Laurent Brossay

Targeting natural killer cells and natural killer T cells in cancer.

Natural killer (NK) cells and natural killer T (NKT) cells are subsets of lymphocytes that share some phenotypical and functional similarities. Both cell types can rapidly respond to the presence of tumour cells and participate in antitumour immune responses. This has prompted interest in the development of innovative cancer therapies that are based on the manipulation of NK and NKT cells. Recent studies have highlighted how the immune reactivity of NK and NKT cells is shaped by the environment in which they develop. The rational use of these cells in cancer immunotherapies awaits a better understanding of their effector functions, migratory patterns and survival properties in humans.

NK cells and NKT cells in innate defense against viral infections

NK cells contribute to innate defense during certain viral infections, but the mechanisms for their regulation and delivery of antiviral effects are incompletely understood. A second NK cell population, from within T cell populations--NKT cells--has a unique potential to initiate cellular effector mechanisms, including those delivered by NK cells, provided that the antigen for their restricted TCR is induced during infection. If elicited, particular innate cytokine responses promote activation of NK cell cytotoxicity or IFN-gamma production. These responses can contribute to defense by mediating antiviral and/or immunoregulatory effects. Roles of positive or negative receptors for target cells in protection against viruses are less clear. Exciting new data indicate that, in at least one system, NK cell receptors that positively signal for activation participate in the recruitment of these cells into antiviral defense mechanisms. Other recent evidence suggests that NKT cells may be important for protection during one viral infection and may be artificially activated by delivery of antigen to promote antiviral defense. Taken together, these recent advances in the characterization of the NK and NKT cell responses are filling in the details of the complex and critical events taking place, at the earliest times after challenge, to promote resistance to viruses.

Immunization with α‐galactosylceramide polarizes CD1‐reactive NK T cells towards Th2 cytokine synthesis

We have compared the immune responses of mice immunized either with α‐galactosylceramide (α‐GalCer), capable of eliciting a CD1‐metiated stimulation of Vα14+ NK Tu2004cells, or with lipoarabinomannan (LAM), a glycophospholipid derived from mycobacteria which is known to be presented by CD1b in humans. Within 24u2004h, α‐GalCer induces a burst of IFN‐γ secretion inu2004vivo, and recall with antigen inu2004vitro leads to the synthesis of IL‐4 and IL‐10 in addition to IFN‐γ. Associated with this inu2004vivo cytokine release is a polyclonal activation of splenic B and Tu2004cells. CD1‐reactive NK Tu2004lymphocytes mediate these events, because none of them are observed in α‐GalCer‐immunized CD1−u2009/u2009− mice. LAM immunization fails to promote similar early responses inu2004vivo. Repeated exposure of mice to α‐GalCer induces splenic Tu2004cells to secrete IL‐4 and IL‐10 but dramatically reduced levels of IFN‐γ. Such a biasin the cytokine balance triggered by NK Tu2004cells stimulated with multiple doses of α‐GalCer suggests that this compound might be useful in the induction of Th2 immune responses and the prevention of chronic inflammatory conditions mediated by Th1 cytokines.

PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae

Neisseria gonorrhoeae, the Gram‐negative aetiological agent of gonorrhoea, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp‐dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal pilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tfp. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in pilT. Piliated pilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence‐specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT‐deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.

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-γ 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-γ production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN-γ upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN-γ 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-γ mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.

Cutting Edge: Inhibitory Functions of the Killer Cell Lectin-Like Receptor G1 Molecule During the Activation of Mouse NK Cells

The killer cell lectin-like receptor G1 (KLRG1) is the mouse homolog of the rat mast cell function-associated Ag and contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. In this study we demonstrate that both pathogenic and nonpathogenic in vivo activation of NK cells induces the expression of KLRG1 on their cell surface. Upon infection with murine CMV, this induction peaks between days 5 and 7 with ∼90% of the NK cells expressing KLRG1. On day 1.5 post-murine CMV infection of C57BL/6 mice, the main producers of IFN-γ are the KLRG1-negative NK cells. This effect has been recapitulated in vitro as we show that engagement of KLRG1 on a transfected NK cell line inhibits both cytokine production and NK cell-mediated cytotoxicity. Taken together, these data illustrate the crucial role played by KLRG1 during the termination of mouse NK cell activation.

Expansion and Contraction of the NK Cell Compartment in Response to Murine Cytomegalovirus Infection

NK cells are capable of responding quickly to infectious challenge and contribute to the early defense against a wide variety of pathogens. Although the innate NK cell response to murine CMV (MCMV) has been extensively characterized, its resolution and the fate of the activated NK cell population remains unexplored. Herein, we characterize both the expansion and contraction phases of the NK cell response to MCMV. We demonstrate that NK cell recruitment into the immune response to MCMV infection is restricted to the first 3 days of infection and as the peripheral NK cell compartment expands, NK cells undergo accelerated phenotypic maturation. During the resolution of the immune response, NK cell compartmental contraction is marked by the selective death of responding NK cells. Additionally, throughout the infection, a naive NK cell pool that remains responsive to additional stimuli is actively maintained. These findings illustrate the plasticity of the NK cell compartment in response to pathogens and underscore the homeostatic maintenance of the resting peripheral NK cell pool.

NK Cell–Like Behavior of Vα14i NK T Cells during MCMV Infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Vα14 invariant natural killer T cell response to MCMV has not been examined. We found that Vα14i NK T cells become activated and produce significant levels of IFN-γ, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Vα14i NK T cells into MCMV-infected CD1d−/− mice demonstrate that CD1d is dispensable for Vα14i NK T cell activation. In contrast, both IFN-α/β and IL-12 are required for optimal activation. Vα14i NK T cell–derived IFN-γ is partially dependent on IFN-α/β but highly dependent on IL-12. Vα14i NK T cells contribute to the immune response to MCMV and amplify NK cell–derived IFN-γ. Importantly, mortality is increased in CD1d−/− mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Vα14i NK T cells that act as effector T cells during bacterial infection, but have NK cell–like behavior during the innate immune response to MCMV infection.

Membrane Lymphotoxin Is Required for the Development of Different Subpopulations of NK T Cells

The development of lymphoid organs requires membrane-bound lymphotoxin (LT), a heterotrimer containing LTα and LTβ, but the effects of LT on T cell function have not been characterized extensively. Upon TCR cross-linking in vitro, splenocytes from both LTα−/− and LTβ−/− mice failed to produce IL-4 and IL-10 due to a reduction in NK T cells. Concordantly, LTα−/− and LTβ−/− mice did not respond to the lipoglycan α-galactosylceramide, which is presented by mouse CD1 to Vα14+ NK T cells. Interestingly, both populations of NK T cells, including those that are mouse CD1 dependent and α-galactosylceramide reactive and those that are not, were affected by disruption of the LTα and LTβ genes. NK T cells were not affected, however, in transgenic mice in which LT signaling is blocked, beginning on day 3 after birth, by expression of a soluble decoy LTβ receptor. This suggests that membrane-bound LT is critical for NK T cells early in ontogeny, but not for the homeostasis of mature cells.

Diverse CD1d-restricted reactivity patterns of human T cells bearing "invariant" AV24BV11 TCR

Human and murine natural T (NT) cells, also referred to as NK1.1+ or NK T cells, express TCR with homologous V regions (hAV24/BV11 and mAV14/BV8, respectively) and conserved “invariant” TCR AVAJ junctional sequences, suggesting recognition of closely related antigens. Murine NT cells recognize CD1‐expressing cells and are activated in a CD1‐restricted fashion by several synthetic α‐glycosylceramides, such as α‐GalCer. Here we studied the reactivity of human T cells against CD1d+ cells pulsed or not with α‐GalCer and other related ceramides. CD1d‐restricted recognition of α‐GalCer was a general and specific feature of T cell clones expressing both BV11 and canonical AV24AJ18 TCR chains. Besides, human and murine NT cells showed the same reactivity patterns against a set of related glycosylceramides, suggesting a highly conserved mode of recognition of these antigens in humans and rodents. We also identified several AV24BV11 T cell clones self reactive against CD1+ cells of both hemopoietic and nonhemopoietic origin, suggesting the existence of distinct NT cell subsets differing by their ability to recognize self CD1d molecules.