Neoadjuvant nivolumab or nivolumab plus LAG-3 inhibitor relatlimab in resectable esophageal/gastroesophageal junction cancer: a phase Ib trial and ctDNA analyses.

Gastroesophageal cancer dynamics and drivers of clinical responses with immune checkpoint inhibitors (ICI) remain poorly understood. Potential synergistic activity of dual programmed cell death protein 1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) inhibition may help improve immunotherapy responses for these tumors. We report a phase Ib trial that evaluated neoadjuvant nivolumab (Arm A, n = 16) or nivolumab-relatlimab (Arm B, n = 16) in combination with chemoradiotherapy in 32 patients with resectable stage II/stage III gastroesophageal cancer together with an in-depth evaluation of pathological, molecular and functional immune responses. Primary endpoint was safety; the secondary endpoint was feasibility; exploratory endpoints included pathological complete (pCR) and major pathological response (MPR), recurrence-free survival (RFS) and overall survival (OS). The study met its primary safety endpoint in Arm A, although Arm B required modification to mitigate toxicity. pCR and MPR rates were 40% and 53.5% for Arm A and 21.4% and 57.1% for Arm B. Most common adverse events were fatigue, nausea, thrombocytopenia and dermatitis. Overall, 2-year RFS and OS rates were 72.5% and 82.6%, respectively. Higher baseline programmed cell death ligand 1 (PD-L1) and LAG-3 expression were associated with deeper pathological responses. Exploratory analyses of circulating tumor DNA (ctDNA) showed that patients with undetectable ctDNA post-ICI induction, preoperatively and postoperatively had a significantly longer RFS and OS; ctDNA clearance was reflective of neoantigen-specific T cell responses. Our findings provide insights into the safety profile of combined PD-1 and LAG-3 blockade in gastroesophageal cancer and highlight the potential of ctDNA analysis to dynamically assess systemic tumor burden during neoadjuvant ICI that may open a therapeutic window for future intervention. ClinicalTrials.gov registration: NCT03044613 .

Deep neural networks predict class I major histocompatibility complex epitope presentation and transfer learn neoepitope immunogenicity.

Identifying neoepitopes that elicit an adaptive immune response is a major bottleneck to developing personalized cancer vaccines. Experimental validation of candidate neoepitopes is extremely resource intensive and the vast majority of candidates are non-immunogenic, creating a needle-in-a-haystack problem. Here we address this challenge, presenting computational methods for predicting class I major histocompatibility complex (MHC-I) epitopes and identifying immunogenic neoepitopes with improved precision. The BigMHC method comprises an ensemble of seven pan-allelic deep neural networks trained on peptide-MHC eluted ligand data from mass spectrometry assays and transfer learned on data from assays of antigen-specific immune response. Compared with four state-of-the-art classifiers, BigMHC significantly improves the prediction of epitope presentation on a test set of 45,409 MHC ligands among 900,592 random negatives (area under the receiver operating characteristic = 0.9733; area under the precision-recall curve = 0.8779). After transfer learning on immunogenicity data, BigMHC yields significantly higher precision than seven state-of-the-art models in identifying immunogenic neoepitopes, making BigMHC effective in clinical settings.

Ablative radiation alone in stage I lung cancer produces an adaptive systemic immune response: insights from a prospective stud.

Stereotactic ablative body radiation (SABR) delivers high rates of local control in early-stage non-small cell lung cancer (NSCLC); however, systemic immune effects are poorly understood. Here, we evaluate the early pathologic and immunologic effects of SABR. Blood/core-needle tumor biopsies were collected from six patients with stage I NSCLC before and 5-7 days after SABR (48 Gy/4 or 50 Gy/5 fractions). Serial blood was collected up to 1-year post-SABR. We used immunohistochemistry to evaluate pathological changes, immune-cell populations (CD8, FoxP3), and PD-L1/PD-1 expression within the tumor. We evaluated T-cell receptor (TCR) profile changes in the tumor using TCR sequencing. We used the MANAFEST (Mutation-Associated Neoantigen Functional Expansion of Specific T-cells) assay to detect peripheral neoantigen-specific T-cell responses and dynamics. At a median follow-up of 40 months, 83% of patients (n=5) were alive without tumor progression. Early post-SABR biopsies showed viable tumor and similar distribution of immune-cell populations as compared with baseline samples. Core-needle samples proved insufficient to detect population-level TCR-repertoire changes. Functionally, neoantigen-specific T-cells were detected in the blood prior to SABR. A subset of these patients had a transient increase in the frequency of neoantigen-specific T-cells between 1 week and 3-6 months after SABR. SABR alone could induce a delayed, transient neoantigen-specific T-cell immunologic response in patients with stage I NSCLC.

Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response.

Regulatory T cells (Treg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-Treg-expressing T cell receptors that are specific for TAA fully develop a distinct TH1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNG, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific TH1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to antitumor responses.

Durvalumab with platinum-pemetrexed for unresectable pleural mesothelioma: survival, genomic and immunologic analyses from the phase 2 PrE0505 trial.

Mesothelioma is a rare and fatal cancer with limited therapeutic options until the recent approval of combination immune checkpoint blockade. Here we report the results of the phase 2 PrE0505 trial ( NCT02899195 ) of the anti-PD-L1 antibody durvalumab plus platinum-pemetrexed chemotherapy for 55 patients with previously untreated, unresectable pleural mesothelioma. The primary endpoint was overall survival compared to historical control with cisplatin and pemetrexed chemotherapy; secondary and exploratory endpoints included safety, progression-free survival and biomarkers of response. The combination of durvalumab with chemotherapy met the pre-specified primary endpoint, reaching a median survival of 20.4 months versus 12.1 months with historical control. Treatment-emergent adverse events were consistent with known side effects of chemotherapy, and all adverse events due to immunotherapy were grade 2 or lower. Integrated genomic and immune cell repertoire analyses revealed that a higher immunogenic mutation burden coupled with a more diverse T cell repertoire was linked to favorable clinical outcome. Structural genome-wide analyses showed a higher degree of genomic instability in responding tumors of epithelioid histology. Patients with germline alterations in cancer predisposing genes, especially those involved in DNA repair, were more likely to achieve long-term survival. Our findings indicate that concurrent durvalumab with platinum-based chemotherapy has promising clinical activity and that responses are driven by the complex genomic background of malignant pleural mesothelioma.

Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.

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.

Functional characterization of CD4+ T cell receptors crossreactive for SARS-CoV-2 and endemic coronaviruses.

BACKGROUNDRecent studies have reported T cell immunity to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in unexposed donors, possibly due to crossrecognition by T cells specific for common cold coronaviruses (CCCs). True T cell crossreactivity, defined as the recognition by a single TCR of more than one distinct peptide-MHC ligand, has never been shown in the context of SARS-CoV-2.METHODSWe used the viral functional expansion of specific T cells (ViraFEST) platform to identify T cell responses crossreactive for the spike (S) glycoproteins of SARS-CoV-2 and CCCs at the T cell receptor (TCR) clonotype level in convalescent COVID-19 patients (CCPs) and SARS-CoV-2-unexposed donors. Confirmation of SARS-CoV-2/CCC crossreactivity and assessments of functional avidity were performed using a TCR cloning and transfection system.RESULTSMemory CD4+ T cell clonotypes that crossrecognized the S proteins of SARS-CoV-2 and at least one other CCC were detected in 65% of CCPs and unexposed donors. Several of these TCRs were shared among multiple donors. Crossreactive T cells demonstrated significantly impaired SARS-CoV-2-specific proliferation in vitro relative to monospecific CD4+ T cells, which was consistent with lower functional avidity of their TCRs for SARS-CoV-2 relative to CCC.CONCLUSIONSOur data confirm, for what we believe is the first time, the existence of unique memory CD4+ T cell clonotypes crossrecognizing SARS-CoV-2 and CCCs. The lower avidity of crossreactive TCRs for SARS-CoV-2 may be the result of antigenic imprinting, such that preexisting CCC-specific memory T cells have reduced expansive capacity upon SARS-CoV-2 infection. Further studies are needed to determine how these crossreactive T cell responses affect clinical outcomes in COVID-19 patients.FUNDINGNIH funding (U54CA260492, P30CA006973, P41EB028239, R01AI153349, R01AI145435-A1, R21AI149760, and U19A1088791) was provided by the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, and the National Institute of Biomedical Imaging and Bioengineering. The Bloomberg~Kimmel Institute for Cancer Immunotherapy, The Johns Hopkins University Provost, and The Bill and Melinda Gates Foundation provided funding for this study.

The strengths and limitations of using biolayer interferometry to monitor equilibrium titrations of biomolecules.

Every method used to quantify biomolecular interactions has its own strengths and limitations. To quantify protein-DNA binding affinities, nitrocellulose filter binding assays with 32 P-labeled DNA quantify Kd values from 10-12 to 10-8 M but have several technical limitations. Here, we considered the suitability of biolayer interferometry (BLI), which monitors association and dissociation of a soluble macromolecule to an immobilized species; the ratio koff /kon determines Kd . However, for lactose repressor protein (LacI) and an engineered repressor protein ("LLhF") binding immobilized DNA, complicated kinetic curves precluded this analysis. Thus, we determined whether the amplitude of the BLI signal at equilibrium related linearly to the fraction of protein bound to DNA. A key question was the effective concentration of immobilized DNA. Equilibrium titration experiments with DNA concentrations below Kd (equilibrium binding regime) must be analyzed differently than those with DNA near or above Kd (stoichiometric binding regime). For ForteBio streptavidin tips, the most frequent effective DNA concentration was ~2 × 10-9 M. Although variation occurred among different lots of sensor tips, binding events with Kd ≥ 10-8 M should reliably be in the equilibrium binding regime. We also observed effects from multi-valent interactions: Tetrameric LacI bound two immobilized DNAs whereas dimeric LLhF did not. We next used BLI to quantify the amount of inducer sugars required to allosterically diminish protein-DNA binding and to assess the affinity of fructose-1-kinase for the DNA-LLhF complex. Overall, when experimental design corresponded with appropriate data interpretation, BLI was convenient and reliable for monitoring equilibrium titrations and thereby quantifying a variety of binding interactions.

A role for proteolytic regulation of δ-catenin in remodeling a subpopulation of dendritic spines in the rodent brain.

Neural wiring and activity are essential for proper brain function and behavioral outputs and rely on mechanisms that guide the formation, elimination, and remodeling of synapses. During development, it is therefore vital that synaptic densities and architecture are tightly regulated to allow for appropriate neural circuit formation and function. δ-Catenin, a component of the cadherin-catenin cell adhesion complex, has been demonstrated to be a critical regulator of synaptic density and function in the developing central neurons. In this study, we identified forms of δ-catenin that include only the N-terminal (DcatNT) or the C-terminal (DcatCT) regions. We found that these δ-catenin forms are differentially expressed in different regions of the male mouse brain. Our results also indicated that in rat primary cortical culture, these forms are generated in an activity-dependent manner by Ca2+-dependent and calpain-mediated cleavage of δ-catenin or in an activity-independent but lysosome-dependent manner. Functionally, loss of the domain containing the calpain-cleavage sites allowing for generation of DcatCT and DcatNT perturbed the density of a subpopulation of dendritic protrusions in rat hippocampal neurons. This subpopulation likely included protrusions that are either in transition toward becoming mature mushroom spines or in the process of being eliminated. By influencing this subpopulation of spines, proteolytic processing of δ-catenin can likely regulate the balance between mature and immature dendritic protrusions in coordination with neural activity. We conclude that by undergoing cleavage, δ-catenin differentially regulates the densities of subpopulations of dendritic spines and contributes to proper neural circuit wiring in the developing brain.

Differential regulation of apical-basolateral dendrite outgrowth by activity in hippocampal neurons.

Hippocampal pyramidal neurons have characteristic dendrite asymmetry, characterized by structurally and functionally distinct apical and basolateral dendrites. The ability of the neuron to generate and maintain dendrite asymmetry is vital, since synaptic inputs received are critically dependent on dendrite architecture. Little is known about the role of neuronal activity in guiding maintenance of dendrite asymmetry. Our data indicate that dendrite asymmetry is established and maintained early during development. Further, our results indicate that cell intrinsic and global alterations of neuronal activity have differential effects on net extension of apical and basolateral dendrites. Thus, apical and basolateral dendrite extension may be independently regulated by cell intrinsic and network neuronal activity during development, suggesting that individual dendrites may have autonomous control over net extension. We propose that regulated individual dendrite extension in response to cell intrinsic and neuronal network activity may allow temporal control of synapse specificity in the developing hippocampus.

Spatially selective photoconductive stimulation of live neurons.

Synaptic activity is intimately linked to neuronal structure and function. Stimulation of live cultured primary neurons, coupled with fluorescent indicator imaging, is a powerful technique to assess the impact of synaptic activity on neuronal protein trafficking and function. Current technology for neuronal stimulation in culture include chemical techniques or microelectrode or optogenetic based techniques. While technically powerful, chemical stimulation has limited spatial resolution and microelectrode and optogenetic techniques require specialized equipment and expertise. We report an optimized and improved technique for laser based photoconductive stimulation of live neurons using an inverted confocal microscope that overcomes these limitations. The advantages of this approach include its non-invasive nature and adaptability to temporal and spatial manipulation. We demonstrate that the technique can be manipulated to achieve spatially selective stimulation of live neurons. Coupled with live imaging of fluorescent indicators, this simple and efficient technique should allow for significant advances in neuronal cell biology.

Mef2 promotes spine elimination in absence of δ-catenin.

δ-Catenin is a component of the cadherin-catenin cell adhesion complex and its loss has been implicated in the mental retardation associated with the Cri du chat syndrome. We have previously demonstrated that loss of δ-catenin in a murine model during development results in excessive spine and synaptic density and function. In order to examine the role of potential molecules that might cooperate with δ-catenin to regulate spine density, we focused on Mef2. Our data demonstrate that while loss of δ-catenin does not alter the expression levels of endogenous Mef2, expression of Mef2 in neurons that are knocked down for δ-catenin promotes spine elimination. These results establish a molecular mechanism by which excessive spines in the absence of δ-catenin may be eliminated and may point toward pharmacological therapy for the Cri du chat syndrome.

Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex.

The ability to culture and maintain postnatal mouse hippocampal and cortical neurons is highly advantageous, particularly for studies on genetically engineered mouse models. Here we present a protocol to isolate and culture pyramidal neurons from the early postnatal (P0-P1) mouse hippocampus and cortex. These low-density dissociated cultures are grown on poly-L-lysine-coated glass substrates without feeder layers. Cultured neurons survive well, develop extensive axonal and dendritic arbors, express neuronal and synaptic markers, and form functional synaptic connections. Further, they are highly amenable to low- and high-efficiency transfection and time-lapse imaging. This optimized cell culture technique can be used to culture and maintain neurons for a variety of applications including immunocytochemistry, biochemical studies, shRNA-mediated knockdown and live imaging studies. The preparation of the glass substrate must begin 5 d before the culture. The dissection and plating out of neurons takes 3-4 h and neurons can be maintained in culture for up to 4 weeks.

Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology.

Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.