Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance.

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

Modulation of myeloid and T cells in vivo by Bruton's tyrosine kinase inhibitor ibrutinib in patients with metastatic pancreatic ductal adenocarcinoma.

BACKGROUND: In preclinical studies of pancreatic ductal adenocarcinoma (PDAC), ibrutinib improved the antitumor efficacy of the standard of care chemotherapy. This led to a phase 1b clinical trial to determine the safety, tolerability, and immunologic effects of ibrutinib treatment in patients with advanced PDAC. METHODS: Previously untreated patients with PDAC were enrolled in a phase 1b clinical trial (ClinicalTrials.gov) to determine the safety, toxicity, and maximal tolerated dose of ibrutinib when administered with the standard regimen of gemcitabine and nab-paclitaxel. To study the immune response to ibrutinib alone, the trial included an immune response arm where patients were administered with ibrutinib daily for a week followed by ibrutinib combined with gemcitabine and nab-paclitaxel. Endoscopic ultrasonography-guided primary PDAC tumor biopsies and blood were collected before and after ibrutinib monotherapy. Changes in abundance and functional state of immune cells in the blood was evaluated by mass cytometry by time of flight and statistical scaffold analysis, while that in the local tumor microenvironment (TME) were assessed by multiplex immunohistochemistry. Changes in B-cell receptor and T-cell receptor repertoire were assessed by sequencing and analysis of clonality. RESULTS: In the blood, ibrutinib monotherapy significantly increased the frequencies of activated inducible T cell costimulator+(ICOS+) CD4+ T cells and monocytes. Within the TME, ibrutinib monotherapy led to a trend in decreased B-cell abundance but increased interleukin-10+ B-cell frequency. Monotherapy also led to a trend in increased mature CD208+dendritic cell density, increased late effector (programmed cell death protein 1 (PD-1-) eomesodermin (EOMES+)) CD8+ T-cell frequency, with a concomitantly decreased dysfunctional (PD-1+ EOMES+) CD8+ T-cell frequency. When ibrutinib was combined with chemotherapy, most of these immune changes were not observed. Patients with partial clinical responses had more diverse T and B cell receptor repertoires prior to therapy initiation. CONCLUSION: Ibrutinib monotherapy skewed the immune landscape both in the circulation and TME towards activated T cells, monocytes and DCs. These effects were not observed when combining ibrutinib with standard of care chemotherapy. Future studies may focus on other therapeutic combinations that augment the immunomodulatory effects of ibrutinib in solid tumors. TRIAL REGISTRATION NUMBER: NCT02562898.

SCITO-seq: single-cell combinatorial indexed cytometry sequencing.

The development of DNA-barcoded antibodies to tag cell surface molecules has enabled the use of droplet-based single-cell sequencing (dsc-seq) to profile protein abundances from thousands of cells simultaneously. As compared to flow and mass cytometry, the high per cell cost of current dsc-seq-based workflows precludes their use in clinical applications and large-scale pooled screens. Here, we introduce SCITO-seq, a workflow that uses splint oligonucleotides (oligos) to enable combinatorially indexed dsc-seq of DNA-barcoded antibodies from over 105 cells per reaction using commercial microfluidics. By encoding sample barcodes into splint oligos, we demonstrate that multiplexed SCITO-seq produces reproducible estimates of cellular composition and surface protein expression comparable to those from mass cytometry. We further demonstrate two modified splint oligo designs that extend SCITO-seq to achieve compatibility with commercial DNA-barcoded antibodies and simultaneous expression profiling of the transcriptome and surface proteins from the same cell. These results demonstrate SCITO-seq as a flexible and ultra-high-throughput platform for sequencing-based single-cell protein and multimodal profiling.

The effect of low-dose IL-2 and Treg adoptive cell therapy in patients with type 1 diabetes.

BACKGROUNDA previous phase I study showed that the infusion of autologous Tregs expanded ex vivo into patients with recent-onset type 1 diabetes (T1D) had an excellent safety profile. However, the majority of the infused Tregs were undetectable in the peripheral blood 3 months postinfusion (Treg-T1D trial). Therefore, we conducted a phase I study (TILT trial) combining polyclonal Tregs and low-dose IL-2, shown to enhance Treg survival and expansion, and assessed the impact over time on Treg populations and other immune cells.METHODSPatients with T1D were treated with a single infusion of autologous polyclonal Tregs followed by one or two 5-day courses of recombinant human low-dose IL-2 (ld-IL-2). Flow cytometry, cytometry by time of flight, and 10x Genomics single-cell RNA-Seq were used to follow the distinct immune cell populations' phenotypes over time.RESULTSMultiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a substantial increase from baseline in a subset of activated NK, mucosal associated invariant T, and clonal CD8+ T cell populations.CONCLUSIONThese data support the hypothesis that ld-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with preexisting active immunity.TRIAL REGISTRATIONClinicalTrials.gov NCT01210664 (Treg-T1D trial), NCT02772679 (TILT trial).FUNDINGSean N. Parker Autoimmune Research Laboratory Fund, National Center for Research Resources.

Deep immune profiling reveals targetable mechanisms of immune evasion in immune checkpoint inhibitor-refractory glioblastoma.

BACKGROUND: Glioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed. METHODS: We used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations. RESULTS: ICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation. CONCLUSIONS: Our data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.

Phenotypic analysis of the unstimulated in vivo HIV CD4 T cell reservoir.

The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence.

There is no cure for the human immunodeficiency virus infection (HIV), but anti-retroviral drugs allow infected people to keep the virus at bay and lead a normal life. These drugs suppress the growth of HIV, but they do not eliminate the virus. If the treatment is interrupted, the virus bounces back within weeks in most individuals. HIV can start growing again because it hides within particular immune cells, called T cells. These infected cells stay in the infected person's body for their whole life in a dormant or "latent" state, and represent the main barrier to an HIV cure. If these cells could be eliminated or prevented from producing more virus without daily treatment, then HIV could be cured. The fact that HIV hides inside T cells has been known for a long time, but it has remained unclear exactly what kinds of T cells the virus prefers. One challenge to characterizing latently-infected cells is that there is no single protein made by them that is not also made by uninfected T cells. The latently-infected T cells are also very rare: HIV mainly attaches to a protein called CD4, but only one in about a million T cells with CD4 contain the virus. To figure out which CD4-carrying T cells in a patient sample are latently infected, the cells are extracted from the patient's body and 'reactivated' so the virus will start growing again. Unfortunately, the mixture of drugs used to reactivate the T cells changes the cells and the proteins they are producing, which obscures the features the latently-infected T cells had before reactivation. Neidleman, Luo et al. developed a new approach to trace the infected, reactivated T cells back to their state before reactivation. Using computational methods and a laboratory technique called mass cytometry, the levels of approximately 40 different proteins were measured in millions of T cells from people living with HIV. These experiments provided an 'atlas' of overall T cell features onto which each reactivated cell could be mapped. The population of latently-infected T cells exhibited common features among all the participants. Selecting a few of the most abundant proteins on the surface of the latently-infected cells allowed these cells to be physically separated from all other immune cells. In the future, this relatively pure population of infected T cells could be used to study how HIV can persist for many decades. The 'map' of these cells' features will provide a valuable resource for HIV researchers and might enable the discovery of new drugs to eliminate the latent T cells.

Lower PDL1, PDL2, and AXL Expression on Lung Myeloid Cells Suggests Inflammatory Bias in Smoking and Chronic Obstructive Pulmonary Disease.

Lung myeloid cells are important in pulmonary immune homeostasis and in the pathogenesis of chronic obstructive pulmonary disease (COPD). Multiparameter immunophenotypic characterization of these cells is challenging because of their autofluorescence and diversity. We evaluated the immunophenotypic landscape of airway myeloid cells in COPD using time of flight mass cytometry. Cells from BAL, which were obtained from never-smokers (n = 8) and smokers with (n = 20) and without (n = 4) spirometric COPD, were examined using a 44-parameter time of flight mass cytometry panel. Unsupervised cluster analysis was used to identify cellular subtypes that were confirmed by manual gating. We identified major populations of CD68+ and CD68- cells with 22 distinct phenotypic clusters, of which 18 were myeloid cells. We found a higher abundance of putative recruited myeloid cells (CD68+ classical monocytes) in BAL from patients with COPD. CD68+ classical monocyte population had distinct responses to smoking and COPD that were potentially related to their recruitment from the interstitium and vasculature. We demonstrate that BAL cells from smokers and subjects with COPD have lower AXL expression. Also, among subjects with COPD, we report significant differences in the abundance of PDL1high and PDL2high clusters and in the expression of PDL1 and PDL2 across several macrophage subtypes suggesting modulation of inflammatory responses. In addition, several phenotypic differences in BAL cells from subjects with history of COPD exacerbation were identified that could inform potential disease mechanisms. Overall, we report several changes to the immunophenotypic landscape that occur with smoking, COPD, and past exacerbations that are consistent with decreased regulation and increased activation of inflammatory pathways.

Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.