Acute and persistent responses after H5N1 vaccination in humans.

To gain insight into how an adjuvant impacts vaccination responses, we use systems immunology to study human H5N1 influenza vaccination with or without the adjuvant AS03, longitudinally assessing 14 time points including multiple time points within the first day after prime and boost. We develop an unsupervised computational framework to discover high-dimensional response patterns, which uncover adjuvant- and immunogenicity-associated early response dynamics, including some that differ post prime versus boost. With or without adjuvant, some vaccine-induced transcriptional patterns persist to at least 100 days after initial vaccination. Single-cell profiling of surface proteins, transcriptomes, and chromatin accessibility implicates transcription factors in the erythroblast-transformation-specific (ETS) family as shaping these long-lasting signatures, primarily in classical monocytes but also in CD8+ naive-like T cells. These cell-type-specific signatures are elevated at baseline in high-antibody responders in an independent vaccination cohort, suggesting that antigen-agnostic baseline immune states can be modulated by vaccine antigens alone to enhance future responses.

Adaptive immune responses to SARS-CoV-2 persist in the pharyngeal lymphoid tissue of children.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection.

Robust, persistent adaptive immune responses to SARS-CoV-2 in the oropharyngeal lymphoid tissue of children.

SARS-CoV-2 infection triggers adaptive immune responses from both T and B cells. However, most studies focus on peripheral blood, which may not fully reflect immune responses in lymphoid tissues at the site of infection. To evaluate both local and systemic adaptive immune responses to SARS-CoV-2, we collected peripheral blood, tonsils, and adenoids from 110 children undergoing tonsillectomy/adenoidectomy during the COVID-19 pandemic and found 24 with evidence of prior SARS-CoV-2 infection, including detectable neutralizing antibodies against multiple viral variants. We identified SARS-CoV-2-specific germinal center (GC) and memory B cells; single cell BCR sequencing showed that these virus-specific B cells were class-switched and somatically hypermutated, with overlapping clones in the adenoids and tonsils. Oropharyngeal tissues from COVID-19-convalescent children showed persistent expansion of GC and anti-viral lymphocyte populations associated with an IFN-γ-type response, with particularly prominent changes in the adenoids, as well as evidence of persistent viral RNA in both tonsil and adenoid tissues of many participants. Our results show robust, tissue-specific adaptive immune responses to SARS-CoV-2 in the upper respiratory tract of children weeks to months after acute infection, providing evidence of persistent localized immunity to this respiratory virus.

Cancer prognosis with shallow tumor RNA sequencing.

Disrupted molecular pathways are often robustly associated with disease outcome in cancer1-3. Although biologically informative transcriptional pathways can be revealed by RNA sequencing (RNA-seq) at up to hundreds of folds reduction in conventionally used coverage4-6, it remains unknown how low-depth sequencing datasets perform in the challenging context of developing transcriptional signatures to predict clinical outcomes. Here we assessed the possibility of cancer prognosis with shallow tumor RNA-seq, which would potentially enable cost-effective assessment of much larger numbers of samples for deeper biological and predictive insights. By statistically modeling the relative risk of an adverse outcome for thousands of subjects in The Cancer Genome Atlas7-13, we present evidence that subsampled tumor RNA-seq data with a few hundred thousand reads per sample provide sufficient information for outcome prediction in several types of cancer. Analysis of predictive models revealed robust contributions from pathways known to be associated with outcomes. Our findings indicate that predictive models of outcomes in cancer may be developed with dramatically increases in sample numbers at low cost, thus potentially enabling the development of more realistic predictive models that incorporate diverse variables and their interactions. This strategy could also be used, for example, in longitudinal analysis of multiple regions of a tumor alongside treatment for quantitative modeling and prediction of outcome in personalized oncology.

The Chemoattractant Receptor Ebi2 Drives Intranodal Naive CD4+ T Cell Peripheralization to Promote Effective Adaptive Immunity.

Lymph nodes (LNs) play critical roles in adaptive immunity by concentrating in one location the antigens, antigen-presenting cells, and antigen-responsive lymphocytes involved in such responses. Recent studies have revealed nonrandom localization of innate and adaptive immune cells within these organs, suggesting that microanatomical positioning optimizes responses involving sparse cooperating cells. Here, we report that the peripheral localization of LN cDC2 dendritic cells specialized for MHC-II antigen presentation is matched by a similarly biased paracortical distribution of CD4+ T cells directed by the chemoattractant receptor Ebi2. In the absence of Ebi2, CD4+ T cells lose their location bias and are delayed in antigen recognition, proliferative expansion, differentiation, direct effector activity, and provision of help for CD8+ T cell-mediated memory responses, limiting host defense and vaccine responses. These findings demonstrate evolutionary selection for distinct niches within the LN that promote cellular responses, emphasizing the critical link between fine-grained tissue organization and host defense.

Foxp3+ regulatory T-cell homeostasis quantitatively differs in murine peripheral lymph nodes and spleen.

Regulatory T (Treg) cells are essential for maintaining self-tolerance and modulating inflammatory immune responses. Treg cells either develop within the thymus or are converted from CD4(+) naive T (Tnaive) cells in the periphery. The Treg-cell population size is tightly controlled and Treg-cell development and homeostasis have been intensively studied; however, quantitative information about mechanisms of peripheral Treg-cell homeostasis is lacking. Here we developed the first mathematical model of peripheral Treg-cell homeostasis, incorporating secondary lymphoid organs as separate entities and encompassing factors determining the size of the Treg-cell population, namely thymic output, homeostatic proliferation, peripheral conversion, transorgan migration, apoptosis, and the Tnaive-cell population. Quantitative data were collected by monitoring Tnaive-cell homeostasis and Treg-cell rebound after selective in vivo depletion of Treg cells. Our model predicted the previously unanticipated possibility that Treg cells regulate migration of Tnaive cells between spleen and peripheral lymph nodes (LNs), whereas migration of Treg cells between these organs can largely be neglected. Furthermore, our simulations suggested that peripheral conversion significantly contributed to the maintenance of the Treg-cell population, especially in LNs. Hence, we provide the first estimation of the peripheral Treg-cell conversion rate and propose additional facets of Treg-cell-mediated immune regulation that may previously have escaped attention.

Advantages of Foxp3(+) regulatory T cell depletion using DEREG mice.

Several mechanisms enable immunological self-tolerance. Regulatory T cells (Tregs) are a specialized T cell subset that prevents autoimmunity and excessive immune responses, but can also mediate detrimental tolerance to tumors and pathogens in a Foxp3-dependent manner. Genetic tools exploiting the foxp3 locus including bacterial artificial chromosome (BAC)-transgenic DEREG mice have provided essential information on Treg biology and the potential therapeutic modulation of tolerance. In DEREG mice, Foxp3(+) Tregs selectively express eGFP and diphtheria toxin (DT) receptor, allowing for the specific depletion of Tregs through DT administration. We here provide a detailed overview about important considerations such as DT toxicity, which affects any mouse strain treated with DT, and Treg rebound after depletion. Additionally, we point out the specific advantages of BAC-transgenic DEREG mice including their suitability to study organ-specific autoimmunity such as type I diabetes. Moreover, we discuss recent insights into the role of Tregs in viral infections. In summary, DEREG mice are an important tool to study Treg-mediated tolerance and its therapeutic circumvention.

Differential control of immune cell homeostasis by Foxp3(+) regulatory T cells in murine peripheral lymph nodes and spleen.

Foxp3(+) regulatory T cells (Tregs) hamper efficient immune responses to tumors and chronic infections. Therefore, depletion of Foxp3(+) Tregs has been proposed as therapeutic option to boost immune responses and to improve vaccinations. Although Treg-mediated control of T cell homeostasis is well established, Foxp3(+) Treg interaction with other immune cell subsets is only incompletely understood. Thus, the present study aimed at examining dynamic effects of experimental Foxp3(+) Treg depletion on a broad range of immune cell subsets, including B cells, natural killer cells, and myeloid cells. Striking differences were observed when peripheral lymph nodes (LN) and spleen were compared. B cells, for example, showed a massive and long-lasting accumulation only in LN but not in spleen of transiently Treg-depleted mice. In contrast, monocyte-derived dendritic cells, which are potent inducers of T cell responses, also accumulated selectively, but only transiently in LN, suggesting that this cell population is under very strict control of Foxp3(+) Tregs. In summary, the observations described here provide insights into the dynamics of immune cells after selective depletion of Foxp3(+) Tregs. This will allow a better prediction of the impact of Treg ablation in translational studies that aim at boosting immune responses and vaccinations.

A mathematical model of immune activation with a unified self-nonself concept.

The adaptive immune system reacts against pathogenic nonself, whereas it normally remains tolerant to self. The initiation of an immune response requires a critical antigen(Ag)-stimulation and a critical number of Ag-specific T cells. Autoreactive T cells are not completely deleted by thymic selection and partially present in the periphery of healthy individuals that respond in certain physiological conditions. A number of experimental and theoretical models are based on the concept that structural differences discriminate self from nonself. In this article, we establish a mathematical model for immune activation in which self and nonself are not distinguished. The model considers the dynamic interplay of conventional T cells, regulatory T cells (Tregs), and IL-2 molecules and shows that the renewal rate ratio of resting Tregs to naïve T cells as well as the proliferation rate of activated T cells determine the probability of immune stimulation. The actual initiation of an immune response, however, relies on the absolute renewal rate of naïve T cells. This result suggests that thymic selection reduces the probability of autoimmunity by increasing the Ag-stimulation threshold of self reaction which is established by selection of a low number of low-avidity autoreactive T cells balanced with a proper number of Tregs. The stability analysis of the ordinary differential equation model reveals three different possible immune reactions depending on critical levels of Ag-stimulation: a subcritical stimulation, a threshold stimulation inducing a proper immune response, and an overcritical stimulation leading to chronic co-existence of Ag and immune activity. The model exhibits oscillatory solutions in the case of persistent but moderate Ag-stimulation, while the system returns to the homeostatic state upon Ag clearance. In this unifying concept, self and nonself appear as a result of shifted Ag-stimulation thresholds which delineate these three regimes of immune activation.