SummaryAging is generally associated with an increased predisposition to infectious diseases and cancers, related in part to the development of immune senescence, a process that affects all cell compartments of the immune system. Although many studies have investigated the effects of age on natural killer (NK) cells, their conclusions remain controversial because the diverse health status of study subjects resulted in discordant findings. To clarify this situation, we conducted the first extensive phenotypic and functional analysis of NK cells from healthy subjects, comparing NK cells derived from newborn (cord blood), middle-aged (18-60 years), old (60-80 years), and very old (80-100 years) subjects. We found that NK cells in cord blood displayed specific features associated with immaturity, including poor expression of KIR and LIR-1 ⁄ ILT-2 and high expression of both NKG2A and IFN-c. NK cells from older subjects, on the other hand, preserved their major phenotypic and functional characteristics, but with their mature features accentuated. These include a profound decline of the CD56 bright subset, a specific increase in LIR-1 ⁄ ILT-2, and a perfect recovering of NK-cell function following IL2-activation in very old subjects. We conclude that the preservation of NK cell features until very advanced age may contribute to longevity and successful aging.

Human NK cells comprise two main subsets, CD56bright and CD56dim cells, which differ in function, phenotype, and tissue localization. To further dissect the differentiation from CD56bright to CD56dim cells, we performed ex vivo and in vitro experiments demonstrating that the CD56brightCD16+ cells are an intermediate stage of NK cell maturation. We observed that the maximal frequency of the CD56brightCD16+ subset among NK cells, following unrelated cord blood transplantation, occurs later than this of the CD56brightCD16− subset. We next performed an extensive phenotypic and functional analysis of CD56brightCD16+ cells in healthy donors, which displayed a phenotypic intermediary profile between CD56brightCD16− and CD56dimCD16+ NK cells. We also demonstrated that CD56brightCD16+ NK cells were fully able to kill target cells, both by Ab-dependent cell cytotoxicity (ADCC) and direct lysis, as compared with CD56brightCD16− cells. Importantly, in vitro differentiation experiments revealed that autologous T cells specifically encourage the differentiation from CD56brightCD16− to CD56brightCD16+ cells. Finally, further investigations performed in elderly patients clearly showed that both CD56brightCD16+ and CD56dimCD16+ mature subsets were substantially increased in older individuals, whereas the CD56brightCD16− precursor subset was decreased. Altogether, these data provide evidence that the CD56brightCD16+ NK cell subset is a functional intermediate between the CD56bright and CD56dim cells and is generated in the presence of autologous T CD3+ cells.

Although anti-CD20 monoclonal antibodies (mAbs) show promise for the treatment of chronic lymphocytic leukemia (CLL), the success of the anti-CD20 mAb rituximab in CLL treatment has been limited. Novel anti-CD20 mAbs with more potent cytotoxic activity have recently been engineered, but so far most have only been tested in vitro with natural killer (NK) cells from healthy donors. Because it is still unclear whether these optimized cytotoxic mAbs will improve NK-cell killing of tumor cells in CLL patients, we characterized the relevant phenotypic and functional features of NK cells from CLL patients in detail. Expression of inhibitory and activating NK-cell receptors and of Fc gamma receptor IIIA (FccRIIIA) is well preserved in CD16 þ CD56 dim cytotoxic NK cells from these patients, independently of disease progression. These cells are fully functional following cytokine stimulation. In addition, the FccRIIIA-optimized LFB-R603 anti-CD20 mAb mediates 100 times greater antibody-dependent cell-mediated cytotoxicity by NK cells from CLL patients and healthy donors than rituximab. Enhanced degranulation against autologous B-CLL cells is observed at lower concentrations of LFB-R603 than rituximab, regardless of CLL prognostic factors. These findings strongly justify further clinical development of anti-CD20 mAbs optimized for FccR engagement in CLL patients.

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Immunotherapy with monoclonal antibodies (mAbs), such as anti-CD20, is used in CLL treatment and represents a promising approach for achieving MRD eradication. Given their FcγRIIIa expression, NK cells are known to be involved in mAb therapy. We previously conducted a complete NK cells phenotypic expertise and functional assays including cytotoxicity against K562 cell line and antibody-dependent cellular cytotoxicity (ADCC) with rituximab, showing no major differences between NK cells from CLL patients and NK cells from healthy donors. We are now interested in functional capacities of NK cells in presence or not of a new anti-CD20 mAb: R603, a chimeric anti-CD20 mAb exhibiting a low fucose content as described for EMAB-6 mAb (C. de Romeuf et al, BJH 2008) in comparison to rituximab. To assess the degranulation of NK cells from CLL patients in response to anti-CD20 mAbs, we examined the surface expression of CD107a (percentage of CD107a+ NK cells) after co-incubation of PBMC from untreated CLL patients (n=8) with Raji cells (E/T ratio: 1/1) at 2 concentrations of each anti-CD20 mAb (10 and 1,000 ng/ml). At the higher mAb dose (1,000 ng/ml), R603 related degranulation of CLL NK cells (median (m): 43.6%; range (r): 27.0–79.8) was similar to the one obtained with rituximab (m: 38.9%; r: 22.4–75.2). At the lower dose (10 ng/ml), R603 related degranulation of CLL NK cells (m: 45.7%; r: 28.7–79.2) was similar to the one obtained with the high mAb concentration, contrary to rituximab related degranulation which was significantly decreased (m: 14.1%; r: 1.4–45.4) (p<0.0001). These results are emphasized by ADCC chromium assay performed with purified CLL NK cells (E/T ratio: 5/1 and 10/1) against Raji cells and with or without anti-CD20 mAbs (at 3 doses: 1, 10 and 1,000 ng/ml). R603 related ADCC levels were high whatever the mAb concentration, contrary to rituximab related ADCC levels which were very low at 1 ng/ml and only reached R603 ADCC levels at 1,000 ng/ml. Similar results were obtained with healthy donors. Without addition of Raji cells (CLL PBMC + mAb), at the lower dose (10 ng/ml), none of the NK cells from CLL patients exhibited degranulation with rituximab, contrary to R603 where 5/8 CLL patients exhibited degranulation (cut-off: more than 10% of CD107a+ NK cells). At the higher dose (1,000 ng/ml), NK cells from 6/8 CLL patients with rituximab and from 7/8 CLL patients with R603 showed degranulation and R603 related degranulation levels (m: 32.3%, r: 0.8–51.0) were significantly superior to rituximab related degranulation levels (m: 12.1%, r: 0.1–30.6) (p=0.0005). These results showed that R603 in the presence of CLL B cells might induce CLL NK degranulation. In conclusion, NK cells from CLL patients appeared to be capable of being efficient in anti-CD20 immunotherapy by the ADCC pathway. Moreover, R603 a new anti-CD20 mAb, induced at low dose a significantly higher in vitro ADCC against Raji cells and autologous CLL B cells, compared to rituximab. This R603 mAb feature may be useful in therapeutic strategy for CLL patients.

Immunotherapy with monoclonal antibodies (mAbs), such as anti-CD20, is used in CLL treatment and represents a promising approach for achieving MRD eradication. Given their FcγRIIIa expression, NK cells are known to be involved in mAb therapy. However, little is known about characteristics of NK cells in CLL patients. For 29 untreated CLL patients and 18 healthy donors, we conducted a complete phenotypic expertise and/or we measured functional capacities of NK cells in presence or not of 2 anti-CD20 mAbs: rituximab or EMAB-6, an optimized chimeric anti-CD20 mAb exhibiting a low fucose content (C. De Romeuf et al, submitted). Four-color FACS analyses were performed on fresh blood cells from 19 CLL patients and 10 healthy donors. NK cells were analyzed on CD3-CD19-CD56+ lymphocyte subset by staining with antibodies against: CD16 (3G8), CD69, activating (NKp30, NKp44, NKp46, NKG2C and NKG2D) and inhibitory (p58a, p58b, p70, NKG2A, ILT2 and LAIR-1) cytotoxicity receptors. We showed that absolute numbers of NK cells were identical or increased in CLL patients (median (m): 410/mm3, range (r): 123–2034) compared to healthy donors (m: 127/mm3, r: 14–314). Immunophenotyping of CLL NK cells revealed a CD16 heterogenous expression. Although no difference in other NK markers expression appeared in patients with normal or high expression of CD16, an heterogeneity and a diminution of activatory cytotoxicity receptors expression were observed on low CD16 NK cells (6/19). In addition, functional tests including NK cell direct cytotoxicity and/or antibody-dependent cellular cytotoxicity (ADCC) were performed simultaneously on NK-enriched PBMC from 10 CLL patients and 8 healthy donors. NK cell direct cytotoxicity was evaluated in a standard 4h 51Cr-release assay against the HLA class-I deficient K562 cell line. NK cells direct cytotoxicity of CLL patients (m: 47%, r: 20–78) was preserved and comparable to that of healthy donors (m: 55%, r: 26–61). Furthermore, preliminary ADCC experiments were performed on 51Cr Raji cells with or without anti-CD20 mAbs at the single dose of 1 μg/ml. Under these conditions, rituximab related ADCC levels obtained with CLL NK cells (m: 45%, r: 25–96) were equal or superior to those obtained with healthy donors NK cells (m: 30%, r: 25–58). EMAB-6 related ADCC levels obtained with CLL NK cells (m: 57%, r: 34–98) were, as for rituximab, also equal or superior to those obtained with healthy donors NK cells (m: 45%, r: 30–61). A moderate enhanced ADCC was observed with EMAB-6 in CLL NK cells or in healthy donors as compared to rituximab, which is in agreement with previous observations (C. De Romeuf et al, submitted). In conclusion, NK cells from CLL patients appeared to be capable of being efficient in anti-CD20 immunotherapy: they are quantitatively and qualitatively similar to those observed in healthy donors, except in a subgroup of patients with low CD16 NK cells, which need to be more investigated. Integrity of ADCC pathway is very encouraging for mAb treatment. Moreover, EMAB-6, an optimized anti-CD20 mAb, induced a higher in vitro ADCC against Raji cells and could be a promising drug candidate for the treatment of CLL.