Deep mutational scanning identifies SARS-CoV-2 Nucleocapsid escape mutations of currently available rapid antigen tests.

The effects of mutations in continuously emerging variants of SARS-CoV-2 are a major concern for the performance of rapid antigen tests. To evaluate the impact of mutations on 17 antibodies used in 11 commercially available antigen tests with emergency use authorization, we measured antibody binding for all possible Nucleocapsid point mutations using a mammalian surface-display platform and deep mutational scanning. The results provide a complete map of the antibodies' epitopes and their susceptibility to mutational escape. Our data predict no vulnerabilities for detection of mutations found in variants of concern. We confirm this using the commercial tests and sequence-confirmed COVID-19 patient samples. The antibody escape mutational profiles generated here serve as a valuable resource for predicting the performance of rapid antigen tests against past, current, as well as any possible future variants of SARS-CoV-2, establishing the direct clinical and public health utility of our system.

Proliferating Transitory T Cells with an Effector-like Transcriptional Signature Emerge from PD-1+ Stem-like CD8+ T Cells during Chronic Infection.

T cell dysfunction is a characteristic feature of chronic viral infection and cancer. Recent studies in chronic lymphocytic choriomeningitis virus (LCMV) infection have defined a PD-1+ Tcf-1+ CD8+ T cell subset capable of self-renewal and differentiation into more terminally differentiated cells that downregulate Tcf-1 and express additional inhibitory molecules such as Tim3. Here, we demonstrated that expression of the glycoprotein CD101 divides this terminally differentiated population into two subsets. Stem-like Tcf-1+ CD8+ T cells initially differentiated into a transitory population of CD101-Tim3+ cells that later converted into CD101+ Tim3+ cells. Recently generated CD101-Tim3+ cells proliferated in vivo, contributed to viral control, and were marked by an effector-like transcriptional signature including expression of the chemokine receptor CX3CR1, pro-inflammatory cytokines, and granzyme B. PD-1 pathway blockade increased the numbers of CD101-Tim3+ CD8+ T cells, suggesting that these newly generated transitional cells play a critical role in PD-1-based immunotherapy.

PD-1 combination therapy with IL-2 modifies CD8+ T cell exhaustion program.

Combination therapy with PD-1 blockade and IL-2 is highly effective during chronic lymphocytic choriomeningitis virus infection1. Here we examine the underlying basis for this synergy. We show that PD-1 + IL-2 combination therapy, in contrast to PD-1 monotherapy, substantially changes the differentiation program of the PD-1+TCF1+ stem-like CD8+ T cells and results in the generation of transcriptionally and epigenetically distinct effector CD8+ T cells that resemble highly functional effector CD8+ T cells seen after an acute viral infection. The generation of these qualitatively superior CD8+ T cells that mediate viral control underlies the synergy between PD-1 and IL-2. Our results show that the PD-1+TCF1+ stem-like CD8+ T cells, also referred to as precursors of exhausted CD8+ T cells, are not fate-locked into the exhaustion program and their differentiation trajectory can be changed by IL-2 signals. These virus-specific effector CD8+ T cells emerging from the stem-like CD8+ T cells after combination therapy expressed increased levels of the high-affinity IL-2 trimeric (CD25-CD122-CD132) receptor. This was not seen after PD-1 blockade alone. Finally, we show that CD25 engagement with IL-2 has an important role in the observed synergy between IL-2 cytokine and PD-1 blockade. Either blocking CD25 with an antibody or using a mutated version of IL-2 that does not bind to CD25 but still binds to CD122 and CD132 almost completely abrogated the synergistic effects observed after PD-1 + IL-2 combination therapy. There is considerable interest in PD-1 + IL-2 combination therapy for patients with cancer2,3, and our fundamental studies defining the underlying mechanisms of how IL-2 synergizes with PD-1 blockade should inform these human translational studies.

Defining HPV-specific B cell responses in patients with head and neck cancer.

Tumours often contain B cells and plasma cells but the antigen specificity of these intratumoral B cells is not well understood1-8. Here we show that human papillomavirus (HPV)-specific B cell responses are detectable in samples from patients with HPV-positive head and neck cancers, with active production of HPV-specific IgG antibodies in situ. HPV-specific antibody secreting cells (ASCs) were present in the tumour microenvironment, with minimal bystander recruitment of influenza-specific cells, suggesting a localized and antigen-specific ASC response. HPV-specific ASC responses correlated with titres of plasma IgG and were directed against the HPV proteins E2, E6 and E7, with the most dominant response against E2. Using intratumoral B cells and plasma cells, we generated several HPV-specific human monoclonal antibodies, which exhibited a high degree of somatic hypermutation, consistent with chronic antigen exposure. Single-cell RNA sequencing analyses detected activated B cells, germinal centre B cells and ASCs within the tumour microenvironment. Compared with the tumour parenchyma, B cells and ASCs were preferentially localized in the tumour stroma, with well-formed clusters of activated B cells indicating ongoing germinal centre reactions. Overall, we show that antigen-specific activated and germinal centre B cells as well as plasma cells can be found in the tumour microenvironment. Our findings provide a better understanding of humoral immune responses in human cancer and suggest that tumour-infiltrating B cells could be harnessed for the development of therapeutic agents.

The Magnitude of IFN-γ Responses Is Fine-Tuned by DNA Architecture and the Non-coding Transcript of Ifng-as1.

Interferon gamma (IFN-γ), critical for host defense and tumor surveillance, requires tight control of its expression. Multiple cis-regulatory elements exist around Ifng along with a non-coding transcript, Ifng-as1 (also termed NeST). Here, we describe two genetic models generated to dissect the molecular functions of this locus and its RNA product. DNA deletion within the Ifng-as1 locus disrupted chromatin organization of the extended Ifng locus, impaired Ifng response, and compromised host defense. Insertion of a polyA signal ablated the Ifng-as1 full-length transcript and impaired host defense, while allowing proper chromatin structure. Transient knockdown of Ifng-as1 also reduced IFN-γ production. In humans, discordant expression of IFNG and IFNG-AS1 was evident in memory T cells, with high expression of this long non-coding RNA (lncRNA) and low expression of the cytokine. These results establish Ifng-as1 as an important regulator of Ifng expression, as a DNA element and transcribed RNA, involved in dynamic and cell state-specific responses to infection.

The structure, function and evolution of proteins that bind DNA and RNA.

Proteins that bind both DNA and RNA typify the ability of a single gene product to perform multiple functions. Such DNA- and RNA-binding proteins (DRBPs) have unique functional characteristics that stem from their specific structural features; these developed early in evolution and are widely conserved. Proteins that bind RNA have typically been considered as functionally distinct from proteins that bind DNA and studied independently. This practice is becoming outdated, in partly owing to the discovery of long non-coding RNAs (lncRNAs) that target DNA-binding proteins. Consequently, DRBPs were found to regulate many cellular processes, including transcription, translation, gene silencing, microRNA biogenesis and telomere maintenance.

An intra-tumoral niche maintains and differentiates stem-like CD8 T cells.

Tumour-infiltrating lymphocytes are associated with a survival benefit in several tumour types and with the response to immunotherapy1-8. However, the reason some tumours have high CD8 T cell infiltration while others do not remains unclear. Here we investigate the requirements for maintaining a CD8 T cell response against human cancer. We find that CD8 T cells within tumours consist of distinct populations of terminally differentiated and stem-like cells. On proliferation, stem-like CD8 T cells give rise to more terminally differentiated, effector-molecule-expressing daughter cells. For many T cells to infiltrate the tumour, it is critical that this effector differentiation process occur. In addition, we show that these stem-like T cells reside in dense antigen-presenting-cell niches within the tumour, and that tumours that fail to form these structures are not extensively infiltrated by T cells. Patients with progressive disease lack these immune niches, suggesting that niche breakdown may be a key mechanism of immune escape.

Longitudinal Analysis of the Phenotype, Transcriptional Profile, and Anatomic Location of Memory CD8 T Cell Subsets after Acute Viral Infection.

Increased demand for novel, highly effective vaccination strategies necessitates a better understanding of long-lived memory CD8 T cell differentiation. To achieve this understanding, we used the mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. We reexamined classical memory CD8 T cell subsets and performed in-depth, longitudinal analysis of their phenotype, transcriptional programming, and anatomic location within the spleen. All analyses were performed at multiple time points from 8 days to 1 year postinfection. Memory subsets are conventionally defined by their expression of KLRG1 and IL-7Rα, as follows: KLRG1+IL-7Rα- terminal effectors (TEs) and KLRG1-IL-7Rα+ memory precursors (MPs). But we also characterized a third KLRG1+IL-7Rα+ subset which we refer to as KLRG1+ MPs. In these analyses, we defined a comprehensive memory phenotype that is associated with higher levels of CD28 expression. We also demonstrated that MPs, KLRG1+ MPs, and TEs have distinct localization programs within the spleen. We found that MPs became preferentially enriched in the white pulp as early as 1 to 2 weeks postinfection, and their predominance in the white pulp was maintained throughout the course of a year. On the other hand, KLRG1+ MPs and TEs localized to the red pulp just as early, and they consistently localized to the red pulp thereafter. These findings indicate that location may be crucial for memory formation and that white pulp-derived signals may contribute to long-term memory survival. Achieving robust memory responses following vaccination may require more deliberate consideration of which memory phenotypes are induced, as well as where they traffic, as these factors could impact their longevity. IMPORTANCE CD8 T cells play a critical role in viral immunity and it is important to understand how memory cells are formed and what processes lead to their long-term maintenance. Here, we use a mouse model of acute infection to perform an in-depth, longitudinal analysis of memory CD8 T cell differentiation, examining the phenotype and location of memory cells out to 1 year postinfection.

PD-1+ stemlike CD8 T cells are resident in lymphoid tissues during persistent LCMV infection.

The migratory patterns of virus-specific CD8 T cells during chronic viral infection are not well understood. To address this issue, we have done parabiosis experiments during chronic lymphocytic choriomeningitis virus (LCMV) infection of mice. We found that despite the high frequency of virus-specific CD8 T cells in both lymphoid and nonlymphoid tissues there was minimal migration of virus-specific CD8 T cells between the chronically infected conjoined parabiont mice. This was in contrast to parabionts between mice that had undergone an acute LCMV infection where virus-specific CD8 T cells established equilibrium demonstrating circulation of memory T cells generated after viral clearance. We have identified a population of PD-1+ TCF1+CXCR5+Tim-3- stemlike virus-specific CD8 T cells that reside in lymphoid tissues and act as resource cells for maintaining the T cell response during chronic infection. These are the cells that proliferate and give rise to the more terminally differentiated PD-1+ CXCR5-Tim-3+ CD8 T cells. Both the stemlike CD8 T cells and their terminally differentiated progeny showed minimal migration during chronic infection and the few LCMV-specific CD8 T cells that were present in circulation were the recently emerging progeny from the stemlike CD8 T cells. The PD-1+ TCF1+CXCR5+ stemlike CD8 T cells were truly resident in lymphoid tissues and did not circulate in the blood. We propose that this residency in specialized niches within lymphoid tissues is a key aspect of their biology and is essential for maintaining their quiescence and stemlike program under conditions of a chronic viral infection.

Distinct Gene Expression Profiles Define Anaplastic Grade in Retinoblastoma.

Morbidity and mortality associated with retinoblastoma have decreased drastically in recent decades, in large part owing to better prediction of high-risk disease and appropriate treatment stratification. High-risk histopathologic features and severe anaplasia both predict the need for more aggressive treatment; however, not all centers are able to assess tumor samples easily for the degree of anaplasia. Instead, identification of genetic signatures that are able to distinguish among anaplastic grades and thus predict high- versus low-risk retinoblastoma would facilitate appropriate risk stratification in a wider patient population. A better understanding of genes dysregulated in anaplasia also would yield valuable insights into pathways underlying the development of more severe retinoblastoma. Here, we present the histopathologic and gene expression analysis of 28 retinoblastoma cases using microarray analysis. Tumors of differing anaplastic grade show clear differential gene expression, with significant dysregulation of unique genes and pathways in severe anaplasia. Photoreceptor and nucleoporin expression in particular are identified as highly dysregulated in severe anaplasia and suggest particular cellular processes contributing to the development of increased retinoblastoma severity. A limited set of highly differentially expressed genes also are able to predict severe anaplasia accurately in our data set. Together, these data contribute to the understanding of the development of anaplasia and facilitate the identification of genetic markers of high-risk retinoblastoma.

Tethering not required: the glucocorticoid receptor binds directly to activator protein-1 recognition motifs to repress inflammatory genes.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that controls the expression of extensive gene networks, driving both up- and down-regulation. GR utilizes multiple DNA-binding-dependent and -independent mechanisms to achieve context-specific transcriptional outcomes. The DNA-binding-independent mechanism involves tethering of GR to the pro-inflammatory transcription factor activator protein-1 (AP-1) through protein-protein interactions. This mechanism has served as the predominant model of GR-mediated transrepression of inflammatory genes. However, ChIP-seq data have consistently shown GR to occupy AP-1 response elements (TREs), even in the absence of AP-1. Therefore, the current model is insufficient to explain GR action at these sites. Here, we show that GR regulates a subset of inflammatory genes in a DNA-binding-dependent manner. Using structural biology and biochemical approaches, we show that GR binds directly to TREs via sequence-specific contacts to a GR-binding sequence (GBS) half-site found embedded within the TRE motif. Furthermore, we show that GR-mediated transrepression observed at TRE sites to be DNA-binding-dependent. This represents a paradigm shift in the field, showing that GR uses multiple mechanisms to suppress inflammatory gene expression. This work further expands our understanding of this complex multifaceted transcription factor.

Human antibody responses after dengue virus infection are highly cross-reactive to Zika virus.

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus of significant public health concern. ZIKV shares a high degree of sequence and structural homology compared with other flaviviruses, including dengue virus (DENV), resulting in immunological cross-reactivity. Improving our current understanding of the extent and characteristics of this immunological cross-reactivity is important, as ZIKV is presently circulating in areas that are highly endemic for dengue. To assess the magnitude and functional quality of cross-reactive immune responses between these closely related viruses, we tested acute and convalescent sera from nine Thai patients with PCR-confirmed DENV infection against ZIKV. All of the sera tested were cross-reactive with ZIKV, both in binding and in neutralization. To deconstruct the observed serum cross-reactivity in depth, we also characterized a panel of DENV-specific plasmablast-derived monoclonal antibodies (mAbs) for activity against ZIKV. Nearly half of the 47 DENV-reactive mAbs studied bound to both whole ZIKV virion and ZIKV lysate, of which a subset also neutralized ZIKV. In addition, both sera and mAbs from the dengue-infected patients enhanced ZIKV infection of Fc gamma receptor (FcγR)-bearing cells in vitro. Taken together, these findings suggest that preexisting immunity to DENV may impact protective immune responses against ZIKV. In addition, the extensive cross-reactivity may have implications for ZIKV virulence and disease severity in DENV-experienced populations.

Distal substitutions drive divergent DNA specificity among paralogous transcription factors through subdivision of conformational space.

Many genomes contain families of paralogs--proteins with divergent function that evolved from a common ancestral gene after a duplication event. To understand how paralogous transcription factors evolve divergent DNA specificities, we examined how the glucocorticoid receptor and its paralogs evolved to bind activating response elements [(+)GREs] and negative glucocorticoid response elements (nGREs). We show that binding to nGREs is a property of the glucocorticoid receptor (GR) DNA-binding domain (DBD) not shared by other members of the steroid receptor family. Using phylogenetic, structural, biochemical, and molecular dynamics techniques, we show that the ancestral DBD from which GR and its paralogs evolved was capable of binding both nGRE and (+)GRE sequences because of the ancestral DBD's ability to assume multiple DNA-bound conformations. Subsequent amino acid substitutions in duplicated daughter genes selectively restricted protein conformational space, causing this dual DNA-binding specificity to be selectively enhanced in the GR lineage and lost in all others. Key substitutions that determined the receptors' response element-binding specificity were far from the proteins' DNA-binding interface and interacted epistatically to change the DBD's function through DNA-induced allosteric mechanisms. These amino acid substitutions subdivided both the conformational and functional space of the ancestral DBD among the present-day receptors, allowing a paralogous family of transcription factors to control disparate transcriptional programs despite high sequence identity.

A Direct Comparison of in Vitro and in Vivo Nucleic Acid Delivery Mediated by Hundreds of Nanoparticles Reveals a Weak Correlation.

Endothelial cells and macrophages play active roles in disease and as a result are important targets for nucleic acid therapies. While thousands of chemically distinct lipid nanoparticles (LNPs) can be synthesized to deliver nucleic acids, studying more than a few LNPs in vivo is challenging. As a result, it is difficult to understand how nanoparticles target these cells in vivo. Using high throughput LNP barcoding, we quantified how well LNPs delivered DNA barcodes to endothelial cells and macrophages in vitro, as well as endothelial cells and macrophages isolated from the lung, heart, and bone marrow in vivo. We focused on two fundamental questions in drug delivery. First, does in vitro LNP delivery predict in vivo LNP delivery? By comparing how 281 LNPs delivered barcodes to endothelial cells and macrophages in vitro and in vivo, we found in vitro delivery did not predict in vivo delivery. Second, does LNP delivery change within the microenvironment of a tissue? We quantified how 85 LNPs delivered barcodes to eight splenic cell populations, and found that cell types derived from myeloid progenitors tended to be targeted by similar LNPs, relative to cell types derived from lymphoid progenitors. These data demonstrate that barcoded LNPs can elucidate fundamental questions about in vivo nanoparticle delivery.

Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes.

The evolution of behavior relies on changes at the level of the genome; yet the ability to attribute a behavioral change to a specific, naturally occurring genetic change is rare in vertebrates. In the white-throated sparrow (Zonotrichia albicollis), a chromosomal polymorphism (ZAL2/2(m)) is known to segregate with a behavioral phenotype. Individuals with the ZAL2(m) haplotype engage in more territorial aggression and less parental behavior than individuals without it. These behaviors are thought to be mediated by sensitivity to sex steroids, and the chromosomal rearrangement underlying the polymorphism has captured a prime candidate gene: estrogen receptor 1 (ESR1), which encodes estrogen receptor α (ERα). We therefore hypothesized that the behavioral effects of the ZAL2(m) rearrangement are mediated by polymorphism in ESR1. We report here that (i) the ESR1 promoter region contains fixed polymorphisms distinguishing the ZAL2(m) and ZAL2 alleles; (ii); those polymorphisms regulate transcription efficiency in vitro and therefore potentially do the same in vivo (iii); the local expression of ERα in the brain depends strongly on genotype in a free-living population; and (iv) ERα expression in the medial amygdala and medial preoptic area may fully mediate the effects of genotype on territorial aggression and parenting, respectively. Thus, our study provides a rare glimpse of how a chromosomal polymorphism has affected the brain and social behavior in a vertebrate. Our results suggest that in this species, differentiation of ESR1 has played a causal role in the evolution of phenotypes with alternative life-history strategies.

Technology meets TILs: Deciphering T cell function in the -omics era.

T cells are at the center of cancer immunology because of their ability to recognize mutations in tumor cells and directly mediate cancer cell killing. Immunotherapies to rejuvenate exhausted T cell responses have transformed the clinical management of several malignancies. In parallel, the development of novel multidimensional analysis platforms, such as single-cell RNA sequencing and high-dimensional flow cytometry, has yielded unprecedented insights into immune cell biology. This convergence has revealed substantial heterogeneity of tumor-infiltrating immune cells in single tumors, across tumor types, and among individuals with cancer. Here we discuss the opportunities and challenges of studying the complex tumor microenvironment with -omics technologies that generate vast amounts of data, highlighting the opportunities and limitations of these technologies with a particular focus on interpreting high-dimensional studies of CD8+ T cells in the tumor microenvironment.

Immune microenvironment remodeling after radiation of a progressing brain metastasis.

Radiation is commonly used in the treatment of many cancers. However, its effects on anti-tumor immune responses are incompletely understood. Here, we present a detailed immunological analysis of two tumors from a patient with multiple non-small cell lung cancer metastases to the brain. One tumor was resected without treatment; the second was irradiated to a total dose of 30 Gy and resected following further progression. Comprehensive single-cell analysis reveals a substantially reduced immune cell fraction in the irradiated tumor, including the depletion of tissue-resident macrophages and infiltration of pro-inflammatory monocytes. Despite the presence of similar somatic mutations in both tumors, radiation is associated with the depletion of exhausted, tumor-resident T cell clones and their replacement by circulating clones unlikely to contribute to tumor-specific immunity. These results provide insight into the local effects of radiation on anti-tumor immunity and raise important considerations for the combination of radiation and immunotherapy.

PD-1 blockade increases the self-renewal of stem-like CD8 T cells to compensate for their accelerated differentiation into effectors.

PD-1+TCF-1+ stem-like CD8 T cells act as critical resource cells for maintaining T cell immunity in chronic viral infections and cancer. In addition, they provide the proliferative burst of effector CD8 T cells after programmed death protein 1 (PD-1)-directed immunotherapy. However, it is not known whether checkpoint blockade diminishes the number of these stem-like progenitor cells as effector cell differentiation increases. To investigate this, we used the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. Treatment of chronically infected mice with either αPD-1 or αPD-L1 antibody not only increased effector cell differentiation from the virus-specific stem-like CD8 T cells but also increased their proliferation so their numbers were maintained. The increased self-renewal of LCMV-specific stem-like CD8 T cells was mTOR dependent. We used microscopy to understand the division of these progenitor cells and found that after PD-1 blockade, an individual dividing cell could give rise to a differentiated TCF-1- daughter cell alongside a self-renewing TCF-1+ sister cell. This asymmetric division helped to preserve the number of stem-like cells. Moreover, we found that the PD-1+TCF-1+ stem-like CD8 T cells retained their transcriptional program and their in vivo functionality in terms of responding to viral infection and to repeat PD-1 blockade. Together, our results demonstrate that PD-1 blockade does not deplete the stem-like population despite increasing effector differentiation. These findings have implications for PD-1-directed immunotherapy in humans.

mTOR regulates T cell exhaustion and PD-1-targeted immunotherapy response during chronic viral infection.

T cell exhaustion is a state of T cell dysfunction associated with expression of programmed death 1 (PD-1). Exhausted CD8+ T cells are maintained by self-renewing stem-like T cells that provide differentiated TIM3+ cells, a part of which possesses effector-like properties. PD-1-targeted therapies enhance T cell response by promoting differentiation of stem-like T cells toward TIM3+ cells, but the role of mTOR during T cell exhaustion remains elusive. Here, we showed that mTOR inhibition has distinct outcomes during the beginning of and after the establishment of chronic viral infection. Blocking mTOR during the T cell expansion phase enhanced the T cell response by causing accumulation of stem-like T cells, leading to improved efficacy of PD-1 immunotherapy; whereas, after exhaustion progressed, mTOR inhibition caused immunosuppression, characterized by decreased TIM3+ cells and increased viral load with minimal changes in stem-like T cells. Mechanistically, a cell-intrinsic mTOR signal was vital for differentiation of stem-like T cells into the TIM3+ state in the early and late phases of chronic infection as well as during PD-1 immunotherapy. Thus, PD-1 blockade worked after cessation of mTOR inhibition, but simultaneous treatment failed to induce functional TIM3+ cells, reducing efficacy of PD-1 immunotherapy. Our data demonstrate that mTOR regulates T cell exhaustion and have important implications for combination cancer therapies with PD-1 blockade.