Mucosal viral infection induces a regulatory T cell activation phenotype distinct from tissue residency in mouse and human tissues.

Regulatory T cells (Tregs) mediate immune homeostasis, yet also facilitate nuanced immune responses during infection, balancing pathogen control while limiting host inflammation. Recent studies have identified Treg populations in non-lymphoid tissues that are phenotypically distinct from Tregs in lymphoid tissues (LT), including performance of location-dependent roles. Mucosal tissues serve as critical barriers to microbes while performing unique physiologic functions, so we sought to identify distinct phenotypical and functional aspects of mucosal Tregs in the female reproductive tract. In healthy human and mouse vaginal mucosa, we found that Tregs are highly activated compared to blood or LT Tregs. To determine if this phenotype reflects acute activation or a general signature of vaginal tract (VT)-residency, we infected mice with HSV-2 to discover that VT Tregs express granzyme-B (GzmB) and acquire a VT Treg signature distinct from baseline. To determine the mechanisms that drive GzmB expression, we performed ex vivo assays to reveal that a combination of type-I interferons and interleukin-2 is sufficient for GzmB expression. Together, we highlight that VT Tregs are activated at steady state and become further activated in response to infection; thus, they may exert robust control of local immune responses, which could have implications for mucosal vaccine design.

Extricating human tumour immune alterations from tissue inflammation.

Immunotherapies have achieved remarkable successes in the treatment of cancer, but major challenges remain1,2. An inherent weakness of current treatment approaches is that therapeutically targeted pathways are not restricted to tumours, but are also found in other tissue microenvironments, complicating treatment3,4. Despite great efforts to define inflammatory processes in the tumour microenvironment, the understanding of tumour-unique immune alterations is limited by a knowledge gap regarding the immune cell populations in inflamed human tissues. Here, in an effort to identify such tumour-enriched immune alterations, we used complementary single-cell analysis approaches to interrogate the immune infiltrate in human head and neck squamous cell carcinomas and site-matched non-malignant, inflamed tissues. Our analysis revealed a large overlap in the composition and phenotype of immune cells in tumour and inflamed tissues. Computational analysis identified tumour-enriched immune cell interactions, one of which yields a large population of regulatory T (Treg) cells that is highly enriched in the tumour and uniquely identified among all haematopoietically-derived cells in blood and tissue by co-expression of ICOS and IL-1 receptor type 1 (IL1R1). We provide evidence that these intratumoural IL1R1+ Treg cells had responded to antigen recently and demonstrate that they are clonally expanded with superior suppressive function compared with IL1R1- Treg cells. In addition to identifying extensive immunological congruence between inflamed tissues and tumours as well as tumour-specific changes with direct disease relevance, our work also provides a blueprint for extricating disease-specific changes from general inflammation-associated patterns.

Convergent clonal selection of donor- and recipient-derived CMV-specific T cells in hematopoietic stem cell transplant patients.

Competition between antigen-specific T cells for peptide:MHC complexes shapes the ensuing T cell response. Mouse model studies provided compelling evidence that competition is a highly effective mechanism controlling the activation of naïve T cells. However, assessing the effect of T cell competition in the context of a human infection requires defined pathogen kinetics and trackable naïve and memory T cell populations of defined specificity. A unique cohort of nonmyeloablative hematopoietic stem cell transplant patients allowed us to assess T cell competition in response to cytomegalovirus (CMV) reactivation, which was documented with detailed virology data. In our cohort, hematopoietic stem cell transplant donors and recipients were CMV seronegative and positive, respectively, thus providing genetically distinct memory and naïve T cell populations. We used single-cell transcriptomics to track donor versus recipient-derived T cell clones over the course of 90 d. We found that donor-derived T cell clones proliferated and expanded substantially following CMV reactivation. However, for immunodominant CMV epitopes, recipient-derived memory T cells remained the overall dominant population. This dominance was maintained despite more robust clonal expansion of donor-derived T cells in response to CMV reactivation. Interestingly, the donor-derived T cells that were recruited into these immunodominant memory populations shared strikingly similar TCR properties with the recipient-derived memory T cells. This selective recruitment of identical and nearly identical clones from the naïve into the immunodominant memory T cell pool suggests that competition is in place but does not interfere with rejuvenating a memory T cell population. Instead, it results in selection of convergent clones to the memory T cell pool.

Pandemic-Related Instructor Talk: How New Instructors Supported Students at the Onset of the COVID-19 Pandemic.

At the same time that COVID-19 cases in the United States first began to increase, fellows in a mentored teaching apprenticeship for postdoctoral scientists began to teach undergraduate seminars. The fellows suddenly needed to support students emotionally and switch to online instruction. They were encouraged to acknowledge and address the pandemic during each class and decided to do so. In this case study, we examined the language fellows used in response to this encouragement, hypothesizing that they would engage in a variety of pandemic-related instructor talk, i.e., language that instructors use in the classroom that is not directly tied to educational content. We analyzed transcripts from 17 2-hour undergraduate biology seminar courses and found 167 instances of pandemic-related instructor talk. We used grounded theory to identify categories that emerged from these quotations: Positive coping mechanisms and self-care; Adjusting to online learning; Compassionate instruction; Personal impacts; COVID-19 and society; Dreaming; and Biology of COVID-19. Talk in these categories may help build relationships among instructors and students. The category about quickly Adjusting to online learning is unique, in that it is unlikely that there will be another time that will require simultaneous and rapid national movement to online instruction. In addition, four of the seven categories are direct consequences of COVID-19 specifically, and thus are unique to this time. Analyzing pandemic-related instructor talk has shed light on how new instructors navigated the trials of teaching in 2020.

AbSeq Protocol Using the Nano-Well Cartridge-Based Rhapsody Platform to Generate Protein and Transcript Expression Data on the Single-Cell Level.

By including oligonucleotide-labeled antibodies into high-throughput single-cell RNA-sequencing protocols, combined transcript and protein expression data can be acquired on the single-cell level. Here, we describe a protocol for the combined analysis of over 40 proteins and 400 genes on over 104 cells using the nano-well based Rhapsody platform. We also include a workflow for sample multiplexing, which uniquely identifies the initial source of cells (such as tissue type or donor) in the downstream analysis after upstream pooling. For complete information on the use and execution of this protocol, please refer to Mair et al. (2020).

A Targeted Multi-omic Analysis Approach Measures Protein Expression and Low-Abundance Transcripts on the Single-Cell Level.

High-throughput single-cell RNA sequencing (scRNA-seq) has become a frequently used tool to assess immune cell heterogeneity. Recently, the combined measurement of RNA and protein expression was developed, commonly known as cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq). Acquisition of protein expression data along with transcriptome data resolves some of the limitations inherent to only assessing transcripts but also nearly doubles the sequencing read depth required per single cell. Furthermore, there is still a paucity of analysis tools to visualize combined transcript-protein datasets. Here, we describe a targeted transcriptomics approach that combines an analysis of over 400 genes with simultaneous measurement of over 40 proteins on 2 × 104 cells in a single experiment. This targeted approach requires only about one-tenth of the read depth compared to a whole-transcriptome approach while retaining high sensitivity for low abundance transcripts. To analyze these multi-omic datasets, we adapted one-dimensional soli expression by nonlinear stochastic embedding (One-SENSE) for intuitive visualization of protein-transcript relationships on a single-cell level.

The human tissue-resident CCR5+ T cell compartment maintains protective and functional properties during inflammation.

CCR5 is thought to play a central role in orchestrating migration of cells in response to inflammation. CCR5 antagonists can reduce inflammatory disease processes, which has led to an increased interest in using CCR5 antagonists in a wide range of inflammation-driven diseases. Paradoxically, these antagonists appear to function without negatively affecting host immunity at barrier sites. We reasoned that the resolution to this paradox may lie in the CCR5+ T cell populations that permanently reside in tissues. We used a single-cell analysis approach to examine the human CCR5+ T cell compartment in the blood, healthy, and inflamed mucosal tissues to resolve these seemingly contradictory observations. We found that 65% of the CD4 tissue-resident memory T (TRM) cell compartment expressed CCR5. These CCR5+ TRM cells were enriched in and near the epithelial layer and not only limited to TH1-type cells but also contained a large TH17-producing and a stable regulatory T cell population. The CCR5+ TRM compartment was stably maintained even in inflamed tissues including the preservation of TH17 and regulatory T cell populations. Further, using tissues from the CHARM-03 clinical trial, we found that CCR5+ TRM are preserved in human mucosal tissue during treatment with the CCR5 antagonist Maraviroc. Our data suggest that the human CCR5+ TRM compartment is functionally and spatially equipped to maintain barrier immunity even in the absence of CCR5-mediated, de novo T cell recruitment from the periphery.

Learning from regeneration research organisms: The circuitous road to scar free wound healing.

The skin is the largest organ in the body and plays multiple essential roles ranging from regulating temperature, preventing infection and ultimately defining who we are physically. It is a highly dynamic organ that constantly replaces the outermost cells throughout life. However, when faced with a major injury, human skin cannot restore a significant lesion to its original functionality, instead a reparative scar is formed. In contrast to this, many other species have the unique ability to regenerate full thickness skin without formation of scar tissue. Here we review recent advances in the field that shed light on how the skin cells in regenerative species react to injury to prevent scar formation versus scar forming humans.

A novel role for SALL4 during scar-free wound healing in axolotl.

The human response to serious cutaneous damage is limited to relatively primitive wound healing, whereby collagenous scar tissue fills the wound bed. Scars assure structural integrity at the expense of functional regeneration. In contrast, axolotls have the remarkable capacity to functionally regenerate full thickness wounds. Here, we identified a novel role for SALL4 in regulating collagen transcription after injury that is essential for perfect skin regeneration in axolotl. Furthermore, we identify miR-219 as a molecular regulator of Sall4 during wound healing. Taken together, our work highlights one molecular mechanism that allows for efficient cutaneous wound healing in the axolotl.

Identification of Conserved and Novel MicroRNAs during Tail Regeneration in the Mexican Axolotl.

The Mexican axolotl salamander (Ambystoma mexicanum) is one member of a select group of vertebrate animals that have retained the amazing ability to regenerate multiple body parts. In addition to being an important model system for regeneration, the axolotl has also contributed extensively to studies of basic development. While many genes known to play key roles during development have now been implicated in various forms of regeneration, much of the regulatory apparatus controlling the underlying molecular circuitry remains unknown. In recent years, microRNAs have been identified as key regulators of gene expression during development, in many diseases and also, increasingly, in regeneration. Here, we have used deep sequencing combined with qRT-PCR to undertake a comprehensive identification of microRNAs involved in regulating regeneration in the axolotl. Specifically, among the microRNAs that we have found to be expressed in axolotl tissues, we have identified 4564 microRNA families known to be widely conserved among vertebrates, as well as 59,811 reads of putative novel microRNAs. These findings support the hypothesis that microRNAs play key roles in managing the precise spatial and temporal patterns of gene expression that ensures the correct regeneration of missing tissues.

In vivo modulation and quantification of microRNAs during axolotl tail regeneration.

The ability to regenerate diseased, injured, or missing complex tissue is widespread throughout lower vertebrates and invertebrates; however, our knowledge of the molecular mechanisms that regulate this amazing ability is still in its infancy. Many recent papers have shown important roles for microRNAs in regulating regeneration in a number of species. The ability to detect and quantify miRNA expression fluctuations at a single cell level in vivo in different cell types during processes like regeneration is very informative. In this chapter, we describe how to use a dual-fluorescent green fluorescent protein (GFP)-reporter/monomeric red fluorescent protein (mRFP)-sensor (DFRS) plasmid to quantitate the dynamics of specific miRNAs over time following miRNA mimic injection as well as during regeneration. In this bicistronic vector, the mRFP allows for verification of miRNA expression, while the GFP functions as an internal control to normalize miRNA expression and thus obtain quantitative results. In addition, we demonstrate how this technique revealed dynamic miR-23a expression and function during tail regeneration.

CD8+ T cell-independent tumor regression induced by Fc-OX40L and therapeutic vaccination in a mouse model of glioma.

Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Although some promising advances have been made, the immune response stimulated as a result of immunotherapeutic protocols has been inefficient at complete tumor elimination, primarily due to our lack of understanding of the necessary effector functions of the immune system. We previously demonstrated that a tumor lysate vaccine/Fc-OX40L therapy is capable of inducing enhanced survival and tumor elimination in the GL261 mouse glioma model. The following experiments were performed to determine the mechanism(s) of action of this therapy that elicits a potent antitumor immune response. The evidence subsequently outlined indicates a CD8(+) T cell-independent and CD4(+) T cell-, NK cell-, and B cell-dependent means of prolonged survival. CD8(+) T cell-independent tumor clearance is surprising considering the current focus of many cancer immunotherapy protocols. These results provide evidence for CD8(+) T cell-independent means of antitumor response and should lead to additional examination of the potential manipulation of this mechanism for future treatment strategies.

Murine thymic selection quantified using a unique method to capture deleted T cells.

Thymic positive and negative selection events generate a T-cell repertoire that is MHC restricted and self-tolerant. The number of T cells undergoing positive and negative selection in normal mice has never been firmly established. We generated mice that lack the proapoptotic molecule Bim (bcl2l11) together with a Nur77(GFP) transgene, which allowed the identification and enumeration of T cells that would normally undergo clonal deletion. Using this method, we report the striking observation that six times more cells undergo negative selection than complete positive selection. Seventy-five percent of the negatively selected cells are deleted at the double positive stage in the thymic cortex, compared with 25% at the single positive stage in the medulla. The fact that more thymocytes are highly reactive to MHC than are weakly reactive is inconsistent with a random model of recognition and suggests that T-cell recognition is MHC biased. Furthermore, Bim(-/-) mice had an increased number of GFP(hi) cells in the peripheral lymphoid tissue and a corresponding increase in antigen experienced or anergic cell phenotype. Our data also show that the CD4+ T cells that are clonally deleted experienced only slightly stronger T-cell receptor signaling than those that developed into regulatory T cells.

An in vivo immunotherapy screen of costimulatory molecules identifies Fc-OX40L as a potent reagent for the treatment of established murine gliomas.

PURPOSE: We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas. EXPERIMENTAL DESIGN: Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide. RESULTS: Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine/Fc-OX40L-treated mice had superior tumoricidal function relative to all other groups. Vaccine/Fc-OX40L-treated mice exhibited a significant increase in proliferation of brain-infiltrating CD4 and CD8 T cells, as indicated by Ki67 staining. Fc-OX40L had single-agent activity in transplanted and spontaneous glioma models, and the pattern of inflammatory gene expression in the tumor predicted the degree of therapeutic response. CONCLUSIONS: These data show that Fc-OX40L has unique and potent activity against experimental gliomas and warrants further testing.