V-CARMA: A tool for the detection and modification of antigen-specific T cells.

T cells promote our body's ability to battle cancers and infectious diseases but can act pathologically in autoimmunity. The recognition of peptides presented by major histocompatibility complex (pMHC) molecules by T cell receptors (TCRs) enables T cell-mediated responses. To modify disease-relevant T cells, new tools to genetically modify T cells and decode their antigen recognition are needed. Here, we present an approach using viruses pseudotyped with peptides loaded on MHC called V-CARMA (Viral ChimAeric Receptor MHC-Antigen) to specifically target T cells expressing cognate TCRs for antigen discovery and T cell engineering. We show that lentiviruses displaying antigens on human leukocyte antigen (HLA) class I and class II molecules can robustly infect CD8+ and CD4+ T cells expressing cognate TCRs, respectively. The infection rates of the pseudotyped lentiviruses (PLVs) are correlated with the binding affinity of the TCR to its cognate antigen. Furthermore, peptide-HLA pseudotyped lentivirus V-CARMA constructs can identify target cells from a mixed T cell population, suppress PD-1 expression on CD8+ T cells via PDCD1 shRNA delivery, and induce apoptosis in autoreactive CD4+ T cells. Thus, V-CARMA is a versatile tool for TCR ligand identification and selective T cell manipulation.

Activated CD4+ T cells and CD14hiCD16+ monocytes correlate with antibody response following influenza virus infection in humans.

The failure to mount an antibody response following viral infection or seroconversion failure is a largely underappreciated and poorly understood phenomenon. Here, we identified immunologic markers associated with robust antibody responses after influenza virus infection in two independent human cohorts, SHIVERS and FLU09, based in Auckland, New Zealand and Memphis, Tennessee, USA, respectively. In the SHIVERS cohort, seroconversion significantly associates with (1) hospitalization, (2) greater numbers of proliferating, activated CD4+ T cells, but not CD8+ T cells, in the periphery during the acute phase of illness, and (3) fewer inflammatory monocytes (CD14hiCD16+) by convalescence. In the FLU09 cohort, fewer CD14hiCD16+ monocytes during early illness in the nasal mucosa were also associated with the generation of influenza-specific mucosal immunoglobulin A (IgA) and IgG antibodies. Our study demonstrates that seroconversion failure after infection is a definable immunological phenomenon, associated with quantifiable cellular markers that can be used to improve diagnostics, vaccine efficacy, and epidemiologic efforts.

Publisher Correction: Exuberant fibroblast activity compromises lung function via ADAMTS4.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exuberant fibroblast activity compromises lung function via ADAMTS4.

Severe respiratory infections can result in acute respiratory distress syndrome (ARDS)1. There are no effective pharmacological therapies that have been shown to improve outcomes for patients with ARDS. Although the host inflammatory response limits spread of and eventually clears the pathogen, immunopathology is a major contributor to tissue damage and ARDS1,2. Here we demonstrate that respiratory viral infection induces distinct fibroblast activation states, which we term extracellular matrix (ECM)-synthesizing, damage-responsive and interferon-responsive states. We provide evidence that excess activity of damage-responsive lung fibroblasts drives lethal immunopathology during severe influenza virus infection. By producing ECM-remodelling enzymes-in particular the ECM protease ADAMTS4-and inflammatory cytokines, damage-responsive fibroblasts modify the lung microenvironment to promote robust immune cell infiltration at the expense of lung function. In three cohorts of human participants, the levels of ADAMTS4 in the lower respiratory tract were associated with the severity of infection with seasonal or avian influenza virus. A therapeutic agent that targets the ECM protease activity of damage-responsive lung fibroblasts could provide a promising approach to preserving lung function and improving clinical outcomes following severe respiratory infections.

Necroptosis restricts influenza A virus as a stand-alone cell death mechanism.

Influenza A virus (IAV) activates ZBP1-initiated RIPK3-dependent parallel pathways of necroptosis and apoptosis in infected cells. Although mice deficient in both pathways fail to control IAV and succumb to lethal respiratory infection, RIPK3-mediated apoptosis by itself can limit IAV, without need for necroptosis. However, whether necroptosis, conventionally considered a fail-safe cell death mechanism to apoptosis, can restrict IAV-or indeed any virus-in the absence of apoptosis is not known. Here, we use mice selectively deficient in IAV-activated apoptosis to show that necroptosis drives robust antiviral immune responses and promotes effective virus clearance from infected lungs when apoptosis is absent. We also demonstrate that apoptosis and necroptosis are mutually exclusive fates in IAV-infected cells. Thus, necroptosis is an independent, "stand-alone" cell death mechanism that fully compensates for the absence of apoptosis in antiviral host defense.

Pediatric patients with acute lymphoblastic leukemia generate abundant and functional neoantigen-specific CD8+ T cell responses.

Cancer arises from the accumulation of genetic alterations, which can lead to the production of mutant proteins not expressed by normal cells. These mutant proteins can be processed and presented on the cell surface by major histocompatibility complex molecules as neoepitopes, allowing CD8+ T cells to mount responses against them. For solid tumors, only an average 2% of neoepitopes predicted by algorithms have detectable endogenous antitumor T cell responses. This suggests that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphoblastic leukemia (ALL) have tumor-associated neoepitope-specific CD8+ T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. These responses include a public neoantigen from the ETV6-RUNX1 fusion that is targeted in seven of nine tested patients. We characterized phenotypic and transcriptional profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at the single-cell level and found a heterogeneous population that included highly functional effectors. Moreover, we observed immunodominance hierarchies among the CD8+ TILs restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in pediatric ALL despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. Furthermore, these data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens.

Lung γδ T Cells Mediate Protective Responses during Neonatal Influenza Infection that Are Associated with Type 2 Immunity.

Compared to adults, infants suffer higher rates of hospitalization, severe clinical complications, and mortality due to influenza infection. We found that γδ T cells protected neonatal mice against mortality during influenza infection. γδ T cell deficiency did not alter viral clearance or interferon-γ production. Instead, neonatal influenza infection induced the accumulation of interleukin-17A (IL-17A)-producing γδ T cells, which was associated with IL-33 production by lung epithelial cells. Neonates lacking IL-17A-expressing γδ T cells or Il33 had higher mortality upon influenza infection. γδ T cells and IL-33 promoted lung infiltration of group 2 innate lymphoid cells and regulatory T cells, resulting in increased amphiregulin secretion and tissue repair. In influenza-infected children, IL-17A, IL-33, and amphiregulin expression were correlated, and increased IL-17A levels in nasal aspirates were associated with better clinical outcomes. Our results indicate that γδ T cells are required in influenza-infected neonates to initiate protective immunity and mediate lung homeostasis.

Metabolic signaling directs the reciprocal lineage decisions of αß and γδ T cells.

The interaction between extrinsic factors and intrinsic signal strength governs thymocyte development, but the mechanisms linking them remain elusive. We report that mechanistic target of rapamycin complex 1 (mTORC1) couples microenvironmental cues with metabolic programs to orchestrate the reciprocal development of two fundamentally distinct T cell lineages, the αß and γδ T cells. Developing thymocytes dynamically engage metabolic programs including glycolysis and oxidative phosphorylation, as well as mTORC1 signaling. Loss of RAPTOR-mediated mTORC1 activity impairs the development of αß T cells but promotes γδ T cell generation, associated with disrupted metabolic remodeling of oxidative and glycolytic metabolism. Mechanistically, we identify mTORC1-dependent control of reactive oxygen species production as a key metabolic signal in mediating αß and γδ T cell development, and perturbation of redox homeostasis impinges upon thymocyte fate decisions and mTORC1-associated phenotypes. Furthermore, single-cell RNA sequencing and genetic dissection reveal that mTORC1 links developmental signals from T cell receptors and NOTCH to coordinate metabolic activity and signal strength. Our results establish mTORC1-driven metabolic signaling as a decisive factor for reciprocal αß and γδ T cell development and provide insight into metabolic control of cell signaling and fate decisions.

Severe Influenza Is Characterized by Prolonged Immune Activation: Results From the SHIVERS Cohort Study.

Background: The immunologic factors underlying severe influenza are poorly understood. To address this, we compared the immune responses of influenza-confirmed hospitalized individuals with severe acute respiratory illness (SARI) to those of nonhospitalized individuals with influenza-like illness (ILI). Methods: Peripheral blood lymphocytes were collected from 27 patients with ILI and 27 with SARI, at time of enrollment and then 2 weeks later. Innate and adaptive cellular immune responses were assessed by flow cytometry, and serum cytokine levels were assessed by a bead-based assay. Results: During the acute phase, SARI was associated with significantly reduced numbers of circulating myeloid dendritic cells, CD192+ monocytes, and influenza virus-specific CD8+ and CD4+ T cells as compared to ILI. By the convalescent phase, however, most SARI cases displayed continued immune activation characterized by increased numbers of CD16+ monocytes and proliferating, and influenza virus-specific, CD8+ T cells as compared to ILI cases. SARI was also associated with reduced amounts of cytokines that regulate T-cell responses (ie, interleukin 4, interleukin 13, interleukin 12, interleukin 10, and tumor necrosis factor ß) and hematopoiesis (interleukin 3 and granulocyte-macrophage colony-stimulating factor) but increased amounts of a proinflammatory cytokine (tumor necrosis factor α), chemotactic cytokines (MDC, MCP-1, GRO, and fractalkine), and growth-promoting cytokines (PDGFBB/AA, VEGF, and EGF) as compared to ILI. Conclusions: Severe influenza cases showed a delay in the peripheral immune activation that likely led prolonged inflammation, compared with mild influenza cases.

New fronts emerge in the influenza cytokine storm.

Influenza virus is a significant pathogen in humans and animals with the ability to cause extensive morbidity and mortality. Exuberant immune responses induced following infection have been described as a "cytokine storm," associated with excessive levels of proinflammatory cytokines and widespread tissue damage. Recent studies have painted a more complex picture of cytokine networks and their contributions to clinical outcomes. While many cytokines clearly inflict immunopathology, others have non-pathological delimited roles in sending alarm signals, facilitating viral clearance, and promoting tissue repair, such as the IL-33-amphiregulin axis, which plays a key role in resolving some types of lung damage. Recent literature suggests that type 2 cytokines, traditionally thought of as not involved in anti-influenza immunity, may play an important regulatory role. Here, we discuss the diverse roles played by cytokines after influenza infection and highlight new, serene features of the cytokine storm, while highlighting the specific functions of relevant cytokines that perform unique immune functions and may have applications for influenza therapy.

Stress Kinase GCN2 Controls the Proliferative Fitness and Trafficking of Cytotoxic T Cells Independent of Environmental Amino Acid Sensing.

GCN2 is one of four "stress kinases" that block translation by phosphorylating eIF2α. GCN2 is thought to bind uncharged tRNAs to "sense" amino acid availability. In mammals, myeloid cells expressing indoleamine dioxygenases locally deplete tryptophan, which is detected by GCN2 in T cells to cause proliferative arrest. GCN2-deficient T cells were reported to ectopically enter the cell cycle when tryptophan was limiting. Using GCN2-deficient strains crossed to T cell receptor (TCR) transgenic backgrounds, we found GCN2 is essential for induction of stress target genes such as CHOP. However, GCN2-deficient CD8+ T cells fail to proliferate in limiting tryptophan, arginine, leucine, lysine, or asparagine, the opposite of what previous studies concluded. In vitro and in vivo proliferation experiments show that GCN2-deficient CD8+ T cells have T cell-intrinsic proliferative and trafficking defects not observed in CD4+ T cells. Thus, GCN2 is required for normal cytotoxic T cell function.

Rapid cloning, expression, and functional characterization of paired αß and γδ T-cell receptor chains from single-cell analysis.

Transgenic expression of antigen-specific T-cell receptor (TCR) genes is a promising approach for immunotherapy against infectious diseases and cancers. A key to the efficient application of this approach is the rapid and specific isolation and cloning of TCRs. Current methods are often labor-intensive, nonspecific, and/or relatively slow. Here, we describe an efficient system for antigen-specific αßTCR cloning and CDR3 substitution. We demonstrate the capability of cloning influenza-specific TCRs within 10 days using single-cell polymerase chain reaction (PCR) and Gibson Assembly techniques. This process can be accelerated to 5 days by generating receptor libraries, requiring only the exchange of the antigen-specific CDR3 region into an existing backbone. We describe the construction of this library for human γδ TCRs and report the cloning and expression of a TRGV9/TRDV2 receptor that is activated by zoledronic acid. The functional activity of these αß and γδ TCRs can be characterized in a novel reporter cell line (Nur77-GFP Jurkat 76 TCRα(-)ß(-)) for screening of TCR specificity and avidity. In summary, we provide a rapid method for the cloning, expression, and functional characterization of human and mouse TCRs that can assist in the development of TCR-mediated therapeutics.

Diverse heterologous primary infections radically alter immunodominance hierarchies and clinical outcomes following H7N9 influenza challenge in mice.

The recent emergence of a novel H7N9 influenza A virus (IAV) causing severe human infections in China raises concerns about a possible pandemic. The lack of pre-existing neutralizing antibodies in the broader population highlights the potential protective role of IAV-specific CD8(+) cytotoxic T lymphocyte (CTL) memory specific for epitopes conserved between H7N9 and previously encountered IAVs. In the present study, the heterosubtypic immunity generated by prior H9N2 or H1N1 infections significantly, but variably, reduced morbidity and mortality, pulmonary virus load and time to clearance in mice challenged with the H7N9 virus. In all cases, the recall of established CTL memory was characterized by earlier, greater airway infiltration of effectors targeting the conserved or cross-reactive H7N9 IAV peptides; though, depending on the priming IAV, each case was accompanied by distinct CTL epitope immunodominance hierarchies for the prominent K(b)PB(1703, D(b)PA(224), and D(b)NP(366) epitopes. While the presence of conserved, variable, or cross-reactive epitopes between the priming H9N2 and H1N1 and the challenge H7N9 IAVs clearly influenced any change in the immunodominance hierarchy, the changing patterns were not tied solely to epitope conservation. Furthermore, the total size of the IAV-specific memory CTL pool after priming was a better predictor of favorable outcomes than the extent of epitope conservation or secondary CTL expansion. Modifying the size of the memory CTL pool significantly altered its subsequent protective efficacy on disease severity or virus clearance, confirming the important role of heterologous priming. These findings establish that both the protective efficacy of heterosubtypic immunity and CTL immunodominance hierarchies are reflective of the immunological history of the host, a finding that has implications for understanding human CTL responses and the rational design of CTL-mediated vaccines.

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study.

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.

[Reconstructed seminiferous tubules from ectopic grafting of testicular cells].

OBJECTIVE: To investigate the ectopic grafts of mouse testicular cells by observing the reconstruction of seminiferous tubules, colonization of spermatogenic cells and spermatogenesis using immunodeficient mice as recipients. METHODS: The testes of newborn male ICR mice were digested to obtain single cell suspension. The cells were then mixed with matrigel and subcutaneously grafted into the dorsal region of the male nude mice. The mice were castrated after the operation and the grafts were dissected from 5 of the nude mice at 4, 6, 8 and 10 weeks, respectively. The success rates of transplantation and the graft diameters were calculated, and the structure of the reconstituted seminiferous tubules, colonization of the germ cells and spermatogenesis were observed by HE staining and immunohistochemistry. RESULTS: All the mice recipients survived after the testicular cell transplantation. Within 10 weeks after the operation, tissue masses could be observed, with the diameter increased from (3.91 +/- 0.71) mm at 4 weeks to (6.69 +/- 0.50) mm. Neovascularization was detected at the surface of the masses and seminiferous tubule structures found in the grafts. The germ cells that developed from spermatogonia to round spermatids were observed, but with no sperm in the tubules. Germ cells, Sertoli cells and Leydig cells were identified by immunochemical detection of Mvh, Gata4 and P450Scc in the grafts at 8 weeks. CONCLUSION: Seminiferous tubules could be ectopically reconstructed from suspension of neonatal mouse testicular cells. Ectopic grafting provided a preferable model for the studies on testis tissue engineering and interactions between testicular cells during testicular development and spermatogenesis.

[Spontaneous differentiation potency of induced pluripotent stem cells into male germ cells in vitro].

OBJECTIVE: To explore the expression profile of male germ cell-associated genes during the spontaneous differentiation of induced pluripotent stem cells (iPS) and assess the potency of their spontaneous differentiation into male germ cells in vitro. METHODS: Embryoid body (EB) formation was used to promote the spontaneous differentiation of iPS into male germ cells, and the expressions of germ cell-associated genes were detected by real-time PCR and PCR. RESULTS: Real-time PCR and PCR revealed different expression levels of relevant genes at different times of iPS spontaneous differentiation into male germ cells. Each of the 9 genes analyzed exhibited one of the four temporal expression patterns: wavelike increase of Oct4, progressive decrease of Dppa3 and Stra8, wavelike decrease of Dazl, and decrease following initial increase of Tex14, Msy2, Scp1, Scp3 and Akap3. CONCLUSION: Induced pluripotent stem cells express male germ cell-associated genes and male haploid genes during their spontaneous differentiation through EB formation, and have the potency of differentiating into male gametes.

[The construction and screening of genomic library based on transformable artificial chromosome (TAC) vector in Lotus japonicus].

Many vectors, especially artificial chromosome vectors, have been developed for genome-scale mapping and sequencing. As the first artificial chromosome, YAC has been extensively used in the genomic library construction and mapping. Shizuya et al. (1992) devised the bacterial artificial chromosome (BAC) originated from F factor of E.coli, which is much easy to handle and isolated with large harboring capacity. Liu et al. (1999) designed the transformation-competent artificial chromosome (TAC) vector, derived from PAC vector. TAC could shuttle a large-scale DNA fragment between bacteria and Agrobaceria. Further, it could integrate a large targetted DNA fragment into plant genome, as being documented in Arabidopsis and rice genome research (Liu et al. 2000, 2002). The time-saving virtue of TAC should be significant in genomics research in Lotus japonicus, a model plant of legume. Using a nuclei-based method of Liu and Whittier, high molecular weight DNA was isolated from Lotus japonicus (Gifu ecotype). The DNA was digested partially with Hind III and size-fractionated in the 10- to 20-kb size range as described (Liu and Whittier 1994). The partially digested and size selected DNA fragments were ligated with Hind III-digested pYLTAC7 and then used for transformation of E. coli DH10B by electroporation. Transformants carrying inserts were selected on LB agar plates containing 25 mg/L kanamycin and 5% sucrose. The library, 6 haploid genome equivalents, was pooled in 12 96-well microtiter plates at about 150 transformants per well. The TAC library was then arrayed in nylon membranes and subjected to screening. The probe, a homolog fragment of CEN gene controlling the structure of inflorescence Antirrhinum, was used for screening. 0.5 muL solution from the positive pool was titered and the secondary screening was conducted in the same way. Finally, these positive clonies were confirmed by Southern blot. These data showed that this genomic library was reliable for further molecular research in Lotus japonicus.

Cloning of LjCYC1 gene and nuclear localization of LjCYC1 protein in Lotus japonicus.

The LjCYC1 (Lotus japonicus Cycloidea-like 1) gene, a homolog of CYC (Cycloidea) belonging to the TCP [TB1(teosinte branched 1), CYC, PCFs (PCF1 and PCF2)] gene family and encoding a predicted transcription factor and being proposed controlling different aspects of plant development, was isolated from the papilionaceous plant Lotus japonicus by screening the genomic DNA library, in order to test the functional conservation and divarication of CYC-like genes in legume. Sequence analyses indicate that LjCYC1 gene contains two exons and one intron and encodes a 370-AA peptide LjCYC1. The putative protein, LjCYC1, contains a TCP domain and an R domain, being a member of the CYC/TB1 subfamily of TCP family, and has 39.0% identity with and 42.6% similarity to CYC. LjCYC1-cDNA was cloned through RT-PCR. Different regions of the LjCYC1-cDNA were fused with the report gene GUS and then the fused constructs were transiently expressed in the onion epidermal cells through particle bombardment. Results of GUS and DAPI staining showed that the chimeric proteins with TCP domain were localized within the nucleus, confirming that LjCYC1 may act as a transcription factor. But the TCP domain itself could not confer the nuclear localization because the chimeric proteins with TCP domain alone were dispersed all over the transformed cells.