XLP1 inhibitory effect by 2B4 does not affect DNAM‐1 and NKG2D activating pathways in NK cells

X‐linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM‐associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B‐cell lymphomas. Here, we show that in the absence of SLAM‐associated protein, co‐engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM‐dependent signaling pathways including activating killer Ig‐like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN‐γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM‐1 and NKG2D triggering receptors. Thus, while CD48⁺ B‐EBV and lymphoma B cells devoid of NKG2D and DNAM‐1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross‐talk of inhibitory 2B4 with triggering NK (and T) receptors.     

Potential pathways for regulation of NK and T cell responses: differential X-linked lymphoproliferative syndrome gene product SAP interactions with SLAM and 2B4

SAP, the gene that is altered or absent in the X-linked lymphoproliferative syndrome (XLP), encodes a small protein that comprises a single SH2 domain and binds to the cell-surface protein SLAM which is present on activated or memory T and B cells. Because defective NK cell activity also has been reported in XLP patients, we studied the SAP gene in NK cells. SAP was induced upon viral infection of SCID mice and shown to be expressed in NK cells by in vitro culturing in the presence of IL-2. Moreover, SAP was expressed in the NK cell lines YT and RNK 16. Because SLAM, the cell-surface protein with which SAP interacts, and 2B4, a membrane protein having sequence homologies with SLAM, also were found to be expressed on the surfaces of activated NK and T cell populations, they may access SAP functions in these populations. Whereas we found that 2B4 also binds SAP, 2B4-SAP interactions occurred only upon tyrosine phosphorylation of 2B4. By contrast, SLAM-SAP interactions were independent of phosphorylation of Y281 and Y327 on SLAM. As CD48, the ligand for 2B4, is expressed on the surface of Epstein-Barr virus (EBV)-infected B cells, it is likely that SAP regulates signal transduction through this pair of cell-surface molecules. These data support the hypothesis that XLP is a result of both defective NK and T lymphocyte responses to EBV. The altered responses may be due to aberrant control of the signaling cascades which are initiated by the SLAM-SLAM and 2B4-CD48 interactions.     

The differential impact of natural killer (NK) cell education via KIR2DL3 and KIR3DL1 on CCL4 secretion in the context of in‐vitro HIV infection

Carriage of certain inhibitory natural killer (NK) cell receptor (iNKR)/HLA ligand pairs is associated with protection from infection and slow time to AIDS implicating NK cells in HIV control. NK cells acquire functional potential through education, which requires the engagement of iNKRs by their human leucocyte antigen (HLA) ligands. HIV infection down‐regulates cell surface HLA‐A/B, but not HLA‐C/E. We investigated how NK cell populations expressing combinations of the iNKRs NKG2A, KIR2DL3 (2DL3) and KIR3DL1 (3DL1) responded to autologous HIV infected CD4 (iCD4) cells. Purified NK cells from HIV‐uninfected individuals were stimulated with autologous HIV iCD4 or uninfected CD4 T cells. Using flow cytometry we gated on each of the 8 NKG2A^+/–2DL3^+/–3DL1^+/‐ populations and analysed all possible combinations of interferon (IFN)‐γ, CCL4 and CD107a functional subsets responding to iCD4 cells. Infected CD4 cells induced differential frequencies of NKG2A^+/–2DL3^+/–3DL1^+/– populations with total IFN‐γ⁺, CCL4⁺ and CD107a⁺ functional profiles. 2DL3⁺NKG2A⁺ NK cells had a higher frequency of responses to iCD4 than other populations studied. A higher frequency of 2DL3⁺ NK cells responded to iCD4 from individuals that were not HLA‐C1 homozygotes. These results show that 2DL3⁺ NK cells are mediators of HIV‐specific responses. Furthermore, responses of NK cell populations to iCD4 are influenced not only by NK cell education through specific KIR/HLA pairs, but also by differential HIV‐mediated changes in HLA expression.     

WhatSAP — 2B4 sends mixed messages in the absence of SAP

2B4 (CD244), a member of the SLAM‐related receptor family, has important immuno‐regulatory functions including coactivating the cytotoxicity and cytokine secretion of NK cells. Immune modulation by 2B4 is dependent on the small intracellular signaling molecule SAP. In patients suffering from X‐linked lymphoproliferative disease (XLP1), SAP is nonfunctional, not only abolishing the activating function of 2B4, but rendering this receptor inhibitory. In this issue of European Journal of Immunology, Meazza et al. [Eur. J. Immunol. 2014. 44: 1526–1534.] demonstrate that 2B4‐mediated inhibition in NK cells from XLP1 patients is selective. While the activation of NK cells via ITAM‐based receptors is blocked by inhibitory 2B4, DNAM‐1, and NKG2D‐dependent NK‐cell activation is not affected by SAP deficiency. These findings provide an important insight into the different defective NK‐cell functions in XLP1 patients and demonstrate the differential integration of redundant receptor signaling pathways in NK cells.     

Recognition of HLA-G by the NK cell receptor KIR2DL4 is not essential for human reproduction

A central issue of reproductive immunology in mammals is why a semi-allogeneic embryo is not rejected by the pregnant mother. This is particularly intriguing since, in different species, the early pregnant uterus is infiltrated by numerous maternal lymphocytes, predominantly NK cells. The human NK cell receptor KIR2DL4 has been implicated in the maternal tolerance to the embryo due to its recognition of HLA-G, a non-classical MHC molecule expressed preferentially in the placenta. Killer cell Ig-like receptors (KIR) are believed to participate in the natural immunity to infection and tumors, but KIR2DL4 has unique structural, functional and genetic features that could confer it a different role. However, we demonstrate here that the KIR2DL4:HLA-G interaction is not essential for human reproduction by showing that a multiparous woman lacks a KIR2DL4 gene.     

NK细胞受体KIR2DL4的结构与功能

KIR2DL4分子是NK细胞受体(NKR)的一种,属于免疫球蛋白样(IgSF)受体家族成员,主要分布在自然杀伤(Natural killer,NK)细胞上。KIR2DL4是HLA-G分子的特异性受体,结构上具备激活性和抑制性受体的双重特点,能够通过不同途径影响NK细胞的活性,具有重要的免疫调节功能。     

Co-evolution of MHC class I and variable NK cell receptors in placental mammals

Shaping natural killer (NK) cell functions in human immunity and reproduction are diverse killer cell immunoglobulin-like receptors (KIRs) that recognize polymorphic MHC class I determinants. A survey of placental mammals suggests that KIRs serve as variable NK cell receptors only in certain primates and artiodactyls. Divergence of the functional and variable KIRs in primates and artiodactyls predates placental reproduction. Among artiodactyls, cattle but not pigs have diverse KIRs. Catarrhine (humans, apes, and Old World monkeys) and platyrrhine (New World monkeys) primates, but not prosimians, have diverse KIRs. Platyrrhine and catarrhine systems of KIR and MHC class I are highly diverged, but within the catarrhines, a stepwise co-evolution of MHC class I and KIR is discerned. In Old World monkeys, diversification focuses on MHC-A and MHC-B and their cognate lineage II KIR. With evolution of C1-bearing MHC-C from MHC-B, as informed by orangutan, the focus changes to MHC-C and its cognate lineage III KIR. Evolution of C2 from C1 and fixation of MHC-C drove further elaboration of MHC-C-specific KIR, as exemplified by chimpanzee. In humans, the evolutionary trajectory changes again. Emerging from reorganization of the KIR locus and selective attenuation of KIR avidity for MHC class I are the functionally distinctive KIR A and KIR B haplotypes.     

The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues

The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2C). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A orCD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.     

Autologous and allogeneic HLA KIR ligand environments and activating KIR control KIR NK‐cell functions

NK‐cell function is regulated by a balance between inhibitory and activating killer cell immunoglobulin‐like receptors (KIR) that specifically recognize HLA class I molecules. Using KIR‐specific mAb to discriminate between KIR2DS1 and KIR2DL1 receptors, we show that KIR2DS1⁺ NK cells are C2‐alloreactive only from C2^? individuals. Moreover, using an in vitro model of NK‐cell expansion, we show here that the frequency of KIR2DL1⁺ NK cells is significantly higher in the absence of C2 ligand on stimulator EBV‐B cells than in its presence. This observation was made regardless of the presence or absence of the autologous C2 ligand, suggesting that the C2^? EBV‐B stimulator cells used in this in vitro model could activate unlicensed KIR2DL1⁺ NK cells. In the case of KIR2DL1⁺/S1⁺ genotyped individuals, KIR2DS1⁺ NK‐cell frequency was increased after stimulation with C2⁺ compared with C2^? stimulator B cells, but only from C2^? individuals. Altogether, these data highlight the C2 alloreactivity of KIR2DS1⁺ NK cells that is only observed in C2^? individuals. These results provide new insights into the way in which NK KIR cell expansion might be regulated in an allogeneic environment.     

PPP1R9B (Neurabin 2): Involvement and dynamics in the NK immunological synapse

The NK immunological synapse (NKIS) is a dynamic structure dependent on the assembly of membrane, cytoskeletal and signaling components. These serve to focus and generate stimuli for adhesion and orientation of the cytoskeleton for targeted cytolytic granule release. Previous studies have demonstrated the importance of the cytoskeleton in these processes. We previously identified PPP1R9B (neurabin 2, spinophilin) as a cytoskeletal component of the NK‐like cell line YTS. We demonstrate that (i) PPP1R9B gradually accumulates at the NKIS in a maturation stage‐dependent manner; (ii) it mimics the early kinetics of actin recruitment to the NKIS but it precedes actin departure from the site; (iii) it is recruited by CD18 stimulation but not by CD28 ligation; (iv) it is required for the maintenance of the cortical F‐actin organization in the YTS cells and knocking down PPP1R9B reduces the frequency of YTS–target cell conjugation, possibly due to the collapsed F‐actin cytoskeleton in these cells. These results indicate that PPP1R9B is required for synapse formation in the NK cells and suggest that it may be involved in the maintenance of cellular architecture by regulation of actin assembly, possibly acting to stabilize the NKIS until granule release is eminent.     

Comparative analysis of NK cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditions

We have characterized the heterogeneity of human blood NK cell subsets defined by expression of KIR, lectin like receptors and NK cell differentiation markers within a cohort of 51 healthy Caucasian individuals. High inter-individual variability in cell surface expression of most NK cell markers is observed. Range values defining NK cell subsets in healthy donors were further used as references to characterize 14 patients with NK-type lymphoproliferative disease of granular lymphocytes (NK-LDGL). Alterations of the KIR repertoire were noted in all NK-LDGL patients. NK cell expansions were classified as oligoclonal KIR(+) or as non-detectable KIR ((nd)KIR) using anti-KIR2DL1/2DS1, anti-KIR2DL2/2DL3/2DS2, anti-KIR3DL1 and anti-KIR2DS4 monoclonal antibodies. A major reduction in the size of the CD56(bright) NK cell subset was a constant feature of NK-LDGL. Altered distribution of CD94(+), CD161(+), and CD162R(+) NK cell subsets was also observed in NK-LDGL patients. Considering the potential role of NK cells in eliminating tumors or virus-infected cells, the reference values defined in this study should be valuable to characterize both quantitative and qualitative alterations of the NK cell repertoire in pathological conditions and to monitor NK cell reconstitution following hematopoietic transplantation.     

Significant NK cell activation associated with decreased cytolytic function in peripheral blood of HIV-1-infected patients

One of the hallmarks of HIV-1 infection is represented by the finding of massive T cell activation in peripheral blood lymphocytes of infected patients. An impairment of NK cell function during HIV-1 infection is also detected, and is associated with decreased expression of natural cytotoxicity receptors (NCR). In this study we tried to determine whether also NK cells are affected by relevant activation and whether this could be associated with decreased NK cell function. In 18 viremic HIV-1-infected patients, freshly drawn purified peripheral NK cells displayed significant levels of activation with an incomplete pattern (HLA-DR(+)CD69(+)CD25(-)NKp44(-)). Activated (HLA-DR(+)CD69(+)) peripheral NK cells expressed an NCR(dull) phenotype as determined by cytofluorometric analysis in all the patients, and did not derive from a homogeneous/oligoclonal expansion in vivo as analyzed by expression of HLA-specific inhibitory NK cell receptors. As determined by cytotoxicity assays, activated NK cells showed a decreased cytolytic function in HIV-1-infected patients. Thus, the decrease in NK cell function observed during HIV-1 infection is associated not only with decreased NCR expression, but also with significant and incomplete NK cell activation in vivo. These results suggest a consistent continuous involvement of the innate immune response in the failure to control viral replication.     

Disordered expression of inhibitory receptors on the NK1-type natural killer (NK) leukaemic cells from patients with hypersensitivity to mosquito bites

Recent studies have revealed the existence of a distinct type of NK cell leukaemia of the juvenile type, which presents with hypersensitivity to mosquito bites (HMB) as an essential clinical manifestation and is infected with clonal Epstein-Barr virus (EBV). This disorder is thus called HMB-EBV-NK disease and has been reported in Orientals, mostly from Japan. We investigated the profile of cytokine production and the expression of both types of NK inhibitory receptors, i.e. CD94 lectin-like dimers and killer-cell immunoglobulin-like receptors, in NK leukaemic cells from three patients with HMB-EBV-NK disease. It was found that freshly isolated NK leukaemic cells expressed mRNA for interferon-gamma (IFN-gamma) and additionally produced IL-10 upon stimulation with IL-2, indicating that the NK cells were of NK1 type. More than 98% of NK cells from the patients bore CD94 at a higher level than did normal NK cells, whereas p70 or NKAT2, belonging to immunoglobulin-like receptor, was not expressed in those NK cells. Freshly isolated leukaemic NK cells transcribed mRNA for CD94-associated molecule NKG2C at an abnormally high level, and upon stimulation with IL-2 and/or IL-12 they expressed NKG2A as well. The disordered expression of these inhibitory receptors not only provides some insights into the pathogenesis of HMB-EBV-NK disease but also can be used as phenotypic markers for the diagnosis of this type of NK cell leukaemia.     

Expression of the HLA-C2-specific activating killer-cell Ig-like receptor KIR2DS1 on NK and T cells

Killer Ig-like receptors (KIRs) are MHC class I-specific receptors expressed by Natural Killer (NK) and T cell subsets. KIRs either inhibit (KIR-L) or activate (KIR-S) lymphocyte functions. Inhibitory KIR2DL1 and activating KIR2DS1 share ligand specificity for the HLA-C2 group, consistent with their almost identical extracytoplasmic domain. This homology hampered the distinction between KIR2DL1 and KIR2DS1. We report here the characterization of the KIR2DS1⁺ subsets among primary human NK and T cells. Regardless of the host HLA-C genotype, around 10% of circulating NK cells expressed KIR2DS1 in absence of KIR2DL1. In HLA-C2 individuals, KIR2DS1 was not able to induce NK cell education (i.e., the acquisition of NK cell competence) nor to interfere with KIR2DL1-induced NK cell education. KIR2DS1 was also present on rare oligoclonal TCRαβ⁺CD8α⁺ and TCRαβ⁺CD4⁻CD8⁻ subsets. As KIR2DS1 has been associated with autoimmunity and hematopoietic stem cell transplantation, these results pave the way to dissect the function of KIR2DS1 in these clinical conditions.     

Altered expression of signalling lymphocyte activation molecule (SLAM) family receptors CS1 (CD319) and 2B4 (CD244) in patients with systemic lupus erythematosus

CS1 (CRACC, CD319) and 2B4 (CD244), members of the signalling lymphocyte activation molecule (SLAM) family receptors, regulate various immune functions. Genes encoding SLAM family receptors are located at 1q23, implicated in systemic lupus erythematosus (SLE). In this study, we have investigated the expression and alternative splicing of CS1 and 2B4 in immune cells from SLE patients. The surface expression of CS1 and 2B4 on total peripheral blood mononuclear cells (PBMCs), T, B, natural killer (NK) cells and monocytes in 45 patients with SLE and 30 healthy individuals was analysed by flow cytometry. CS1‐positive B cell population was increased significantly in SLE patients. Because CS1 is a self‐ligand and homophilic interaction of CS1 induces B cell proliferation and autocrine cytokine secretion, this could account for autoreactive B cell proliferation in SLE. The proportion of NK cells and monocytes expressing 2B4 on their surface was significantly lower in patients with SLE compared to healthy controls. Our study demonstrated altered expression of splice variants of CS1 and 2B4 that mediate differential signalling in PBMC from patients with SLE.     

NK cell activation by KIR-binding antibody 1-7F9 and response to HIV-infected autologous cells in viremic and controller HIV-infected patients

Natural killer (NK) cells may be protective in HIV infection and are inhibited by killer cell immunoglobulin-like receptors (KIRs) interacting with MHC class I molecules, including HLA-C. Retention of HLA-C despite downregulation of other MHC class I molecules on HIV infected cells might protect infected cells from NK cell recognition in vitro. To assess the role of inhibitory HLA-C ligands in the capacity of NK cells to recognize autologous infected T cells, we measured NK cell degranulation in vitro in viremic patients, controllers with low viremia, and healthy donors. No difference in NK cell response to uninfected compared to HIV-1_IIIB infected targets was observed. Activation of NK cells was regulated by KIRs, because NK cell degranulation was increased by 1-7F9, a human antibody that binds KIR2DL1/L2/L3 and KIR2DS1/S2, and this effect was most pronounced in KIR haplotype B individuals.     

Soluble HLA‐I‐mediated secretion of TGF‐β1 by human NK cells and consequent down‐regulation of anti‐tumor cytolytic activity

Soluble HLA class I (sHLA‐I) molecules can regulate survival of NK cells and their anti‐tumor killing activity. Herein, we have analysed whether interaction of sHLA‐I with CD8 and/or different isoforms of killer Ig‐like receptors (KIR) induced secretion of transforming growth factor (TGF)‐β1. CD8⁺KIR^? NK cell clones secreted TGF‐β1 upon the interaction of sHLA‐I with CD8 molecule. sHLA‐Cw4 or sHLA‐Cw3 alleles engaging inhibitory isoforms of KIR, namely KIR2DL1 or KIR2DL2, strongly downregulated TGF‐β1 production elicited through CD8. On the other hand, sHLA‐Cw4 or sHLA‐Cw3 alleles induced secretion of TGF‐β1 by ligation of stimulatory KIR2DS1 or KIR2DS2 isoforms. TGF‐β1 strongly reduced NK cell‐mediated tumor cell lysis and production of pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. Also, TGF‐β1 inhibited NK cell cytolysis induced by the engagement of stimulatory receptors including NKG2D, DNAM1, 2B4, CD69, NKp30, NKp44 and NKp46. The IL‐2‐dependent surface upregulation of some of these receptors was prevented by TGF‐β1. Furthermore, TGF‐β1 hampered IL‐2‐induced NK cell proliferation but not IL‐2‐mediated rescue from apoptosis of NK cells. Depletion of TGF‐β1 restored all the NK cell‐mediated functional activities analysed. Taken together these findings suggest that sHLA‐I antigens may downregulate the NK cell‐mediated innate response by inducing TGF‐β1 release.     

CMV drives clonal expansion of NKG2C+ NK cells expressing self-specific KIRs in chronic hepatitis patients

Natural killer (NK) cells are affected by infection with human cytomegalovirus (HCMV) manifested by increased expression of the HLA-E binding activating receptor NKG2C. We here show that HCMV seropositivity was associated with a profound expansion of NKG2C(+) CD56(dim) NK cells in patients with chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Multi-color flow cytometry revealed that the expanded NKG2C(+) CD56(dim) NK cells displayed a highly differentiated phenotype, expressed high amounts of granzyme B and exhibited polyfunctional responses (CD107a, IFN-γ, and TNF-α) to stimulation with antibody-coated as well as HLA-E expressing target cells but not when stimulated with IL-12/IL-18. More importantly, NKG2C(+) CD56(dim) NK cells had a clonal expression pattern of inhibitory killer cell immunoglobulin-like receptors (KIRs) specific for self-HLA class I molecules, with predominant usage of KIR2DL2/3. KIR engagement dampened NKG2C-mediated activation suggesting that such biased expression of self-specific KIRs may preserve self-tolerance and limit immune-pathology during viral infection. Together, these findings shed new light on how the human NK-cell compartment adjusts to HCMV infection resulting in clonal expansion and differentiation of educated and polyfunctional NK cells.