KIR Gene and HLA-Cw Mismatches Affect on NK Cell Killer Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4926-4926
Author(s):  
Xiangshan Cao ◽  
Jianyong Li
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Target Cell ◽  
Nk Cell ◽  
Leukemic Cells ◽  
Target Cells ◽  
Cytotoxicity Assays ◽  
Control Target ◽  
Nk Cytotoxicity ◽  
Kir Gene

Abstract The KIRs were knew as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. We investigated NK cell activation on the number of matches between cell killer immunoglobulin-like receptor (KIR) gene and HLA-Cw, and the level of inhibitory KIRs expressed on NK cell surface and the cytotoxicity of NK cell against AML leukemic cells in vitro. NK cell were isolated and purified from 27 healthy donors by isolation kit, Target cells were blasts derived from bone marrow of 30 patients with AML.Inhibitory KIRs expression knew as CD158a, CD158b was analyzed by flow cytometry to estimate the percentage of NK cells that could be inhibited by the HLA-Cw ligands..KIR and HLA gene typing were performed by PCR –SSP. from donors and patients respectively. NK cytotoxicity against AML leukemic cells demonstrated by MTT which showed the correlation between NK cytotoxicity and the number of KIR/HLA matches. the NK-susceptible K562 cell line which lacks HLA class I expression, was used as a positive control target in all cytotoxicity assays, autologous non-NK cell was used as negative control target cell. the cytotoxicity assays was performed in E:T 50:1 20:1 10:1 5:1 2.5:1. Results demonstrated the less number of KIR/HLA-Cw matches, the more NK cells are activated..0 match of NK cell/target cell KIR/HLA-Cw, cytotoxicity was (50.66±8.40)%,1 match and 2 matches were (38.28±6.71)%, (19.74±4.15)%, F=20.226, P<0.001. NK cells expressed KIRs also had relationship with cytotoxicity, inhibitory KIRs expressed >50%, the cytotoxicity is 10%, inhibitory KIRs expressed 20%–50%, the cytotoxicity is 20%, inhibitory KIRs <25%, the cytotoxicity is 55%, F=16.276,p<0.001. Therefore these data indicate NK cell kill AML leukemic cells mechanism follow KIR/HLA-Cw mismatch theory, the level of inhibitory KIRs expressed on NK cell surface showed the percentage of NK cells that could be inhibited by the HLA-Cw ligands. Key words: KIR NK cell CD158 HLA-Cw

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

Thermal stresses reduce natural killer cell cytotoxicity

1995 ◽  
Vol 79 (3) ◽  
pp. 732-737 ◽  
Author(s):  
S. J. Won ◽  
M. T. Lin
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Cell Activity ◽  
Cortisol Concentration ◽  
Target Cells ◽  
Nk Cell Activity ◽  
Colonic Temperature ◽  
Effector Target

The effects of different ambient temperatures (Ta) on the splenic natural killer (NK) cell activity, effector-target cell conjugation activity, and NK cell numbers were assessed in male inbred C3H/HeNCrj mice (7–10 wk old). The splenic NK cytotoxic activities were examined in a 4-h 51Cr release assay in mouse spleen cells that were obtained 1, 2, 4, 8, or 16 days after exposure to Ta of 22, 4, or 35 degrees C. The percentage of conjugating lymphocytes was calculated by counting the number of single lymphocytes bound to single target cells per 400 effector cells. The numbers of NK cells were expressed by the percentage of 5E6-positive cells. The 5E6 identifies only a subset of NK cells. It was found that the splenic NK cell activity, the effector-target cell conjugation activity, or the NK cell number began to fall 1 day after cold (Ta 4 degrees C) or heat (Ta 35 degrees C) stress. After a 16-day period of either cold or heat exposure, the fall in the splenic NK cell activity, the effector-target cell conjugation activity, or the number of 5E6-positive subsets of NK cells was still evident. Compared with those of the control group (Ta 22 degrees C), the cold-stressed mice had higher adrenal cortisol concentration and lower colonic temperature, whereas the heat-stressed animals had higher adrenal cortisol concentration and higher colonic temperature during a 16-day period of thermal exposure. However, neither cold nor heat stress affected both the body weight gain and the spleen weight in our mice.

Molecular analysis of the methylprednisolone-mediated inhibition of NK-cell function: evidence for different susceptibility of IL-2– versus IL-15–activated NK cells

Blood ◽  
2007 ◽  
Vol 109 (9) ◽  
pp. 3767-3775 ◽  
Author(s):  
Laura Chiossone ◽  
Chiara Vitale ◽  
Francesca Cottalasso ◽  
Sara Moretti ◽  
Bruno Azzarone ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Surface Expression ◽  
Steroid Treatment ◽  
Cross Linking ◽  
Target Cells ◽  
Activating Receptors ◽  
And Function ◽  
Nk Cytotoxicity

Abstract Steroids have been shown to inhibit the function of fresh or IL-2–activated natural killer (NK) cells. Since IL-15 plays a key role in NK-cell development and function, we comparatively analyzed the effects of methylprednisolone on IL-2– or IL-15–cultured NK cells. Methylprednisolone inhibited the surface expression of the major activating receptors NKp30 and NKp44 in both conditions, whereas NK-cell proliferation and survival were sharply impaired only in IL-2–cultured NK cells. Accordingly, methylprednisolone inhibited Tyr phosphorylation of STAT1, STAT3, and STAT5 in IL-2–cultured NK cells but only marginally in IL-15–cultured NK cells, whereas JAK3 was inhibited under both conditions. Also, the NK cytotoxicity was similarly impaired in IL-2– or IL-15–cultured NK cells. This effect strictly correlated with the inhibition of ERK1/2 Tyr phosphorylation, perforin release, and cytotoxicity in a redirected killing assay against the FcRγ+ P815 target cells upon cross-linking of NKp46, NKG2D, or 2B4 receptors. In contrast, in the case of CD16, inhibition of ERK1/2 Tyr phosphorylation, perforin release, and cytotoxicity were not impaired. Our study suggests a different ability of IL-15–cultured NK cells to survive to steroid treatment, thus offering interesting clues for a correct NK-cell cytokine conditioning in adoptive immunotherapy.

scholarly journals Understanding the Synergy of NKp46 and Co-Activating Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 753 ◽  
Author(s):  
Loris Zamai ◽  
Genny Del Zotto ◽  
Flavia Buccella ◽  
Sara Gabrielli ◽  
Barbara Canonico ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Response ◽  
Cell Activation ◽  
Nk Cell ◽  
High Sensitivity ◽  
Leukemic Cells ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Oncologic Patients ◽  
The Way

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

scholarly journals Human natural killer cells can inhibit clonogenic growth of fresh leukemic cells

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 596-599 ◽  
Author(s):  
M Beran ◽  
M Hansson ◽  
R Kiessling
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Colony Growth ◽  
Inhibitory Effect ◽  
Leukemic Cells ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Release Assay

Abstract The effect of allogenic human natural killer (NK) cells on fresh leukemic cells from three patients was investigated. The low levels of leukemic target cell lysis in the conventional 51Cr-release assay contrasted with a pronounced inhibitory effect on the colony growth of the clonogeneic leukemic target cells (L-CFC). The ability of allogeneic lymphocytes to inhibit L-CFC increased if they were pretreated with interferon (IFN), which also increased their NK activity, monitored in parallel cytotoxicity assay, against K562. Furthermore, cell separation procedures, based on differences in density among nonadherent lymphocytes, revealed that only NK cell containing fractions were inhibitory. We have also compared the susceptibility to NK-mediated L-CFC inhibition of IFN pretreated leukemic target cells with that of nontreated target cells. As in the case of NK lysis in general, this pretreatment of target cells abolished the presumably NK-mediated L-CFC inhibition. In conclusion, these data provide the first indication that NK cells can inhibit the in vitro growth of fresh clonogenic leukemia cells from patients with nonlymphocytic leukemia. The identity of NK cells as effector is strongly suggested by Percoll separation and responsiveness to interferon; the final proof awaits more sophisticated purification of these cells.

scholarly journals Specificity of HLA class I antigen recognition by human NK clones: evidence for clonal heterogeneity, protection by self and non-self alleles, and influence of the target cell type.

1993 ◽  
Vol 178 (4) ◽  
pp. 1321-1336 ◽  
Author(s):  
V Litwin ◽  
J Gumperz ◽  
P Parham ◽  
J H Phillips ◽  
L L Lanier
Keyword(s):  
Nk Cells ◽  
Mhc Class I ◽  
Target Cell ◽  
Nk Cell ◽  
Hla Class I ◽  
Class I ◽  
Target Cells ◽  
Clonal Heterogeneity ◽  
Multiple Alleles ◽  
Wide Range

Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Christopher M Borges ◽  
Kevin Wasko ◽  
Jared M Nasser ◽  
Kelly Donahue ◽  
Amanda Pfautz ◽  
...  
Keyword(s):  
Cell Growth ◽  
Nk Cells ◽  
Target Cell ◽  
Nk Cell ◽  
Hematologic Malignancies ◽  
Target Cells ◽  
Publicly Traded ◽  
Killing Assay ◽  
Serial Killing

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Fc-Engineered NKG2D-IgG1 Fusion Proteins Target Leukemia Cells for Antibody-Dependent Cellular Cytotoxicity (ADCC) of NK Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1537-1537 ◽  
Author(s):  
Julia Hilpert ◽  
Katrin Baltz-Ghahremanpour ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
Gundram Jung ◽  
...  
Keyword(s):  
Nk Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Antibody Dependent Cellular Cytotoxicity

Abstract Abstract 1537 The capability of anti-tumor antibodies to recruit Fc-receptor (FcR) bearing effector cells like NK cells, a feature considered critical for therapeutic success, can be markedly improved by modifications of the human IgG1 part. At present, Fc-engineered antibodies targeting leukemia cells are yet not available. The various ligands of the NK cell-activating immunoreceptor NKG2D (NKG2DL) are generally absent on healthy cells but upregulated on malignant cells of various origins including leukemia. We aimed to take advantage of the tumor-restricted expression of NKG2DL by using them as target-antigens for Fc-optimized NKG2D-IgG1 fusion proteins targeting leukemia cells for antibody-dependent cellular cytotoxicity (ADCC) and IFN-g production of NK cells. NKG2D-IgG1 fusion proteins with distinct modifications in their Fc portion were generated as previously described (Lazar 2006; Armour 1999). Compared to wildtype NKG2D-Fc (NKG2D-Fc-WT), the mutants (S239D/I332E and E233P/L234V/L235A/DG236/A327G/A330S) displayed highly enhanced (NKG2D-Fc-ADCC) and abrogated (NKG2D-Fc-KO) affinity to the NK cell FcgRIIIa receptor but comparable binding to NKG2DL-expressing target cells. Functional analyses with allogenic NK cells and leukemia cell lines as well as primary leukemic cells of AML and CLL patients revealed that NKG2D-Fc-KO significantly (p<0.05, Mann-Whitney U test) reduced NK cytotoxicity and IFN-g production (about 20% and 30% reduction, respectively), which can be attributed to blockade of NKG2DL-mediated activating signals. Treatment with NKG2D-Fc-WT significantly (p<0.05, Mann-Whitney U test) enhanced NK reactivity (about 20% and 100% increase in cytotoxicity and cytokine production, respectively). The effects observed upon treatment with NKG2D-Fc-ADCC by far exceeded that of NKG2D-Fc-WT resulting in at least doubled NK ADCC and IFN-g production compared to NKG2D-Fc-WT. When applied in combination with Rituximab in analyses with CLL cells, a clear additive effect resulting in a more than four-fold increase of ADCC and FcgRIIIa-induced IFN-g production was observed. The NKG2D-Fc fusion proteins did not induce NK reactivity against healthy blood cells, which is in line with the tumor-restricted expression of NKG2DL. Of note, treatment with NKG2D-Fc-ADCC also significantly (p<0.05, Mann-Whitney U test) enhanced reactivity (up to 70% increase) of NK cells against NKG2DL-positive AML and CLL cells among patient PBMC in an autologous setting. Together, our results demonstrate that Fc-engineered NKG2D-Fc-ADCC fusion proteins can effectively target NKG2DL-expressing leukemia cells for NK anti-tumor reactivity. In line with the hierarchically organized potential of the various activating receptors governing NK reactivity and due to their highly increased affinity to the FcgRIIIa receptor, NKG2D-Fc-ADCC potently enhances NK anti-leukemia reactivity despite the inevitable reduction of activating signals upon binding to NKG2DL. Due to the tumor-restricted expression of NKG2DL, Fc-modified NKG2D-Ig may thus constitute an attractive means for immunotherapy of leukemia. Disclosures: No relevant conflicts of interest to declare.

CD30/CD16A Tandab AFM13-Induced Target Cell Lysis By NK-Cells Is Enhanced By CD137 Co-Stimulation and Blocking PD-1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2747-2747 ◽  
Author(s):  
Xing Zhao ◽  
Narendiran Rajasekaran ◽  
Uwe Reusch ◽  
Jens-Peter Marschner ◽  
Martin Treder ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Target Cell ◽  
Tumor Size ◽  
Target Cells ◽  
In Vivo Models ◽  
Effector Cells ◽  
Cytotoxicity Assays

Abstract Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated. Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC. To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58. Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines. Conclusions: The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

2009 ◽  
Vol 284 (24) ◽  
pp. 16463-16472 ◽  
Author(s):  
Pedro Roda-Navarro ◽  
Hugh T. Reyburn
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Cell Activation ◽  
Cell Function ◽  
Nk Cell ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Receptor Complex ◽  
Secretory Lysosomes

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and “resting” NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of ∼50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.

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