Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics.

The activation of T cells by the T cell antigen receptor (TCR) results in the formation of signaling protein complexes (signalosomes), the composition of which has not been analyzed at a systems level. Here, we isolated primary CD4+ T cells from 15 gene-targeted mice, each expressing one tagged form of a canonical protein of the TCR-signaling pathway. Using affinity purification coupled with mass spectrometry, we analyzed the composition and dynamics of the signalosomes assembling around each of the tagged proteins over 600 s of TCR engagement. We showed that the TCR signal-transduction network comprises at least 277 unique proteins involved in 366 high-confidence interactions, and that TCR signals diversify extensively at the level of the plasma membrane. Integrating the cellular abundance of the interacting proteins and their interaction stoichiometry provided a quantitative and contextual view of each documented interaction, permitting anticipation of whether ablation of a single interacting protein can impinge on the whole TCR signal-transduction network.

The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation.

Natural killer (NK) cells play critical roles defending against tumors and pathogens. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis.

Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor.

Consistent with their function in immune surveillance, natural killer (NK) cells are distributed throughout lymphoid and nonlymphoid tissues. However, the mechanisms governing the steady-state trafficking of NK cells remain unknown. The lysophospholipid sphingosine 1-phosphate (S1P), by binding to its receptor S1P1, regulates the recirculation of T and B lymphocytes. In contrast, S1P5 is detected in the brain and regulates oligodendrocyte migration and survival in vitro. Here we show that S1P5 was also expressed in NK cells in mice and humans and that S1P5-deficient mice had aberrant NK cell homing during steady-state conditions. In addition, we found that S1P5 was required for the mobilization of NK cells to inflamed organs. Our data emphasize distinct mechanisms regulating the circulation of various lymphocyte subsets and raise the possibility that NK cell trafficking may be manipulated by therapies specifically targeting S1P5.

Cutting Edge: Eomesodermin Is Sufficient To Direct Type 1 Innate Lymphocyte Development into the Conventional NK Lineage.

Type 1 innate lymphocytes comprise two developmentally divergent lineages, type 1 helper innate lymphoid cells (hILC1s) and conventional NK cells (cNKs). All type 1 innate lymphocytes (ILCs) express the transcription factor T-bet, but cNKs additionally express Eomesodermin (Eomes). We show that deletion of Eomes alleles at the onset of type 1 ILC maturation using NKp46-Cre imposes a substantial block in cNK development. Formation of the entire lymphoid and nonlymphoid type 1 ILC compartment appears to require the semiredundant action of both T-bet and Eomes. To determine if Eomes is sufficient to redirect hILC1 development to a cNK fate, we generated transgenic mice that express Eomes when and where T-bet is expressed using Tbx21 locus control to drive expression of Eomes codons. Ectopic Eomes induces cNK-like properties across the lymphoid and nonlymphoid type 1 ILC compartments. Subsequent to their divergent lineage specification, hILC1s and cNKs thus possess substantial developmental plasticity.

Cutting edge: CXCR4 is critical for CD8+ memory T cell homeostatic self-renewal but not rechallenge self-renewal.

Central memory (CM) CD8(+) T cells "remember" prior encounters because they maintain themselves through cell division in the absence of ongoing challenge (homeostatic self-renewal), as well as reproduce the CM fate while manufacturing effector cells during secondary Ag encounters (rechallenge self-renewal). We tested the consequence of conditional deletion of the bone marrow homing receptor CXCR4 on antiviral T cell responses. CXCR4-deficient CD8(+) T cells have impaired memory cell maintenance due to defective homeostatic proliferation. Upon rechallenge, however, CXCR4-deficient T cells can re-expand and renew the CM pool while producing secondary effector cells. The critical bone marrow-derived signals essential for CD8(+) T cell homeostatic self-renewal appear to be dispensable to yield self-renewing, functionally asymmetric cell fates during rechallenge.

Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor.

NKp46 is a cell surface receptor expressed on natural killer (NK) cells, on a minute subset of T cells, and on a population of innate lymphoid cells that produce IL-22 and express the transcription factor retinoid-related orphan receptor (ROR)-γt, referred to as NK cell receptor (NKR)(+)ROR-γt(+) cells. Here we describe Nkp46(iCre) knock-in mice in which the gene encoding the improved Cre (iCre) recombinase was inserted into the Nkp46 locus. This mouse was used to noninvasively trace cells expressing NKp46 in vivo. Fate mapping experiments demonstrated the stable expression of NKp46 on NK cells and allowed a reappraisal of the sequential steps of NK cell maturation. NKp46 genetic tracing also showed that gut NKR(+)ROR-γt(+) and NK cells represent two distinct lineages. In addition, the genetic heterogeneity of liver NK cells was evidenced. Finally, Nkp46(iCre) mice also represent a unique mouse model of conditional mutagenesis specifically in NKp46(+) cells, paving the way for further developments in the biology of NKp46(+) NK, T, and NKR(+)ROR-γt(+) cells.

Prior SARS-CoV-2 infection enhances and reshapes spike protein-specific memory induced by vaccination.

The diversity of vaccination modalities and infection history are both variables that have an impact on the immune memory of individuals vaccinated against SARS-CoV-2. To gain more accurate knowledge of how these parameters imprint on immune memory, we conducted a long-term follow-up of SARS-CoV-2 spike protein-specific immune memory in unvaccinated and vaccinated COVID-19 convalescent individuals as well as in infection-naïve vaccinated individuals. Here, we report that individuals from the convalescent vaccinated (hybrid immunity) group have the highest concentrations of spike protein-specific antibodies at 6 months after vaccination. As compared with infection-naïve vaccinated individuals, they also display increased frequencies of an atypical mucosa-targeted memory B cell subset. These individuals also exhibited enhanced TH1 polarization of their SARS-CoV-2 spike protein-specific follicular T helper cell pool. Together, our data suggest that prior SARS-CoV-2 infection increases the titers of SARS-CoV-2 spike protein-specific antibody responses elicited by subsequent vaccination and induces modifications in the composition of the spike protein-specific memory B cell pool that are compatible with enhanced functional protection at mucosal sites.

Maturation of mouse NK cells is a 4-stage developmental program.

Surface density of CD27 and CD11b subdivides mouse natural killer (NK) cells into 4 subsets: CD11b(low)CD27(low), CD11b(low)CD27(high), CD11b(high)CD27(high), and CD11b(high)CD27(low). To determine the developmental relationship between these 4 subsets, we used several complementary approaches. First, we took advantage of NDE transgenic mice that express enhanced green fluorescent protein (EGFP) and diphtheria toxin receptor specifically in NK cells. Diphtheria toxin injection leads to a transient depletion of NK cells, allowing the monitoring of the phenotype of developing EGFP+ NK cells after diphtheria toxin injection. Second, we evaluated the overall proximity between NK-cell subsets based on their global gene profile. Third, we compared the proliferative capacity of NK-cell subsets at steady state or during replenishment of the NK-cell pool. Fourth, we performed adoptive transfers of EGFP+ NK cell subsets from NDE mice into unirradiated mice and followed the fate of transferred cells. The results of these various experiments collectively support a 4-stage model of NK-cell maturation CD11b(low)CD27(low) --> CD11b(low)CD27(high) --> CD11b(high)CD27(high) --> CD11b(high)CD27(low). This developmental program appears to be associated with a progressive acquisition of NK-cell effector functions.

Natural killer cells: from CD3(-)NKp46(+) to post-genomics meta-analyses.

The original definition of NK cells was based on their 'natural' cytolytic response against tumor cells and virus-infected cells in the absence of specific immunization. However, the term 'natural killer' reflects neither the education/maturation requirements before NK cells can kill nor the entirety of their biological functions. In light of new functional assays, genetic models and genomics analysis, we propose a more accurate definition of NK cells. This definition includes the phenotypical identification of NK cells as CD3(-)NKp46(+) cells across mammalian species. In general, this attempt to redefine NK cells also highlights the need to update the operational definition of cell types in the post-genomic area.

Cutting edge: Priming of NK cells by IL-18.

Recent evidence suggests that NK cells require priming to display full effector activity. In this study, we demonstrate that IL-18 contributed to this phenomenon. IL-18 signaling-deficient NK cells were found to be unable to secrete IFN-gamma in response to ex vivo stimulation with IL-12. This was not due to a costimulatory role of IL-18, because blocking IL-18 signaling during the ex vivo stimulation with IL-12 did not alter IFN-gamma production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN-gamma upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN-gamma transcription by NK cells was comparable in IL-18 signaling-deficient and -sufficient NK cells. This suggests that priming by IL-18 leads to an improved translation of IFN-gamma mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.

Characterization and analytical validation of a new antigenic rapid diagnostic test for Ebola virus disease detection.

Hemorrhagic fever outbreaks are difficult to diagnose and control in part because of a lack of low-cost and easily accessible diagnostic structures in countries where etiologic agents are present. Furthermore, initial clinical symptoms are common and shared with other endemic diseases such as malaria or typhoid fever. Current molecular diagnostic methods such as polymerase chain reaction require trained personnel and laboratory infrastructure, hindering diagnostics at the point of need, particularly in outbreak settings. Therefore, rapid diagnostic tests such as lateral flow can be broadly deployed and are typically well-suited to rapidly diagnose hemorrhagic fever viruses, such as Ebola virus. Early detection and control of Ebola outbreaks require simple, easy-to-use assays that can detect very low amount of virus in blood. Here, we developed and characterized an immunoassay test based on immunochromatography coupled to silver amplification technology to detect the secreted glycoprotein of EBOV. The glycoprotein is among the first viral proteins to be detected in blood. This strategy aims at identifying infected patients early following onset of symptoms by detecting low amount of sGP protein in blood samples. The limit of detection achieved by this sGP-targeted kit is 2.2 x 104 genome copies/ml in plasma as assayed in a monkey analytical cohort. Clinical performance evaluation showed a specificity of 100% and a sensitivity of 85.7% when evaluated with plasma samples from healthy controls and patients infected with Zaire Ebola virus from Macenta, Guinea. This rapid and accurate diagnostic test could therefore be used in endemic countries for early detection of infected individuals in point of care settings. Moreover, it could also support efficient clinical triage in hospitals or clinical centers and thus reducing transmission rates to prevent and better manage future severe outbreaks.

Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection.

Natural killer (NK) cell maturation is a tightly controlled process that endows NK cells with functional competence and the capacity to recognize target cells. Here, we found that the transcription factor (TF) Zeb2 was the most highly induced TF during NK cell maturation. Zeb2 is known to control epithelial to mesenchymal transition, but its role in immune cells is mostly undefined. Targeted deletion of Zeb2 resulted in impaired NK cell maturation, survival, and exit from the bone marrow. NK cell function was preserved, but mice lacking Zeb2 in NK cells were more susceptible to B16 melanoma lung metastases. Reciprocally, ectopic expression of Zeb2 resulted in a higher frequency of mature NK cells in all organs. Moreover, the immature phenotype of Zeb2(-/-) NK cells closely resembled that of Tbx21(-/-) NK cells. This was caused by both a dependence of Zeb2 expression on T-bet and a probable cooperation of these factors in gene regulation. Transgenic expression of Zeb2 in Tbx21(-/-) NK cells partially restored a normal maturation, establishing that timely induction of Zeb2 by T-bet is an essential event during NK cell differentiation. Finally, this novel transcriptional cascade could also operate in human as T-bet and Zeb2 are similarly regulated in mouse and human NK cells.

Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46.

Natural killer (NK) cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. However, our understanding of NK cell biology is severely limited by the lack of consensus phenotypic definition of these cells across species, by the lack of specific marker to visualize them in situ, and by the lack of a genetic model where NK cells may be selectively ablated. NKp46/CD335 is an Ig-like superfamily cell surface receptor involved in human NK cell activation. In addition to human, we show here that NKp46 is expressed by NK cells in all mouse strains analyzed, as well as in three common monkey species, prompting a unifying phenotypic definition of NK cells across species based on NKp46 cell surface expression. Mouse NKp46 triggers NK cell effector function and allows the detection of NK cells in situ. NKp46 expression parallels cell engagement into NK differentiation programs because it is detected on all NK cells from the immature CD122(+)NK1.1(+)DX5(-) stage and on a minute fraction of NK-like T cells, but not on CD1d-restricted NKT cells. Moreover, human NKp46 promoter drives NK cell selective expression both in vitro and in vivo. Using NKp46 promoter, we generated transgenic mice expressing EGFP and the diphtheria toxin (DT) receptor in NK cells. DT injection in these mice leads to a complete and selective NK cell ablation. This model paves a way for the in vivo characterization and preclinical assessment of NK cell biological function.

Temporal cross-talk between TCR and STAT signals for CD8 T cell effector differentiation.

The strength and duration of signaling through surface receptors is a primary means of controlling cell fate decisions. In adaptive immunity, Ag-initiated T cell stimulation is secondarily regulated by cytokines. We here summarize evidence for temporal control of a gene expression program in naive CD8 T cells. It is initiated in response to TCR engagement but relies on secondary signaling from cytokine receptors to be sustained and to allow development of full effector capacity. This mechanism permits cytokine receptor signaling to rescue abortive TCR signaling, such as that induced in response to weak or partial TCR agonists. Indeed, limiting TCR-initiated signaling on the Ras/ERK pathway may be complemented by STAT activation. Thus, TCR- and cytokine-driven activation of transcription factors and epigenetic modifications may act in concert in a temporally staggered process to establish the functional program of effector CD8 T cells. Based on gene expression profiling, molecular targets whose activation or inactivation may boost or dampen CD8 T cell effectors are also identified. Manipulation of these targets may, respectively, increase anti-tumor responses or prevent graft-versus-host reactions.