Human CD3+CD56+NKT-like cells express a range of complement receptors and C3 activation has negative effects on these cell activity and effector function

CD3⁺CD56⁺NKT-like cells are a rare population of lymphocytes that serve important roles in various types of immune-related diseases, and particularly in cancer. The complement system regulates inflammatory and immune responses by interacting with complement receptors expressed on a range of immune cells. However, whether CD3⁺CD56⁺NKT-like cells are regulated by the complement system has still not been definitively determined. In the present study, the expression of complement receptors and regulators in gated CD3⁺CD56⁺NKT-like cells isolated from human peripheral blood was assessed using PCR and flow cytometry. The results showed that human CD3⁺CD56⁺NKT-like cells expressed a range of complement receptors and regulators, such as CR3, C3aR, C5aR, C5L2, CD46 and CD55. Furthermore, the presence of complement component 3 (C3), a key component in complement activation in culture supernatant, mitigated the activity, IFN-γ production and killing function of CD3⁺CD56⁺NKT-like cells. The present study provides evidences supporting the relationship between complement activation and functional modulation of CD3⁺CD56⁺NKT-like cells, expanding our knowledge of the complement regulatory network, and also highlighting a potential target for treatment of numerous immune-related diseases, particularly NKT cell-based tumor adoptive immunotherapy.     

Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble β-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein–Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble β-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I⁺ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcγRIIIA (CD16).     

CR3 (CD11b/CD18) expressed by cytotoxic T cells and natural killer cells is upregulated in a manner similar to neutrophil CR3 following stimulation with various activating agents

CR3 (CD11b/CD18) functions both as an iC3b-receptor and as an adhesion molecule for cellular ligands such as ICAM-1. Although CR3 has been well characterized on phagocytic cells, much less is known about CR3 on lymphocytes. In this study, the expression of CR3 was examined on resting and stimulated B, T, and natural killer (NK) cells by three-color flow cytometry. Biotinylated anti-CR3 mAb and streptavidin-FITC were used in combination with anti-CD3 mAb conjugated with peridinin chlorophyll-a protein (PerCP) and phycoerythrinlabeled mAbs to CD4, CD8, CD19, or CD56. Among resting lymphocytes, CR3 was expressed on nearly all NK cells (CD56⁺CD3^–), 1% of CD4⁺CD3⁺ helper T cells, 7% of CD8⁺CD3⁺ cytotoxic T cells, and 20% of B cells (CD19⁺). Among the 5% of T cells (CD3⁺) expressing CR3, the majority was CD56⁺. Incubation of PBMC for 30 min with PMA induced a three- to fivefold increase in CR3 expression on NK cells and a twofold increase on T cells but did not change the expression of CR3 on B cells. This effect of PMA was not blocked by the presence of cycloheximide, suggesting the presence of cytoplasmic (granule) stores of CR3 in these lymphoid cells resembling those previously reported in neutrophils and monocytes. When PBMC were incubated with rIFN-, rIL-2, -glucan, or high concentrations of LPS, expression of CR3 on NK cells increased significantly, but 4 hr of stimulation was required. Other cytokines (rIFN-, rIL-1, rIL-4, rIL-6, TNF-) and rC5a had no significant effect on CR3 expression. Among NK cells, both the CD56^bright and the CD56^dim cells expressed CR3, and the expression of CR3 on both of these NK cell subsets was increased in a similar manner by PMA. However, rIL-2 stimulated a greater increase in CR3 expression on CD56^bright cells than on CD56^dim cells. These studies suggest that CR3 expressed by NK cells or cytotoxic T cells resembles phagocyte CR3 in that cellular activation stimulates increased surface expression of CR3 derived from cytoplasmic reserves of the receptor.     

Murine CXCR3+CD27bright NK cells resemble the human CD56bright NK‐cell population

Human NK cells can be subdivided into CD56^dim and CD56^bright NK cells, which exhibit different phenotypical and functional characteristics. As murine NK cells lack CD56 or a distinct correlate, direct comparative studies of NK cells in mice and humans are limited. Although CD27 is currently proposed as a feasible subset marker in mice, we assume that the usage of this marker alone is insufficient. We rather investigated the expression of the chemokine receptor CXCR3 for its suitability for distinguishing murine NK‐cell subsets with simultaneous consideration of CD27. Compared with CXCR3^? NK cells, exerting stronger cytotoxic capability, CXCR3⁺ NK cells displayed an activated phenotype with a lower expression of Ly49 receptors, corresponding to human CD56^bright NK cells. Also in common with human CD56^bright NK cells, murine CXCR3⁺ NK cells exhibit prolific expansion as well as robust IFN‐γ, TNF‐α and MIP‐1α production. We additionally demonstrated changes in both CXCR3 and CD27 expression upon NK‐cell activation. In summary, CXCR3 serves as an additional applicable marker for improved discrimination of functionally distinct murine NK‐cell subsets that comply with those in humans.     

Functional impairment in circulating and intrahepatic NK cells and relative mechanism in hepatocellular carcinoma patients

Functional defects in natural killer (NK) cells have been proposed to be responsible for the failure of anti-tumor immune responses. Whether and how NK cells are impaired in hepatocellular carcinoma (HCC) patients remain unknown. In this study, we found that HCC patients displayed a dramatic reduction in peripheral CD56^dimCD16^pos NK subsets compared with healthy subjects. A significant reduction of CD56^dimCD16^pos NK subsets was also found in tumor regions compared with non-tumor regions in the livers of these HCC patients. Both these peripheral and tumor-infiltrating NK cells exhibited poorer capacity to produce IFN-γ and kill K562 targets, which was further found to be associated with increased CD4⁺CD25⁺ T regulatory cells as we previously-described in HCC patients. Addition of Tregs from HCC patients efficiently inhibited the anti-tumor ability of autologous NK cells in vitro. These findings are helpful for understanding the mechanism of NK cell-mediated anti-tumor immune responses in HCC patients.     

Complement regulates conventional DC-mediated NK-cell activation by inducing TGF-β1 in Gr-1+ myeloid cells

Complement activation modulates DC-mediated T-cell activation, but whether complement affects DC-mediated priming of NK cells is unknown. Here, we demonstrated that conventional DCs (cDCs) from C3(-/-) and C5aR(-/-) mice are hyperresponsive to polyI:C, a TLR3 ligand, leading to enhanced NK-cell activation. We found that cDCs lack C5a receptor (C5aR) and do not respond to C5a directly. Depletion of Gr-1(+) myeloid cells augments polyI:C-induced cDC activation in WT but not in C3(-/-) or C5aR(-/-) mice, indicating that the effect of complement activation on cDCs is indirectly mediated through C5aR-expressing Gr-1(+) myeloid cells. We further demonstrated that the mechanism by which Gr-1(+) myeloid cells regulate the activity of cDCs involves C5a-dependent TGF-β1 production in Gr-1(+) myeloid cells. C5a enhances and blocking C5aR decreases TGF-β1 production in cultured bone marrow Gr-1(+) CD11b(+) cells. C5aR deficiency is associated with reduced circulating TGF-β1 levels, while depleting Gr-1(+) myeloid cells abrogates this difference between WT and C5aR(-/-) mice. Lastly, we showed that enhanced cDC-NK-cell activity in C3(-/-) mice led to delayed melanoma tumor growth. Thus, complement activation indirectly regulates cDC-NK-cell activation in response to inflammatory stimuli such as TLR3 by promoting TGF-β1 production in Gr-1(+) myeloid cells at steady state.     

Contribution of CR3, CD11b/CD 18 to cytolysis by human NK cells

The complement receptor CR3 molecule functions in direct intercellular contacts mediated by its beta chain, CD18. Similarly to the Fc receptor (CD16), CR3 is a marker of human natural killer cells. We have shown that opsonization of NK targets with iC3b leads to their increased lytic sensitivity. Opsonization could be achieved by incubating certain B and T cell lines in human serum. The expression of CR2 was a prerequisite for C3 fragment fixation. The CR2 negative cell line, P3HR1 could be opsonized by incubation in human serum when induced to express the EBV envelope glycoprotein gp350. C3b or iC3b could also be deposited artificially on cell surfaces by chemical coupling to surface reactive antibodies. Similarly to the function of macrophages and monocytes, contact with opsonized targets exclusively through the iC3b binding site of CR3 did not seem to trigger NK function. We attempted to clarify the functional role of other CR3 ligands. The beta chain of the molecule, CD18, was essential to the NK effect. The NK targets did not seem to interact with the beta-glucan binding epitope on the alpha chain of CR3, CD11b. On the other hand, the cytolytic function could be enhanced through this epitope with the appropriate ligand.     

The immune potential of decidua-resident CD16+CD56+ NK cells in human pregnancy

Human CD56⁺CD3^? NK cells can be subdivided into two different subsets according to the expression pattern of CD56 and CD16. CD56^+/brightCD16^? (CD16^?) NK cells are prominently cytokine producers with little cytotoxicity whereas CD56^+/dimCD16⁺ (CD16⁺) NK cells are efficient killers with poorer cytokine production potential. In human pregnancy, CD56⁺ decidual (d)NK cells accumulate in the maternal fetal interface to regulate placental immunity and development. Unlike peripheral blood (pb)NK cells, the majority of dNK cells are CD56 positive with limited CD16 reactivity. Our results demonstrated that in normal and pathological pregnancies, CD16⁺ dNK cells are a unique population in comparison to CD16^? dNK subset. The expression of NK activation receptors CD335, CD336, CD244 and CD314 on CD16⁺ dNK subpopulation was lower than that on CD16^? dNK cells. Upon cytokine stimulation with rhIL-12/15/18 or TGFβ blockade, the CD16⁺ dNK subset exhibited more robust response on the expression of IFNG, IL-8 and CD107a, compared to that of the CD16^? dNK subpopulation. Functions of the CD16⁺ dNK subset were shown to be independent of cellular interaction with trophoblast cells. Studies of preeclamptic patients revealed lower proportions of CD16⁺ dNK cells, suggesting potential protective roles of these cells during normal gestations.. Therefore, we suggest that the CD16⁺ dNK subset, through compensating CD16^? dNK cell function, is an indispensable component to regulate decidual immune response and to support placentation.     

Enhanced binding of immune complexes processed by erythrocyte CR1 (CD 35) receptors to purified CR2 (CD 21) receptors from tonsillar mononuclear cells

The binding of immune complexes (IC) opsonized by serum complement (C) and IC processed by CR1 (CD 35) receptors on human erythrocytes (E) to purified CR2 (CD 21) receptors was compared. Soluble CR2 was prepared from tonsillar mononuclear cells and purified by antibody affinity chromatography. Solid phase CR2 as well as CR2 subjected to PAGE and blotted onto nitro-cellulose membranes bound 125I-labelled BSA anti-BSA IC which had been opsonized by C and processed by CR1 up to ten times more efficiently than IC reacted with serum only. Radiolabelled monomeric C3d also bound to solid phase CR2. The binding of IC to purified and solid phase bound CR2 could be inhibited by anti-CR2 antibodies or by preincubation of the IC with polyclonal antibodies reacting with C3d or C3b/iC3b. Thus, both C3dg and iC3b appeared to mediate binding of IC to CR2. Preincubation of solid phase CR2 with purified monomeric C3d did not inhibit the subsequent binding of E-CR1 processed IC. The data indicate that E-CR1 have an important role in generating IC which bind effectively to CR2 receptors on B lymphocytes.     

Dysregulation of regulatory CD56bright NK cells/T cells interactions in multiple sclerosis

Recent evidence has shown that CD56^bright NK cells, a subset of NK cells abundant in lymph nodes, may have an immunoregulatory function. In multiple sclerosis (MS), expansion of CD56^bright NK cells has been associated to successful response to different treatments and to remission of disease during pregnancy; how whether they exert immunoregulation in physiologic conditions and whether this is impaired in MS is not known. We dissected the immunoregulatory role of CD56^bright NK cells function in healthy subjects (HS) and compared it with that of untreated MS subjects or patients with clinically isolated syndrome suggestive of MS (CIS). We found that CD56^bright NK cells from HS acquire, upon inflammatory cues, the capability of suppressing autologous CD4+T cell proliferation through direct cytotoxicity requiring engagement of natural cytotoxicity receptors (NCRs) and secretion of granzyme B. CD56^bright NK cells from patients with MS/CIS did not differ in frequency and share a similar phenotype but displayed a significantly lower ability to inhibit autologous T cell proliferation. This impairment was not related to deficient expression of NCRs or granzyme B by CD56^bright NK cells, but to increased HLA-E expression on T cells from MS/CIS subjects, which could enhance the inhibitory effect mediated by NKG2A that is homogeneously expressed on CD56^bright NK cells. The defect in controlling autologous T cells by CD56^bright NK cells in MS/CIS might contribute to the excess of autoimmune response that is associated to disease development.     

Binding and catabolism of aggregated immunoglobulins bearing C3b or iC3b by U937 cells.

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CR1 and CR3 of human monocyte cell line U937 on the complement-mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgG to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgG to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes.     

An interaction between CD16 and CR3 enhances iC3b binding to CR3 but is lost during differentiation of monocytes into dendritic cells

The receptor for the iC3b fragment of complement, CR3, is involved in monocytes/macrophages and neutrophils phagocytosis. CR3 is known to interact with the low affinity receptor for Ig (CD16) and previous studies have suggested that this cooperation modulates CR3 functions. Herein we have studied the effect of CD16 on the ability of human monocytes CR3 to bind to iC3b. We show that iC3b binding to CR3 is inhibited by several reagents that are known to dissociate the CD16/CR3 complex. In addition, treatment of monocytes with soluble CD16 inhibited iC3b binding to CR3. Together, these data indicate that iC3b binding to monocyte CR3 is up-regulated by an interaction between membrane CD16 and CR3. The implication of CD16 in CR3 binding to iC3b was also analyzed after monocyte differentiation into dendritic cells (DC). Differentiation of monocytes into DC abrogates the cooperation between CD16 and CR3, due to a loss of CD16/CR3 interaction. In accordance, this phenomenon is associated with a lack of iC3b binding to DC. As a consequence, deposition of iC3b on apoptotic cells does not modify their phagocytosis by DC. In conclusion, we demonstrate a cooperation between CD16 and CR3 that favors iC3b binding to CR3 but is lost on DC.     

TGF‐β affects development and differentiation of human natural killer cell subsets

Human peripheral blood NK cells may be divided into two main subsets: CD56^brightCD16^? and CD56^dimCD16⁺. Since TGF‐β is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34⁺Lin^? hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF‐β represses development of NK cells from CD34⁺ progenitors and inhibits differentiation of CD16⁺ NK cells. Moreover, TGF‐β also results in conversion of a minor fraction of CD56^brightCD16⁺ cells found in peripheral blood into CD56^brightCD16^? cells, highlighting a possible role of the former as a developmental intermediate and of TGF‐β in influencing the genesis of NK subsets found in blood.     

Human T cell derived, cell-bound complement iC3b is integrally involved in T cell activation

Although the complement system is thought to be mainly involved in innate immunity and in the humoral arm of adaptive responses, evidence implicating that complement impacts T cell responses are accumulating recently. The role of the various activation products of the major complement component C3 were mainly studied so far in animal systems, and investigations regarding the effect of different C3-fragments on human T cells are sparse. Here we show that anti-CD3 activated human T lymphocytes derived from the blood and tonsil of healthy individuals produce C3, and the major cleavage fragment that appears on the T cell surface is iC3b. Based on studies carried out in allogenic system we demonstrate that the T cell membrane bound iC3b binds to the CR3 and probably to CR4 receptors expressed on monocyte-derived dendritic cells, and this interaction leads to significantly enhanced T-cell proliferation. Since neither C3aR and nor C3a binding could be detected on the membrane of anti-CD3 activated T cells, our findings indicate that in humans – in contrast to mice – the C3a peptide is most probably not involved directly in the T cell activation process.