Release Of Cytolytic Granules Research Articles

CXCR4 has a dual role in improving the efficacy of BCMA-redirected CAR-NK cells in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disease with a preferential bone marrow (BM) tropism. Enforced expression of tissue-specific chemokine receptors has been shown to successfully guide adoptively-transferred CAR NK cells towards the malignant milieu in solid cancers, but also to BM-resident AML and MM. For redirection towards BM-associated chemokine CXCL12, we armored BCMA CAR-NK-92 as well as primary NK cells with ectopic expression of either wildtype CXCR4 or a gain-of-function mutant CXCR4R334X. Our data showed that BCMA CAR-NK-92 and -primary NK cells equipped with CXCR4 gained an improved ability to migrate towards CXCL12 in vitro. Beyond its classical role coordinating chemotaxis, CXCR4 has been shown to participate in T cell co-stimulation, which prompted us to examine the functionality of CXCR4-cotransduced BCMA-CAR NK cells. Ectopic CXCR4 expression enhanced the cytotoxic capacity of BCMA CAR-NK cells, as evidenced by the ability to eliminate BCMA-expressing target cell lines and primary MM cells in vitro and through accelerated cytolytic granule release. We show that CXCR4 co-modification prolonged BCMA CAR surface deposition, augmented ZAP-70 recruitment following CAR-engagement, and accelerated distal signal transduction kinetics. BCMA CAR sensitivity towards antigen was enhanced by virtue of an enhanced ZAP-70 recruitment to the immunological synapse, revealing an increased propensity of CARs to become triggered upon CXCR4 overexpression. Unexpectedly, co-stimulation via CXCR4 occurred in the absence of CXCL12 ligand-stimulation. Collectively, our findings imply that co-modification of CAR-NK cells with tissue-relevant chemokine receptors affect adoptive NK cell therapy beyond improved trafficking and retention within tumor sites.

Bone Marrow Stroma Impairs CAR-T Cell Proliferation and Function: Mechanistic Insights

Chimeric Antigen Receptor (CAR)-T cells have proven highly successful in the treatment of some haemopoietic neoplasms. However, in several cases the treatment fails and disease does not respond and/or relapses. Amongst the different factors impacting on a negative clinical outcome, an unfavourable immunosuppressive microenvironment has been proposed as playing a critical role, particularly in the context of acute myeloid leukemia. We hypothesised that bone marrow mesenchymal stromal cells (BM-MSC) acquire immunosuppressive properties after exposure to the by-stander effects of activated CAR-T cells against leukemic cells. We used a CAR-T cell consisting of a CD33 binder and CD28z-OX40 costimulatory moiety. BM-MSC were exposed to the CAR following in vitro activation on CD33-coated plates for 24 hours. To model the different ways by which stromal cells can license their immunosuppressive activity, BM-MSC were exposed to the supernatant harvested from the cultures of activated CARs or in direct contact with the activated CARs themselves. In the first case, after incubation with the CAR-conditioned medium (CAR-cm), BM-MSCs were investigated for their transcriptome by RNASeq. Gene ontology analysis of the transcriptome revealed a strong NF-κB and STAT1 signature in BM-MSC exposed to the CAR-cm compared to those which were not exposed. Amongst the most upregulated molecules, we identified several chemokines (CXCL10, CCL8, CXCL8, and CXCL9), PDL1 and CECAM1, IL-6, indoleamine-2,3 dyoxigenase (IDO), and most prominently PTGES and PTGS2. We then interrogated the immune functional profile by incubating by CAR-cm licensed BM-MSC with fresh CD33-activated CAR-T cells. We observed that CAR proliferation, cytotoxicity and cytokine production was significantly inhibited. CAR function was restored if Rel-A - a REL-associated protein critically involved in NF-κB heterodimer formation, nuclear translocation and activation - was inhibited in BM-MSC by shRNA before exposure to CAR-cm. We previously described that BM-MSC undergo apoptosis when in contact with activated cytotoxic T cells and that this also triggers their immunosuppressive capability. Therefore, we evaluated the impact of cell contact dependent mechanisms on BM-MSC licensing. CD33-activated CAR-T cells were incubated with BM-MSC and the proportion of annexin-V positive BM-MSC were assessed after 4 hours. We observed that a large fraction of BM-MSC underwent apoptosis and that this was dependent on caspase activation, but not RIP kinase 3, and the release of cytolytic granules in a contact-dependent fashion. The supernatant of apoptotic BM-MSC but not the terminally apoptotic and neither the live cells exhibited a strong inhibitory activity on CAR-T cell proliferation, cytotoxicity and effector cytokine production. Similarly to what described with the CAR-cm induced profile, BM-MSC immunosuppression required NF-κB and was effected via the COX2 pathway. We finally tested the impact of apoMSC in vivo in a xenograft model of AML (Fig. 1). CD33-positive KG1 cells were injected in NSG mice and 2 weeks later, CD33-specific CAR-T were administered with or without (controls) apoMSC. Peripheral blood samples were collected 7, 14, and 21 days after. We observed an inhibitory effect of apoMSc on the in vivo expansion of CAR-T cells which was accompanied by disease progression. Accordingly, the survival of the mice treated with apoMSC was markedly reduced compared to those receiving CAR-T cells only. We conclude that BM-MSC can be induced to acquire immunosuppressive properties by activated CAR-T cells through 2 different but overlapping mechanisms, both dependent on NF-κB and COX2 signalling pathway (Fig. 2). Our work provides critical information to overcome resistance to CAR-T cell treatment by neutralising the negative effects of the stromal microenvironment.

Cytotoxic T lymphocytes require transcription for infiltration but not target cell lysis.

Effector cytotoxic T lymphocytes (CTLs) are critical for ridding the body of infected or cancerous cells. CTL T cell receptor (TCR) ligation not only drives the delivery and release of cytolytic granules but also initiates a new wave of transcription. In order to address whether TCR-induced transcriptomic changes impact the ability of CTLs to kill, we asked which genes are expressed immediately after CTLs encounter targets and how CTL responses change when inhibiting transcription. Our data demonstrate that while expression of cytokines/chemokines and transcriptional machinery depend on transcription, cytotoxic protein expression and cytolytic activity are relatively robust to transcription blockade, with CTLs lysing nearby target cells for several hours after actinomycin D treatment. Monitoring CTL movement among target cells after inhibiting transcription demonstrates an infiltration defect that is not rectified by provision of exogenous cytokine/chemokine gradients, indicating a cell-intrinsic transcriptional requirement for infiltration. Together, our results reveal differential molecular control of CTL functions, separating recruitment and infiltration from cytolysis.

GRISCELLI SYNDROME TYPE 2 –CLINICAL APPROACH AND CASE REPORT

Griscelli syndrome type 2 is rare authosomal ressesive disorder caused by a defect in the RAB27A gene, which affects a melanosome-anchoring complex in melanocytes, affecting release of cytolytic granules from T and NK cells. Children with GS type 2 develop an uncontrolled T-lymphocyte and macrophage activation syndrome known as hemophagocytic syndrome (HS) or hemophagocytic lymphohistiocytosis (HLH). We describe a 3 years old girl patient classic features of Griscelli syndrome type 2 A 3-year-old girl was admitted to hematology with complaints of LAP, hepatosplenomegaly and pancytopenia (WBC- 3080/µl, Hb-7.6 g/dl, Neutr.-350/µl, PLT - 165000/µl). The patient's condition was below the percentile, her skin was bronze, her hair was silver-grey. HLH criteria were met (triglycerides 458 mg/mL, ferritin 3445 ng/mL, fibrinogen 180 mg/dl). Morphology of the bone marrow was hypocellular, signs of dyserythropoiesis (stage I) and megakaryocytes were reduced According to the clinical and laboratory data(hepatosplenomegaly, increased ferritin, hypertriglyceridemia, pancytopenia, hyperthermia resistant to antimicrobial therapy, silver-gray hair, pigment balls of hair seen light microscope) and the death of another undiagnosed child in the family, suggested likely primary HLH and GS. As a result of genetic analysis (homozygous mutation c.514_ 518delCAAGC(p.GLN172Asnfs*,rs767481076)1 in the RAB27A gene), the diagnosis of GS type 2 was confirmed. The patient was treated according to the HLH 2004 protocol. CSA levels were measured once a week. IVIG support was given based on IgG levels. HSCT was planned from patient's healthy HLA-matched sibling, but HSCT was delayed because the brother was infant. After 45 weeks of maintenance therapy, etoposide was discontinued, dose of dexamethasone was reduced to 5 mg/kg, but CSA was continued at the same dose. Control studies are carried out once a week. As far as possible HSCT is planning The prognosis of patients with Griscelli syndrome is poor. It is usually rapidly fatal within 1-4 years without aggressive treatment and bone marrow transplantation at onset of an accelerated phase. HSCT is more successful when implemented early course of the disease. Palliative care includes treatment and prophylaxis care infections and immunosupressor therapy in accelerated phases. Some patients have died after transplantation, but others have had lasting remissions

Gold-seaurchin based immunomodulator enabling photothermal intervention and αCD16 transfection to boost NK cell adoptive immunotherapy

Despite huge potentials of NK cells in adoptive cell therapy (ACT), formidable physical barriers of the tumor tissue and deficiency of recognizing signals on tumor cells severely prevent NK cell infiltrating, activating and killing performances. Herein, a nano-immunomodulator AuNSP@αCD16 (CD16 antibody encoding plasmid) is explored to remodel the tumor microenvironment (TME) for improving the antitumor effects of adoptive NK cells. The as-prepared AuNSP, with a seaurchin-like gold core and a cationic polymer shell, exhibited a high gene transfection efficiency and a stable NIR-II photothermal capacity. The AuNSP could trigger mild photothermal intervention to partly destroy tumors and collapse the dense physical barriers, making a permeable TME for NK cell infiltration. What's more, the AuNSP could achieve αCD16 gene transfection to modify tumor surface with CD16 antibody, marking a unique structure on tumor cells for NK cell recognition and then lead to strong NK cell activation by CD16-mediated antibody-dependent cellular cytotoxicity (ADCC). As expected, the designed AuNSP@αCD16 induced an immune-favorable TME for NK cell performing killing functions against solid tumors, increasing the release of cytolytic granules and proinflammatory cytokines, which ultimately achieved a robustly boosted NK cell-based immunotherapy. Hence, the AuNSP@αCD16-mediated TME reconstituting strategy provides a substantial perspective for NK-based ACT on solid tumors. Statement of significanceIn adoptive cell therapy (ACT), natural killer (NK) cells exhibit greater off-the-shelf utility and improved safety comparing with T cells, but the efficacy of NK cell therapy is severely compromised by formidable physical barriers of the tumor tissue and deficiency of NK cell recognizing signals on tumor cells. Herein, a nano-immunomodulator AuNSP@αCD16, with the abilities of inducing mild photothermal intervention and modifying the tumor cell surface with αCD16, is explored to reconstruct an infiltration-favorable and activation-facilitating tumor microenvironment for NK cells to perform killing functions. Such a simple and safe strategy is believed as a very promising candidate for future NK-based ACT.

Efficient killing of tumor cells by CAR-T cells requires greater number of engaged CARs than TCRs

Although CAR-T cells are widely used to treat cancer, efficiency of CAR-T cell cytolytic responses has not been carefully examined. We engineered CAR specific for HMW-MAA (high-molecular-weight melanoma-associated antigen) and evaluated potency of CD8+ CAR-T cells to release cytolytic granules and to kill tissue-derived melanoma cells, which express different levels of HMW-MAA. CAR-T cells efficiently killed melanoma cells expressing high level of HMW-MAA, but not melanoma cells with lower levels of HMW-MAA. The same melanoma cells presenting significantly lower level of stimulatory peptide-MHC ligand were readily lysed by T cells transduced with genes encoding α,β-TCR specific for the peptide-MHC ligand. The data suggest that higher level of targeted molecules is required to engage a larger number of CARs than TCRs to induce efficient cytolytic granule release and destruction of melanoma cells. Understanding the difference in molecular mechanisms controlling activation thresholds of CAR- versus TCR-mediated responses will contribute to improving efficiency of CAR T cells required to eliminate solid tumors presenting low levels of targeted molecules.

Tuning Antibody Presentation to Enhance T-Cell Activation for Downstream Cytotoxicity

Cytotoxic effector cells are an integral component of the immune response against pathogens and diseases such as cancer and thus of great interest to researchers who wish to enhance the native immune response. Although researchers routinely use particles to stimulate cytotoxic T cells, few studies have comprehensively investigated: (1) beyond initial activation responses (i.e., proliferation and CD25/CD69 expression) to downstream cancer-killing effects and (2) how to drive cytotoxic T-cell responses by adjusting biomolecular and physical properties of particles. In this study, we designed particles displaying an anti-CD3 antibody to activate cytotoxic T cells and study their downstream cytotoxic effects. We evaluated the effect of antibody immobilization, particle size, molecular surface density of an anti-CD3 antibody, and the inclusion of an anti-CD28 antibody on cytolytic granule release by T cells. We found that immobilizing the anti-CD3 antibody onto smaller nanoparticles elicited increased T-cell activation products for an equivalent delivery of the anti-CD3 antibody. We further established that the mechanism behind increased cancer cell death was associated with the proximity of T cells to cancer cells. Functionalizing particles additionally with the anti-CD28 antibody at an optimized antibody density caused increased T-cell proliferation and T-cell binding but we observed no effective increase in cytotoxicity. Meaningfully, our results are discussed within the context of commercially available and widely used anti-CD3/28 Dynabeads. These results showed that T-cell activation and cytotoxicity can be optimized with a molecular presentation on smaller particles and thus, offer exciting new possibilities to engineer T-cell activation responses for effective outcomes.

Comparison of abacavir-specific effector and proliferating functions of CD8 T cells in abacavir-treated HIV-1 patients.

Susceptibility to abacavir hypersensitivity (ABH) in HIV-1-positive patients is strongly linked to the carriage of HLA-B*57:01 and the potential mechanism includes drug-specific activation of cytokine producing CD8 T cells exclusively in individuals carrying HLA-B*57:01. Here, we report a detailed characterization of abacavir-induced functional response of CD8 T cells in HLA-B*57:01pos individuals. Peripheral blood mononuclear cells (PBMNCs) from HLA-B*57:01pos ABHpos and HLA-B*57:01neg ABHneg individuals were stimulated with abacavir. Multicolor flow cytometry was performed to assess the cytokine (IFNγ) production and degranulation (CD107a expression) after 6-18 hr culture and to enumerate proliferating CD4/CD8 T cells by culturing carboxyfluorescein diacetate succinimidyl ester-loaded PBMNCs for 7 days. CD8 T cells from HLA-B*57:01pos ABHpos individuals were multifunctional: proliferating, IFNγ producing, degranulating (CD107apos ), and both degranulating and IFNγ producing (CD107apos IFNγpos ). Degranulating CD8 T cells in general and both degranulating and IFNγ producing CD8 T cells in particular dominated abacavir-specific immune response. All functional responses were partially blocked by addition of HLA-B*57:01-reactive Bw4 mAb, but not by non-HLA-B*57:01-reactive Bw6 mAb. In conclusion, the study demonstrates that abacavir-specific CD8 T-cell-restricted immune response in HLA-B*57:01pos ABHpos HIV-1 patients has multiple effector and proliferating functions, where the primary effector response appears to be the release of cytolytic granules. The findings have implications for immunotherapy of HLA-related drug hypersensitivities.

Natural Killer (NK) Cell Assays in Immunotoxicity Testing.

It is well known that natural killer (NK) cells are involved in defense against viruses and some tumors. NK cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes, and granulysin. It is increasingly important to evaluate NK cell function in immunotoxicity testing. NK cell function can be evaluated by determining cytolytic activity against target tumor cells by the 51Cr-release assay and also by determining the number of NK cells in peripheral blood in humans and in the spleen in animals using flow cytometry. Recently, the intracellular levels of perforin, granzymes, and granulysin determined by flow cytometry have also been used in the evaluation of NK cell function. In addition, chemical-induced apoptosis in NK cells also has been applied to evaluate the immunotoxicity of chemicals. This chapter will describe the methods for NK cell assays in immunotoxicity testing.

A Novel Function for the Streptococcus pneumoniae Aminopeptidase N: Inhibition of T Cell Effector Function through Regulation of TCR Signaling.

Streptococcus pneumoniae (Spn) causes a variety of disease states including fatal bacterial pneumonia. Our previous finding that introduction of Spn into an animal with ongoing influenza virus infection resulted in a CD8+ T cell population with reduced effector function gave rise to the possibility of direct regulation by pneumococcal components. Here, we show that treatment of effector T cells with lysate derived from Spn resulted in inhibition of IFNγ and tumor necrosis factor α production as well as of cytolytic granule release. Spn aminopeptidase N (PepN) was identified as the inhibitory bacterial component and surprisingly, this property was independent of the peptidase activity found in this family of proteins. Inhibitory activity was associated with reduced activation of ZAP-70, ERK1/2, c-Jun N-terminal kinase, and p38, demonstrating the ability of PepN to negatively regulate TCR signaling at multiple points in the cascade. These results reveal a novel immune regulatory function for a bacterial aminopeptidase.

Efficient Killing of High Risk Neuroblastoma Using Natural Killer Cells Activated by Plasmacytoid Dendritic Cells.

High-risk neuroblastoma (NB) remains a major therapeutic challenge despite the recent advent of disialoganglioside (GD2)-antibody treatment combined with interleukin (IL)-2 and granulocyte monocyte-colony stimulating factor (GM-CSF). Indeed, more than one third of the patients still die from this disease. Here, we developed a novel approach to improve the current anti-GD2 immunotherapy based on NK cell stimulation using toll-like receptor (TLR)-activated plasmacytoid dendritic cells (pDCs). We demonstrated that this strategy led to the efficient killing of NB cells. When the expression of GD2 was heterogeneous on NB cells, the combination of pDC-mediated NK-cell activation and anti-GD2 treatment significantly increased the cytotoxicity of NK cells against NB cells. Activation by pDCs led to a unique NK-cell phenotype characterized by increased surface expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), with increased expression of CD69 on CD56dim cytotoxic cells, and strong interferon-γ production. Additionally, NB-cell killing was mediated by the TRAIL death-receptor pathway, as well as by the release of cytolytic granules via the DNAX accessory molecule 1 pathway. NK-cell activation and lytic activity against NB was independent of cell contact, depended upon type I IFN produced by TLR-9-activated pDCs, but was not reproduced by IFN-α stimulation alone. Collectively, these results highlighted the therapeutic potential of activated pDCs for patients with high-risk NB.

Abstract A092: Surpassing the emerging immune-checkpoint inhibitor market

Abstract Antibodies to checkpoint inhibitors like PD-1, PD-L1 and CTLA4 activate resident Cytotoxic T-Lymphocytes to attack and kill tumor cells. The main mechanism that they employ to kill the cancer cells is through the release of cytolytic granules that contain perforin and Granzyme-A (GrA) protease. Perforin punches holes in the tumor cell membrane facilitating entry of GrA into the cytoplasm where it selectively degrades the SET oncoprotein, which is at the core of the SET complex composed of the SET oncoprotein and at least 5 different tumor suppressor proteins. Upon GrA mediated degradation of the SET oncoprotein, the tumor suppressors including nm23-H1 and TREX-1 deoxyribonucleases (DNAse's) are released into the nucleus where genomic DNA is degraded leading to cancer cells apoptosis. Importantly, we found that the SET oncoprotein is over-expressed in more than 90% of all leukemias and in 50 to 75% of all human solid tumor biopsies ranging from breast cancer to pancreatic cancer and others. As an inhibitor of tumor suppressors, SET overexpression in cancer helps to explain why tumor suppressors fail to effectively function to kill cancer cells. We discovered that OP449, a peptide mimetic of apolipoprotein-E (apoE), specifically antagonizes SET to release tumor suppressors that then act to kill the cancer cells. Initially we found that OP449 antagonized SET in MDA-MB-231 breast cancer cells to release the nm23-H1 DNAse tumor suppressor, which accumulated in the nucleus where it actively participated in degrading genomic DNA. Using MDA-MB-231 cells, OP449 caused a dose dependent death in cultures and in orthotopic xenografts with NSG mice with severe T-cell deficiency. Interestingly, using MDA-MB-231 cells labeled with green fluorescent protein to produce xenografts, the number of metastases in brain and lung were significantly reduced in OP449 treated animals compared to controls. MDA-MB-231, MDA-MB-436 and MDA-MB-468 tumor growth was significantly impaired in xenografts treated with OP449 or with small hairpin DNA (shDNA) constructs used to reduce levels of the SET protein, but not with empty shDNA vector controls. Using a genetically engineered mouse model (GEMM) of breast cancer, tumor volumes in Myc Neu: Blg CRE (Her2/Myc) mice treated with OP449 were significantly less than their control counterparts and similar to animals treated with Lapatinib alone. In contrast, combining OP449 plus Lapatinib caused tumor regression to the limit of measurement in this GEMM model. Additional xenografts with CAPAN1 pancreatic cancer cell lines similarly showed significant reductions of tumor volumes in OP449 treated animals compared to vehicle controls, and with shDNA constructs that reduce SET levels and reduce tumor volumes. These experiments demonstrate that in the presence or absence of T-cells, that OP449 can provide effective control of tumor growth. These results provide the groundwork for a next-generation immuno-oncology approach that directly antagonizes the SET oncoprotein to mimic proteolytic degradation of SET by Granzyme-A resulting from immune cell activation and cytolytic granule action. While clinical use of checkpoint inhibitor antibodies has proven very effective in treating deadly cancers, the rate of unwanted side effects of uncontrolled immune activation is also increasing with more widespread use of these antibodies. In contrast, SET antagonists like OP449 targeted to the cancer cells do not require the presence of activated immune cells, but do implicate the release and re-activation of endogenous tumor suppressors in the tumor tissue to kill cancer cells in vitro and in vivo. Citation Format: Michael P. Vitek. Surpassing the emerging immune-checkpoint inhibitor market. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A092.

The two‐pore domain K2P channel TASK2 drives human NK‐cell proliferation and cytolytic function

Natural killer (NK) cells are a subset of cytotoxic lymphocytes that recognize and kill tumor- and virus-infected cells without prior stimulation. Killing of target cells is a multistep process including adhesion to target cells, formation of an immunological synapse, and polarization and release of cytolytic granules. The role of distinct potassium channels in this orchestrated process is still poorly understood. The current study reveals that in addition to the voltage-gated KV 1.3 and the calcium-activated KCa 3.1 channels, human NK cells also express the two-pore domain K2 P channel TASK2 (TWIK-related acid-sensitive potassium channel). Expression of Task2 varies among NK-cell subsets and depends on their differentiation and activation state. Despite its different expression in TASK2(high) CD56(bright) CD16(-) and TASK2(low) CD56(dim) CD16(+) NK cells, TASK2 is involved in cytokine-induced proliferation and cytolytic function of both subsets. TASK2 is crucial for leukocyte functional antigen (LFA-1) mediated adhesion of both resting and cytokine-activated NK cells to target cells, an early step in killing of target cells. With regard to the following mechanism, TASK2 plays a role in release of cytotoxic granules by resting, but not IL-15-induced NK cells. Taken together, our data exhibit two-pore potassium channels as important players in NK-cell activation and effector function.

TRAIL-mediated killing of acute lymphoblastic leukemia by plasmacytoid dendritic cell-activated natural killer cells.

Acute lymphoblastic leukemia (ALL) still frequently recurs after hematopoietic stem cell transplantation (HSCT), underscoring the need to improve the graft-versus-leukemia (GvL) effect. Natural killer (NK) cells reconstitute in the first months following HSCT when leukemia burden is at its lowest, but ALL cells have been shown to be resistant to NK cell-mediated killing. We show here that this resistance is overcome by NK cell stimulation with TLR-9-activated plasmacytoid dendritic cells (pDCs). NK cell priming with activated pDCs resulted in TRAIL and CD69 up-regulation on NK cells and IFN-γ production. NK cell activation was dependent on IFN-α produced by pDCs, but was not reproduced by IFN-α alone. ALL killing was further enhanced by inhibition of KIR engagement. We showed that ALL lysis was mainly mediated by TRAIL engagement, while the release of cytolytic granules was involved when ALL expressed NK cell activating receptor ligands. Finally, adoptive transfers of activated-pDCs in ALL-bearing humanized mice delayed the leukemia onset and cure 30% of mice. Our data therefore demonstrate that TLR-9 activated pDCs are a powerful tool to overcome ALL resistance to NK cell-mediated killing and to reinforce the GvL effect of HSCT. These results open new therapeutic avenues to prevent relapse in children with ALL.

Therapeutic Potential and Challenges of Natural Killer Cells in Treatment of Solid Tumors

Natural killer (NK) cells are innate lymphoid cells that hold tremendous potential for effective immunotherapy for a broad range of cancers. Due to the mode of NK cell killing, requiring one-to-one target engagement and site-directed release of cytolytic granules, the therapeutic potential of NK cells has been most extensively explored in hematological malignancies. However, their ability to precisely kill antibody coated cells, cancer stem cells, and genotoxically altered cells, while maintaining tolerance to healthy cells makes them appealing therapeutic effectors for all cancer forms, including metastases. Due to their release of pro-inflammatory cytokines, NK cells may potently reverse the anti-inflammatory tumor microenvironment (TME) and augment adaptive immune responses by promoting differentiation, activation, and/or recruitment of accessory immune cells to sites of malignancy. Nevertheless, integrated and coordinated mechanisms of subversion of NK cell activity against the tumor and its microenvironment exist. Although our understanding of the receptor ligand interactions that regulate NK cell functionality has evolved remarkably, the diversity of ligands and receptors is complex, as is their mechanistic foundations in regulating NK cell function. In this article, we review the literature and highlight how the TME manipulates the NK cell phenotypes, genotypes, and tropism to evade tumor recognition and elimination. We discuss counter strategies that may be adopted to augment the efficacy of NK cell anti-tumor surveillance, the clinical trials that have been undertaken so far in solid malignancies, critically weighing the challenges and opportunities with this approach.

Natural Killer Cells: A Potential Therapy for Paediatric Brain Tumours

Natural killer cells are innate immune cells that are naturally involved in tumour immunosurveillance. These cells have direct cytotoxic activity and also secrete proinflammatory cytokines upon activation. The activation of natural killer cells’ cytolytic activity is dependent on a balance between inhibitory signals and activating signals provided by the host cells. Natural killer cells do not exert their activity on healthy cells, due to interactions of inhibitory receptors with self major histocompatibility class I. Inhibition may be overcome when the cell binds tumour-associated antigens on the surface of tumour cells, resulting in the polarized release of cytolytic granules toward the target cell. Natural killer cells demonstrate particular promise for cellular therapy, as they can identify and eliminate tumours that have altered major histocompatibility class I expression. Central nervous system tumours are the second most common paediatric cancer and are associated with a high mortality rate. Many of these high-grade paediatric brain tumours have very dismal prognoses, despite standard treatment regimens, and patients would substantially benefit from a novel type of therapy. Natural killer cells have been shown to be effective against many human malignancies; however, less is known about their efficacy against brain tumours. Treatment of medulloblastoma and adult glioblastoma with natural killer cells has been investigated previously and yielded promising results. Natural killer cells represent a small fraction of peripheral blood, which poses a limitation for their widespread use in adoptive cell therapy. Current research is focussed on establishing an optimal protocol for the ex vivo expansion and activation of natural killer cells. In the future, paediatric brain tumours such as glioblastoma multiforme, ependymoma and atypical teratoid/rhabdoid tumours should be examined for their susceptibility to natural killer cells.

Chimeric Antigen Receptor-Redirected T Cells Display Multifunctional Capacity and Enhanced Tumor-Specific Cytokine Secretion Upon Secondary Ligation of Chimeric Receptor

The aim of the current study was to fully elucidate the functions of T cells genetically modified with an erbB2-specific chimeric antigen receptor (CAR). In this study, key functional parameters of CAR T cells were examined following antigen-specific stimulation of the chimeric anti-erbB2 receptor. Gene-modified T cells produced the cytokines IFN-γ, IL-2, IL-4, IL-10, TNF-α and IL-17, and the chemokine RANTES upon CAR ligation. A multifunctional capacity of these CAR T cells was also demonstrated, where 13.7% of cells were found to simultaneously express IFN-γ and CD107a, indicative of cytolytic granule release. In addition, the CAR T cells were able to respond to a greater degree on the second ligation of CAR, which has not been previously shown. IFN-γ secretion levels were significantly higher on second ligation than those secreted following initial ligation. CAR-expressing T cells were also demonstrated to lyze erbB2-expressing tumor cells in the absence of activity against bystander cells not expressing the erbB2 antigen, thereby demonstrating exquisite specificity. This study demonstrates the specificity of CAR gene-engineered T cells and their capacity to deliver a wide range of functions against tumor cells with an enhanced response capability after initial receptor engagement.

Hemophagocytic Lymphohistiocytosis in B-Cell Lymphoproliferative Disorder: Report of a Rare Association

Hemophagocytic Lymphohistiocytosis in B-Cell Lymphoproliferative Disorder: Report of a Rare Association

Minimal requirement for induction of natural cytotoxicity and intersection of activation signals by inhibitory receptors

AbstractNatural killer (NK) cells provide innate control of infected and neoplastic cells. Multiple receptors have been implicated in natural cytotoxicity, but their individual contribution remains unclear. Here, we studied the activation of primary, resting human NK cells by Drosophila cells expressing ligands for receptors NKG2D, DNAM-1, 2B4, CD2, and LFA-1. Each receptor was capable of inducing inside-out signals for LFA-1, promoting adhesion, but none induced degranulation. Rather, release of cytolytic granules required synergistic activation through coengagement of receptors, shown here for NKG2D and 2B4. Although engagement of NKG2D and 2B4 was not sufficient for strong target cell lysis, collective engagement of LFA-1, NKG2D, and 2B4 defined a minimal requirement for natural cytotoxicity. Remarkably, inside-out signaling induced by each one of these receptors, including LFA-1, was inhibited by receptor CD94/NKG2A binding to HLA-E. Strong inside-out signals induced by the combination of NKG2D and 2B4 or by CD16 could overcome CD94/NKG2A inhibition. In contrast, degranulation induced by these receptors was still subject to inhibition by CD94/NKG2A. These results reveal multiple layers in the activation pathway for natural cytotoxicity and that steps as distinct as inside-out signaling to LFA-1 and signals for granule release are sensitive to inhibition by CD94/NKG2A.

2B4 (CD244) signaling by recombinant antigen-specific chimeric receptors costimulates natural killer cell activation to leukemia and neuroblastoma cells.

Novel natural killer (NK) cell-directed strategies in cancer immunotherapy aim at specifically modulating the balance between NK cell receptor signals toward tumor-specific activation. The signaling lymphocyte activation molecule-related receptor 2B4 (CD244) is an important regulator of NK cell activation. We investigated whether 2B4-enhanced activation signals can redirect the cytolytic function of human NK cells to NK cell-resistant and autologous leukemia and tumor targets. In vitro-stimulated NK cells from healthy donors and pediatric leukemia patients were gene modified with CD19 or G(D2)-specific chimeric receptors containing either the T-cell receptor zeta or 2B4 endodomain alone or combined. Chimeric 2B4 signaling alone failed to induce interleukin-2 receptor up-regulation and cytokine secretion but triggered a specific degranulation response. Integration of the 2B4 endodomain into T-cell receptor zeta chimeric receptors significantly enhanced all aspects of the NK cell activation response to antigen-expressing leukemia or neuroblastoma cells, including CD25 up-regulation, secretion of IFN-gamma and tumor necrosis factor-alpha, release of cytolytic granules, and growth inhibition, and overcame NK cell resistance of autologous leukemia cells while maintaining antigen specificity. These data indicate that the 2B4 receptor has a potent costimulatory effect in NK cells. Antigen-specific 2B4zeta-expressing NK cells may be a powerful new tool for adoptive immunotherapy of leukemia and other malignancies.