Lytic Effector Research Articles

Effect of indoximod-based chemo-immunotherapy in patients with pediatric brain tumors on activation and clonal proliferation of a circulating population of early non-exhausted stem-like CD8+ T cells whose on-treatment expansion is predictive of long-term outcome.

2566 Background: Combination chemo-immunotherapy is a promising strategy, but it is difficult to determine whether clinical responses are associated with on-target immune activation or just due to chemotherapy. The field lacks well-accepted readouts of on-treatment T cell activation/expansion to answer this question. Methods: We analyzed longitudinal blood samples from 30 patients with pediatric brain tumors treated with the IDO-inhibitor drug indoximod combined with either chemotherapy (n=27, NCT02502708, NCT04049669) or chemotherapy plus ibrutinib (n=3, NCT05106296). Patients in this “Training Set” were selected to include multiple trials, various histologies/molecular risk factors, and a range of outcomes (overall survival (OS) range 6-55 months). Longitudinal blood samples (2-11 samples/patient) were obtained over 4-36 months, depending on duration of treatment, and analyzed by single-cell RNA and TCR sequencing (scRNAseq/TCRseq). TCR clonotypes of interest were defined as having at least 2-fold expansion (or appearance de novo) during treatment compared to baseline. To calculate a “Clonal Expansion Index” (CEI), the total number of T cells belonging to treatment-expanded clonotypes was summed for each sample and expressed as a percentage of total T cells. The peak CEI value for each patient was used to stratify subjects into “Immune Responders” vs “Non-responders”, based on an optimized cutoff established by Receiver Operating Characteristic (ROC) analysis with Youden’s J statistic. Results: In these patients, CEI ranged from <1% to >60% of all circulating T cells. The optimized cut-point was found to be a CEI of 8.6%, producing a sensitivity of 91% and specificity of 77% for this dataset. Kaplan-Meier analysis showed a highly significant 3-fold difference in median OS for the CEI-High patients (26.5 months) compared to CEI-Low (8.9 months, p=0.0003). The CEI metric was far superior as a predictor of long-term outcome than radiographic response by RANO, RAPNO or iRANO criteria, which were not predictive. UMAP clustering and Monocle trajectory analysis of the treatment-expanded T cells revealed that the majority arose from a population of early stem-like cells (TCF7+ LEF1+ FOXP1+), progressing through a more activated stem-like population (HOBIT/ZNF683+) to attain proliferation and effector maturation states. Throughout this sequence, the T cells showed minimal markers of exhaustion (PDCD1, HAVCR2, TOX) and acquired a lytic effector phenotype. Conclusions: We hypothesize that clonal expansion of this population of non-exhausted stem-like T cells, as quantitated by the CEI assay, provides a mechanistically based pharmacodynamic readout of T cell response to indoximod-based (and perhaps other) chemo-immunotherapy.

Targeting terminal pathway reduces brain complement activation, amyloid load and synapse loss, and improves cognition in a mouse model of dementia

Complement is dysregulated in the brain in Alzheimer’s Disease and in mouse models of Alzheimer’s disease. Each of the complement derived effectors, opsonins, anaphylatoxins and membrane attack complex (MAC), have been implicated as drivers of disease but their relative contributions remain unclarified. Here we have focussed on the MAC, a lytic and pro-inflammatory effector, in the AppNL−G−F mouse amyloidopathy model. To test the role of MAC, we back-crossed to generate AppNL−G−F mice deficient in C7, an essential MAC component. C7 deficiency ablated MAC formation, reduced synapse loss and amyloid load and improved cognition compared to complement-sufficient AppNL−G−F mice at 8–10 months age. Adding back C7 caused increased MAC formation in brain and an acute loss of synapses in C7-deficient AppNL−G−F mice. To explore whether C7 was a viable therapeutic target, a C7-blocking monoclonal antibody was administered systemically for one month in AppNL−G−F mice aged 8–9 months. Treatment reduced brain MAC and amyloid deposition, increased synapse density and improved cognitive performance compared to isotype control-treated AppNL−G−F mice. The findings implicate MAC as a driver of pathology and highlight the potential for complement inhibition at the level of MAC as a therapy in Alzheimer’s disease.

NK cell-induced damage to P.falciparum-infected erythrocytes requires ligand-specific recognition and releases parasitophorous vacuoles that are phagocytosed by monocytes in the presence of immune IgG.

Natural killer (NK) cells lyse virus-infected cells and transformed cells through polarized delivery of lytic effector molecules into target cells. We have shown that NK cells lyse Plasmodium falciparum-infected red blood cells (iRBC) via antibody-dependent cellular cytotoxicity (ADCC). A high frequency of adaptive NK cells, with elevated intrinsic ADCC activity, in people chronically exposed to malaria transmission is associated with reduced parasitemia and resistance to disease. How NK cells bind to iRBC and the outcome of iRBC lysis by NK cells has not been investigated. We applied gene ablation in inducible erythrocyte precursors and antibody-blocking experiments with iRBC to demonstrate a central role of CD58 and ICAM-4 as ligands for adhesion by NK cells via CD2 and integrin αMβ2, respectively. Adhesion was dependent on opsonization of iRBC by IgG. Live imaging and quantitative flow cytometry of NK-mediated ADCC toward iRBC revealed that damage to the iRBC plasma membrane preceded damage to P. falciparum within parasitophorous vacuoles (PV). PV were identified and tracked with a P.falciparum strain that expresses the PV membrane-associated protein EXP2 tagged with GFP. After NK-mediated ADCC, PV were either found inside iRBC ghosts or released intact and devoid of RBC plasma membrane. Electron microscopy images of ADCC cultures revealed tight NK-iRBC synapses and free vesicles similar in size to GFP+ PV isolated from iRBC lysates by cell sorting. The titer of IgG in plasma of malaria-exposed individuals that bound PV was two orders of magnitude higher than IgG that bound iRBC. This immune IgG stimulated efficient phagocytosis of PV by primary monocytes. The selective NK-mediated damage to iRBC, resulting in release of PV, and subsequent phagocytosis of PV by monocytes may combine for efficient killing and removal of intra-erythrocytic P.falciparum parasite. This mechanism may mitigate the inflammation and malaria symptoms during blood-stage P. falciparum infection.

Lipopolysaccharide Primes Human Macrophages for Noncanonical Inflammasome-Induced Extracellular Vesicle Secretion.

Human macrophages secrete extracellular vesicles (EVs) loaded with numerous immunoregulatory proteins. Vesicle-mediated protein secretion in macrophages is regulated by poorly characterized mechanisms; however, it is now known that inflammatory conditions significantly alter both the quantities and protein composition of secreted vesicles. In this study, we employed high-throughput quantitative proteomics to characterize the modulation of EV-mediated protein secretion during noncanonical caspase-4/5 inflammasome activation via LPS transfection. We show that human macrophages activate robust caspase-4-dependent EV secretion upon transfection of LPS, and this process is also partially dependent on NLRP3 and caspase-5. A similar effect occurs with delivery of the LPS with Escherichia coli-derived outer membrane vesicles. Moreover, sensitization of the macrophages through TLR4 by LPS priming prior to LPS transfection dramatically augments the EV-mediated protein secretion. Our data demonstrate that this process differs significantly from canonical inflammasome activator ATP-induced vesiculation, and it is dependent on the autocrine IFN signal associated with TLR4 activation. LPS priming preceding the noncanonical inflammasome activation significantly enhances vesicle-mediated secretion of inflammasome components caspase-1, ASC, and lytic cell death effectors GSDMD, MLKL, and NINJ1, suggesting that inflammatory EV transfer may exert paracrine effects in recipient cells. Moreover, using bioinformatics methods, we identify 15-deoxy-Δ12,14-PGJ2 and parthenolide as inhibitors of caspase-4-mediated inflammation and vesicle secretion, indicating new therapeutic potential of these anti-inflammatory drugs.

CAR T Cells Targeting Membrane-Bound Hsp70 on Tumor Cells Mimic Hsp70-Primed NK Cells.

Strategies to boost anti-tumor immunity are urgently needed to treat therapy-resistant late-stage cancers, including colorectal cancers (CRCs). Cytokine stimulation and genetic modifications with chimeric antigen receptors (CAR) represent promising strategies to more specifically redirect anti-tumor activities of effector cells like natural killer (NK) and T cells. However, these approaches are critically dependent on tumor-specific antigens while circumventing the suppressive power of the solid tumor microenvironment and avoiding off-tumor toxicities. Previously, we have shown that the stress-inducible heat shock protein 70 (Hsp70) is frequently and specifically expressed on the cell surface of many different, highly aggressive tumors but not normal tissues. We could take advantage of tumors expressing Hsp70 on their membrane (‘mHsp70’) to attract and engage NK cells after in vitro stimulation with the 14-mer Hsp70 peptide TKDNNLLGRFELSG (TKD) plus low dose interleukin (IL)-2. However, a potential limitation of activated primary NK cells after adoptive transfer is their comparably short life span. T cells are typically long-lived but do not recognize mHsp70 on tumor cells, even after stimulation with TKD/IL-2. To combine the advantages of mHsp70-specificity with longevity, we constructed a CAR having specificity for mHsp70 and retrovirally transduced it into primary T cells. Co-culture of anti-Hsp70 CAR-transduced T cells with mHsp70-positive tumor cells stimulates their functional responsiveness. Herein, we demonstrated that human CRCs with a high mHsp70 expression similarly attract TKD/IL-2 stimulated NK cells and anti-Hsp70 CAR T cells, triggering the release of their lytic effector protein granzyme B (GrB) and the pro-inflammatory cytokine interferon (IFN)-γ, after 4 and 24 hours, respectively. In sum, stimulated NK cells and anti-Hsp70 CAR T cells demonstrated comparable anti-tumor effects, albeit with somewhat differing kinetics. These findings, together with the fact that mHsp70 is expressed on a large variety of different cancer entities, highlight the potential of TKD/IL-2 pre-stimulated NK, as well as anti-Hsp70 CAR T cells to provide a promising direction in the field of targeted, cell-based immunotherapies which can address significant unmet clinical needs in a wide range of cancer settings.

Granulomatous bacterial diseases in fish: An overview of the host's immune response

Bacterial diseases represent the main impediment to the development of fish aquaculture. Granulomatous diseases caused by bacteria lead to fish culture losses by high mortality rates and slow growth. Bacteria belonging to genera Streptococcus spp., Mycobacterium sp., Nocardia sp., Francisella sp., and Staphylococcus sp. have been implicated in the development of granulomatous processes. The granuloma formation and the fish's immune response continue to be the subject of scientific research. In fish, the first defense line is constituted by non-specific humoral factors through growth-inhibiting substances such as transferrin and antiproteases, or lytic effectors as lysozyme and antimicrobial peptides, and linking with non-specific phagocyte responses. If the first line is breached, fish produce antibody constituents for a specific humoral defense inhibiting bacterial adherence, as well as the mobilization of non-phagocytic host cells and counteracting toxins from bacteria. However, bacteria causing granulomatous diseases can be persistent microorganisms, difficult to eliminate that can cause chronic diseases, even using some immune system components to survive. Understanding the infectious process leading to granulomatosis and how the host's immune system responds against granulomatous diseases is crucial to know more about fish immunology and develop strategies to overcome granulomatous diseases.

Live cell imaging of lytic granule motility in anti-ErbB2 CAR NK cells and FcR NK cells plus Herceptin towards ErbB2+ breast cancer cells

Background & Aim Upon encountering a susceptible target, NK cells mediate directed cytotoxicity by exocytosis of lytic effector molecules such as perforin and granzymes. The steps leading to NK cell granule exocytosis are highly regulated. Granule exocytosis is preceded by convergence of granules to the microtubule organizing center (MTOC) and subsequent polarization of the MTOC and granules to the immunological synapse (IS). In case of antibody-dependent cell-mediated cytotoxicity (ADCC), it has been shown that signaling through the Fc receptor is critical to polarize MTOC and granules to the IS with otherwise resistant targets. Methods, Results & Conclusion Here we used spinning disk confocal microscopy for live cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR expressing NK-92 cells (NK-92/5.28.z) and research-grade high affinity FcR expressing NK-92 cells plus Herceptin™ towards ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. Interestingly, unmodified NK-92 cells in combination with MDA-MB-453 cells showed granule convergence to the MTOC, but failed to polarize MTOC and granules to the IS. In contrast, retargeting by either CAR or mAb/FcR towards the ErbB2 antigen on MDA-MB-453 enabled granule polarization to the IS resulting in highly effective cytotoxicity. Granule polarization was rapid in both the CAR and high affinity FcR expressing NK-92 cells after cell-cell contact was initiated. These observations suggest that retargeting of NK-92 cells by either transgenic CAR or high affinity FcR expression in combination with tumor-specific antibodies confers tumor cell lysis by enabling the otherwise impaired MTOC and granule polarization to the IS which resembles the physiological exocytosis cascade observed in naturally occurring ADCC.

Recognition of Apoptotic Cells by Viruses and Cytolytic Lymphocytes: Target Selection in the Fog of War.

Viruses and cytolytic lymphocytes operate in an environment filled with dying and dead cells, and cell fragments. For viruses, irreversible fusion with doomed cells is suicide. For cytotoxic T lymphocyte and natural killer cells, time and limited lytic resources spent on apoptotic targets is wasteful and may result in death of the host. We make the case that the target membrane cytoskeleton is the best source of information regarding the suitability of potential targets for engagement for both viruses and lytic effector cells, and we present experimental evidence for detection of apoptotic cells by HIV, without loss of infectivity.

The Pyroptotic Cell Death Effector Gasdermin D Is Activated by Gout-Associated Uric Acid Crystals but Is Dispensable for Cell Death and IL-1β Release.

The pyroptotic cell death effector gasdermin D (GSDMD) is required for murine models of hereditary inflammasome-driven, IL-1β-dependent, autoinflammatory disease, making it an attractive therapeutic target. However, the importance of GSDMD for more common conditions mediated by pathological IL-1β activation, such as gout, remain unclear. In this study, we address whether GSDMD and the recently described GSDMD inhibitor necrosulfonamide (NSA) contribute to monosodium urate (MSU) crystal-induced cell death, IL-1β release, and autoinflammation. We demonstrate that MSU crystals, the etiological agent of gout, rapidly activate GSDMD in murine macrophages. Despite this, the genetic deletion of GSDMD or the other lytic effector implicated in MSU crystal killing, mixed lineage kinase domain-like (MLKL), did not prevent MSU crystal-induced cell death. Consequently, GSDMD or MLKL loss did not hinder MSU crystal-mediated release of bioactive IL-1β. Consistent with in vitro findings, IL-1β induction and autoinflammation in MSU crystal-induced peritonitis was not reduced in GSDMD-deficient mice. Moreover, we show that the reported GSDMD inhibitor, NSA, blocks inflammasome priming and caspase-1 activation, thereby preventing pyroptosis independent of GSDMD targeting. The inhibition of cathepsins, widely implicated in particle-induced macrophage killing, also failed to prevent MSU crystal-mediated cell death. These findings 1) demonstrate that not all IL-1β-driven autoinflammatory conditions will benefit from the therapeutic targeting of GSDMD, 2) document a unique mechanism of MSU crystal-induced macrophage cell death not rescued by pan-cathepsin inhibition, and 3) show that NSA inhibits inflammasomes upstream of GSDMD to prevent pyroptotic cell death and IL-1β release.

RORγ Agonists Enhance the Sustained Antitumor Activity through Intrinsic Tc17 Cytotoxicity and Tc1 Recruitment.

Activation of RORγ with synthetic small-molecule agonists has been shown to enhance type 17 effector (CD4+ Th17 and CD8+ Tc17 cells) cell functions and decrease immunosuppressive mechanisms, leading to improved antitumor efficacy in adoptive cell transfer and syngeneic murine tumor models. However, whether Tc17 cells possess intrinsic cytotoxicity and the mechanism they use to lyse target cells is controversial. We report here that Tc17 cells were lytic effectors dependent on perforin and granzyme A. In contrast to Tc1 cells, Tc17 cells resisted activation-induced cell death and maintained granzyme A levels, which conferred the ability to lyse target cells in serial encounters. Thus, although the acute lytic capacity of Tc17 cells could be inferior to Tc1 cells, comparable lysis was achieved over time. In addition to direct lytic activity, Tc17 cells infiltrated early into the tumor mass, recruited other CD8+ T cells to the tumor, and enhanced the survival and lytic capability of these cells during repeated target encounters. Synthetic RORγ agonists further augmented Tc17 survival and lytic activity in vitro and in vivo, controlling tumor growth not only through direct cytotoxicity, but also through recruitment and improved function of other effector cells in the tumor microenvironment, which suggests complementary and cooperate activities for effective immunotherapy.

The Role of Type VI Secretion System Effectors in Target Cell Lysis and Subsequent Horizontal Gene Transfer

Bacteria use type VI secretion systems (T6SSs) to manipulate host cells during pathogenesis or to kill competing bacteria, which, in some cases, increases horizontal gene transfer. These functions largely depend on T6SS regulation, dynamics, and the set of effectors that the system delivers into the target cells. Here, we show that Acinetobacter baylyi ADP1 assembles a highly dynamic T6SS capable of killing and lysing bacterial cells. T6SS function depends on conserved T6SS components as well as Acinetobacter-specific genes of unknown function. Five different effectors, encoded next to VgrG or PAAR proteins and their cognate immunity proteins, cause distinct changes in the prey cells, resulting in various degrees of their lysis. Prey lysis correlates with the rate of DNA transfer from prey to predator, suggesting that lytic effectors are required for efficient T6SS-dependent horizontal gene transfer in naturally competent bacteria.

Predicting the impact of CD8+ T cell polyfunctionality on HIV disease progression.

During the chronic phase of HIV-1 infection, polyfunctional CD8+ T cell responses, which are characterized by a high frequency of cells able to secrete multiple cytokines simultaneously, are associated with lower virus loads and slower disease progression. This relationship may arise for different reasons. Polyfunctional responses may simply be stronger. Alternatively, it could be that the increased functional diversity in polyfunctional responses leads to lower virus loads and slower disease progression. Lastly, polyfunctional responses could contain more CD8+ T cells that mediate a specific key function that is primarily responsible for viral control. Disentangling the influences of overall strength, functional diversity, and specific function on viral control and disease progression is very relevant for the rational design of vaccines and immunotherapy using cellular immune responses. We developed a mathematical model to study how polyfunctional CD8+ T cell responses mediating lytic and nonlytic effector functions affect the CD4+ T cell count and plasma viral load. We based our model on in vitro data on the efficacy of gamma interferon (IFN-γ) and macrophage inflammatory protein 1β (MIP-1β)/RANTES against HIV. We find that the strength of the response is a good predictor of disease progression, while functional diversity has only a minor influence. In addition, our model predicts for realistic levels of cytotoxicity that immune responses dominated by nonlytic effector functions most positively influence disease outcome. It is an open question in HIV research why polyfunctional CD8+ T cell responses are associated with better viral control, while individual functional correlates of protection have not been identified so far. Identifying the role of CD8+ T cells in HIV-1 infection has important implications for the potential development of effective T cell-based vaccines. Our analysis provides new ways to think about a causative role of CD8+ T cells by studying different hypotheses regarding why polyfunctional CD8+ T cells might be more advantageous. We identify measurements that have to be obtained in order to evaluate the role of CD8+ T cells in HIV-1 infection. In addition, our method shows how individual cell functionality data can be used in population-based virus dynamics models.

135 HIV-associated immunodeficiency despite potent antiretroviral therapy with CD4 T cell reconstitution

CD4 T cell reconstitution in patients with HIV is associated with increased responses to conventional vaccines and improved resistance to opportunistic infections. However, prolonged therapy fails to replenish a capacity for immune control over HIV and patients who interrupt therapy inevitably rebound to pre-treatment viral burden in plasma. Only elite patients, termed NVS or natural virus suppressors (1), control viremia to undetectable levels in the absence of therapy. Our earlier studies on NVS patients showed that this group was distinguished from all other HIV patients by the presence of normal levels of CD56+ V 2V 2 T cells in blood (2) reflecting reconstitution of the T cell receptor repertoire through new cell synthesis (3). The CD56 marker identifies cytotoxic effector lymphocytes including NK, CD8, T and NKT subsets, but it’s function remains unknown. We have now shown persistent depression of CD56 expression on CD8 T cells from HIV+ individuals except for NVS patients where the levels approach those found in normal controls. The CD56+ CD8 T cell subset is highly responsive to stimulus, expresses high levels of perforin/granzyme and the small population remaining in HIV patients on therapy, also express the immune exhaustion marker TIM-3, indicating cells may be lost through an apoptosis mechanism. The persisting defect in CD56 expression for 2 T cells subsets is consistent with a lack of cytolytic effector function and is present in patients with complete virus suppression for many years due to treatment, long after the T cell population is refreshed by new cell synthesis. The block to lytic effector function may explain why treated patients fail to eradicate viral reservoirs; this mechanism is a key target for new therapies designed to cure HIV disease.1. M. M. Sajadi, A. Heredia, N. Le, N. T. Constantine, R. R. Redfield, HIV-1 natural viral suppressors: control of viral replication in the absence of therapy. AIDS 21, 517 (2007).2. D. J. Riedel et al. Natural viral suppressors of HIV-1 have a unique capacity to maintain gamma delta T cells. AIDS 23, 1955 (2009).3. S. Chaudhry, C. Cairo, V. Venturi, C. D. Pauza, The gamma delta T cell receptor repertoire is reconstituted in HIV patients after prolonged antiretroviral therapy. AIDS epub ahead of print, (2013).

Levels of CD56+TIM-3- Effector CD8 T Cells Distinguish HIV Natural Virus Suppressors from Patients Receiving Antiretroviral Therapy

Prolonged antiretroviral therapy (ART) with effective HIV suppression and reconstitution of CD4 T cells, fails to restore CD8 T cell lytic effector function that is needed to eradicate the viral reservoir. Better understanding of the phenotype and function of circulating CD8 cells in HIV patients will contribute to new targeted therapies directed at increasing CD8 T cell lytic effector function and destruction of the viral reservoir. We show that CD8 T cells from ART treated patients had sharply reduced expression of CD56 (neural cell adhesion molecule-1), a marker associated with cytolytic function whereas elite patients who control HIV in the absence of ART had CD56+ CD8 T cell levels similar to uninfected controls. The CD56+ CD8 T cells had higher perforin upregulation as well as degranulation following stimulation with HIV gag peptides compared with CD56 negative CD8 T cells. Elite patients had the highest frequencies of perforin producing CD56+ CD8 T cells among all HIV+ groups. In patients receiving ART we noted high levels of the exhaustion marker TIM-3 on CD56+ CD8 T cells, implying that defective effector function was related to immune exhaustion. CD56+ CD8 T cells from elite or treated HIV patients responded to PMA plus ionomycin stimulation, and expressed transcription factors T-bet and EOMES at levels similar to uninfected controls. Consequently, the lytic effector defect in chronic HIV disease is due to immune exhaustion and quantitative loss of CD56+ CD8 T cells and this defect is not repaired in patients where viremia is suppressed and CD4 T cells are recovered after ART.Reconstituting the cytotoxic CD56+ subset of CD8+ T cells through new interventions might improve the lytic effector capacity and contribute to reducing the viral reservoir. Our initial studies indicate that IL-15 treatment partly reverses the CD56 defect, implying that myeloid cell defects could be targeted for immune therapy during chronic HIV disease.

Viral Escape from CD8+ T Cells: Evidence for a Lytic Effector Mechanism?

Recent evidence suggests that, in SIV and HIV-1 infection, CD8+ T cells mediate anti-viral control predominantly via non-cytolytic mechanisms. This in apparent conflict with the observation that SIV and HIV-1 variants that escape CD8+ T cell surveillance are frequently and reproducibly selected.We use a cellular automata model that describes spatial and temporal HIV-1 dynamics to address the question is the observation of escape variants evidence that CD8+ T cells kill HIV-1 infected cells?

Complete Global Dynamics of a Delayed Viral Infection Model with Lytic and Nonlytic Effectors

We perform the global stability analysis for a viral infection model with two types of immune response. The model is formulated as a system of delay differential equations. We determine the global dynamics in terms of the basic reproduction number using Lyapunov functionals. Our results on the global stability of the infected equilibrium give an affirmative answer to the conjecture offered in the paper [K. Wang, W. Wang and X. Liu, Comput. Math. Appl. 51 (2006), 1593-1610].

Novel mutation in STXBP2 prevents IL-2–induced natural killer cell cytotoxicity

We have identified dizygotic twins with a novel syntaxin-binding protein 2 (STXBP2) mutation, where cytotoxicity cannot be restored with IL-2. This defines STXBP2 as an absolute requirement for NK cell cytotoxic function.

Lenalidomide Treatment Enhances Immunological Synapse Formation of Cord Blood Natural Killer Cells with B Cells Derived From Chronic Lymphocytic Leukemia

Abstract 1794Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL) including suppressed humoral and cell-mediated immune responses. The immunomodulatory agent lenalidomide has shown effective clinical activity against CLL, but its mechanism of action is poorly understood. Previous work has demonstrated that the T cell immunological synapse and functional defects in CLL can be reversed following lenalidomide treatment (J Clin Invest. 2008; 118). Polymerization of F-actin at the NK cell immunological synapse with tumor cells is required for signaling molecules to assemble and regulate NK cell activation and effector function. Confocal microscopy was used to visualize and analyze F-actin polymerization at the immune synapse between NK cells and CLL cells. The impaired immune synapse defect identified in CLL could result from not only the defects of CLL B cells but also defects in the CLL NK cells or a combination of both factors. To investigate the contribution of each factor, we examined synapse formation in experiments using CLL B cells with autologous CLL NK cells or healthy allogeneic NK cells. Conjugates formed with healthy NK cells and CLL B cells exhibited a strong band of F-actin at the immune synapse. In contrast, significantly less actin polymerization at the synapse was observed in autologous CLL NK cells and CLL B cells (P < 0.01). These results indicate CLL B cells, together with CLL NK cells contributed to the immune dysfunction in CLL. As autologous NK cell function in CLL is suppressed, we investigated the utility of CB as a potential functional source of NK cells for CLL immunotherapy. We examined the effect of lenalidomide on NK cell immune synapse function with CLL B cells acting as APCs. We demonstrated that ex vivo treatment of CLL cells with lenalidomide (500 ng/ml) for 48 hours caused a significant increase in the ability of autologous CLL NK cells to form F-actin immune synapses with CLL B cells. The same treatment of CLL B cells also significantly increased the ability of CB-NK cells to form F-actin immunological synapses with these treated CLL B cells compared to untreated CLL B cells (33.6% to 67.3%, P < 0.01, n=6).Our results also show that lenalidomide treatment of autologous NK cells from CLL patients enhanced synapse formation with treated CLL cells compared to experiments using untreated NK cells, but with reduced function compared to CB NK cells. Of note, lenalidomide treatment was shown to increase the recruitment of the signaling molecule Lck to NK cell:CLL cell synapse site, that is known to regulate lytic synapse function. Importantly, lenalidomide treatment significantly increased CB-NK killing of CLL B cells compared to untreated CLL B cells (20.5% versus 48.2%, E:T ratio of 10:1, n = 6, p < 0.001). These results provide insight into the potential mechanism of action of lenalidomide’s anti-leukemic function – priming CLL tumor cells for enhanced NK cell lytic synapse formation and effector function. In addition, the data suggests that immunotherapeutic strategies utilizing a combination of CB-NK cells and lenalidomide has an enhanced clinical efficacy in CLL. Disclosures:Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Antigen-specific versus Antigen-nonspecific Immunotherapeutic Approaches for Human Melanoma: The Need for Integration for Optimal Efficacy?

Due to its immunogenecity and evidence of immune responses resulting in tumor regression, metastatic melanoma has been the target for numerous immunotherapeutic approaches. Unfortunately, based on the clinical outcomes, even the successful induction of tumor-specific responses does not correlate with efficacy. Immunotherapies can be divided into antigen-specific approaches, which seek to induce T cells specific to one or several known tumor associated antigens (TAA), or with antigen-nonspecific approaches, which generally activate T cells to become nonspecifically lytic effectors. Here the authors critically review the different immunotherapeutic approaches in melanoma.

Perforin activates clathrin- and dynamin-dependent endocytosis, which is required for plasma membrane repair and delivery of granzyme B for granzyme-mediated apoptosis

AbstractCytotoxic T lymphocytes and natural killer cells destroy target cells via the polarized exocytosis of lytic effector proteins, perforin and granzymes, into the immunologic synapse. How these molecules enter target cells is not fully understood. It is debated whether granzymes enter via perforin pores formed at the plasma membrane or whether perforin and granzymes are first endocytosed and granzymes are then released from endosomes into the cytoplasm. We previously showed that perforin disruption of the plasma membrane induces a transient Ca2+ flux into the target cell that triggers a wounded membrane repair response in which lysosomes and endosomes donate their membranes to reseal the damaged membrane. Here we show that perforin activates clathrin- and dynamin-dependent endocytosis, which removes perforin and granzymes from the plasma membrane to early endosomes, preserving outer membrane integrity. Inhibiting clathrin- or dynamin-dependent endocytosis shifts death by perforin and granzyme B from apoptosis to necrosis. Thus by activating endocytosis to preserve membrane integrity, perforin facilitates granzyme uptake and avoids the proinflammatory necrotic death of a membrane-damaged cell.