Metabolic and immune effects of immunotherapy with proinsulin peptide in human new-onset type 1 diabetes

Mohammad Alhadj Ali1,*, Yuk-Fun Liu2,3,*, Sefina Arif2, Danijela Tatovic1, Hina Shariff2, Vivienne B. Gibson2, Norkhairin Yusuf2, Roman Baptista2,4, Martin Eichmann2, Nedyalko Petrov4, Susanne Heck4, Jennie H. M. Yang2, Timothy I. M. Tree2, Irma Pujol-Autonell2, Lorraine Yeo2, Lucas R. Baumard2, Rachel Stenson1, Alex Howell1, Alison Clark1, Zoe Boult5, Jake Powrie3, Laura Adams3, Florence S. Wong1, Stephen Luzio6, Gareth Dunseath6, Kate Green7, Alison O’Keefe7, Graham Bayly7, Natasha Thorogood7, Robert Andrews7, Nicola Leech8, Frank Joseph9, Sunil Nair9, Susan Seal9, HoYee Cheung9, Craig Beam10, Robert Hills11, Mark Peakman2,4,12,†,‡ and Colin M. Dayan1,‡


Immunotherapy using short immunogenic peptides of disease-related autoantigens restores immune tolerance in preclinical disease models. We studied safety and mechanistic effects of injecting human leukocyte antigen–DR4(DRB1*0401)–restricted immunodominant proinsulin peptide intradermally every 2 or 4 weeks for 6 months in newly diagnosed type 1 diabetes patients. Treatment was well tolerated with no systemic or local hypersensitivity. Placebo subjects showed a significant decline in stimulated C-peptide (measuring insulin reserve) at 3, 6, 9, and 12 months versus baseline, whereas no significant change was seen in the 4-weekly peptide group at these time points or the 2-weekly group at 3, 6, and 9 months. The placebo group’s daily insulin use increased by 50% over 12 months but remained unchanged in the intervention groups. C-peptide retention in treated subjects was associated with proinsulin-stimulated interleukin-10 production, increased FoxP3 expression by regulatory T cells, low baseline levels of activated β cell–specific CD8 T cells, and favorable β cell stress markers (proinsulin/C-peptide ratio). Thus, proinsulin peptide immunotherapy is safe, does not accelerate decline in β cell function, and is associated with antigen-specific and nonspecific immune modulation.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target

Nature (2017) doi:10.1038/nature23270Nature (2017) doi:10.1038/nature23270
Robert T. Manguso, Hans W. Pope, Margaret D. Zimmer, Flavian D. Brown, Kathleen B. Yates, Brian C. Miller, Natalie B. Collins, Kevin Bi, Martin W. LaFleur, Vikram R. Juneja, Sarah A. Weiss, Jennifer Lo, David E. Fisher, Diana Miao, Eliezer Van Allen, David E. Root, Arlene H. Sharpe, John G. Doench & W. Nicholas Haining

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR–Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy

Nature Biotechnology (2017) doi:10.1038/nbt.3892

Yongxin Zhao, Octavian Bucur, Humayun Irshad, Fei Chen, Astrid Weins, Andreea L Stancu, Eun-Young Oh, Marcello DiStasio, Vanda Torous, Benjamin Glass, Isaac E Stillman, Stuart J Schnitt, Andrew H Beck & Edward S Boyden

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ~70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.

Massively Multiplexed Cloning Rides Herd on Long Target Sequences

Stampeding DNA sequences can overwhelm clonehands who would like to capture not just individual sequences, but entire herds of them. The task is particularly daunting if the DNA sequences of interest include kilobase-sized fragments. Fortunately, there’s a new class of oligonucleotide probe in town. The probe, called LASSO (for long adapter single-stranded oligonucleotide), has demonstrated, in a proof-of-concept study, that it can achieve highly multiplexed cloning of long target sequences.

LASSO was developed by scientists at Johns Hopkins, Rutgers, the University of Trento, and Harvard Medical School. According to these scientists, LASSO can be used to isolate thousands of long DNA sequences at the same time, more than ever before possible. The new technology, they say, speeds up the creation of proteins, the final products of genes, and is likely to lead to far more rapid discovery of new medicines and biomarkers for scores of diseases.

LASSO’s gene-wrangling ways appeared June 26 in the journal Nature Biomedical Engineering, in an article entitled “Long-Adapter Single-Strand Oligonucleotide Probes for the Massively Multiplexed Cloning of Kilobase Genome Regions.” Besides describing how LASSO was used to corral over 3000 bacterial open reading frames (ORFs), the article suggested that LASSO could facilitate downstream sequencing or expression, helping to close the widening gap from “sequencing to significance.”

“Targets were enriched up to a median of around 60-fold compared with non-targeted genomic regions,” wrote the article’s authors. “At a cutoff of three times the median non-target reads per kilobase of genetic element per million reads, around 75% of the targeted ORFs were successfully captured.”

The article also described how LASSO probes were used to clone human ORFs from complementary DNA and to clone an ORF library from a human–microbiome sample.

Historically, figuring out what a gene does by cloning its DNA and expressing its protein was done one gene at a time. Approaches do exist, however, that can accomplish the parallel amplification of hundreds of DNA targets, provided that they are not too long. For example, a method called molecular inversion probes (MIPs) can capture hundreds of DNA targets in a single reaction, but only if the targets are no longer than about 200 bases of DNA.

With LASSO, each target gene sequence can be up to a few thousand DNA base pairs long, which is the typical size of a gene’s protein-coding sequence. Also, collections of these LASSO probes can be used to grab desired DNA sequences—much like a rope lasso is used to capture cattle—but in this case thousands at a time in a single effort.

“Our goal is to make it cheap and easy for any researcher in any field to clone and express the entire set of proteins from any organism,” said Ben Larman, Ph.D., an assistant professor of pathology at the Johns Hopkins University School of Medicine and the study’s co-senior author. “Until now, such a prospect was only realistic for high-powered research consortia studying model organisms like fruit flies or mice.”

Importantly, the researchers suggested in their paper, DNA sequences may be captured by LASSO in a way that permits scientists to analyze what the genes’ proteins do, as demonstrated by conferring antibiotic resistance to an otherwise susceptible cell.

“We’re very excited about all the potential applications for LASSO cloning,” added Dr. Larman. “Our hope is that by greatly expanding the number of proteins that can be expressed and screened in parallel, the road to interesting biology and new therapeutic biomolecules will be dramatically shortened for many researchers.”


Massively parallel cloning of long target sequences has been achieved with a new kind of probe. The probe, called LASSO (for long adapter single-stranded oligonucleotide), can bind target genome regions for functional cloning and analysis. Collections of probes can be used to corral many DNA fragments in a single reaction. [Jennifer E. Fairman/Johns Hopkins University]

Team Modifies Molecular Inversion Probes to Capture Kilobase-Sized DNA for Cloning, Sequencing

NEW YORK (GenomeWeb) – A new method that uses long oligonucleotide probes to capture and clone large numbers of kilobase-sized DNA fragments in parallel promises to enable high-throughput functional studies of proteins and genomic elements, target preparation for long-read sequencing, and other applications.

The approach, which uses long-adapter single-strand oligonucleotide (LASSO) probes, was published earlier this week in Nature Biomedical Engineering by a team of researchers at Massachusetts General Hospital and elsewhere.

For their study, they cloned more than 3,000 open reading frames — up to about 4 kilobases in size — from the Escherichia coli genome in parallel. They also used LASSO probes to clone human ORFs from cDNA, and to generate an E. coli ORF library from a complex human microbiome sample.

According to Ben Larman, an assistant professor of pathology at Johns Hopkins University and one of the corresponding authors, he and co-senior author Biju Parekkadan developed the method in order to make bacterial protein expression libraries for functional screening assays. Originally, they had planned to encode the proteins on bacteriophages, “but we realized that we could not really encode long enough protein fragments to construct a library that would be likely to yield interesting proteins,” he said.

Instead, they came up with the idea to use modified molecular inversion probes (MIPs), which have been widely used to capture and amplify short DNA targets in parallel, to tackle larger targets. Traditional MIPs are short, single-stranded oligos about 150 base pairs in size, with target sequences at each end that bind to the ends of a DNA target. When the probe binds, it forms a circle and the space between the target sequences is filled in by DNA polymerase. Padlock probes are related to MIPs but do not have a gap in the target sequence that needs to be filled in.

“Seeing the ease of padlock- or molecular inversion-probe-based assays, this paper shows an interesting alternative to hybridization-based assays” for capturing larger DNA targets, said Alexander Hoischen, assistant professor of immuno-genomics at Radboud University Medical Center in the Netherlands, in an email. His lab has used MIPs to target shorter DNA regions for sequencing.

Previous studies have shown that the target DNA size for MIPs can be increased by making the adapter between the two ends of the probe longer, “but there was not a way to make such probes in high throughput,” Larman explained. “That’s when we did some brainstorming and methods development and came up with a way to convert, in a single-pot reaction, oligo libraries into capture probes that have very long adapters.”

In essence, the researchers fuse two DNA molecules — one that contains the two targeting sequences and another that serves as a long adapter — by overlap-extension PCR. They then circularize the resulting molecule and conduct an inverted PCR reaction to yield linear DNA that has a target sequence at each end. After removing the PCR priming sites and making the molecules single-stranded, they can be used as DNA capture probes.

Initially, the researchers tested individual LASSO probes that targeted DNA of four different sizes, including a 4-kilobase target, in the M13 bacteriophage genome and were able to capture all of them.

They then made a set of more than 3,100 LASSO probes with two different adapter lengths to target most ORFs in the E. coli genome and compared the results to a set of conventional MIPs targeting the same ORFs. Overall, about 75 percent of the targeted ORFs were successfully captured by the LASSO probes, whereas the MIPs capture fewer full-length ORFs.

They also used LASSO probes to capture two full-length ORFs from human cDNA libraries, and they employed their E. coli LASSO library to capture more than a thousand E. coli ORFs from DNA extracted from a human stool sample.

In contrast to probe hybridization methods, which also target kilobase-sized DNA fragments, the LASSO strategy allows researchers to clone DNA in the correct reading frame and to insert it directly into a protein expression vector, Larman said.

The advantage over existing cDNA-based methods is that those typically only yield a small number of protein-expressing clones. “We’re able to make expression libraries where a very large fraction of the clones are full-length proteins, which gives you a tremendous advantage over traditional cDNA-based protein libraries,” he said.

While the largest LASSO library the researchers used in their paper had about 3,000 probes, and the largest target was about 5 kilobases, it should be possible to generate tens of thousands of LASSO probes in parallel, and to tackle even longer targets.

Parekkadan, who is a faculty member in the Department of Surgery at Massachusetts General Hospital and at Rutgers University, said that the team is currently working on a new library with more than 8,000 LASSO probes. “We will see where the ceiling is in terms of number of probes as well as the length that can be captured,” he said.

The material cost of making a library with tens of thousands of LASSO probes is around $2,000 to $3,000, he said, and there are “tremendous economies of scales” for making even larger libraries. “The issue of cost was on our mind” when developing the method, he said, and the team’s hope is that it will be cheap enough for academic researchers to adopt.

The researchers have filed a patent application for the technology but have not licensed it out yet. “We’re exploring all options,” Parekkadan said, noting that discussions with potential licensees are ongoing.

One way to improve the method is to increase the uniformity with which targets are captured, and Parekkadan said his team has some ideas for how to do that. They also plan to modify the method so it can capture not only DNA but also RNA targets.

In addition, they want to improve the design of the LASSO probes, so more targets can be captured successfully. For the E. coli library, for example, they had some design constraints, so they were not able to generate probes for all ORFs, and during the capture, not all probes worked.

Larman said that for their published study, the researchers used conservative cutoffs for the probe design, excluding, for example, ORFs shorter than 400 base pairs in order to avoid amplification bias of small targets. “Future work will involve characterizing these effects, so we know where we can be less conservative in our thresholds,” he said. Also, one way to avoid amplification bias might be to subdivide the target library into fractions that each cover a certain target size range.

Larman said he plans to use the new method to identify protein targets of immune responses, while Parekkadan said he primarily plans to employ it as a platform to test and discover new drugs.

“We’re very interested in using this in the space of autoimmune disease, by going into a target tissue and cloning all the expressed proteins, and then screening them against an autoimmune patient’s immune system to identify what the molecular targets driving their disease are,” Larman explained. “We do that now using a phage-based system that displays peptides, but peptides don’t always contain the epitope information present in full-length proteins.”

The method could also be useful for researchers working with model organisms for which open reading frames have not been cloned yet, he said.

Besides making protein expression libraries, a potential application of LASSO probes is the capture of large DNA targets for long-read sequencing, for example, with Pacific Biosciences’ or Oxford Nanopore’s platforms. “As novel long-read sequencing technologies emerge, there is an increasing need for novel target enrichment methods that allow highly multiplexed enrichment of kilobase-sized DNA,” Hoischen said.

Others have explored using hybridization-based methods, for example, capture probes from Roche NimbleGen or Integrated DNA Technologies, to target DNA up to 8 kilobases in size for sequencing with PacBio’s system, and “it remains to be seen how these approaches compare to the LASSO method,” Hoischen said. The standard MIP workflow his lab uses is easy and allows many samples to be processed in parallel, “so I can imagine that a MIP-based assay for long reads can be attractive,” he said.

“Ultimately, it will be very interesting whether LASSO will allow even longer captures,” Hoischen said. Large DNA fragments have already been enriched using CRISPR/Cas9, he noted, but that method currently has low throughput at the moment and has not been highly multiplexed yet.

Another potential application, which the researchers did not explore in their paper, is to target non-protein-coding DNA from eukaryotic genomes. “It will be interesting to see whether this also works on human genomic DNA and will allow even larger targets to be enriched,” Hoischen said. “There is a huge need for sequencing entire genomic regions of genes, or entire regulatory regions including enhancers and promoters, in a highly multiplexed fashion.”

Larman agreed that cloning large fragments of human DNA is an interesting application, but his team has not explored this yet. “I don’t see why that would not work,” he said.

Phenome-wide scanning identifies multiple diseases and disease severity phenotypes associated with HLA variants

Phenome-wide scanning identifies multiple diseases and disease severity phenotypes associated with HLA variants

Jason H. Karnes1,*, Lisa Bastarache2,*, Christian M. Shaffer3, Silvana Gaudieri4,5,6, Yaomin Xu7,8, Andrew M. Glazer3, Jonathan D. Mosley3, Shilin Zhao8, Soumya Raychaudhuri9,10,11,12,13,14, Simon Mallal5,6,15, Zhan Ye16, John G. Mayer16, Murray H. Brilliant17, Scott J. Hebbring17, Dan M. Roden2,3,18, Elizabeth J. Phillips3,15 and Joshua C. Denny2,3,†

Although many phenotypes have been associated with variants in human leukocyte antigen (HLA) genes, the full phenotypic impact of HLA variants across all diseases is unknown. We imputed HLA genomic variation from two populations of 28,839 and 8431 European ancestry individuals and tested association of HLA variation with 1368 phenotypes. A total of 104 four-digit and 92 two-digit HLA allele phenotype associations were significant in both discovery and replication cohorts, the strongest being HLA-DQB1*03:02 and type 1 diabetes. Four previously unidentified associations were identified across the spectrum of disease with two- and four-digit HLA alleles and 10 with nonsynonymous variants. Some conditions associated with multiple HLA variants and stronger associations with more severe disease manifestations were identified. A comprehensive, publicly available catalog of clinical phenotypes associated with HLA variation is provided. Examining HLA variant disease associations in this large data set allows comprehensive definition of disease associations to drive further mechanistic insights.



Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire

Ryan O Emerson, William S DeWitt, Marissa Vignali, Jenna Gravley, Joyce K Hu, Edward J Osborne, Cindy Desmarais, Mark Klinger, Christopher S Carlson, John A Hansen, Mark Rieder & Harlan S Robins

An individual’s T cell repertoire dynamically encodes their pathogen exposure history. To determine whether pathogen exposure signatures can be identified by documenting public T cell receptors (TCRs), we profiled the T cell repertoire of 666 subjects with known cytomegalovirus (CMV) serostatus by immunosequencing. We developed a statistical classification framework that could diagnose CMV status from the resulting catalog of TCRβ sequences with high specificity and sensitivity in both the original cohort and a validation cohort of 120 different subjects. We also confirmed that three of the identified CMV-associated TCRβ molecules bind CMV in vitro, and, moreover, we used this approach to accurately predict the HLA-A and HLA-B alleles of most subjects in the first cohort. As all memory T cell responses are encoded in the common format of somatic TCR recombination, our approach could potentially be generalized to a wide variety of disease states, as well as other immunological phenotypes, as a highly parallelizable diagnostic strategy.

Combined antiangiogenic and anti–PD-L1 therapy stimulates tumor immunity through HEV formation

Combined antiangiogenic and anti–PD-L1 therapy stimulates tumor immunity through HEV formation

  1. Elizabeth Allen1,*,
  2. Arnaud Jabouille2,*,
  3. Lee B. Rivera2,
  4. Inge Lodewijckx1,
  5. Rindert Missiaen1,
  6. Veronica Steri2,
  7. Kevin Feyen1,
  8. Jaime Tawney2,
  9. Douglas Hanahan3,
  10. Iacovos P. Michael3 and
  11. Gabriele Bergers1,2,


Inhibitors of VEGF (vascular endothelial growth factor)/VEGFR2 (vascular endothelial growth factor receptor 2) are commonly used in the clinic, but their beneficial effects are only observed in a subset of patients and limited by induction of diverse relapse mechanisms. We describe the up-regulation of an adaptive immunosuppressive pathway during antiangiogenic therapy, by which PD-L1 (programmed cell death ligand 1), the ligand of the negative immune checkpoint regulator PD-1 (programmed cell death protein 1), is enhanced by interferon-γ–expressing T cells in distinct intratumoral cell types in refractory pancreatic, breast, and brain tumor mouse models. Successful treatment with a combination of anti-VEGFR2 and anti–PD-L1 antibodies induced high endothelial venules (HEVs) in PyMT (polyoma middle T oncoprotein) breast cancer and RT2-PNET (Rip1-Tag2 pancreatic neuroendocrine tumors), but not in glioblastoma (GBM). These HEVs promoted lymphocyte infiltration and activity through activation of lymphotoxin β receptor (LTβR) signaling. Further activation of LTβR signaling in tumor vessels using an agonistic antibody enhanced HEV formation, immunity, and subsequent apoptosis and necrosis in pancreatic and mammary tumors. Finally, LTβR agonists induced HEVs in recalcitrant GBM, enhanced cytotoxic T cell (CTL) activity, and thereby sensitized tumors to antiangiogenic/anti–PD-L1 therapy. Together, our preclinical studies provide evidence that anti–PD-L1 therapy can sensitize tumors to antiangiogenic therapy and prolong its efficacy, and conversely, antiangiogenic therapy can improve anti–PD-L1 treatment specifically when it generates intratumoral HEVs that facilitate enhanced CTL infiltration, activity, and tumor cell destruction.

Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer

Landscape of immunogenic tumor antigens in successful immunotherapy of virally induced epithelial cancer

  1. Sanja Stevanović1,*,
  2. Anna Pasetto2,
  3. Sarah R. Helman1,
  4. Jared J. Gartner2,
  5. Todd D. Prickett2,
  6. Bryan Howie3,
  7. Harlan S. Robins3,4,
  8. Paul F. Robbins2,
  9. Christopher A. Klebanoff5,6,
  10. Steven A. Rosenberg2,
  11. Christian S. Hinrichs1,*


Immunotherapy has clinical activity in certain virally associated cancers. However, the tumor antigens targeted in successful treatments remain poorly defined. We used a personalized immunogenomic approach to elucidate the global landscape of antitumor T cell responses in complete regression of human papillomavirus–associated metastatic cervical cancer after tumor-infiltrating adoptive T cell therapy. Remarkably, immunodominant T cell reactivities were directed against mutated neoantigens or a cancer germline antigen, rather than canonical viral antigens. T cells targeting viral tumor antigens did not display preferential in vivo expansion. Both viral and nonviral tumor antigen–specific T cells resided predominantly in the programmed cell death 1 (PD-1)–expressing T cell compartment, which suggests that PD-1 blockade may unleash diverse antitumor T cell reactivities. These findings suggest a new paradigm of targeting nonviral antigens in immunotherapy of virally associated cancers.

Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI)


Single-cell genomics is important for biology and medicine. However, current whole-genome amplification (WGA) methods are limited by low accuracy of copy-number variation (CNV) detection and low amplification fidelity. Here we report an improved single-cell WGA method, Linear Amplification via Transposon Insertion (LIANTI), which outperforms existing methods, enabling micro-CNV detection with kilobase resolution. This allowed direct observation of stochastic firing of DNA replication origins, which differs from cell to cell. We also show that the predominant cytosine-to-thymine mutations observed in single-cell genomics often arise from the artifact of cytosine deamination upon cell lysis. However, identifying single-nucleotide variations (SNVs) can be accomplished by sequencing kindred cells. We determined the spectrum of SNVs in a single human cell after ultraviolet radiation, revealing their nonrandom genome-wide distribution.