Exhausted intratumoral Vδ2- γδ T cells in human kidney cancer retain effector function.

Gamma delta (γδ) T cells reside within human tissues including tumors, but their function in mediating antitumor responses to immune checkpoint inhibition is unknown. Here we show that kidney cancers are infiltrated by Vδ2- γδ T cells, with equivalent representation of Vδ1+ and Vδ1- cells, that are distinct from γδ T cells found in normal human tissues. These tumor-resident Vδ2- T cells can express the transcriptional program of exhausted αß CD8+ T cells as well as canonical markers of terminal T-cell exhaustion including PD-1, TIGIT and TIM-3. Although Vδ2- γδ T cells have reduced IL-2 production, they retain expression of cytolytic effector molecules and co-stimulatory receptors such as 4-1BB. Exhausted Vδ2- γδ T cells are composed of three distinct populations that lack TCF7, are clonally expanded and express cytotoxic molecules and multiple Vδ2- T-cell receptors. Human tumor-derived Vδ2- γδ T cells maintain cytotoxic function and pro-inflammatory cytokine secretion in vitro. The transcriptional program of Vδ2- T cells in pretreatment tumor biopsies was used to predict subsequent clinical responses to PD-1 blockade in patients with cancer. Thus, Vδ2- γδ T cells within the tumor microenvironment can contribute to antitumor efficacy.

Stealth Killing by Uterine NK Cells for Tolerance and Tissue Homeostasis.

Human natural killer (NK) cells are critical for innate defense against pathogens through direct cytotoxicity of infected cells and are the predominant immune cell at the maternal-fetal interface. In this issue of Cell, Crespo et al. show that human NK cells in the decidual region of the uterus can clear a bacterial infection from the developing fetus by infusion of granulysin into placental trophoblast cells via nanotubes, thus removing the intracellular pathogen without damage to the placental cell. These findings reveal a mechanism for targeted immune protection of the developing fetus that maintains tolerance at the maternal-fetal interface.

Tissue Determinants of Human NK Cell Development, Function, and Residence.

Immune responses in diverse tissue sites are critical for protective immunity and homeostasis. Here, we investigate how tissue localization regulates the development and function of human natural killer (NK) cells, innate lymphocytes important for anti-viral and tumor immunity. Integrating high-dimensional analysis of NK cells from blood, lymphoid organs, and mucosal tissue sites from 60 individuals, we identify tissue-specific patterns of NK cell subset distribution, maturation, and function maintained across age and between individuals. Mature and terminally differentiated NK cells with enhanced effector function predominate in blood, bone marrow, spleen, and lungs and exhibit shared transcriptional programs across sites. By contrast, precursor and immature NK cells with reduced effector capacity populate lymph nodes and intestines and exhibit tissue-resident signatures and site-specific adaptations. Together, our results reveal anatomic control of NK cell development and maintenance as tissue-resident populations, whereas mature, terminally differentiated subsets mediate immunosurveillance through diverse peripheral sites. VIDEO ABSTRACT.

Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum.

Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.

Site-specific development and progressive maturation of human tissue-resident memory T cells over infancy and childhood.

Infancy and childhood are critical life stages for generating immune memory to protect against pathogens; however, the timing, location, and pathways for memory development in humans remain elusive. Here, we investigated T cells in mucosal sites, lymphoid tissues, and blood from 96 pediatric donors aged 0-10 years using phenotypic, functional, and transcriptomic profiling. Our results revealed that memory T cells preferentially localized in the intestines and lungs during infancy and accumulated more rapidly in mucosal sites compared with blood and lymphoid organs, consistent with site-specific antigen exposure. Early life mucosal memory T cells exhibit distinct functional capacities and stem-like transcriptional profiles. In later childhood, they progressively adopt proinflammatory functions and tissue-resident signatures, coincident with increased T cell receptor (TCR) clonal expansion in mucosal and lymphoid sites. Together, our findings identify staged development of memory T cells targeted to tissues during the formative years, informing how we might promote and monitor immunity in children.

De Novo Epigenetic Programs Inhibit PD-1 Blockade-Mediated T Cell Rejuvenation.

Immune-checkpoint-blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA-methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells, and approaches to reverse these programs improved T cell responses and tumor control during ICB. These data establish de novo DNA-methylation programming as a regulator of T cell exhaustion and barrier of ICB-mediated T cell rejuvenation.

Longitudinal profiling of respiratory and systemic immune responses reveals myeloid cell-driven lung inflammation in severe COVID-19.

Immune response dynamics in coronavirus disease 2019 (COVID-19) and their severe manifestations have largely been studied in circulation. Here, we examined the relationship between immune processes in the respiratory tract and circulation through longitudinal phenotypic, transcriptomic, and cytokine profiling of paired airway and blood samples from patients with severe COVID-19 relative to heathy controls. In COVID-19 airways, T cells exhibited activated, tissue-resident, and protective profiles; higher T cell frequencies correlated with survival and younger age. Myeloid cells in COVID-19 airways featured hyperinflammatory signatures, and higher frequencies of these cells correlated with mortality and older age. In COVID-19 blood, aberrant CD163+ monocytes predominated over conventional monocytes, and were found in corresponding airway samples and in damaged alveoli. High levels of myeloid chemoattractants in airways suggest recruitment of these cells through a CCL2-CCR2 chemokine axis. Our findings provide insights into immune processes driving COVID-19 lung pathology with therapeutic implications for targeting inflammation in the respiratory tract.

Distinct Localization, Transcriptional Profiles, and Functionality in Early Life Tonsil Regulatory T Cells.

CD4+ regulatory T cells (Tregs) are key orchestrators of the immune system, fostering the establishment of protective immunity while preventing deleterious responses. Infancy and childhood are crucial periods of rapid immunologic development, but how Tregs mediate immune responses at these earliest timepoints of human life is poorly understood. In this study, we compare blood and tissue (tonsil) Tregs across pediatric and adult subjects to investigate age-related differences in Treg biology. We observed increased FOXP3 expression and proportions of Tregs in tonsil compared with paired blood samples in children. Within tonsil, early life Tregs accumulated in extrafollicular regions with cellular interactions biased toward CD8+ T cells. Tonsil Tregs in both children and adults expressed transcriptional profiles enriched for lineage defining signatures and canonical functionality compared with blood, suggesting tissue as the primary site of Treg activity. Early life tonsil Tregs transcriptional profiles were further defined by pathways associated with activation, proliferation, and polyfunctionality. Observed differences in pediatric tonsil Treg transcriptional signatures were associated with phenotypic differences, high proliferative capacity, and robust production of IL-10 compared with adult Tregs. These results identify tissue as a major driver of Treg identity, provide new insights into developmental differences in Treg biology across the human lifespan, and demonstrate unique functional properties of early life Tregs.

Effector CD8 T cells dedifferentiate into long-lived memory cells.

Memory CD8 T cells that circulate in the blood and are present in lymphoid organs are an essential component of long-lived T cell immunity. These memory CD8 T cells remain poised to rapidly elaborate effector functions upon re-exposure to pathogens, but also have many properties in common with naive cells, including pluripotency and the ability to migrate to the lymph nodes and spleen. Thus, memory cells embody features of both naive and effector cells, fuelling a long-standing debate centred on whether memory T cells develop from effector cells or directly from naive cells. Here we show that long-lived memory CD8 T cells are derived from a subset of effector T cells through a process of dedifferentiation. To assess the developmental origin of memory CD8 T cells, we investigated changes in DNA methylation programming at naive and effector cell-associated genes in virus-specific CD8 T cells during acute lymphocytic choriomeningitis virus infection in mice. Methylation profiling of terminal effector versus memory-precursor CD8 T cell subsets showed that, rather than retaining a naive epigenetic state, the subset of cells that gives rise to memory cells acquired de novo DNA methylation programs at naive-associated genes and became demethylated at the loci of classically defined effector molecules. Conditional deletion of the de novo methyltransferase Dnmt3a at an early stage of effector differentiation resulted in reduced methylation and faster re-expression of naive-associated genes, thereby accelerating the development of memory cells. Longitudinal phenotypic and epigenetic characterization of the memory-precursor effector subset of virus-specific CD8 T cells transferred into antigen-free mice revealed that differentiation to memory cells was coupled to erasure of de novo methylation programs and re-expression of naive-associated genes. Thus, epigenetic repression of naive-associated genes in effector CD8 T cells can be reversed in cells that develop into long-lived memory CD8 T cells while key effector genes remain demethylated, demonstrating that memory T cells arise from a subset of fate-permissive effector T cells.

Generating long-lived CD8(+) T-cell memory: Insights from epigenetic programs.

T-cell-based immunological memory has the potential to provide the host with life-long protection against pathogen reexposure and thus offers tremendous promise for the design of vaccines targeting chronic infections or cancer. In order to exploit this potential in the design of new vaccines, it is necessary to understand how and when memory T cells acquire their poised effector potential, and moreover, how they maintain these properties during homeostatic proliferation. To gain insight into the persistent nature of memory T-cell functions, investigators have turned their attention to epigenetic mechanisms. Recent efforts have revealed that many of the properties acquired among memory T cells are coupled to stable changes in DNA methylation and histone modifications. Furthermore, it has recently been reported that the delineating features among memory T cells subsets are also linked to distinct epigenetic events, such as permissive and repressive histone modifications and DNA methylation programs, providing exciting new hypotheses regarding their cellular ancestry. Here, we review recent studies focused on epigenetic programs acquired during effector and memory T-cell differentiation and discuss how these data may shed new light on the developmental path for generating long-lived CD8(+) T-cell memory.

Novel Human Cytomegalovirus Viral Chemokines, vCXCL-1s, Display Functional Selectivity for Neutrophil Signaling and Function.

Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (ß-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.

A little cooperation helps murine cytomegalovirus (MCMV) go a long way: MCMV co-infection rescues a chemokine salivary gland defect.

Cytomegaloviruses (CMVs) produce chemokines (vCXCLs) that have both sequence and functional homology to host chemokines. Assessment of vCXCL-1's role in CMV infection is limited to in vitro and in silico analysis due to CMVs species specificity. In this study, we used the murine CMV (MCMV) mouse model to evaluate the function of vCXCL-1 in vivo. Recombinant MCMVs expressing chimpanzee CMV vCXCL-1 (vCXCL-1CCMV) or host chemokine, mCXCL1, underwent primary dissemination to the popliteal lymph node, spleen and lung similar to the parental MCMV. However, neither of the recombinants expressing chemokines was recovered from the salivary gland (SG) at any time post-infection although viral DNA was detected. This implies that the virus does not grow in the SG or the overexpressed chemokine induces an immune response that leads to suppressed growth. Pointing to immune suppression of virus replication, recombinant viruses were isolated from the SG following infection of immune-ablated mice [i.e. SCID (severe combined immunodeficiency), NSG (non-obese diabetic SCID gamma) or cyclophosphamide treated]. Depletion of neutrophils or NK cells does not rescue the recovery of chemokine-expressing recombinants in the SG. Surprisingly we found that co-infection of parental virus and chemokine-expressing virus leads to the recovery of the recombinants in the SG. We suggest that parental virus reduces the levels of chemokine expression leading to a decrease in inflammatory monocytes and subsequent SG growth. Therefore, aberrant expression of the chemokines induces cells of the innate and adaptive immune system that curtail the growth and dissemination of the recombinants in the SG.

Efbalropendekin Alfa enhances human natural killer cell cytotoxicity against tumor cell lines in vitro.

IL-15 has shown preclinical activity by enhancing the functional maturation of natural killer (NK) cells. Clinical evaluation of the potential anticancer activity of most cytokines, including IL-15, has been limited by low tolerability and rapid in vivo clearance. Efbalropendekin Alfa (XmAb24306) is a soluble IL15/IL15-receptor alpha heterodimer complex fused to a half-life extended Fc domain (IL15/IL15Rα-Fc), engineered with mutations to reduce IL-15 affinity for CD122. Reduced affinity drives lower potency, leading to prolonged pharmacodynamic response in cynomolgus monkeys. We show that in vitro, human NK cells treated with XmAb24306 demonstrate enhanced cytotoxicity against various tumor cell lines. XmAb24306-treated NK cells also exhibit enhanced killing of 3D colorectal cancer spheroids. Daratumumab (dara), a monoclonal antibody (mAb) that targets CD38 results in antibody-dependent cellular cytotoxicity (ADCC) of both multiple myeloma (MM) cells and NK cells. Addition of XmAb24306 increases dara-mediated NK cell ADCC against various MM cell lines in vitro. Because NK cells express CD38, XmAb24306 increases dara-mediated NK cell fratricide, but overall does not negatively impact the ADCC activity against a MM cell line likely due to increased NK cell activity of the surviving cells. These data show that XmAb24306 increases direct and ADCC-mediated human NK cell cytotoxicity in vitro.

Inhaled particulate accumulation with age impairs immune function and architecture in human lung lymph nodes.

Older people are particularly susceptible to infectious and neoplastic diseases of the lung and it is unclear how lifelong exposure to environmental pollutants affects respiratory immune function. In an analysis of human lymph nodes (LNs) from 84 organ donors aged 11-93 years, we found a specific age-related decline in lung-associated, but not gut-associated, LN immune function linked to the accumulation of inhaled atmospheric particulate matter. Increasing densities of particulates were found in lung-associated LNs with age, but not in the corresponding gut-associated LNs. Particulates were specifically contained within CD68+CD169- macrophages, which exhibited decreased activation, phagocytic capacity, and altered cytokine production compared with non-particulate-containing macrophages. The structures of B cell follicles and lymphatic drainage were also disrupted in lung-associated LNs with particulates. Our results reveal that the cumulative effects of environmental exposure and age may compromise immune surveillance of the lung via direct effects on immune cell function and lymphoid architecture.

Immune and epithelial determinants of age-related risk and alveolar injury in fatal COVID-19.

Respiratory failure in COVID-19 is characterized by widespread disruption of the lung's alveolar gas exchange interface. To elucidate determinants of alveolar lung damage, we performed epithelial and immune cell profiling in lungs from 24 COVID-19 autopsies and 43 uninfected organ donors ages 18-92 years. We found marked loss of type 2 alveolar epithelial (T2AE) cells and increased perialveolar lymphocyte cytotoxicity in all fatal COVID-19 cases, even at early stages before typical patterns of acute lung injury are histologically apparent. In lungs from uninfected organ donors, there was also progressive loss of T2AE cells with increasing age, which may increase susceptibility to COVID-19-mediated lung damage in older individuals. In the fatal COVID-19 cases, macrophage infiltration differed according to the histopathological pattern of lung injury. In cases with acute lung injury, we found accumulation of CD4+ macrophages that expressed distinctly high levels of T cell activation and costimulation genes and strongly correlated with increased extent of alveolar epithelial cell depletion and CD8+ T cell cytotoxicity. Together, our results show that T2AE cell deficiency may underlie age-related COVID-19 risk and initiate alveolar dysfunction shortly after infection, and we define immune cell mediators that may contribute to alveolar injury in distinct pathological stages of fatal COVID-19.

Intrathymic differentiation of natural antibody-producing plasma cells in human neonates.

The thymus is a central lymphoid organ primarily responsible for the development of T cells. A small proportion of B cells, however, also reside in the thymus to assist negative selection of self-reactive T cells. Here we show that the thymus of human neonates contains a consistent contingent of CD138+ plasma cells, producing all classes and subclasses of immunoglobulins with the exception of IgD. These antibody-secreting cells are part of a larger subset of B cells that share the expression of signature genes defining mouse B1 cells, yet lack the expression of complement receptors CD21 and CD35. Data from single-cell transcriptomic, clonal correspondence and in vitro differentiation assays support the notion of intrathymic CD138+ plasma cell differentiation, alongside other B cell subsets with distinctive molecular phenotypes. Lastly, neonatal thymic plasma cells also include clones reactive to commensal and pathogenic bacteria that commonly infect children born with antibody deficiency. Thus, our findings point to the thymus as a source of innate humoral immunity in human neonates.

Heterogeneity of human anti-viral immunity shaped by virus, tissue, age, and sex.

The persistence of anti-viral immunity is essential for protection and exhibits profound heterogeneity across individuals. Here, we elucidate the factors that shape maintenance and function of anti-viral T cell immunity in the body by comprehensive profiling of virus-specific T cells across blood, lymphoid organs, and mucosal tissues of organ donors. We use flow cytometry, T cell receptor sequencing, single-cell transcriptomics, and cytokine analysis to profile virus-specific CD8+ T cells recognizing the ubiquitous pathogens influenza and cytomegalovirus. Our results reveal that virus specificity determines overall magnitude, tissue distribution, differentiation, and clonal repertoire of virus-specific T cells. Age and sex influence T cell differentiation and dissemination in tissues, while T cell tissue residence and functionality are highly correlated with the site. Together, our results demonstrate how the covariates of virus, tissue, age, and sex impact the anti-viral immune response, which is important for targeting, monitoring, and predicting immune responses to existing and emerging viruses.

SARS-CoV-2 infection generates tissue-localized immunological memory in humans.

Adaptive immune responses to SARS-CoV-2 infection have been extensively characterized in blood; however, most functions of protective immunity must be accomplished in tissues. Here, we report from examination of SARS-CoV-2 seropositive organ donors (ages 10 to 74) that CD4+ T, CD8+ T, and B cell memory generated in response to infection is present in the bone marrow, spleen, lung, and multiple lymph nodes (LNs) for up to 6 months after infection. Lungs and lung-associated LNs were the most prevalent sites for SARS-CoV-2­specific memory T and B cells with significant correlations between circulating and tissue-resident memory T and B cells in all sites. We further identified SARS-CoV-2­specific germinal centers in the lung-associated LNs up to 6 months after infection. SARS-CoV-2­specific follicular helper T cells were also abundant in lung-associated LNs and lungs. Together, the results indicate local tissue coordination of cellular and humoral immune memory against SARS-CoV-2 for site-specific protection against future infectious challenges.

Analysis of respiratory and systemic immune responses in COVID-19 reveals mechanisms of disease pathogenesis.

Immune responses to respiratory viruses like SARS-CoV-2 originate and function in the lung, yet assessments of human immunity are often limited to blood. Here, we conducted longitudinal, high-dimensional profiling of paired airway and blood samples from patients with severe COVID-19, revealing immune processes in the respiratory tract linked to disease pathogenesis. Survival from severe disease was associated with increased CD4 + T cells and decreased monocyte/macrophage frequencies in the airway, but not in blood. Airway T cells and macrophages exhibited tissue-resident phenotypes and activation signatures, including high level expression and secretion of monocyte chemoattractants CCL2 and CCL3 by airway macrophages. By contrast, monocytes in blood expressed the CCL2-receptor CCR2 and aberrant CD163 + and immature phenotypes. Extensive accumulation of CD163 + monocyte/macrophages within alveolar spaces in COVID-19 lung autopsies suggested recruitment from circulation. Our findings provide evidence that COVID-19 pathogenesis is driven by respiratory immunity, and rationale for site-specific treatment and prevention strategies.

Antibody responses to SARS-CoV2 are distinct in children with MIS-C compared to adults with COVID-19.

Clinical manifestations of COVID-19 caused by the novel coronavirus SARS-CoV-2 are associated with age. While children are largely spared from severe respiratory disease, they can present with a SARS-CoV-2-associated multisystem inflammatory syndrome (MIS-C) similar to Kawasaki's disease. Here, we show distinct antibody (Ab) responses in children with MIS-C compared to adults with severe COVID-19 causing acute respiratory distress syndrome (ARDS), and those who recovered from mild disease. There was a reduced breadth and specificity of anti-SARS-CoV-2-specific antibodies in MIS-C patients compared to the COVID patient groups; MIS-C predominantly generated IgG Abs specific for the Spike (S) protein but not for the nucleocapsid (N) protein, while both COVID-19 cohorts had anti-S IgG, IgM and IgA Abs, as well as anti-N IgG Abs. Moreover, MIS-C patients had reduced neutralizing activity compared to COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children and adults who develop severe disease, with implications for optimizing treatments based on symptom and age.