The dual-functional capability of cytokine-induced killer cells and application in tumor immunology

Cytokine-induced killer (CIK) cells represent a heterogeneous cell population, including a large majority of CD3+CD56+ cells, a relatively minor fractions of typical T cells (CD3+CD56−), and natural killer (NK) cells (CD3−CD56+). In order to elucidate the tumor killing mechanism of these three subpopulations of CIK cells, this review summarized the concordances and differences among CD3+CD56+ CIK cells, CD3−CD56+ NK cells and CD3+CD56− T cells to the following aspects: the effects of cell surface molecules, mechanisms of tumor killing, and clinical applications of these cells in immunotherapy. NK cells can be classified into CD56brightCD16− NK cells, which produce cytokines in response to monokine co-stimulation, and the CD56dimCD16+ NK cells, which contribute to lysing susceptible target. Also, the immunity of NK cells is mainly regulated by several immune-receptors, such as ACR, ICR, NCR and KIRs. T cells require TCR and co-stimulatory molecules for initiation of T cell activation. The CD3+CD56+ CIK cells co-express with T-cell marker CD3 and NK cell marker CD56 to appear the most potent cytotoxicity and high impact on adoptive cellular immunotherapy. These CIK subpopulations share some similar tumor killing mechanisms. LFA-1 not only mediates the binding of NK cells to target cells through its ligand ICAM-1 to localize actin accumulation but also acts as a co-stimulatory receptor on NK cells. LFA-1 also functions as co-stimulatory receptor for T cells to transmit intracellular signals from the TCR to LFA-1. Furthermore, cytotoxic effect of CD3+CD56+ CIK cells is blocked by antibodies directly against LFA-1 and its counter receptor, ICAM-1. Clinically, antibody-dependent cell-mediated cytotoxicity (ADCC) is shown in both NK cells and T cells for tumor killing while dendritic cells are another main regulator for the activation of three subpopulations. In summary, CD3+CD56+ CIK cells have dual-functional capability as T-cell subsets which acquire NK cells function and reserve TCR-mediated specific cytotoxicity. Meanwhile, CIK cells play important roles in tumor immunology. It paves the way to more effective immunotherapies for various tumors.     

CD3+CD56+ NK T cells are significantly decreased in the peripheral blood of patients with psoriasis

Psoriasis is a chronic, inflammatory, hyperproliferative skin disease, in which autoimmunity plays a great role. Natural killer T cells (NK T cells), are suggested to be involved in the pathogenesis of different autoimmune diseases. To examine the involvement of CD3+CD56+ NK T cells in the pathogenesis of psoriasis, we investigated the lymphocyte subpopulations obtained from blood samples of psoriatic patients before and after treatment, and of healthy controls, using two-colour flow cytometry. We found no significant differences between total T cells, total B cells, T helper cells, T cytotoxic cells and NK cells in patients with psoriasis before and after treatment and in controls. Increased percentage of memory T cells and decreased percentage of naive T cells was detected in psoriatic patients compared to controls, but these changes were not statistically significant. The CD3+CD56+ cells of psoriatic patients were significantly decreased relative to controls. The percentage of CD3+CD56+ cells increased after different antipsoriatic therapies, but remained significantly lower than those found in controls. CD3+CD56+ cells of healthy controls were capable of rapid activation, while in psoriatic patients activated NK T cells were almost absent. The decrease in the number of CD3+CD56+ cells may represent an intrinsic characteristic feature of patients with psoriasis, which is supported by the fact that after treatment NK T cells do not reach the values found in controls. In conclusion our results suggest that CD3+CD56+ NK T cells could be actively involved in the development of Th1 mediated autoimmune diseases.     

Characterization of equine natural killer and IL-2 stimulated lymphokine activated killer cell populations.

Natural killer (NK) cells are an important component of the innate immune system. Though intensively studied in humans and rodents. NK cells remain less well characterized in other species. Studies are often limited by the lack of specific cell markers; however, the mAb NK-5C6 has been suggested to recognize an evolutionarily conserved molecule on NK cells and reacts with cells from several species. This mAb was used in the current investigation to identify and characterize equine NK cells, and was found to label approximately 10% of peripheral blood lymphocytes (PBL). Two-color flow cytometry analysis identified the NK-5C6+ cell population as being CD3-CD4- and CD8-, but positive for MHC class I and LFA-1 expression. Depletion of CD3+ T cells increased the percent NK-5C6+ cells in PBL; this enriched population demonstrated a specific cytotoxic response against a major histocompatibility complex (MHC) deficient NK target cell line (K-562), but not MHC+ target cells (EqT8888). These results provide evidence for an equine NK cell population, which exhibits endogenous lytic activity and a phenotype similar to that of human and mouse NK cells. Stimulation of peripheral blood mononuclear cells (PBMC) with IL-2 promoted the development of LAK cells. These cells were predominantly CD3+ T cells, demonstrated intracellular perforin expression, and effectively lysed both K-562 and EqT8888 target cells. Hence, equine NK cells can be identified by the NK-5C6 mAb and distinguished from IL-2 stimulated LAK cells by their cytotoxic response to specific target cell lines.     

Cytotoxicity of human natural killer (NK) cell subsets for Plasmodium falciparum erythrocytic schizonts: stimulation by cytokines and inhibition by neomycin.

Quantification of human peripheral blood NK subsets has been made in a group of Kenyan adults and children with acute P. falciparum malaria. Results were compared with data obtained from three age- and sex-matched control cohorts: parasitaemic but asymptomatic children; aparasitaemic children and adults; and adult Caucasians with no previous history of malaria. Separated NK subsets were tested in vitro for cytotoxicity to erythrocytic schizonts of P. falciparum in the presence and absence of cytokines. There was a statistically significant quantitative and qualitative depression of the CD3-CD56+ subset in patients with acute malaria and this was accompanied by an expansion of the 'non-functional' CD3-CD57+CD16-CD56- subset. Both CD3-CD16+ and CD3-CD56+ NK cells from all patients and donors lysed schizonts, and this cytotoxicity was enhanced by the addition of recombinant interferon-alpha and/or IL-2, notably with the CD3-CD56+ subset. Interestingly, asymptomatic donors had the highest levels of CD3-CD56+ NK cells, which also demonstrated an enhanced response to cytokine stimulation. Cytotoxicity to schizonts was accompanied by the release of soluble NK cell lytic factors. Neomycin suppressed cytotoxicity in a dose-dependent manner, indicating that the lysis of schizonts by NK cells involves phospholipase C-mediated phosphoinositide metabolism. Our findings define a role for NK cells in immunity to malaria through the lysis of infected erythrocytes as a first-line defence against the parasite.     

Humoral and cellular autoimmunity in autoimmune bullous skin disorders

Autoimmune bullous skin diseases, such as pemphigus vulgaris (PV) and bullous pemphigoid (BP), are severe, frequently life-threatening skin disorders. Immunologically, they are characterized by the presence of serum autoantibodies (auto-Ab) targeting distinct adhesion molecules of the epidermis or dermoepidermal basement membrane zone. Antibody (Ab) binding interferes with the adhesive function of these molecules, leading to detachment and subsequently blister formation. PV is the classical example of an Ab-mediated autoimmune disease affecting epidermal adhesion. Auto-Ab against the desmosomal adhesion molecule, desmoglein 3 (Dsg3), are critical in the pathogenesis of this disease, since the transfer of serum IgG Ab reactive with Dsg3 into newborn mice induces a bullous skin disease resembling PV. Autoreactive T cell responses to Dsg3 may be critical in the pathogenesis of PV because: (1) Ab production generally requires T cell help; (2) the involvement of CD4+ T lymphocytes in PV has been suggested by the strong association with distinct HLA class II alleles, and (3) T cell recognition of epitopes of Dsg3 may be crucial for the initiation and perpetuation of the production of Dsg3-specific auto-Ab by B cells. In PV and BP, autoreactive CD4+ T cells recognize distinct epitopes of the extracellular portions of Dsg3 and BP180 [BP antigen 2 (BPAG2) or type XVII collagen], respectively, and produce preferentially T helper type 2 (TH2) cytokines. Auto-Ab of the TH2-dependent IgG4 subtype are preferentially seen in the active stages of both PV and BP, while auto-Ab of the TH1-dependent IgG1 subclass are predominant during the chronic course of these disorders. These observations suggest that autoreactive TH2 cells may provide targets to specifically modulate the T cell-dependent production of pathogenic auto-Ab in these disorders.     

Phenotype analysis of tumour-infiltrating lymphocytes and lymphocytes in peripheral blood in patients with renal carcinoma

INTRODUCTION: When checking tumour growth, a number of observations indicate that the immune system plays a significant role in patients with renal cell carcinoma (RCC). Infiltration by lymphocytes (tumour infiltrating lymphocytes, TILs) is more prevalent in RCC than any other tumours. T lymphocytes are the dominant population of TIL cells. Views concerning the role of T lymphocytic subpopulations, B lymphocytes and NK cells in an anti-tumour response are not established. AIM: The aim is to determine the phenotype and activation of T and B lymphocytic subpopulations and NK cells and to compare their representation in tumour stroma and peripheral blood lymphocytes (PBL) in patients with RCC. MATERIAL AND METHODS: Samples of peripheral blood taken from the cubital and renal veins and tumour stroma cells were obtained from 44 patients in the course of their surgeries carried out due to primary RCC. TILs were isolated from mechanically disintegrated tumour tissue. Immunophenotype multiparametric analysis of PBL and TILs was carried out. Their surface and activation characteristics were determined by means of flow cytometer. RESULTS: CD3+ T lymphocytes (69.7%) were the main population of TILs. The number of CD3+/CD8+ T lymphocytes was significantly higher in TILs, 42.6% (p < 0.01), while CD4+ T lymphocytes were the majority population in peripheral blood, 41.35% (p < 0.001). The representation of CD3+/69+ T lymphocytes was significantly higher in TILs, 32.9%, compared to PBL (p < 0.001). On the contrary, the numbers of CD3+/CD25+, CD8+/57+ and CD4+/RA+ (naive CD4+ T lymphocytes) were higher in PBL (p < 0.001). The differences in representation of (CD3-/16+56+) NK cells and CD3+/DR+ T cells in TILs and PBL were not significant. CONCLUSION: The above-mentioned results prove that the characteristics and intensity of anti-tumour responses are different in compared compartments (tumour/PBL). CD3+/CD8+ T lymphocytes are the dominant lymphocytic population of TILs. The knowledge of the phenotype and functions of effector cells, which are responsible for anti-tumour response, are the basic precondition for understanding the anti-tumour immune response and the cause of its failure.     

Cognate Th2–B Cell Interaction is Essential for the Autoantibody Production in Pemphigus Vulgaris

Pemphigus vulgaris (PV) is a Th2-dominant autoimmune skin disease. We showed that indeed active PV patients had a biased Th2 response and specific IgG4 autoantibodies were dominant. To further investigate the role of antigen-specific Th2 cells in the regulation of pathogenic Dsg3-IgG antibody production, we used recombined Dsg3 protein to immunize wild-type C57BL/6 mice with aluminum hydroxide or complete Freund’s adjuvant as adjuvant. CD4⁺ T cells from Dsg3-immunized mice were adoptively transferred into TCR-β chain deficient mice. The transferred CD4⁺ T cells were readily seen in the peripheral blood and spleen, and interacted with B cells, resulting in B-cell activation. Furthermore, transferred CD4⁺ T cells from mice immunized with Dsg3 plus Alum with Th2 phenotype were able to render unprimed B cells to secrete Dsg3-specific IgG1 antibody in vivo. Taken together, these results provide the first demonstration of direct role of Dsg3-reactive CD4⁺ T (Th2) cells in the regulation of pathologic anti-Dsg3 antibody production.     

Analysis of the CD161-expressing cell quantities and CD161 expression levels in peripheral blood natural killer and T cells of systemic lupus erythematosus patients

Expressed on the cell surface of most of NK cells and some T cells, CD161 has been shown to deliver inhibitory signal in human NK cells. To determine whether the CD161-expressing cell quantities and the cell surface expression levels of CD161 in NK and T cells were altered in systemic lupus erythematosus (SLE) patients, we analyzed the CD3, CD56 and CD161 expression patterns of peripheral blood lymphocytes by flow cytometric analysis to identify different NK and T cell subpopulations. The cell surface expression levels of CD161 were estimated by the mean florescence intensities (MFIs) of CD161. It was found that SLE patients had lower frequencies of CD161+CD56+CD3? and CD161+CD56+CD3+ cells among the lymphocyte population than normal controls, whereas the frequencies of CD161?CD56+CD3? and CD161+CD56?CD3+ cells were not statistically different between two groups. In addition, SLE patients also had decreased absolute counts of all CD161-expressing NK cells and T cells and had reduced frequencies of CD161+ cells in CD56+CD3?, CD56+CD3+ and CD56?CD3+ cell populations. Moreover, SLE patients had reduced MFIs of CD161 in CD161+CD56+CD3+ and CD161+CD56?CD3+, but not CD161+CD56+CD3?, cell populations. Our results indicated that CD161-expressing cell frequency and the CD161 expression levels were reduced in some NK and T cell subpopulations of SLE patients, suggesting possible important role of CD161 and CD161-expressing immune cells in the SLE pathogenesis.     

NK cells, displaying early activation, cytotoxicity and adhesion molecules, are associated with mild dengue disease

During the innate immune response against infections, Natural Killer (NK) cells are as important effector cells as are Cytotoxic T lymphocytes (CTL) generated after antigenic stimulation in the adaptative response. NK cells increase in numbers, after viral infection or vaccination. We investigated the NK cell and CD8 T lymphocyte status in 55 dengue infected patients. The NK (CD56+CD3-) and CD56+ T cell (CD56+CD3+) rates rise during the acute phase of disease. The majority of NK cells from dengue patients display early markers for activation (CD69, HLA-DR, and CD38) and cell adhesion molecules (CD44, CD11a) during the acute phase of disease. The intracellular cytotoxic granule, TIA-1, is also up-regulated early in NK cells. Most of these markers appear also on CD8+ T lymphocytes but during the late acute phase. Circulating IL-15 is elevated in a significant number of patients during early acute infection and its values were statistically correlated with NK frequencies and cytotoxic markers on NKs. We have therefore shown that dengue virus infection is very likely stimulating a cytotoxic response that may be efficient in controlling the virus in synergism with CD8+ T lymphocytes. Interestingly, the heightened CD56+CD3-, CD56+CD3+, CD56+TIA-1+ and CD56+CD11a+ cell rates are associated with mild dengue clinical manifestations and might indicate a good prognosis of the disease.     

Role of decidual natural killer (NK) cells in patients with missed abortion: differences between cases with normal and abnormal chromosome.

In order to study the mechanism of abortion, the proportions of NK cells in the peripheral blood and decidual lymphocytes were evaluated in both chromosomally normal and abnormal missed abortions. In normal pregnancy, CD56+16-3- NK cells are a major element of decidual lymphocytes. The percentages of CD56+16-3-NK cells of peripheral lymphocytes in normal pregnancies were not statistically significantly different from those of chromosomally normal and abnormal abortions. In the decidua, the percentages of CD56+16-3- NK cells of decidual lymphocytes showed no statistically significant differences between normal pregnancies and chromosomally abnormal abortions. However, the percentages of CD56+16-3-NK cells of chromosomally normal abortions were lower than those of chromosomally abnormal (P = 0.0025). Moreover, the percentages of CD56+16- NK cells in abortions with normal chromosomes were lower than those in normal pregnancies or abortions with abnormal chromosomes (P = 0.0037, P = 0.0025). However, when the proportion of CD56+ NK cells expressing CD16 was evaluated, there were no statistically significant differences in the percentages of CD56+16+ NK cells in normal pregnancies and missed abortions with normal chromosomes and abnormal chromosomes. We conclude that the expression of decidual CD56+16-3- NK cells in missed abortions with normal chromosomes is different from abortions with abnormal chromosomes and that this phenomenon may depend on an abnormal immune response of the maternal side.     

Antigen-independent development of Foxp3+ regulatory T cells suppressing autoantibody production in experimental pemphigus vulgaris

The CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) play suppressive roles in various types of autoimmunity. It has been reported that Tregs develop in the thymus after high-affinity interaction of their TCR with self-peptide/MHC ligands mostly utilizing TCR-transgenic system. In this study, we examined whether the specific antigen is involved in the development of polyclonal Tregs in pemphigus vulgaris (PV), an autoimmune blistering disease caused by anti-desmoglein 3 (Dsg3) IgG antibodies, as a model system. Adoptive transfer of splenocytes of Dsg3(-)(/-) mice immunized with recombinant mouse Dsg3 to Rag2(-)(/-) recipient mice expressing Dsg3 resulted in the stable production of anti-Dsg3 IgG and the development of PV phenotypes. We show here that Tregs control anti-Dsg3 antibody production in PV model mice: the adoptive transfer of Tregs and the depletion of endogenous Tregs suppressed and augmented, respectively, the anti-Dsg3 antibody production. To examine whether the endogenous expression of Dsg3 is involved in the generation of these PV-relevant Tregs, we compared the potential of wild-type Tregs with that of Tregs from Dsg3(-)(/-) mice. Polyclonal Tregs from Dsg3(-)(/-) mice were more potent than that of wild-type mice, in both adoptive transfer and Treg-depletion experiments, while suppressive activities against IgG production against an irrelevant antigen were similar between Tregs from wild-type and Dsg3(-)(/-) mice. Our observation implies that Tregs capable of suppressing T(h) cells that drive autoantibody production can develop in the absence of the target antigen.     

CD56(+dim) and CD56(+bright) cell activation and apoptosis in hepatitis C virus infection

CD3- CD56(+dim) natural killer (NK) cells, which are cytotoxic against virally infected cells, may be important in hepatitis C virus (HCV)-infected patients who are successfully treated with pegylated interferon (PEG-IFN)-alpha. We used flow cytometry to enumerate activated (CD69+) and apoptotic (annexin-V+) dim (CD3- CD56(+dim)) and bright (CD3- CD56(+bright)) NK cells obtained from HCV-infected patients before treatment (n=16) and healthy controls (n=15) in the absence and presence of pegylated interferon (PEG-IFN)-alpha-2b. A subset of HCV-infected patients, subsequently treated with PEG-IFN-alpha-2b in vivo, was determined to have a sustained virological response (SVR, n=6) or to not respond (NR) to treatment (n=5). In the absence of IFN, activated dim (CD3- CD56(+dim) CD69+) NK cells were significantly decreased (P=0.04) while activated apoptotic dim (CD3- CD56(+dim)CD69+ annexin-V+) NK cells tended to be increased (P=0.07) in SVR patients compared with NR patients. Activated bright (CD3-CD56(+bright)CD69+) and activated apoptotic bright (CD3- CD56(+bright)CD69+ annexin-V+) NK cells were significantly correlated (P=0.02 and P=0.01, respectively) with increasing hepatic inflammation. These findings suggest that in the absence of PEG-IFN, activated dim (CD3- CD56(+dim)CD69+) NK cell turnover may be enhanced in SVR compared with NR patients and that activated bright (CD3- CD56(+bright)CD69+) NK cells may play a role in liver inflammation.     

Differential expression of the interleukin 2 receptor beta (p75) chain on human peripheral blood natural killer subsets.

The interleukin-2 receptor (IL-2R) is composed of at least two subunits, the IL-2R alpha (P55, CD25/Tac) and IL-2R beta (p70-75) chains. In the present study we have identified the quantitative expression of IL-2R beta on subsets of NK cells in peripheral blood using flow cytometry and a recently established mAb against IL-2R beta (TU27). We demonstrate that NK cells are not a homogeneous population but that phenotypic subsets exist, and that the levels of IL-2R beta expression correlate with these subsets. The levels of IL-2R beta expression on NK subsets are CD3- CD16- CD56bright NK cells (immature NK) greater than CD16+CD4- NK cells (mature NK) greater than CD16+CD57+ NK cells (more mature NK) in adult peripheral blood lymphocytes. The level of IL-2R beta expression on CD16+ NK cells in cord blood is significantly higher than that in adult PBL. These findings suggest that there may be a inverse relationship between IL-2R beta expression and the differentiation of NK cells. IL-2R beta, preferentially expressed on the NK cells, may play an important role in differentiation and maturation of NK cells.     

Bone marrow and peripheral blood natural killer cell activity in lymphomas. Its response to IL-2.

Natural killer (NK) cytotoxic activity was simultaneously investigated in bone marrow mononuclear cells (BMMC) and peripheral blood lymphocytes (PBL) from nine Hodgkin's disease (HD) and 15 non-Hodgkin lymphoma (NHL) untreated patients. Twenty-five PBL samples and seven bone marrow specimens from healthy individuals were also included as control group (C). NK cell activity was evaluated in basal condition and post-stimulation with human recombinant IL-2 (rIL-2). Data were expressed in K values (number of BMMC or PBL needed to lyse 50% of the target cells). In basal condition, both HD and NHL patients showed a NK cell activity comparable to the C group, both in BMMC (HD, K = 2.48 +/- 1.3; NHL, K = 3.8 +/- 2.0; C, K = 3.2 +/- 0.7) and PBL (HD, K = 2.0 +/- 1.0; NHL, K = 2.3 +/- 1.0; C, K = 2.2 +/- 0.2). Stimulation with rIL-2 induced a significant and comparable enhancement of the NK activity in PBL from HD, NHL and C while the response to rIL-2 of the BMMC in most of the HD and NHL patients was significantly greater than the C group. Responder cells were characterized by negative selection with specific MoAb plus complement as a CD3-, CD16+, CD56+ cytotoxic cell and further confirmed by flow cytometry. We postulate that IL-2 activation of bone marrow NK cell precursors, in addition to enhancing the activity of circulating NK, may be of value for the therapeutic rationale of IL-2 in patients with lymphoma.     

Changes in natural immunity during the course of HIV-1 infection.

The role of natural killer (NK) and lymphokine-activated killer (LAK) cell-mediated cytotoxicity in AIDS has yet to be established. The objective of this study was to determine inducible LAK cell responses at different stages of HIV-1 infection, and specifically to establish the participation of CD8 lymphocytes in these responses. Peripheral blood lymphocytes (PBL) were isolated from healthy seronegative (CDC-0) subjects and HIV-1+ individuals who were clinically asymptomatic (Centre for Disease Control group 2, CDC-2) or symptomatic (CDC-4) with regard to secondary opportunistic infection (OI). LAK cells were generated upon incubation of PBL with IL-2 and their cytolysis of K562 and U-937 targets was determined using chromium release assays. The role of CD8+ lymphocytes as progenitors and effectors of these LAK cell responses was determined by immunomagnetic depletion of CD8+ cells from precursor PBL and LAK cells, respectively. LAK cell-mediated cytotoxicities in HIV-1-infected individuals were reduced compared with seronegative controls without any corresponding changes in the relative proportions of CD56+ (NK) cells among groups. Depletions of CD8+ subsets from either PBL or LAK cells dramatically reduced total LAK cytotoxic responses and LAK activities per unit CD56+ cell in the OI-/CDC-2 seropositive population. No corresponding changes in LAK activities in seronegative control or HIV+/OI+/CDC-4 groups were observed. Levels of LAK activity against K562 targets in CDC-0/HIV- and CDC-4/HIV+ groups correlated with the percentage of CD56+ LAK cells; corresponding LAK activity in the CDC-2/HIV+ group correlated with the percentage of both CD56+ and CD8+ subsets. These findings suggest that adaptive changes in non-MHC restricted cytotoxic responses occur in HIV-1 individuals at early stages post-HIV infection, before the onset of opportunistic infection.     

Systematic characterization of human CD8+ T cells with natural killer cell markers in comparison with natural killer cells and normal CD8+ T cells

We investigated the function of CD56+ CD8+ T cells (CD56+ T cells) and CD56- CD57+ CD8+ T cells (CD57+ T cells; natural killer (NK)-type T cells) and compared them with those of normal CD56- CD57- CD8+ T cells (CD8+ T cells) and CD56+ NK cells from healthy volunteers. After the stimulation with immobilized anti-CD3 antibodies, both NK-type T cells produced much larger amounts of interferon-gamma (IFN-gamma) than CD8+ T cells. Both NK-type T cells also acquired a more potent cytotoxicity against NK-sensitive K562 cells than CD8+ T cells while only CD56+ T cells showed a potent cytotoxicity against NK-resistant Raji cells. After the stimulation with a combination of interleukin (IL)-2, IL-12 and IL-15, the IFN-gamma amounts produced were NK cells > or = CD56+ T cells > or = CD57+ T cells > CD8+ T cells. The cytotoxicities against K562 cells were NK cells > CD56+ T cells > or = CD57+ T cells > CD8+ T cells while cytotoxicities against Raji cells were CD56+ T cells > CD57+ T cells > or = CD8+ T cells > or = NK cells. However, the CD3-stimulated proliferation of both NK-type T cells was smaller than that of CD8+ T cells partly because NK-type T cells were susceptible to apoptosis. In addition to NK cells, NK-type T cells but not CD8+ T cells stimulated with cytokines, expressed cytoplasmic perforin and granzyme B. Furthermore, CD3-stimulated IFN-gamma production from peripheral blood mononuclear cells (PBMC) correlated with the proportions of CD57+ T cells in PBMC from donors. Our findings suggest that NK-type T cells play an important role in the T helper 1 responses and the immunological changes associated with ageing.     

Suppression of natural killer cells in the presence of CD34+ blood progenitor cells and peripheral blood lymphocytes.

Abstract Mobilization of CD34 + peripheral blood progenitor cells (PBPCs) with granulocyte-colony stimulating factor (G-CSF) may induce functional alterations in peripheral blood lymphocyte (PBL) subsets. We and others have shown that natural killer (NK) cells from PBPC collections are less expandable in vitro than those obtained during steady-state hematopoiesis. We show here that the extent of this proliferation deficit is related to the number of circulating CD34 + cells in vivo at the time of PBPC apheresis. Likewise, addition of autologous CD34 + cells to unseparated PBL reduced the expansion of the NK-cell subset by 22.2% +/- 6.0% (n = 10; P <.005). In contrast, when using purified NK cells, their proliferation remained unimpaired by autologous CD34 + cells. Supernatants from CD34 + cells cultured with autologous PBLs had an inhibitory effect on proliferation of purified NK cells (n = 16; P =.03), indicating that an interaction between CD34 + cells and lymphocytes is essential for the suppressive effect on NK cells. To investigate the role of T cells in this interaction, intracellular cytokines were determined in T cells cultured for 7 days with or without autologous CD34 + cells. When cultured with CD34 + cells, the frequency of IL-2-producing CD4 + and CD8 + T cells was reduced by 19% and 24%, respectively, compared with T cells cultured alone (n = 7; P =.016). Interferon-gamma-producing T cells were slightly reduced ( P = not statistically significant [ns]). Finally, the influence of T cells and NK cells on the recovery of myeloid colony-forming cells (CFU-GMs) from purified CD34 + cells was examined. In the presence of T cells, 16% +/- 6% of the input CFU-GM recovered after 7 days, compared with 5% +/- 4% in the presence of NK cells (n = 5; P = ns). Our findings point to an inhibition of NK-cell proliferation mediated by an interaction of CD34 + cells and T cells occurring during PBPC mobilization with G-CSF.     

Mistletoe Lectin I-Induced Effects on Human Cytotoxic Lymphocytes. I. Synergism with Il-2 in the Induction of Enhanced Lak Cytotoxicity

This report demonstrates that in vitro activation of human cells with the β-galactoside-specific lectin from mistletoe (ML-I) or interleukin-2 (IL-2) results in different patterns of activation and function of cytotoxic cells. It is now well established that natural killer (NK) and lymphokine-activated killer (LAK) cytotoxicity is mainly mediated by resting (NK) and IL-2-activated (LAK) CD56-positive (+) cells respectively. Culture of peripheral blood lymphocytes (PBL) for 3 days with ML-I led to expansion and activation of T cells which demonstrated NK-and LAK-like cytotoxicity. T lymphocyte subset analysis revealed that in total PBL, ML-I preferentially stimulated and expanded CD8+ T cells which mediated the cytotoxic effect. Incubation of highly purified CD8+ T cells alone with ML-I did not lead to induction of cytotoxicity, which required the presence of both CD4+ and CD 14+ (monocytes) cells, suggesting that ML-I does not exert a direct effect on CD8+ T cells. Activation of PBL with both ML-I and IL-2 resulted in simultaneous induction of T and CD56+ cell-mediated NK and LAK cytotoxicity. These data suggest that treatment with ML-I and DL-2 might provide an approach to induce maximum cytotoxicity against tumors and to recruit both T and NK cells for tumor therapy.     

Relationship of large and small CD3- CD56+ lymphocytes mediating NK-associated activities.

We have defined a population of CD3-, CD56+ small lymphocytes (SLs) that exhibit the same phenotype and lytic capacity as natural killer (NK) cells. NK cells characteristically express the surface markers CD16 and CD56, mediate non-major histocompatibility complex (MHC)-restricted lysis, and have been equated with CD3- large granular lymphocytes (LGLs). In the present study we extended the observation that CD3-, CD56+ SLs can mediate NK- and antibody-dependent cellular cytotoxicity activity by studying the activation signals and lytic mechanisms that might be utilized by CD3-, CD56+ SLs in comparison to CD3- CD56+ LGLs. Our results show that CD3- SLs, similar to CD3- LGLs, exhibited activated killing in response to interleukin-2 (IL-2). In addition, after IL-2 activation, the CD3- SLs exhibited morphologic changes, including increases in size and granularity, and both morphologically and phenotypically became virtually indistinguishable from CD3- LGLs. Similar to CD3- LGLs, CD3- SLs could be directly activated by IL-2 alone to secrete significant quantities of interferon-gamma (IFN-gamma) and to express IL-2 receptor (IL-2R) p55. Examination of serine esterases and pore-forming protein (PFP) demonstrated that these cells exhibited a cytoplasmic distribution of perforin, which, unlike that of CD3- LGLs, was not associated with dense cytoplasmic azurophilic granules. Serine esterase levels were similar. However, after IL-2 activation PFP was concentrated in dense cytoplasmic granules, similar or identical to the situation in CD3-, CD56+ LGLs. These CD3-, CD56+ subsets appear to represent a continuum of activated cells that might represent various states of maturation of NK cells.     

Induction of CD16+ CD56bright NK cells with antitumour cytotoxicity not only from CD16- CD56bright NK Cells but also from CD16- CD56dim NK cells

The aim of this study was to examine the effect of cytokines on different subsets of NK cells, while especially focusing on CD16(-) CD56(dim) cells and CD16(-) CD56(bright) cells. When human peripheral blood mononuclear cells (PBMC) were cultured with a combination of IL-2, IL-12 and IL-15 for several days, a minor population of CD56(bright) NK cells expanded up to 15%, and also showed potent cytotoxicities against various cancer cells. Sorting experiments revealed that unconventional CD16(-) CD56(+) NK cells (CD16(-) CD56(dim) NK cells and CD16(-) CD56(bright) NK cells, both of which are less than 1% in PBMC) much more vigorously proliferated after cytokine stimulation, whereas predominant CD16(+) CD56(dim) NK cells proliferated poorly. In addition, many of the resting CD16(-) CD56(bright) NK cells developed into CD16(+) CD56(bright) NK cells, and CD16(-) CD56(dim) NK cells developed into CD16(-) CD56(bright) NK cells and also further into CD16(+) CD56(bright) NK cells by the cytokines. CSFE label experiments further substantiated the proliferation capacity of each subset and the developmental process of CD16(+) CD56(bright) NK cells. Both CD16(-) CD56(dim) NK cells and CD16(-) CD56(bright) NK cells produced large amounts of IFN-gamma and Fas-ligands. The CD16(+) CD56(bright) NK cells showed strong cytotoxicities against not only MHC class I (-) but also MHC class I (+) tumours regardless of their expression of CD94/NKG2A presumably because they expressed NKG2D as well as natural cytotoxicity receptors. The proliferation of CD16(+) CD56(bright) NK cells was also induced when PBMC were stimulated with penicillin-treated Streptococcus pyogenes, thus suggesting their role in tumour immunity and bacterial infections.