SUMMARY During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants controlling CD8+ T lymphocyte fate specification. These findings suggest a model of terminal effector cell differentiation initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, highlighting the power and necessity of single-cell approaches.

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IntroductionSince their discovery 15 years ago, 1 it is now well established that CD25 ϩ regulatory T cells (Tregs) are indispensable for immune homeostasis and self-tolerance. Tregs suppress the activation, proliferation, and effector functions of a wide range of immune cells via multiple mechanisms. 2 FOXP3 has been identified as a master transcription factor, controlling both Treg development and functionality. 3,4 In addition, human Tregs can be identified by high CD25 and low IL-7 receptor (CD127) expression. 5,6 A critical role of Tregs in controlling autoimmune responses is demonstrated in various animal models of autoimmune disease. 7 Furthermore, lack of functional Tregs leads to severe, systemic autoimmunity in humans. 8,9 Because of their unique function, Tregs are considered important for the treatment of autoimmune disease, and several strategies are now being explored to target these cells for therapeutic purposes. 10 However, there is still an ongoing debate whether the numbers and/or function of Tregs are changed in patients suffering from chronic autoimmune inflammation. 11 In rheumatoid arthritis (RA) and multiple sclerosis, similar Treg numbers,12,13 or even enhanced numbers in RA, 14 were observed in peripheral blood (PB) of patients compared with healthy controls (HCs). Thus, it appears that Treg numbers are not reduced in patients suffering from autoimmune inflammation. In addition, it remains unclear whether Treg function is impaired; some studies report reduced functioning of Tregs in PB of patients, 12,13,15 whereas others have found no difference. 14,16 In addition to these discrepancies concerning Treg numbers and function in the periphery, characterization of Tregs functionality at the site of autoimmune inflammation in humans is missing. High levels of Tregs have been found at the inflammatory sites in patients with arthritis and inflammatory bowel disease and these cells can suppress CD4 ϩ CD25 Ϫ effector cells in vitro. 17 Also at the site of inflammation in juvenile idiopathic arthritis (JIA), one of the most common childhood autoimmune diseases, we have previously shown that Tregs are present in high numbers and suppress proliferation of CD4 ϩ CD25 Ϫ effector cells in vitro. 18 However, in vivo inflammation persists despite the large numbers of Tregs present, suggesting that these cells are defective in their ability to control the ongoing autoimmune response. This may result from the local proinflammatory environment, because in vitro experiments have shown that pro-inflammatory cytokines can affect both Treg function 15,[19][20][21] as well as effector T-cell responses. 22,23 These data suggest that increasing Treg numbers or enhancing function for therapeutic purposes might be less effective in a chronic inflammatory environment. However, ex vivo data from patients with autoimmune disease are required to clarify the role of Tregs at the site of inflammation in humans.Here, we studied Treg function at the site of inflammation in patients with JIA and compared their inhibitory p...

Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone Reprints and permissions information is available at www.nature.com/reprints.

Since the discovery of FOXP3+ regulatory T (T(REG)) cells over 15 years ago, intensive research has focused on their presence, phenotype and function in autoimmune disease. Whether deficiencies in T(REG) cells underlie autoimmune pathology and whether, or how, therapeutic approaches based on these cells might be successful is still the subject of debate. The potential role of T(REG)-cell extrinsic factors, such as proinflammatory cytokines and resistance of effector T cells to suppression, as the cause of regulatory defects in chronic autoimmune inflammation is an intensive area of research. It is now clear that, at the site of inflammation, antigen presenting cells (APCs) and proinflammatory cytokines drive effector T cell skewing and plasticity, and that these T cells can become unresponsive to regulation. In addition, expansion and function of T(REG) cells is affected by the inflammatory environment; indeed, new data suggest that, in certain conditions, T(REG) cells promote inflammation. This Review summarizes the latest findings on changes in effector T cell homeostasis in autoimmune disease and focuses on how mechanisms that normally regulate these cells are affected in the inflamed joints of patients with arthritis. These findings have important clinical implications and will affect the development of new therapeutic strategies for autoimmune arthritis.

Key Points Autologous HSCT induces functional renewal of regulatory T cells as well as a strong Treg TCR diversification in autoimmune patients. Adding regulatory T cells to the graft does not lead to additional clinical improvement but results in delayed donor T-cell reconstitution.

Although characterization of T cell exhaustion has unlocked powerful immunotherapies, the mechanisms sustaining adaptations of short-lived innate cells to chronic inflammatory settings remain unknown. During murine chronic viral infection, we found that concerted events in bone marrow and spleen mediated by type I interferon (IFN-I) and Toll-like receptor 7 (TLR7) maintained a pool of functionally exhausted plasmacytoid dendritic cells (pDCs). In the bone marrow, IFN-I compromised the number and the developmental capacity of pDC progenitors, which generated dysfunctional pDCs. Concurrently, exhausted pDCs in the periphery were maintained by self-renewal via IFN-I- and TLR7-induced proliferation of CD4 subsets. On the other hand, pDC functional loss was mediated by TLR7, leading to compromised IFN-I production and resistance to secondary infection. These findings unveil the mechanisms sustaining a self-perpetuating pool of functionally exhausted pDCs and provide a framework for deciphering long-term exhaustion of other short-lived innate cells during chronic inflammation.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.