Expansion of the HSV-2-specific T cell repertoire in skin after immunotherapeutic HSV-2 vaccine.

The skin at the site of HSV-2 reactivation is enriched for HSV-2-specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2-reactive CD4+ T cells from PBMCs by T cell receptor (TCR) ß chain (TRB) sequencing before and after vaccination with a replication-incompetent whole-virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. In one participant, a switch in immunodominance occurred with the emergence of a TCR αß pair after vaccination that was not detected in blood. This TCRαß was shown to be HSV-2 reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possessed an oligoclonal TRB repertoire that was distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2-specific TRB repertoire requires tissue-based evaluation.

Expansion of the HSV-2-specific T cell repertoire in skin after immunotherapeutic HSV-2 vaccine.

The skin at the site of HSV-2 reactivation is enriched for HSV-2-specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2-reactive CD4+ T cells from peripheral blood mononuclear cells (PBMCs) by T cell receptor ß (TRB) sequencing before and after vaccination with a replication-incompetent whole virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T-cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. In one participant a switch in immunodominance occurred with the emergence of a T cell receptor (TCR) αß pair after vaccination that was not detected in blood. This TCRαß was shown to be HSV-2-reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possesses an oligoclonal TRB repertoire that is distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2-specific TRB repertoire requires tissue-based evaluation.

Repeated mRNA vaccination sequentially boosts SARS-CoV-2-specific CD8+ T cells in persons with previous COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hybrid immunity is more protective than vaccination or previous infection alone. To investigate the kinetics of spike-reactive T (TS) cells from SARS-CoV-2 infection through messenger RNA vaccination in persons with hybrid immunity, we identified the T cell receptor (TCR) sequences of thousands of index TS cells and tracked their frequency in bulk TCRß repertoires sampled longitudinally from the peripheral blood of persons who had recovered from coronavirus disease 2019 (COVID-19). Vaccinations led to large expansions in memory TS cell clonotypes, most of which were CD8+ T cells, while also eliciting diverse TS cell clonotypes not observed before vaccination. TCR sequence similarity clustering identified public CD8+ and CD4+ TCR motifs associated with spike (S) specificity. Synthesis of longitudinal bulk ex vivo single-chain TCRß repertoires and paired-chain TCRÉ‘ß sequences from droplet sequencing of TS cells provides a roadmap for the rapid assessment of T cell responses to vaccines and emerging pathogens.

CD8+ T cell clonotypes from prior SARS-CoV-2 infection predominate during the cellular immune response to mRNA vaccination.

Almost three years into the SARS-CoV-2 pandemic, hybrid immunity is highly prevalent worldwide and more protective than vaccination or prior infection alone. Given emerging resistance of variant strains to neutralizing antibodies (nAb), it is likely that T cells contribute to this protection. To understand how sequential SARS-CoV-2 infection and mRNA-vectored SARS-CoV-2 spike (S) vaccines affect T cell clonotype-level expansion kinetics, we identified and cross-referenced TCR sequences from thousands of S-reactive single cells against deeply sequenced peripheral blood TCR repertoires longitudinally collected from persons during COVID-19 convalescence through booster vaccination. Successive vaccinations recalled memory T cells and elicited antigen-specific T cell clonotypes not detected after infection. Vaccine-related recruitment of novel clonotypes and the expansion of S-specific clones were most strongly observed for CD8+ T cells. Severe COVID-19 illness was associated with a more diverse CD4+ T cell response to SARS-CoV-2 both prior to and after mRNA vaccination, suggesting imprinting of CD4+ T cells by severe infection. TCR sequence similarity search algorithms revealed myriad public TCR clusters correlating with human leukocyte antigen (HLA) alleles. Selected TCRs from distinct clusters functionally recognized S in the predicted HLA context, with fine viral peptide requirements differing between TCRs. Most subjects tested had S-specific T cells in the nasal mucosa after a 3rd mRNA vaccine dose. The blood and nasal T cell responses to vaccination revealed by clonal tracking were more heterogeneous than nAb boosts. Analysis of bulk and single cell TCR sequences reveals T cell kinetics and diversity at the clonotype level, without requiring prior knowledge of T cell epitopes or HLA restriction, providing a roadmap for rapid assessment of T cell responses to emerging pathogens.

The Anticancer Potential of T Cell Receptor-Engineered T Cells.

Adoptively transferred T cell receptor (TCR)-transgenic T cells (TCR-T cells) are not restricted by cell surface expression of their targets and are therefore poised to become a main pillar of cellular cancer immunotherapies. Addressing clinical and laboratory data, we discuss emerging features for the efficient deployment of novel TCR-T therapies, such as selection of ideal TCRs targeting validated epitopes with well-characterized cancer cell expression and processing, enhancing TCR-T effector function, trafficking, expansion, persistence, and memory formation by strategic selection of substrate cells, and gene-engineering with synthetic co-stimulatory circuits. Overall, a better understanding of the relevant mechanisms of action and resistance will help prioritize the vast array of potential TCR-T optimizations for future clinical products.

Use of eltrombopag in aplastic anemia in Europe.

Eltrombopag (ELT), an oral thrombopoietin receptor agonist, has recently emerged as a promising new drug for the treatment of aplastic anemia (AA). How ELT is used outside of clinical trials in the real-world setting and results of this treatment are not known. We conducted therefore a retrospective survey on the use of ELT in AA among EBMT member centers. We analyzed the 134 patients reported in our survey together with 46 patients recently published by Lengline et al. The median follow-up from start of ELT treatment was 15.3 months, with 85.6% patients alive at last follow-up. Importantly, only 28.9% of our patients received ELT according to the FDA/EMA label as monotherapy in the relapsed/refractory setting, whereas 16.7% received ELT upfront. The overall response rate in our cohort was 62%, very similar to the results of the pivotal ELT trial. In multivariate analysis, combination therapy with ELT/cyclosporine/ATG and response to previous therapy were associated with response. Overall survival was favorable with a 1-year survival from ELT start of 87.4%. We identified age, AA severity before ELT start and response to ELT as variables significantly associated with OS. Two patients transformed to MDS; other adverse events were mostly benign. In sum, ELT is used widely in Europe to treat AA patients, mostly in the relapsed/refractory setting. Response to ELT is similar to the clinical trial data across different age groups, treatment lines, and treatment combinations and results in favorable survival.

Anti-thymocyte globulin-induced hyperbilirubinemia in patients with myelofibrosis undergoing allogeneic hematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only curative treatment option for myelofibrosis (MF) despite the emergence of novel targeted therapies. To reduce graft rejection and graft-versus-host disease (GvHD), current allo-HCT protocols often include in vivo T lymphocyte depletion using polyclonal anti-thymocyte globulin (ATG). Shortly after ATG administration, an immediate inflammatory response with fever, chills, and laboratory alterations such as cytopenias, elevation of serum C-reactive protein, bilirubin, and transaminases can develop. Here, we explore whether MF patients, who commonly exhibit extramedullary hematopoiesis in the liver, might be particularly susceptible to ATG-induced liver toxicity. To test this hypothesis, we analyzed 130 control and 94 MF patients from three transplant centers treated with or without ATG during the allo-HCT conditioning regimen. Indeed, hyperbilirubinemia was found in nearly every MF patient treated with ATG (MF-ATG 54/60 = 90 %) as compared to non-ATG treated MF (MF-noATG 15/34 = 44.1 %, p < 0.001) and respectively ATG-treated non-MF patients of the control group (control-ATG, 43/77 = 56 %, p < 0.001). In contrast, transaminases were only inconsistently elevated. Hyperbilirubinemia was in most cases self-limiting and not predictive of increased incidence of non-relapse mortality, hepatic sinusoidal obstruction syndrome (SOS) or liver GvHD. In sum, awareness of this stereotypic bilirubin elevation in MF patients treated with ATG provides a relatively benign explanation for hyperbilirubinemia occurring in these patients during the early transplant. However, attention to drug levels of biliary excreted drugs is warranted, since altered bile flow may influence their clearance and enhance toxicity (e.g., busulfan, antifungal agents).

A genetic interactome of the let-7 microRNA in C. elegans.

The heterochronic pathway controls temporal patterning during Caenorhabditis elegans larval development. The highly conserved let-7 microRNA (miRNA) plays a key role in this pathway, directing the larval-to-adult (L/A) transition. Hence, knowledge of the genetic interactome of let-7 has the potential to provide insight into both control of temporal cell fates and mechanisms of regulation and function of miRNAs. Here, we report the results of a genome-wide, RNAi-based screen for suppressors of let-7 mutant vulval bursting. The 201 genetic interaction partners of let-7 thus identified include genes that promote target silencing activity of let-7, seam cell differentiation, or both. We illustrate the suitability of our approach by uncovering the mitotic cyclin-dependent kinase CDK-1 as a downstream effector of let-7 that affects both seam cell proliferation and differentiation, and by identifying a core set of candidate modulators of let-7 activity, which includes all subunits of the condensin II complex. We propose that the genes identified in our screen thus constitute a valuable resource for studies of the heterochronic pathway and miRNAs.

The let-7 microRNA directs vulval development through a single target.

The let-7 microRNA (miRNA) regulates stemness in animals ranging from worms to humans. However, the cause of the dramatic vulval rupturing phenotype of let-7 mutant C. elegans has remained unknown. Consistent with the notion that miRNAs function by coordinately tuning the expression of many targets, bursting may result from joint dysregulation of several targets, possibly in the epidermis. Alternatively, overexpression of LET-60/RAS, a key vulva development gene and a phylogenetically conserved target of let-7, may be responsible. Here, we show that let-7 functions in the vulval-uterine system to ensure vulval integrity but that regulation of most targets of let-7, including LET-60/RAS, is dispensable. Using CRISPR-Cas9 to edit endogenous let-7 target sites, we found that regulation of LIN-41/TRIM71 alone is necessary and sufficient to prevent vulval rupturing. Hence, let-7 does not function to reduce gene expression noise broadly, but to direct vulval development through extensive regulation of a single, defined target.

LIN-41/TRIM71: emancipation of a miRNA target.

lin-41 (lineage variant 41)/TRIM71 (tripartite motif 71) is well known for being a conserved target of the let-7 (lethal 7) microRNA (miRNA), a regulatory relationship found in animals evolutionarily as distant as Caenorhabditis elegans and humans. It has thus been studied extensively as a model for miRNA-mediated gene silencing. In contrast, the developmental and molecular functions of LIN41 have historically received less attention. However, LIN41 proteins are now emerging as important regulators of cell proliferation and differentiation in stem and progenitor cells. Moreover, LIN41's functions appear to involve two distinct molecular activities; namely, protein ubiquitylation and post-transcriptional silencing of mRNAs. Thus, LIN41 is ready for a scientific life of its own.