30 Gbit/s PAM4 transmission using an 8-GHz directly modulated multi-section laser.

This paper describes the characterization of a novel directly modulated multi-section laser with a master-slave configuration. Amplitude and phase noise measurements show relative intensity noise values of around -150 dB/Hz and a 3-dB linewidth of around 3 MHz. The laser's suitability for optical access networks, enabled by the chirp reduction from the external injection locking, is shown by demonstrating unamplified 30 Gbit/s C-band transmission over 25 km and 50 km of single mode fiber using PAM4, as well as 30 Gbit/s PAM4 and PAM8 amplified transmission over 75 km.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost 1 million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. RESULTS: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io ( https://doi.org/10.17504/protocols.io.bvntn5en ). SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. The final version of Mini-XT has been used to sequence 4384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. CONCLUSIONS: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes eightfold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

Dietary Lactobacillus-Derived Exopolysaccharide Enhances Immune-Checkpoint Blockade Therapy.

Microbes and their byproducts have been reported to regulate host health and immune functions. Here we demonstrated that microbial exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (EPS-R1) induced CCR6+ CD8+ T cells of mice and humans. In mice, ingestion of EPS-R1 augmented antitumor effects of anti-CTLA-4 or anti-PD-1 monoclonal antibody against CCL20-expressing tumors, in which infiltrating CCR6+ CD8+ T cells were increased and produced IFNγ accompanied by a substantial immune response gene expression signature maintaining T-cell functions. Of note, the antitumor adjuvant effect of EPS-R1 was also observed in germ-free mice. Furthermore, the induction of CCR6 expression was mediated through the phosphorylated structure in EPS-R1 and a lysophosphatidic acid receptor on CD8+ T cells. Overall, we find that dietary EPS-R1 consumption induces CCR6+ CD8+ T cells in Peyer's patches, favoring a tumor microenvironment that augments the therapeutic effect of immune-checkpoint blockade depending on CCL20 production by tumors. SIGNIFICANCE: Gut microbiota- and probiotic-derived metabolites are attractive agents to augment the efficacy of immunotherapies. Here we demonstrated that dietary consumption of Lactobacillus-derived exopolysaccharide induced CCR6+ CD8+ T cells in Peyer's patches and improved the tumor microenvironment to augment the therapeutic effects of immune-checkpoint blockade against CCL20-producing tumors. See related commentary by Di Luccia and Colonna, p. 1189. This article is highlighted in the In This Issue feature, p. 1171.

NKG7 Is Required for Optimal Antitumor T-cell Immunity.

Tumor antigen-specific CD8+ T cells play a critical role in antitumor immunity. Clinical trials reinvigorating the immune system via immune checkpoint blockade (ICB) have shown remarkable clinical promise. Numerous studies have identified an association between NKG7 expression and patient outcome across different malignancies. However, aside from these correlative observations, very little is known about NKG7 and its role in antitumor immunity. Herein, we utilized single-cell RNA sequencing (scRNA-seq) datasets, NKG7-deficient mice, NKG7-reporter mice, and mouse tumor models to investigate the role of NKG7 in neoantigen-mediated tumor rejection and ICB immunotherapy. scRNA-seq of tumors from patients with metastatic melanoma or head and neck squamous cell carcinoma revealed that NKG7 expression is highly associated with cytotoxicity and specifically expressed by CD8+ T cells and natural killer (NK) cells. Furthermore, we identified a key role for NKG7 in controlling intratumor T-cell accumulation and activation. NKG7 was upregulated on intratumor antigen-specific CD8+ T cells and NK cells and required for the accumulation of T cells in the tumor microenvironment. Accordingly, neoantigen-expressing mouse tumors grew faster in Nkg7-deficient mice. Strikingly, efficacy of single or combination ICB was significantly reduced in Nkg7-deficient mice.See related article by Wen et al., p. 162.

Prolonged tedizolid use in cutaneous non-tuberculous mycobacterial infection.

Cutaneous non-tuberculous mycobacterial (NTM) infections are an emerging infectious disease and require a protracted course of combination antibiotics. Antimicrobial choice is limited by resistance and toxicity. Tedizolid is a new oxazolidinone antibiotic with in vitro activity against some NTM, but its use in the management of extrapulmonary NTM has not been described. We report on the utility of prolonged tedizolid use (8 months), after linezolid intolerance, in combination therapy in a case of difficult Mycobacterium chelonae cutaneous infection. Although tedizolid contributed to clinical remission, it may have also contributed to a multifocal peripheral neuropathy. Its efficacy may also have been limited by continued immunosuppression, with evidence to suggest disease relapse or treatment failure after completion of combination therapy. Tedizolid can be considered, with caution, in combination therapy for difficult cases of cutaneous NTM infection.

Characterization of CD8 T Cell-Mediated Mutations in the Immunodominant Epitope GP33-41 of Lymphocytic Choriomeningitis Virus.

Cytotoxic T lymphocytes (CTLs) represent key immune effectors of the host response against chronic viruses, due to their cytotoxic response to virus-infected cells. In response to this selection pressure, viruses may accumulate escape mutations that evade CTL-mediated control. To study the emergence of CTL escape mutations, we employed the murine chronic infection model of lymphocytic choriomeningitis virus (LCMV). We developed an amplicon-based next-generation sequencing pipeline to detect low frequency mutations in the viral genome and identified non-synonymous mutations in the immunodominant LCMV CTL epitope, GP33-41, in infected wildtype mice. Infected Rag2-deficient mice lacking CTLs did not contain such viral mutations. By using transgenic mice with T cell receptors specific to GP33-41, we characterized the emergence of viral mutations in this epitope under varying selection pressure. We investigated the two most abundant viral mutations by employing reverse genetically engineered viral mutants encoding the respective mutations. These experiments provided evidence that these mutations prevent activation and expansion of epitope-specific CD8 T cells. Our findings on the mutational dynamics of CTL escape mutations in a widely-studied viral infection model contributes to our understanding of how chronic viruses interact with their host and evade the immune response. This may guide the development of future treatments and vaccines against chronic infections.

ATP and cancer immunosurveillance.

While intracellular adenosine triphosphate (ATP) occupies a key position in the bioenergetic metabolism of all the cellular compartments that form the tumor microenvironment (TME), extracellular ATP operates as a potent signal transducer. The net effects of purinergic signaling on the biology of the TME depend not only on the specific receptors and cell types involved, but also on the activation status of cis- and trans-regulatory circuitries. As an additional layer of complexity, extracellular ATP is rapidly catabolized by ectonucleotidases, culminating in the accumulation of metabolites that mediate distinct biological effects. Here, we discuss the molecular and cellular mechanisms through which ATP and its degradation products influence cancer immunosurveillance, with a focus on therapeutically targetable circuitries.

BET inhibition blocks inflammation-induced cardiac dysfunction and SARS-CoV-2 infection.

Cardiac injury and dysfunction occur in COVID-19 patients and increase the risk of mortality. Causes are ill defined but could be through direct cardiac infection and/or inflammation-induced dysfunction. To identify mechanisms and cardio-protective drugs, we use a state-of-the-art pipeline combining human cardiac organoids with phosphoproteomics and single nuclei RNA sequencing. We identify an inflammatory "cytokine-storm", a cocktail of interferon gamma, interleukin 1ß, and poly(I:C), induced diastolic dysfunction. Bromodomain-containing protein 4 is activated along with a viral response that is consistent in both human cardiac organoids (hCOs) and hearts of SARS-CoV-2-infected K18-hACE2 mice. Bromodomain and extraterminal family inhibitors (BETi) recover dysfunction in hCOs and completely prevent cardiac dysfunction and death in a mouse cytokine-storm model. Additionally, BETi decreases transcription of genes in the viral response, decreases ACE2 expression, and reduces SARS-CoV-2 infection of cardiomyocytes. Together, BETi, including the Food and Drug Administration (FDA) breakthrough designated drug, apabetalone, are promising candidates to prevent COVID-19 mediated cardiac damage.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Innate myeloid cells in the tumor microenvironment.

Cancer immunotherapies are receiving increasing approval in the clinic, but still only a fraction of patients benefit long-term. Understanding the most important mechanisms of immunotherapeutic resistance is critical for broader utility and benefit of cancer immunotherapy. While the tumor microenvironment (TME) is made up of many cell types, immunosuppressive monocytes/macrophages, granulocytes and myeloid derived suppressor cells interact with, and play a critical role in regulating the anti-tumor lymphocyte effector cells that mediate effective immunotherapies. Herein, we discuss the latest research that has identified and compared the importance of pro-tumor and immunosuppressive mechanisms that tumor infiltrating myeloid cells employ. Exploiting this new information may help to develop totally novel therapies to boost contemporary cancer immunotherapy.

Systemic administration of IL-33 induces a population of circulating KLRG1hi type 2 innate lymphoid cells and inhibits type 1 innate immunity against multiple myeloma.

Type 2 innate lymphoid cells (ILC2s) are important producers of type 2 cytokines whose role in hematological cancers remains unclear. ILC2s are a heterogeneous population encompassing distinct subsets with different tissue localization and cytokine responsiveness. In this study, we investigated the role of bone marrow (BM) ILC2s and interleukin (IL)-33-stimulated ILC2s in multiple myeloma, a plasma cell malignancy that develops in the BM. We found that myeloma growth was associated with phenotypic and functional alterations of BM ILC2s, characterized by an increased expression of maturation markers and reduced cytokine response to IL-2/IL-33. We identified a population of KLRG1hi ILC2s that preferentially accumulated in the liver and spleen of Il2rg-/- Rag2-/- mice reconstituted with BM ILC2s. A similar population of KLRG1hi ILC2s was observed in the blood, liver and spleen of IL-33-treated wild-type mice. The presence of KLRG1hi ILC2s in ILC2-reconstituted Il2rg-/- Rag2-/- mice or in IL-33-treated wild-type mice was associated with increased eosinophil numbers but had no effect on myeloma progression. Interestingly, while decreased myeloma growth was observed following treatment of Rag-deficient mice with the type 1 cytokines IL-12 and IL-18, this protection was reversed when mice received a combined treatment of IL-33 together with IL-12 and IL-18. In summary, our data indicate that IL-33 treatment induces a population of circulating inflammatory KLRG1hi ILC2s and inhibits type 1 immunity against multiple myeloma. These results argue against therapeutic administration of IL-33 to myeloma patients.

Genomic epidemiology of superspreading events in Austria reveals mutational dynamics and transmission properties of SARS-CoV-2.

Superspreading events shaped the coronavirus disease 2019 (COVID-19) pandemic, and their rapid identification and containment are essential for disease control. Here, we provide a national-scale analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading during the first wave of infections in Austria, a country that played a major role in initial virus transmissions in Europe. Capitalizing on Austria's well-developed epidemiological surveillance system, we identified major SARS-CoV-2 clusters during the first wave of infections and performed deep whole-genome sequencing of more than 500 virus samples. Phylogenetic-epidemiological analysis enabled the reconstruction of superspreading events and charts a map of tourism-related viral spread originating from Austria in spring 2020. Moreover, we exploited epidemiologically well-defined clusters to quantify SARS-CoV-2 mutational dynamics, including the observation of low-frequency mutations that progressed to fixation within the infection chain. Time-resolved virus sequencing unveiled viral mutation dynamics within individuals with COVID-19, and epidemiologically validated infector-infectee pairs enabled us to determine an average transmission bottleneck size of 103 SARS-CoV-2 particles. In conclusion, this study illustrates the power of combining epidemiological analysis with deep viral genome sequencing to unravel the spread of SARS-CoV-2 and to gain fundamental insights into mutational dynamics and transmission properties.

Concomitant or delayed anti-TNF differentially impact on immune-related adverse events and antitumor efficacy after anti-CD40 therapy.

BACKGROUND: Concomitant tumor necrosis factor (TNF) neutralization in combination with immune checkpoint inhibitors (ICIs) reduces clinical immune-related adverse events (irAEs) and appears to improve antitumor efficacy in preclinical tumor models. Agonistic antibodies targeting costimulatory receptors such as CD40 represent an additional strategy to boost antitumor immune response and potentiate the activity of ICIs. However, the dose-limiting toxicities observed in anti-CD40-treated cancer patients have hindered its clinical development. METHODS: We previously described a mouse model to assess both antitumor activity and irAEs induced by various effective combination immunotherapies. Using the BALB/c and C57BL/6 strains of FoxP3-GFP-DTR (FoxP3DTR) mice, transient depletion of T regulatory cells (Tregs) prior to immunotherapy with additional immunomodulatory antibodies, lowered immune self-tolerance, resulting in the development of a spectrum of physical and biochemical irAEs similar to that reported clinically. In MC38 and 4T1.2 tumor models, following transient Treg depletion, we evaluated the impact of anti-CD40 on antitumor efficacy and the development of irAEs and the impact of concomitant or delayed TNF blockade on both these parameters. Physical irAEs were scored and biochemical irAEs were measured in the serum (ALT and cytokine levels). Histopathological liver and colon tissue analysis were performed to assess immune cell infiltration and tissue damage. RESULTS: Similar to early clinical trials of CD40 agonists, in our tumor models we observed liver toxicities and rapid release of proinflammatory cytokines (TNF, interleukin 6, interferon-γ). In the BALB/c strain, anti-CD40 induced severe physical and biochemical irAEs. Concomitant anti-TNF treatment abrogated weight loss, liver damage and colitis, which consequently resulted in an improved clinical score. However, concomitant anti-TNF impaired antitumor response in a proportion of anti-CD40-treated C57BL/6 FoxP3DTR mice. Delaying TNF blockade in these mice reduced biochemical but not physical irAEs while preserving antitumor efficacy. CONCLUSIONS: Our results suggest concomitant rather than delayed anti-TNF is most effective in reducing biochemical and physical irAEs induced by anti-CD40, although it had the potential to negatively impact antitumor efficacy. Furthermore, our findings highlight the utility of our mouse model to assess the severity of irAEs induced by novel immunotherapeutic agents and evaluate whether their toxicity and antitumor efficacy can be uncoupled.