Intratumor Regulatory Noncytotoxic NK Cells in Patients with Hepatocellular Carcinoma

Previous studies support the role of natural killer (NK) cells in controlling hepatocellular carcinoma (HCC) progression. However, ambiguity remains about the multiplicity and the role of different NK cell subsets, as a pro-oncogenic function has been suggested. We performed phenotypic and functional characterization of NK cells infiltrating HCC, with the corresponding nontumorous tissue and liver from patients undergoing liver resection for colorectal liver metastasis used as controls. We identified a reduced number of NK cells in tumors with higher frequency of CD56BRIGHTCD16− NK cells associated with higher expression of NKG2A, NKp44, and NKp30 and downregulation of NKG2D. Liver-resident (CXCR6+) NK cells were reduced in the tumors where T-bethiEomeslo expression was predominant. HCCs showed higher expression of CD49a with particular enrichment in CD49a+Eomes+ NK cells, a subset typically represented in the decidua and playing a proangiogenic function. Functional analysis showed reduced TNF-α production along with impaired cytotoxic capacity that was inversely related to CXCR6−, T-bethiEomeslo, and CD49a+Eomes+ NK cells. In conclusion, we identified a subset of NK cells infiltrating HCC, including non-liver-resident cells that coexpressed CD49a and Eomes and showed reduced cytotoxic potential. This NK cell subset likely plays a regulatory role in proangiogenic function.

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An Anti-MICA/B Antibody and IL-15 Rescue Altered NKG2D-Dependent NK Cell Responses in Hepatocellular Carcinoma

Cancers ◽

10.3390/cancers12123583 ◽

2020 ◽

Vol 12 (12) ◽

pp. 3583

Author(s):

Stefania Mantovani ◽

Stefania Varchetta ◽

Dalila Mele ◽

Matteo Donadon ◽

Guido Torzilli ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Protein A ◽

Ligand Interaction ◽

Cell Responses ◽

Hcc Cells

Natural killer (NK) cells play a pivotal role in cancer immune surveillance, and activating the receptor/ligand interaction may contribute to control the development and evolution of hepatocellular carcinoma (HCC). We investigated the role of the natural killer group 2 member D (NKG2D) activating receptor and its ligand, the major histocompatibility complex class I chain-related protein A and B (MICA/B) in patients with cirrhosis and HCC subjected to surgical resection, patients with cirrhosis and no HCC, and healthy donors (HD). The NKG2D-mediated function was determined in peripheral blood (PB), in tumor-infiltrating lymphocytes (NK-TIL), and in matched surrounding liver tissue (NK-LIL). A group of patients treated with sorafenib because of clinically advanced HCC was also studied. A humanized anti-MICA/B monoclonal antibody (mAb) was used in in vitro experiments to examine NK cell-mediated antibody-dependent cellular cytotoxicity. Serum concentrations of soluble MICA/B were evaluated by ELISA. IL-15 stimulation increased NKG2D-dependent activity which, however, remained dysfunctional in PB NK cells from HCC patients, in line with the reduced NKG2D expression on NK cells. NK-TIL showed a lower degranulation ability than NK-LIL, which was restored by IL-15 stimulation. Moreover, in vitro IL-15 stimulation enhanced degranulation and interferon-γ production by PB NK from patients at month one of treatment with sorafenib. Anti-MICA/B mAb associated with IL-15 was able to induce PB NK cytotoxicity for primary HCC cells in HD and patients with HCC, who also showed NK-TIL degranulation for autologous primary HCC cells. Our findings highlight the key role of the NKG2D-MICA/B axis in the regulation of NK cell responses in HCC and provide evidence in support of a potentially important role of anti-MICA/B mAb and IL-15 stimulation in HCC immunotherapy.

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Characterization of the defective interaction between a subset of natural killer cells and dendritic cells in HIV-1 infection

Journal of Experimental Medicine ◽

10.1084/jem.20060894 ◽

2006 ◽

Vol 203 (10) ◽

pp. 2339-2350 ◽

Cited By ~ 129

Author(s):

Domenico Mavilio ◽

Gabriella Lombardo ◽

Audrey Kinter ◽

Manuela Fogli ◽

Andrea La Sala ◽

...

Keyword(s):

Dendritic Cells ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Cell Subset ◽

Interferon Γ ◽

And Function ◽

Hiv 1

In this study, we demonstrate that the in vitro interactions between a CD56neg/CD16pos (CD56neg) subset of natural killer (NK) cells and autologous dendritic cells (DCs) from HIV-1–infected viremic but not aviremic individuals are markedly impaired and likely interfere with the development of an effective immune response. Among the defective interactions are abnormalities in the process of reciprocal NK–DC activation and maturation as well as a defect in the NK cell–mediated editing or elimination of immature DCs (iDCs). Notably, the lysis of mature DCs (mDCs) by autologous NK cells was highly impaired even after the complete masking of major histocompatibility complex I molecules, suggesting that the defective elimination of autologous iDCs is at the level of activating NK cell receptors. In this regard, the markedly impaired expression/secretion and function of NKp30 and TNF-related apoptosis-inducing ligand, particularly among the CD56neg NK cell subset, largely accounts for the highly defective NK cell–mediated lysis of autologous iDCs. Moreover, mDCs generated from HIV-1 viremic but not aviremic patients are substantially impaired in their ability to secrete interleukin (IL)-10 and -12 and to prime the proliferation of neighboring autologous NK cells, which, in turn, fail to secrete adequate amounts of interferon-γ.

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Phenotypic and Functional Characterization of NK Cells in αβT-Cell and B-Cell Depleted Haplo-HSCT to Cure Pediatric Patients with Acute Leukemia

Cancers ◽

10.3390/cancers12082187 ◽

2020 ◽

Vol 12 (8) ◽

pp. 2187

Author(s):

Raffaella Meazza ◽

Michela Falco ◽

Fabrizio Loiacono ◽

Paolo Canevali ◽

Mariella Della Chiesa ◽

...

Keyword(s):

Acute Leukemia ◽

Nk Cells ◽

Nk Cell ◽

Functional Characterization ◽

Cell Subset ◽

Cell Phenotype ◽

Cell Repertoire ◽

Hematopoietic Stem ◽

Γδt Cells ◽

Effector Cells

NK cells can exert remarkable graft-versus-leukemia (GvL) effect in HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT). Here, we dissected the NK-cell repertoire of 80 pediatric acute leukemia patients previously reported to have an excellent clinical outcome after αβT/B-depleted haplo-HSCT. This graft manipulation strategy allows the co-infusion of mature immune cells, mainly NK and γδT cells, and hematopoietic stem cells (HSCs). To promote NK-cell based antileukemia activity, 36/80 patients were transplanted with an NK alloreactive donor, defined according to the KIR/KIR-Ligand mismatch in the graft-versus-host direction. The analysis of the reconstituted NK-cell repertoire in these patients showed relatively high proportions of mature and functional KIR+NKG2A−CD57+ NK cells, including the alloreactive NK cell subset, one month after HSCT. Thus, the NK cells adoptively transfused with the graft persist as a mature source of effector cells while new NK cells differentiate from the donor HSCs. Notably, the alloreactive NK cell subset was endowed with the highest anti-leukemia activity and its size in the reconstituted repertoire could be influenced by human cytomegalovirus (HCMV) reactivation. While the phenotypic pattern of donor NK cells did not impact on post-transplant HCMV reactivation, in the recipients, HCMV infection/reactivation fostered a more differentiated NK-cell phenotype. In this cohort, no significant correlation between differentiated NK cells and relapse-free survival was observed.

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Adaptive Subsets Limit the Anti-Tumoral NK-Cell Activity in Hepatocellular Carcinoma

Cells ◽

10.3390/cells10061369 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1369

Author(s):

Charlotte Rennert ◽

Catrin Tauber ◽

Pia Fehrenbach ◽

Kathrin Heim ◽

Dominik Bettinger ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cirrhosis ◽

Nk Cells ◽

Nk Cell ◽

Cell Activity ◽

Cell Subset ◽

Cell Repertoire ◽

Healthy Donors ◽

B Virus ◽

The Impact

Hepatocellular carcinoma (HCC) is a global health burden with increasing incidence, poor prognosis and limited therapeutic options. Natural killer (NK) cells exhibit potent anti-tumoral activity and therefore represent potential targets for immunotherapeutic approaches in HCC treatment. However, the anti-tumoral activity of NK cells in HCC associated with different etiologies, and the impact of the heterogeneous NK cell subset, e.g., adaptive and conventional subsets, are not understood in detail. By comparatively analyzing the NK-cell repertoire in 60 HCC patients, 33 liver cirrhosis patients and 36 healthy donors (HD), we show in this study that the NK-cell repertoire is linked to HCC etiology, with increased frequencies of adaptive NK cells in Hepatitis B virus (HBV)-associated HCC. Adaptive NK cells exhibited limited anti-tumoral activity toward liver cancer cells; however, this was not a result of a specific NK-cell impairment in HCC but rather represented an intrinsic feature, since the characteristics of circulating and intra-tumoral adaptive NK cells were conserved between HD, HCC and liver cirrhosis patients. Hence, the expansion of adaptive NK cells with reduced anti-tumoral activity, detectable in HBV-associated HCC, may have implications for tumor surveillance and therapy.

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Adaptive subsets limit the anti-tumoral NK-cell activity in Hepatocellular Carcinoma

10.1101/2021.04.16.440140 ◽

2021 ◽

Author(s):

Charlotte Rennert ◽

Catrin Tauber ◽

Pia Fehrenbach ◽

Kathrin Heim ◽

Dominik Bettinger ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Cirrhosis ◽

Nk Cells ◽

Nk Cell ◽

Cell Activity ◽

Cell Subset ◽

Cell Repertoire ◽

Healthy Donors ◽

B Virus ◽

Infiltrating Lymphocytes

Background and Aims: Hepatocellular carcinoma (HCC) is a global health burden with increasing incidence, poor prognosis and limited therapeutic options. Natural killer (NK) cells exhibit potent anti-tumoral activity and therefore represent potential targets for immunotherapeutic approaches in HCC treatment. However, the human NK-cell repertoire is highly diverse including conventional and adaptive NK cells that differ in phenotype and effector function. Adaptive NK-cell frequencies are increased in association with HCMV (human cytomegalovirus) seropositivity that is also common in HCC patients. In this study, we aimed to gain a better understanding of the NK-cell repertoire and the associated anti-tumoral activity in HCC patients. Methods: In-depth phenotypic and functional flow-cytometry analyses of the HCMV-associated NK cell-repertoire obtained from 57 HCC patients, 33 liver cirrhosis patients and 36 healthy donors (HD). Results: First, adaptive subsets are present in all three cohorts with conserved characteristics in patients with liver cirrhosis and HCC. Second, adaptive NK cells can be isolated from HCC tissue however lack features of tissue-residency and thus probably represent circulating/infiltrating lymphocytes. Third, the anti-tumoral activity by adaptive NK cells is reduced compared to conventional NK-cell subsets, also in HCC. Lastly, frequencies of adaptive NK cells were increased in patients suffering from Hepatitis B virus-associated HCC providing a link between etiology and the NK-cell repertoire in HCC. Conclusion: Adaptive NK cells limit the anti-tumoral activitity of NK cells in HCC, especially in association with HBV infection that is accompanied by an expansion of this NK cell subset.

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Key Role of the CD56lowCD16low Natural Killer Cell Subset in the Recognition and Killing of Multiple Myeloma Cells

Cancers ◽

10.3390/cancers10120473 ◽

2018 ◽

Vol 10 (12) ◽

pp. 473 ◽

Cited By ~ 12

Author(s):

Elisabetta Vulpis ◽

Helena Stabile ◽

Alessandra Soriani ◽

Cinzia Fionda ◽

Maria Petrucci ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Subset ◽

Cytolytic Activity ◽

Hematopoietic Stem ◽

Natural Killer Cell Subset

Natural Killer (NK) cells play a pivotal role in the immunosurveillance of Multiple Myeloma (MM), but it is still undefined whether the NK cell functional properties underlying their protective activity against MM are confined to distinct NK cell populations. Interestingly, herein we report that the CD56lowCD16low NK cell subset displayed higher cytolytic activity compared to the other NK cell subsets (i.e., CD56highCD16+/−, CD56lowCD16high) against MM cells and its activity was impaired in MM patients. Decreased DNAM-1 expression levels were observed on the CD56lowCD16low NK cells during MM progression. Evaluating NK cell subset frequency after autologous hematopoietic stem cell transplantation, we found that CD56lowCD16low NK cells recovered earlier after transplantation. Overall, our data denote a key role of CD56lowCD16low subpopulation in the killing of MM cells and suggest that the reconstitution of CD56lowCD16low subpopulation after HSCT could be a useful approach of adoptive immunotherapy in the treatment of relapsed/refractory MM patients.

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Heterogeneity Among Ly-49C Natural Killer (NK) Cells: Characterization of Highly Related Receptors with Differing Functions and Expression Patterns

Journal of Experimental Medicine ◽

10.1084/jem.184.6.2085 ◽

1996 ◽

Vol 184 (6) ◽

pp. 2085-2090 ◽

Cited By ~ 77

Author(s):

Jack Brennan ◽

Suzanne Lemieux ◽

J. Douglas Freeman ◽

Dixie L. Mager ◽

Fumio Takei

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Expression Patterns ◽

Flow Cytometric Analysis ◽

Cdna Clones ◽

The Novel ◽

Color Flow ◽

Flow Cytometric

Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2d bone marrow by F1 mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49CB6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49CBALB at only 4 residues, whereas the previously reported Ly-49CB6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49CB6. Two-color flow cytometric analysis detected 5E6+4LO3311− as well as 5E6+4LO3311+ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49CB6 was indistinguishable from that of Ly-49CBALB, whereas no binding was detectable with previously reported Ly-49CB6. These results demonstrate that the newly described Ly-49CB6, not the previously reported Ly-49CB6, is the probable B6 allelic form of Ly49C. The previously reported Ly-49CB6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.

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Expansion of CD56dimCD16neg NK Cell Subset and Increased Inhibitory KIRs in Hospitalized COVID-19 Patients

Viruses ◽

10.3390/v14010046 ◽

2021 ◽

Vol 14 (1) ◽

pp. 46

Author(s):

José L. Casado ◽

Elisa Moraga ◽

Pilar Vizcarra ◽

Héctor Velasco ◽

Adrián Martín-Hondarza ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Subset ◽

Innate Response ◽

High Frequencies ◽

Innate Defense ◽

Kir Receptors ◽

Ifn Γ ◽

The One

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) infection induces elevated levels of inflammatory cytokines, which are mainly produced by the innate response to the virus. The role of NK cells, which are potent producers of IFN-γ and cytotoxicity, has not been sufficiently studied in the setting of SARS-CoV-2 infection. We confirmed a different distribution of NK cell subsets in hospitalized COVID-19 patients despite their NK cell deficiency. The impairment of this innate defense is mainly focused on the cytotoxic capacity of the CD56dim NK cells. On the one hand, we found an expansion of the CD56dimCD16neg NK subset, lower cytotoxic capacities, and high frequencies of inhibitory 2DL1 and 2DL1/S1 KIR receptors in COVID-19 patients. On the other hand, the depletion of CD56dimCD16dim/bright NK cell subsets, high cytotoxic capacities, and high frequencies of inhibitory 2DL1 KIR receptors were found in COVID-19 patients. In contrast, no differences in the distribution of CD56bright NK cell subsets were found in this study. These alterations in the distribution and phenotype of NK cells might enhance the impairment of this crucial innate line of defense during COVID-19 infection.

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Changes in NK Cell Subsets and Receptor Expressions in HIV-1 Infected Chronic Patients and HIV Controllers

Frontiers in Immunology ◽

10.3389/fimmu.2021.792775 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhi Zhang ◽

Ying Zhou ◽

Jing Lu ◽

Yuan-Fang Chen ◽

Hai-Yang Hu ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Subset ◽

Chronic Patients ◽

Activating Receptors ◽

Healthy Donors ◽

Tnf Α ◽

Plasma Cytokines ◽

Hiv Controllers ◽

Hiv 1

Natural killer (NK) cells are major effectors of the innate immune response and purported to play an influential role in the spontaneous control of HIV infection. In the present study, we compared the phenotypes of NK cells in the peripheral blood of three groups of subjects with chronic HIV-1 infection, HIV controllers, and healthy donors. The results showed that CD56+/CD16- NK cell subsets decreased in chronic patients and remained unchanged in controllers. Notably, we found that people living with chronic HIV-1 infection had suppressed NKp80, NKp46, and NKG2D expressions on NK cells compared to healthy donors, while HIV controllers remained unchanged. In contrast, NKG2D expression was substantially higher in controllers than in chronic patients (M=97.67, p<0.001). There were no significant differences in inhibitory receptors KIR3DL1 and KIR2DL1 expressions. In addition, plasma cytokine IFN-γ, TNF-α and IL-12showed higher levels in HIV controllers compared to chronic patients. Overall, our study revealed that, as compared to chronic patients, HIV controllers show an increased activating receptors expression and higher number ofCD56+/CD16-NK cell subset, with increased expression levels of plasma cytokines, suggesting that higher immune activation in controllers may have a key role in killing and suppressing HIV.

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The Role of CD137 and Its Ligand in Chronic Lymphocytic Leukemia (CLL)

Blood ◽

10.1182/blood.v112.11.3130.3130 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3130-3130

Author(s):

Tina Baessler ◽

Corina Buechele ◽

Matthias Krusch ◽

Benjamin J Schmiedel ◽

Lothar Kanz ◽

...

Keyword(s):

Nk Cells ◽

Tumor Immunity ◽

Nk Cell ◽

Cell Subset ◽

Surrogate Marker ◽

Lymphocytic Leukemia ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Cell Reactivity

Abstract Tumor immunosurveillance is dependent on the reciprocal interaction between tumor cells and anti-tumor immunity mediated e.g. by NK cells. This has led to the concept of tumor immunoediting, which incorporates the multitude of mechanisms underlying this dual tumor- and immune-sculpting interaction. Various members of the TNF/TNFR family modulate differentiation, proliferation, activation, and death of both tumor and immune effector cells. Very recently the TNFR family member CD137 has been shown to be induced on NK cells by Fc receptor triggering indicating that not only the Fab region but also the Fc part of a given antibody may be responsible for effects attributed to CD137 modulation (Lin et al., Blood2008, 112: 699). Thus we here studied the role of human CD137 and its cognate counterpart, the CD137 ligand (CD137L) in the interaction of CLL with NK cells. High levels of CD137L expression were detected on B-CLL cells in all investigated patients (n=40). Incubation of CLL cells in the presence of an immobilized CD137-Ig fusionprotein significantly induced the release of the immunoregulatory cytokine TNF demonstrating that CLL-expressed CD137L was capable to transduce bidirectional signals. Furthermore, we found that NK cells of CLL patients displayed substantial CD137 expression. While being absent on resting NK cells, CD137 expression was upregulated on the CD56dimCD16+ but not the CD56brightCD16− NK cell subset of healthy donors upon activation e.g. with IL-2 or IL-15. In addition, CD137 was also induced on NK cells after incubation in supernatants of PBMC of CLL patients. Surprisingly, disruption of CD137-CD137L interaction in cocultures of allogenic NK cells with patient CLL cells by blocking CD137 antibody caused a significant increase in NK cell cytotoxicity. The observed inhibitory effect of CD137L on NK cell reactivity was confirmed in cytotoxicity assays using CD137L-transfectants with mock-transfectants as control. Furthermore, blocking CD137-CD137L interaction also substantially enhanced Rituximab-induced antibody dependent cellular cytotoxicity in an allogenic setting. Importantly, CD137 blockade also substantially enhanced CD107a expression as a surrogate marker for granule mobilization on autologous NK cells within PBMC of B-CLL patients, and this effect was observed both in the absence and more pronounced in the presence of Rituximab. Thus, expression of functional CD137L by CLL cells impairs anti-tumor immunity by diminishing both direct and antibody-dependent cellular cytotoxicity of allogenic and autologous NK cells. Modulation of the CD137-CD137L system might therefore be a suitable therapeutic approach in strategies like antibody therapy which rely on a sufficient NK cell anti-tumor response.

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