ImmunoGlobe: enabling systems immunology with a manually curated intercellular immune interaction network.

BACKGROUND: While technological advances have made it possible to profile the immune system at high resolution, translating high-throughput data into knowledge of immune mechanisms has been challenged by the complexity of the interactions underlying immune processes. Tools to explore the immune network are critical for better understanding the multi-layered processes that underlie immune function and dysfunction, but require a standardized network map of immune interactions. To facilitate this we have developed ImmunoGlobe, a manually curated intercellular immune interaction network extracted from Janeway's Immunobiology textbook. RESULTS: ImmunoGlobe is the first graphical representation of the immune interactome, and is comprised of 253 immune system components and 1112 unique immune interactions with detailed functional and characteristic annotations. Analysis of this network shows that it recapitulates known features of the human immune system and can be used uncover novel multi-step immune pathways, examine species-specific differences in immune processes, and predict the response of immune cells to stimuli. ImmunoGlobe is publicly available through a user-friendly interface at www.immunoglobe.org and can be downloaded as a computable graph and network table. CONCLUSION: While the fields of proteomics and genomics have long benefited from network analysis tools, no such tool yet exists for immunology. ImmunoGlobe provides a ground truth immune interaction network upon which such tools can be built. These tools will allow us to predict the outcome of complex immune interactions, providing mechanistic insight that allows us to precisely modulate immune responses in health and disease.

Accelerated, but not conventional, radiotherapy of murine B-cell lymphoma induces potent T cell-mediated remissions.

Conventional local tumor irradiation (LTI), delivered in small daily doses over several weeks, is used clinically as a palliative, rather than curative, treatment for chemotherapy-resistant diffuse large B-cell lymphoma (DLBCL) for patients who are ineligible for hematopoietic cell transplantation. Our goal was to test the hypothesis that accelerated, but not conventional, LTI would be more curative by inducing T cell-mediated durable remissions. We irradiated subcutaneous A20 and BL3750 lymphoma tumors in mice with a clinically relevant total radiation dose of 30 Gy LTI, delivered in 10 doses of 3 Gy over 4 days (accelerated irradiation) or as 10 doses of 3 Gy over 12 days (conventional irradiation). Compared with conventional LTI, accelerated LTI resulted in more complete and durable tumor remissions. The majority of these mice were resistant to rechallenge with lymphoma cells, demonstrating the induction of memory antitumor immunity. The increased efficacy of accelerated LTI correlated with higher levels of tumor cell necrosis vs apoptosis and expression of "immunogenic cell death" markers, including calreticulin, heat shock protein 70 (Hsp70), and Hsp90. Accelerated LTI-induced remissions were not seen in immunodeficient Rag-2 -/- mice, CD8+ T-cell-depleted mice, or Batf-3 -/- mice lacking CD8α+ and CD103+ dendritic cells. Accelerated, but not conventional, LTI in immunocompetent hosts induced marked increases in tumor-infiltrating CD4+ and CD8+ T cells and MHCII+CD103+CD11c+ dendritic cells and corresponding reductions in exhausted PD-1+Eomes+CD8+ T cells and CD4+CD25+FOXP3+ regulatory T cells. These findings raise the possibility that accelerated LTI can provide effective immune control of human DLBCL.

The Predictive Value of Inflammation-Related Peripheral Blood Measurements in Cancer Staging and Prognosis.

In this review, we discuss the interaction between cancer and markers of inflammation (such as levels of inflammatory cells and proteins) in the circulation, and the potential benefits of routinely monitoring these markers in peripheral blood measurement assays. Next, we discuss the prognostic value and limitations of using inflammatory markers such as neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios and C-reactive protein measurements. Furthermore, the review discusses the benefits of combining multiple types of measurements and longitudinal tracking to improve staging and prognosis prediction of patients with cancer, and the ability of novel in silico frameworks to leverage this high-dimensional data.

Longitudinal Monitoring of Antibody Responses against Tumor Cells Using Magneto-nanosensors with a Nanoliter of Blood.

Each immunoglobulin isotype has unique immune effector functions. The contribution of these functions in the elimination of pathogens and tumors can be determined by monitoring quantitative temporal changes in isotype levels. Here, we developed a novel technique using magneto-nanosensors based on the effect of giant magnetoresistance (GMR) for longitudinal monitoring of total and antigen-specific isotype levels with high precision, using as little as 1 nL of serum. Combining in vitro serologic measurements with in vivo imaging techniques, we investigated the role of the antibody response in the regression of firefly luciferase (FL)-labeled lymphoma cells in spleen, kidney, and lymph nodes in a syngeneic Burkitt's lymphoma mouse model. Regression status was determined by whole body bioluminescent imaging (BLI). The magneto-nanosensors revealed that anti-FL IgG2a and total IgG2a were elevated and sustained in regression mice compared to non-regression mice (p < 0.05). This platform shows promise for monitoring immunotherapy, vaccination, and autoimmunity.

A Robust Protocol for Protein Extraction and Digestion.

Proteins play a key role in all aspects of cellular homeostasis. Proteomics, the large-scale study of proteins, provides in-depth data on protein properties, including abundances and post-translational modification states, and as such provides a rich avenue for the investigation of biological and disease processes. While proteomic tools such as mass spectrometry have enabled exquisitely sensitive sample analysis, sample preparation remains a critical unstandardized variable that can have a significant impact on downstream data readouts. Consistency in sample preparation and handling is therefore paramount in the collection and analysis of proteomic data.Here we describe methods for performing protein extraction from cell culture or tissues, digesting the isolated protein into peptides via in-solution enzymatic digest, and peptide cleanup with final preparations for analysis via liquid chromatography-mass spectrometry. These protocols have been optimized and standardized for maximum consistency and maintenance of sample integrity.

High-dimensional analysis of the aging immune system: verification of age-associated differences in immune signaling responses in healthy donors.

BACKGROUND: Single-cell network profiling (SCNP) is a multiparametric flow cytometry-based approach that simultaneously measures evoked signaling in multiple cell subsets. Previously, using the SCNP approach, age-associated immune signaling responses were identified in a cohort of 60 healthy donors. METHODS: In the current study, a high-dimensional analysis of intracellular signaling was performed by measuring 24 signaling nodes in 7 distinct immune cell subsets within PBMCs in an independent cohort of 174 healthy donors [144 elderly (>65 yrs); 30 young (25-40 yrs)]. RESULTS: Associations between age and 9 immune signaling responses identified in the previously published 60 donor cohort were confirmed in the current study. Furthermore, within the current study cohort, 48 additional immune signaling responses differed significantly between young and elderly donors. These associations spanned all profiled modulators and immune cell subsets. CONCLUSIONS: These results demonstrate that SCNP, a systems-based approach, can capture the complexity of the cellular mechanisms underlying immunological aging. Further, the confirmation of age associations in an independent donor cohort supports the use of SCNP as a tool for identifying reproducible predictive biomarkers in areas such as vaccine response and response to cancer immunotherapies.

Genomic relationship between SINE retrotransposons, Pol III-Pol II transcription, and chromatin organization: the journey from junk to jewel.

A typical eukaryotic genome harbors a rich variety of repetitive elements. The most abundant are retrotransposons, mobile retroelements that utilize reverse transcriptase and an RNA intermediate to relocate to a new location within the cellular genomes. A vast majority of the repetitive mammalian genome content has originated from the retrotransposition of SINE (100-300 bp short interspersed nuclear elements that are derived from the structural 7SL RNA or tRNA), LINE (7kb long interspersed nuclear element), and LTR (2-3 kb long terminal repeats) transposable element superfamilies. Broadly labeled as "evolutionary junkyard" or "fossils", this enigmatic "dark matter" of the genome possesses many yet to be discovered properties.

Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal.

Cellular aging is linked to deficiencies in efficient repair of DNA double strand breaks and authentic genome maintenance at the chromatin level. Aging poses a significant threat to adult stem cell function by triggering persistent DNA damage and ultimately cellular senescence. Senescence is often considered to be an irreversible process. Moreover, critical genomic regions engaged in persistent DNA damage accumulation are unknown. Here we report that 65% of naturally occurring repairable DNA damage in self-renewing adult stem cells occurs within transposable elements. Upregulation of Alu retrotransposon transcription upon ex vivo aging causes nuclear cytotoxicity associated with the formation of persistent DNA damage foci and loss of efficient DNA repair in pericentric chromatin. This occurs due to a failure to recruit of condensin I and cohesin complexes. Our results demonstrate that the cytotoxicity of induced Alu repeats is functionally relevant for the human adult stem cell aging. Stable suppression of Alu transcription can reverse the senescent phenotype, reinstating the cells' self-renewing properties and increasing their plasticity by altering so-called "master" pluripotency regulators.