Understanding the tumor immune microenvironment (TIME) promises to be key for optimal cancer therapy, especially in triple-negative breast cancer (TNBC). Integrating spatial resolution of immune cells with laser capture microdissection gene expression profiles, we defined distinct TIME stratification in TNBC, with implications for current therapies including immune checkpoint blockade. TNBCs with an immunoreactive microenvironment exhibited tumoral infiltration of granzyme B + CD8 + T cells (GzmB + CD8 + T cells), a type 1 IFN signature, and elevated expression of multiple immune inhibitory molecules including indoleamine 2,3-dioxygenase (IDO) and programmed cell death ligand 1 (PD-L1), and resulted in good outcomes. An "immune-cold" microenvironment with an absence of tumoral CD8 + T cells was defined by elevated expression of the immunosuppressive marker B7-H4, signatures of fibrotic stroma, and poor outcomes. A distinct poor-outcome immunomodulatory microenvironment, hitherto poorly characterized, exhibited stromal restriction of CD8 + T cells, stromal expression of PD-L1, and enrichment for signatures of cholesterol biosynthesis. Metasignatures defining these TIME subtypes allowed us to stratify TNBCs, predict outcomes, and identify potential therapeutic targets for TNBC.

SummaryHuman immunodeficiency virus type 1 (HIV-1) Gag selects for and mediates genomic RNA (vRNA) encapsidation into progeny virus particles. The host protein, Staufen1 interacts directly with Gag and is found in ribonucleoprotein (RNP) complexes containing vRNA, which provides evidence that Staufen1 plays a role in vRNA selection and encapsidation. In this work, we show that Staufen1, vRNA and Gag are found in the same RNP complex. These cellular and viral factors also colocalize in cells and constitute novel Staufen1 RNPs (SHRNPs) whose assembly is strictly dependent on HIV-1 expression. SHRNPs are distinct from stress granules and processing bodies, are preferentially formed during oxidative stress and are found to be in equilibrium with translating polysomes. Moreover, SHRNPs are stable, and the association between Staufen1 and vRNA was found to be evident in these and other types of RNPs. We demonstrate that following Staufen1 depletion, apparent supraphysiologic-sized SHRNP foci are formed in the cytoplasm and in which Gag, vRNA and the residual Staufen1 accumulate. The depletion of Staufen1 resulted in reduced Gag levels and deregulated the assembly of newly synthesized virions, which were found to contain several-fold increases in vRNA, Staufen1 and other cellular proteins. This work provides new evidence that Staufen1-containing HIV-1 RNPs preferentially form over other cellular silencing foci and are involved in assembly, localization and encapsidation of vRNA.

Human immunodeficiency virus type 1 (HIV-1) co-opts host proteins and cellular machineries to its advantage at every step of the replication cycle. Here we show that HIV-1 enhances heterogeneous nuclear ribonucleoprotein (hnRNP) A1 expression and promotes the relocalization of hnRNP A1 to the cytoplasm. The latter was dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of the FG-repeat nuclear pore glycoprotein p62. hnRNP A1 and vRNA remain colocalized in the cytoplasm supporting a post-nuclear function during the late stages of HIV-1 replication. Consistently, we show that hnRNP A1 acts as an internal ribosomal entry site trans-acting factor up-regulating internal ribosome entry site-mediated translation initiation of the HIV-1 vRNA. The up-regulation and cytoplasmic retention of hnRNP A1 by HIV-1 would ensure abundant expression of viral structural proteins in cells infected with HIV-1.During the late stages of the HIV-1 3 replication cycle, the full-length HIV-1 viral RNA (vRNA) must be exported from the nucleus and both translated and packaged into new viral particles (1). The orchestration of these events is directed by a diversity of viral and host proteins that interact with each other and with the vRNA to form HIV-1 ribonucleoprotein (RNP) complexes that originate in the nucleus and persist in the cytoplasm (2). Investigations into the composition and functions of the HIV-1 RNP will reveal new information about innate immunity as well as identify new potential therapeutic targets (3-6).The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a predominantly nuclear protein engaged in a number of cellular and viral RNPs for RNA-processing activities, including splicing regulation, nuclear export, microRNA processing, mRNA stability, telomere maintenance, and IRES-mediated translation initiation (7-12). What enables hnRNP A1 to have such broad functions in RNA metabolism is its ability to bind both nuclear and cytoplasmic RNAs (10). In addition to its well characterized role in nuclear RNA processing, hnRNP A1 binds to purine-rich sequences of mRNAs for mRNA turnover and translation (13,14). Close examination of the HIV-1 vRNA reveals many AG-and AU-rich sequences closely resembling hnRNP A1 binding motifs (15). In fact, hnRNP A1 binds to a number of sequences on the HIV-1 vRNA such as the cis-acting repressive sequences, instability elements, and exonic splicing silencer elements, and indeed, hnRNP A1 is implicated in the fate of HIV-1 RNA, including splicing regulation, nucleocytoplasmic export, and cytoplasmic stability (16 -21).Our previous work demonstrated that hnRNP A1 efficiently immunoprecipitated with the HIV-1 vRNA (22) and that siRNA-mediated knockdown of hnRNP A1 caused a dramatic decrease in HIV-1 structural protein, pr55Gag (herein termed Gag) expression and virus production with little effect on steady-state levels of the three HIV-1 RNA species (i.e. 9-, 4-, and 2-kb RNAs) (23). In this work, we show that HIV-1 ...

The modification of glutamic acid residues to ␥-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme ␥-glutamylcarboxylase. Despite ubiquitous expression of the ␥-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized ␥-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus ␥-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, ␤ig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent ␥-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro.

Tumor immunology has changed the landscape of cancer treatment. Yet, not all patients benefit as cancer immune responsiveness (CIR) remains a limitation in a considerable proportion of cases. The multifactorial determinants of CIR include the genetic makeup of the patient, the genomic instability central to cancer development, the evolutionary emergence of cancer phenotypes under the influence of immune editing, and external modifiers such as demographics, environment, treatment potency, co-morbidities and cancer-independent alterations including immune homeostasis and polymorphisms in the major and minor histocompatibility molecules, cytokines, and chemokines. Based on the premise that cancer is fundamentally a disorder of the genes arising within a cell biologic process, whose deviations from normality determine the rules of engagement with the host’s response, the Society for Immunotherapy of Cancer (SITC) convened a task force of experts from various disciplines including, immunology, oncology, biophysics, structural biology, molecular and cellular biology, genetics, and bioinformatics to address the complexity of CIR from a holistic view. The task force was launched by a workshop held in San Francisco on May 14–15, 2018 aimed at two preeminent goals: 1) to identify the fundamental questions related to CIR and 2) to create an interactive community of experts that could guide scientific and research priorities by forming a logical progression supported by multiple perspectives to uncover mechanisms of CIR. This workshop was a first step toward a second meeting where the focus would be to address the actionability of some of the questions identified by working groups. In this event, five working groups aimed at defining a path to test hypotheses according to their relevance to human cancer and identifying experimental models closest to human biology, which include: 1) Germline-Genetic, 2) Somatic-Genetic and 3) Genomic-Transcriptional contributions to CIR, 4) Determinant(s) of Immunogenic Cell Death that modulate CIR, and 5) Experimental Models that best represent CIR and its conversion to an immune responsive state. This manuscript summarizes the contributions from each group and should be considered as a first milestone in the path toward a more contemporary understanding of CIR. We appreciate that this effort is far from comprehensive and that other relevant aspects related to CIR such as the microbiome, the individual’s recombined T cell and B cell receptors, and the metabolic status of cancer and immune cells were not fully included. These and other important factors will be included in future activities of the taskforce. The taskforce will focus on prioritization and specific actionable approach to answer the identified questions and implementing the collaborations in the follow-up workshop, which will be held in Houston on September 4–5, 2019.

Abstract:The success of promising anti-cancer adoptive cell therapies relies on the abilities of the perfused CD8 + T lymphocytes to gain access to and persist within the tumor microenvironment to carry out their cytotoxic functions. We propose a new method for their local delivery as a living concentrate, which may not only reduce the numbers of cells required for treatment but also enhance their site-specific mobilization. Using combinations of sodium hydrogen carbonate and phosphate buffer as gelling agents, novel injectable chitosan-based biocompatible thermogels (CTGels) having excellent mechanical properties and cytocompatibility have been developed. Three thermogel formulations with acceptable physicochemical properties, such as physiological pH and osmolality, macroporosity, and gelation rates were compared.The CTGel2 formulation outperformed the others by providing an environment suitable for the encapsulation of viable CD8 + T lymphocytes, supporting their proliferation and gradual release. In addition, the encapsulated T cell phenotypes were influenced by surrounding conditions and by tumor cells, while maintaining their capacity to kill tumor cells. This strongly suggests that cells encapsulated in this formulation retain their anti-cancer functions, and that this locally injectable hydrogel may be further developed to complement a wide variety of existing immunotherapies.

Highlights d Retroviral Gag proteins have conserved intrinsically disordered prion-like domains d Pan-retrovirus family nucleocapsid proteins induce liquidliquid phase separation d Nucleocapsid protein phase separation and stress granule assembly is Zn 2+ dependent d Zn 2+ chelators and ejectors induce nuclear repositioning of the genomic RNA

The precursor group-specific antigen (pr55Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55Gag expression, the synthesis of other HIV-1 proteins, including that of pr160Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55Gag for virus assembly and production.