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T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8 + memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8 + memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8 + memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines. The identity of stem-cell memory progenitor cells has been unclear. Lugli and colleagues use high-dimensional approaches to identify two new progenitor populations of human T cells—one giving rise to a functional lineage, the other to an exhausted-like one.

γδ T cells are considered to be innate-like lymphocytes that respond rapidly to stress without clonal selection and differentiation. Here we use next-generation sequencing to probe how this paradigm relates to human Vδ2 neg T cells, implicated in responses to viral infection and cancer. The prevalent Vδ1 T cell receptor (TCR) repertoire is private and initially unfocused in cord blood, typically becoming strongly focused on a few high-frequency clonotypes by adulthood. Clonal expansions have differentiated from a naive to effector phenotype associated with CD27 downregulation, retaining proliferative capacity and TCR sensitivity, displaying increased cytotoxic markers and altered homing capabilities, and remaining relatively stable over time. Contrastingly, Vδ2 + T cells express semi-invariant TCRs, which are present at birth and shared between individuals. Human Vδ1 + T cells have therefore evolved a distinct biology from the Vδ2 + subset, involving a central, personalized role for the γδ TCR in directing a highly adaptive yet unconventional form of immune surveillance. γδ T cells are generally considered innate‐like lymphocytes. Here the authors sequence human γδ T cell receptors (TCR) to show focusing of the private Vδ1 TCR repertoire, suggesting that, unlike Vδ2 T cells, the Vδ1 T cell compartment has adaptive attributes.

The fetus is thought to be protected from exposure to foreign antigens, yet CD45RO + T cells reside in the fetal intestine. Here we combined functional assays with mass cytometry, single-cell RNA sequencing and high-throughput T cell antigen receptor (TCR) sequencing to characterize the CD4 + T cell compartment in the human fetal intestine. We identified 22 CD4 + T cell clusters, including naive-like, regulatory-like and memory-like subpopulations, which were confirmed and further characterized at the transcriptional level. Memory-like CD4 + T cells had high expression of Ki-67, indicative of cell division, and CD5, a surrogate marker of TCR avidity, and produced the cytokines IFN-γ and IL-2. Pathway analysis revealed a differentiation trajectory associated with cellular activation and proinflammatory effector functions, and TCR repertoire analysis indicated clonal expansions, distinct repertoire characteristics and interconnections between subpopulations of memory-like CD4 + T cells. Imaging mass cytometry indicated that memory-like CD4 + T cells colocalized with antigen-presenting cells. Collectively, these results provide evidence for the generation of memory-like CD4 + T cells in the human fetal intestine that is consistent with exposure to foreign antigens. Koning and colleagues used mass cytometry, single-cell RNA-seq and high-throughput TCR sequencing to characterize the CD4 + T cell compartment in the human fetal intestine.

The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

Single-cell RNA sequencing (scRNA-Seq) provides valuable insights into cell biology. However, current scRNA-Seq analytic approaches do not distinguish between spliced and unspliced mRNA at the level of dimensionality reduction. RNA velocity paradigm suggests that the presence of unspliced mRNA reflects transitional cell states, informative for studies of dynamic processes such as embryogenesis or tissue regeneration. Alternatively, stable cell subsets may also maintain translationally repressed spliced mRNA (e.g., in P-bodies) and/or unspliced mRNA reservoirs for prompt initiation of transcription-independent expression. Thus, functional cell subsets may differ not only in the current levels of actively produced mRNAs, but also in which mRNAs and in what forms are stored in the nucleus and cytoplasm. To enable splicing-aware analysis of scRNA-Seq data, we developed a method called SANSARA (Splicing-Aware scrNa-Seq AppRoAch). We employed SANSARA to characterize peripheral blood regulatory T cell (T reg ) subsets, revealing a complementary interplay between the FOXP3 and Helios master transcription factors and high levels of spliced  IL10RA , LGALS3 , FCRL3 , CD38 , ITGAL , and LEF1 mRNAs in effector T reg s. Among Th1 and cytotoxic CD4 + T cell subsets, SANSARA also revealed substantial splicing heterogeneity across subset-specific genes. SANSARA is straightforward to implement in current data analysis pipelines and opens new dimensions for scRNA-Seq-based discoveries.

Inhibitory CD4 + T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity. However, the mechanisms that underpin the development of these regulatory cells, especially in the context of ongoing antigen exposure, have remained obscure. To address this knowledge gap, we undertook a comprehensive functional, phenotypic, and transcriptomic analysis of interleukin (IL)-10-producing CD4 + T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated T H 1-like cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L -arginine. Mice lacking Arg1-expressing CD4 + T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4 + lineage suppressed the development of these inhibitory cells and also enhanced immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4 + T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the site-specific delivery of Arg1.

T stem cell-like memory cells (T SCM cells) are considered to be essential for the maintenance of immune memory. The T SCM population has been shown to have the key properties of a stem cell population: multipotency, self-renewal and clonal longevity. Here we show that no single population has all these stem cell properties, instead the properties are distributed. We show that the human T SCM population consists of two distinct cell subpopulations which can be distinguished by the level of their CD95 expression (CD95int and CD95hi). Crucially, using long-term in vivo labelling of human volunteers, we establish that these are distinct populations rather than transient states of the same population. These two subpopulations have different functional profiles ex vivo , different transcriptional patterns, and different tissue distributions. They also have significantly different TREC content indicating different division histories and we find that the frequency of CD95hi T SCM increases with age. Most importantly, CD95hi and CD95int T SCM cells also have very different dynamics in vivo with CD95hi cells showing considerably higher proliferation but significantly reduced clonal longevity compared with CD95int T SCM . While both T SCM subpopulations exhibit considerable multipotency, no single population of T SCM cells has both the properties of self-renewal and clonal longevity. Instead, the “stemness” of the T SCM population is generated by the complementary dynamic properties of the two subpopulations: CD95int T SCM which have the property of clonal longevity and CD95hi T SCM which have the properties of expansion and self-renewal. We suggest that together, these two populations function as a stem cell population.

Active immunization using tumor antigen-loaded dendritic cells holds promise for the adjuvant treatment of cancer to eradicate or control residual disease, but so far, most dendritic cell trials have been performed in end-stage cancer patients with high tumor loads. Here, in a phase I/II trial, we investigated the effect of autologous dendritic cell vaccination in 10 patients with acute myeloid leukemia (AML). The Wilms' tumor 1 protein (WT1), a nearly universal tumor antigen, was chosen as an immunotherapeutic target because of its established role in leukemogenesis and superior immunogenic characteristics. Two patients in partial remission after chemotherapy were brought into complete remission after intradermal administration of full-length WT1 mRNA-electroporated dendritic cells. In these two patients and three other patients who were in complete remission, the AML-associated tumor marker returned to normal after dendritic cell vaccination, compatible with the induction of molecular remission. Clinical responses were correlated with vaccine-associated increases in WT1-specific CD8⁺ T cell frequencies, as detected by peptide/HLA-A*0201 tetramer staining, and elevated levels of activated natural killer cells postvaccination. Furthermore, vaccinated patients showed increased levels of WT1-specific IFN-γ–producing CD8⁺ T cells and features of general immune activation. These data support the further development of vaccination with WT1 mRNA-loaded dendritic cells as a postremission treatment to prevent full relapse in AML patients.

Adaptive immunity relies on the generation and maintenance of memory T cells to provide protection against repeated antigen exposure. It has been hypothesised that a self-renewing population of T cells, named stem cell-like memory T (TSCM) cells, are responsible for maintaining memory. However, it is not clear if the dynamics of TSCM cells in vivo are compatible with this hypothesis. To address this issue, we investigated the dynamics of TSCM cells under physiological conditions in humans in vivo using a multidisciplinary approach that combines mathematical modelling, stable isotope labelling, telomere length analysis, and cross-sectional data from vaccine recipients. We show that, unexpectedly, the average longevity of a TSCM clone is very short (half-life < 1 year, degree of self-renewal = 430 days): far too short to constitute a stem cell population. However, we also find that the TSCM population is comprised of at least 2 kinetically distinct subpopulations that turn over at different rates. Whilst one subpopulation is rapidly replaced (half-life = 5 months) and explains the rapid average turnover of the bulk TSCM population, the half-life of the other TSCM subpopulation is approximately 9 years, consistent with the longevity of the recall response. We also show that this latter population exhibited a high degree of self-renewal, with a cell residing without dying or differentiating for 15% of our lifetime. Finally, although small, the population was not subject to excessive stochasticity. We conclude that the majority of TSCM cells are not stem cell-like but that there is a subpopulation of TSCM cells whose dynamics are compatible with their putative role in the maintenance of T cell memory.

Aims/hypothesisThe aim of this study was to characterise islet autoantibody profiles and immune cell phenotypes in slow progressors to type 1 diabetes.MethodsImmunological variables were compared across peripheral blood samples obtained from slow progressors to type 1 diabetes, individuals with newly diagnosed or long-standing type 1 diabetes, and healthy individuals. Polychromatic flow cytometry was used to characterise the phenotypic attributes of B and T cells. Islet autoantigen-specific B cells were quantified using an enzyme-linked immunospot (ELISpot) assay and islet autoantigen-specific CD8+ T cells were quantified using peptide–HLA class I tetramers. Radioimmunoassays were used to detect islet autoantibodies. Sera were assayed for various chemokines, cytokines and soluble receptors via ELISAs.ResultsIslet autoantibodies were lost over time in slow progressors. Various B cell subsets expressed higher levels of CD95 in slow progressors, especially after polyclonal stimulation, compared with the corresponding B cell subsets in healthy donors (p < 0.05). The phenotypic characteristics of CD4+ and CD8+ T cells were similar in slow progressors and healthy donors. Lower frequencies of CD4+ T cells with a central memory phenotype (CD27int, CD127+, CD95int) were observed in slow progressors compared with healthy donors (mean percentage of total CD4+ T cells was 3.00% in slow progressors vs 4.67% in healthy donors, p < 0.05). Autoreactive B cell responses to proinsulin were detected at higher frequencies in slow progressors compared with healthy donors (median no. of spots was 0 in healthy donors vs 24.34 in slow progressors, p < 0.05) in an ELISpot assay. Islet autoantigen-specific CD8+ T cell responses were largely absent in slow progressors and healthy donors. Serum levels of DcR3, the decoy receptor for CD95L, were elevated in slow progressors compared with healthy donors (median was 1087 pg/ml in slow progressors vs 651 pg/ml in healthy donors, p = 0.06).Conclusions/interpretationIn this study, we found that slow progression to type 1 diabetes was associated with a loss of islet autoantibodies and a distinct B cell phenotype, consistent with enhanced apoptotic regulation of peripheral autoreactivity via CD95. These phenotypic changes warrant further studies in larger cohorts to determine their functional implications.