 17% of non-U.S. human stools and 3 to 55% of non-U.S. meat samples tested, in both human and animal diseases is now facilitated by knowledge of their genomes.

The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in Ն3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.[Supplemental material is available online at www.genome.org. The raw data from this study are available online at http://dbpartners.stanford.edu/454/pub.] Dideoxynucleotide (Sanger) sequencing of non-clonal PCR products (direct PCR sequencing) of plasma viral cDNA is widely used to detect more than 50 drug-resistance mutations in the molecular targets of HIV-1 therapy-reverse transcriptase (RT) and protease-in clinical settings (US Department of Health and Human Services Panel on Clinical Practices for Treatment of HIV Infection 2006). A major limitation of direct PCR sequencing, however, is its inability to detect low proportions of drug-resistant variants in the heterogeneous virus population existing in a patient's plasma sample (Palmer et al. 2005). Several studies have shown that minor drug-resistant variants that are not detected by population-based sequencing are clinically relevant in that they are often responsible for the virological failure of a new antiretroviral treatment regimen (Jourdain et al. 2004;Kapoor et al. 2004;Lecossier et al. 2005;Palmer et al. 2006b). Multiple approaches have been developed to detect minor HIV-1 variants in research settings; however, no single approach has proved useful for clinical settings.The 454 Life Sciences GS20 sequencing platform allows massively parallel picoliter-scale amplification and pyrosequencing of individual DNA molecules (Margulies et al. 2005). Simons and colleagues described two cases in which ultra-deep pyrosequencing detected minority variant drug-resistance mutations in a previously treated patient in whom mutations were no longer detectable by standard direct PCR sequencing (Simons et al. 2005). Tsibris and colleagues demonstrated that ultra-deep pyrosequencing could accurately quantify a mixture of three HIV-1 envelope variants pooled in defined proportions of 89%, 10%, and 1% (Tsibris et al. 2006). Here, we systematically investigate the potential...