Cellules "tueuses naturelles" = cellules NK (Natural Killer)

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1 Cellules "tueuses naturelles" = cellules NK (Natural Killer)
3e population de lymphocytes … aux frontières de l'immunité innée et de l'immunité adaptative

2 renouvellement ~ bimensuel
Cellules NK CD3+ CD19+ B T 5 - 15% renouvellement ~ bimensuel NK Grands lymphocytes granuleux Sang, Lymphe, Organes Lymphoïdes Foie, Poumon, Intestin, Utérus … Foyers inflammatoires "ni T ni B" : CD3nég CD19nég ~2x109 most peripheral blood NK cells express a large set of activating NK cell receptors expression of any given MHC class I specific inhibitory receptor is restricted to subsets of NK cells.  2 sous-populations 2 marqueurs : intensité d'expression // fonctionnalité : CD56 CD16 = FcRIII 2 types de récepteurs (non réarrangés)  activation : 0 – 1 – quelques R inhibiteurs (nombre et type ≠ selon les clones) plusieurs R activateurs ( pour la plupart des cellules de la sous-population)

3 Cellules NK : Récepteurs CD56 et CD16
>90% des NK des organes lymphoïdes CD56+fort CD16nég/+faible 90% des NK circulantes CD56+faible CD16+ 2 sous-populations CD56 Héparane-sulfate … IgG CD56 (NCAM) 5 domaines Ig and 2 domaines fibronectine type III FcyRIII … FcRy or CD3ζ +- CD56 Récepteur d'adhérence CD16 = FcRIII

4 Récepteurs pour le CMH classe I
KIR Killer Ig-like Receptors 0/1/2/3 … selon les clones principalement clones CD56+faible CD94-NKG2 ~ tous les clones CD56+fort certains clones CD56+faible HLA-B (Bw4) (quelques HLA-A) KIR2DL4 … HLA-G KIR2DL …C1 KIR2 D1 … C2 2 3 L S HLA-C (Asn/Lys80) 94 NKG2 HLA-E Hétérodimères Lectines C +- 2D 3D Killer cell Ig-like Receptors and Leukocyte Immunoglobulin-like Receptors / Ig-like transcripts  Nomenclature of KIR is based upon the number of extracellular immunoglobulin-like domains and the length of the cytoplasmic domain. Receptors with long cytoplasmic domains contain one or two immunoreceptor tyrosine-based inhibitory motif sequences that provide inhibitory function. The short cytoplasmic KIR are activating receptors, which contain a basic amino acid within the transmembrane domain that interacts with an acidic amino acid within the transmembrane domains of a signalling adaptor protein.  2DL2 2DL3  HLA-C1 (80N) no corresponding activating receptor 2DL1 2DS1  HLA-C2 (80K) 3DL1 3DS1  Bw4 A24; 40% of all HLA-B allotypes contain the Bw4 motif. In general, Bw4 allotypes with isoleucine 80 are better ligands for most KIR3DL1 allotypes 3DL2  A11 2DL4  HLA-G ILT2/LIR1  HLA-G > all class I (NK, DN mature, T CD8) The D1 and D2 domains contact the the HLA class I molecule at a site that straddles the C-terminal end of the bound antigenic peptide; certain bound peptides can decrease the affinity of inhibitory KIRs for MHC-I The activating KIR binds HLA class I with an affinity much weaker than that of the paired inhibitory KIR Heterodimeric C-lectins CD94-NKG2A/B/C/E  HLA-E NKG2-A and NKG2-B, are alternative splicing products of a single gene, expressed on ~1/2 of all blood NK cells and a subset of CD8+ T cells ou 1er à être exprimé S L

5 Lectine des ¢ myéloïdes activées (AICL) Homodimères Lectines C
Récepteurs de détresse Protéines de stress ou Protéines tumorales ou Hémagglutinines ou autres Protéines virales NKp80 Lectine des ¢ myéloïdes activées (AICL) NKG2D Protéines de stress apparentées au CMH Homodimères Lectines C +- ~ tous les clones clones activés NKp46 NKp44 NKp30 +- Natural Cytoxicity Receptors NKp30/NCR3  BAT3 (stress), CMV pp65, mycobact, B7-H6 (tumor) NKp44/NCR2 is expressed only by activated NK cells NKp46/NCR1  hemaglutinin potent activation receptors linked to the ITAM-bearing CD3ζ, FcRγ, or DAP12 molecules Homodimeric C-lectins NKG2D  stress-inducible MICA/B (MHC class I chain-related), ULBP1/2/3/4 (UL16-binding proteins) NKp80  AICL (activation-induced C-type lectin on myeloid cells) DNAX accessory molecule-1 (DNAM-1, CD226) an adhesion receptor with 2 Ig-like domains expressed on the majority of NK cells, T cells and monocytes and associated with LFA-1. Activating signal through Fyn and PKC. Recognizes members of the Nectin family: the poliovirus receptor (PVR, CD155) and Nectin-2 (CD112) expressed on healthy neuronal, epithelial, endothelial cells and fibroblasts, and highly expressed on tumor cells  NCR Natural Cytotoxicity Receptors

6 Cellules NK : Intégration des signaux
R activateurs expression sur la plupart des clones NKp46 sur tous les clones R inhibiteurs expression propre au clone KIR principalement sur clones CD56+faible CD16+ Motif ITAM  Tyrosine-Kinases Intrinsèque ou Protéine adaptatrice associée Motif ITIM  Tyrosine-Phosphatases effet prédominant si interaction R - Ligand … et signaux : R associés à l'adhérence (ex. DNAM-1  intégrines, nectines) R de Cytokines : IL-2 IL-15  prolifération, différenciation IFN-/ IL-12 IL-18 IL-1 R de Danger the balance of signals from activating and inhibitory receptors that determines the outcome of NK cell function

7     Tolérance au "soi" Rejet du "non soi" "soi altéré" ou "non soi
"soi manquant"  "soi altéré" ou "non soi activant"  ¢ saine : expression du CMH classe I pas/peu de ligands des R activateurs  Les sous marins de Klas Kärre : un biologiste suédois, qui s’est inspiré d’une tactique adoptée au début des années 80 : on avait demandé assistance aux pêcheurs locaux pour repérer des sous-marins étrangers. Comme il y avait trop de submersibles étrangers susceptibles de se retrouver dans l’espace maritime suédois, on a appris aux pêcheurs à ne distinguer que les sous-marins suédois et à signaler tous les autres. Kärre en déduisit qu’il y avait deux façons de discerner le soi du non-soi : «l’activation après la reconnaissance du non-soi ou l’inhibition après la reconnaissance du soi».  Virus, Cancer … Allogreffe … GR : pas/peu d'expression du CMH pas/peu de ligands des R activateurs

8 CMH classe I : 3 gènes classiques … et d'autres
MICA/B NKG2D Intestin ¢ Dendritiques activées Cancers CD94-NKG2 Fixe le peptide signal de HLA-A/B/C = reflet d'expression du CMH classe I pathologie : CMH et peptide manquant ou remplacé par : Protéines de stress ou d'agents infectieux E 6 NKp30 NKp44 DPA DQA DRA DPB DQB DRB B C A G KIR2DL4 LIR1 Placenta Cancers DP DQ DR B C A Allèles Bw6 C1/C2 The extracellular portions of ULBP and MIC proteins also share homology with the α1/α2 peptide-binding domains of MHC class I molecules. MIC (MHC class I polypeptide-related sequence) proteins do not associate with β2m and do not bind peptide ; expressed on gut epithelium and on tumors UL16-BP are GPI-linked proteins HLA-E  nucleated cells, high levels on neoplastic cells; binds signal peptides from classical HLA class I proteins; stress  hsp-derived peptides; intra-cellular bacteria and viruses encode binding peptides HLA-G  extravillous cytotrophoblasts, tumors … Bw4 L 1/2 S 1 L 1 S 1 KIR3D KIR2D

9 Ségrégation indépendante des gènes des récepteurs
12 NKG2A/B/C/D/E CD94 NKp80 6 NKp30 NKp44 HLA 19 LRC : Complexe des Récepteurs Leucocytaires Leukocyte Receptor Complex Protéines Adaptatrices associées aux R activateurs HLA CLASS I AND KIR INTERACT FUNCTIONALLY BUT SEGREGATE INDEPENDENTLY IN HUMAN POPULATIONS CD94 – NKG2A C D on chr. 12p13. The Leukocyte receptor complex (LRC) on chr. 19q13.4 encodes KIR receptors: Two main haplotypes that vary considerably in terms of differences in gene number, gene content, and allelic polymorphism Group A haplotypes = only inhibitory KIR Group B haplotypes are more variable with 1 or more activating KIR.  LIR receptors (Leukocyte Ig-like Receptors (LILRs/ILTs) NKp46 DAP10 DAP12 FcR and FcRn The TREM gene cluster (Triggering receptors expressed on myeloid cells) in MHC class III on chr. 6p21.33 TREM-1 on neutrophils/monocytes TREM2 on DCs NKp30 NKp44 The most common A-haplotype confers maximal inhibition for all 3 HLA ligands C1, C2 and Bw4 Récepteurs LIR Récepteurs KIR NKp46 2 groupes d'haplotypes : A : uniquement KIR inhibiteurs ( C1, C2 et Bw4) B : KIR inhibiteurs et KIR activateur ( C2 ou Bw4) … et variation du nombre de gènes, plusieurs allèles/gène AA AB BB ~30%

10 au contact des ¢ Dendritiques et Lymphocytes T
Développement des cellules NK Développement Homéostasie Dissémination Pro-NK au contact des ¢ Dendritiques et Lymphocytes T IL-2 IL-15 90% CD56+fort CD16nég CCR7+ NK T IL-2  NCRs, CD16 90% CD56+faible CD16+ Organes Foyer inflammatoire Niche tumorale … CSH IL-15 human NK-cell development. NK cells are generated, are maintained and perform their biological functions within the context of environmental niches CD56bright PB NK cells give rise to CD56dim cells (they appear first after HSCT) BM–derived CD34+CD45RA+ HPCs circulate in the blood  HEVs  parafollicular space: 1–5% of mononuclear cells in lymph nodes are NK cells Blockade of the interaction between CD56  FGF receptor-1 inhibits the generation of CD56dim NK cells CD56dim NK cells return to the circulation via the efferent lymph (> 2x109 circulating, CD62Lneg) CD56bright NK cells express CD62L (and CCR7 in response to IL-18), which direct them to the T-cell area of lymph nodes. Some CD56bright NK cells remain in close association with DC and can produce cytokines within minutes but display poor cytotoxic capacity.  immune response / tumor / pregnancy niches

11  "Education" des cellules NK CD94-NKG2  ± KIR(s) > IL-15 CSH
Activation si la ¢ n'exprime pas la molécule CMH correspondante, même si d'autres molécules CMH sont normalement exprimées CSH Pro-NK IL-15 Pro-NK anergique NK cells are educated by self-MHC-I at an immature stage of development in the bone marrow (but peripheral NK cells from MHC-I-deficient mice become responsive upon transfer into a MHC-I-sufficient host, and  some human NK cells can acquire MHC-I receptors in vitro upon cytokine stimulation).  In lymph nodes, NK cells express high levels of CD94/NKG2A and KIR acquisition in LN appears to be confined to NK cells that have been previously subjected to the education process upon recognition of HLA-E MHC-I recognition by inhibitory receptors improves the function of activating NK cell receptors:  ‘disarming’ model (unopposed activation by normal host cells induces NK cell hyporesponsiveness) ‘licensing’ or ‘arming’ model (signals delivered by self-MHC-I receptors render activation pathways competent to respond to stimulation) cis interaction’ model (expression of an unengaged MHC-I receptor dampens the response of activation receptors to stimulation) ? the reactivity of mature NK cells has to be maintained by continuous recognition of self-MHC-I NK cell distinguish normal healthy cells from sensitive target cells by a balance between signals from activating and inhibitory receptors  NK cell ‘tuning’  (related to how many inhibitory self-MHC-I receptors NK cells express, combined with the affinity of these receptors for MHC-I)  frequency of individual NK cells and magnitude of their response NK cell memory ? following viral infection  longevity (>2 months), improved cytolytic function and cytokine production and robust recall expansion Activating stimuli may be delivered to NK cells through triggering via TLRs (TLR2, TLR3, TLR7/8, TLR9) and interleukin receptors (IL-1, IL-2, IL-12, IL-15, IL-18, IFNAR…)

12 Cellules NK : des clones à expression ≠ de R inhibiteurs et 2 sous-populations à fonction partiellement ≠ >90% des NK des organes lymphoïdes, de l'utérus 90% des NK circulantes CD56+fort CD16nég ≠ clones KIRnég/(+) mais CD94-NKG2A+ CD56+faible CD16+ ≠ clones portant des KIR ≠ et beaucoup portant NKG2D expression ~ de R activateurs IL-2 IL-15 IL-12 IL-18 IFN-/ IL-1 Cytotoxicité +++ Cytokines Chimiokines Soi manquant, Soi altéré ou Non Soi CD56bright NK cells are more prominent cytokine and chemokine producers than CD56dim  NK cells CD56bright NK cells are poorly cytolytic, do not express inhibitory KIRs, LIR1/ILT2, FcγR, or perforin, but CD94/NKG2A is expressed at a higher density on all CD56bright NK cells. NKp46 is usually at higher density on the CD56bright population Cytokines Chimiokines +++

13 Cytotoxicité cellulaire Ac-dépendante
CD56+faible CD16+ contact (intégrines, DNAM-1…) polarisation dégranulation  TNF Perforine Granzymes Ligands mortifères FasL TRAIL apoptose NK Naturelle Cytotoxicité directe NK CD16 = FcRIII Ac IgG Cytotoxicité cellulaire Ac-dépendante Lytic granules are secretory lysosomes that contain a dense core, including perforin and granzymes. The combined function of these proteins leads to the generation of pores on the plasma membrane and activation of the caspase cascade after cleavage of caspase-3. The core of lytic granules is surrounded by a lipid bilayer that contains Fas ligand  engagement of integrin LFA-1 by ICAM-1 signals for polarization; whereas co-engagement of activating receptors by their ligands triggers degranulation. In activated NK cells the responsiveness of the natural cytotoxicity receptors is strongly upregulated

14 IFN- Production de Cytokines TNF
CD56+fort CD16nég > CD56+faible CD16+ IFN- GM-CSF NK IL-10 TNF Chimiokines CCL3/4/5 IL-8 The intensity and the quality of NK cell cytotoxic and cytokine responses depend on the cytokine microenvironment and interactions with T cells, DCs and macrophages NK cell activation by DCs requires direct cell-to-cell contact, including NKG2D recognition of MICA/B stress ligands upregulated on IFN-α-stimulated mDCs cytokines produced by NK cells are TNF and IFN-γ + several immunoregulatory cytokines: IL-10, IL-13, GM-CSF, and chemokines IL-8, CCL3/4/5 (MIP-1α, MIP-1β, RANTES). … dépendante du contexte d'activation

15 Recrutement des NK  foyer inflammatoire
Chimiokines inflammatoires Intégrines 1 et 2 DNAM-1 … CD56+fort NK Cytokines Chimiokines NK CD16+ CD56+faible  NCRs  Perforine granzymes IL-15 IL-12 IL-18 IFN- Cytotoxicité A crucial role in the transendothelial migration process of NK cells is played by DNAM. Its ligands, PVR (poliovirus R) and Nectin-2, are present at the cell junctions on vascular endothelial cells. NK cell recruitment involves IL-8, CCL3 (MIP1-) and CX3CL1.Most CD56low CD16+ NK cells express CXCR1 and CX3CR1 in sites of peripheral inflammation, CD56bright NK cells represent 40–60% of all NK cells. Upon stimulation by IL-12, IL-15, IL-18, they produce IFN-γ and increase significantly the percentage of TNF-producing monocytes thru a bidirectional direct physical contact Upregulation of NK-mediated killing is greatly influenced by the type of cytokines released by bystander cells during innate immune responses. The apoptotic/necrotic material or heat-shock proteins that are generated as a result of the NK-mediated killing of tumors or virus-infected cells can modulate the function of DC or other bystander cells and can prime DCs to promote Th1 responses and induce cross-presentation/cross-priming of CTLs NK cells recruited to sites of inflammation acquire the capability of killing immature myeloid DCs, since immature DCs underexpress HLA-class I molecules. That may serve to keep in check the quality and the quantity of DCs undergoing maturation (‘editing’ process). NK cell receptors involved in DC recognition and killing are NKp30 and DNAM-1. Killing of DCs can be inhibited by TGF-β, that downregulates the surface expression of NKp30. Conversely, activated NK cells can induce cell-contact dependent maturation of DCs via their secretion of IFN-γ and TNF. The cross talk between NK and DCs might change when DCs are subjected to viral infection. Influenza-infected human DCs express the ligands for both NKG2D (ULBP) and NKp46 (hemagglutinin) activating receptors and the upregulation of NK cell-mediated cytotoxicity mainly depends on IFN-α, whereas IL12 is only necessary for enhancing IFN-γ production by NK cells. Débris cellulaires  R de Danger   capture d'Ag

16 Production de Cytokines ± Cytotoxicité
 réplication virale  infection (VIH) MΦ activation MΦ  microbicidie  MΦ infectés (CMH non ) Neu IL-8 IFN- TNF CCL3/4/5 IFN- TNF CCL3/4/5 IL-12 IL-15 IFN-/  prolifération tumorale NK IFN- TNF D  maturation (NKp30 NKG2D)  DC immatures (CMH non ) TNF GM-CSF T CD4  polarisation Th1/Tc1  recrutement LcT CCR5+ T CD8 IFN- NK cell–mediated cytotoxicity and cytokine production impact DC, macrophages, and neutrophils and endow NK cells with regulatory function affecting subsequent antigen-specific T and B cell responses.  NK cells as regulatory cells NK cells can kill immature DC in humans. NK cell-mediated elimination of immature DCs is prompted by low expression of MHC class I and consequent low amounts of HLA-E expression, is mediated by KIR-NKG2A+ NK cells through the activating receptor NKp30 Conversely, the killing of target cells by NK cells can lead to the cross-presentation of target cell Ags by subsets of DCs. NK cell-mediated maturation of DCs is cell-contact dependent, and might involve interaction between NKp30 with its still undefined cellular ligand on the surface of the immature DCs Their early cytokines, such as IFN-γ and TNF ensure that DC maturation is skewed towards a Th1 response IL-2 IFN- IL-12 IL-15 IFN-

17 Principales fonctions des cellules NK dans l'immunité
allogreffes de cellules souches hématopoïétiques (≠ allotypes CMH) Rejet effet anti-leucémique Interférons  et  NK IFN- Immunité adaptative virus jours NK NK CD56+CD16neg HLA-C HLA-G HLA-E trophoblaste Protection Cytotoxicité, IFN-, Chimiokines Tumeurs NK cells and viruses Only a few cases of selective NK cell deficiencies have been reported, but these are consistent with a role for NK cells in defense against human herpesvirus infections. However, half the patients with NK- SCID (IL2RG or JAK3 deficiency) who undergo allogeneic HCT have no or very few circulating NK cells for up to 30 yrs, yet they do not develop any detectable sensitivity to viruses (or tumors). In HIV infection, NK cell counts and function decrease with AIDS progression; expression of KIR3DS1 + HLA-Bw4-80I is associated with protection. In chronic HBV infection, a subset of NK cells contributes to liver inflammation by inducing hepatocyte death through a TRAIL-dependent mechanism NK cells also control macrophage activation, preventing the appearance of hemophagocytosis lymphohistiocytosis (HLH). NK cells and cancer low NK cell activity is associated with increased cancer risk In allogeneic HCT after AML, the development of donor NK cells in patients lacking donor KIR ligands can lead to improved post-transplant survival NK cells and reproduction During the proliferative phase preceding ovulation, the endometrium becomes infiltrated with agranular NK cells (< 70% of leukocytes). During the secretory phase, uNK cells proliferate and differentiate. If an embryo does not implant, NK cells die off before menstruation uNK ~50–90% of the leukocytes during the first 2 trimesters and may differentiate in the presence of TGF-β from CD16+ peripheral NK cells or from an immature NK precursor within the decidua Trophoblast cells have a unique pattern of HLA class I expression that includes invariant HLA-G and HLA-E and polymorphic HLA-C, but not highly polymorphic HLA-A and HLA-B. Interactions between fetal trophoblast and maternal uterine NK (uNK) cells influence placentation in human pregnancy. Maternal activating KIRs (LRC B haplotype) protect against reproductive failure (preeclampsia, IUGR and recurrent miscarriage) by fetal HLA-C2 Cytokines inhibitrices Lc T régulateurs induits

18 R.I. innée + adaptative : Complémentarité – Interactivité :
Les cellules dendritiques et les cellules NK à l'interface inné adaptatif Cellules tissulaires Cellules sentinelles Cellules "relai" Lymphocytes à Récepteur réarrangé pour l'Ag T IL-15 IL-12 IFN- Cellules spécialisées TNF GM-CSF IL-2 NK cells play a central role in alerting DCs to an infection, and in promoting their functional maturation.  NK cell recognition of infected cells results in cytokine release, aimed at recruiting innate immune cells, including DCs, to the site of the antiviral response. NK-cell activation in secondary lymphoid tissues by mature DCs allows for the activation of innate lymphocytes, which limit pathogen replication prior to priming of adaptive immune responses by the same APC subsets at the same sites NK-cell distribution appears to be adjusted in T-cell zones of secondary lymphoid organs to ensure efficient activation by DCs to rapidly mobilize the NK-cell compartment during the initial phase of immune responses to infections IFN- B NK Ac IgG IL-15 IL-12 Prépare - Oriente Relai Renforce - Complète

19 R.I. innée + adaptative : Complémentarité – Interactivité :
Les cellules dendritiques et les cellules NK à l'interface inné adaptatif Cellules tissulaires Cellules sentinelles Cellules "relai" Lymphocytes à Récepteur réarrangé pour l'Ag NKT Glycolipides Sphingolipides + CD1d T Phospho-Ag Protéines de stress D Cellules spécialisées NK NK cells play a central role in alerting DCs to an infection, and in promoting their functional maturation.  NK cell recognition of infected cells results in cytokine release, aimed at recruiting innate immune cells, including DCs, to the site of the antiviral response. NK-cell activation in secondary lymphoid tissues by mature DCs allows for the activation of innate lymphocytes, which limit pathogen replication prior to priming of adaptive immune responses by the same APC subsets at the same sites NK-cell distribution appears to be adjusted in T-cell zones of secondary lymphoid organs to ensure efficient activation by DCs to rapidly mobilize the NK-cell compartment during the initial phase of immune responses to infections Peptide + CMH T CD4 T CD8 "relai" B Prépare – Oriente la réponse immune Renforce Complète Protéine Polysaccharide

20 Cellules NK et lymphocytes T CD8 cytotoxiques :
complémentarité, ressemblances et différences T CD8 Activation Différenciation par l'Ag IL-2 IL-15 IL-12 IL-2 IL-12 KIR stress NCR NK T CD8 TCR   IFN- TNF Chimiokines IFN- TNF Chimiokines Grossesse Cytotoxicité Perforine Granzymes TNF … Cytotoxicité Perforine Granzymes TNF … Virus, cancers, greffes …