Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit Type I interferon responses critical for antimicrobial defense1–3. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis (TB) requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)4 over-expressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-β, elicits increased macrophage autophagy, and exhibits significant attenuation in mice. We show that c-di-AMP-mediated IFN-β induction during M. tuberculosis infection requires stimulator of interferon genes (STING)5-signaling. We observed that c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acid receptor cyclic-GMP-AMP (cGAMP) synthase (cGAS)6–7, but cGAS nevertheless contributes substantially to the overall IFN-β response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen associated molecular pattern (PAMP) driving host Type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against TB.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a ‘barcode’ to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein–Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic KrasG12D from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by KrasG12D expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. KrasG12D HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. KrasG12D expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted KrasG12D HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in KrasG12D mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.

Mycobacterium tuberculosis infection leads to cytosolic release of the bacterial cyclic dinucleotide (CDN) c-di-AMP and a host-generated CDN, cGAMP, both of which trigger type I interferon (IFN) expression in a STING-dependent manner. Here we report that M. tuberculosis has developed a mechanism to inhibit STING activation and the type I IFN response via the bacterial phosphodiesterase (PDE) CdnP, which mediates hydrolysis of both bacterial-derived c-di-AMP and host-derived cGAMP. Mutation of cdnP attenuates M. tuberculosis virulence, as does loss of a host CDN PDE known as ENPP1. CdnP is inhibited by both US Food and Drug Administration (FDA)-approved PDE inhibitors and nonhydrolyzable dinucleotide mimetics specifically designed to target the enzyme. These findings reveal a crucial role of CDN homeostasis in governing the outcome of M. tuberculosis infection as well as a unique mechanism of subversion of the host's cytosolic surveillance pathway (CSP) by a bacterial PDE that may serve as an attractive antimicrobial target.

Active tuberculosis (TB) often presents with advanced pulmonary disease, including irreversible lung damage and cavities. Cavitary pathology contributes to antibiotic failure, transmission, morbidity and mortality. Matrix metalloproteinases (MMPs), in particular MMP-1 are implicated in TB pathogenesis. We explored the mechanisms relating MMP/TIMP imbalance to cavity formation in a modified rabbit model of cavitary TB. Our model results in consistent progression of consolidation to human-like cavities (100% by day 28) with resultant bacillary burdens (>107 CFU/g) far greater than those found in matched granulomatous tissue (105 CFU/g). Using a novel, breath-hold computerized tomography scanning and image analysis protocol. We show that cavities develop rapidly from areas of densely consolidated tissue. Radiological change correlated with a decrease in functional lung tissue as estimated by changes in lung density during controlled pulmonary expansion (R2=0.6356, p<0.0001). We demonstrated that the expression of interstitial collagenase (MMP-1) is specifically greater in cavitary compared to granulomatous lesions (p<0.01), and that TIMP-3 significantly decreases at the cavity surface. Our findings demonstrate that an MMP-1/TIMP imbalance, is associated with the progression of consolidated regions to cavities containing very high bacterial burdens. Our model provided mechanistic insight, correlating with human disease at the pathological, microbiological and molecular levels,. It also provides a strategy to investigate therapeutics in the context of complex TB pathology. We used these findings to predict a MMP/TIMP balance in active TB; and confirmed this in human plasma, revealing the potential of MMP/TIMP levels as key components of a diagnostic matrix aimed at distinguishing active from latent TB (PPV=92.9%; 95%CI 66.1–99.8%, NPV=85.6%; 95%CI 77.0–91.9%).

Src homology 2 domain-containing phosphatase 2 (Shp2), encoded by Ptpn11, is a member of the nonreceptor proteintyrosine phosphatase family, and functions in cell survival, proliferation, migration, and differentiation in many tissues. Here we report that loss of IntroductionHematopoiesis is maintained by a small number of multipotent long-term hematopoietic stem cells (LT-HSCs) with extensive self-renewal capability. These cells give rise to lineage-committed progenitors, which produce various types of mature blood cells. Cytokine and growth factor signaling is critical for the sustained production of HSCs and progenitors and directs cellular survival, migration, and differentiation. 1,2 Src homology 2 (SH2) domain-containing phosphatase 2 (Shp2) is a ubiquitously expressed nonreceptor protein-tyrosine phosphatase (PTP), encoded by the Ptpn11 gene, which regulates signaling networks and cell fates in many organisms. Shp2 is composed of 2 SH2 domains (N-SH2 and C-SH2), a PTP domain, a C-terminal tail with tyrosyl phosphorylation sites that modulate some RTK signaling pathways, and a proline-rich motif of unknown function. In multiple tissues, Shp2 positively regulates Ras/Erk signaling downstream of receptor tyrosine kinases (RTKs) and cytokine receptors. 3 Shp2 also appears to have cell-type-and/or agonistdependent effects on the activation of Akt, Jnk, NF-B, Rho, and Nfat. 3 Germline PTPN11 mutations underlie approximately 50% of Noonan syndrome, which is characterized by short stature, skeletal abnormalities, cardiac defects, learning disabilities, and a predisposition to hematologic abnormalities, particularly juvenile myelomonocytic leukemia. Somatic gain-of-function mutations in PTPN11 also are the most common cause of sporadic juvenile myelomonocytic leukemia. 4 Although Shp2 is required for normal Ras/Erk activation in many contexts, 3 the underlying mechanism shows great diversity and depends on the precise cell type and stimulus involved. Epistasis studies in Caenorhabditis elegans and Drosophila show that activated Ras/let-60/ras1 suppresses the effects of Shp2/ptp-2/ csw loss, 5,6 arguing that Shp2 acts upstream of Ras in these signaling pathways. However, in C elegans vulva development and Drosophila R7 photoreceptors and muscle precursors, Shp2/ptp-2/ csw appears to act downstream of Ras/let-60/ras1 in some contexts. 5,7-9 Furthermore, in the EGL-15/Egfr signaling pathway, ptp-2 may act parallel to let-60. 10 In mice, expression of activated Kras (Kras G12D ) does not completely restore defective lens proliferation and lacrimal gland development caused by the loss of Ptpn11. 11 Collectively, these results indicate that Shp2 can act upstream, downstream, or parallel to Ras in different systems, and imply that the consequences and the mediators of Shp2 action must be specifically delineated in particular biologic contexts.Homozygous inactivation of murine Shp2 results in early embryonic lethality 12-14 because of a critical role of Shp2 in trophoblast stem cell survival. 14 Loss of Shp2 also reduces...

CONFLICTS OF INTERESTDMP and KNS have filed for patent protection on a subset of the technologies described herein (US provisional patent application no. 62/407,820). AD, ALC, FD, DMP, JB, and KNS have filed for patent protection on a subset of the technologies described herein (US provisional patent application no. 63/135,534). SZ is a founder of, holds equity in, and serves as a consultant to Personal Genome Diagnostics. SZ holds equity in Thrive Earlier

Denileukin diftitox (DAB-IL-2, Ontak) is a diphtheria-toxin–based fusion protein that depletes CD25-positive cells including regulatory T cells and has been approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB-IL-2(V6A), which elicits 50-fold less human umbilical vein endothelial cell monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB-IL-2. Additionally, to overcome aggregation problems, we developed a production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully folded, biologically active, monomeric s-DAB-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB-IL-2(V6A) and s-DAB-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB-IL-2(V6A) could provide a superior activity window. In a sequential dual-therapy study in tumors that have progressed for 10 days, both s-DAB-IL-2(V6A) and s-DAB-IL-2 given before checkpoint inhibition with anti–programmed cell death-1 (anti–PD-1) antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria-toxin–based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.