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Natural killer (NK) cells decrease in function during chronic myelogenous leukemia (CML) progression from chronic phase to blast crisis, and they can become BCR/ABL(+) late in the disease course. To study this altered function, NK92 cells were transduced with the BCR/ABL oncogene. In contrast to the parental cells, which died when deprived of inter...
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Context 1
... cells are IL-2 dependent and start dying within 72 hours after removal of IL-2. To explore the role of the BCR/ABL gene on NK cell function in CML, NK92 cells were transduced with an MSCV retrovirus containing p210 BCR/ABL -eGFP (p210) or eGFP alone (control). After sorting for eGFP ϩ cells, NK92 cells were cultured in media with and without IL-2. In the presence of IL-2 all cells survive and grow like the parental NK92 cell line. When IL-2 is removed, both the parental and eGFP-transduced control cells die within 7 days. However, p210-transduced cells survive and become IL-2 independent ( Figure 1). To determine whether p210- transduced cells had a competitive advantage versus untransduced cells, eGFP and p210-transduced NK92 cells were cultured without prior sorting, in the presence or absence of IL-2. Fourteen days later, phenotypic analysis for eGFP showed no survival of control (eGFP ϩ ) cells in the absence of IL-2, whereas the p210 ϩ cells survived and grew inde fi nitely, resulting in outgrowth of eGFP ϩ cells (data not shown). These data prove that the IL-2 withdrawal selects for p210 ϩ NK92 cells, and that p210 transduction induces the IL-2 independence of the NK92 cells. To test for the effect of BCR/ABL on NK cell function, the cytotoxicity of the parental and transduced NK92 cells was measured against K562 targets. Both parental and control eGFP ϩ NK92 cells exhibited potent cytotoxic activity. In contrast, p210 ϩ NK92 cells killed K562 cells signi fi cantly less well, regardless of whether IL-2 was present ( P Ͻ .01) (Figure 2). NK cells function not only as direct cytotoxic cells but they also produce cytokines important in the immune response. 32,33 NK92 cells were therefore analyzed for production of IL-2, IFN- ␥ , and TNF- ␣ by intracellular cytokine (IC) staining. There was no signi fi cant difference between the cytokine pro fi les of the parental, eGFP ϩ control, and the p210 ϩ NK92 cells (data not shown), demonstrating that their IL-2 independence is not due to autocrine production of IL-2. The selective inhibitor of the BCR/ABL tyrosine kinase, imatinib mesylate, leads to growth inhibition of BCR/ABL -expressing myeloid cells and induces a variable degree of apoptosis in human CML cell lines. 35 To assess the speci fi c effect on BCR/ABL ϩ NK cells, untransduced and eGFP or p210-eGFP – transduced NK92 cells were cultured in the presence or absence of imatinib mesylate, with or without IL-2. Parental NK92 cells expanded 60-fold regardless of whether imatinib mesylate was present. In contrast, imatinib mesylate inhibited the growth of the IL-2 – independent p210 ϩ cells in a dose-dependent manner (Figure 3A). Furthermore, in the presence of imatinib mesylate, the addition of IL-2 rescued p210 ϩ NK92 cells from death and allowed continuous expansion. This fi nding demonstrates that imatinib mesylate ’ s effects are reversible. To determine whether imatinib mesylate inhibited proliferation or promoted apoptosis of the p210 ϩ NK92 cells, we tested short-term proliferation using thymidine incorporation and moni- tored apoptosis using annexin-V. As expected, parental and eGFP ϩ control NK92 cells proliferated signi fi cantly less in the absence of IL-2, and imatinib mesylate further contributed to this effect. When IL-2 was present, imatinib mesylate had no effect on proliferation of the parental or control eGFP ϩ NK92 cells (Figure 3B). In marked contrast, the IL-2 – independent p210 ϩ NK92 cells demonstrated a signi fi cant dose-dependent decrease in proliferation when imatinib mesylate was present ( P Ͻ .0001), and the addition of IL-2 restored their proliferation close to baseline (Figure 3B). Imatinib mesylate potently inhibited BCR/ABL -kinase activity both in the presence or absence of IL-2, as demonstrated by the reduced phosphorylation of cellular proteins, including p210 BCR/ABL itself (Figure 4). Imatinib mesylate has been shown to induce apoptosis of BCR/ABL ϩ myeloid cell lines, 34,35 partly by blocking the antiapoptotic signaling pathways and also by stimulating BCR/ABL nuclear entry. 36 K562 cells underwent signi fi cant apoptosis when imatinib mesylate was present (15% increase from baseline), whereas little effect was observed on parental and eGFP ϩ control NK92 cells maintained in IL-2. In contrast, p210 ϩ NK92 cells in the absence of IL-2 showed a major increase in apoptosis in the presence of imatinib mesylate ( P Ͻ .001), which was reversed by the addition of IL-2 (Figure 5). These fi ndings suggest that the growth inhibitory effect of imatinib mesylate on the IL-2 – independent p210 ϩ NK92 cells is due both to reduced proliferation (90% reduction from 72 hours of treatment with 10 M imatinib mesylate; Figure 3B) and increase in apoptosis (30% after 96 hours of treatment; Figure 5). Using this assay, no signi fi cant apoptosis was detected for the p210 ϩ NK92 cells with less than 96-hour exposure to imatinib mesylate, suggesting that the imatinib mesylate in fl uence in 3 H-thymidine assays may be due to an antiproliferative effect. Because p210 BCR/ABL decreases NK92-mediated natural cytotoxicity against K562 targets, we thought that imatinib mesylate might improve their reduced function back to that of control eGFP ϩ NK92 cells. However, after 48 hours of incubation (before apoptosis can be detected), imatinib mesylate decreased the lytic function of IL-2 – independent p210 ϩ NK92 cells (6.5% lysis at E/T ratio 20:1) (n ϭ 3), and the addition of IL-2 restored their killing rates back to baseline (36% lysis) ( P ϭ .01), but not to the level of control cells without p210. NK cell effector function is regulated by the balance between inhibitory and activating receptors, which endow NK cells with cytokine secretion and cytotoxicity against various target cells. It is currently unknown what determines the NK cell receptor reper- toire, but data suggest that the receptor acquisition occurs after NK-cell lineage commitment. 37 The NK92 parental cell line lacks the expression of most KIR (except CD158d [KIR2DL4]), but expresses NCR and lectin-type (CD94, NKG2, etc) receptors. 38 Serial phenotypic analysis of p210 ϩ NK92 clones for KIR expression detected small subpopulations that preferentially expressed CD158b/j (KIR2DL2/L3/S2 recognized by the monoclonal antibody GL183). Subclones of CD158b/j ϩ cells maintained receptor expression inde fi nitely. CD158e- (KIR3DL1, recognized by the monoclonal antibody DX9) or CD158i- (KIR2DS4, recognized by the monoclonal antibody FES172) expressing cells were not found from the original KIR-negative p210-transduced cells. However, all CD158b/j ϩ clones also expressed CD158i (data not shown). In addition, the CD158b/j ϩ p210 ϩ clones continued to acquire new KIRs, and subclones expressing CD158e have been established (Figure 6). The CD158b/j /CD158e , CD158b/j /CD158e , and CD158b/ j Ϫ /CD158e Ϫ p210 ϩ cell populations were derived from the same clone, as determined by Southern blot analysis, showing a distinct dominant band after probing for the presence of eGFP in the genomic DNA (Figure 7). This fi nding shows that KIR acquisition occurred in the progeny of a single cell marked by a retrovirus with a unique random integration site. KIR ϩ NK92 cells from the parental or control eGFP ϩ NK92 cells were dif fi cult to fi nd. However, after a year of subcloning from the parental clones, a CD158b/j ϩ /CD158e Ϫ subclone has been established, suggesting that BCR/ABL is not speci fi cally required for KIR acquisition in the NK92 cell line. The CD158b/j receptor acquired by the p210 ϩ clones can be activating (CD158j, KIR2DS2) or inhibitory (CD158b, KIR2DL2/ L3). To determine the function of the CD158b/j surface expression, we performed reverse ADCC assays against the 51 Cr-labeled P815 targets. The GL183 mAb mediated a speci fi c increase in lysis of the P815 cells (60%-80% lysis) by CD158b/j ϩ p210 ϩ NK92 cells, whereas no signi fi cant effect ( Ͻ 10% lysis) was seen without antibody, with control anti-CD56 mAb, or with NK92 cells lacking CD158b/j surface expression. The GL183 mAb induced signi fi cant lysis (58%) by the CD158b/j ϩ /CD158e ϩ p210 ϩ effector cells, and this was suppressed by the concomitant addition of the DX9 mAb (38% lysis). Therefore, reverse ADCC identi fi es the CD158b/j ϩ cells as expressing the activating receptor CD158j (KIR2DS2) and suggests that CD158e (KIR3DL1) has at least partial, but not complete, overriding inhibitory function. Therefore, in contrast to natural cytotoxicity, BCR/ABL does not block KIR function. Findings in NK92 cells may be limited by the immortalized nature of cell lines. Therefore, we designed experiments to test the effects of BCR/ABL in primary NK cells. CD56 ϩ bright and CD56 ϩ dim NK cells from healthy donors were transduced with eGFP (control) or p210-eGFP. We were unable to transduce CD56 ϩ dim NK cells at a frequency suf fi cient for further testing. In contrast, CD56 ϩ bright NK cells were transduced, albeit at low frequency, after IL-2 prestimulation, and CD56 ϩ /eGFP ϩ cells were sorted and expanded using conditions with feeders described for NK cell cloning. 29 Unlike NK92 cells, primary p210 ϩ NK cells killed K562 targets similar to control cells (data not shown). However, primary p210 ϩ NK cells gave similar results to NK92 cells with an altered response to IL-2 and increased KIR acquisition. When IL-2 was removed from 3-week expanded transduced cells, eGFP ϩ control cells had ...
Context 2
... cells are IL-2 dependent and start dying within 72 hours after removal of IL-2. To explore the role of the BCR/ABL gene on NK cell function in CML, NK92 cells were transduced with an MSCV retrovirus containing p210 BCR/ABL -eGFP (p210) or eGFP alone (control). After sorting for eGFP ϩ cells, NK92 cells were cultured in media with and without IL-2. In the presence of IL-2 all cells survive and grow like the parental NK92 cell line. When IL-2 is removed, both the parental and eGFP-transduced control cells die within 7 days. However, p210-transduced cells survive and become IL-2 independent ( Figure 1). To determine whether p210- transduced cells had a competitive advantage versus untransduced cells, eGFP and p210-transduced NK92 cells were cultured without prior sorting, in the presence or absence of IL-2. Fourteen days later, phenotypic analysis for eGFP showed no survival of control (eGFP ϩ ) cells in the absence of IL-2, whereas the p210 ϩ cells survived and grew inde fi nitely, resulting in outgrowth of eGFP ϩ cells (data not shown). These data prove that the IL-2 withdrawal selects for p210 ϩ NK92 cells, and that p210 transduction induces the IL-2 independence of the NK92 cells. To test for the effect of BCR/ABL on NK cell function, the cytotoxicity of the parental and transduced NK92 cells was measured against K562 targets. Both parental and control eGFP ϩ NK92 cells exhibited potent cytotoxic activity. In contrast, p210 ϩ NK92 cells killed K562 cells signi fi cantly less well, regardless of whether IL-2 was present ( P Ͻ .01) (Figure 2). NK cells function not only as direct cytotoxic cells but they also produce cytokines important in the immune response. 32,33 NK92 cells were therefore analyzed for production of IL-2, IFN- ␥ , and TNF- ␣ by intracellular cytokine (IC) staining. There was no signi fi cant difference between the cytokine pro fi les of the parental, eGFP ϩ control, and the p210 ϩ NK92 cells (data not shown), demonstrating that their IL-2 independence is not due to autocrine production of IL-2. The selective inhibitor of the BCR/ABL tyrosine kinase, imatinib mesylate, leads to growth inhibition of BCR/ABL -expressing myeloid cells and induces a variable degree of apoptosis in human CML cell lines. 35 To assess the speci fi c effect on BCR/ABL ϩ NK cells, untransduced and eGFP or p210-eGFP – transduced NK92 cells were cultured in the presence or absence of imatinib mesylate, with or without IL-2. Parental NK92 cells expanded 60-fold regardless of whether imatinib mesylate was present. In contrast, imatinib mesylate inhibited the growth of the IL-2 – independent p210 ϩ cells in a dose-dependent manner (Figure 3A). Furthermore, in the presence of imatinib mesylate, the addition of IL-2 rescued p210 ϩ NK92 cells from death and allowed continuous expansion. This fi nding demonstrates that imatinib mesylate ’ s effects are reversible. To determine whether imatinib mesylate inhibited proliferation or promoted apoptosis of the p210 ϩ NK92 cells, we tested short-term proliferation using thymidine incorporation and moni- tored apoptosis using annexin-V. As expected, parental and eGFP ϩ control NK92 cells proliferated signi fi cantly less in the absence of IL-2, and imatinib mesylate further contributed to this effect. When IL-2 was present, imatinib mesylate had no effect on proliferation of the parental or control eGFP ϩ NK92 cells (Figure 3B). In marked contrast, the IL-2 – independent p210 ϩ NK92 cells demonstrated a signi fi cant dose-dependent decrease in proliferation when imatinib mesylate was present ( P Ͻ .0001), and the addition of IL-2 restored their proliferation close to baseline (Figure 3B). Imatinib mesylate potently inhibited BCR/ABL -kinase activity both in the presence or absence of IL-2, as demonstrated by the reduced phosphorylation of cellular proteins, including p210 BCR/ABL itself (Figure 4). Imatinib mesylate has been shown to induce apoptosis of BCR/ABL ϩ myeloid cell lines, 34,35 partly by blocking the antiapoptotic signaling pathways and also by stimulating BCR/ABL nuclear entry. 36 K562 cells underwent signi fi cant apoptosis when imatinib mesylate was present (15% increase from baseline), whereas little effect was observed on parental and eGFP ϩ control NK92 cells maintained in IL-2. In contrast, p210 ϩ NK92 cells in the absence of IL-2 showed a major increase in apoptosis in the presence of imatinib mesylate ( P Ͻ .001), which was reversed by the addition of IL-2 (Figure 5). These fi ndings suggest that the growth inhibitory effect of imatinib mesylate on the IL-2 – independent p210 ϩ NK92 cells is due both to reduced proliferation (90% reduction from 72 hours of treatment with 10 M imatinib mesylate; Figure 3B) and increase in apoptosis (30% after 96 hours of treatment; Figure 5). Using this assay, no signi fi cant apoptosis was detected for the p210 ϩ NK92 cells with less than 96-hour exposure to imatinib mesylate, suggesting that the imatinib mesylate in fl uence in 3 H-thymidine assays may be due to an antiproliferative effect. Because p210 BCR/ABL decreases NK92-mediated natural cytotoxicity against K562 targets, we thought that imatinib mesylate might improve their reduced function back to that of control eGFP ϩ NK92 cells. However, after 48 hours of incubation (before apoptosis can be detected), imatinib mesylate decreased the lytic function of IL-2 – independent p210 ϩ NK92 cells (6.5% lysis at E/T ratio 20:1) (n ϭ 3), and the addition of IL-2 restored their killing rates back to baseline (36% lysis) ( P ϭ .01), but not to the level of control cells without p210. NK cell effector function is regulated by the balance between inhibitory and activating receptors, which endow NK cells with cytokine secretion and cytotoxicity against various target cells. It is currently unknown what determines the NK cell receptor reper- toire, but data suggest that the receptor acquisition occurs after NK-cell lineage commitment. 37 The NK92 parental cell line lacks the expression of most KIR (except CD158d [KIR2DL4]), but expresses NCR and lectin-type (CD94, NKG2, etc) receptors. 38 Serial phenotypic analysis of p210 ϩ NK92 clones for KIR expression detected small subpopulations that preferentially expressed CD158b/j (KIR2DL2/L3/S2 recognized by the monoclonal antibody GL183). Subclones of CD158b/j ϩ cells maintained receptor expression inde fi nitely. CD158e- (KIR3DL1, recognized by the monoclonal antibody DX9) or CD158i- (KIR2DS4, recognized by the monoclonal antibody FES172) expressing cells were not found from the original KIR-negative p210-transduced cells. However, all CD158b/j ϩ clones also expressed CD158i (data not shown). In addition, the CD158b/j ϩ p210 ϩ clones continued to acquire new KIRs, and subclones expressing CD158e have been established (Figure 6). The CD158b/j /CD158e , CD158b/j /CD158e , and CD158b/ j Ϫ /CD158e Ϫ p210 ϩ cell populations were derived from the same clone, as determined by Southern blot analysis, showing a distinct dominant band after probing for the presence of eGFP in the genomic DNA (Figure 7). This fi nding shows that KIR acquisition occurred in the progeny of a single cell marked by a retrovirus with a unique random integration site. KIR ϩ NK92 cells from the parental or control eGFP ϩ NK92 cells were dif fi cult to fi nd. However, after a year of subcloning from the parental clones, a CD158b/j ϩ /CD158e Ϫ subclone has been established, suggesting that BCR/ABL is not speci fi cally required for KIR acquisition in the NK92 cell line. The CD158b/j receptor acquired by the p210 ϩ clones can be activating (CD158j, KIR2DS2) or inhibitory (CD158b, KIR2DL2/ L3). To determine the function of the CD158b/j surface expression, we performed reverse ADCC assays against the 51 Cr-labeled P815 targets. The GL183 mAb mediated a speci fi c increase in lysis of the P815 cells (60%-80% lysis) by CD158b/j ϩ p210 ϩ NK92 cells, whereas no signi fi cant effect ( Ͻ 10% lysis) was seen without antibody, with control anti-CD56 mAb, or with NK92 cells lacking CD158b/j surface expression. The GL183 mAb induced signi fi cant lysis (58%) by the CD158b/j ϩ /CD158e ϩ p210 ϩ effector cells, and this was suppressed by the concomitant addition of the DX9 mAb (38% lysis). Therefore, reverse ADCC identi fi es the CD158b/j ϩ cells as expressing the activating receptor CD158j (KIR2DS2) and suggests that CD158e (KIR3DL1) has at least partial, but not complete, overriding inhibitory function. Therefore, in contrast to natural cytotoxicity, BCR/ABL does not block KIR function. Findings in NK92 cells may be limited by the immortalized nature of cell lines. Therefore, we designed experiments to test the effects of BCR/ABL in primary NK cells. CD56 ϩ bright and CD56 ϩ dim NK cells from healthy donors were transduced with eGFP (control) or p210-eGFP. We were unable to transduce CD56 ϩ dim NK cells at a frequency suf fi cient for further testing. In contrast, CD56 ϩ bright NK cells were transduced, albeit at low frequency, after IL-2 prestimulation, and CD56 ϩ /eGFP ϩ cells were sorted and expanded using conditions with feeders described for NK cell cloning. 29 Unlike NK92 cells, primary p210 ϩ NK cells killed K562 targets similar to control cells (data not shown). However, primary p210 ϩ NK cells gave similar results to NK92 cells with an altered response to IL-2 and increased KIR acquisition. When IL-2 was removed from 3-week expanded transduced cells, eGFP ϩ control cells had no growth after an additional 14 days in culture, whereas the p210 ϩ NK cells expanded up to 6-fold (similar results from 2 separate experiments). BCR/ABL -transduced cells did not proliferate further after 14 days of IL-2 withdrawal, showing that BCR/ABL alone does not lead to inde fi nite growth like the ...
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In this study, we have characterized reconstitution of the natural killer (NK) cell repertoire after haploidentical CD34(+) selected hematopoietic stem cell transplantation (HSCT) for high-risk hematologic malignancies. Analysis focused on alloreactive single-KIR(+) NK cells, which reportedly are potent antileukemic effectors. One month after HSCT,...
Here we discuss CD160 an essential NK cell activating receptor that remains poorly understood. CD160 receptor exhibits a number of unique structural and functional characteristics that are not common to other killer immunoglobulin-like receptors that recognize major histocompatibility complex (MHC) class I molecules: (1) In addition to humans and m...
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Citations
... К числу рецепторов, регулирующих функциональную активность NK-клеток, относятся иммуноглобулиноподобные рецепторы киллерных клеток (killer cell immunoglobulin-like receptors, KIR), лектиноподобные кальций-зависимые рецепторы (C-type lectin KIR-генетические факторы при ХМЛ receptors, CLR), рецепторы естественной цитотоксичности (natural cytotoxicity receptors, NCR) и др. [12]. ...
... На поздних стадиях заболевания возможно появление BCR/ABL-позитивных NK-клеток [23]. Эксперименты in vitro демонстрируют, что трансформация NKклеток онкогеном BCR/ABL лишает их зависимости от интерлейкина-2, повышает экспрессию KIR и снижает естественную цитотоксичность [12]. ...
The development of tyrosine kinase inhibitors (TKIs) and their introduction into clinical practice considerably improved the prognosis for chronic myeloid leukemia (CML) patients. About 50 % of patients with achieved deep molecular response are eligible for safe TKI discontinuation. Despite these advances, no reliable biomarkers are known to predict a response and sustaining treatment-free remission after TKI withdrawal. As TKIs do not destroy leukemic stem cells, which can be responsible for relapse, critical importance in CML is attached to natural killers (NK-cells) having antitumor activity. Functional activity of NK-cells is evaluated by expression level and repertoire of killer cell immunoglobulin-like receptors (KIR). Current studies demonstrate that a patient’s KIR genotype affects the probability of achieving early and deep molecular responses to first- and second-generation TKIs, progression-free and overall survivals, and sustaining treatment-free remission. On that ground, KIR-genetic factors can be regarded as promising predictors of response to TKI therapy in CML. Early clinical studies, which dealt with monoclonal antibodies blocking the inhibitory KIR in order to increase NK-cell activity, revealed an acceptable safety profile and efficacy in some hematological diseases (such as acute myeloid leukemia, multiple myeloma, Т-cell lymphoma) if used in combination with cytostatic drugs or antitumor monoclonal antibodies. KIR genotype determination can contribute to the development of effective therapies of this malignant hematological tumor.
... NK cell activity, which is mostly cytotoxic against tumor cells, seems to decrease gradually as the disease progresses. 5 The NK cell receptor repertoire prevents NK cell autoreactivity against healthy cells, enabling the killing of target cells with altered HLA class I expression and up-regulated stress-inducible antigens. 6 Several studies focusing largely on killer cell immunoglobulin receptors (KIR) and their HLA class I ligands have suggested that CML onset and progression may be influenced by the number and combination of NK receptors. ...
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm treated with tyrosine kinase inhibitors (TKIs). Although survival rates have improved, response to these treatments is highly heterogeneous. Variations in response rates may be due to different causes such as, treatment adherence, mutations in the BCR-ABL1 gene, clonal evolution and amplification of the BCR-ABL1 gene, but innate immune response is also considered to play a very important role and, specifically, NK cell activity through their receptors and ligands, could be determinant.
The aim of this retrospective study was to explore the role of different activating and inhibiting KIR genes as well as the activating NKG2D receptor, present in NK cells, and also their respective ligands, HLA-A, -B, -C, -G, -F, MICA and MICB, in the progression of 190 patients with CML and treated at two hospitals from Barcelona between 2000 and 2019. Early molecular response (EMR), major molecular response (MMR) or MR3.0 and deep molecular response (DMR) or MR4.0 were correlated. As control samples, healthy donors from the Barcelona Blood Bank were analyzed.
The presence of KIR2DL2/KIR2DS2 was associated with the achievement of EMR, MR3.0 and MR4.0. Carriers of the higher expression NKG2D variant and MICA*009:01 were also likely to achieve molecular response (MR). The most remarkable difference between CML patients and controls was a higher frequency of the lower expression NKG2D variant in CML patients.
In summary, our results showed that activating NK receptor phenotypes might help to achieve MR and DMR in CML patients treated with TKIs although confirmatory studies are necessary.
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... To evaluate the impact of BCR/ABL1 on NK cell differentiation and function, BCR/ABL1 was introduced into cord blood CD34 + cells and the NK92 NK cell line (111). Enforced BCR/ABL1 expression in cord blood CD34 + cells resulted in altered NK cell differentiation (110), and in NK92 cells, a decreased cytotoxicity was observed (112). Consistent with these findings, BCR/ABL1 + NK cells from CML patients grown in culture had reduced cytotoxic and proliferative capacity (113). ...
Natural killer (NK) cells are large granular lymphocytes involved in our defense against certain virus-infected and malignant cells. In contrast to T cells, NK cells elicit rapid anti-tumor responses based on signals from activating and inhibitory cell surface receptors. They also lyse target cells via antibody-dependent cellular cytotoxicity, a critical mode of action of several therapeutic antibodies used to treat cancer. A body of evidence shows that NK cells can exhibit potent anti-tumor activity against chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myelodysplastic syndromes (MDS). However, disease-associated mechanisms often restrain the proper functions of endogenous NK cells, leading to inadequate tumor control and risk for disease progression. Although allogeneic NK cells can prevent leukemia relapse in certain settings of stem cell transplantation, not all patients are eligible for this type of therapy. Moreover, remissions induced by adoptively infused NK cells are only transient and require subsequent therapy to maintain durable responses. Hence, new strategies are needed to trigger full and durable anti-leukemia responses by NK cells in patients with myeloid malignancies. To achieve this, we need to better understand the interplay between the malignant cells, their microenvironment, and the NK cells. This review focuses on mechanisms that are involved in suppressing NK cells in patients with myeloid leukemia and MDS, and means to restore their full anti-tumor potential. It also discusses novel molecular targets and approaches, such as bi- and tri-specific antibodies and immune checkpoint inhibitors, to redirect and/or unleash the NK cells against the leukemic cells.
... A sizable number of clinical and experimental data underscore the dysregulation of innate immune components in CML. These regulatory elements comprise dendritic cells (19), NK cells (20,21), and invariant natural killer T (iNKT) cells (22), an innate T cell subset co-expressing activated/memory and NK markers, which is well-recognized for its antitumor activity (23)(24)(25)(26). In accordance with this notion, we recently reported CML immune subversion of iNKT-cell activities in CML patients at diagnosis (22,27), including reduced or suppressed expression of perforin, CD95L, and (PLZF), a transcription factor required for maintenance of iNKT cell functions (28,29). ...
We recently identified a new human subset of NK-like [KIR/NKG2A(+)] CD8(+) T cells with a marked/memory phenotype, high Eomesodermin expression, potent antigen-independent cytotoxic activity, and the capacity to generate IFN-γ rapidly after exposure to pro-inflammatory cytokines. These features support the hypothesis that this new member of the innate T cell family in humans, hereafter referred to as innate CD8(+) T cells, has a role in cancer immune surveillance analogous to invariant natural killer T (iNKT) cells. Here, we report the first quantitative and functional analysis of innate CD8(+) T cells in a physiopathological context in humans, namely chronic myeloid leukemia (CML), a well-characterized myeloproliferative disorder. We have chosen CML based on our previous report that IL-4 production by iNKT cells was deficient in CML patients at diagnosis and considering the recent evidence in mice that IL-4 promotes the generation/differentiation of innate CD8(+) T cells. We found that the pool of innate CD8(+) T cells was severely reduced in the blood of CML patients at diagnosis. Moreover, like iNKT and NK cells, innate CD8(+) T cells were functionally impaired, as attested by their loss of antigen-independent cytotoxic activity and IFN-γ production in response to innate-like stimulation with IL-12 + IL-18. Remarkably, as previously reported for IL-4 production by iNKT cells, both quantitative and functional deficiencies of innate CD8(+) T cells were at least partially corrected in patients having achieved complete cytogenetic remission following tyrosine kinase inhibitor therapy. Finally, direct correlation between the functional potential of innate CD8(+) T and iNKT cells was found when considering all healthy donors and CML patients in diagnosis and remission, in accordance with the iNKT cell-dependent generation of innate CD8(+) T cells reported in mice. All in all, our data demonstrate that CML is associated with deficiencies of innate CD8(+) T cells that are restored upon remission, thereby suggesting their possible contribution to disease control. More generally, our study strongly supports the existence of an innate iNKT/innate CD8(+) T-cell axis in humans and reveals its potential contribution to the restoration of tumor immune surveillance.
... The NK-like T cells demonstrate cytotoxic killing but are not restricted by MHC recognition (12). NK cell activity has been studied in various malignancies including breast carcinomas (13), non-Hodgkin lymphoma (14)(15)(16), Hodgkin lymphoma (17), multiple myeloma (18), and myeloid neoplasms (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31) and evasion of NK immunosurveillance may be important for disease progression. Most studies evaluating NK cells in myeloid neoplasms have focused on their functional abnormalities, with few addressing their relative or absolute numbers (21,23,25,30,31). ...
The impact of the immune microenvironment on the behavior and therapeutic strategies for hematopoietic and lymphoid neoplasms is being increasingly recognized. Many functional studies of natural killer (NK) cell cytotoxic responses in myelodysplasia (MDS) and acute myeloid leukemia (AML) exist, but with limited data on these lymphocyte proportions and related T-cell subsets. The proportions of these cells and their prognostic implications were therefore investigated in 89 AML, 51 MDS, and 20 control marrows by flow cytometry. The median proportion of NK cells (relative to the total lymphocytes) was lower in AML versus controls (p=0.01). Among AML, a lower proportion of NK cells predicted better survival, whereas a higher NK cell proportion was associated with the poor prognostic AML category (p=0.002). NK cell proportions were similar in MDS, MDS subgroups and control marrows. The relative proportion of the mature NK cell subset (CD56(dim) CD16/57(bright) ) was lower in AML and MDS versus controls (p=0.006, p=0.0002 respectively). The proportion of mature NK cells was not a prognostic indicator although fewer were seen in poor prognosis AML. In contrast, a lower proportion of mature NK cells correlated with worse survival in MDS (p=0.027). A higher proportion of NK-like T-cells (of total lymphoid cells) was found in MDS compared to controls (p=0.01). A lower proportion of NK-like T-cells predicted better survival in AML but not in MDS. Thus, the proportions of NK, NK-cell subsets and NK-like T-cells vary in myeloid neoplasms, may potentially impact immunomodulatory therapies, and may impact outcome. This article is protected by copyright. All rights reserved.
... Chronic myeloid leukemia (CML) is a MPD characterized by a gain of function due to constitutive tyrosine-kinase properties of the bcr-abl protein. In this disease, decreased NK cytotoxicity is linked to an abnormal expression of inhibitory killer immunoglobulin-like receptors (KIRs) [8]. ...
... The JAK2 mutation was detected at a significant level in all samples, we concluded to its presence in PV-NK cells, in accordance with the notion that this mutation occurs in very primitive cells explaining the multi-lineage abnormalities observed in JAK2-mutated MPD [12,19]. We then tested the expression of both stimulating and inhibitory molecules on PV-NK in comparison with HD-NK, since the observation of increased KIRs expression in CML suggested that the JAK2 gain of function mutation, which has common point with the BCR/ABL gain of function, could lead to comparable results [8]. Regarding the activating and co-activating receptors expression (NCRs, NKG2D and 2B4) we failed to detect a significant difference in surface expression between PV-NK and HD-NK, although RT-qPCR showed a trend for decreased genes expression of NKp30/NCR3 and NKG2D in PV patients, but without impact on protein level, suggesting that post-transcriptional regulation plays a role in the level of expression of these molecules. ...
... Of note, the small sample size does not allow us to exclude we failed to detect significant differences in molecule or gene expression. The study of Chiorena et al. [8] has showed that the BCR/ABL fusion protein induced decreased NK cytotoxicity. This prompted us to indirectly test this property by flow cytometry detection of NK degranulation. ...
Natural killer cells (NK) are pivotal cells of innate immunity. They are potent antileukemic cytotoxic effectors. A defect in their cytotoxicity has been described in some hematopoietic malignancies such as acute myeloid leukemia, multiple myeloma and myelodysplastic syndromes. This defect is at least partially linked to a decreased or absent expression of some activating NK cells molecules, more particularly the so-called natural cytotoxicity receptors. In the present study, we more particularly focused our attention on NK cells of polycythemia vera, a myeloproliferative disease characterized by the presence of mutated JAK2 tyrosine kinase. The polymerase chain reaction analysis of NK cells from patients showed that they expressed the mutated form of JAK2. In polycythemia vera the proportion of NK was increased compared to healthy donors. The proliferative and cytotoxic abilities of NK cells from patients were similar to healthy donors. Expression of activating or inhibitory receptors was comparable in patients and donors, with nonetheless an imbalance for the inhibitory form of the CD158a,h couple of receptors in patients. Finally, the transcriptomic profile analysis clearly identified a discriminant signature between NK cells from patients and donors that could putatively be the consequence of abnormal continuous activation of mutated JAK2.
... However, the natural cytotoxicity receptor (NCR) family only possesses activating receptors. (2,6,20) Inhibitory receptors recognize MHC-I, which is often expressed in healthy cells but rarely found in cancerous cells, while the activating receptors of NK cells recognize structures that are present in both normal and tumor cells. (21) The influence of the inhibitory routes is greater when MHC-I is recognized compared to the activating routes. ...
Chronic myeloid leukemia is a neoplasia resulting from a translocation between chromosomes 9 and 22 producing the BCR-ABL hybrid known as the Philadelphia chromosome (Ph). In chronic myeloid leukemia a proliferation of malignant myeloid cells occurs in the bone marrow due to excessive tyrosine kinase activity. In order to maintain homeostasis, natural killer cells, by means of receptors, identify the major histocompatibility complex on the surface of tumor cells and subsequently induce apoptosis. The NKG2D receptor in the natural killer cells recognizes the transmembrane proteins related to major histocompatibility complex class I chain-related genes A and B (MICA and MICB), and it is by the interaction between NKG2D and MICA that natural killer cells exert cytotoxic activity against chronic myeloid leukemia tumor cells. However, in the case of chronic exposure of the NKG2D receptor, the MICA ligand releases soluble proteins called sMICA from the tumor cell surface, which negatively modulate NKG2D and enable the tumor cells to avoid lysis mediated by the natural killer cells. Blocking the formation of sMICA may be an important antitumor strategy. Treatment using tyrosine kinase inhibitors induces modulation of NKG2DL expression, which could favor the activity of the natural killer cells. However this mechanism has not been fully described in chronic myeloid leukemia. In the present study, we analyze the role of natural killer cells to reduce proliferation and in the cellular death of tumor cells in chronic myeloid leukemia.
... We confirmed positive fluorescence and obtained viral supernatants after 48 and 72 hours as described [29]. Following preactivation UCB CD34 + progenitors were transduced twice, 24 hours apart, with 3 mL of supernatant containing eGFP and ICN retrovirus in Fibronectin (Retronectin, Tarka) coated transwells at a density of 300,000 cells/well [30]. ...
... NK cell development proceeds through an orderly sequence of maturational stages governed by signals provided by a supportive microenvironment [30,31,34]. Using an approach where Notch is constitutively activated, we found that Notch1 signaling results in brisk acquisition of the CD7 antigen resulting in a bipotent CD7 + lymphoid precursor, brightly expressing CD45RA + and CD62L + receptors capable of NK cell lineage commitment. ...
Natural Killer (NK) cells are powerful effectors of cytotoxicity against "stressed" cells. They also produce cytokines and chemokines to activate the adaptive immune response. Understanding NK cell development and maturation may have implications for cancer therapy and for immunity against infections. We hypothesized that Notch signaling, critical for hematopoesis, would be involved in NK cell development. The role of constitutively activated Notch1 (ICN) on NK cell maturation was studied using human umbilical cord blood (UCB) progenitors cultured on a murine embryonic liver stroma cell line (EL08-1D2) and human cytokines. UCB CD34(+)/ICN(+) sorted cells resulted in a population of CD7(+) early lymphoid precursors and subsequent NK lineage commitment independent of stroma or IL-15. Early expression of L-selectin on ICN(+) precursors suggested their homing competence. These precursors further committed to the NK lineage, and were capable of producing cytokines and chemokines such as interleukin (IL)-13, granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), yet poorly acquired NK inhibitory receptors and cytotoxic effector function. In the presence of stroma, ICN(+) precursors also gave rise to a population of early T lineage committed cells characterized by expression of cytoplasmic CD3 gamma, epsilon, and delta chains, RAG1/2, and production of IL-2, suggesting bona fide Th1 commitment. Importantly, signals from EL08-1D2 stroma were required for this development process. In conclusion, sustained Notch signaling can replace stroma in differentiation of a common CD7(+) lymphoid precursor from UCB CD34(+) progenitors and induce NK cell commitment. However, these NK cells are immature in their cytokine production profile, are hyporesponsive, and poorly acquire NK cell receptors involved in self-tolerance and effector function.
... Moreover, because of their hematogeneous route of primary tumor and metastasis formation, leukemic and lymphoma cells are targeted by NK cells which are often distributed in the peripheral blood as well as in secondary and tertiary lymphoid sites (i.e., lung, liver, spleen, bone marrow and lymph nodes) [67]. The role of NK cells in controlling leukemic cells is supported by the association between a decrease in NK cell function and progression of chronic myelogenous leukemia (CML) from chronic phase to blast crisis686970. Therefore, the high HLA class I expression found in leukemia and lymphoma cells may result from the outgrowth of tumor cells with high HLA class I expression because of their reduced susceptibility to NK cellmediated lysis. ...
Changes in classical and nonclassical HLA class I as well as HLA class II antigens have been identified in malignant lesions. These changes, which are described in this review are believed to play a major role in the clinical course of the disease since both HLA class I and class II antigens are critical to the interaction between tumor cells and components of both innate and adaptive immune system. Abnormalities in HLA antigen expression in malignant cells, which range in frequency from 0-90%, are caused by distinct mechanisms. They include defects in beta(2)-microglobulin (beta(2)m) synthesis, loss of the gene(s) encoding HLA antigen heavy chain(s), mutations, which inhibit HLA antigen heavy chain transcription or translation, defects in the regulatory mechanisms, which control HLA antigen expression and/or abnormalities in one or more of the antigen processing, machinery (APM) components. More recently, epigenetic events associated with tumor development and progression have been found to underlie changes in HLA antigen, APM component, costimulatory molecule and tumor antigen (TA) expression in malignant cells. The types of epigenetic modifications that may occur in normal and malignant cells as well as their role in changes in HLA antigen expression by malignant cells have been reviewed. The epigenetic events associated with alterations in HLA antigen expression may be clinically relevant as, in some cases, they have been shown to impair the recognition of tumor cells by components of the adaptive immune system. The functional relevance and potential clinical significance of these epigenetic alterations have been addressed. Finally, unlike genetic alterations, epigenetic modifications can, in some cases, be reversed with pharmacologic agents that induce DNA hypomethylation or inhibit histone deacetylation. Therefore, strategies to overcome epigenetic modifications underlying changes in HLA antigen expression in malignant cells have been discussed.
... 4,5 Substantial numbers of NK cells in an advanced phase of the disease were found to be positive for t (9;22) translocation-related breakpoint cluster region-abelson (BCR/ ABL) in contrast to those in the chronic phase. 5,13 It has been demonstrated that BCR/ABL can increase KIR expression and so influence the activity of NK cells. 13 The importance of inhibitory KIR-HLA class I interactions in the innate immune response against leukemic cells has been further underscored in allogeneic HLA-mismatched HSCT. ...
... 5,13 It has been demonstrated that BCR/ABL can increase KIR expression and so influence the activity of NK cells. 13 The importance of inhibitory KIR-HLA class I interactions in the innate immune response against leukemic cells has been further underscored in allogeneic HLA-mismatched HSCT. In the setting of KIR ligandmismatched HSCT, NK cell alloreactions are directed toward allogeneic cells that lack inhibitory HLA class I ligands. ...
Human natural killer (NK) cells are built to kill abnormal cells but to preserve autologous normal cells. To accomplish this task, they are equipped with a large number of inhibiting and activating receptors. Ligation with corresponding ligands will determine whether the NK cell becomes activated to destroy the abnormal cell. This review will focus on the abnormalities of NK cell receptors and their putative ligands found in patients with leukemia, which can lead to an inadequate function of NK cells allowing these malignant cells to escape from NK cell destruction. In recent years it has become clear that NK cells in the haploidentical hematopoietic stem cell transplantation (HSCT) setting are very effective in eliminating residual acute myeloid, but not acute lymphoid, leukemic cells. In this regard, we also reviewed published studies of retrospective cohorts of HSCT investigating the potential beneficial effect of killer-cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) ligands on NK alloreactivity. Manipulating NK cell inhibition or activation could lead to new forms of immunotherapy, ultimately leading to the elimination of resistant leukemic cells.
